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**Provisional chapter**

## **Hypoxia and Pulmonary Hypertension Hypoxia and Pulmonary Hypertension**

Nicoletta Charolidi and Veronica A. Carroll Nicoletta Charolidi and Veronica A. Carroll Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/67151

#### **Abstract**

Vasoconstriction in response to low oxygen tension (hypoxia) in pulmonary arteries is an important physiological adaptation to reroute blood flow to areas of higher oxygenation for effective gaseous exchange. However, chronic hypoxia is a common feature of lung disease, such as chronic obstructive pulmonary disease (COPD). Hypoxic stress triggers cellular phenotypic alterations including increased proliferation and migration of vascular smooth muscle cells (VSMCs), as well as synthesis of extracellular matrix (ECM) proteins that remodel lung vasculature. Remodelling of vessels increases the risk of pulmonary hypertension (PH)—elevated pulmonary arterial pressure—and eventually right heart failure. This chapter will summarise the major pathways and mechanisms involved in hypoxia-driven pulmonary hypertension (PH).

**Keywords:** hypoxia, pulmonary hypertension, HIF-1α, HIF-2α, mTOR, VHL

## **1. Introduction**

The main function of the cardiovascular system is to circulate and deliver oxygen to metabolically active tissues of the body. At physiologically normal oxygen levels, the pulmonary vasculature of healthy individuals is highly distensible, allowing the cardiac output to adjust to levels of activity. In varying degrees of oxygen availability, as in different altitudes, adaptive cardiovascular responses are employed. In acute hypoxia (short, transient reduction in oxygen tension), the pulmonary vascular bed constricts rapidly [1]. When oxygen levels are restored, it dilates again in a swift and reversible manner. With a sustained hypoxic exposure (hours to days), the response is different. There is a loss of pulmonary distensibility, increased arterial pressure, tachycardia and increased workload for the right cardiac ventricle. In return to normoxic conditions, there is, at least in the short term, a limited reversibility of these effects. The Operation Everest II study [2] demonstrated this phenomenon by monitoring the pulmonary vascular pressure of healthy individuals who were exposed to progressive

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

partially pressured oxygen over a period of a few weeks. However, for high-altitude populations, such as the Tibetans, this is not the case. Due to natural selection and adaptation over many thousands of years living under low oxygen conditions, Tibetans have altered oxygensensing mechanisms and pulmonary vascular resistance to sustained hypoxia (discussed later in this chapter) [3].

Healthy, native sea-level dwellers, who move to high altitude, develop high pulmonary arterial pressure, but with time, in the majority of cases, it stabilises and becomes well tolerated [4]. By contrast, people with pre-existing lung pathologies, such as chronic obstructive pulmonary disease (COPD), cystic fibrosis, idiopathic pulmonary fibrosis, bronchiectasis or restrictive chest wall abnormalities, are at risk of developing pulmonary hypertension (PH). Chronic PH lowers quality of life and decreases life expectancy for the affected individuals [5–8].

The pathophysiology of hypoxia-associated PH is characterised by extensive vascular remodelling that leads to arterial narrowing rather than reversible vessel vasoconstriction (**Figure 1**). Processes that take place include endothelial cell dysfunction, muscularisation of normally non-muscular arteries, phenotypic switching and proliferation of vascular smooth muscle cells (VSMCs), increased extracellular matrix deposition and erythrocytosis [7, 9, 10]. In this chapter, recent developments in mechanistic aspects underlying hypoxia-induced pathophysiological changes in PH will be briefly summarised.

**Figure 1.** Schematic representation of pulmonary arterial responses to normoxia, acute hypoxia and chronic hypoxia. With acute and chronic hypoxia, the pulmonary artery undergoes vasoconstriction. In the case of acute hypoxia, the artery can reversibly dilate. But in chronic hypoxia, the artery undergoes nonreversible vascular remodelling characterised by intimal thickening due to VSMC dedifferentiation (loss of contractility, hypertrophy and hyperplasia). Additionally, there is distal muscularisation of non-muscular vessels, a settled-in endothelial cell dysfunction and erythrocytosis. Activation of HIF-1α and HIF-2α as well as over-activation of mTORC1 contributes to VSMC dedifferentiation and the establishment of hypoxic PH. Abbreviations: HIF-1α, hypoxia-inducible factor 1α; HIF-2α, hypoxia-inducible factor 2α; mTORC1, mechanistic target of rapamycin complex 1; PH, pulmonary hypertension.

## **2. Role of endothelial cell dysfunction**

partially pressured oxygen over a period of a few weeks. However, for high-altitude populations, such as the Tibetans, this is not the case. Due to natural selection and adaptation over many thousands of years living under low oxygen conditions, Tibetans have altered oxygensensing mechanisms and pulmonary vascular resistance to sustained hypoxia (discussed later

Healthy, native sea-level dwellers, who move to high altitude, develop high pulmonary arterial pressure, but with time, in the majority of cases, it stabilises and becomes well tolerated [4]. By contrast, people with pre-existing lung pathologies, such as chronic obstructive pulmonary disease (COPD), cystic fibrosis, idiopathic pulmonary fibrosis, bronchiectasis or restrictive chest wall abnormalities, are at risk of developing pulmonary hypertension (PH). Chronic PH lowers quality of life and decreases life expectancy for the affected indi-

The pathophysiology of hypoxia-associated PH is characterised by extensive vascular remodelling that leads to arterial narrowing rather than reversible vessel vasoconstriction (**Figure 1**). Processes that take place include endothelial cell dysfunction, muscularisation of normally non-muscular arteries, phenotypic switching and proliferation of vascular smooth muscle cells (VSMCs), increased extracellular matrix deposition and erythrocytosis [7, 9, 10]. In this chapter, recent developments in mechanistic aspects underlying hypoxia-induced patho-

**Figure 1.** Schematic representation of pulmonary arterial responses to normoxia, acute hypoxia and chronic hypoxia. With acute and chronic hypoxia, the pulmonary artery undergoes vasoconstriction. In the case of acute hypoxia, the artery can reversibly dilate. But in chronic hypoxia, the artery undergoes nonreversible vascular remodelling characterised by intimal thickening due to VSMC dedifferentiation (loss of contractility, hypertrophy and hyperplasia). Additionally, there is distal muscularisation of non-muscular vessels, a settled-in endothelial cell dysfunction and erythrocytosis. Activation of HIF-1α and HIF-2α as well as over-activation of mTORC1 contributes to VSMC dedifferentiation and the establishment of hypoxic PH. Abbreviations: HIF-1α, hypoxia-inducible factor 1α; HIF-2α, hypoxia-inducible factor 2α;

mTORC1, mechanistic target of rapamycin complex 1; PH, pulmonary hypertension.

physiological changes in PH will be briefly summarised.

in this chapter) [3].

212 Hypoxia and Human Diseases

viduals [5–8].

Endothelial cells in pulmonary vessels first sense hypoxic stress. Having a role in maintaining homeostasis, endothelial cells contribute to reducing the vascular tone in order for vasoconstriction to take place and regulate vessel adaptation to increased blood flow [11]. In healthy individuals, the endothelium is responsible for the balanced expression of vasoactive mediators that have either vasodilator ability, such as nitric oxide (NO) and prostacyclin (PGI2 ), or vasoconstrictive properties, such as endothelin-1 (ET-1) [11–14]. ET-1 is released abluminally and triggers vasoconstriction through binding to its VSMC receptors ETA and ETB [15]. However, when ET-1 binds to its endothelial ETB receptor, it can induce vasodilation through NO and PGI2 recruitment [15], while this route also serves for ET-1 clearance from the lung [16].

In pathological PH, as in COPD, endothelial cell dysfunction is one of the major contributing factors for the progression of the condition. It has been found that endothelial NO synthase (eNOS), the enzyme responsible for NO production, as well as prostacyclin synthase, the enzyme responsible for PGI2 production, is markedly diminished in patients with COPD [12, 17]. Furthermore, ET-1 has been reported to have an increased expression in the lungs of patients with PH and is a therapeutic target [14]. ET-1, as well as being a potent vasoconstrictor, is also a VSMC mitogen, acting through smooth muscle ETA and ETB receptors [15]. So in effect, during hypoxic endothelial dysregulation, the pathogenic excess of ET-1 maintains vessel constriction and VSMC proliferation.

## **3. Phenotypic switching of vascular smooth muscle cells**

In hypoxia, the highly plastic VSMCs switch from a contractile to a synthetic phenotype, which is characterised by increased proliferation and extracellular matrix deposition [18]. Differentiated smooth muscle cells express a repertoire of contractile proteins, signalling molecules and receptors for their primary function of vessel contraction. These contractile VSMCs have little capacity for proliferation, protein synthesis or migration [18]. However, pulmonary VSMCs, under chronic hypoxic stimulation, switch to a synthetic state exhibiting hypertrophy, hyperplasia, loss of contractility and migration, contributing to the enlargement of the arterial intimal layer (**Figure 1**) and in the muscularisation of non-muscular pulmonary vessels [9]. Additionally, there is a deposition of collagen and elastic fibres. In extreme cases, the excessive VSMC proliferation can progress from vascular lesions to calcification. These phenomena seem to correlate with the degree of PH extent and COPD severity [19–21].

The endothelial dysfunction that takes place in PH may also contribute to the dedifferentiation and proliferation of VSMCs [22]. Specifically, dysregulated endothelial cells can cause alterations in AKT signalling in VSMCs, which in turn triggers their phenotypic switch [23]. This pathway is also affected by aberrant regulation of the mechanistic target of rapamycin (mTOR) pathway (discussed later in this chapter).

## **4. Hypoxia and pulmonary hypertension**

The major cellular oxygen-sensing mechanism implicated in hypoxia-induced pulmonary hypertension is the hypoxia-inducible factor (HIF) pathway. HIFs are transcription factors that induce the activation of some several hundred genes in response to hypoxia [24]. Initially identified as regulators of erythropoietin (EPO), the hormone responsible for increased red blood cells in response to low oxygen levels, HIFs have since been found to regulate expression of genes that are important for angiogenesis, cellular metabolism, cardiovascular development and cardiovascular control [24–26].

In low oxygen conditions, HIFs bind DNA as heterodimeric complexes of alpha (HIF-α) and beta (HIF-β) subunits, with HIF-α being the subunit regulated by oxygen tension [27]. Higher animals have a series of isoforms for each of the HIF subunits as a result of gene evolutionary duplications [24]. In humans, there are three paralogs of HIF-α—HIF-1α, HIF-2α and HIF-3α—with the first two members being the best characterised [24, 25]. The expression of HIF-1α and HIF-2α is differentially regulated, while their balance is believed to be important for tissue-specific differences in oxygen sensing [25]. They both bind to the same DNA consensus (RCGTG) in hypoxia-response elements of the genome, but they only induce partially overlapping sets of genes [27, 28].

In normoxic conditions, the HIF-α subunit is hydroxylated by Fe(II) prolyl hydroxylase domain (PHD) enzymes (PHD1, PHD2 and PHD3 or otherwise known as Egln2, Egln1 and Egln3) that use 2-oxoglutarate and Fe2+ as substrates [29]. After hydroxylation by PHDs, HIF-α is recognised and bound by the von Hippel-Lindau (VHL) protein, a ubiquitin E3 ligase, which marks HIF-α for proteasomal degradation. In hypoxia, PHD enzymes are inactive allowing HIF-α subunits to translocate to the nucleus and activate HIF target genes. HIFs are further regulated by factor-inhibiting HIF (FIH)-mediated asparaginyl hydroxylation, which impairs their recruitment to transcriptional complexes [30].

Mouse models of HIF-1α and HIF-2α have illustrated that the HIF pathway is critically important for the pulmonary hypoxic response and the development of PH. Heterozygous deficiency of either HIF-1α or HIF-2α allele in mice does not affect their life span, and these animals are largely normal in unstressed, normal oxygen conditions. In response to chronic hypoxia (10% for 3 weeks), HIF-1α+/− mice exhibit an attenuated PH with a low rise in right ventricular pressure and right ventricular hypertrophy [31]. Interestingly, heterozygous HIF-2α+/− mice, exposed to 10% oxygen for 10 weeks, showed a complete lack of any PH manifestation [32]. Of note, animals with hetero- or homozygous mutations in stabilising HIF-2α spontaneously developed progressive PH [33]. These studies all indicate a pathological role of both HIF-α subunits in PH development.

Cell-type-specific inactivation of HIF-α with the use of a variety of promoters has also been studied but with some variable results, which may be due to the method of HIF-α manipulations and/or the use of different mouse strains [34–36]. Nevertheless, there seems to be a clear link between HIFs and PH, since studies from human genetics, including several populations that have adapted to different altitudes, have demonstrated the importance of HIF-2α in pulmonary response to hypoxia and PH pathophysiology [37].

The Tibetans, who have lived for at least 25,000 years in 4000 m elevation and continuously inspired partially pressured oxygen (~80 mmHg), have been identified to have a number of single-nucleotide polymorphisms in close-to-one-another loci near the gene *EPAS1*, which encodes HIF-2α [38]. HIF-2α is the subunit responsible for EPO regulation and in turn erythropoiesis. Tibetans manifest blunted PH and reduced erythropoiesis at high altitude. At sea level, they manifest a lower pulmonary arterial pressure in response to hypoxia when compared with other populations [39, 40]. Recently, a missense mutation in PHD2 (*EGLN1*) was identified which allows for increased PHD2 activity under hypoxic conditions, thereby decreasing HIF-α stabilisation and reducing erythropoiesis at altitude [41].

Further evidence for a role for HIF-2α in PH comes from another human genetic study, which showed that an activating HIF-2α mutation (G→A substitution in position 2097) caused erythrocytosis with elevated total red cell volume and PH in an affected family [42].

## **5. VHL and pulmonary hypertension**

**4. Hypoxia and pulmonary hypertension**

214 Hypoxia and Human Diseases

opment and cardiovascular control [24–26].

their recruitment to transcriptional complexes [30].

of both HIF-α subunits in PH development.

monary response to hypoxia and PH pathophysiology [37].

The major cellular oxygen-sensing mechanism implicated in hypoxia-induced pulmonary hypertension is the hypoxia-inducible factor (HIF) pathway. HIFs are transcription factors that induce the activation of some several hundred genes in response to hypoxia [24]. Initially identified as regulators of erythropoietin (EPO), the hormone responsible for increased red blood cells in response to low oxygen levels, HIFs have since been found to regulate expression of genes that are important for angiogenesis, cellular metabolism, cardiovascular devel-

In low oxygen conditions, HIFs bind DNA as heterodimeric complexes of alpha (HIF-α) and beta (HIF-β) subunits, with HIF-α being the subunit regulated by oxygen tension [27]. Higher animals have a series of isoforms for each of the HIF subunits as a result of gene evolutionary duplications [24]. In humans, there are three paralogs of HIF-α—HIF-1α, HIF-2α and HIF-3α—with the first two members being the best characterised [24, 25]. The expression of HIF-1α and HIF-2α is differentially regulated, while their balance is believed to be important for tissue-specific differences in oxygen sensing [25]. They both bind to the same DNA consensus (RCGTG) in hypoxia-response

elements of the genome, but they only induce partially overlapping sets of genes [27, 28].

In normoxic conditions, the HIF-α subunit is hydroxylated by Fe(II) prolyl hydroxylase domain (PHD) enzymes (PHD1, PHD2 and PHD3 or otherwise known as Egln2, Egln1 and Egln3) that use 2-oxoglutarate and Fe2+ as substrates [29]. After hydroxylation by PHDs, HIF-α is recognised and bound by the von Hippel-Lindau (VHL) protein, a ubiquitin E3 ligase, which marks HIF-α for proteasomal degradation. In hypoxia, PHD enzymes are inactive allowing HIF-α subunits to translocate to the nucleus and activate HIF target genes. HIFs are further regulated by factor-inhibiting HIF (FIH)-mediated asparaginyl hydroxylation, which impairs

Mouse models of HIF-1α and HIF-2α have illustrated that the HIF pathway is critically important for the pulmonary hypoxic response and the development of PH. Heterozygous deficiency of either HIF-1α or HIF-2α allele in mice does not affect their life span, and these animals are largely normal in unstressed, normal oxygen conditions. In response to chronic hypoxia (10% for 3 weeks), HIF-1α+/− mice exhibit an attenuated PH with a low rise in right ventricular pressure and right ventricular hypertrophy [31]. Interestingly, heterozygous HIF-2α+/− mice, exposed to 10% oxygen for 10 weeks, showed a complete lack of any PH manifestation [32]. Of note, animals with hetero- or homozygous mutations in stabilising HIF-2α spontaneously developed progressive PH [33]. These studies all indicate a pathological role

Cell-type-specific inactivation of HIF-α with the use of a variety of promoters has also been studied but with some variable results, which may be due to the method of HIF-α manipulations and/or the use of different mouse strains [34–36]. Nevertheless, there seems to be a clear link between HIFs and PH, since studies from human genetics, including several populations that have adapted to different altitudes, have demonstrated the importance of HIF-2α in pulThe VHL protein is a tumour suppressor and an essential component for the clearance of HIF-α through the ubiquitin-proteasomal degradation pathway [24, 43]. A number of VHL mutations have been described that result in aberrant induction of HIF target genes, due to the loss of function of VHL and in turn to the loss of HIF-α regulation. VHL mutations are associated with VHL syndrome, which is a hereditary condition, characterised by highly vascularised tumours within specific tissues, including the renal, retinal and central nervous system [44]. However, a small number of VHL mutations (R200W, D126N, S183L, D126N) are associated with development of Chuvash polycythemia (CP) [45–47]. CP is a rare autosomal recessive condition that is endemic to the population in Chuvashia, Russia and in the island of Ischia, Italy [46, 48]. Chuvash patients manifest increased haemoglobin and haematocrit with elevated levels of EPO, as well as increased expression of vascular endothelial growth factor (VEGF) and ET-1, which are HIF-α target genes [45–49]. In addition, these patients are highly susceptible to both arterial and venous thrombosis and can develop mild to severe PH [45–49].

The importance of HIF-2α isoform in the regulation of pulmonary vascular control has also been demonstrated by the use of a mouse model of CP [50]. This model carries a hypomorphic VHL allele (with an R200W substitution) and recapitulates all symptoms of the human CP phenotype. Interestingly, when these mice are crossed with HIF-2α+/− or HIF-1α+/− strains for heterozygous deficiency in either of the two HIF-α, they manifest an ameliorated PH phenotype for suppressed HIF-2α, but not for HIF-1α.

Comparison of CP and HIF-2α gain-of-function mutation human phenotypes has additionally shown that the latter condition somehow manifests more moderate symptoms than the first. The explanation for this may be that, in CP, both HIF-α subunits are upregulated, and therefore, there may be an additive effect [51]. Furthermore, VHL has a number of HIF-αindependent functions that may also play a role in the CP phenotype.

## **6. New advances: hypoxic induction of zinc transporters**

Zinc, an essential dietary element, plays an important cytoprotective role for the lung by sheltering the pulmonary epithelium from extrinsic activation of apoptotic pathways following acute lung injury [52]. Zinc transporters are responsible for zinc cellular uptake and homeostasis [53]. A recent linkage analysis study that compared a PH-resistant rat strain, Fisher 344 (F344), with the Wistar Kyoto (WKY) strain identified the gene *Slc39a12*, which encodes the ZIP12 zinc transporter, as a major regulator of hypoxia-induced pulmonary vascular remodelling [53]. In the F344 strain, this gene lacks a crucial thymidine, which leads to a frameshift mutation in exon 11 and renders translation of the protein redundant. ZIP12 is normally expressed in endothelial, interstitial and VSMCs, but its expression increases in remodelled pulmonary vessels following hypoxia-induced PH [53]. ZIP12 is likely a HIF target gene since both HIF-1α and HIF-2α were detected bound to ZIP12 hypoxia-response element. The investigators of this study further generated a ZIP12−/− rat model for comparison with the original F344 and WKY strains and found that genetic disruption of ZIP12 recapitulates the phenotype of the PH-resistant F344 strain under conditions of hypoxia.

Zinc-binding motifs have been considered as potential PH drug-therapeutic targets with phosphodiesterase type 5 (PDE5) and histone deacetylases as examples [54, 55]. Zinc is a structural component of a number of intracellular enzymes, transcription factors, other proteins and cofactors and is a putative drug target for PH.

## **7. Role of hypoxia-inducible microRNAs in pulmonary hypertension**

MicroRNAs (miRNAs) are small non-coding RNA molecules (about 21 nucleotides long) that regulate gene expression post-transcriptionally. Hypoxic stimulation of a variety of human cell types has shown induction of more than 90 miRNAs [56], with altered expression of some of these miRNAs involved in VSMC remodelling and endothelial cell dysfunction in PH [57].

MiRNAs that have been causally implicated in PH include miR-204, miR-138, miR-21 and miR-130/miR-301, among others (**Table 1**). MiR-204 has been shown to be downregulated in VSMCs of patients suffering from PH, as well as in mouse models of the disease [58, 59]. The degree of miR-204 suppression has been found to be inversely proportional to the degree of pulmonary artery resistance and pressure, while compensating for the loss of miR-204 through nebulisation in PH patients has been shown to reverse the VSMC proliferative and anti-apoptotic phenotype [59]. MiR-204 is involved in the activation of the nuclear factor of activated T cell (NFAT) pathway, the Rho pathway, VSMC proliferation and resistance to apoptosis, as well as downregulation of transcripts such as bone morphogenetic protein receptor type II (BMPR2) and interleukin-6 (IL-6) [60–62]. Also, miR-204 regulates the expression of the Runt-related transcription factor 2 (RUNX2), which has been shown to stabilise HIF-1α in chondrocytes by competing with VHL [20, 63]. In the context of hypoxia, RUNX2 is upregulated, since miR-204 is downregulated, and therefore sustains HIF-1α activation,


**Table 1.** MicroRNAs that are causally implicated in PH.

**6. New advances: hypoxic induction of zinc transporters**

teins and cofactors and is a putative drug target for PH.

tions of hypoxia.

216 Hypoxia and Human Diseases

Zinc, an essential dietary element, plays an important cytoprotective role for the lung by sheltering the pulmonary epithelium from extrinsic activation of apoptotic pathways following acute lung injury [52]. Zinc transporters are responsible for zinc cellular uptake and homeostasis [53]. A recent linkage analysis study that compared a PH-resistant rat strain, Fisher 344 (F344), with the Wistar Kyoto (WKY) strain identified the gene *Slc39a12*, which encodes the ZIP12 zinc transporter, as a major regulator of hypoxia-induced pulmonary vascular remodelling [53]. In the F344 strain, this gene lacks a crucial thymidine, which leads to a frameshift mutation in exon 11 and renders translation of the protein redundant. ZIP12 is normally expressed in endothelial, interstitial and VSMCs, but its expression increases in remodelled pulmonary vessels following hypoxia-induced PH [53]. ZIP12 is likely a HIF target gene since both HIF-1α and HIF-2α were detected bound to ZIP12 hypoxia-response element. The investigators of this study further generated a ZIP12−/− rat model for comparison with the original F344 and WKY strains and found that genetic disruption of ZIP12 recapitulates the phenotype of the PH-resistant F344 strain under condi-

Zinc-binding motifs have been considered as potential PH drug-therapeutic targets with phosphodiesterase type 5 (PDE5) and histone deacetylases as examples [54, 55]. Zinc is a structural component of a number of intracellular enzymes, transcription factors, other pro-

**7. Role of hypoxia-inducible microRNAs in pulmonary hypertension**

MicroRNAs (miRNAs) are small non-coding RNA molecules (about 21 nucleotides long) that regulate gene expression post-transcriptionally. Hypoxic stimulation of a variety of human cell types has shown induction of more than 90 miRNAs [56], with altered expression of some of these miRNAs involved in VSMC remodelling and endothelial cell dysfunction in PH [57]. MiRNAs that have been causally implicated in PH include miR-204, miR-138, miR-21 and miR-130/miR-301, among others (**Table 1**). MiR-204 has been shown to be downregulated in VSMCs of patients suffering from PH, as well as in mouse models of the disease [58, 59]. The degree of miR-204 suppression has been found to be inversely proportional to the degree of pulmonary artery resistance and pressure, while compensating for the loss of miR-204 through nebulisation in PH patients has been shown to reverse the VSMC proliferative and anti-apoptotic phenotype [59]. MiR-204 is involved in the activation of the nuclear factor of activated T cell (NFAT) pathway, the Rho pathway, VSMC proliferation and resistance to apoptosis, as well as downregulation of transcripts such as bone morphogenetic protein receptor type II (BMPR2) and interleukin-6 (IL-6) [60–62]. Also, miR-204 regulates the expression of the Runt-related transcription factor 2 (RUNX2), which has been shown to stabilise HIF-1α in chondrocytes by competing with VHL [20, 63]. In the context of hypoxia, RUNX2 is upregulated, since miR-204 is downregulated, and therefore sustains HIF-1α activation, which in turn contributes to aberrant VSMC proliferation, resistance to apoptosis and their transdifferentiation to osteoblast-like cells [20].

MiR-138 is upregulated by hypoxia and suppresses HIF-1α [64]. However, its upregulation also contributes to endothelial cell dysfunction in PH by downregulating the small EF-hand Ca2+-binding protein S100A1 that relays Ca2+ oscillations, controlling vascular tone responses [64].

MiR-21 expression has been found to be upregulated in both pulmonary VSMC and endothelial cells during hypoxic conditions [61, 65]. This upregulation, in turn, leads to downregulation of programmed cell death protein 4 (PDCD4), sprouty homolog 2 (SPRY2) and peroxisome proliferator-activated receptor-α (PPARα), which when dysregulated play a role in the increased proliferation and resistance to apoptosis [65–67]. Treatment of mice with anti-miR-21 during hypoxia showed an improvement in distal pulmonary artery muscularisation [69]. However, miR-21 has also been shown to have a protective effect during PH [61]. Using VHL-null mice, IL-6 transgenic mice, pulmonary vessels from patients with PH as well as deficient (miR-21−/−) or miR-21 overexpression (miR-21+/+) mouse models, it has been demonstrated that miR-21 loss of function causes onset of PH [61]. Specifically, miR-21 deletion showed exaggerated pulmonary vascular remodelling, whereas in mice overexpressing miR-21, these disease-associated phenotypes were abolished [61].

The family of miR-130/301 is also upregulated in pulmonary VSMCs and the endothelium in hypoxia, as well as in the lungs of mice with PH due to chronic hypoxic exposure [68]. This upregulation is mediated by HIF-2α and Oct-4. MiR-130/301 is a master regulator miRNA subordinating other miRNA pathways, and, for instance, it suppresses miR-204 [68].

miR-223, miR-17, miR-130, miR-145, miR-424 and miR503 are also involved in the pathophysiology of PH (reviewed in Ref. [70]). So far, PH animal models have helped greatly in these studies, but the exact role and balance for each of these miRNAs in human PH have not been fully elucidated.

## **8. mTOR signalling in hypoxia-induced pulmonary hypertension**

Mechanistic target of rapamycin (mTOR) is a cellular hub that controls growth factor signalling and nutrient sensing to regulate cell growth, proliferation, metabolism and survival [71]. mTOR is a protein kinase that is the catalytic component of two functionally distinct complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) [72, 73]. mTORC1 is composed of mTOR, Raptor, LST8/GβL, PRAS40 and DEP domain containing mTOR-interacting protein (DEPTOR), and its activity is stimulated by growth factor signals to regulate protein synthesis through 4E-BP1/BP2 and the S6 kinases, S6K1 and S6K2 [74, 75]. By contrast, mTORC2, which comprises mTOR, Rictor, LST8/GβL, DEPTOR, SIN1 and PRR5, regulates cytoskeletal organisation [76, 77] and has a role in phosphorylation of protein kinase C (PKC), protein kinase B (PKB) and serum- and glucocorticoid-induced protein kinase (SGK) to promote cell survival and cell cycle progression [78–80].

Aberrant mTOR activity has a well-characterised role in promoting proliferative diseases including cancer and smooth muscle cell pathologies [71]. mTORC1 signalling is activated following vascular injury promoting Vinhibitor, rapamycin, promotes smooth muscle cell (SMC) remodelling. Accordingly, mTOR inhibitors are widely used in drug-eluting stents to prevent restenosis. In addition, mTOR also regulates the differentiation state of VSMCs since the mTOR inhibitor, rapamycin, promotes SMC differentiation and expression of contractile proteins [81]. mTORC1 activity is low in differentiated contractile VSMCs but becomes activated by growth factors and is thought to contribute to the change towards a synthetic phenotype that is characterised by increased SMC proliferation and migration. As such, rapamycin analogues may have therapeutic potential for treating PH.

The relationship between hypoxic conditions and mTOR is complex and depends, in part, on cellular context. Many cell types respond to prolonged periods of hypoxia by inactivating energy-intensive processes such as protein synthesis and proliferation, and accordingly mTOR is downregulated [82]. By contrast, the vasculature responds to long-term hypoxia by promoting new blood vessel growth—angiogenesis, which in turn, restores O2 to deprived tissues. Hypoxic stress is a key driving force in the vascular remodelling observed in pulmonary hypertension, and HIFs activate pulmonary artery endothelial and smooth muscle cell proliferation, which is mediated by both mTORC1 and mTORC2 [83–85]. Currently, the mechanisms by which hypoxia/HIFs signal to activate mTOR in ECs and VSMCs are poorly understood [86–90].

## **9. Conclusion**

Severe PH associated with hypoxic lung disease is a life-threatening condition with poor survival rates. Despite significant advances in targeted therapeutics for PH, randomised clinical trial data for this particular group of patients are scarce, and it is not clear whether endothelin receptor antagonists will benefit patients with hypoxia-associated PH. Importantly, recent genetic studies identifying mutations in the oxygen-sensing machinery have provided new mechanistic insights into the aetiology of PH. Further studies are required to determine whether specific targeting of HIF-2α will provide additional therapeutic benefit for this complex disease.

## **Author details**

**8. mTOR signalling in hypoxia-induced pulmonary hypertension**

mote cell survival and cell cycle progression [78–80].

analogues may have therapeutic potential for treating PH.

understood [86–90].

218 Hypoxia and Human Diseases

**9. Conclusion**

Mechanistic target of rapamycin (mTOR) is a cellular hub that controls growth factor signalling and nutrient sensing to regulate cell growth, proliferation, metabolism and survival [71]. mTOR is a protein kinase that is the catalytic component of two functionally distinct complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) [72, 73]. mTORC1 is composed of mTOR, Raptor, LST8/GβL, PRAS40 and DEP domain containing mTOR-interacting protein (DEPTOR), and its activity is stimulated by growth factor signals to regulate protein synthesis through 4E-BP1/BP2 and the S6 kinases, S6K1 and S6K2 [74, 75]. By contrast, mTORC2, which comprises mTOR, Rictor, LST8/GβL, DEPTOR, SIN1 and PRR5, regulates cytoskeletal organisation [76, 77] and has a role in phosphorylation of protein kinase C (PKC), protein kinase B (PKB) and serum- and glucocorticoid-induced protein kinase (SGK) to pro-

Aberrant mTOR activity has a well-characterised role in promoting proliferative diseases including cancer and smooth muscle cell pathologies [71]. mTORC1 signalling is activated following vascular injury promoting Vinhibitor, rapamycin, promotes smooth muscle cell (SMC) remodelling. Accordingly, mTOR inhibitors are widely used in drug-eluting stents to prevent restenosis. In addition, mTOR also regulates the differentiation state of VSMCs since the mTOR inhibitor, rapamycin, promotes SMC differentiation and expression of contractile proteins [81]. mTORC1 activity is low in differentiated contractile VSMCs but becomes activated by growth factors and is thought to contribute to the change towards a synthetic phenotype that is characterised by increased SMC proliferation and migration. As such, rapamycin

The relationship between hypoxic conditions and mTOR is complex and depends, in part, on cellular context. Many cell types respond to prolonged periods of hypoxia by inactivating energy-intensive processes such as protein synthesis and proliferation, and accordingly mTOR is downregulated [82]. By contrast, the vasculature responds to long-term hypoxia by

tissues. Hypoxic stress is a key driving force in the vascular remodelling observed in pulmonary hypertension, and HIFs activate pulmonary artery endothelial and smooth muscle cell proliferation, which is mediated by both mTORC1 and mTORC2 [83–85]. Currently, the mechanisms by which hypoxia/HIFs signal to activate mTOR in ECs and VSMCs are poorly

Severe PH associated with hypoxic lung disease is a life-threatening condition with poor survival rates. Despite significant advances in targeted therapeutics for PH, randomised clinical trial data for this particular group of patients are scarce, and it is not clear whether endothelin receptor antagonists will benefit patients with hypoxia-associated PH. Importantly, recent genetic studies identifying mutations in the oxygen-sensing machinery have provided new mechanistic insights into the aetiology of PH. Further studies are required to determine whether specific targeting of HIF-2α will provide additional therapeutic benefit for this complex disease.

to deprived

promoting new blood vessel growth—angiogenesis, which in turn, restores O2

Nicoletta Charolidi and Veronica A. Carroll\*

\*Address all correspondence to: vcarroll@sgul.ac.uk

Vascular Biology Research Centre, Molecular and Clinical Sciences Research Institute, St George's, University of London, London, UK

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**Provisional chapter**

## **Stage-Specific Effects of Hypoxia on Interstitial Lung Disease Lung Disease**

**Stage-Specific Effects of Hypoxia on Interstitial** 

Sandeep Artham and Payaningal R. Somanath Payaningal R. Somanath

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/66817

Sandeep Artham and

#### **Abstract**

Interstitial lung disease (ILD) comprises a group of lung diseases principally affecting the pulmonary interstitium, for example, pulmonary fibrosis. Following acute lung injury (ALI), the fate of an injured lung progressing towards either injury resolution or pulmonary fibrosis is dictated by hypoxia at various stages during the disease progression. Hypoxia that is tissue destructive at one stage of lung injury becomes beneficial at a different stage, with each hypoxic stage involving a different scheme of molecular pathways, cellular interplay and tissue remodeling. In this chapter, we provide a detailed account of hypoxia during the different stages of lung injury in ILDs, delineate the cellular and molecular mechanisms mediating tissue remodeling in the hypoxic lungs as well as the basic and clinical findings in this field with an emphasis on future therapeutics to modulate hypoxia to treat ILD.

**Keywords:** acute lung injury, wound resolution, hypoxia, interstitial lung disease, PAH

## **1. Introduction**

Interstitial lung disease (ILD) comprises a group of lung diseases principally affecting the pulmonary interstitium, for example, pulmonary fibrosis [1]. An injured lung as a result of infection, inhalation of chemical, and other harmful substances either resolves over time or progresses into irreversible damage and fibrosis. Therefore, lung injury as in acute respiratory distress syndrome (ARDS), due to conditions like hypoxia can progress to interstitial lung damage or fibrosis similar to ILD-associated pulmonary fibrosis. Yet, another important pulmonary pathological condition associated with hypoxia is the pulmonary arterial hypertension (PAH) [2]. The ARDS is a devastating clinical syndrome of acute lung injury (ALI) that affects both medical and surgical patients [3]. The official definition of ARDS was first published in 1994 by

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

American-European Consensus conference (AECC), according to which ARDS is characterized by arterial partial pressure of oxygen to fraction of inspired oxygen [PaO2/FIO2] ≤200 mm Hg with bilateral infiltrates on frontal chest radiograph, with no evidence of left atrial hypertension. A new entity—ALI was also introduced as a condition of less severe hypoxemia [PaO2/ FIO2] ≤300 mm Hg. Arterial hypoxemia that is refractory to treatment with supplemental oxygen is a characteristic feature of acute lung injury. ALI is characterized by alveolar-capillary injury, inflammation with neutrophil accumulation and release of pro-inflammatory cytokines leading to alveolar edema [3]. Patients with ALI develop hypoxia. The term ALI was eventually removed in 2011 in the updated Berlin definition of ARDS. According to Berlin definition, ARDS was classified into three mutually exclusive categories based on the degree of hypoxemia; mild (200 mm Hg < PaO2/FIO2 ≤ 300 mm Hg), moderate (100 mm Hg < PaO2/FIO2 ≤ 200 mm Hg) and severe (PaO2/FIO2 ≤ 100 mm Hg) [4]. Hypoxia may be a consequence of ALI leading to deviation in lung function and preventing repair. Hypoxia induces destructive exudative changes within the lung parenchyma, which include the following: (1) increased alveolar paracellular permeability due to hypoxia disrupted alveolar epithelial cell (AEC) cytoskeleton and tight junction (TJ) protein organization; (2) Prolonged hypoxia induces loss of stress fibers such as actin (including breakdown of spectrin), internalization of TJ protein occludin and a decrease in zona occludens-1 (ZO-1) protein levels that are associated with trans-epithelial permeability; (3) reduced efficacy of AEC to clear alveolar edema fluid as a result of decreased expression of two major proteins, the apical epithelial sodium channel (ENaC) and the basolateral Na/K-ATPase channel which are involved in transcellular sodium (Na) transport. Thus, hypoxia-mediated effects not only enhance alveolar edema but also impair alveolar edema clearance contributing to reduced alveolar gaseous exchange capacity in ALI [5].

## **2. Hypoxia in alveolar edema and fluid clearance in the lungs**

The mechanism by which hypoxia promotes pulmonary edema is not completely understood and is still under scrutiny. Alveolar edema accumulation is a result of enhanced pulmonary vascular permeability. Vascular endothelial growth factor (VEGF) is a potent inducer of endothelial dysfunction and thus can play a crucial role in vascular permeability [6]. Since VEGF is induced in hypoxic conditions and recovery from hypoxia, its role in pulmonary vascular remodeling and enhanced alveolar edema is prominent [7]. The source of VEGF in the inflammatory milieu of lung injury includes monocytes, eosinophils and aggregated platelets. Research on hypoxia-induced VEGF expression as a cause for pathological conditions has been carried out for more than two decades now. Studies have shown that both acute and chronic hypoxia induce an upregulation in the gene expression of VEGF, and its receptors (KDR/Flk and Flt) in the animal models of prolonged hypoxia-induced pulmonary hypertension [8]. In fact, the increase in the VEGF gene expression was seen as early as 2 h upon hypoxic challenge in isolated and perfused rat lungs while chronic hypoxia resulted in greater upregulation of the VEGF receptor genes. These studies also scrutinized the mechanism by which hypoxia induces VEGF expression by examining the role of nitric oxide synthase (NOS) and hypoxia inducible factors (HIFs) as the downstream regulators [8, 9]. Studies on transcriptional regulation of VEGF by hypoxia have revealed a functional HIF-1 binding site on the rat VEGF 5′-flanking region as a possible transcriptional activator of VEGF gene by hypoxia [9]. Further studies have shown the involvement of specific regions in 3′-untranslated region (UTR) of VEGF gene in the stability of VEGF mRNA induced by hypoxia [10]. This has led to investigation of proteins that bind to this specific region to control the posttranscriptional regulation of VEGF expression. One such protein is HuR, a member of Elav-like protein family (Elav is a *Drosophila* RNA-binding protein required for neuronal differentiation). HuR was found to post-transcriptionally regulate VEGF expression by binding within four nucleotides of a canonical nonameric instability element in the VEGF AU-rich element [10]. Thus, hypoxia regulates VEGF at both transcriptional and posttranscriptional levels. Transcriptional regulation is by the hypoxia-induced transcription factor HIF-1 which activates VEGF transcription by binding to specific promoter sequences. A study exploring possible mechanisms involved in securing efficient translation of VEGF during hypoxic stress showed that internal ribosome entry site (IRES) present in the 5′-UTR of VEGF gene functions as an alternative to cap-dependent translation during such stressful conditions [11].

American-European Consensus conference (AECC), according to which ARDS is characterized by arterial partial pressure of oxygen to fraction of inspired oxygen [PaO2/FIO2] ≤200 mm Hg with bilateral infiltrates on frontal chest radiograph, with no evidence of left atrial hypertension. A new entity—ALI was also introduced as a condition of less severe hypoxemia [PaO2/ FIO2] ≤300 mm Hg. Arterial hypoxemia that is refractory to treatment with supplemental oxygen is a characteristic feature of acute lung injury. ALI is characterized by alveolar-capillary injury, inflammation with neutrophil accumulation and release of pro-inflammatory cytokines leading to alveolar edema [3]. Patients with ALI develop hypoxia. The term ALI was eventually removed in 2011 in the updated Berlin definition of ARDS. According to Berlin definition, ARDS was classified into three mutually exclusive categories based on the degree of hypoxemia; mild (200 mm Hg < PaO2/FIO2 ≤ 300 mm Hg), moderate (100 mm Hg < PaO2/FIO2 ≤ 200 mm Hg) and severe (PaO2/FIO2 ≤ 100 mm Hg) [4]. Hypoxia may be a consequence of ALI leading to deviation in lung function and preventing repair. Hypoxia induces destructive exudative changes within the lung parenchyma, which include the following: (1) increased alveolar paracellular permeability due to hypoxia disrupted alveolar epithelial cell (AEC) cytoskeleton and tight junction (TJ) protein organization; (2) Prolonged hypoxia induces loss of stress fibers such as actin (including breakdown of spectrin), internalization of TJ protein occludin and a decrease in zona occludens-1 (ZO-1) protein levels that are associated with trans-epithelial permeability; (3) reduced efficacy of AEC to clear alveolar edema fluid as a result of decreased expression of two major proteins, the apical epithelial sodium channel (ENaC) and the basolateral Na/K-ATPase channel which are involved in transcellular sodium (Na) transport. Thus, hypoxia-mediated effects not only enhance alveolar edema but also impair alveolar edema

228 Hypoxia and Human Diseases

clearance contributing to reduced alveolar gaseous exchange capacity in ALI [5].

**2. Hypoxia in alveolar edema and fluid clearance in the lungs**

The mechanism by which hypoxia promotes pulmonary edema is not completely understood and is still under scrutiny. Alveolar edema accumulation is a result of enhanced pulmonary vascular permeability. Vascular endothelial growth factor (VEGF) is a potent inducer of endothelial dysfunction and thus can play a crucial role in vascular permeability [6]. Since VEGF is induced in hypoxic conditions and recovery from hypoxia, its role in pulmonary vascular remodeling and enhanced alveolar edema is prominent [7]. The source of VEGF in the inflammatory milieu of lung injury includes monocytes, eosinophils and aggregated platelets. Research on hypoxia-induced VEGF expression as a cause for pathological conditions has been carried out for more than two decades now. Studies have shown that both acute and chronic hypoxia induce an upregulation in the gene expression of VEGF, and its receptors (KDR/Flk and Flt) in the animal models of prolonged hypoxia-induced pulmonary hypertension [8]. In fact, the increase in the VEGF gene expression was seen as early as 2 h upon hypoxic challenge in isolated and perfused rat lungs while chronic hypoxia resulted in greater upregulation of the VEGF receptor genes. These studies also scrutinized the mechanism by which hypoxia induces VEGF expression by examining the role of nitric oxide synthase (NOS) and hypoxia inducible factors (HIFs) as the downstream regulators [8, 9]. Studies on transcriptional regulation of VEGF by hypoxia have revealed a functional HIF-1 binding Becker et al. studied hypoxia-induced VEGF's role in enhancing pulmonary vascular permeability. They showed that ischemia/hypoxia-induced upregulation of VEGF mRNA and protein was associated with increased pulmonary vascular permeability [12]. Their study was also supported by several other studies which have reported an increase in vascular permeability due to exogenously administered VEGF in skin, muscle, GI tract and airways. In their study, hypoxic ischemia-enhanced VEGF expression, which was associated with increased HIF-1α protein expression and redistribution of VEGF protein to alveolar septae as demonstrated by immunohistochemical staining. This distribution of VEGF protein in the alveolar septae was further associated with increased pulmonary vascular permeability, suggesting its role in acute lung injury and alveolar edema [12]. The enhanced pulmonary vascular permeability effect of VEGF was also confirmed by another study in a sepsis-induced lung injury model, which showed that enhanced plasma VEGF level was accompanied by increased expression of vascular permeability-mediating VEGF receptor, Flt-1 and not the angiogenic-mediating receptor, Flk-1. As a result, enhanced lung edema was observed confirming the role of VEGF in causing alveolar edema [13].

Na,K-ATPase channels present in the alveolar epithelial cells play a major role in edema clearance from the alveoli [14]. Hypoxia-induced pulmonary edema also disrupts their function and inhibits edema clearance. Studies have shown that hypoxia generated reactive oxygen species (ROS) activates PKCζ (Protein Kinase C Zeta is a key regulator of critical intracellular signaling pathways induced by various extracellular stimuli), which in turn, phosphorylates the α1-subunit of Na,K-ATPase at Ser-18 site leading to its endocytosis through a clathrindependent mechanism and eventually to lysosomal degradation. With the loss of Na,K-ATPase, edema reabsorption is impaired and thus hypoxia not only promotes pulmonary edema but also inhibits its clearance as observed in conditions like ALI [14].

## **3. Hypoxia in pulmonary aquaporin's expression and edema**

Aquaporins (AQPs) comprise a group of cell membrane water-transporting proteins that are involved in physiological as well as pathological fluid transport. They have been identified in the lung and are believed to play a major role in pulmonary edema [15]. AQPs can bidirectionally transport fluid across the alveolar epithelium and hence are involved in both edema formation and clearance of edema from alveoli (thus injury resolution). About 6 (AQP-1, -3, -4, -5, -8 and -9) of the 13 different AQPs are distributed in lung tissue, and it is very interesting to study how hypoxia regulates the expression of these AQPs and thus pulmonary edema formation or clearance of edema. AQPs expression could play a major role in the pathological condition of hypoxia-induced enhanced pulmonary edema and ALI [15]. Several studies have scrutinized the role of aquaporins in pulmonary edema, and the results are controversial, yet intriguing. For example, Wu et al. studied the role of AQP-1 [expressed on pulmonary endothelial cells (ECs) and alveolar type II cells] and AQP-4 (expressed throughout the airways epithelial cells) in relation to high-altitude hypoxia lung injury. They found that hypoxiainduced pulmonary edema was associated with a decreased expression of AQP-1 and no change in the expression of AQP-4 [16]. They went on to reason that hypoxia resulted in pulmonary edema as a consequence of decreased function of AQP-1, which plays a regulatory role in water clearance around the bronchi and vessels. However, the relation of AQP-1 expression and pulmonary edema, as a result of hypoxia was only correlative and the study did not use knockout models to confirm the relationship between these effects of hypoxia. On the contrary, Su et al. showed that depletion of AQP-1 does not affect isosmolar fluid clearance and had no effect on lung edema. Nevertheless, depletion of AQP-1 resulted in a 10-fold decrease in the alveolar-capillary osmotic water permeability. They concluded that depletion of AQP-1 did not have any effect on lung edema formation and resolution [17]. Several other reports have also ruled out the role of AQP-1, -4 and -5 in physiological clearance of water in the lung or the accumulation of edema in the injured lung. Another report using gene knockout mouse model of AQP5 in hypoxic conditions showed a significant increase in pulmonary edema with the loss of AQP-5 [18]. As aforementioned, a few other reports also demonstrated that upregulation and downregulation of AQPs expression is related to pulmonary edema in different kinds of lung injuries. AQP-1 has also been shown to facilitate stabilization of HIF and has been speculated that besides its role as water transporter, it could also be involved in oxygen transport [19]. Therefore, the effect of hypoxia on AQPs expression especially in the lung and its effect on pulmonary edema warrants further studies before arriving at a conclusion [16–20].

## **4. Hypoxia in pulmonary arterial hypertension (PAH)**

Prolonged lung injury can lead to lung fibrosis as well as PAH. Hypoxia is a well-studied trigger for pulmonary vascular remodeling and PAH development [2]. In fact, hypoxiainduced PAH is an established animal model for studying the pathophysiology and therapeutic management of PAH. PAH is a refractory disease characterized by uncontrolled vascular remodeling involving enhanced proliferation and differentiation of pulmonary vascular ECs and pulmonary vascular smooth muscle cells [2]. This vascular remodeling ensues enhanced pulmonary arterial pressure (≥25 mm Hg on right heart catheterization) due to increased pulmonary vasoconstriction and increased pulmonary vascular resistance and eventually right ventricular failure [2]. Chronic hypoxia is a well-known trigger for the abovementioned events. The mechanism by which hypoxia induces PAH has been extensively studied and involves several molecular signaling pathways. Leptin, a non-glycosylated protein, synthesized and secreted by adipocytes is encoded by obese (ob) gene, which is hypoxia sensitive. HIF-1 induces the expression of ob gene in adipocytes, and clinical studies have suggested an association between plasma leptin levels and severity of PAH [21]. Results of studies scrutinizing the role of leptin signaling in hypoxia-induced PAH show that hypoxia-induced leptin expression results in pulmonary arterial smooth muscle cells (PASMCs) proliferation through ERK, STAT and AKT pathways [21]. These results were further confirmed in ob/ob mice. Obese gene knockout mice subjected to hypoxia showed an attenuated hypoxia-induced PAH that was gauged in terms of reduced right ventricular systolic pressure (RVSP) and right ventricular hypertrophy index (RVHI) when compared to wild-type (WT) mice. Thus, leptin signaling could be a potential therapeutic target to treat hypoxia-induced PAH [21]. In hypoxia-induced pulmonary hypertension, iron supplementation has been found to be beneficial [22]. A study involving human subjects in an acute model of mountain sickness has shown that iron supplementation was associated with a decrease in pulmonary arterial systolic pressure (PASP) while progressive development of iron deficiency correlated with worsening of pulmonary arterial pressure determined by echocardiography, thus suggesting a causal relationship between iron deficiency and acute hypoxic PAH [23]. Recent studies speculate that iron deficiency may worsen hypoxic pulmonary hypertension through HIFs signaling [24].

in the lung and are believed to play a major role in pulmonary edema [15]. AQPs can bidirectionally transport fluid across the alveolar epithelium and hence are involved in both edema formation and clearance of edema from alveoli (thus injury resolution). About 6 (AQP-1, -3, -4, -5, -8 and -9) of the 13 different AQPs are distributed in lung tissue, and it is very interesting to study how hypoxia regulates the expression of these AQPs and thus pulmonary edema formation or clearance of edema. AQPs expression could play a major role in the pathological condition of hypoxia-induced enhanced pulmonary edema and ALI [15]. Several studies have scrutinized the role of aquaporins in pulmonary edema, and the results are controversial, yet intriguing. For example, Wu et al. studied the role of AQP-1 [expressed on pulmonary endothelial cells (ECs) and alveolar type II cells] and AQP-4 (expressed throughout the airways epithelial cells) in relation to high-altitude hypoxia lung injury. They found that hypoxiainduced pulmonary edema was associated with a decreased expression of AQP-1 and no change in the expression of AQP-4 [16]. They went on to reason that hypoxia resulted in pulmonary edema as a consequence of decreased function of AQP-1, which plays a regulatory role in water clearance around the bronchi and vessels. However, the relation of AQP-1 expression and pulmonary edema, as a result of hypoxia was only correlative and the study did not use knockout models to confirm the relationship between these effects of hypoxia. On the contrary, Su et al. showed that depletion of AQP-1 does not affect isosmolar fluid clearance and had no effect on lung edema. Nevertheless, depletion of AQP-1 resulted in a 10-fold decrease in the alveolar-capillary osmotic water permeability. They concluded that depletion of AQP-1 did not have any effect on lung edema formation and resolution [17]. Several other reports have also ruled out the role of AQP-1, -4 and -5 in physiological clearance of water in the lung or the accumulation of edema in the injured lung. Another report using gene knockout mouse model of AQP5 in hypoxic conditions showed a significant increase in pulmonary edema with the loss of AQP-5 [18]. As aforementioned, a few other reports also demonstrated that upregulation and downregulation of AQPs expression is related to pulmonary edema in different kinds of lung injuries. AQP-1 has also been shown to facilitate stabilization of HIF and has been speculated that besides its role as water transporter, it could also be involved in oxygen transport [19]. Therefore, the effect of hypoxia on AQPs expression especially in the lung and its effect on pulmonary edema warrants further studies before arriving at a conclu-

sion [16–20].

230 Hypoxia and Human Diseases

**4. Hypoxia in pulmonary arterial hypertension (PAH)**

Prolonged lung injury can lead to lung fibrosis as well as PAH. Hypoxia is a well-studied trigger for pulmonary vascular remodeling and PAH development [2]. In fact, hypoxiainduced PAH is an established animal model for studying the pathophysiology and therapeutic management of PAH. PAH is a refractory disease characterized by uncontrolled vascular remodeling involving enhanced proliferation and differentiation of pulmonary vascular ECs and pulmonary vascular smooth muscle cells [2]. This vascular remodeling ensues enhanced pulmonary arterial pressure (≥25 mm Hg on right heart catheterization) due to increased pulmonary vasoconstriction and increased pulmonary vascular resistance and eventually right ventricular failure [2]. Chronic hypoxia is a well-known trigger HIFs are transcription factors comprising of an O<sup>2</sup> -sensitive α-subunit, mainly HIF-1α and HIF-2α and a constitutively expressed β-subunit which are responsible for mediating adaptive responses to hypoxia and ischemia [25]. HIF-α and HIF-β form heterodimer and induce the transcription of over 100 genes that affect cellular functions ranging from metabolism, survival, proliferation, migration and angiogenesis among several others [25]. While HIF-1α is more ubiquitously expressed, HIF-2α expression is predominant in the lung tissue [25]. Several studies have shown the mechanistic role of HIF-2α in hypoxia-induced PAH. In hypoxia-induced PAH studies, even partial deficiency of either HIF1α (HIF1α+/−) or HIF2α (HIF2α+/−), achieved using murine models, significantly decreased pulmonary arterial pressure and right ventricular hypertrophy induced by chronic hypoxia in comparison with wildtype mice that did not have any alteration in HIF1α or HIF2α expression [26]. The role of HIFs in hypoxia-induced PAH was further scrutinized and deficiency in HIFs-related beneficial effects in PAH was at least partly due to the reduced pulmonary vascular remodeling observed in these animals. Further *in vitro* analysis on PASMCs showed that HIF-1-dependent smooth muscle hypertrophy contributed to pulmonary vascular remodeling during hypoxia [26]. HIF1α is involved in hypoxia-induced PASMC depolarization, reduction in K<sup>+</sup> channel expression and activity and elevated intracellular calcium concentration and pH. This eventually results in altered PASMC ion homeostasis contributing to a more contractile, apoptosis resistant, proliferative and migratory phenotype [26]. Furthermore, in human PAH patients and mouse models of PAH, dysregulation of HIF pathway was reported and it has been associated with HIF-2α mutations, which was confirmed by studies where loss of one copy of HIF-2α gene was sufficient to attenuate hypoxia-induced PAH in these animal models [27]. On the other hand, HIF-2α gain of functions is associated with PAH. Studies scrutinizing the mechanism by which HIF-2α regulates hypoxic PAH have found several ways by which it mediates the hypoxic effects. In human PASMC, hypoxia increases expression of transcription factor forkhead box M1 (FoxM1), through HIF-2α, to promote PASMC proliferation [27]. Secreted matricellular protein thrombospondin-1 (TSP-1) is believed to play an important role in vascular health and disease via inhibition of vasodilation in part by limiting NO production and signaling [28]. Vascular remodeling in PAH involves the proliferation of both pulmonary artery smooth muscle cells (PASMCs) and fibroblasts apart from endothelial dysfunction. In a recent study published from our laboratory, we showed that hypoxia-induced pulmonary rarefaction and fibrosis in mice lung, and mechanistically, we found that hypoxia-induced Akt1 expression in fibroblasts was associated with enhanced TSP-1 expression resulting in fibroproliferation and fibrosis [29]. Another study has shown that hypoxia, in a HIF-2αdependent manner, increases the expression of TSP-1 in pulmonary tissue and pulmonary artery cells which in turn contributes to enhanced endothelial permeability (mediated in part by changes in cell-cell adhesion) and accompanied by increased fibroblast and PASMC proliferation which is at least partially due to restricted adhesion of these cells in their mouse model of hypoxia-induced PAH. Also it was speculated that TSP-1 could promote hypoxic pulmonary artery contraction through enhanced TSP-1–induced endothelin-1 expression [28].

Prolyl hydroxylase domain-containing enzymes (PHDs) use molecular O<sup>2</sup> as a substrate to hydroxylate-specific proline residues of HIF-α which subsequently promotes HIF-α binding to von Hippel-Lindau (VHL protein) and ubiquitin E3 ligase, resulting in ubiquitination and proteasomal degradation [27]. In patients with idiopathic pulmonary fibrosis (IPF), PHD2 expression is diminished in ECs of obliterative pulmonary vessels [27]. A study using mouse model of endothelial and hematopoietic cells-specific knockdown of gene encoding PHD2 has shown that these mice spontaneously develop PAH with obliterative vascular remodeling as seen in human PAH [27]. They found that PHD2 deficiency in ECs promoted HIF-2α-mediated (and not HIF-1α) expression of CXCL12 (also known as stromal cell-derived factor 1α) that had a paracrine effect on PASMC proliferation contributing to the pathogenesis of severe PAH in this mouse model. PHD2 deficiency in ECs also promoted endothelin-1 expression that resulted in pulmonary artery-vasoconstriction. Thus, HIF-2α-mediated vascular remodeling and plexiform-like lesions formation (due to PASMC proliferation) resulted in PAH in this mouse model [27, 28]. As discussed above, prevention of PASMC apoptosis along with enhanced proliferation is an important pathological event in hypoxic PAH. Another study showed the mechanism by which hypoxia mediates this effect. In PASMCs, hypoxia induces opening of mitochondrial ATP-sensitive potassium channels (mitoKATP), which results in calcium-dependent increase in mitochondrial permeability or mitochondrial membrane transition (MPT). MPT eventually leads to loss of mitochondrial membrane potential (denoted by ΔΨm), thus preventing the cytochrome C release from mitochondria and inhibition of cytochrome C–caspase 9 pathway induced PASMC apoptosis [30]. The involvement of mitoKATP channels in hypoxia-induced PASMC apoptosis resistance was further confirmed by administering 5-hydroxydecanoate (5-HD), a compound that prevents opening of mitoKATP channels abolishes these effects of hypoxia to a certain extent and prevents mitoKATP channels opening and PASMC apoptosis. Hypoxia-induced opening of mitoKATP was not only associated with prevention of PASMC apoptosis but also increased the production of H<sup>2</sup> O2 in mitochondria. The effect of this ROS production was an increased transcriptional activity of AP-1, which is responsible for the proliferation of PASMCs. Thus, hypoxia through mitoKATP opening prevented apoptosis and enhanced proliferation of PASMCs. As discussed, apart from proliferation of PASMCs, hypoxia-induced prevention of PASMC apoptosis also plays a major role in PAH. Another mechanism involves inhibition of the mitochondrial pro-apoptotic Bax protein expression and induction of the anti-apoptotic Bcl-2 expression, thus preventing the release of mitochondrial cytochrome C into cytoplasm and eventually inhibiting cleavage of caspase 9 resulting in PASMC apoptosis [31]. Therefore, hypoxia-HIF signaling is a potential therapeutic target to treat PAH, and several in vivo studies have demonstrated this [30–32].

mechanism by which HIF-2α regulates hypoxic PAH have found several ways by which it mediates the hypoxic effects. In human PASMC, hypoxia increases expression of transcription factor forkhead box M1 (FoxM1), through HIF-2α, to promote PASMC proliferation [27]. Secreted matricellular protein thrombospondin-1 (TSP-1) is believed to play an important role in vascular health and disease via inhibition of vasodilation in part by limiting NO production and signaling [28]. Vascular remodeling in PAH involves the proliferation of both pulmonary artery smooth muscle cells (PASMCs) and fibroblasts apart from endothelial dysfunction. In a recent study published from our laboratory, we showed that hypoxia-induced pulmonary rarefaction and fibrosis in mice lung, and mechanistically, we found that hypoxia-induced Akt1 expression in fibroblasts was associated with enhanced TSP-1 expression resulting in fibroproliferation and fibrosis [29]. Another study has shown that hypoxia, in a HIF-2αdependent manner, increases the expression of TSP-1 in pulmonary tissue and pulmonary artery cells which in turn contributes to enhanced endothelial permeability (mediated in part by changes in cell-cell adhesion) and accompanied by increased fibroblast and PASMC proliferation which is at least partially due to restricted adhesion of these cells in their mouse model of hypoxia-induced PAH. Also it was speculated that TSP-1 could promote hypoxic pulmonary artery contraction through enhanced TSP-1–induced endothelin-1 expression [28].

232 Hypoxia and Human Diseases

Prolyl hydroxylase domain-containing enzymes (PHDs) use molecular O<sup>2</sup>

hydroxylate-specific proline residues of HIF-α which subsequently promotes HIF-α binding to von Hippel-Lindau (VHL protein) and ubiquitin E3 ligase, resulting in ubiquitination and proteasomal degradation [27]. In patients with idiopathic pulmonary fibrosis (IPF), PHD2 expression is diminished in ECs of obliterative pulmonary vessels [27]. A study using mouse model of endothelial and hematopoietic cells-specific knockdown of gene encoding PHD2 has shown that these mice spontaneously develop PAH with obliterative vascular remodeling as seen in human PAH [27]. They found that PHD2 deficiency in ECs promoted HIF-2α-mediated (and not HIF-1α) expression of CXCL12 (also known as stromal cell-derived factor 1α) that had a paracrine effect on PASMC proliferation contributing to the pathogenesis of severe PAH in this mouse model. PHD2 deficiency in ECs also promoted endothelin-1 expression that resulted in pulmonary artery-vasoconstriction. Thus, HIF-2α-mediated vascular remodeling and plexiform-like lesions formation (due to PASMC proliferation) resulted in PAH in this mouse model [27, 28]. As discussed above, prevention of PASMC apoptosis along with enhanced proliferation is an important pathological event in hypoxic PAH. Another study showed the mechanism by which hypoxia mediates this effect. In PASMCs, hypoxia induces opening of mitochondrial ATP-sensitive potassium channels (mitoKATP), which results in calcium-dependent increase in mitochondrial permeability or mitochondrial membrane transition (MPT). MPT eventually leads to loss of mitochondrial membrane potential (denoted by ΔΨm), thus preventing the cytochrome C release from mitochondria and inhibition of cytochrome C–caspase 9 pathway induced PASMC apoptosis [30]. The involvement of mitoKATP channels in hypoxia-induced PASMC apoptosis resistance was further confirmed by administering 5-hydroxydecanoate (5-HD), a compound that prevents opening of mitoKATP channels abolishes these effects of hypoxia to a certain extent and prevents mitoKATP channels opening and PASMC apoptosis. Hypoxia-induced opening of mitoKATP was not only associated with prevention of PASMC apoptosis but also increased the production of H<sup>2</sup>

as a substrate to

O2 in

## **5. Hypoxia and alveolar epithelial-to-mesenchymal transition (EMT)**

Several groups have studied the role of hypoxia in disease progression and pathogenesis of ILDs such as pulmonary fibrosis [33, 34]. Activated myofibroblasts play an important role in the production of collagen and ECM proteins during pulmonary fibrosis. The source of these myofibroblasts are numerous, which include resident stromal fibroblasts, bone marrow-derived fibroblasts, and mesenchymal transition of epithelial and ECs [33]. Epithelial-tomesenchymal transition (EMT) is a cellular process during which epithelial cells lose many of their epithelial characteristics such as cell-cell interaction and apicobasal polarity and acquire properties typical to mesenchymal cells. EMT is driven by a cytokine, transforming growth factor-β1 (TGF-β1) and is characterized by changes in cell morphology and acquisition of mesenchymal markers including α-smooth muscle actin (α-SMA) and vimentin as well as loss of epithelial markers such as E-cadherin [33, 34]. Active TGF-β1 binds to its receptors (transmembrane serine-threonine kinase receptor I and II), which leads to a downstream activation of the transcription factor Smad, whose target genes include α-SMA and vimentin [33]. Increasing evidence over the years has highlighted the critical role of EMT in pathological conditions such as fibrosis apart from its well-known involvement in tissue development during embryogenesis. Exposure to hypoxia during ALI could promote phenotypic changes in AEC consistent with EMT. In vitro studies on rat AEC cultured on semipermeable filters showed that prolonged hypoxic exposure (1.5% O2 for up to 12 days) induced profound changes in AEC phenotype consistent with EMT including change in cell morphology, decrease in transepithelial resistance and in the expression of epithelial markers such as zona occludens (ZO-1), E-cadherin, AQP-5, TTF-1, together with an increase in mesenchymal markers such as vimentin and α-SMA. Supporting this phenotypical switch, expression of transcription factors driving EMT such as SNAIL1, ZEB1 and TWIST1 increased after 2, 24 and 48 h of hypoxia, respectively. Hypoxia also induced expression and secretion of two EMT inducers TGF-β1 and connective tissue growth factor (CTGF) [35].

Similarly, Zhou et al. investigated the effect of hypoxia on the induction of EMT in AEC. Results from this study suggest that hypoxia induces EMT in transformed human, rat and mouse AEC lines, and freshly isolated rat type II AECs [36]. They also scrutinized the mechanism by which hypoxia induces EMT in AEC and showed the involvement of hypoxia-induced mitochondrial ROS production and HIF-1α stabilization in TGF-β1 production, resulting in EMT [37]. Treatment of cells with ROS scavenger Euk-134 or using mitochondria-deficient cells prevented hypoxia-induced EMT illustrating their importance in this cellular process. Moreover, although ROS is known to stabilize HIF-1α, their results showed that normoxic stabilization of HIF-1α failed to induce α-SMA expression, suggesting that HIF alone is not sufficient to induce EMT in AEC. Their data suggest that ROS and HIF-1α stabilization are upstream of TGF-β1 production in hypoxia-induced EMT in AEC. However, TGF-β1 can also increase ROS production and HIF-1α stabilization. TGF-β1 can either directly activate NADPH (Nicotinamide adenine dinucleotide phosphate) oxidase or upregulate gene expression of Nox4 NADPH oxidase to generate ROS [38, 39]. TGF-β1 decreases mitochondrial complex IV activity resulting in disruption of mitochondrial membrane potential and ROS production [40]. TGF-β1 was reported to stabilize HIF-1α through selective inhibition of PHD2 (a HIF-1α prolyl hydroxylase) expression thus reducing HIF-1α prolyl hydroxylation leading to its stabilization [41]. Therefore, TGF-β1 and ROS/HIF may form a feedback loop to maintain a prolonged signaling cascade initiated by either ROS/HIF or TGF-β1 leading to hypoxia-induced EMT in AECs [36].

In one interesting study, investigators evaluated the possible role of tissue hypoxia in the development of fibrotic lesions in lung fibrosis [42]. In this study, they used animal models of ALI/ARDS, in which severe inflammation progresses into the early (exudative) phase of ALI and sequentially fibrosis develops as the late (fibrotic) phase of ALI. They found intriguing effects of acute versus persistent hypoxia as seen in exudative and fibrotic phases of ALI, respectively. Acute hypoxia induced de novo Surfactant Protein-D (SP-D) expression in AECs followed by stabilization of HIF-1α expression [42]. Contrastingly, persistent hypoxiainduced HIF-1α stabilization repressed SP-D expression and enhanced the mRNA levels of an EMT-driving transcription factor TWIST, but not SNAIL. This was accompanied by phenotypic switch in the AECs exposed to persistent hypoxia (72-h hypoxia for in vitro studies) as seen by decreased E-cadherin expression and enhanced vimentin expression. SP-D is mainly derived from alveolar epithelial cells and therefore loss of its expression during persistent hypoxia along with enhanced EMT transcription factor expression clearly indicates phenotypic switch of these alveolar epithelial cells to more proliferative phenotype contributing to lung fibrosis [42].

Endothelial-to-mesenchymal transition (EndMT) is similar to EMT, which is characterized by a loss of endothelial cell-cell junctions, the acquisition of migratory properties, and phenotypic switch involving loss of endothelial-specific markers such as CD31 and vascular endothelial (VE)-cadherin expression, and the acquisition of mesenchymal markers α-SMA, and vimentin [43]. EndMT also contributes to fibrosis. The role of EndMT in pulmonary fibrosis involves phenotypic switch in the pulmonary EC lining the pulmonary capillaries. Radiation-induced pulmonary fibrosis (RIPF) may involve hypoxia-mediated EndMT as an initial pathological insult leading to fibrosis [13]. Fleckenstein and colleagues have shown that radiation during thoracic radiotherapy for lung cancer induces tissue hypoxia, in part, due to enhanced oxygen consumption by Macrophages. These macrophages are activated because of radiation-induced reduction in blood perfusion in the lungs contributing to lung injury [44]. This suggests that hypoxia plays a major role in the radiation-induced lung injury. Fleckenstein et al. also reported that hypoxia is important in triggering continuous production of fibrogenic cytokines and perpetuation of late lung tissue injury [44]. However, the precise mechanism by which hypoxia affects radiation-induced fibrosis remains elusive. EndMT of the pulmonary ECs was shown as a possible consequence of radiation-induced hypoxia resulting in lung fibrosis and injury by Choi et al. [43]. They investigated the reason behind fibrotic effects of radiation in a mouse model of RIPF and in *in vitro* studies on human pulmonary ECs. Since fibrosis is a longterm event, their investigation aimed at elucidating the mechanisms behind the early damage to ECs by radiation and its link to the later observed fibrosis. Their results indicate ECs specifically expressing hypoxic marker, CA9, just prior to the substantial fibrogenesis. They went on to show that radiation-induced vascular hypoxia-triggered EndMT in vascular ECs, and in fact, this was observed prior to the onset of alveolar EMT and thus could be a trigger to EMT as well. Thus, EndMT contributed to chronic tissue fibrosis and targeting EndMT was speculated to be a potential therapeutic target to treat RIPF [43, 44].

hypoxia-induced mitochondrial ROS production and HIF-1α stabilization in TGF-β1 production, resulting in EMT [37]. Treatment of cells with ROS scavenger Euk-134 or using mitochondria-deficient cells prevented hypoxia-induced EMT illustrating their importance in this cellular process. Moreover, although ROS is known to stabilize HIF-1α, their results showed that normoxic stabilization of HIF-1α failed to induce α-SMA expression, suggesting that HIF alone is not sufficient to induce EMT in AEC. Their data suggest that ROS and HIF-1α stabilization are upstream of TGF-β1 production in hypoxia-induced EMT in AEC. However, TGF-β1 can also increase ROS production and HIF-1α stabilization. TGF-β1 can either directly activate NADPH (Nicotinamide adenine dinucleotide phosphate) oxidase or upregulate gene expression of Nox4 NADPH oxidase to generate ROS [38, 39]. TGF-β1 decreases mitochondrial complex IV activity resulting in disruption of mitochondrial membrane potential and ROS production [40]. TGF-β1 was reported to stabilize HIF-1α through selective inhibition of PHD2 (a HIF-1α prolyl hydroxylase) expression thus reducing HIF-1α prolyl hydroxylation leading to its stabilization [41]. Therefore, TGF-β1 and ROS/HIF may form a feedback loop to maintain a prolonged signaling cascade initiated by either ROS/HIF or TGF-β1 leading to hypoxia-induced EMT

In one interesting study, investigators evaluated the possible role of tissue hypoxia in the development of fibrotic lesions in lung fibrosis [42]. In this study, they used animal models of ALI/ARDS, in which severe inflammation progresses into the early (exudative) phase of ALI and sequentially fibrosis develops as the late (fibrotic) phase of ALI. They found intriguing effects of acute versus persistent hypoxia as seen in exudative and fibrotic phases of ALI, respectively. Acute hypoxia induced de novo Surfactant Protein-D (SP-D) expression in AECs followed by stabilization of HIF-1α expression [42]. Contrastingly, persistent hypoxiainduced HIF-1α stabilization repressed SP-D expression and enhanced the mRNA levels of an EMT-driving transcription factor TWIST, but not SNAIL. This was accompanied by phenotypic switch in the AECs exposed to persistent hypoxia (72-h hypoxia for in vitro studies) as seen by decreased E-cadherin expression and enhanced vimentin expression. SP-D is mainly derived from alveolar epithelial cells and therefore loss of its expression during persistent hypoxia along with enhanced EMT transcription factor expression clearly indicates phenotypic switch of these alveolar epithelial cells to more proliferative phenotype contributing to

Endothelial-to-mesenchymal transition (EndMT) is similar to EMT, which is characterized by a loss of endothelial cell-cell junctions, the acquisition of migratory properties, and phenotypic switch involving loss of endothelial-specific markers such as CD31 and vascular endothelial (VE)-cadherin expression, and the acquisition of mesenchymal markers α-SMA, and vimentin [43]. EndMT also contributes to fibrosis. The role of EndMT in pulmonary fibrosis involves phenotypic switch in the pulmonary EC lining the pulmonary capillaries. Radiation-induced pulmonary fibrosis (RIPF) may involve hypoxia-mediated EndMT as an initial pathological insult leading to fibrosis [13]. Fleckenstein and colleagues have shown that radiation during thoracic radiotherapy for lung cancer induces tissue hypoxia, in part, due to enhanced oxygen consumption by Macrophages. These macrophages are activated because of radiation-induced reduction in blood perfusion in the lungs contributing to lung injury [44]. This suggests

in AECs [36].

234 Hypoxia and Human Diseases

lung fibrosis [42].

In conclusion, current evidences suggest that the pathogenesis of human pulmonary fibrosis might involve the recruitment of fibroblasts derived from AECs through hypoxia-induced EMT as well as fibroblasts derived from pulmonary ECs through hypoxia-induced EndMT, apart from the bone marrow-derived precursors forming the fibrotic lesions. Thus, hypoxia could contribute to the formation of fibrotic lesions in the lung and hence the pathogenesis of pulmonary fibrosis (see **Figure 1**).

**Figure 1.** Summary of the effect of hypoxia on pulmonary tissue and vasculature. Hypoxia induces pulmonary edema by enhancing vascular permeability and decreasing the ability of alveolar fluid clearance. Hypoxia induces pulmonary vascular EndMT and alveolar EMT that result in myofibroblast proliferation ensuing pulmonary fibrosis. Hypoxiainduced PAH is a result of enhanced proliferation and survival of PASMCs. ALI and PAH can eventually progress to pulmonary fibrosis.

## **6. Hypoxia in lung injury resolution (fate of hypoxia as a consequence of pathological conditions)**

While in the early stages of ALI, hypoxia plays a major role in the progression of lung injury, intriguingly in chronic pulmonary pathological conditions that ensue hypoxic milieu, and hypoxia has also been found to be involved in enhancing injury resolution. Studies indicate a protective and anti-inflammatory role of HIFs such as HIF-1α in lung protection during the early exudative phase of ALI [45–47]. As mentioned above, hypoxia inactivates PHDs and stabilizes HIF-1α [45–47]. During the acute stage of ALI, inflammation, including enhanced neutrophil activity within the alveoli, leads to an increased alveolar edema and decreased alveolar gaseous exchange capacity. HIF stabilization has been shown to have anti-inflammatory role in conditions like intestinal inflammation. The protective role of HIF activators in the treatment of inflammatory bowel disease or ischemia and reperfusion injury of several organs has been shown in several studies [48–50]. Interestingly, Eckle et al. showed the beneficial role of normoxic HIF1A stabilization in lung protection during ALI, where HIF-dependent control of alveolar-epithelial glucose metabolism function as an endogenous feedback loop to dampen lung inflammation [51]. In vivo HIF-1α increased glycolysis, lactate production and glucose flux rates in alveolar epithelium. Overall, this normoxic stabilization of HIF-1α in alveolar epithelium increased glycolytic capacity and TCA flux thus optimizing mitochondrial respiration to enhance ATP production. This HIFdependent protection of mitochondrial function in ALI not only enhanced ATP production but also concomitantly prevented ROS accumulation and lung inflammation [51]. Hence, the role of hypoxia and subsequent HIF stabilization in reducing inflammation is prominent in resolution of ALI.

#### **6.1. Hypoxia and adenosine signaling in lung injury resolution**

Emigration of polymorphonucleated neutrophils (PMNs) through the endothelial barrier in an injured lung creates a potential for vascular fluid leakage leading to edema and decreased oxygenation [52]. The vascular endothelial adaptations to hypoxia include enhanced extracellular adenosine production during limited oxygen availability. In the vascular ECs, hypoxia induces enhanced expression of surface ectonucleotidases, CD39 that converts ATP/ADP to AMP (ectoapyrase), as well as CD73 that is involved in phosphohydrolysis of AMP to adenosine thus forming the source for extracellular adenosine production [52]. This enhanced extracellular adenosine can then signal through four different G-protein-coupled adenosine receptors, all of which are present on vascular endothelia thus enhancing adenosine signaling that is implicated in tissue protection in different models of injury including ALI. Several studies, notably couple of them from Eltzschig, H.K., et al. [52, 53], have shown the role of extracellular adenosine and its signaling in attenuating hypoxia-induced vascular leakage. They also showed that the source of ATP in hypoxic milieu is the PMNs. Hypoxia induces the production of ATP by PMNs, however, the exact mechanism by which ATP is produced still needs to be explored. This ATP is then phosphohydrolyzed as mentioned above to produce extracellular adenosine [53]. Enhanced adenosine concentrations activate adenosine receptor, (AdoRA2A/A2B on ECs, which when activated increases intracellular cyclic AMP (cAMP) and activates protein kinase A (PKA) to induce resealing of the endothelial-barrier [54]. The resealing of endothelial-barrier during PMN transmigration was obviated by inhibition of cAMP formation. This resealing effect is mediated by PKA-induced phosphorylation of vasodilator-stimulated phosphoprotein, a protein responsible for changes in the geometry of actin filaments and distribution of junctional proteins as a result affecting the characteristics of junctional proteins and increasing barrier function [54]. Intriguingly, adenosine not only activates the endothelial A2B receptor, but also neutrophil A<sup>2</sup> adenosine receptor which has been shown to play an important role in limitation and termination of PMN mediated systemic inflammatory responses. Few others have also demonstrated that PMN A<sup>2</sup> adenosine receptor stimulation decreased leukocyte adherence and transmigration which might contribute to attenuated vascular leak associated with leukocyte accumulation [53–55]. Thus, hypoxia-induced adenosine signaling in vascular ECs and PMNs contributes to decreased vascular leak and inflammation, both of which are beneficial in inflammatory conditions such as ALI (see **Figure 2**).

**6. Hypoxia in lung injury resolution (fate of hypoxia as a** 

**6.1. Hypoxia and adenosine signaling in lung injury resolution**

While in the early stages of ALI, hypoxia plays a major role in the progression of lung injury, intriguingly in chronic pulmonary pathological conditions that ensue hypoxic milieu, and hypoxia has also been found to be involved in enhancing injury resolution. Studies indicate a protective and anti-inflammatory role of HIFs such as HIF-1α in lung protection during the early exudative phase of ALI [45–47]. As mentioned above, hypoxia inactivates PHDs and stabilizes HIF-1α [45–47]. During the acute stage of ALI, inflammation, including enhanced neutrophil activity within the alveoli, leads to an increased alveolar edema and decreased alveolar gaseous exchange capacity. HIF stabilization has been shown to have anti-inflammatory role in conditions like intestinal inflammation. The protective role of HIF activators in the treatment of inflammatory bowel disease or ischemia and reperfusion injury of several organs has been shown in several studies [48–50]. Interestingly, Eckle et al. showed the beneficial role of normoxic HIF1A stabilization in lung protection during ALI, where HIF-dependent control of alveolar-epithelial glucose metabolism function as an endogenous feedback loop to dampen lung inflammation [51]. In vivo HIF-1α increased glycolysis, lactate production and glucose flux rates in alveolar epithelium. Overall, this normoxic stabilization of HIF-1α in alveolar epithelium increased glycolytic capacity and TCA flux thus optimizing mitochondrial respiration to enhance ATP production. This HIFdependent protection of mitochondrial function in ALI not only enhanced ATP production but also concomitantly prevented ROS accumulation and lung inflammation [51]. Hence, the role of hypoxia and subsequent HIF stabilization in reducing inflammation is prominent

Emigration of polymorphonucleated neutrophils (PMNs) through the endothelial barrier in an injured lung creates a potential for vascular fluid leakage leading to edema and decreased oxygenation [52]. The vascular endothelial adaptations to hypoxia include enhanced extracellular adenosine production during limited oxygen availability. In the vascular ECs, hypoxia induces enhanced expression of surface ectonucleotidases, CD39 that converts ATP/ADP to AMP (ectoapyrase), as well as CD73 that is involved in phosphohydrolysis of AMP to adenosine thus forming the source for extracellular adenosine production [52]. This enhanced extracellular adenosine can then signal through four different G-protein-coupled adenosine receptors, all of which are present on vascular endothelia thus enhancing adenosine signaling that is implicated in tissue protection in different models of injury including ALI. Several studies, notably couple of them from Eltzschig, H.K., et al. [52, 53], have shown the role of extracellular adenosine and its signaling in attenuating hypoxia-induced vascular leakage. They also showed that the source of ATP in hypoxic milieu is the PMNs. Hypoxia induces the production of ATP by PMNs, however, the exact mechanism by which ATP is produced still needs to be explored. This ATP is then phosphohydrolyzed as mentioned above to produce extracellular adenosine [53]. Enhanced adenosine concentrations activate adenosine receptor, (AdoRA2A/A2B on ECs, which when activated increases intracellular cyclic AMP (cAMP)

**consequence of pathological conditions)**

in resolution of ALI.

236 Hypoxia and Human Diseases

**Figure 2.** Hypoxia and adenosine signaling in the lungs. Hypoxia-induced extracellular adenosine production acts through adenosine receptors on ECs to enhance intracellular cAMP and PKA production. PKA catalyzes the phosphorylation of VASP, which integrates into stress fibers and helps seal the endothelial barrier by enhancing expression of AJs, TJs and also focal adhesion. PKA also enhances HIF-1A expression, which translocates into nucleus and enhances adenosine receptor transcription. Extracellular adenosine also acts on A2-receptors on PMNs and prevents their adhesion, rolling and infiltration into lung tissue. Thus, hypoxia-induced extracellular adenosine seals endothelial junctions, prevents PMN infiltration and protects lung tissue by preventing alveolar edema accumulation. PKA, protein kinase-A; PMN-polymorphonuclear neutrophils; ATP, adenosine triphosphate; AMP, adenosine monophosphate; A2bR, adenosine 2b receptor; cAMP, cyclic AMP; VASP, vasodilator-stimulated phosphoprotein; AJ, adherent junction; TJ, tight junction and ECM, extracellular matrix.

When adenosine signaling was inhibited in transgenic mice with targeted disruption of CD73 that were subjected to hypoxia, fulminant vascular leakage, associated with severe edema and inflammation was seen [56]. Recently, studies have shown three other mechanisms by which hypoxia enhances extracellular adenosine levels, including hypoxia-mediated repression of the equilibrative nucleoside transporters (ENT-1 and ENT-2) that are responsible for adenosine transport across the membrane into the cytoplasm; HIF-1α mediated inhibition of intracellular adenosine kinase that converts intracellular adenosine to AMP and transcriptional induction of AdoRA2B receptor [57]. These studies indicate the protective role of adenosine signaling during hypoxia, especially in the pulmonary tissue [37]. On the other hand, chronically increased adenosine levels are detrimental as seen in pathological conditions, such as asthma and chronic obstructive pulmonary disease (COPD), and they also correlate with degree of inflammation in COPD. In order to regulate excessive adenosine signaling, chronic exposure to hypoxia eventually induces endothelial CD26 and extracellular adenosine deaminase (ADA). CD26 on EC surface acts as the ADA-complexing protein and localizes ADA accumulation on EC surface limiting extracellular adenosine accumulation during prolonged hypoxia [55].

#### **6.2. Hypoxia and lung inflammation**

Uncontrolled inflammation is one of the major players in ALI and suppression of inflammation is beneficial for injury resolution [58, 59]. Interestingly, as mentioned above, hypoxiainduced, HIF-1–mediated enhanced expression of Adenosine A<sup>2</sup> receptor on different types of immune cells, along with enhanced extracellular adenosine levels, which activate these receptors, are responsible for anti-inflammatory and tissue-protecting effects of hypoxia [58, 59]. This anti-inflammatory effect is attributed to elevated intracellular cAMP levels through activation of adenylyl cyclase. Even pharmacological immunosuppressive molecules, such as catecholamines, neuropeptides, histamine and prostaglandins are known to have their effects through elevation of cAMP levels [59]. Therefore, this extracellular adenosine serves to report excessive collateral immune damage and prevents further damage by suppressing-activated immune cells. Adenosine triggers high-affinity A2A adenosine receptors on activated immune cells resulting in enhanced intracellular cAMP levels to suppress these immune cells. Few studies also show that hypoxia inhibits adenosine kinase, an enzyme responsible for re-phosphorylation of adenosine to AMP, to maximize the anti-inflammatory effect [60].

#### **6.3. Adenosine receptors in inflammation**

Adenosine receptors are a family of heptahelical transmembrane G-protein-coupled purinergic receptors that are classified into four types based on the potency of agonists with respect to the intracellular production of cAMP [37]. They are A1, A2A, A2B and A3 receptors. Extracellular agonists signal through these G protein receptors and can either stimulate (Gs) or inhibit (Gi) adenylyl cyclase, an enzyme that catalyzes the formation of cAMP. Cloning experiments show that high-affinity A2A and low-affinity A2B receptors activate adenylyl cyclase (Gs) enhancing the levels of intracellular cAMP, whereas high-affinity A1 and lowaffinity A3 receptors inhibit (Gi) adenylyl cyclase [37].

#### **6.4. Hypoxia induced adenosine signaling in individual immune cells**

and inflammation was seen [56]. Recently, studies have shown three other mechanisms by which hypoxia enhances extracellular adenosine levels, including hypoxia-mediated repression of the equilibrative nucleoside transporters (ENT-1 and ENT-2) that are responsible for adenosine transport across the membrane into the cytoplasm; HIF-1α mediated inhibition of intracellular adenosine kinase that converts intracellular adenosine to AMP and transcriptional induction of AdoRA2B receptor [57]. These studies indicate the protective role of adenosine signaling during hypoxia, especially in the pulmonary tissue [37]. On the other hand, chronically increased adenosine levels are detrimental as seen in pathological conditions, such as asthma and chronic obstructive pulmonary disease (COPD), and they also correlate with degree of inflammation in COPD. In order to regulate excessive adenosine signaling, chronic exposure to hypoxia eventually induces endothelial CD26 and extracellular adenosine deaminase (ADA). CD26 on EC surface acts as the ADA-complexing protein and localizes ADA accumulation on EC surface limiting extracellular adenosine accumulation during pro-

Uncontrolled inflammation is one of the major players in ALI and suppression of inflammation is beneficial for injury resolution [58, 59]. Interestingly, as mentioned above, hypoxia-

of immune cells, along with enhanced extracellular adenosine levels, which activate these receptors, are responsible for anti-inflammatory and tissue-protecting effects of hypoxia [58, 59]. This anti-inflammatory effect is attributed to elevated intracellular cAMP levels through activation of adenylyl cyclase. Even pharmacological immunosuppressive molecules, such as catecholamines, neuropeptides, histamine and prostaglandins are known to have their effects through elevation of cAMP levels [59]. Therefore, this extracellular adenosine serves to report excessive collateral immune damage and prevents further damage by suppressing-activated immune cells. Adenosine triggers high-affinity A2A adenosine receptors on activated immune cells resulting in enhanced intracellular cAMP levels to suppress these immune cells. Few studies also show that hypoxia inhibits adenosine kinase, an enzyme responsible for re-phosphorylation of adenosine to AMP, to maximize the anti-inflammatory

Adenosine receptors are a family of heptahelical transmembrane G-protein-coupled purinergic receptors that are classified into four types based on the potency of agonists with

Extracellular agonists signal through these G protein receptors and can either stimulate (Gs) or inhibit (Gi) adenylyl cyclase, an enzyme that catalyzes the formation of cAMP. Cloning experiments show that high-affinity A2A and low-affinity A2B receptors activate adenylyl cyclase (Gs) enhancing the levels of intracellular cAMP, whereas high-affinity A1 and low-

receptor on different types

A2A, A2B and A3 receptors.

induced, HIF-1–mediated enhanced expression of Adenosine A<sup>2</sup>

respect to the intracellular production of cAMP [37]. They are A1,

affinity A3 receptors inhibit (Gi) adenylyl cyclase [37].

longed hypoxia [55].

238 Hypoxia and Human Diseases

effect [60].

**6.2. Hypoxia and lung inflammation**

**6.3. Adenosine receptors in inflammation**


in T- and B-cells [67, 68]. This enhanced extracellular adenosine signals through A2A receptor and induces apoptosis in a subset of immature thymocytes through its cAMP elevating effects. In peripheral T-cells, activation of extracellular adenosine-mediated A2A receptor inhibits TCR-triggered IL-2 receptor upregulation, thereby inhibiting T-cell proliferation [69]. Other effects of adenosine signaling in CD8<sup>+</sup> cytotoxic T-lymphocytes include inhibition of inflammatory cytokine production, lethal hit delivery by granule exocyotosis, as well as FasL mRNA upregulation. It is interesting to note that in human blood peripheral leukocytes, more CD4<sup>+</sup> than CD8<sup>+</sup> T-cells express A2A receptor, but on activation of T-cells increased A2A receptor expression is predominantly observed in CD8+ T-cells. These studies suggest the variable expression of A2A receptors on T-cell subset and how they favor the production of anti-inflammatory cytokines over inflammatory cytokines. Compared to T-lymphocytes, not much is known about the effects of A2A receptor signaling in B-cell development, activation, antibody-production and class switching, and cytokine secretion [70].

However, it is very important to note that all the above mentioned effects of extracellular adenosine on immune cells were mostly observed in pharmacological experiments and is yet to be explored whether there are sufficient levels of extracellular adenosine in vivo to signal through A2A receptor on immune cells. So far, there is no evidence of physiological downregulation of immune cells by extracellular adenosine in vivo. However, hypoxia-induced extracellular adenosine may have anti-inflammatory effects even in in vivo similar to in vitro studies [67, 71, 72].

## **7. Conclusions and future directions**

Hypoxia, either as a consequence of the pathological condition during ILDs or as an etiology for ILDs has several roles in modulating the severity of the disease condition. Most of the effects of hypoxia are regulated through HIFs. Interestingly, stabilization of HIFs at various stages of lung injury can have different consequences either favoring injury resolution or worsening the condition. This complicates to provide a potential therapeutic target against HIFs to treat ILDs. Targeting hypoxia signaling was speculated to have therapeutic importance in inflammatory and ischemic conditions, such as inflammatory bowel disease, myocardial ischemic-reperfusion injury, ALI and so on. However, most of the clinical trials for drug discovery examined HIF inhibitors in the context of cancer treatment. Some of the examples include pharmacological HIF inhibitors such as dutasteride152 (ClinicalTrials.gov identifier: NCT00880672), topotecan153 (ClinicalTrials.gov identifier: NCT00117013), PX-478 (ClinicalTrials.gov identifier: NCT00522652) or digoxin13 (ClinicalTrials.gov identifier: NCT01763931) or the antisense oligonucleotide HIF inhibitor EZN-2968 (ClinicalTrials.gov identifier: NCT01120288). Apart from HIF inhibitors, HIF-stabilizing agents such as PHD inhibitors are also being studied as potential therapeutic targets in conditions where HIF stabilization is beneficial, such as, conditions which require enhanced angiogenesis (HIF activates VEGF and enhances angiogenesis) like bronchopulmonary dysplasia, a chronic disease effecting preterm neonates in which enhanced angiogenesis improves lung growth and function. Favoring the plethora of evidence from preclinical studies, in future, we can expect more clinical trials targeting PHD-HIF pathway as a potential therapy for ILDs and several other ischemic conditions.

## **Author details**

in T- and B-cells [67, 68]. This enhanced extracellular adenosine signals through A2A receptor and induces apoptosis in a subset of immature thymocytes through its cAMP elevating effects. In peripheral T-cells, activation of extracellular adenosine-mediated A2A receptor inhibits TCR-triggered IL-2 receptor upregulation, thereby inhibiting T-cell proliferation [69].

inflammatory cytokine production, lethal hit delivery by granule exocyotosis, as well as FasL mRNA upregulation. It is interesting to note that in human blood peripheral leukocytes,

variable expression of A2A receptors on T-cell subset and how they favor the production of anti-inflammatory cytokines over inflammatory cytokines. Compared to T-lymphocytes, not much is known about the effects of A2A receptor signaling in B-cell development, activation,

However, it is very important to note that all the above mentioned effects of extracellular adenosine on immune cells were mostly observed in pharmacological experiments and is yet to be explored whether there are sufficient levels of extracellular adenosine in vivo to signal through A2A receptor on immune cells. So far, there is no evidence of physiological downregulation of immune cells by extracellular adenosine in vivo. However, hypoxia-induced extracellular adenosine may have anti-inflammatory effects even in in vivo similar to in vitro studies [67, 71, 72].

Hypoxia, either as a consequence of the pathological condition during ILDs or as an etiology for ILDs has several roles in modulating the severity of the disease condition. Most of the effects of hypoxia are regulated through HIFs. Interestingly, stabilization of HIFs at various stages of lung injury can have different consequences either favoring injury resolution or worsening the condition. This complicates to provide a potential therapeutic target against HIFs to treat ILDs. Targeting hypoxia signaling was speculated to have therapeutic importance in inflammatory and ischemic conditions, such as inflammatory bowel disease, myocardial ischemic-reperfusion injury, ALI and so on. However, most of the clinical trials for drug discovery examined HIF inhibitors in the context of cancer treatment. Some of the examples include pharmacological HIF inhibitors such as dutasteride152 (ClinicalTrials.gov identifier: NCT00880672), topotecan153 (ClinicalTrials.gov identifier: NCT00117013), PX-478 (ClinicalTrials.gov identifier: NCT00522652) or digoxin13 (ClinicalTrials.gov identifier: NCT01763931) or the antisense oligonucleotide HIF inhibitor EZN-2968 (ClinicalTrials.gov identifier: NCT01120288). Apart from HIF inhibitors, HIF-stabilizing agents such as PHD inhibitors are also being studied as potential therapeutic targets in conditions where HIF stabilization is beneficial, such as, conditions which require enhanced angiogenesis (HIF activates VEGF and enhances angiogenesis) like bronchopulmonary dysplasia, a chronic disease effecting preterm neonates in which enhanced angiogenesis improves lung growth and function. Favoring the plethora of evidence from preclinical studies, in future, we can expect more clinical trials targeting PHD-HIF path-

T-cells express A2A receptor, but on activation of T-cells increased A2A

cytotoxic T-lymphocytes include inhibition of

T-cells. These studies suggest the

Other effects of adenosine signaling in CD8<sup>+</sup>

**7. Conclusions and future directions**

receptor expression is predominantly observed in CD8+

antibody-production and class switching, and cytokine secretion [70].

way as a potential therapy for ILDs and several other ischemic conditions.

than CD8<sup>+</sup>

more CD4<sup>+</sup>

240 Hypoxia and Human Diseases

Sandeep Artham<sup>1</sup> and Payaningal R. Somanath1, 2\*

\*Address all correspondence to: sshenoy@augusta.edu

1 Program in Clinical and Experimental Therapeutics, College of Pharmacy, University of Georgia and the Charlie Norwood VA Medical Center, Augusta, GA, USA

2 Department of Medicine, Vascular Biology Center and Cancer Center, Augusta University, Augusta, GA, USA

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244 Hypoxia and Human Diseases

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#### **Hypoxia Modulates the Adenosinergic Neural Network Hypoxia Modulates the Adenosinergic Neural Network**

Susana P. Gaytán and Rosario Pasaro Susana P. Gaytán and Rosario Pasaro

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/65242

#### **Abstract**

[69] Huang, S., et al., Role of A2a extracellular adenosine receptor-mediated signaling in adenosine-mediated inhibition of T-cell activation and expansion. Blood, 1997. **90**(4): p.

[70] Kojima, H., et al., Abnormal B lymphocyte development and autoimmunity in hypoxiainducible factor 1alpha-deficient chimeric mice. Proc Natl Acad Sci U S A, 2002. **99**(4):

[71] Hale, L.P., et al., Hypoxia in the thymus: role of oxygen tension in thymocyte survival.

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Am J Physiol Heart Circ Physiol, 2002. **282**(4): p. H1467–77.

1600–10.

246 Hypoxia and Human Diseases

p. 2170–4.

p. 6140–9.

The aim of this study was to review the latest findings about the neural plasticity on the adenosinergic neural network after the exposition to hypoxia. Identification of the neuromorphology that supports the physiological adaptations underlying the response of organisms to environmental factors including injurious exposures (specifically hypoxia) has been one of the major research challenges in biomedicine. To know these responses would connect the metabolic needs and the vegetative neuronal networks in an integrated way. Hypoxia refers to a state in which oxygen supply is insufficient and several neural cardiorespiratory structures are responsible for correcting and prevent‐ ing its effects. Although hypoxia is often a pathological condition, variations in arterial oxygen concentrations can be part of the normal physiological responses, for example, during hypoventilation training or strenuous physical exercise. Also, hypoxia is a serious consequence of preterm birth in the neonate. Neural plasticity is a persistent change in the morphology and/or function based on prior experiences, and it is crucial for understanding its effects. Plasticity is well evident when the triggering experience occurs early in life; but in the case of respiratory control plasticity, could also be present in adult life. The regulation of adenosinergic neural network maturation, especially in central cardiorespiratory areas, could provide new perspectives in respiratory new‐born distress symptoms.

**Keywords:** hypoxia, purinergic network, adenosine, central respiratory control, neu‐ ronal plasticity

## **1. Introduction**

One of the main functions of the cardiorespiratory system is to guarantee that all tissues are adequately oxygenated at all time, maintaining the normal mitochondrial oxidative process and ATP production. The most common electron acceptor is molecular oxygen (O2), and when O2 is

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

present, the mitochondria will undergo aerobic respiration. To maintain O2 levels, in healthy animals, ventilation is tightly controlled by a system that must maintain the precise constancy of alveolar and arterial blood gases and acid‐base status, as well as minimizing the work and metabolic cost of breathing. Deviations from these normal values lead to hypoxic tissue environments [1–6].

In mammals, a lack of O2 (hypoxia) induces acute reflexes including increasing ventilation and sympathetic tone in order to almost immediately improve the uptake and distribution of O2 to all tissues of the organism. When the conditions of hypoxia were prolonged, during hours or days, as a defence mechanism it would induce the expression of different genes that, consequently, would modify the ATP metabolism. Indeed, all the homeostatic control of the animal would be affected and other changes would be induced such as increased ventilation, erythropoiesis and angiogenesis, all of that would results in improved O2 tissue levels. In order to produce the complete response to hypoxia, a group of specialized cells are key to mediate these fast reflex responses. These cells are crucial because they are capable of sensing small variations of O2, and this information is crucial to maintain O2 homeostasis. Among the organs that respond acutely to hypoxia, the carotid body (CB) is currently attracting renewed medical interest, as its over‐activation seems to be involved in the autonomous dysfunction that accompanies numerous highly prevalent disorders such as sleep apnoea, diabetes, hyperten‐ sion and chronic heart failure [1–6].

The aim of this study was to review the latest findings about the neural plasticity of the adenosinergic neural network implicated in the central regulation of breathing after the exposition to hypoxia. Four types of hypoxia are currently known: first, the hypoxemic type, in which the blood O2 levels fall down and they could not saturate the molecules of haemo‐ globin. Secondly, in the anaemic type, low concentrations of functional haemoglobin avoid the erythrocytes that could make an effective transportation of O2.Thirdly, the stagnant type, in which hemodynamic is altered and the velocity and volume of the blood flow is diminished, as that occurs in shock or syncope. Finally, the histotoxic hypoxia was referred to a reduction due to a deficiency in the utilization of O2 by the cells. To compensate for hypoxia, cardiovas‐ cular and respiratory functions are implemented increasing the cardiac rhythm, causing hypertension, modifying the ventilatory rhythm and increasing the activity of the accessory breathing muscles of neck and upper chest. As the hypoxia continues to worsen, these compensatory mechanisms would begin to fail [1–6].

All of these systemic responses are controlled by specific brain areas that integrate the information about the hypoxic conditions and conduct the changes during the hypoxic insult to initiate an adaptive process. As in other systems, the neural plasticity is a central cue for the cardiovascular and respiratory responses to the hypoxia functional adaptation. Neural plasticity implies a persistent change in the morphology and/or function based on prior experiences and it is crucial for understanding the changes in the central control of cardior‐ espiratory functions. Plasticity is well evident when the triggering experience occurs early in life; but in the case of respiratory central control, plasticity could also be present in adult life [3, 5, 7–11]. Since ATP production is compromised during exposure to hypoxia, it is interesting to try to discuss the possible role of the purinergic signalling, in general, and the neuronal adenosinergic network, among other neural structures, responsible of the defence response against hypoxia. This interplay could confer emergent properties to the central respiratory control system. Understanding these mechanisms and their interactions may enable us to optimize hypoxia‐induced plasticity as a way to improve treatments for patients that suffer from different ventilatory impairments or other related pathologies [1–6]. On the other hand, the hypoxic hypometabolism differs in adults or young animals. Indeed, it would have a more evident effect in mammals when the levels of O2 consumption are higher (i.e. in small or young animals when they are exposed to cold). It is clear that a good strategical adaptation to low O2 levels (hypoxia) requires coordinated down‐regulation of metabolic demand, as well as tissue supply, in order to prevent a mismatch in ATP utilization and production that might end in a bioenergetic collapse. In this way, substantial experimental evidence suggests that common integrative structures are probably involved in the metabolic and ventilatory responses to hypoxia [12–15]. The synthesis of adenosine is related to the cellular ratio AMP/ ATP (**Figure 1**) and, obviously, to the energy metabolism of cells. In addition, in the central nervous system (CNS), an increase in neuronal activity needs a higher expense of energy and, for this reason, the extracellular levels of adenosine would be modified. The adenosinergic system acts, in CNS, to bind adenosine to one of the different adenosine receptors (A‐Rs). Usually, the increased high levels of extracellular adenosine would induce a decrease in neuronal activity. Because the cells reduce its activity, its need for energy falls down too. This nucleoside usually acts via receptor‐dependent mechanisms, and could also use receptor‐ independent mechanisms. Anyway, its complex and wide range of actions imply that adenosine could have a significant role in the defence against cell damage in areas of increased energy requirements, in tissues as well as in recovering the normal/physiological state from a pathologic one [6, 12–22].

present, the mitochondria will undergo aerobic respiration. To maintain O2 levels, in healthy animals, ventilation is tightly controlled by a system that must maintain the precise constancy of alveolar and arterial blood gases and acid‐base status, as well as minimizing the work and metabolic cost of breathing. Deviations from these normal values lead to hypoxic tissue

In mammals, a lack of O2 (hypoxia) induces acute reflexes including increasing ventilation and sympathetic tone in order to almost immediately improve the uptake and distribution of O2 to all tissues of the organism. When the conditions of hypoxia were prolonged, during hours or days, as a defence mechanism it would induce the expression of different genes that, consequently, would modify the ATP metabolism. Indeed, all the homeostatic control of the animal would be affected and other changes would be induced such as increased ventilation, erythropoiesis and angiogenesis, all of that would results in improved O2 tissue levels. In order to produce the complete response to hypoxia, a group of specialized cells are key to mediate these fast reflex responses. These cells are crucial because they are capable of sensing small variations of O2, and this information is crucial to maintain O2 homeostasis. Among the organs that respond acutely to hypoxia, the carotid body (CB) is currently attracting renewed medical interest, as its over‐activation seems to be involved in the autonomous dysfunction that accompanies numerous highly prevalent disorders such as sleep apnoea, diabetes, hyperten‐

The aim of this study was to review the latest findings about the neural plasticity of the adenosinergic neural network implicated in the central regulation of breathing after the exposition to hypoxia. Four types of hypoxia are currently known: first, the hypoxemic type, in which the blood O2 levels fall down and they could not saturate the molecules of haemo‐ globin. Secondly, in the anaemic type, low concentrations of functional haemoglobin avoid the erythrocytes that could make an effective transportation of O2.Thirdly, the stagnant type, in which hemodynamic is altered and the velocity and volume of the blood flow is diminished, as that occurs in shock or syncope. Finally, the histotoxic hypoxia was referred to a reduction due to a deficiency in the utilization of O2 by the cells. To compensate for hypoxia, cardiovas‐ cular and respiratory functions are implemented increasing the cardiac rhythm, causing hypertension, modifying the ventilatory rhythm and increasing the activity of the accessory breathing muscles of neck and upper chest. As the hypoxia continues to worsen, these

All of these systemic responses are controlled by specific brain areas that integrate the information about the hypoxic conditions and conduct the changes during the hypoxic insult to initiate an adaptive process. As in other systems, the neural plasticity is a central cue for the cardiovascular and respiratory responses to the hypoxia functional adaptation. Neural plasticity implies a persistent change in the morphology and/or function based on prior experiences and it is crucial for understanding the changes in the central control of cardior‐ espiratory functions. Plasticity is well evident when the triggering experience occurs early in life; but in the case of respiratory central control, plasticity could also be present in adult life [3, 5, 7–11]. Since ATP production is compromised during exposure to hypoxia, it is interesting to try to discuss the possible role of the purinergic signalling, in general, and the neuronal

environments [1–6].

248 Hypoxia and Human Diseases

sion and chronic heart failure [1–6].

compensatory mechanisms would begin to fail [1–6].

Furthermore, the above‐mentioned hypometabolism is mediated by an activation of the chemoreceptors by depletion in the arterial O2 partial pressure (PaO2 ) among other factors. The

sensing of the PaO2 is the principal afferent pathway to modify the alveolar ventilation, which

assure the O2 supply. Thus, arterial chemoreceptors (aortic bodies and CBs) serve an important role in the control of alveolar ventilation, but they also exert a powerful influence on cardio‐ vascular function. Aortic bodies sense likewise the levels of arterial carbon dioxide partial pressure (PaCO2 ) to regulate the depth and rhythm of breathing, but not changes in the blood

H+ concentration ([H+ ]). To detect this last factor it is necessary to understand the role of the CB that detects all the previously described arterial variables, and, as its major quality, they do not desensitize. Finally, central chemoreceptors located on the ventrolateral surface of medulla oblongata detect changes in cerebrospinal fluid [H+ ] (**Figure 1**) [5–7, 9–11].

It is obvious then that the hypoxic response is a complex effect that must be studied at different levels, including the central areas where the respiratory rhythm and pattern is generated, as well as newly described functions of the CB, the integrative nature of central chemoreceptors and the interaction between peripheral and central chemoreception. Furthermore, it must be also taken into account the metabolic signalling influence of purinergic control, in general, and, in particular, the adenosinergic influence [1, 13, 17, 18, 23, 24].

**Figure 1.** Schematic diagram of the hypoxic response generated at the ventrolateral medulla after the integration of peripheral and central chemoreceptors, including the role of metabolic signalling within the central neuronal network. Abbreviations: 12, hypoglossal nucleus; py, pyramidal tract; Sp5, spinal trigeminal nucleus.

## **2. Brain, hypoxia and pathophysiology**

Normal breathing must be continuously adjusted to maintain homeostasis of arterial blood gases by means of feedback, feedforward and adaptive control strategies that depend of the brainstem respiratory network. How this process is centrally controlled is still under discus‐ sion, despite the advances (especially thanks to the development of *in vitro* preparations) that have been recently made [2, 3, 6, 7]. The precise mechanisms (cellular, synaptic and molecular) that underlie the generation and modulation of respiratory rhythm/pattern still remain largely unknown. This lack of fundamental knowledge in the field of neural control of respiration, and its relationship with other neurovegetative controls, is likely due to the complexity of the mammalian brain where synaptic connectivity between central cardiorespiratory neurons, motoneurons and their peripheral counterparts, to the present day, cannot be reliably mapped [2, 3, 7, 8].

Adaptive responses have evolved in different animal species to guarantee a sufficient supply of O2 to tissues and to facilitate the survival of cells under transient or sustained conditions of limited O2 availability. Although hypoxia is often related to a pathological condition, it is of great importance to recognize that variations in PaO2 can be part of normal situations that require strenuous physiological responses, for example, during hypoventilation training or intense physical exercise [4, 5, 16, 21, 25–28]. It has to be also taken into account the condition of hypercapnia that results from an excess of PaCO2 , which results in acidification of blood and

tissues. The respiratory central medullary rhythm/pattern generator must respond to these chemosensory cues to maintain O2 and carbon dioxide (CO2) homeostasis in the blood and tissues. To do so, sensorial cells located in the periphery and CNS monitors the PaO2 and PaCO2

and initiates respiratory and autonomic reflex adjustments during hypoxic and hypercapnic. Activation of either the hypoxic or hypercapnic chemoreflex elicits both hyperventilation and sympathetic activation [4, 5, 16, 25–28]. However, the hypoxic insult is a fundamental drive to increase respiratory rate.

Traditionally, physiological research has been focused on the effect of a chronic sustained hypoxia (CH), but relatively few works were directed to the effect of periods of intermittent hypoxia that is maintained chronically. However, the different protocols resemble several pathological states that occur when patients suffer discontinuous expositions to hypoxia by malfunction of the ventilatory system. Nevertheless, these chronic intermittent hypoxia (CIH) laboratory protocols vary greatly between researches in lifespan of hypoxic exposure periods, numbers of hypoxic episodes *per day* and the total number of days of exposure. In any case, and in spite of the lack of a uniform definition, most of the recent data suggest that animals exposed to CIH would present multiple long‐term pathophysiological consequences that are similar to those observed in clinic and, for that, it would be a good animal model to study different respiratory pathologies [4, 5, 16, 21, 25–28].

#### **2.1. The role of carotid body as chemoreceptor**

**Figure 1.** Schematic diagram of the hypoxic response generated at the ventrolateral medulla after the integration of peripheral and central chemoreceptors, including the role of metabolic signalling within the central neuronal network.

Normal breathing must be continuously adjusted to maintain homeostasis of arterial blood gases by means of feedback, feedforward and adaptive control strategies that depend of the brainstem respiratory network. How this process is centrally controlled is still under discus‐ sion, despite the advances (especially thanks to the development of *in vitro* preparations) that have been recently made [2, 3, 6, 7]. The precise mechanisms (cellular, synaptic and molecular) that underlie the generation and modulation of respiratory rhythm/pattern still remain largely unknown. This lack of fundamental knowledge in the field of neural control of respiration, and its relationship with other neurovegetative controls, is likely due to the complexity of the mammalian brain where synaptic connectivity between central cardiorespiratory neurons, motoneurons and their peripheral counterparts, to the present day, cannot be reliably mapped

Adaptive responses have evolved in different animal species to guarantee a sufficient supply of O2 to tissues and to facilitate the survival of cells under transient or sustained conditions of limited O2 availability. Although hypoxia is often related to a pathological condition, it is of

can be part of normal situations that

Abbreviations: 12, hypoglossal nucleus; py, pyramidal tract; Sp5, spinal trigeminal nucleus.

**2. Brain, hypoxia and pathophysiology**

great importance to recognize that variations in PaO2

[2, 3, 7, 8].

250 Hypoxia and Human Diseases

O2 sensing is necessary for the activation of cardiorespiratory reflexes that permit the survival of individuals under hypoxic environments, like high altitude or pathological conditions (with reduced capacity for gas exchange between the lung alveoli and the blood). Changes are detected by the arterial chemoreceptors, in particular CB, to facilitate rapid adaptations to hypoxia including hyperventilation and sympathetic activation. The CB is located at the carotid bifurcation although its precise location varies between mammalian species. The CB is composed of functional units named glomeruli, which are clusters of cells separated by a profuse network of small capillaries and connective tissue. Each glomerulus (in close contact with blood vessels and nerve fibres) contains neuron‐like glomus (or type I) cells, which can be easily identified because they are strongly dopaminergic. Glomus cells are surrounded by processes of sustentacular (type II) cells that are positive for antibodies against glial fibrillary acidic protein and other glial markers. It has been shown that type II cells, or a subpopulation of them, are quiescent stem cells that are activated under hypoxia to proliferate and differen‐ tiate into glomus and other cell types [5, 9–11]. Glomus neuron‐like cells contain O2‐sensitive K+ channels, which are inhibited by hypoxia acting through several mechanisms, including release of gaseous transmitters (NO, CO, H2S), AMP‐activated protein kinases and/or reactive oxygen species. Finally, it has been demonstrated that CBs are polymodal receptors that would respond not only to modifications in PaO2 , PaCO2 and H+ , but also to stimuli as K+ , several neurotransmitters (i.e. norepinephrine), changes in temperature and osmolarity, as well as variations in the levels of glucose or insulin. Furthermore, reductions in CB blood flow (in addition to a decrease in PaO2 ) also provide powerful CB stimulation and remodelling over

#### time [5, 9–11].

The feedback from the CB is sent to the cardiorespiratory centres in the medulla oblongata via the afferent branches of the glossopharyngeal nerve. The afferent neurons to CB have their somas in the petrosal ganglion. This ganglion is anatomically distinct in several species of mammals like cat and rabbit, but in others (i.e. rat) it is part of a structure that includes the jugular and nodose ganglia. Their afferent fibres project to the commissural or medial subnu‐ clei of the nucleus tractus solitarius (NTS) (**Figure 1**) that convey sensory information regarding cardiorespiratory homeostasis in the form of graded action potential frequencies in fibres of the carotid sinus branch of the ninth cranial (glossopharyngeal) nerve. The efferent innervation arises primarily from the sympathetic fibres originating from the superior cervical ganglion constituting the ganglio‐glomerular nerve. Efferent innervation may best be considered as a modulating influence affecting the CB chemosensitivity largely, but not solely, via a modula‐ tion of CB blood flow [5, 7–11, 29, 30].

#### **2.2. Central integrative chemoreception process**

The brainstem is the central structure that operates the integrative process of the different chemoreceptors and baroreceptors inputs and which also generates the respiratory rhythm/ pattern. From these structures it should be outlined that the NTS is composed of a series of clusters of neuronal cell bodies forming a vertical column of grey matter embedded in the dorsal medulla oblongata. The NTS projects to, among other regions, the reticular formation, parasympathetic preganglionic neurons, hypothalamus and thalamus, conforming circuits that contribute to autonomic regulation (**Figure 1**). Anatomical and physiological experiments have shown that the dorsomedial part of the NTS is the primary termination site of glosso‐ pharyngeal and vagal baroreceptors, integrating the baroreceptor afferents, while the midline area, caudal to the *calamus scriptorius*, has been identified as a primary central termination site for CB afferents. The NTS neurons are stimulated by hypoxia or hypercapnia, and most profoundly by a combination of both. Under normal or pathological conditions, CB informa‐ tion reaches the respiratory pattern generator neuronal network via NTS glutamatergic neurons, which also target the rostral ventrolateral medulla oblongata (RVLM) presympathetic neurons, thereby raising sympathetic nerve activity (**Figure 1**). For that, NTS second‐order neurons could induce chemoreceptor reflex responses that include hyperpnoea, bradycardia and a sympathetically mediated vasoconstriction for a long‐term acclimatization to hypoxia [5, 7, 9–11, 29, 30].

Other group of neurons to be highlighted is the RVLM, containing several functionally distinct types of neurons, which control and orchestrate cardiovascular and respiratory responses to hypoxia and hypercapnia (**Figure 1**) [3, 7, 8, 29, 30]. At this level, chemoreceptors regulate presympathetic neurons and cardiovagal preganglionic neurons indirectly via inputs from the neurons related to the respiratory pattern generator. Secondary effects of chemoreceptors on the autonomic outflows result from changes in lung stretch afferent and baroreceptor activity [3, 7, 8, 29, 30].

neurotransmitters (i.e. norepinephrine), changes in temperature and osmolarity, as well as variations in the levels of glucose or insulin. Furthermore, reductions in CB blood flow (in

The feedback from the CB is sent to the cardiorespiratory centres in the medulla oblongata via the afferent branches of the glossopharyngeal nerve. The afferent neurons to CB have their somas in the petrosal ganglion. This ganglion is anatomically distinct in several species of mammals like cat and rabbit, but in others (i.e. rat) it is part of a structure that includes the jugular and nodose ganglia. Their afferent fibres project to the commissural or medial subnu‐ clei of the nucleus tractus solitarius (NTS) (**Figure 1**) that convey sensory information regarding cardiorespiratory homeostasis in the form of graded action potential frequencies in fibres of the carotid sinus branch of the ninth cranial (glossopharyngeal) nerve. The efferent innervation arises primarily from the sympathetic fibres originating from the superior cervical ganglion constituting the ganglio‐glomerular nerve. Efferent innervation may best be considered as a modulating influence affecting the CB chemosensitivity largely, but not solely, via a modula‐

The brainstem is the central structure that operates the integrative process of the different chemoreceptors and baroreceptors inputs and which also generates the respiratory rhythm/ pattern. From these structures it should be outlined that the NTS is composed of a series of clusters of neuronal cell bodies forming a vertical column of grey matter embedded in the dorsal medulla oblongata. The NTS projects to, among other regions, the reticular formation, parasympathetic preganglionic neurons, hypothalamus and thalamus, conforming circuits that contribute to autonomic regulation (**Figure 1**). Anatomical and physiological experiments have shown that the dorsomedial part of the NTS is the primary termination site of glosso‐ pharyngeal and vagal baroreceptors, integrating the baroreceptor afferents, while the midline area, caudal to the *calamus scriptorius*, has been identified as a primary central termination site for CB afferents. The NTS neurons are stimulated by hypoxia or hypercapnia, and most profoundly by a combination of both. Under normal or pathological conditions, CB informa‐ tion reaches the respiratory pattern generator neuronal network via NTS glutamatergic neurons, which also target the rostral ventrolateral medulla oblongata (RVLM) presympathetic neurons, thereby raising sympathetic nerve activity (**Figure 1**). For that, NTS second‐order neurons could induce chemoreceptor reflex responses that include hyperpnoea, bradycardia and a sympathetically mediated vasoconstriction for a long‐term acclimatization to hypoxia

Other group of neurons to be highlighted is the RVLM, containing several functionally distinct types of neurons, which control and orchestrate cardiovascular and respiratory responses to hypoxia and hypercapnia (**Figure 1**) [3, 7, 8, 29, 30]. At this level, chemoreceptors regulate presympathetic neurons and cardiovagal preganglionic neurons indirectly via inputs from the neurons related to the respiratory pattern generator. Secondary effects of chemoreceptors on

) also provide powerful CB stimulation and remodelling over

addition to a decrease in PaO2

tion of CB blood flow [5, 7–11, 29, 30].

**2.2. Central integrative chemoreception process**

time [5, 9–11].

252 Hypoxia and Human Diseases

[5, 7, 9–11, 29, 30].

On the other hand, central respiratory chemosensitivity is caused by direct effects of cerebro‐ spinal [H+ ] on neurons and indirect effects of CO2 via astrocytes. Central respiratory chemo‐ receptors are not definitively identified but several brainstem areas have been demonstrated to have a role as chemoreceptor. First, the retrotrapezoid nucleus (RTN), located at the rostral end of RVLM, is a particularly strong candidate (**Figure 1**). Indeed, the absence of RTN likely causes severe central apnoeas in congenital central hypoventilation syndrome. The RTN chemoreceptor neurons provide a CO2/H+ ‐dependent drive to breathe and serve as an integrator centre of convergence of chemosensory information from other central and periph‐ eral sites, including the CBs. Finally, the RTN chemosensitive neurons also appear to serve as important sites of integration of several stimuli, as these neurons are significantly modulated by inputs from vagal‐mediated pulmonary stretch receptors and from the hypothalamus [29, 30].

Another cluster of RVLM cells (constituted by a population of C1 catecholaminergic neurons) controls sympathetic vasomotor tone in resting and in hypoxic and hypercapnic conditions, including the peripheral chemoreflex [29, 30]. The increased sympathetic outflow elicited by peripheral chemoreceptors is mediated primarily by activation of the presympathetic neurons of the RVLM, the majority of which are C1 neurons. In fact, the cardiorespiratory effects of peripheral chemoreceptors are mediated in part by the direct glutamatergic inputs from the NTS to C1 neurons (**Figure 1**) [2, 3, 7, 8, 29, 30].

Recently, the description of the structures related to the respiratory rhythmogenesis has improved with the advent of the *in vitro* neonatal rodent brainstem preparation [31]. This recording technique has allowed for precise identification of specific medullary sites for separate but coupled rhythm generation or "oscillators". These neurons reside in the pre‐ Bötzinger complex and in the parafacial respiratory group (pFRG) located in the RVLM [29, 30]. The most exciting result so far was the finding that some inspiratory neurons in RVLM act as inspiratory pacemakers; they continue to produce rhythmic bursts of potentials even when the synaptic connections are blocked [2]. Although the inspiratory pacemaker neurons do not constitute a well‐defined group within the medulla, this group of neurons named pre‐ Bötzinger complex certainly play an important role in the generation and/or modulation of the breathing rhythm [2]. Of the several models proposed for generating respiratory rhythm, the most promising appears to be a hybrid model, which combines emergent properties of networks of synaptic connections and intrinsic membrane properties of individual neurons together with independent pacemaker‐type neurons [1– 3, 7, 8, 23, 29, 30].

Furthermore, several facts support that the pFRG/RTN complex is likely to be the major site of central CO2 chemo‐responsiveness. First, pFRG/RTN is characterized by glutamatergic interneurons that strongly express Phox2b (that codes for the homeodomain transcription factor expressed exclusively in the nervous system, in most neurons that control the viscera, like cardiovascular, digestive and respiratory systems). Besides, the Phox2b neurons are part of an uninterrupted chain of neurons in a circuit that includes the CBs and their afferents as well as the NTS projections to the RTN. The functional consequences of this linkage are that stimulation of the peripheral chemoreceptors enhances the slope of the central CO2 ventilatory response,and conversely, inhibition of the CBs reduces the slope of the central CO2 response [1–3, 7, 8, 23, 29, 30].

Another interesting central chemosensitive area is the caudal parapyramidal (Ppy), located near the ventral surface of the medulla, at the level of the pyramidal decussation and may function as well as the pFRG/RTN complex (**Figure 1**). Furthermore, medullary neurons activated in response to hypercapnia were only found in the Ppy area. Nevertheless, neurons in both regions, RTN and PPy, could belong to the same cell population based on their histochemical and physiological properties and their location, near the medullary surface that facilitates the sensing of the arterial composition [1, 23].

In any case, the brainstem cardiorespiratory control areas are connected with other areas such as periaqueductal gray (PAG), hypothalamus, amygdala, cortex and cerebellum (**Figure 1**). These areas also exert influences over the respiratory rhythm/pattern generator. In this way, it has been found, from data obtained by clinical evidences in patients submitted to deep brain stimulation (by means of stimulating electrodes that recorded field potentials during neuro‐ surgical procedures), that the PAG and the subthalamic nucleus have a key role in activating the central command of cardiorespiratory responses to stress. The PAG is an integrative structure that maintains a wide network of connectivity with different neural systems, such as prefrontal cortex, hypothalamus and nociceptive pathways. Moreover, the PAG efferent projections also addressed to the medullary cardiorespiratory control areas. Finally, anatom‐ ical evidences support the connectivity to amygdala and cortex from RVLM and neurons of the respiratory pattern generator that supports, among others effects, the vegetative correlate of emotions or learning (**Figure 1**) [3, 7, 8, 30, 31].

All of the above described structures are part of an extended neuronal network that participates in the regulation and integration of cardiovascular and respiratory functions. From all of the neurotransmitters shared by this complex neuronal network, the purinergic network is one of the choices to regulate the physiologic responses to hypoxia. Recent evidence suggests that ATP‐mediated purinergic signalling at the level of the RVLM coordinates cardiorespiratory responses triggered by hypoxia and hypercapnia by activating RTN and C1 neurons, respec‐ tively. For all of that, the role of ATP‐mediated signalling in the RVLM must be critical for cardiovascular and respiratory activities (**Figure 1**) [3, 7, 8, 29, 30].

#### **2.3. Pathophysiological responses to hypoxia**

Since the condition in which the whole body (or a region) was exposed to variations in arterial O2 concentrations can be part of the normal physiology, a mild and non‐damaging intermittent hypoxia (IH) is used intentionally, for example, during altitude training to develop an athletic performance adaptation at both the systemic and cellular level [4, 5, 16, 21, 26–28]. However, hypoxia is a deprivation of adequate O2 supply at the tissue level and, often, a pathological condition with very serious consequences of preterm birth in the neonate, for example. The main cause for this pathology is that the lungs of the human foetus are among the last organs to develop during pregnancy. The perinatal hypoxic‐ischemic cerebral injuries found in the clinic are a main problem of paediatrics because of its severe consequences for the posterior development of the infants, such as the appearances of cerebral paralysis. Accumulating evidence points to an evolving process of brain injury after intrapartum hypoxia‐ischemia, initiated *in utero* and extending into a recovery period. This process in the neonate originates numerous functional deficits, such as impaired resting ventilation and ventilatory response to hypoxia [17, 28, 31, 32].

stimulation of the peripheral chemoreceptors enhances the slope of the central CO2 ventilatory response,and conversely, inhibition of the CBs reduces the slope of the central CO2 response

Another interesting central chemosensitive area is the caudal parapyramidal (Ppy), located near the ventral surface of the medulla, at the level of the pyramidal decussation and may function as well as the pFRG/RTN complex (**Figure 1**). Furthermore, medullary neurons activated in response to hypercapnia were only found in the Ppy area. Nevertheless, neurons in both regions, RTN and PPy, could belong to the same cell population based on their histochemical and physiological properties and their location, near the medullary surface that

In any case, the brainstem cardiorespiratory control areas are connected with other areas such as periaqueductal gray (PAG), hypothalamus, amygdala, cortex and cerebellum (**Figure 1**). These areas also exert influences over the respiratory rhythm/pattern generator. In this way, it has been found, from data obtained by clinical evidences in patients submitted to deep brain stimulation (by means of stimulating electrodes that recorded field potentials during neuro‐ surgical procedures), that the PAG and the subthalamic nucleus have a key role in activating the central command of cardiorespiratory responses to stress. The PAG is an integrative structure that maintains a wide network of connectivity with different neural systems, such as prefrontal cortex, hypothalamus and nociceptive pathways. Moreover, the PAG efferent projections also addressed to the medullary cardiorespiratory control areas. Finally, anatom‐ ical evidences support the connectivity to amygdala and cortex from RVLM and neurons of the respiratory pattern generator that supports, among others effects, the vegetative correlate

All of the above described structures are part of an extended neuronal network that participates in the regulation and integration of cardiovascular and respiratory functions. From all of the neurotransmitters shared by this complex neuronal network, the purinergic network is one of the choices to regulate the physiologic responses to hypoxia. Recent evidence suggests that ATP‐mediated purinergic signalling at the level of the RVLM coordinates cardiorespiratory responses triggered by hypoxia and hypercapnia by activating RTN and C1 neurons, respec‐ tively. For all of that, the role of ATP‐mediated signalling in the RVLM must be critical for

Since the condition in which the whole body (or a region) was exposed to variations in arterial O2 concentrations can be part of the normal physiology, a mild and non‐damaging intermittent hypoxia (IH) is used intentionally, for example, during altitude training to develop an athletic performance adaptation at both the systemic and cellular level [4, 5, 16, 21, 26–28]. However, hypoxia is a deprivation of adequate O2 supply at the tissue level and, often, a pathological condition with very serious consequences of preterm birth in the neonate, for example. The main cause for this pathology is that the lungs of the human foetus are among the last organs to develop during pregnancy. The perinatal hypoxic‐ischemic cerebral injuries found in the clinic are a main problem of paediatrics because of its severe consequences for the posterior

[1–3, 7, 8, 23, 29, 30].

254 Hypoxia and Human Diseases

facilitates the sensing of the arterial composition [1, 23].

of emotions or learning (**Figure 1**) [3, 7, 8, 30, 31].

**2.3. Pathophysiological responses to hypoxia**

cardiovascular and respiratory activities (**Figure 1**) [3, 7, 8, 29, 30].

On the other hand, abnormalities or mutations of the medullary neuronal breathing rhythm/ pattern networks may also have a great impact on the progress of human diseases in children or adults. Failures in the breathing pattern with severe consequences are well‐documented. These problems, often cause CO2 retention in awake, and in particular, in sleeping subjects, that could be associated to neurodegenerative diseases such as Parkinson's disease, amyotro‐ phic lateral sclerosis or post‐polio syndrome. It is also been proved that these breathing alterations are often associated to medullary and multiple system atrophy of patients. These syndromes have been linked to deficits in neurons related with the respiratory control in the pre‐Bötzinger complex, pontine raphe and adjacent areas. Obviously, an understanding of how the response to hypoxia is organized and when or why the system become maladapted and could induce cell damage is extremely important for knowing how to fight against the diseases in the future [4, 5, 16, 21, 26–28].

It is well known that disruption of the drive to breathe is thought to contribute to the mortality of certain pathologies, including stroke or epilepsy, and it is the cause of sudden infant death syndrome (SIDS) [1–6]. In the case of SIDS, it has generally been accepted that, in the absence of trauma, children death occurs to either respiratory or circulatory failure. The events appear to be a sequential process, first hypoxia occurs, and then there must be a failure to recover from hypoxia. The failure to recover could occur when the infant does not arouse from sleep and/or self‐resuscitation mechanisms fail. For that, it has been proposed that there should be three necessary components for development of SIDS: congenital or acquired vulnerability, a critical "time‐window" during maturational development and an acute stressor. The arousal response is essential for avoiding the hypoxic conditions due to certain microenvironments that could cause the loss of consciousness or the risk of dying. A failure of the neural system that would induce the arousal response from sleep, in the hypoxic condition, could be related to the progress or, in certain cases, the fatal result in diagnosed SIDS. However, these kinds of malfunctions do not explain all the process that should appear in SIDS. In fact, it seems clear that an initial respiratory failure and hypoxemia ignites the sequence of responses that, dramatically, may cause the death. Respiratory chemoreceptor studies on infants at risk for SIDS have suggested that a decreased sensitivity to CO2 could play a causal role in these deaths [1–6, 33].

Concerning CB function, there is a significant increase in sensitivity of the peripheral chemo‐ receptors during the first few weeks of life and it has been frequently shown that CB dener‐ vation in animal models is followed by hypoventilation and sudden death later on. Therefore, these denervated animals for the most part are markedly symptomatic prior to death. The principal problem in translating these results to humans is that SIDS infants do not appear to have any symptoms before death. This fact implies that it could be a problem related to the central integration of CB information in SIDS, but its role is still under discussion. However, CB must be taken into account in the pathogeny of SIDS, because a partial decrease in the sensitivity to hypercapnia or hypoxemia would be a causal role in this syndrome [2, 33].

Several studies indicate that changes in the strength and/or pattern of respiratory‐sympathetic coupling may have pathological implications in the control of arterial pressure levels. Such dysfunctions can be observed in the experimental condition of CIH, and also is commonly observed in patients suffering from obstructive sleep apnoea (OSA). OSA consists of a repetitive obstruction of the upper airways during sleep. Each obstruction causes an episode of hypoxia leading to a picture of CIH causing a fall in the PaO2 and arterial haemoglobin

saturation. OSA is characterized by repetitive collapse or near collapse of the upper airway during sleep, and these repetitive events impose substantial adverse effects on multiple organ systems. As a result of these mechanical changes in the airway, hypoxemia and hypercapnia develop, which further stimulate respiratory effort. Without airway opening the increased drive is ineffective at increasing ventilation. Hypoxic episodes stimulate the CB, triggering an increased motor muscles towards the inspiratory output and an arousal reaction, which together solve the obstruction [1–6]. Following even very brief periods of IH interspersed with normoxia, hyperventilation and increased sympathetic activity are sustained over an hour or more (i.e. the so‐called long‐term facilitation). Central adaptive responses occur following CIH in the persistent elevation of tonic hyperactivity of neurons at the level of the hypothalamus and other structures [4, 16, 21, 26–28]. As OSA progresses, it frequently generates a syndrome with associated pathologies at different systems: cardiovascular (hypertension and augmented acute vascular accidents), hepato‐metabolic (insulin resistance, glucose intolerance, fatty liver disease) and neuropsychiatric (anxiety, depression and cognitive‐executive deficits). Clinical and experimental studies indicate that CIH is an important event in the occurrence of OSA‐ associated pathologies because it causes CB sensitization [1–6]. The process probably includes increasing CB chemoreceptor input to the brainstem leading to an exaggerated sympathetic tone, which generates hypertension and subsequent cardiovascular and metabolic pathologies. In OSA patients, the repetitive respiratory events lead to IH and CO2 retention, both of which can augment sympathetic nerve activity via stimulation of central and peripheral chemore‐ ceptors. Conditions of hypoxia, both chronic and intermittent lack of O2, seem to induce CNS plasticity of respiratory and sympathetic functions neuronal networks and metabolic changes that could also lead to pathological states [4, 5, 27, 28].

## **3. Purinergic neuronal networks and hypoxia**

ATP is released in an activity‐dependent manner from different cell types in the brain, fulfilling different roles as a neurotransmitter, neuromodulator, in astrocyte‐to‐neuron communication, propagating astrocytic responses and modulating microglia responses. So, purinergic signal‐ ling has been found to contribute at all levels of the nervous system, including enteric, autonomic and central [34–38]. The term purinergic receptor was classically introduced to name specific classes of membrane receptors that mediate the release of ATP (P2 receptors) or adenosine (P1 receptors). The group of adenosine P1 receptors (A1‐R, A2a‐R, A2b‐R, A3‐R) are expressed on presynaptic and postsynaptic neurons, on astrocytes, microglia and mature and precursor oligodendrocytes. The mechanisms of ATP signalling are equally diverse, acting by means of P2 receptors, including ionotropic (P2X‐R) and metabotropic (P2Y‐R) subtypes, as well as varying methods of transmission, including vesicular, volume‐regulated anion channel and gap junction hemichannel release of ATP from neuronal and non‐neuronal cells [34–38].

CB must be taken into account in the pathogeny of SIDS, because a partial decrease in the sensitivity to hypercapnia or hypoxemia would be a causal role in this syndrome [2, 33].

Several studies indicate that changes in the strength and/or pattern of respiratory‐sympathetic coupling may have pathological implications in the control of arterial pressure levels. Such dysfunctions can be observed in the experimental condition of CIH, and also is commonly observed in patients suffering from obstructive sleep apnoea (OSA). OSA consists of a repetitive obstruction of the upper airways during sleep. Each obstruction causes an episode

saturation. OSA is characterized by repetitive collapse or near collapse of the upper airway during sleep, and these repetitive events impose substantial adverse effects on multiple organ systems. As a result of these mechanical changes in the airway, hypoxemia and hypercapnia develop, which further stimulate respiratory effort. Without airway opening the increased drive is ineffective at increasing ventilation. Hypoxic episodes stimulate the CB, triggering an increased motor muscles towards the inspiratory output and an arousal reaction, which together solve the obstruction [1–6]. Following even very brief periods of IH interspersed with normoxia, hyperventilation and increased sympathetic activity are sustained over an hour or more (i.e. the so‐called long‐term facilitation). Central adaptive responses occur following CIH in the persistent elevation of tonic hyperactivity of neurons at the level of the hypothalamus and other structures [4, 16, 21, 26–28]. As OSA progresses, it frequently generates a syndrome with associated pathologies at different systems: cardiovascular (hypertension and augmented acute vascular accidents), hepato‐metabolic (insulin resistance, glucose intolerance, fatty liver disease) and neuropsychiatric (anxiety, depression and cognitive‐executive deficits). Clinical and experimental studies indicate that CIH is an important event in the occurrence of OSA‐ associated pathologies because it causes CB sensitization [1–6]. The process probably includes increasing CB chemoreceptor input to the brainstem leading to an exaggerated sympathetic tone, which generates hypertension and subsequent cardiovascular and metabolic pathologies. In OSA patients, the repetitive respiratory events lead to IH and CO2 retention, both of which can augment sympathetic nerve activity via stimulation of central and peripheral chemore‐ ceptors. Conditions of hypoxia, both chronic and intermittent lack of O2, seem to induce CNS plasticity of respiratory and sympathetic functions neuronal networks and metabolic changes

ATP is released in an activity‐dependent manner from different cell types in the brain, fulfilling different roles as a neurotransmitter, neuromodulator, in astrocyte‐to‐neuron communication, propagating astrocytic responses and modulating microglia responses. So, purinergic signal‐ ling has been found to contribute at all levels of the nervous system, including enteric, autonomic and central [34–38]. The term purinergic receptor was classically introduced to name specific classes of membrane receptors that mediate the release of ATP (P2 receptors) or adenosine (P1 receptors). The group of adenosine P1 receptors (A1‐R, A2a‐R, A2b‐R, A3‐R) are

and arterial haemoglobin

of hypoxia leading to a picture of CIH causing a fall in the PaO2

256 Hypoxia and Human Diseases

that could also lead to pathological states [4, 5, 27, 28].

**3. Purinergic neuronal networks and hypoxia**

ATP is involved in central respiratory control and may mediate changes in the activity of medullary respiratory neurons during hypercapnia. The P2 receptor family comprises seven ionotropic P2X‐R subunits (P2X1‐7), forming both homomeric or heteromeric receptors and eight metabotropic P2Y‐R subtypes (P2Y1, 2, 4, 6, 11, 12, 13, 14). The brain displays a robust mRNA expression, an intense binding, and immunoreactivity for both P2X‐R and P2Y‐R in neuronal and non‐neuronal elements, although the role of central P2‐R remains ill defined. The ATP‐mediated signalling in respiratory control and central chemoreception is associated to the profile of the P2X2‐R subunit. This subunit is expressed, by physiologically identified respiratory neurons, in areas of the ventral medulla, the pontine locus coeruleus, the NTS, and the raphe nuclei. There are several evidences that sustain the hypothesis that purinergic signalling could play a central role in the mechanisms underlying the chemosensitivity of RVLM (**Figure 1**). It has been demonstrated the responses evoked by ATP in neurons express‐ ing P2X‐Rs to changes in extracellular [H+ ]. In that way, this evidence supports the putative mechanism of chemosensitivity of RVLM cells, and it would be necessary the tonic release of ATP. This may be the case, when P2‐R blockade reduces the baseline firing of RVLM respiratory neurones. The modulation of P2X2‐R function, evoked by acidification of the extracellular environment during hypercapnia, contributes to the changes in activity of the RVLM respira‐ tory neurones that express these receptors [34–38].

Furthermore, it has been shown that several medullary areas may have chemosensitive responses mediated by ATP. In this way, experiments made in brain slices using cell‐attached recordings of membrane potentials have shown that CO2/H+ ‐receptive NTS neurons are activated by focal ATP applications. However, it has been evidenced that purinergic P2‐R blockade did not affect their CO2/H+ responsiveness [38]. On the other hand, CO2/H+ ‐sensitive raphe neurons were unaffected by ATP or P2‐R blockade [34, 38]. When the experiments where realized *in vivo*, ATP injection into the NTS increased cardiorespiratory activity; however, injections of a P2‐R antagonist into this area did not change the baseline breathing or the CO2/ H+ responsiveness [34, 38]. Indeed, a significant proportion of respiratory neurones located in the vicinity of the Bötzinger and pre‐Bötzinger areas express the P2X2‐R subunit and respond with an increase in discharge during ATP application. This fact could mean that purinergic signalling plays an additional role in the generation and shaping of central respiratory output, as well as premotoneurons that are responsible for transmitting this rhythm to the spinal motoneurons controlling the diaphragm and intercostal muscles [34–38].

Finally, as above stated, RVLM contributes to peripheral chemoreceptor modulation of breathing and blood pressure, by chemosensitive RTN neurons and presympathetic C1 neurons, respectively, and these neurons are activated by purinergic agonists. In contrast, the blockade of P2‐Rs in the RVLM blunted cardiorespiratory responses to peripheral che‐ moreceptor activation in anesthetized rats [34–38]. RTN neuronal activity was found to be independent of temperature and stimulus strength and was wholly retained when synap‐ tic activity was blocked using high‐Mg++, low‐Ca++ solution. In the RTN, mechanisms of chemoreception involved direct H+ ‐mediated activation of chemosensitive neurons and in‐ direct modulation by purinergic signalling. This modulation implies a CO2/H+ ‐evoked ATP release by RTN astrocytes, contributing to respiratory drive. ATP injection into the RTN increased breathing and blood pressure by a P2‐R dependent mechanism, at the cellular and systems level [38]. However, because the results using antagonists of P2‐R and focal injections did not elucidate the cells that were responsible, it is necessary more experimen‐ tal evidence to determine the putative chemoreceptors and, if it is the case, the above ob‐ served effects could be indirect ones. Nevertheless, purinergic signalling also modulates the activity of CO2/H+ ‐sensitive neurons at least in two other brainstem regions thought to contribute to central chemoreception (i.e. the caudal NTS and medullary raphe). In any case, these evidences suggest that purinergic signalling is a unique feature of RTN chemo‐ reception and point out to a unique CO2/H+ sensing mechanism in the RTN [34–38].

## **4. Adenosine receptors, brain development and pathophysiological hypoxic response**

The role of adenosine, as an extracellular signalling molecule, was defined after the observa‐ tions of the ability of purines to control the functioning of the heart. Adenosine modulates the activity of the nervous system at cellular level both presynaptically by inhibiting or facilitat‐ ing transmitter release, and postsynaptically by hyperpolarising or depolarising neurons, as well as exerting non‐synaptic effects (i.e. on glial cells). It is usually assumed that adenosiner‐ gic signalling provides a neuroprotective role. However, several researches have shown that, under determined circumstances, changes in the levels of adenosine could have the opposite effects, contributing to neuronal damage and cell death [6, 12–15, 19–22]. These two ways of actions could be determined by the union of adenosine to different subtypes of A‐Rs. Further‐ more, changes in the levels of expression of the different subtypes, interactions between these receptors, differential actions on neuronal and glial cells and several "time‐windows" (that are critical during development) could also provide different actions at different events, as well as adenosinergic agonist and antagonist compounds administration. Moreover, adeno‐ sine do not work isolated, and, in spite of this, it is still unclear if the role of A‐R subtypes (A1‐ R and A2‐R) in the control of neuroprotection is mostly due to the control of glutamatergic transmission. Another possible role of adenosine is that its protection is mediated by one of the homeostatic roles of its receptors, such as control of metabolism, neuroglial communica‐ tion, inflammatory response, neurogenesis or mechanism of action of growth factors [6, 12– 15, 19–22].

Adenosine acts in parallel as a neuromodulator and as a homeostatic modulator in the CNS [6, 12–22]. The adenosine role as a neuromodulator is especially important around the time of birth and is involved in the suppression of foetal and neonatal breathing, particularly during hypoxia when extracellular levels of this nucleoside rapidly increase. Apnoea of prematurity, defined as cessation of breathing lasting longer than 15 s and accompanied by bradycardia or hypoxia, is common occurring in 85% of infants born less than 34‐week gestation. Preterm birth constitutes approximately 6–12% of all births in industrialized countries and accounts for 70% of neonatal mortality and 75% of neonatal morbidity [6, 12–21]. Depending on gestational age and birth weight, preterm infants present a wide range of abnormal physio‐ logical responses due to their immature organ systems [17, 28, 31, 32]. During development A1‐Rs are especially important, being the earliest receptors expressed in the embryonic brain and heart. A1‐R activation potently inhibits the development of axons and can lead to leuko‐ malacia [18, 32].

independent of temperature and stimulus strength and was wholly retained when synap‐ tic activity was blocked using high‐Mg++, low‐Ca++ solution. In the RTN, mechanisms of

release by RTN astrocytes, contributing to respiratory drive. ATP injection into the RTN increased breathing and blood pressure by a P2‐R dependent mechanism, at the cellular and systems level [38]. However, because the results using antagonists of P2‐R and focal injections did not elucidate the cells that were responsible, it is necessary more experimen‐ tal evidence to determine the putative chemoreceptors and, if it is the case, the above ob‐ served effects could be indirect ones. Nevertheless, purinergic signalling also modulates

contribute to central chemoreception (i.e. the caudal NTS and medullary raphe). In any case, these evidences suggest that purinergic signalling is a unique feature of RTN chemo‐

The role of adenosine, as an extracellular signalling molecule, was defined after the observa‐ tions of the ability of purines to control the functioning of the heart. Adenosine modulates the activity of the nervous system at cellular level both presynaptically by inhibiting or facilitat‐ ing transmitter release, and postsynaptically by hyperpolarising or depolarising neurons, as well as exerting non‐synaptic effects (i.e. on glial cells). It is usually assumed that adenosiner‐ gic signalling provides a neuroprotective role. However, several researches have shown that, under determined circumstances, changes in the levels of adenosine could have the opposite effects, contributing to neuronal damage and cell death [6, 12–15, 19–22]. These two ways of actions could be determined by the union of adenosine to different subtypes of A‐Rs. Further‐ more, changes in the levels of expression of the different subtypes, interactions between these receptors, differential actions on neuronal and glial cells and several "time‐windows" (that are critical during development) could also provide different actions at different events, as well as adenosinergic agonist and antagonist compounds administration. Moreover, adeno‐ sine do not work isolated, and, in spite of this, it is still unclear if the role of A‐R subtypes (A1‐ R and A2‐R) in the control of neuroprotection is mostly due to the control of glutamatergic transmission. Another possible role of adenosine is that its protection is mediated by one of the homeostatic roles of its receptors, such as control of metabolism, neuroglial communica‐ tion, inflammatory response, neurogenesis or mechanism of action of growth factors [6, 12–

Adenosine acts in parallel as a neuromodulator and as a homeostatic modulator in the CNS [6, 12–22]. The adenosine role as a neuromodulator is especially important around the time of birth and is involved in the suppression of foetal and neonatal breathing, particularly during hypoxia when extracellular levels of this nucleoside rapidly increase. Apnoea of prematurity, defined as cessation of breathing lasting longer than 15 s and accompanied by bradycardia or

**4. Adenosine receptors, brain development and pathophysiological**

direct modulation by purinergic signalling. This modulation implies a CO2/H+

‐mediated activation of chemosensitive neurons and in‐

sensing mechanism in the RTN [34–38].

‐sensitive neurons at least in two other brainstem regions thought to

‐evoked ATP

chemoreception involved direct H+

reception and point out to a unique CO2/H+

the activity of CO2/H+

258 Hypoxia and Human Diseases

**hypoxic response**

15, 19–22].

The most common method of treatment of the apnoeas of prematurity is continuous positive airway pressure and administration of a methylxanthine. The family of methyl‐ xanthines includes caffeine (1, 3, 7‐trimethylxanthine), one of the most popular human stimulants, and all of them derivate from xanthine, that is a purine present in human and other organism's tissues and fluids. This group of alkaloids has therapeutically been used for their effects stimulating respiratory function by means of its excitatory effects on the CNS, because of its capacity to suppress respiratory depression, reduce periodic breathing and enhance diaphragmatic activity. Caffeine also increases ventilatory drive and im‐ proves sensitivity and/or responsiveness to changes in the level of PaO2 [6, 12–22]. The

discovery that methylxanthines acted as antagonists of adenosine receptors represented a crucial step to establish the idea that adenosine indeed acted as an extracellular signalling molecule operating on selective receptors. Caffeine, at high doses, can also inhibit phos‐ phodiesterases, block GABAa receptors or cause a release of intracellular Ca++. Further‐ more, caffeine acts on the respiratory cycle by antagonizing the actions of endogenous A1‐R, A2a‐R or A2b‐R [6, 12–22]. Studies on A1‐R have demonstrated that these receptors are found at high density in the brainstem and hypothalamus while A2a‐Rs are widely distributed in the medulla [14, 17, 18]. Animal studies have shown that caffeine treatment alters A‐R expression and distribution, cause transient motor impairments and could also be neurotoxic to the newborn. In rats, limited exposure to therapeutic doses of caffeine during early life (postnatal days 3–6, P3–P6) changes the distribution, density and sensi‐ tivity of A1‐Rs in several regions of the CNS; these changes could persist until adulthood. Caffeine treatment at P2–P6 mimics the clinical use of caffeine in human neonates. Since the relative level of maturation of the CNS in newborn rats in the first week of life is similar to that of a premature newborn human between 20 and 40 weeks postconception, newborn rats could serve as a suitable animal model to test the potential impact of peri‐ natal caffeine treatment on the adenosinergic system. The oral administration of caffeine in critical periods of newborn rat and immunohistological experiments showed an in‐ crease of A1‐R labelling in restricted cardiorespiratory related areas. These labelled struc‐ tures were the anterior hypothalamic area, ventromedial hypothalamic nucleus, parabrachial complex and ventrolateral medulla of the caffeine‐treated group at P6. For the subtype A2a‐R, it was found a moderate increase of immunolabelling in pontome‐ dullary and other hypothalamic areas also related to vegetative functions. Indeed, in‐ creased A1‐R and A2a‐R gene expression was observed in both the brainstem and hypothalamus at P5. These results showed an up‐regulation of adenosinergic maturation in central cardiorespiratory areas when the animals were caffeine treated in the neonatal period and could explain the pharmacological effects observed in caffeine treated prema‐ ture infants, and it would also imply that caffeine mediated a modification of the post‐ natal development of the adenosinergic system during a critical period or "time‐window" [6, 12–22]. To date, human data show that such caffeine treatment has no major side effects on neurodevelopmental outcome in children in the 38–42 weeks following birth and up to 2 years after the treatment. However, further research is required to determine the long‐term pathologic and functional effects of caffeine and the combination of caffeine and other substances on the developing immature brain [6, 12–22].

Anyway, adenosine, is not only crucial in development, it also mediates multifactorial forms of ventilatory responses. The reduced hypoxic ventilatory response could be attributed to depressed adenosinergic peripheral excitatory mechanisms and to enhanced adenosinergic central depression mechanisms, both of which contribute to the blunted ventilatory response in different metabolic states (**Figure 1**). Several important groups of clinical studies, in which the adenosinergic network role has been demonstrated, are related to OSA, asthma and inter‐ stitial lung disease such as idiopathic pulmonary fibrosis (IPF) [1–6]. Levels of adenosine re‐ ceptors are altered in the lungs of asthmatics and OSA patients and a recent study has shown that the A2b‐R is increased in remodelled airway epithelial cells of rapidly progressing IPF patients [6, 12–22]. Furthermore, CIH (as an experimental OSA model) elicits phrenic long‐ term facilitation by an adenosine‐dependent mechanism [2, 6, 12–22]. All of the above are interesting evidences about the mechanisms that support and induce inflammatory and tis‐ sue remodelling processes in these pathological states; however, it is necessary to do more research on the pathways that provoke their progressive and chronic evolution. For example, there are already implemented several models of deregulated or overactive wound healing pathways to explain how these processes contribute to an excessive remodelling response such as seen in chronic lung disease [2, 6, 12–22]. Consistent with this, adenosine levels are elevated in the lungs of patients with chronic lung disease, where it is hypothesized that ade‐ nosine regulates the balance between tissue repair and excessive airway remodelling. Fur‐ thermore, it has been demonstrated that exogenous adenosine treatment can elicit acute bronchoconstriction in patients with asthma or OSA [1–6]. In contrast, the administration of adenosine to healthy subject did not affect them, suggesting a fundamental difference with respect to adenosinergic signalling in the treated patients. The differential response could be mediated by the activation of A‐Rs that would modify the activity of different cell types that play a central role in chronic lung disease. These groups of possible targets include mast cells, eosinophils, macrophages, airway epithelial cells, pulmonary fibroblasts and airway smooth muscle cells. Indeed, recent studies directly demonstrate that adenosine is involved in the regulation of pulmonary fibrosis. Lastly, there are correlations between the degree of inflam‐ mation and damage and adenosine accumulations in adenosine deaminase‐deficient individ‐ uals. Furthermore, purinergic metabolism and signalling components are altered in a manner that promotes adenosine production in tissue samples from patients with OSA and IPF. These modifications were related to the very important changes found in the expression of the pro‐ moter molecules of inflammatory process that could be induced by A2b‐R signalling. Finally, it was interesting to point out that it has been demonstrated that activation of A2b‐Rs can influence the production of inflammatory and fibrotic mediators from macrophages isolated from these patients [6, 12–22].

All of the above findings suggest that adenosine‐based therapeutics may be beneficial in the treatment of chronic lung diseases such as OSA and IPF. On the other hand, it is known that inflammation‐induced release of prostaglandin E2 changes breathing patterns and the re‐ sponse to CO2 levels. This bioactive eicosanoid regulates many biologically important proc‐ esses as a potent activator of several signalling pathways, through four distinct G‐protein‐ coupled receptors. All of this alters neural network activity in the pre‐Bötzinger rhythm‐ generating complex and in the chemosensitive brainstem respiratory regions, thereby increasing sigh frequency and the depth of inspiration with implications for inspiration and sighs throughout life, and the ability to autoresuscitate when breathing fails [2, 6, 7, 12–15, 19– 22, 29, 30].

## **5. Conclusion**

in central cardiorespiratory areas when the animals were caffeine treated in the neonatal period and could explain the pharmacological effects observed in caffeine treated prema‐ ture infants, and it would also imply that caffeine mediated a modification of the post‐ natal development of the adenosinergic system during a critical period or "time‐window" [6, 12–22]. To date, human data show that such caffeine treatment has no major side effects on neurodevelopmental outcome in children in the 38–42 weeks following birth and up to 2 years after the treatment. However, further research is required to determine the long‐term pathologic and functional effects of caffeine and the combination of caffeine

Anyway, adenosine, is not only crucial in development, it also mediates multifactorial forms of ventilatory responses. The reduced hypoxic ventilatory response could be attributed to depressed adenosinergic peripheral excitatory mechanisms and to enhanced adenosinergic central depression mechanisms, both of which contribute to the blunted ventilatory response in different metabolic states (**Figure 1**). Several important groups of clinical studies, in which the adenosinergic network role has been demonstrated, are related to OSA, asthma and inter‐ stitial lung disease such as idiopathic pulmonary fibrosis (IPF) [1–6]. Levels of adenosine re‐ ceptors are altered in the lungs of asthmatics and OSA patients and a recent study has shown that the A2b‐R is increased in remodelled airway epithelial cells of rapidly progressing IPF patients [6, 12–22]. Furthermore, CIH (as an experimental OSA model) elicits phrenic long‐ term facilitation by an adenosine‐dependent mechanism [2, 6, 12–22]. All of the above are interesting evidences about the mechanisms that support and induce inflammatory and tis‐ sue remodelling processes in these pathological states; however, it is necessary to do more research on the pathways that provoke their progressive and chronic evolution. For example, there are already implemented several models of deregulated or overactive wound healing pathways to explain how these processes contribute to an excessive remodelling response such as seen in chronic lung disease [2, 6, 12–22]. Consistent with this, adenosine levels are elevated in the lungs of patients with chronic lung disease, where it is hypothesized that ade‐ nosine regulates the balance between tissue repair and excessive airway remodelling. Fur‐ thermore, it has been demonstrated that exogenous adenosine treatment can elicit acute bronchoconstriction in patients with asthma or OSA [1–6]. In contrast, the administration of adenosine to healthy subject did not affect them, suggesting a fundamental difference with respect to adenosinergic signalling in the treated patients. The differential response could be mediated by the activation of A‐Rs that would modify the activity of different cell types that play a central role in chronic lung disease. These groups of possible targets include mast cells, eosinophils, macrophages, airway epithelial cells, pulmonary fibroblasts and airway smooth muscle cells. Indeed, recent studies directly demonstrate that adenosine is involved in the regulation of pulmonary fibrosis. Lastly, there are correlations between the degree of inflam‐ mation and damage and adenosine accumulations in adenosine deaminase‐deficient individ‐ uals. Furthermore, purinergic metabolism and signalling components are altered in a manner that promotes adenosine production in tissue samples from patients with OSA and IPF. These modifications were related to the very important changes found in the expression of the pro‐ moter molecules of inflammatory process that could be induced by A2b‐R signalling. Finally, it was interesting to point out that it has been demonstrated that activation of A2b‐Rs can

and other substances on the developing immature brain [6, 12–22].

260 Hypoxia and Human Diseases

Identification of the neurophysiological mechanisms underlying the response of organisms to environmental factors, in particular, to injurious exposures like hypoxia, represents one of the most important research problems in biomedicine. Neural plasticity, as a persistent change in the morphology and/or function based on prior experiences, is crucial for understanding the effects of O2 supply changes over neuronal networks. Plasticity is well evident when the triggering experience occurs early in life; but in the case of respiratory control plasticity, could also be present in adult life. The regulation of adenosinergic neural network maturation, especially in central cardiorespiratory areas, could provide new perspectives in respiratory newborn distress symptoms. Adenosine acts as an extracellular signalling molecule operating on selective receptors. Regulation of adenosinergic maturation in central cardiorespiratory areas in caffeine‐treated neonatal mammals could explain the pharmacological effects of caffeine observed in premature infants. Anyhow, the neuroplasticity observed in the cardior‐ espiratory network is fundamental to maintain life in many adverse conditions.

The central and peripheral chemical drive to breathe is associated with several widespread autonomic disorders. Deficits in central chemical drive are associated with central sleep apnoea, a debilitating disease with few therapies besides constant positive airway pressure. In addition, disruption of the drive to breathe is thought to contribute to mortality of certain pathologies, including SIDS, stroke and epilepsy. Finally, in OSA, certain forms of hypertension and heart failure, it has been observed sensitization of peripheral chemoreceptor drive, particularly the sympathetic component and this over‐activity is thought to contribute to the pathology.

Purinergic signalling has been proposed to be an excellent system to target for therapies of numerous pathologies, mainly due to novel pharmacological agents being developed. As more detailed understanding of the purinergic mechanisms involved in the chemical drive to breathe are uncovered, these would allow to possible pharmacological treatments of the aforemen‐ tioned pathologies with the newly developed purinergic agents.

## **Author details**

Susana P. Gaytán\* and Rosario Pasaro

\*Address all correspondence to: sgaytan@us.es

Department of Physiology, University of Seville, Sevilla, Spain

## **References**


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**Author details**

262 Hypoxia and Human Diseases

Susana P. Gaytán\*

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Department of Physiology, University of Seville, Sevilla, Spain

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#### **The HIF System Response to ESA Therapy in CKD‐ Anemia** The HIF System Response to ESA Therapy in CKD-Anemia

Sandra Ribeiro, Luís Belo, Flávio Reis and Alice Santos‐Silva Sandra Ribeiro, Luís Belo, Flávio Reis and Alice Santos-Silva

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/64611

#### Abstract

Anemia is a common complication of chronic kidney disease (CKD) associated with disease progression and increased mortality. This anemia is mainly due to inadequate production of erythropoietin (EPO) by the failing kidneys, resulting from the reduction in renal EPO-producing cells (REPC) or from dysregulation of the hypoxia-inducible factor (HIF) system that regulates several genes related to hypoxia, angiogenesis, fibrosis and glucose metabolism, among others. In this chapter, we present a review on the HIF system in CKD-anemia, the HIF response to erythropoiesis-stimulating agents (ESA) therapy and its potential involvement in the development of ESA resistance by enhancing kidney fibrosis and inflammation. Due to concerns related to ESA use, new drugs to correct anemia are under study, being the prolyl hydroxylase inhibitors the most promising candidates.

Keywords: chronic kidney disease, erythropoietin resistance, fibrosis, HIF system, Hypoxia, inflammation

#### 1. Introduction

Anemia is a common complication of chronic kidney disease (CKD) that often develops early in the course of the disease, and its frequency and severity increase with the decline of renal function [1]. This condition is associated with a decreased quality of life [2, 3], increased hospitalizations and comorbidities [4, 5], progression of renal dysfunction [6–8], enhanced cardiovascular complications [9, 10] and mortality [11–13]. The main cause for anemia in CKD patients is erythropoietin (EPO) deficit, due to decreased hormone production by the failing kidneys; other factors can also contribute to the development or worsening of CKDanemia, such as iron deficiency, inflammation and uremic toxins, among others [14].

© The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited.

EPO is a glycoprotein that presents several functions acting as a hormone, cytokine or growth factor on target cells that express the EPO receptors (EPOR), through different pathways. In the bone marrow, EPO controls cell proliferation, differentiation and death of erythroid cells.

During fetal life, the majority of EPO is produced by the liver; after birth there is a switch to renal production, and in the adulthood, 90% of this hormone is produced by the kidneys, whereas the liver is a secondary site of production [15]. EPO is also expressed in the brain, spleen, lung and testis, but its contribution to serum EPO levels is not clarified [16]. The kidney cells responsible for EPO production are still under debate, but several studies showed that renal EPO-producing cells (REPC) include the peritubular fibroblast-like interstitial cells in the inner cortex and in the outer medulla [17, 18], the proximal and distal convoluted tubules and cortical collecting ducts [19]. REPC are sensitive to changes in oxygen (O2) tension, and in conditions of hypoxia, the kidney responds increasing the number of REPC capable of producing EPO [20].

In CKD, the severity of the disease defines the kidney capacity to produce EPO [21, 22]. Indeed, patients with GFR >30mL/min/1.73m2 are still able to induce a physiologic response to anemia, showed by the normal or even elevated serum EPO levels [23, 24]. Nevertheless, serum EPO levels may not be sufficient for the degree of anemia; actually, anemic patients with normal renal function may present a 10-fold to 100-fold increase in serum EPO levels [25, 26], to achieve correction of anemia.

The kidney is the major site of EPO production in the adults; however, it is possible that extrarenal sites contribute for the marked rise in plasma EPO in end-stage renal disease (ESRD) patients [27], as already showed in animal models of kidney injury [28, 29]. It was also reported that patients with anemia can switch EPO production from the kidney to the liver [30, 31], as can be shown by glycoform analysis of EPO. Indeed, the posttranslational EPO glycosylation is specific of the synthesizing cells, giving rise to different EPO glycoforms that can be used to localize EPO synthesis [30, 32].

Hypoxia regulates the EPO gene through the hypoxia-inducible factor (HIF) system [20]. This HIF system includes O2-dependent HIF-1α, HIF-2α (also known as endothelial PAS domaincontaining protein 1) and HIF-3α subunits, and the constitutively expressed HIF-1β and HIF-2β subunits (also known as aryl hydrocarbon receptor nuclear translocator). The HIF-α subunits are hydroxylated in specific proline residues, by the prolyl-4-hydroxylase (PHD) proteins that require O2 as a co-substrate (Figure 1). The hydroxylated HIF-α subunit targets the von Hippel-Lindau tumor suppressor protein (VHL) to be recognized by an ubiquitin ligase complement that will induce a rapid ubiquitination and proteasomal degradation of HIF-α subunits. Under normoxia, HIF-α subunits are almost undetectable, but in hypoxic conditions, the hydroxylation by PHD proteins is inhibited; thus, the HIF-α accumulates in the cytoplasm, is translocated to the nucleus and binds to the HIF-β subunit, forming a complex that recruits the coactivators P300/CBP and activates the transcription of several genes [20].

Several genes are regulated by the HIF-1α and HIF-2α subunits (Figure 1), but recent studies showed that HIF-2α is the main regulator of EPO synthesis in the kidney and liver [33–35] and is also important for the regulation of several factors involved in iron homeostasis, as iron is an important element for hemoglobin (Hb) synthesis [36]. The HIF-1α subunit activates the transcription of glucose metabolism, angiogenesis and fibrosis related genes to promote wound healing [37]. The role of HIF-3α is still ambiguous and under current investigation. It is known that HIF-3α presents several isoforms with different roles [38]; the up-regulation of some HIF-3α isoforms appears to act as a negative feedback mechanism to regulate HIF-1α and/or HIF-2α subunits; however, recent studies showed that HIF-3α might share with HIF-1α the regulation of some genes [39].

EPO is a glycoprotein that presents several functions acting as a hormone, cytokine or growth factor on target cells that express the EPO receptors (EPOR), through different pathways. In the bone marrow, EPO controls cell proliferation, differentiation and death of erythroid cells. During fetal life, the majority of EPO is produced by the liver; after birth there is a switch to renal production, and in the adulthood, 90% of this hormone is produced by the kidneys, whereas the liver is a secondary site of production [15]. EPO is also expressed in the brain, spleen, lung and testis, but its contribution to serum EPO levels is not clarified [16]. The kidney cells responsible for EPO production are still under debate, but several studies showed that renal EPO-producing cells (REPC) include the peritubular fibroblast-like interstitial cells in the inner cortex and in the outer medulla [17, 18], the proximal and distal convoluted tubules and cortical collecting ducts [19]. REPC are sensitive to changes in oxygen (O2) tension, and in conditions of hypoxia, the kidney responds increasing the number of REPC capable of pro-

In CKD, the severity of the disease defines the kidney capacity to produce EPO [21, 22]. Indeed, patients with GFR >30mL/min/1.73m2 are still able to induce a physiologic response to anemia, showed by the normal or even elevated serum EPO levels [23, 24]. Nevertheless, serum EPO levels may not be sufficient for the degree of anemia; actually, anemic patients with normal renal function may present a 10-fold to 100-fold increase in serum EPO levels [25, 26],

The kidney is the major site of EPO production in the adults; however, it is possible that extrarenal sites contribute for the marked rise in plasma EPO in end-stage renal disease (ESRD) patients [27], as already showed in animal models of kidney injury [28, 29]. It was also reported that patients with anemia can switch EPO production from the kidney to the liver [30, 31], as can be shown by glycoform analysis of EPO. Indeed, the posttranslational EPO glycosylation is specific of the synthesizing cells, giving rise to different EPO glycoforms that

Hypoxia regulates the EPO gene through the hypoxia-inducible factor (HIF) system [20]. This HIF system includes O2-dependent HIF-1α, HIF-2α (also known as endothelial PAS domaincontaining protein 1) and HIF-3α subunits, and the constitutively expressed HIF-1β and HIF-2β subunits (also known as aryl hydrocarbon receptor nuclear translocator). The HIF-α subunits are hydroxylated in specific proline residues, by the prolyl-4-hydroxylase (PHD) proteins that require O2 as a co-substrate (Figure 1). The hydroxylated HIF-α subunit targets the von Hippel-Lindau tumor suppressor protein (VHL) to be recognized by an ubiquitin ligase complement that will induce a rapid ubiquitination and proteasomal degradation of HIF-α subunits. Under normoxia, HIF-α subunits are almost undetectable, but in hypoxic conditions, the hydroxylation by PHD proteins is inhibited; thus, the HIF-α accumulates in the cytoplasm, is translocated to the nucleus and binds to the HIF-β subunit, forming a complex that recruits the

Several genes are regulated by the HIF-1α and HIF-2α subunits (Figure 1), but recent studies showed that HIF-2α is the main regulator of EPO synthesis in the kidney and liver [33–35] and is also important for the regulation of several factors involved in iron homeostasis, as iron is an

coactivators P300/CBP and activates the transcription of several genes [20].

ducing EPO [20].

268 Hypoxia and Human Diseases

to achieve correction of anemia.

can be used to localize EPO synthesis [30, 32].

Figure 1. Regulation of hypoxia-inducible system. (A) In conditions of normoxia, the HIF-α subunits are hydroxylated, in specific proline residues by prolyl-4-hydroxylase (PHD) proteins, which recruit the von Hippel-Lindau tumor suppressor protein (VHL) a signal for rapid ubiquitination and proteasomal degradation of HIF-α subunits. PHD inhibitors are under development, as they might impair the degradation of the HIF-α subunits, improving anemia. (B) Under hypoxic conditions, the PHD proteins are inhibited, and consequently, the HIF-α subunits are not targeted by VHL protein for degradation, translocating to the nucleus and binding to the HIF-β subunit, forming a complex that recruit the coactivators P300/CBP, leading to the transcription of several genes that will depend on the type of HIF-α subunit (HIF-1α or HIF-2a) that binds to the target gene sequences. There is a crosstalk between hypoxia and inflammation, leading to the activation of the nuclear factor kappa beta (NF-κB) pathway that can also induce HIF-1α accumulation.

This chapter reviews the HIF response to erythropoiesis-stimulating agents (ESA) therapy focusing on its potential involvement in the development of ESA resistance, by enhancing kidney fibrosis and inflammation.

## 2. Hypoxia and progression of renal disease

Renal hypoxia is well known as an important contributor for the progression of renal disease. A study conducted in a rat model of diabetic nephropathy reported that intrarenal hypoxia develops early in the course of the disease and precedes the alterations in circulating biomarkers of kidney damage [40]. Irrespective of the initial cause of CKD, the histopathological

analysis of renal biopsies showed that fibrosis is the common final pathway [41]. The underlying mechanisms are still debatable.

Glomerular injury leads to a reduction in glomerular blood flow and consequently limits blood flow into peritubular capillaries, causing hypoxia and tubulointerstitial injury [42]. After an initial injury, the tubular cells will attempt to correct and repair the injury by recruiting and activating several cells, such as macrophages, fibroblasts and epithelial tubular cells that will release pro-inflammatory cytokines and fibrosis factors, and contribute to excessive interstitial extracellular matrix (ECM) accumulation and expansion. Transforming growth factor beta (TGF-β), a recognized pro-fibrotic factor, appears to be central for fibroblast activation, proliferation and transdifferentiation, contributing to ECM deposition [43]. TGF-β also presents immunomodulatory effects on macrophages and monocyte recruitment, leading to the production of inflammatory cytokines [44]. In early renal injuries, M2-type macrophages are recruited to promote tissue remodeling; however, if the injury is continuous, more inflammatory monocytes will be recruited differentiating their phenotype into M1-type macrophages, responsible for the release of pro-inflammatory cytokines (such as tumor necrosis factor [TNFα], interferon [IFN]-γ, interleukin (IL)-1β and IL-6) and cell apoptosis [45]. The release of these pro-inflammatory cytokines leads to the activation of the nuclear factor kappa B (NF-κB) pathway, thus amplifying the inflammatory process [44]. The continuous activation of this system will culminate with the formation of scar tissue or fibrosis. The presence of fibrotic tissue reduces the diffusion of O2, which will further aggravate the hypoxic environment.

Anemia caused by inadequate EPO production by the kidneys also contributes to renal hypoxia. However, the mechanisms underlying the reduced capacity for EPO production by the REPC are not well understood. It has been proposed that after renal injury, REPC can suffer a transdifferentiation, called epithelial to mesenchymal transition (EMT), into myofibroblasts, losing their capacity to synthesize EPO and increasing the synthesis of collagen, contributing to the expansion of ECM [46]. Nevertheless, this EMT phenomenon was never proved in humans. The residual capacity to increase serum EPO levels when subjected to hypoxic environment or high altitudes by renal patients, even those on dialysis [47], indicates that a dysregulation of the HIF system, more than a complete loss of REPC cells, could be responsible for the reduced EPO production. Moreover, the pharmacological inhibition of the PHD in CKD patients stimulates endogenous EPO production further supporting a deranged oxygen sensing [27]. A recent study in mice by Souma et al. [48] also strengthened this hypothesis, by showing that inflammatory cytokines and/or fibrosis factors suppress HIF activation through the over-activation of PHD even under pathologic hypoxic conditions, and that the inhibition of PHD restores EPO production.

### 3. Erythropoiesis-stimulating agents in CKD-anemia

The standard treatment for CKD-anemia is based on pharmacological intervention, using ESA and/or iron supplementation, in order to correct and maintain Hb concentration in the range of 10–11.5g/dL [49]. ESA are medicines produced by recombinant DNA technology with similar structure and biological activity of EPO. They differ from EPO by the different patterns of glycosylation that increases their half-life.

analysis of renal biopsies showed that fibrosis is the common final pathway [41]. The underly-

Glomerular injury leads to a reduction in glomerular blood flow and consequently limits blood flow into peritubular capillaries, causing hypoxia and tubulointerstitial injury [42]. After an initial injury, the tubular cells will attempt to correct and repair the injury by recruiting and activating several cells, such as macrophages, fibroblasts and epithelial tubular cells that will release pro-inflammatory cytokines and fibrosis factors, and contribute to excessive interstitial extracellular matrix (ECM) accumulation and expansion. Transforming growth factor beta (TGF-β), a recognized pro-fibrotic factor, appears to be central for fibroblast activation, proliferation and transdifferentiation, contributing to ECM deposition [43]. TGF-β also presents immunomodulatory effects on macrophages and monocyte recruitment, leading to the production of inflammatory cytokines [44]. In early renal injuries, M2-type macrophages are recruited to promote tissue remodeling; however, if the injury is continuous, more inflammatory monocytes will be recruited differentiating their phenotype into M1-type macrophages, responsible for the release of pro-inflammatory cytokines (such as tumor necrosis factor [TNFα], interferon [IFN]-γ, interleukin (IL)-1β and IL-6) and cell apoptosis [45]. The release of these pro-inflammatory cytokines leads to the activation of the nuclear factor kappa B (NF-κB) pathway, thus amplifying the inflammatory process [44]. The continuous activation of this system will culminate with the formation of scar tissue or fibrosis. The presence of fibrotic tissue reduces the diffusion of O2, which will further aggravate the hypoxic environment.

Anemia caused by inadequate EPO production by the kidneys also contributes to renal hypoxia. However, the mechanisms underlying the reduced capacity for EPO production by the REPC are not well understood. It has been proposed that after renal injury, REPC can suffer a transdifferentiation, called epithelial to mesenchymal transition (EMT), into myofibroblasts, losing their capacity to synthesize EPO and increasing the synthesis of collagen, contributing to the expansion of ECM [46]. Nevertheless, this EMT phenomenon was never proved in humans. The residual capacity to increase serum EPO levels when subjected to hypoxic environment or high altitudes by renal patients, even those on dialysis [47], indicates that a dysregulation of the HIF system, more than a complete loss of REPC cells, could be responsible for the reduced EPO production. Moreover, the pharmacological inhibition of the PHD in CKD patients stimulates endogenous EPO production further supporting a deranged oxygen sensing [27]. A recent study in mice by Souma et al. [48] also strengthened this hypothesis, by showing that inflammatory cytokines and/or fibrosis factors suppress HIF activation through the over-activation of PHD even under pathologic hypoxic conditions, and that the inhibition

The standard treatment for CKD-anemia is based on pharmacological intervention, using ESA and/or iron supplementation, in order to correct and maintain Hb concentration in the range of 10–11.5g/dL [49]. ESA are medicines produced by recombinant DNA technology with similar

ing mechanisms are still debatable.

270 Hypoxia and Human Diseases

of PHD restores EPO production.

3. Erythropoiesis-stimulating agents in CKD-anemia

The use of ESA has beneficial effects by correcting anemia and their associated symptoms and improving patients' quality of life [50, 51]. However, the effects of ESA on the progression of renal function are controversial. Some studies showed that after starting ESA therapy and correction of anemia, renal function declines at a slower rate, delaying the need for dialysis in pre-dialysis patients [52–54]; in opposition, other studies reported that ESA do not significantly affect renal function [55, 56].

ESA were designed to correct anemia, but some evidences showed that these drugs (and EPO) may act beyond hematopoiesis. Pleiotropic effects have been attributed to EPO and ESA, such as cytoprotection, anti-apoptosis, anti-inflammatory and angiogenesis [57]. These non-hematopoietic actions appear to result from the activation of another EPOR, a heterodimeric receptor constituted by the EPOR homodimer complexed with CD131, the common beta receptor (βCR) that is involved in granulocyte macrophage colony-stimulating factor, IL-3 and IL-5 signaling [58]. The two EPOR present different affinities for EPO; in erythroid cells picomolar concentrations of EPO are sufficient to trigger activation of the EPOR homodimer, whereas on other cells and tissues high local EPO concentrations are needed to activate EPOR heterodimer [59]. This receptor was detected in several cells and tissues, such as brain (neurons, astrocytes and microglia), kidney, female reproductive system organs, vascular endothelial cells, cardiomyocytes, lymphocytes and monocytes, among others [57].

The slower progression of renal dysfunction observed in some CKD patients may result from renoprotection of ESA therapy. Several studies on acute kidney injury (AKI) reported that a single dose of recombinant human EPO (rHuEPO) reduces kidney dysfunction through antiapoptotic mechanisms and increases NO production, only in intact vessels [60]. ESA therapy also exerts renoprotective effects by reducing the production of pro-inflammatory cytokines (e. g., IL-1β and TNF-α), acute phase proteins [e.g., C-reactive protein (CRP)], pro-fibrotic factors (e.g., TGF-β) and oxidative stress [61]. However, these effects appear to be only achieved with low doses of ESA, as high doses increase hematocrit and may activate platelets, increasing their adhesion to the injured endothelium, contributing to hemorheologic changes [60]. Indeed, other side effects are associated with ESA therapy, namely hypertension [62] and thrombotic events [63].

Despite the benefits of ESA therapy, some concerns have emerged from studies reporting a high incidence of cardiovascular events and mortality in CKD patients treated with ESA [63, 64], independently of the type of ESA used [65, 66]. Since the introduction of ESA therapy, several clinical trials aimed to define the better Hb target/ESA dose associated with lower cardiovascular risk. Indeed, recent studies reported increased cardiovascular risk and death in patients treated with high ESA doses to achieve higher Hb levels [9, 67–69].

The need for new drugs with lower associated cardiovascular risk opened a growing area of research. The most promising are the PHD inhibitors (Table 1) with several compounds already under evaluation in clinical trials. Some of these compounds showed to be well tolerated, corrected anemia in non-dialysis CKD and incident dialysis patients without increasing blood pressure, and also reduced serum hepcidin levels [70–73]. However, regarding their effects in reducing cardiovascular events and slowing the progression of the renal disease, no data are still available from human studies. Yu et al. [22] showed that the administration of PHD inhibitors in a more advanced stage of CKD in the rat reduced renal fibrosis and protected renal function, whereas the administration in an early stage of CKD promoted renal fibrosis and exacerbated renal dysfunction. In another strategy to induce EPO production, the hydrodynamic gene transfer of a plasmid encoding for EPO in a rat model overexpressing TGF-β showed that this therapy increased Hb levels but had no effect on kidney fibrosis or function [74].


Table 1. Prolyl-4-hydroxylase (PHD) inhibitors in clinical trials.

## 4. Hyporesponsiveness to erythropoiesis-stimulating agents in CKD

The majority of CKD patients respond adequately to the currently available ESA therapy, but 5–10% of them do not respond properly, developing hyporesponsiveness to these drugs [75]. According to the KDIGO guidelines [49], CKD patients can present initial or acquired ESA hyporesponsiveness; in primary hyporesponsiveness patients, after one month of treatment with adequate weight-based ESA dose, the target Hb concentration is not achieved; in acquired ESA hyporesponsiveness, after effective treatment with stable ESA dose, achieving the target Hb concentration, the patient requires two consecutive increases (up to 50% beyond the stable dose) in ESA dose. Hyporesponsiveness (also widely referred as resistance) to ESA therapy is associated with a poor outcome, progression of renal disease, sudden death, infectious complications, sudden death and all-cause mortality, mainly due to cardiovascular events in dialysis patients [76–79]. Several causes are associated with poor response to ESA therapy, including iron deficiency, inflammation, malnutrition and hyperparathyroidism, among others [80–82].

#### 4.1. Inflammation

A pro-inflammatory state is a hallmark of CKD, which is due to increased uremic toxins that induce the production of inflammatory cytokines. Additionally, active infections, the vascular access for hemodialysis (HD) procedure and surgery-related inflammation (vascular surgery included) can also contribute to inflammation.

increasing blood pressure, and also reduced serum hepcidin levels [70–73]. However, regarding their effects in reducing cardiovascular events and slowing the progression of the renal disease, no data are still available from human studies. Yu et al. [22] showed that the administration of PHD inhibitors in a more advanced stage of CKD in the rat reduced renal fibrosis and protected renal function, whereas the administration in an early stage of CKD promoted renal fibrosis and exacerbated renal dysfunction. In another strategy to induce EPO production, the hydrodynamic gene transfer of a plasmid encoding for EPO in a rat model overexpressing TGF-β showed that this therapy increased Hb levels but had no effect on

PHD inhibitor Route administration ClinicalTrials.gov Identifier

• NCT01887600

• NCT02648347 • NCT02680574

Molidustat(BAY85-3934) Oral • NCT02064426 Roxadustat(FG-4592) Oral • NCT01630889

Vadadustat(AKB-6548) Oral • NCT01906489

GSK1278863 Oral • NCT02689206

4. Hyporesponsiveness to erythropoiesis-stimulating agents in CKD

The majority of CKD patients respond adequately to the currently available ESA therapy, but 5–10% of them do not respond properly, developing hyporesponsiveness to these drugs [75]. According to the KDIGO guidelines [49], CKD patients can present initial or acquired ESA hyporesponsiveness; in primary hyporesponsiveness patients, after one month of treatment with adequate weight-based ESA dose, the target Hb concentration is not achieved; in acquired ESA hyporesponsiveness, after effective treatment with stable ESA dose, achieving the target Hb concentration, the patient requires two consecutive increases (up to 50% beyond the stable dose) in ESA dose. Hyporesponsiveness (also widely referred as resistance) to ESA therapy is associated with a poor outcome, progression of renal disease, sudden death, infectious complications, sudden death and all-cause mortality, mainly due to cardiovascular events in dialysis patients [76–79]. Several causes are associated with poor response to ESA therapy, including iron deficiency, inflammation, malnutrition and hyperparathyroidism,

A pro-inflammatory state is a hallmark of CKD, which is due to increased uremic toxins that induce the production of inflammatory cytokines. Additionally, active infections, the vascular

kidney fibrosis or function [74].

272 Hypoxia and Human Diseases

Table 1. Prolyl-4-hydroxylase (PHD) inhibitors in clinical trials.

among others [80–82].

4.1. Inflammation

The activation of inflammatory cells is also associated with increased oxidative stress, favoring alterations in red blood cells (RBC) membrane, namely increased phosphatidylserine exposure, increased membrane bound Hb and increased membrane protein band 3 aggregation, all markers for RBC phagocytosis by macrophages and, thus, for a premature RBC removal [83, 84]. Uremic toxins and pro-inflammatory cytokines also inhibit erythropoiesis, through the inhibitory effect of IL-1β, TNF-α and IFN-γ on early erythroid cell stages in the bone marrow [85]. The macrophages of the bone marrow can also be stimulated to increase local proinflammatory cytokines, amplifying the effects of systemic inflammation [86]. In CKD patients, hepcidin synthesis is enhanced, due to the increase in Il-6, contributing for the limited iron availability for erythropoiesis [87]. Indeed, CKD patients often present with replete or even higher iron stores, alongside with inflammation and anemia. A disturbance in the crosstalk between inflammation, iron metabolism and erythropoiesis may, therefore, favor ESA hyporesponsiveness. The best predictors for ESA response appear to be IL-6 and CRP [88, 89]. Studies conducted by our group showed that HD patients with poorer response to ESA present higher levels of pro-inflammatory cytokines [90, 91]; moreover, in studies using a rat model of chronic renal failure, we found that the severity of the inflammatory state was related to the reduction in the rHuEPO response [92].

#### 4.2. HIF system in the hyporesponsiveness to erythropoiesis-stimulating agents

Hyporesponsive patients to ESA therapy will develop anemia, and as already referred, it will promote the progression of renal disease. Tissue hypoxia is amplified according to the severity of anemia that will reduce O2 availability to body tissues and organs. Within the kidney, the hypoxic environment leads to the activation of the HIF system, promoting the transcription of several target genes. In the hypoxic kidney, HIF-1α is essentially expressed in tubular and glomerular epithelial cells, whereas HIF-2α expression is limited to endothelial and interstitial cells [93]. The localization of these HIF-α subunits is related to their target genes.

Renal biopsies from CKD patients showed that increased expression of HIF-1α in tubular epithelial cells is correlated with the stage of renal disease [94]. It was reported that HIF-α activation in CKD rats presents dynamic changes, as it is activated in early CKD stages and suppressed in the moderate and end-stage of CKD [95]. Thus, the administration of PHD inhibitors may improve renal function in more advanced stages of CKD, while in earlier stages, the PHD inhibitors may increase renal fibrosis due to upregulation of the HIF-1α subunit [22].

HIF-1α subunit is involved in the activation of pro-fibrotic genes (Figure 1), including the connective tissue growth factor (CTGF) gene [96]; indeed, the plasma levels of CTGF appear as a good marker for staging diabetic nephropathy progression [97]. CTGF is a potent pro-fibrotic factor and a marker of renal fibrosis, increasing ECM production, promoting EMT, stimulating fibroblasts and potentiating TGF-β signaling [94, 98]. CTGF and TGF-β present similar effects, but TGF-β also presents immunomodulatory actions [44], recruiting macrophages to reduce the injury; however, a continuous macrophage activation leads to excessive ECM accumulation and increased release of pro-inflammatory cytokines promoting fibrosis. A study by Basu et al. [99] suggested that TGF-β can in turn induce HIF-1α activation, which would amplify cell collagen expression contributing to the progression of fibrosis.

There is also a crosstalk between HIF-1α and inflammation (Figure 1). Inflammation favor tissue hypoxia by several mechanisms including: impaired EPO response, iron mobilization and bone marrow erythropoiesis, reduced RBC lifespan and also increased demand for O2 by the inflammatory cells in order to increase pro-inflammatory cytokines. However, it was also reported that NF-κB can induce HIF-1α activation due to the presence of responsive elements in the promoter of HIF-1α gene [100]. Another mechanism is the interaction of PHD with some effectors of the NF-κB pathway, though the exact proteins involved remain unknown [101].

The majority of the studies report a beneficial effect of ESA on renal fibrosis through several mechanisms [29, 102]. However, recently Gobe et al. [103] reported that in rat model of AKI the use of higher rHuEPO doses was associated with increased TGF-β expression, oxidative stress and stimulation of fibroblasts and EMT, contributing to the progression of the disease and gradual development of CKD in the long term. In this study, the expression of HIF-α subunits was not reported, as well as the linking between HIF activation and the alterations observed. Further studies regarding this issue are warranted.

Despite the underlying mechanism, a continuous inflammatory response favoring fibrosis and a disturbance in the HIF system creates a vicious cycle, contributing to the progression of renal disease and aggravation of renal anemia [92], and reducing the response to ESA therapy creating a scenario of hyporesponsiveness to EPO.

## 5. Conclusions

Anemia is a common complication in CKD patients that can be corrected by the treatment with ESA. However, the development of a hyporesponse to this therapy was associated with (i) the progression of the renal disease, due to the amplification of fibrosis and inflammation through a mechanism involving activation of HIF-1α pathway; (ii) increased risks in the development of cardiovascular disorder events and all-cause mortality in patients treated with higher doses, opened a new research field, focused on the design of more effective agents to control anemia in CKD patients, with less side effects. The use of PHD inhibitors is promising, but further is needed to confirm their effects in the reduction of cardiovascular events and progression of renal disease.

## Acknowledgements

This work received financial support from FCT/MEC through national funds and co-financed by FEDER, under the Partnership Agreement PT2020 (UID/MULTI/04378/2013—POCI/01/0145/ FERDER/007728, UID/NEU/04539/2013) and Norte Portugal Regional Coordination and Development Commission (CCDR-N)/NORTE2020/Portugal 2020 (Norte-01-0145-FEDER-000024).

## Author details

excessive ECM accumulation and increased release of pro-inflammatory cytokines promoting fibrosis. A study by Basu et al. [99] suggested that TGF-β can in turn induce HIF-1α activation, which would amplify cell collagen expression contributing to the progression of fibrosis.

There is also a crosstalk between HIF-1α and inflammation (Figure 1). Inflammation favor tissue hypoxia by several mechanisms including: impaired EPO response, iron mobilization and bone marrow erythropoiesis, reduced RBC lifespan and also increased demand for O2 by the inflammatory cells in order to increase pro-inflammatory cytokines. However, it was also reported that NF-κB can induce HIF-1α activation due to the presence of responsive elements in the promoter of HIF-1α gene [100]. Another mechanism is the interaction of PHD with some effectors of the NF-κB pathway, though the exact proteins involved remain unknown [101].

The majority of the studies report a beneficial effect of ESA on renal fibrosis through several mechanisms [29, 102]. However, recently Gobe et al. [103] reported that in rat model of AKI the use of higher rHuEPO doses was associated with increased TGF-β expression, oxidative stress and stimulation of fibroblasts and EMT, contributing to the progression of the disease and gradual development of CKD in the long term. In this study, the expression of HIF-α subunits was not reported, as well as the linking between HIF activation and the alterations observed.

Despite the underlying mechanism, a continuous inflammatory response favoring fibrosis and a disturbance in the HIF system creates a vicious cycle, contributing to the progression of renal disease and aggravation of renal anemia [92], and reducing the response to ESA therapy

Anemia is a common complication in CKD patients that can be corrected by the treatment with ESA. However, the development of a hyporesponse to this therapy was associated with (i) the progression of the renal disease, due to the amplification of fibrosis and inflammation through a mechanism involving activation of HIF-1α pathway; (ii) increased risks in the development of cardiovascular disorder events and all-cause mortality in patients treated with higher doses, opened a new research field, focused on the design of more effective agents to control anemia in CKD patients, with less side effects. The use of PHD inhibitors is promising, but further is needed to confirm their effects in the reduction of cardiovascular events and progression of

This work received financial support from FCT/MEC through national funds and co-financed by FEDER, under the Partnership Agreement PT2020 (UID/MULTI/04378/2013—POCI/01/0145/ FERDER/007728, UID/NEU/04539/2013) and Norte Portugal Regional Coordination and Development Commission (CCDR-N)/NORTE2020/Portugal 2020 (Norte-01-0145-FEDER-000024).

Further studies regarding this issue are warranted.

creating a scenario of hyporesponsiveness to EPO.

5. Conclusions

274 Hypoxia and Human Diseases

renal disease.

Acknowledgements

Sandra Ribeiro<sup>1</sup> , Luís Belo<sup>1</sup> , Flávio Reis2,3 and Alice Santos-Silva1 \*

\*Address all correspondence to: assilva@ff.up.pt

1 Research Unit on Applied Molecular Biosciences (UCIBIO), REQUIMTE, Department of Biological Sciences, Laboratory of Biochemistry, Faculty of Pharmacy, University of Porto, Porto, Portugal

2 Laboratory of Pharmacology and Experimental Therapeutics, Institute for Biomedical Imaging and Life Sciences (IBILI), Faculty of Medicine, University of Coimbra, Coimbra, Portugal

3 Center for Neuroscience and Cell Biology, Institute for Biomedical Imaging and Life Sciences (CNC.IBILI) Research Unit, University of Coimbra, Coimbra, Portugal

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## **Role of the Hypoxia-Inducible Factor in Periodontal Inflammation Role of the Hypoxia-Inducible Factor in Periodontal Inflammation**

Xiao Xiao Wang, Yu Chen and Wai Keung Leung Leung

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

Xiao Xiao Wang, Yu Chen and Wai Keung

http://dx.doi.org/10.5772/66037

#### **Abstract**

pivotal role in renal fibrogenesis. J Am Soc Nephrol. 2005;16(1):133–43. doi:10.1681/

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ASN.2004040339

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18110

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(2):213–5. doi:10.1038/ki.2015.89

Human periodontitis is a chronic inflammatory disease induced by opportunistic Gramnegative anaerobic bacteria at the tooth-supporting apparatus. Within the gingivitisaffected sulcus or periodontal pocket, the resident anaerobic bacteria interact with the host inflammatory reactions leading to a lower oxygen or hypoxic environment. A cellular/tissue oxygen-sensing mechanism and its appropriate regulation are needed to assist tissue adaptation to natural/pathology-induced variations in oxygen availability. In this chapter, we reviewed the biological relevance of hypoxia in periodontal/oral cellular development, epithelial barrier function, periodontal inflammation, and immunity. The role of hypoxia-inducible factor-1α in pathogen-host cross talk and alveolar bone homeostasis was also discussed. The naturally occurring pathophysiological process of hypoxia appeared to entail fundamental relevance for periodontal defense and regeneration.

**Keywords:** cell hypoxia, chronic periodontitis, hypoxia-inducible factor-1, alpha subunit

## **1. Introduction**

Regardless of the oxygen sources, when an animal acquires oxygen through its breathing apparatus, the oxygen will have to pass under a reducing partial oxygen pressure (pO<sup>2</sup> ) gradient from the source via circulation to different organs and then tissues and cells. In mammals, such as rats, inspired pO<sup>2</sup> is around 21.3 kPa at sea level. When blood flows through the alveolar capillaries, it drops to approximately 14 kPa and is then progressively reduced to 2.1, 1.3, and 0.27–3.3 kPa in the spleen, thymus, and retina, respectively [1, 2], while in the brain, it may be as low as 0.05–1.07 kPa, depending on the cranial location [3].

and reproduction in any medium, provided the original work is properly cited.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Due to the colonization by subgingival biofilm, oxygen is persistently consumed to various extents by the facultative anaerobic microbes within the periodontal sulcus (2.33–8.40 kPa). In the gingivitis-affected sulcus or periodontal pocket, the inflammation induced by the residential anaerobic bacteria with or without microulcerations or wounding leads to an even lower oxygen tension [4]. At the tissue level, the availability of oxygen is dependent on the distance from the oxygen-supplying blood vessels. Although the diffusion distance of oxygen *in vivo* is estimated to be 100–200 μm, a pO<sup>2</sup> of almost zero has been recorded in tissues 100 μm away from the nourishing blood vessels [5]. Therefore, a cellular/tissue oxygen-sensing mechanism is needed to assist tissue adaptation to nature/pathology-induced variations in oxygen availability.

In humans, a drop in oxygen concentration in the atmosphere is sensed by the carotid body at the bifurcation of the carotid arteries, which then increases the rate and depth of breathing. At the levels of tissues and cells, including the human periodontium, such adaptive responses to low oxygen tension or hypoxia are mainly mediated through a key cellular transcription factor named the hypoxia-inducible factor (HIF) [6].

## **2. Hypoxia in the oral/periodontal environment**

Oxygen is an essential molecule for survival. Mammals—including humans—depend on oxygen for electron transport, oxidative phosphorylation, and energy generation. Variations in tissue oxygen needs are attributed to a number of physiological or pathological states, meaning that the tissues concerned have to be able to adapt to various O<sup>2</sup> environments including hypoxia. To survive, mammalian cells evolved in such a way that cellular O<sup>2</sup> availability or homeostasis could be monitored and tightly regulated [7]. This is made possible by a cellular HIF system. Cellular hypoxia, or a lower than "normal" concentration of O<sup>2</sup> in cells, occurs commonly and could induce significant changes, immediate or delayed, on cellular processes, including cell growth and apoptosis, cell proliferation and survival, pH regulation and energy metabolism, cell migration, matrix and barrier function, angiogenesis, and vasomotor regulation [8–13]. These biological processes involve active responses by the body to secure an additional oxygen supply via circulation. Such dynamic processes of cellular/tissue oxygen monitoring, O<sup>2</sup> consumption, and delivery, corresponding to the respective cellular/ tissue functional state, are tightly controlled to ensure proper survival of the multicellular organism concerned [14].

As described earlier, the normal tissue/cellular pO<sup>2</sup> levels in mammals are dependent on their location and physiology and, hence, vary among different human body compartments and cell/tissue conditions [15]. Hypoxia in oral cells/tissues is, in fact, a common occurrence [6]. The local hypoxic microenvironment is considered a consequence of growth/development, wound healing, smoking habits, or concurrent oral inflammation/infection/diseases.

Taking oral cellular development/regeneration as an example, the blood vessel network is promoted by vascular endothelial growth factors (VEGF) secreted by stem cells from apical papilla under hypoxia, suggesting a role of hypoxia in pulp revascularization and bioengineered pulp replacements [16]. On the other hand, it is reported that extreme hypoxia-like response induced by the chemical cobalt chloride (CoCl<sup>2</sup> ) could stimulate periodontal ligament (PDL) stem cell cytotoxicity through mitochondria-apoptotic and autophagic pathways involving HIF-1α [17]. During pathological processes, it is reported that a low oxygen level may regulate cell migration of oral cancer cells, thus influencing the invasion and metastasis in malignant oral lesions [18].

With reference to growth, increasing evidence shows that certain hormonal regulation could be interfered with hypoxia or HIF [19]. For instance, it is reported that growth hormone expression in lymphocytes and parathyroid hormone-related protein in articular chondrocytes can be induced by hypoxia [20, 21]. We postulate that if similar biology could be expressed in the head and neck region, HIF or hypoxia may bring profound effects on the growth and development of orofacial structures.

## **3. Hypoxia and chronic periodontal inflammation**

Due to the colonization by subgingival biofilm, oxygen is persistently consumed to various extents by the facultative anaerobic microbes within the periodontal sulcus (2.33–8.40 kPa). In the gingivitis-affected sulcus or periodontal pocket, the inflammation induced by the residential anaerobic bacteria with or without microulcerations or wounding leads to an even lower oxygen tension [4]. At the tissue level, the availability of oxygen is dependent on the distance from the oxygen-supplying blood vessels. Although the diffusion distance of oxygen *in vivo* is

from the nourishing blood vessels [5]. Therefore, a cellular/tissue oxygen-sensing mechanism is needed to assist tissue adaptation to nature/pathology-induced variations in oxygen

In humans, a drop in oxygen concentration in the atmosphere is sensed by the carotid body at the bifurcation of the carotid arteries, which then increases the rate and depth of breathing. At the levels of tissues and cells, including the human periodontium, such adaptive responses to low oxygen tension or hypoxia are mainly mediated through a key cellular transcription

Oxygen is an essential molecule for survival. Mammals—including humans—depend on oxygen for electron transport, oxidative phosphorylation, and energy generation. Variations in tissue oxygen needs are attributed to a number of physiological or pathological states, mean-

homeostasis could be monitored and tightly regulated [7]. This is made possible by a cel-

occurs commonly and could induce significant changes, immediate or delayed, on cellular processes, including cell growth and apoptosis, cell proliferation and survival, pH regulation and energy metabolism, cell migration, matrix and barrier function, angiogenesis, and vasomotor regulation [8–13]. These biological processes involve active responses by the body to secure an additional oxygen supply via circulation. Such dynamic processes of cellular/tissue

tissue functional state, are tightly controlled to ensure proper survival of the multicellular

location and physiology and, hence, vary among different human body compartments and cell/tissue conditions [15]. Hypoxia in oral cells/tissues is, in fact, a common occurrence [6]. The local hypoxic microenvironment is considered a consequence of growth/development,

Taking oral cellular development/regeneration as an example, the blood vessel network is promoted by vascular endothelial growth factors (VEGF) secreted by stem cells from apical papilla under hypoxia, suggesting a role of hypoxia in pulp revascularization and bioengineered pulp

wound healing, smoking habits, or concurrent oral inflammation/infection/diseases.

consumption, and delivery, corresponding to the respective cellular/

levels in mammals are dependent on their

of almost zero has been recorded in tissues 100 μm away

environments including

availability or

in cells,

estimated to be 100–200 μm, a pO<sup>2</sup>

factor named the hypoxia-inducible factor (HIF) [6].

As described earlier, the normal tissue/cellular pO<sup>2</sup>

**2. Hypoxia in the oral/periodontal environment**

ing that the tissues concerned have to be able to adapt to various O<sup>2</sup>

hypoxia. To survive, mammalian cells evolved in such a way that cellular O<sup>2</sup>

lular HIF system. Cellular hypoxia, or a lower than "normal" concentration of O<sup>2</sup>

availability.

286 Hypoxia and Human Diseases

oxygen monitoring, O<sup>2</sup>

organism concerned [14].

Metabolic shifts under hypoxia are common occurrences in the periodontal inflammatory process as a result of the imbalance between the tissue oxygen supply and consumption [22]. The accumulation of intracellular HIF-1 promotes the transcription of a spectrum of genes to maintain cellular homeostasis. Hypoxia induces the expression of a number of angiogenic factors to improve the blood supply in needed areas including inflamed periodontium [6]. These include VEGF, platelet-derived growth factor (PDGF), and angioprotein-1 and -2. Related genes produce controlling perfusion, such as the PDGF-β receptor, cyclooxygenase-2, and nitric oxide synthase (NOS), of which NOS modulates vascular smooth muscle cells' functions and reacts to changes in the cellular HIF-1 level [23]. Moreover, HIF activation promotes a metabolic switch to reduce oxygen consumption by shifting energy metabolism from aerobic respiration to glycolysis. Activation of HIF also upregulates the expression of pyruvate dehydrogenase kinase, which reduces the incorporation of pyruvate into the citric acid cycle [24]. This metabolic switch is essential for the hosts' defense because such HIF-1α–regulated glycolytic metabolism is required in B cell development [25] and T cell metabolism [26].

Under a chronic inflammatory state, hypoxia induces protective cellular responses or a local defense. However, if the cause of inflammation cannot be eradicated, such hypoxic cell/tissue reactions contribute to the pathophysiology of inflammation and, hence, disease pathogenesis [27]. A similar scenario can be observed within the human periodontium in periodontitis. Periodontitis is characterized by chronic inflammation of the tooth-supporting tissues, initiated by a multitude of Gram-negative anaerobic pathogens including *Aggregatibacter actinomycetemcomitan*s, *Porphyromonas gingivalis*, *Tannerella forsythia*, *Treponema denticola*, and so on [28]. At sites where a chronic inflammatory reaction could be found, oxygen consumption is elevated and blood perfusion is stimulated, but the actual local microcirculation could be compromised [29]. This local tissue pO<sup>2</sup> change is partly due to increased oxygen consumption, including oxygen usage by both resident cells and infiltrated defense cells, and partly because of diminished oxygen availability due to endothelial damage and vasoconstricted microcirculation.

Local hypoxia in periodontitis in turn enhances the anaerobic Gram-negative pathogens' survival and further lowers the oxygen tension at the vicinity. The tissue hypoxia in periodontal disease has been characterized by increased HIF-1α protein that is detectable in periodontitisaffected tissue biopsies using Western blot and anti–HIF-1α immunostaining [6, 30]. Myeloid cell lineage of HIF-1α–/– (deprived) mice had impaired immune effector molecules, such as nitric oxide (NO) and tumor necrosis factor-alpha (TNF-α) production, thus reducing their bactericidal capability [31]. Therefore, the ability to adapt to a reduced oxygen supply, which maintains immune cell surveillance capability in all tissue environments, is important and necessary in the successful elimination of pathogens [32].

Proinflammatory cytokines and matrix metalloproteinases (MMPs) act as mediators for the inflammation process or play a role in extracellular matrix degradation, respectively. Researchers often investigate the levels of such biological markers in the periodontium in attempts to gauge the severity of periodontal disease and monitor periodontal treatment outcomes [33]. Recent studies reported that a hypoxic environment may upregulate proinflammatory cytokines and MMPs' expression from host cells during periodontal disease [34]. The idea was that hypoxia further encourages lipopolysaccharide (LPS)-induced TNF-α, interleukin-1β, and interleukin 6 (IL-6) expressions via LPS toll-like receptor (TLR) interaction that, in turn, activates the nuclear factor kappa B (NF-κB) pathway in human PDL cells upon exposure to the aforementioned Gram-negative bacterial surface component [35–37].

At the collagen destruction front, periodontal epithelial cells could produce MMPs in response to bacteria-induced activation of pathogen-associated molecular patterns (PAMP) including TLRs. These host enzymes contribute to the extracellular matrix degradation that accommodates local inflammatory reactions, as well as the later tissue remodeling that ensues once inflammation stops [38, 39]. Inhibition of HIF-1α activity by chetomin, a *Chaetomium* metabolite that can incapacitate tumor cells' hypoxic adaptation or knockdown HIF-1α gene expression by small, interfering RNA, could markedly attenuate the production of LPS- and nicotine-stimulated MMPs and prostaglandin E<sup>2</sup> from PDL cells. Such observations suggest the possibility of HIF-1α being a potential target in periodontal tissue destruction associated with smoking and dental plaque [40]. Further supporting the idea that hypoxia may be one of the key biological responses in periodontal inflammation.

Certain periodontopathogens, other than acting as effective mediators of periodontal inflammation, are capable of doing more harm to a host under a low pO<sup>2</sup> environment. For instance, *P. gingivalis* LPS under hypoxia increases PDL fibroblasts' oxidative stress and induces a reduction of catalase, indicating a collapse of the protective machinery favoring the increase in reactive oxygen species (ROS) and the progression of inflammatory oral diseases [41].

Considering the healing of oral wounds, several studies reported that the biological process in general could be enhanced or accelerated under hypoxia via HIF-1 [42, 43]. For example, the wound healing of rat palatal mucosa was enhanced by the hydroxylase inhibitor dimethyloxalylglycine, a HIF-1α stabilizer, under a hypoxic environment, and this enzyme was reported to induce hypoxia-mimetic angiogenesis [44]. With reference to hard tissue healing, CoCl<sup>2</sup> triggered the expression of angiogenic mediators and bone turnover-related genes, which promoted fracture healing and repair *in vivo* [45]. The research report also indicated that, during distraction osteogenesis, an angiogenic effect and bone healing could be promoted by conditioned media collected from dental pulp cells under hypoxia [46]. These findings implied the possibility that a low tissue oxygen level may act as a biological signal, promoting soft and hard tissue healing, including that of the orofacial regions, mediated through inflammation.

## **4. Hypoxia and periodontal immunity**

Local hypoxia in periodontitis in turn enhances the anaerobic Gram-negative pathogens' survival and further lowers the oxygen tension at the vicinity. The tissue hypoxia in periodontal disease has been characterized by increased HIF-1α protein that is detectable in periodontitisaffected tissue biopsies using Western blot and anti–HIF-1α immunostaining [6, 30]. Myeloid cell lineage of HIF-1α–/– (deprived) mice had impaired immune effector molecules, such as nitric oxide (NO) and tumor necrosis factor-alpha (TNF-α) production, thus reducing their bactericidal capability [31]. Therefore, the ability to adapt to a reduced oxygen supply, which maintains immune cell surveillance capability in all tissue environments, is important and

Proinflammatory cytokines and matrix metalloproteinases (MMPs) act as mediators for the inflammation process or play a role in extracellular matrix degradation, respectively. Researchers often investigate the levels of such biological markers in the periodontium in attempts to gauge the severity of periodontal disease and monitor periodontal treatment outcomes [33]. Recent studies reported that a hypoxic environment may upregulate proinflammatory cytokines and MMPs' expression from host cells during periodontal disease [34]. The idea was that hypoxia further encourages lipopolysaccharide (LPS)-induced TNF-α, interleukin-1β, and interleukin 6 (IL-6) expressions via LPS toll-like receptor (TLR) interaction that, in turn, activates the nuclear factor kappa B (NF-κB) pathway in human PDL cells upon exposure to the aforementioned Gram-negative bacterial surface compo-

At the collagen destruction front, periodontal epithelial cells could produce MMPs in response to bacteria-induced activation of pathogen-associated molecular patterns (PAMP) including TLRs. These host enzymes contribute to the extracellular matrix degradation that accommodates local inflammatory reactions, as well as the later tissue remodeling that ensues once inflammation stops [38, 39]. Inhibition of HIF-1α activity by chetomin, a *Chaetomium* metabolite that can incapacitate tumor cells' hypoxic adaptation or knockdown HIF-1α gene expression by small, interfering RNA, could markedly attenuate the production of LPS- and

the possibility of HIF-1α being a potential target in periodontal tissue destruction associated with smoking and dental plaque [40]. Further supporting the idea that hypoxia may be one of

Certain periodontopathogens, other than acting as effective mediators of periodontal inflam-

*P. gingivalis* LPS under hypoxia increases PDL fibroblasts' oxidative stress and induces a reduction of catalase, indicating a collapse of the protective machinery favoring the increase in reactive oxygen species (ROS) and the progression of inflammatory oral diseases [41].

Considering the healing of oral wounds, several studies reported that the biological process in general could be enhanced or accelerated under hypoxia via HIF-1 [42, 43]. For example, the wound healing of rat palatal mucosa was enhanced by the hydroxylase inhibitor dimethyloxalylglycine, a HIF-1α stabilizer, under a hypoxic environment, and this enzyme was reported to induce hypoxia-mimetic angiogenesis [44]. With reference to hard tissue

from PDL cells. Such observations suggest

environment. For instance,

necessary in the successful elimination of pathogens [32].

nicotine-stimulated MMPs and prostaglandin E<sup>2</sup>

the key biological responses in periodontal inflammation.

mation, are capable of doing more harm to a host under a low pO<sup>2</sup>

nent [35–37].

288 Hypoxia and Human Diseases

Hypoxic responses or HIF is reported to be strongly related to innate human responses, with low oxygen modulating energy metabolism and various genes' expression within defense cells that, in turn, dictate the immune performance and the host protection outcomes [47]. The biological impact of low pO<sup>2</sup> on T cells' functions was reflected by the HIF-1– and adenosine receptor–modulated effects [48]. Indeed, both lymphocytes and myeloid cells were affected and the hypoxia-induced adaptive immune response changes would interfere or affect the innate immunity. The relevance of hypoxia in pathological processes was well established upon the appreciation that wounds, infectious loci, and tumor growth each involved extremely low oxygen tension [1].

It has been well appreciated that low oxygen tension is common at inflamed periodontitis sites [4]; thus, the corresponding local immune responses must adapt to the hypoxic challenges. As mentioned above, hypoxia plays an important role in modulating the cellular activities of innate and adaptive immunity, so the impact of low pO<sup>2</sup> in periodontal immune responses is quite significant.

Oral innate immunity is the first line of defense against periodontopathogens, which functions to recognize, attenuate, and eliminate the nonself invaders and to trigger downstream immune responses. Granulocytes and monocytes/macrophages are the main cell types for innate periodontal immunity [49, 50]. When extensive inflammation takes place, these cells have to travel into the tissue compartment with low pO<sup>2</sup> (i.e., the infected area) to provide defense and wall-off the invasion. To prevent the invasion, intense energy metabolism has to occur within the involved innate defense cells. An appropriate hypoxic cellular reaction and adaptation is, therefore, very important in the periodontal innate immune cells, which develop functional and survival responses regulated by the oxygen sensor HIF [51].

Defense cells rely heavily on glycolysis for the production of ATP to compensate for the limited oxidative metabolism in hypoxia. Immune cell energy metabolism appeared to significantly influence its corresponding response. As a critical modulator for the expression of glycolytic enzymes, the absence of HIF-1α leads to a significant reduction of ATP availability in myeloid cells [52]. It was reported that a knockdown of HIF-1α protein led to a nullified IL-6 production when exposed to LPS, suggesting that HIF-1α supported the LPS-dependent expression of IL-6 that, in turn, prevented the depletion of ATP and, therefore, protected myeloid cells against LPS/TLR4-induced apoptosis [53]. In human monocytes, LPS and hypoxia synergistically activated HIF-1 through p44/42 mitogen-activated protein kinases (MAPK) and NF-κB; however, repetitive exposure to LPS could induce tolerance to bacterial endotoxins and, hence, impair corresponding HIF-1α induction, which reduces the ability of monocytic cells to survive and function under low oxygen [54, 55].

To combat invading pathogens, HIF also promotes polymorphonuclear neutrophil (PMN) recruitment via the restoration of blood flow at inflamed tissues and enhances neovascularization. With hypoxia, the HIF restored perfusion also facilitates PMNs' diapedesis [56, 57]. Furthermore, PMN apoptosis was attenuated under hypoxia, with HIF-1α reported to be a protective factor in the regulation of its functional longevity [58]. Such longevity regulation involved NF-κB signaling that was found to be essential in constitutive HIF-1 protein translation [58, 59].

The cellular stress-related transcription factor NF-κB is closely related to hypoxia despite the fact that the relationship is not yet completely understood. It was reported that classical or canonical NF-κB activation under the stress of hypoxia often involves the activation of transforming growth factor-B-activating kinase and the inhibitor of κB kinase (IKK) complex [60]. In addition to classical NF-κB signaling, the noncanonical NF-κB pathway could be activated by hypoxia independent of HIF-1α via NF-κB-inducing kinase and IKK homodimer activation [61]. ROS, a key inflammatory regulator in chronic periodontal inflammation, is confirmed to mediate HIF-1α induction dependent on NF-κB [62].

Dendritic cells (DCs), a group of professional antigen-presenting cells, are key members that enable cross talk between the innate and adaptive immune systems. They present an antigen to activate naive lymphocytes and assist in the development of specific adaptive immune responses to pathogens. Hypoxia has been found to play an important role in the maturation and cytokines release of DCs, but the mechanism of the related divergent effects still remains controversial [63]. Studies found that the knockdown of HIF-1α in DCs inhibited their maturation and significantly impaired their capability to stimulate allogeneic T cells, probably because of the reliance on the HIF-controlled glycolysis [64, 65]. In contrast, it is reported that low oxygen tension inhibited the DCs' defense against LPS, but strongly upregulated the production of proinflammatory cytokines in the cells involved [66]. Similar results can be observed in the human antifungal response: hypoxia at the site of *Aspergillus fumigatus* infection inhibited the full activation and function of DCs [67]. These findings suggest that hypoxia may function as a regulator against DCs' mediated immune overreaction.

Lymphocytes are known to be involved in periodontal tissues' health homeostasis, and their functional upset was believed to be associated with periodontal pathogenesis. An HIF-1α deficiency was associated with abnormal B cell development, which led to autoimmunity in a mouse model [68]. A recent study also indicated that T cells' HIF-1α regulation played a critical role in avoiding cardiac damage in diabetic mice [69]. We postulated that a similar protection mechanism may be called to function in diabetic periodontium. Therefore, hypoxia or HIF-1α regulation in DCs and lymphocytes may confer a marked impact on the innate and adaptive cellular immunity in periodontal tissues, with the exact mechanism yet to be elucidated.

## **5. HIF and epithelial barrier function**

of IL-6 that, in turn, prevented the depletion of ATP and, therefore, protected myeloid cells against LPS/TLR4-induced apoptosis [53]. In human monocytes, LPS and hypoxia synergistically activated HIF-1 through p44/42 mitogen-activated protein kinases (MAPK) and NF-κB; however, repetitive exposure to LPS could induce tolerance to bacterial endotoxins and, hence, impair corresponding HIF-1α induction, which reduces the ability of monocytic cells

To combat invading pathogens, HIF also promotes polymorphonuclear neutrophil (PMN) recruitment via the restoration of blood flow at inflamed tissues and enhances neovascularization. With hypoxia, the HIF restored perfusion also facilitates PMNs' diapedesis [56, 57]. Furthermore, PMN apoptosis was attenuated under hypoxia, with HIF-1α reported to be a protective factor in the regulation of its functional longevity [58]. Such longevity regulation involved NF-κB signaling that was found to be essential in constitutive HIF-1 protein transla-

The cellular stress-related transcription factor NF-κB is closely related to hypoxia despite the fact that the relationship is not yet completely understood. It was reported that classical or canonical NF-κB activation under the stress of hypoxia often involves the activation of transforming growth factor-B-activating kinase and the inhibitor of κB kinase (IKK) complex [60]. In addition to classical NF-κB signaling, the noncanonical NF-κB pathway could be activated by hypoxia independent of HIF-1α via NF-κB-inducing kinase and IKK homodimer activation [61]. ROS, a key inflammatory regulator in chronic periodontal inflammation, is con-

Dendritic cells (DCs), a group of professional antigen-presenting cells, are key members that enable cross talk between the innate and adaptive immune systems. They present an antigen to activate naive lymphocytes and assist in the development of specific adaptive immune responses to pathogens. Hypoxia has been found to play an important role in the maturation and cytokines release of DCs, but the mechanism of the related divergent effects still remains controversial [63]. Studies found that the knockdown of HIF-1α in DCs inhibited their maturation and significantly impaired their capability to stimulate allogeneic T cells, probably because of the reliance on the HIF-controlled glycolysis [64, 65]. In contrast, it is reported that low oxygen tension inhibited the DCs' defense against LPS, but strongly upregulated the production of proinflammatory cytokines in the cells involved [66]. Similar results can be observed in the human antifungal response: hypoxia at the site of *Aspergillus fumigatus* infection inhibited the full activation and function of DCs [67]. These findings suggest that hypoxia

Lymphocytes are known to be involved in periodontal tissues' health homeostasis, and their functional upset was believed to be associated with periodontal pathogenesis. An HIF-1α deficiency was associated with abnormal B cell development, which led to autoimmunity in a mouse model [68]. A recent study also indicated that T cells' HIF-1α regulation played a critical role in avoiding cardiac damage in diabetic mice [69]. We postulated that a similar protection mechanism may be called to function in diabetic periodontium. Therefore, hypoxia or HIF-1α regulation in DCs and lymphocytes may confer a marked impact on the innate and adaptive cellular immunity in periodontal tissues, with the exact mechanism yet

to survive and function under low oxygen [54, 55].

firmed to mediate HIF-1α induction dependent on NF-κB [62].

may function as a regulator against DCs' mediated immune overreaction.

tion [58, 59].

290 Hypoxia and Human Diseases

to be elucidated.

The human periodontium is a unique environment for microorganisms. One special characteristic is the nonshedding tooth's hard tissue surface, allowing microorganisms to remain *in situ*. To counter the invasion of possible pathogens, the corresponding epithelial tissues build up an effective barrier against the colonizing microbes [70]. With appropriate daily oral hygiene, the continued host-bacteria interaction maintains the periodontium in health or low grade/subclinical inflammation. Those who have inadequate oral hygiene tip the balance toward a proinflammatory state, resulting in inflammatory responses that present clinically as gingivitis. Due to poor oral hygiene and inherited or acquired risks, approximately 20% of the human population, develop chronic periodontal inflammation with tissue destruction resulting in what is known as periodontitis [71]. Regardless of the host-parasitic interaction outcomes, humans and their complex residential microflora have coevolved over time [72].

TLRs on the periodontal/gingival epithelial cells recognize the conserved molecular patterns on pathogenic bacteria that are also known as PAMP, limiting invasion of the microbes, and help to maintain oral health [73]. Other than providing a physical barrier to the outside world, the skin and mucosal membrane produce a number of antimicrobial peptides (AMPs). The AMPs have a broad activity spectrum against both Gram-negative and Gram-positive bacteria colonization, enveloped viruses, fungi, and even transformed or cancerous cells.

It has become clear that AMPs, such as defensins and the cathelicidins family of peptides especially LL-37, play important roles independently or together in maintaining oral health, including antimicrobial effects and mediating chemotaxis of the immune cells [74, 75]. Researchers reported that a deficit of cathelicidin allowed infection by *A. actinomycetemcomitans* and the development of severe periodontitis [76].

The epithelial cells in both oral mucosa and the gut are relatively hypoxic [4, 77]. The corresponding oxygen gradient between the epithelium and subepithelial perfusion in turn provides a matching cellular HIF-1α gradient in the tissues involved and perhaps the respective physiological function in cellular homeostasis. In human intestine, when the oxygen supply was impaired due to stasis of the local perfusion, the affected site would be left with increased susceptibility to infection [78]. As such, appropriate adaptive response to hypoxia at the epithelial barriers is vital. HIF-1α functions as an intracellular pO<sup>2</sup> sensor, enabling appropriate adaptive responses for cell survival. Using prolyl hydroxylase inhibitor or AKB-4924, a HIF-1α stabilizing agent, production of cathelicidin and β-defensin in uroepithelial cells was significantly enhanced, and *Escherichia coli* infection was deterred [79]. On the other hand, a deletion of HIF-1α in skin keratinocytes decreased the production of cathelicidin and led to increased susceptibility of infection by a group of *A*. *Streptococcus* [80]. Naturally occurring low-grade hypoxic reaction and hence HIF-1 accumulation in gut/urogenital/skin epithelia followed by corresponding HIF-1 downstream genes expression were recently postulated to be a key concept that underpin biological barrier function of intestinal epithelium [77, 81]. If in case the same biological process is also in action at the dentogingival junction, HIF-1 would contribute in the periodontal epithelial barrier function that maintains periodontal health and prevents oral pathogenic microorganism invasion.

Besides AMPs, there are also many factors regulated by HIF at the periodontal epithelial barrier. For instance, trefoil factors (TFF), secreted molecules from mucous epithelia, were involved in oral protection against tissue damage and immune response [82, 83]. Their expression was influenced by cellular pO<sup>2</sup> levels. It was reported that HIF-1 mediated the induction of TFF gene expression and provided an adaptive link for the maintenance of the barrier function during hypoxia of gastric/intestinal lining cells [84, 85]. Salivary mucins form a protective layer on the oral surfaces including that of oral sulcular and junctional epithelia, which serve as a physical barrier against bacterial invasion and function as essential antimicrobial macromolecules [86, 87]. Similar to TFF, mucins' production was upregulated in hypoxia [88]. This evidence indicated that the epithelial barrier cells' HIF regulation may constitute an important defense mechanism. Such oral protective machinery could contribute an additional local defense mechanism against periodontal diseases.

## **6. HIF in the periodontopathogen-host cross talk**

Hypoxia is common in the inflammatory microenvironment, and appropriate cellular responses to hypoxia contribute to mucosal defense through the oxygen-sensitive transcription regulator HIF-1α. Hypoxia increases the expression of certain TLRs on human gingival keratinocytes [89], the interaction of low oxygen with appropriate bacteria ligands *in vivo* could potentially enhance the production of cytokines and antimicrobial peptides and thus, in theory, could help to eliminate or reduce the pathogen-related concerns.

The human periodontium is persistently exposed to risks of infection; the source is the commensal and pathogenic oral microorganisms constituting the dental plaque adhering onto teeth. Bacterial components, such as LPS and peptidoglycans, released by bacteria recognized by TLRs on the surface of host cells could instigate the inflammatory reaction cascade [38]. Under steady-state conditions, activation of TLRs by commensal bacteria is critical for the maintenance of oral health [73]. Thus, TLRs provide the first line of defense in periodontal health maintenance. When stimulated, such as via TLRs recognition, PMNs exhibit increased chemotaxis and proinflammatory cytokine production [90].

Our group previously reported that bacterial components may induce HIF-1α accumulation during periodontal disease pathogenesis independent of hypoxia [91]. An immunoprecipitation experiment showed that human gingival fibroblasts' HIF-1α accumulation was induced by LPS in the dose- and time-dependent manner. The accumulation of HIF-1α may be modulated by TLRs and pattern recognition in certain ways, since a TLR4 neutralizing antibody could attenuate such an effect from *E. coli* LPS. Moreover, the expression of TLR4, CD14, and MD-2 in both human gingival keratinocytes and fibroblasts is confirmed, and the TLR4 protein expression in periodontal epithelial compartments appeared different *in vivo*, indicating that LPS sensing in the dentogingival front in health could be heterogeneous in nature [92].

A recent study on oral squamous cell carcinoma provided a novel mechanism of HIF-1 and TLRs' interplay. It was reported that the activation of TLR3 and TLR4 stimulated the expression of HIF-1 through NF-κB, while HIF-1 accumulation increased the expression of TLR3 and TLR4 through direct promoter binding [93]. This observation provided evidence that the TLR3/4-NF-κB pathway may form a positive feedback loop with HIF-1, which theoretically could also happen in the periodontal tissue. Further investigations are needed to confirm such a postulation.

## **7. HIF and bone homeostasis**

Besides AMPs, there are also many factors regulated by HIF at the periodontal epithelial barrier. For instance, trefoil factors (TFF), secreted molecules from mucous epithelia, were involved in oral protection against tissue damage and immune response [82, 83]. Their expres-

of TFF gene expression and provided an adaptive link for the maintenance of the barrier function during hypoxia of gastric/intestinal lining cells [84, 85]. Salivary mucins form a protective layer on the oral surfaces including that of oral sulcular and junctional epithelia, which serve as a physical barrier against bacterial invasion and function as essential antimicrobial macromolecules [86, 87]. Similar to TFF, mucins' production was upregulated in hypoxia [88]. This evidence indicated that the epithelial barrier cells' HIF regulation may constitute an important defense mechanism. Such oral protective machinery could contribute an additional local

Hypoxia is common in the inflammatory microenvironment, and appropriate cellular responses to hypoxia contribute to mucosal defense through the oxygen-sensitive transcription regulator HIF-1α. Hypoxia increases the expression of certain TLRs on human gingival keratinocytes [89], the interaction of low oxygen with appropriate bacteria ligands *in vivo* could potentially enhance the production of cytokines and antimicrobial peptides and thus,

The human periodontium is persistently exposed to risks of infection; the source is the commensal and pathogenic oral microorganisms constituting the dental plaque adhering onto teeth. Bacterial components, such as LPS and peptidoglycans, released by bacteria recognized by TLRs on the surface of host cells could instigate the inflammatory reaction cascade [38]. Under steady-state conditions, activation of TLRs by commensal bacteria is critical for the maintenance of oral health [73]. Thus, TLRs provide the first line of defense in periodontal health maintenance. When stimulated, such as via TLRs recognition, PMNs exhibit increased

Our group previously reported that bacterial components may induce HIF-1α accumulation during periodontal disease pathogenesis independent of hypoxia [91]. An immunoprecipitation experiment showed that human gingival fibroblasts' HIF-1α accumulation was induced by LPS in the dose- and time-dependent manner. The accumulation of HIF-1α may be modulated by TLRs and pattern recognition in certain ways, since a TLR4 neutralizing antibody could attenuate such an effect from *E. coli* LPS. Moreover, the expression of TLR4, CD14, and MD-2 in both human gingival keratinocytes and fibroblasts is confirmed, and the TLR4 protein expression in periodontal epithelial compartments appeared different *in vivo*, indicating that LPS sensing in the dentogingival front in health could be heterogeneous in nature [92].

A recent study on oral squamous cell carcinoma provided a novel mechanism of HIF-1 and TLRs' interplay. It was reported that the activation of TLR3 and TLR4 stimulated the expression of HIF-1 through NF-κB, while HIF-1 accumulation increased the expression of TLR3

levels. It was reported that HIF-1 mediated the induction

sion was influenced by cellular pO<sup>2</sup>

292 Hypoxia and Human Diseases

defense mechanism against periodontal diseases.

**6. HIF in the periodontopathogen-host cross talk**

chemotaxis and proinflammatory cytokine production [90].

in theory, could help to eliminate or reduce the pathogen-related concerns.

HIF appears to play important functional roles in bone homeostasis. The regulatory system seemed complex because HIF is known to stimulate both bone resorption and regeneration, the two essential biological processes in bone homeostasis/repair.

It is reported that a lack of oxygen in periodontal tissues may contribute to alveolar bone resorption and, in theory, accelerated periodontitis [94]. Chromatin immunoprecipitation showed that HIF-1α binds to the receptor activator of the NF-κB ligand (RANKL) promoter region, and mutations of the putative HIF-1α binding site prevented hypoxia-induced RANKL transcriptional promotion, thus suggesting that HIF-1α mediates hypoxia-induced upregulation of RANKL expression and enhanced osteoclastogenesis [95]. Furthermore, it was reported that hypoxia triggered the differentiation of peripheral mononuclear blood cells into functional osteoclasts in a HIF-dependent manner [96].

Conversely, in recent studies, HIF-1α was considered to be a critical mediator of neoangiogenesis required for bone regeneration. Exposure of PDL stem cells to hypoxia improved their osteogenic potential, mineralization and paracrine release, and the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase, and p38 MAPK signaling pathways were involved [97–99]. It was suggested that HIF, HIF mimicking agents, or HIF stabilizing agents were considered triggers for the initiation and promotion of angiogenic-osteogenic coupling [100, 101]. A recent animal study reported new bone and vessels formation induced by the overexpression of HIF-1α via adenovirus, leading to enhanced alveolar bone defect regeneration [102]. A similar result was reported from a study investigating bone loss arrest in ovariectomized C57BL/6 J mice via activated HIF-1α and Wnt/β-catenin signaling pathways [103]. Cementoblastic differentiation of human dental stem cells, a key cellular mechanism concerning periodontal regeneration, was reported to be stimulated by hypoxia in an HIF-1 dependent manner [104].

Taken together, these reports suggested that HIF-1α plays a part in alveolar bone homeostasis, resorption, or periodontal regeneration, while the exact nature of HIF-1α's roles in these processes and the way in which the related pathophysiological processes were regulated warrants further investigations.

## **8. Conclusions**

It seems that tissue/cellular hypoxia, or more specifically, expression of HIF-1α, is involved in periodontal inflammation. HIF-1 not only mediates the host's immune response, providing defense against microbial invaders and maintaining periodontal health, but also could facilitate periodontal-supporting tissue breakdown and, hence, the progression of periodontitis.

Putting all currently available information together, it appears that hypoxia could bring either beneficial or detrimental effects on periodontal health. At the present juncture, we hypothesize that similar to the intestines, a low-grade hypoxia or low level of HIF-1 is expressed in the human periodontium for baseline defense or to act as a surveillance "alarm" against significant invasion or periodontitis. A successful immune response that associates with appropriate HIF-1 mediated biological reactions would result in periodontal health maintenance. Over- or underactivation of the immune system with or without the corresponding dysregulation of HIF-1 biology in tissues as well as alveolar bone, however, could give rise to periodontal tissue damages. We also postulate that other risk indicators related to progression of periodontitis, such as smoking and diabetes mellitus, under the influence of periodontal plaque biofilm, may exert their harmful effects via inappropriate activation of the HIF pathway. Effects of these risks indicators are particular relevant as they often undermine proper periodontal healing/regeneration after therapy [105].

The mechanisms underlying the role of HIF-1 and periodontal defense/pathogenesis, however, remain elusive. Further investigations are, therefore, required in these directions to decipher what leads to the unfavorable immune reactions in periodontal inflammation and the reasons why that came about. Such new knowledge not only fosters the further understanding of human periodontal disease pathogenesis, but may provide novel therapeutic strategies that take advantage of the new understandings of periodontal HIF biology, an important element relevant for periodontal defense and regeneration.

## **Acknowledgments**

The work described in this paper was substantially supported by grants from the Research Grants Council of the Hong Kong Special Administrative Region, China (HKU 17113114), the University of Hong Kong Small Project Funding (201007176307, 201109176129), and Seed Funding (200911159126).

## **Author details**

Xiao Xiao Wang1 , Yu Chen2,† and Wai Keung Leung2,\*

\*Address all correspondence to: ewkleung@hku.hk

1 Guanghua School of Stomatology, Provincial Key Laboratory of Stomatology, Sun Yat-sen University, Guangzhou, Guangdong, PR China

2 Faculty of Dentistry, The University of Hong Kong, Hong Kong SAR, PR China

† Current address: Department of Periodontology, Nanjing Stomatology Hospital, Nanjing, Jiangsu Province, PR China

## **References**

defense against microbial invaders and maintaining periodontal health, but also could facilitate periodontal-supporting tissue breakdown and, hence, the progression of periodontitis.

Putting all currently available information together, it appears that hypoxia could bring either beneficial or detrimental effects on periodontal health. At the present juncture, we hypothesize that similar to the intestines, a low-grade hypoxia or low level of HIF-1 is expressed in the human periodontium for baseline defense or to act as a surveillance "alarm" against significant invasion or periodontitis. A successful immune response that associates with appropriate HIF-1 mediated biological reactions would result in periodontal health maintenance. Over- or underactivation of the immune system with or without the corresponding dysregulation of HIF-1 biology in tissues as well as alveolar bone, however, could give rise to periodontal tissue damages. We also postulate that other risk indicators related to progression of periodontitis, such as smoking and diabetes mellitus, under the influence of periodontal plaque biofilm, may exert their harmful effects via inappropriate activation of the HIF pathway. Effects of these risks indicators are particular relevant as they often undermine proper periodontal heal-

The mechanisms underlying the role of HIF-1 and periodontal defense/pathogenesis, however, remain elusive. Further investigations are, therefore, required in these directions to decipher what leads to the unfavorable immune reactions in periodontal inflammation and the reasons why that came about. Such new knowledge not only fosters the further understanding of human periodontal disease pathogenesis, but may provide novel therapeutic strategies that take advantage of the new understandings of periodontal HIF biology, an important

The work described in this paper was substantially supported by grants from the Research Grants Council of the Hong Kong Special Administrative Region, China (HKU 17113114), the University of Hong Kong Small Project Funding (201007176307, 201109176129), and Seed

1 Guanghua School of Stomatology, Provincial Key Laboratory of Stomatology, Sun Yat-sen

† Current address: Department of Periodontology, Nanjing Stomatology Hospital, Nanjing,

2 Faculty of Dentistry, The University of Hong Kong, Hong Kong SAR, PR China

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294 Hypoxia and Human Diseases

**Acknowledgments**

Funding (200911159126).

Jiangsu Province, PR China

**Author details**

Xiao Xiao Wang1

element relevant for periodontal defense and regeneration.

, Yu Chen2,† and Wai Keung Leung2,\*

\*Address all correspondence to: ewkleung@hku.hk

University, Guangzhou, Guangdong, PR China


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#### **Interplay between Hypoxia, Inflammation and Adipocyte Remodeling in the Metabolic Syndrome Interplay between Hypoxia, Inflammation and Adipocyte Remodeling in the Metabolic Syndrome**

Ana Marina Andrei, Anca Berbecaru-Iovan, Felix Rareş Ioan Din-Anghel, Camelia Elena Stănciulescu, Sorin Berbecaru-Iovan, Ileana Monica Baniţă and Cătălina Gabriela Pisoschi Ana Marina Andrei, Anca Berbecaru-Iovan, Felix Rareş Ioan Din-Anghel, Camelia Elena Stănciulescu, Sorin Berbecaru-Iovan, Ileana Monica Baniţă and Cătălina Gabriela Pisoschi

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/65491

#### **Abstract**

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302 Hypoxia and Human Diseases

genesis. PLoS One. 2014;9(11):e112744.

Obesity, a major social and health problem in many countries, is due to the accumulation of white adipose tissue in subcutaneous and visceral depots. The discovery of adipocytes capacity of synthesis of numerous adipocytokines and growth factors and the cross talk between adipocytes and cells of the adipose stromo-vascular fraction had highlighted the role of adipose tissue dysfunction in obesity. In visceral obesity the unbalanced synthesis of pro- and anti-inflammatory adipocytokines contributes to the development of the metabolic syndrome which cumulates the factors that increase the risk for ischemic heart disease and cerebral stroke. Adipose tissue accumulation is associated with a state of chronic inflammation, and local hypoxia is considered its underlying cause due to the hypertrophic or/and the hyperplasic growth of the fat pad. Adipose tissue hypoxia is one of the first pathophysiological changes and was placed as a missing link between obesity and low-grade inflammation present in the metabolic syndrome. Hypoxia is a major trigger for adipose tissue remodeling including adipocyte death, inflammation, tissue fibrosis, and angiogenesis. Recently, the role of hypoxia in brown adipose tissue dysfunction, a tissue presumed as the biologic counterbalance of the metabolic disturbances in human obesity, is discussed.

**Keywords:** adipose tissue, hypoxia, inflammation, metabolic syndrome, fibrosis, angiogenesis

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **1. Introduction**

Until two decades ago, the adipose tissue has been considered one of the least dynamic structures of the mammalian organism involved exclusively in fat storage. Some key events have changed this mechanistic point of view, and now the whole fat of an organism is viewed as a complex organ composed of at least two main varieties of adipose tissues: the white adipose tissue (WAT) containing unilocular adipocytes and the brown adipose tissue (BAT) formed by multilocular adipocytes. Besides this different type of adipocytes, both tissues contain a non-adipocitary stromo-vascular fraction that includes undifferentiated cells, preadipocytes, fibroblasts, inflammatory cells, and various amounts of vessels and nerves. The adult adipose organ is divided into two types of depots: subcutaneous/peripheral and visceral/central constituted of lobules of unilocular adipocytes sustained by the stromo-vascular fraction well vascularized and innervated [1–5].

A significant development in the knowledge of adipose tissue is related to its function as an endocrine organ, both types of tissues being able to elaborate adipocytokines, humoral factors with various metabolic, vascular, pro-inflammatory, and anti-inflammatory roles [2, 4].

The accumulation of WAT in physiological depots leads to obesity characterized by an increase of the body mass index (BMI) over 30 kg/m2 [6]. Obesity became a major social and health problem in many countries (between one quarter and one third of the population), a recent report of the World Health Organization (WHO) mentioning more than 1.9 billion of adult overweight subjects worldwide, more than 600 millions being obese [7]. From a pathogenically point of view, the quality and the distribution of adipose tissue seem to be more important in triggering the metabolic syndrome than the quantity of the fat per se. A direct relationship is accepted between abdominal/visceral fat accumulation—apple-shaped obesity—and the emergence and development of the metabolic syndrome or abdominal and pelvic cancers. Unbalanced synthesis of pro- and anti-inflammatory adipocytokines in visceral obesity contributes to the development of many features of the metabolic syndrome which cumulates the factors that increase the risk for ischemic heart disease and cerebral stroke: apple-shaped fat deposition, impaired glucose metabolism, dyslipidemia, and high blood pressure [8–10]. Pear-shaped obesity—i.e., subcutaneous fat accumulation—has a minimal risk for the development of such pathologies even at the same BMI greater than 30 kg/m2 [11–13].

Hypoxia is one of the mechanisms responsible for the development of the metabolic changes and the pro-inflammatory milieu of white adipose tissue in obesity [3].

Tissue partial O2 pressure (pO2) reflects the balance between O2 delivery and consumption, and continuous, chronic low O2 tension occurs as a tissue inability to provide adequate compensatory vascular supply [3, 14, 15]. Obviously, adipose tissue hypoxia has a polymorphic feature since it depends on the adipose tissue blood flow regulation, different between the adipose phenotype WAT or BAT, the adipose fat pad localization, subcutaneous or visceral, and the delay of time between the onset of hypoxia and its quantification.

In healthy lean young adult, the pO2 in adipose tissue is considered 55–60 mm Hg [14, 16] similar to the general tissue oxygenation [4], but important differences were reported in obese subjects. Oxygen supply was found markedly lower (44.7 mm Hg) in obese subjects in fasting and postprandial status than in lean subjects (55.4 mmHg) [14, 17], but Goossens et al. [18] found that in WAT of obese subjects the pO2 was even higher (67.4 mmHg) than lean (46.8 mmHg). Of note, the pO2 is not in a direct relation with the surface of the vascular network in the adipose pad. Capillary density for both subcutaneous and visceral depots is lower in obese human than in lean, but in lean subjects, the density is greater in visceral location [14, 19]. Even if BAT adipose tissue is more vascularized than WAT, it was indicated that obesity also causes BAT hypoxia, the same response being noted in multilocular adipocytes that became larger in obese animals [20]. Interestingly, BAT hypoxia seems to be temperaturedependent. Xue et al. proved that there is no hypoxia in mice housed at 30°C, but it appears in animals living at 4°C [21].

**1. Introduction**

304 Hypoxia and Human Diseases

and innervated [1–5].

of the body mass index (BMI) over 30 kg/m2

Until two decades ago, the adipose tissue has been considered one of the least dynamic structures of the mammalian organism involved exclusively in fat storage. Some key events have changed this mechanistic point of view, and now the whole fat of an organism is viewed as a complex organ composed of at least two main varieties of adipose tissues: the white adipose tissue (WAT) containing unilocular adipocytes and the brown adipose tissue (BAT) formed by multilocular adipocytes. Besides this different type of adipocytes, both tissues contain a non-adipocitary stromo-vascular fraction that includes undifferentiated cells, preadipocytes, fibroblasts, inflammatory cells, and various amounts of vessels and nerves. The adult adipose organ is divided into two types of depots: subcutaneous/peripheral and visceral/central constituted of lobules of unilocular adipocytes sustained by the stromo-vascular fraction well vascularized

A significant development in the knowledge of adipose tissue is related to its function as an endocrine organ, both types of tissues being able to elaborate adipocytokines, humoral factors with various metabolic, vascular, pro-inflammatory, and anti-inflammatory roles [2, 4].

The accumulation of WAT in physiological depots leads to obesity characterized by an increase

problem in many countries (between one quarter and one third of the population), a recent report of the World Health Organization (WHO) mentioning more than 1.9 billion of adult overweight subjects worldwide, more than 600 millions being obese [7]. From a pathogenically point of view, the quality and the distribution of adipose tissue seem to be more important in triggering the metabolic syndrome than the quantity of the fat per se. A direct relationship is accepted between abdominal/visceral fat accumulation—apple-shaped obesity—and the emergence and development of the metabolic syndrome or abdominal and pelvic cancers. Unbalanced synthesis of pro- and anti-inflammatory adipocytokines in visceral obesity contributes to the development of many features of the metabolic syndrome which cumulates the factors that increase the risk for ischemic heart disease and cerebral stroke: apple-shaped fat deposition, impaired glucose metabolism, dyslipidemia, and high blood pressure [8–10]. Pear-shaped obesity—i.e., subcutaneous fat accumulation—has a minimal risk for the devel-

Hypoxia is one of the mechanisms responsible for the development of the metabolic changes

Tissue partial O2 pressure (pO2) reflects the balance between O2 delivery and consumption, and continuous, chronic low O2 tension occurs as a tissue inability to provide adequate compensatory vascular supply [3, 14, 15]. Obviously, adipose tissue hypoxia has a polymorphic feature since it depends on the adipose tissue blood flow regulation, different between the adipose phenotype WAT or BAT, the adipose fat pad localization, subcutaneous or visceral,

In healthy lean young adult, the pO2 in adipose tissue is considered 55–60 mm Hg [14, 16] similar to the general tissue oxygenation [4], but important differences were reported in obese

opment of such pathologies even at the same BMI greater than 30 kg/m2

and the pro-inflammatory milieu of white adipose tissue in obesity [3].

and the delay of time between the onset of hypoxia and its quantification.

[6]. Obesity became a major social and health

[11–13].

Adipose tissue is one of the most plastic organs in adults gifted with the ability of a continuous remodeling—extends or regresses depending on nutrient intake. The plasticity of any tissue is due to its capacity of extending vasculature which requires the cross talk between adipocytes and stromal and endothelial cells in the case of adipose tissue.

There are several arguments in favor of this "hypoxia concept." Normally, each adipocyte is surrounded by capillaries, and it is widely accepted that WAT is poorly oxygenated in obese individuals because adipocytes may be up to 200 μm, so larger than the normal diffusion distance of oxygen within tissues. As adipose tissue mass rapidly increases, clusters of unilocular adipocyte distance from the vessels and pockets of hypoxia are generated [22]. Another cause presumed for adipose tissue hypoxia is the loss of endothelial cells usually associated with the damage of parenchymal cells in other tissues. Recently, the interest in brown adipose tissue (BAT) increased, and studies have indicated that obesity determines also BAT hypoxia and the loss of its thermogenic capacity [20].

Chronic adipose tissue hypoxia has been suggested to be part of the pathogenesis of adipocyte dysfunction [14, 23, 24]. Local hypoxia triggers the generation of reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress [25] and initiates the inflammatory response able to regulate the balance between angiogenic factors and inhibitors in order to stimulate angiogenesis and increase blood flow. The paucity of endothelial barrier is associated with the release of profibrogenic and pro-inflammatory cytokines and an augmented influx of inflammatory cells [26]. There is considerable evidence that obese adipose tissue is markedly infiltrated with macrophages which participate in the inflammatory pathways and are very important in adipose tissue remodeling, macrophage infiltration being signalized by lipid-overloaded adipocytes necrosis. Numerous reports emphasized that visceral adipose tissue in obese individuals is more fibrotic than that of lean subjects [27–29].

Normally, BAT and WAT produce various pro-angiogenic factors and cytokines able to induce remodeling of the vasculature, and as a response to hypoxia, an unbalanced production of these multiple bioactive pro-angiogenic and antiapoptotic growth factors synthesized by the adipose stromal cells may occur. Local hypoxia in obese is the underlying cause of an increase of macrophage cell number accompanied by the state of chronic inflammation and impaired adipokine secretion. Hypoxia promotes the delivery of many adipocytokines related to inflammation and tissue remodeling needed for angiogenesis to the ischemic tissue, such as macrophage migration inhibitory factor (MIF), granulocyte-macrophage colony-stimulating factor (GM-CSF), matrix metalloproteinases MMP-2 and MMP-9, transforming growth factor (TGF)-β, vascular endothelial growth factor (VEGF), interleukins (IL-1, IL-6, IL-10), tumor necrosis factor (TNF)-α, angiopoietin-like (Angptl)-4, and leptin [4, 15, 22, 30–32].

This chapter summarizes the potential links between hypoxia, inflammation, adipocyte hypertrophy, and macrophage infiltration of adipose tissue and the effects of inflammatory mediators on its remodeling.

## **2. Essentials of adipose organ structure and functions**

The adult adipose organ is composed by two types of adipose depots divided into adipose lobules of (i) unilocular adipose tissue (WAT—white adipose tissue) composed of unilocular cells and (ii) brown adipose tissue (BAT), formed by multilocular adipocytes (**Figure 1a** and **b**).

**Figure 1.** (a) Human adult subcutaneous WAT (hematoxylin and eosin staining, ob. ×40) and (b) human newborn visceral adipose depot with unilocular WAT and multilocular BAT adipocytes (hematoxylin and eosin staining, ob. ×20).

Both types of cells organized into adipose lobules are sustained by the stromo-vascular fraction well vascularized and innervated [11, 33]. Anatomically, WAT depots are located primarily in two major areas—subcutaneous/peripheral and visceral/central, which differ in the composition of the stromo-vascular fraction [34, 35]. Although at a first view the adipose tissue looks quite simple, a deeper molecular analysis revealed a high heterogeneity of cells. With respect to adipose cells, recent research identified both in rodent and men, the third type of adipocytes with common features of WAT and BAT adipocytes named "brite" or "beige" cells.

BAT, so named because of its yellow-brown color in vivo due to a very rich vascularization, is distinguishable morphologically from WAT by its cytoplasmic multiple droplets of stored triglycerides, while WAT contains a single large droplet. The multilocular cells are rich in mitochondria containing the uncoupling protein (UCP)1 which is uniquely present in BAT and therefore considered a marker for it (**Figure 2**).

**Figure 2.** Newborn human brown adipocytes labeled with UCP1 (IHC, ob. ×40).

macrophage migration inhibitory factor (MIF), granulocyte-macrophage colony-stimulating factor (GM-CSF), matrix metalloproteinases MMP-2 and MMP-9, transforming growth factor (TGF)-β, vascular endothelial growth factor (VEGF), interleukins (IL-1, IL-6, IL-10), tumor

This chapter summarizes the potential links between hypoxia, inflammation, adipocyte hypertrophy, and macrophage infiltration of adipose tissue and the effects of inflammatory

The adult adipose organ is composed by two types of adipose depots divided into adipose lobules of (i) unilocular adipose tissue (WAT—white adipose tissue) composed of unilocular cells and (ii) brown adipose tissue (BAT), formed by multilocular adipocytes (**Figure 1a** and **b**).

**Figure 1.** (a) Human adult subcutaneous WAT (hematoxylin and eosin staining, ob. ×40) and (b) human newborn visceral adipose depot with unilocular WAT and multilocular BAT adipocytes (hematoxylin and eosin staining, ob. ×20).

Both types of cells organized into adipose lobules are sustained by the stromo-vascular fraction well vascularized and innervated [11, 33]. Anatomically, WAT depots are located primarily in two major areas—subcutaneous/peripheral and visceral/central, which differ in the composition of the stromo-vascular fraction [34, 35]. Although at a first view the adipose tissue looks quite simple, a deeper molecular analysis revealed a high heterogeneity of cells. With respect to adipose cells, recent research identified both in rodent and men, the third type of adipocytes

BAT, so named because of its yellow-brown color in vivo due to a very rich vascularization, is distinguishable morphologically from WAT by its cytoplasmic multiple droplets of stored triglycerides, while WAT contains a single large droplet. The multilocular cells are rich in

with common features of WAT and BAT adipocytes named "brite" or "beige" cells.

necrosis factor (TNF)-α, angiopoietin-like (Angptl)-4, and leptin [4, 15, 22, 30–32].

**2. Essentials of adipose organ structure and functions**

mediators on its remodeling.

306 Hypoxia and Human Diseases

In humans, two types of BAT are present: (i) the classical (or constitutive BAT—cBAT) that is fully developed at birth and then reduced to remain in human adult only in a symmetrical cervical position and around the clavicles as very recently localized by PET/CT scanning, and the second type of brown adipocytes named "beige" or "brite" (brown in white), inducible or recruitable BAT (rBAT). This is composed of isolated brown multilocular cells resident between white cells mainly in subcutaneous depots [36, 37]. WAT is recognized as the site of fat storage, while BAT acts, as in rodents, as a heat-generating tissue through uncoupled oxidative phosphorylation which involves the action of UCP1 [38].

The functional complexity of adipose tissue is also due to the heterogeneity of cell phenotypes located in the non-adipocitary stromo-vascular fraction that includes undifferentiated or mesenchymal cells, preadipocytes, fibroblasts, and inflammatory cells (macrophages, lymphocytes, and mast cells). These cells are surrounded by a very complex network of vessels and nerves. The vascular network is more developed and branched in BAT than in WAT [39]. Normal metabolic functions and their imbalance involve a cross talk between adipocytes and the cells from the stromo-vascular fraction mediated by the components of adipose tissue extracellular matrix (ECM).

*WAT secretoma*. The discovery of leptin by Friedman in 1994 initiated the recognition of white adipocytes as major endocrine cells that secrete numerous bioactive molecules: lipids (such as free fatty acids mobilized in lipolysis, prostaglandins, and endocannabinoids) and proteins (termed "adipokines" or "adipocytokines" with metabolic and pro-/anti-inflammatory functions) [40]. Several adipocytokines are listed in **Table 1**. Impaired production of adipokines is associated with the pathogenesis of obesity-related disorders—type 2 diabetes mellitus, metabolic syndrome, cardiovascular diseases, and certain types of cancer [2, 41–44]. Generally, blood adipocytokine levels rise with the increase of fat mass except for adiponectin and omentin levels which are reported to be lower in obese and overweight subjects [31, 45, 46].


**Table 1.** Adipocytokines and their main biological effects (adapted with permission from [31]).

Leptin and adiponectin are the most important hormones secreted by white adipocytes with multiple metabolic roles (regulating appetite and energy balance, insulin sensitivity) but also encompass angiogenic and anti-inflammatory actions [2, 4]. Leptin increases the vascular permeability in adipose tissue and influences microvessels density [47].

Adiponectin is regarded as a link between obesity and related metabolic disorders because it improves glucose and lipid metabolism and prevents inflammation [15]. There are many other members of the "adipokinome" involved in the inflammatory response: tumor necrosis factor (TNF)-α, interleukins (IL-6, IL-8, IL-10), monocyte chemoattractant protein (MCP)-1, and macrophage migration inhibitory factor (MIF) [4, 15, 22]. Besides the adipocytes, many other cells from the stromo-vascular fraction secrete inflammatory cytokines and chemokines in response to adipocyte hypertrophy or hypoxic conditions. Other adipokines related to inflammation include several crucial angiogenic factors, such as vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF)-1, angiopoetin-2, nerve growth factor (NGF), plasminogen activator inhibitor (PAI)-1, apelin, and adipsin [4, 30–32]. The release of numerous inflammatory adipocytokines is markedly increased in obesity-related diseases. Subcutaneous and visceral adipose tissue display differences in their adipokinome. Even if the results of in vitro and in vivo studies are controversial, it can be assumed, for example, that leptin and adiponectin are mainly produced in vivo by the subcutaneous adipocytes, while others (angiotensinogen, A-fatty acid-binding protein (FABP)-4, IL-6) are secreted at higher levels in visceral adipose tissue [48–51].

## **3. Adipose tissue dysfunction and hypoxia**

**Adipocytokine Function**

308 Hypoxia and Human Diseases

Vaspin (visceral adipose tissue-derived serpin) Insulin resistance Omentin Insulin resistance Retinol-binding protein (RBP)-4 Insulin resistance

Cholesteryl ester transfer protein (CETP) Lipid metabolism Lipoprotein lipase (LPL) Lipid metabolism Adipocyte fatty acid-binding protein (A-FABP)-4 Lipid metabolism Perilipin Lipid metabolism

Tumor necrosis factor (TNF)-α Pro-inflammatory Interleukin 6 (IL-6) Pro-inflammatory C-reactive protein (CRP) Pro-inflammatory

Fibroblast growth factor (FGF)-2 Pro-angiogenic Hepatocyte growth factor (HGF) Pro-angiogenic Platelet-derived growth factor (PDGF) Pro-angiogenic Vascular endothelial growth factor (VEGF) Pro-angiogenic Transforming growth factor (TGF)-β Inflammation, fibrosis

Plasminogen activator inhibitor (PAI)-1 Fibrinolysis, pro-angiogenic Monocyte chemoattractant protein (MCP)-1 Macrophage activation Intercellular adhesion molecule (ICAM)-1 Macrophage activation

Leptin Feeding behavior, fat mass, pro-angiogenic

Visfatin (pre-B-cell colony-enhancing factor, PBEF) Insulin resistance, pro-inflammatory

Serum amyloid A Insulin resistance, pro-inflammatory

Apelin Vasodilatation, pro-angiogenic Angiotensinogen Regulation of blood pressure Angiotensin II Regulation of blood pressure

Adipsin (adipocyte trypsin/complement factor D) Lipid and glucose metabolism, inflammation

Matrix metalloproteinases (MMPs) Pro- and antiangiogenic, ECM remodeling

Leptin and adiponectin are the most important hormones secreted by white adipocytes with multiple metabolic roles (regulating appetite and energy balance, insulin sensitivity) but also encompass angiogenic and anti-inflammatory actions [2, 4]. Leptin increases the vascular

Tissue inhibitor of metalloproteinases (TIMPs) Antiangiogenic, ECM remosdeling

**Table 1.** Adipocytokines and their main biological effects (adapted with permission from [31]).

permeability in adipose tissue and influences microvessels density [47].

Adiponectin Insulin sensitivity, anti-inflammatory, pro-angiogenic Resistin Insulin resistance, pro-inflammatory, antiangiogenic

> Adipocyte capacity of synthesis corroborated with the clinical observation that a proportion of obese individuals seem to be protected against metabolic syndrome [52] had highlighted the role of adipose tissue dysfunction in obesity. Obesity is so long considered a genetic predisposition that promotes the excess of energy intake or the scarce energy expenditure.

> In humans, the adipose tissue from the two main locations (subcutaneous and visceral) shows anatomical and functional differences (in contrast to subcutaneous adipose tissue, abdominal depots drain directly onto the portal circulation [31]) and different gene expressions.

> Oxygen is a main nutritional factor without which oxidation of nutrients in aerobic tissues cannot take place. The decrease of oxygen level in various tissues can occur even if the total amount provided to the organism is not reduced [20]. Evinced hypoxia that follows low oxygen tension has numerous implications for cellular metabolism and transcriptional program [27]. Recent research suggests that adipose tissue hypoxia occurs in obese mice and even in human subjects. In obese rodents the existence of hypoxia was demonstrated by qualitative reaction (using hypoxic cell markers, such as pimonidazole—PIMO) or quantitative technique using needle-type oxygen sensors [3, 15, 22, 23]. In human obese subjects, the results are more controversial since normoxia and even hyperoxia have been reported in various experiments [18, 20, 53].

> Chronic hypoxia has been suggested to be part of the pathogenic pathways leading to adipose tissue dysfunction [14, 23, 24].

> Local hypoxia triggers the main alterations defining the adipose tissue dysfunction: generation of ROS and oxidative stress [54], ER stress [25], adipocyte death [55], inhibition of adiponectin

expression [55, 56], and leptin hyperproduction [57] and initiates the inflammatory response able to regulate the balance between angiogenic and inhibitor factors in order to stimulate angiogenesis and increase blood flow.

More causes of adipose tissue hypoxia are discussed, this concept being related to the histological changes of the adipose obese tissue—hyperplasia and adipocyte hypertrophy. Reduction of blood supply in adipose pads is a common mechanism of tissue hypoxia. Reduced adipose tissue blood flow in obese rats and humans was reported many years ago (Larsen et al, 1966, West et al., 1987 cited by [26]), being associated with insulin resistance in obese individuals [17, 18]. Adipose tissue angiogenesis is insufficient to maintain normoxia in the growing number of fat-storing cells in adipose depots as they are in obesity. Histological analysis has demonstrated a scarce capillary network in abdominal subcutaneous depots in obese subjects compared to the leans [14, 18].

A second cause is related to the increased size of adipocytes—hypertrophy—reaching in obese subjects a diameter larger than 150–200 μm [45]. This exceeds the normal capacity of oxygen diffusion through the tissue (100–200 μm), and oxygenation of adipose tissue will be compromised [58].

Hypoxia-inducible factor (HIF)-1 is the key transcriptional factor involved in response to hypoxia, which moves into the nucleus and binds to hypoxia-response elements from a myriad of target genes to initiate their transcription [3]. Both murine and human adipocytes exhibit extensive functional changes in culture in response to HIF-1, which alters the expression of up to 1300 genes [59]. These include genes encoding key adipokines, such as leptin, apelin, visfatin, TNF-α, IL-1, IL-6, VEGF, angiopoietin-like protein (Angptl)-4, MIF, PAI-1, and matrix metalloproteinases 2 and 9 (MMP-2, MMP-9), which are upregulated, and adiponectin, peroxisome proliferator-activated receptor (PPAR)-γ which is downregulated [3, 20, 55, 60, 61].

Hypoxia alters genes encoding key proteins for metabolic processes: glucose uptake, glycolysis, oxidative metabolism, lipolysis, and lipogenesis. Glucose uptake into adipocytes is stimulated by hypoxia because the expression of GLUT transporters is upregulated [20, 55, 62]. A switch from aerobic to anaerobic metabolism in hypoxic adipocytes is sustained by the increased activity of some glycolytic enzymes (e.g., phosphofructokinase [63]) and a net lactate release. Many studies focused on hypoxia-induced derangements of lipid metabolism reporting an increased lipolysis rather than unchanged but reduced lipogenesis in hypoxic adipocytes [64–66].

It seems that various degrees of adipose tissue hypoxia have different metabolic effects, and it is supposed also that subcutaneous and visceral adipocytes respond differently to factors that mediate tissue hypoxia. A recent study demonstrated that hypercaloric diet induces more severe hypoxia in mesenteric adipose tissue of mice than in the subcutaneous one [67].

Another direct effect of hypoxia is induction of insulin resistance via the upregulation of certain adipokines, the impairment of insulin-signaling pathway being a key change for white adipocyte dysfunction in obese subjects [3, 4].

Adipose tissue is one of the most plastic entities of an organism in terms of growth in the childhood and even in the adulthood in normal and pathological conditions, responding rapidly and dynamically to nutrient excess or starvation. Nor normal or pathological tissue, therefore nor the adipose tissue, is able to grow, develop, and function in the absence of an appropriate vascular network. Therefore, the hypoxia-induced expression of VEGF, the main angiogenic factor, and of certain adipokines, such as angiopoietin-2, Angptl-4, and leptin, sustains the stimulation of angiogenesis in obese adipose tissue [68–70]. Experimental data emphasize the induction of a pro-fibrotic switch of the transcriptional program in hypoxic adipocytes, fibrosis being another feature of adipose tissue dysfunction in obesity [29, 71]. Preadipocytes, pro-inflammatory cells, and fibroblasts from WAT as well as adipocytes respond to hypoxic conditions, favoring cellular events that lead to inflammation and fibrosis. Biostatistical analysis of WAT transcriptome had demonstrated a positive correlation between fat mass, degree of inflammation, and synthesis of ECM in obesity complications [72].

## **4. WAT hypoxia: a link between obesity and inflammation**

expression [55, 56], and leptin hyperproduction [57] and initiates the inflammatory response able to regulate the balance between angiogenic and inhibitor factors in order to stimulate

More causes of adipose tissue hypoxia are discussed, this concept being related to the histological changes of the adipose obese tissue—hyperplasia and adipocyte hypertrophy. Reduction of blood supply in adipose pads is a common mechanism of tissue hypoxia. Reduced adipose tissue blood flow in obese rats and humans was reported many years ago (Larsen et al, 1966, West et al., 1987 cited by [26]), being associated with insulin resistance in obese individuals [17, 18]. Adipose tissue angiogenesis is insufficient to maintain normoxia in the growing number of fat-storing cells in adipose depots as they are in obesity. Histological analysis has demonstrated a scarce capillary network in abdominal subcutaneous depots in

A second cause is related to the increased size of adipocytes—hypertrophy—reaching in obese subjects a diameter larger than 150–200 μm [45]. This exceeds the normal capacity of oxygen diffusion through the tissue (100–200 μm), and oxygenation of adipose tissue will be compro-

Hypoxia-inducible factor (HIF)-1 is the key transcriptional factor involved in response to hypoxia, which moves into the nucleus and binds to hypoxia-response elements from a myriad of target genes to initiate their transcription [3]. Both murine and human adipocytes exhibit extensive functional changes in culture in response to HIF-1, which alters the expression of up to 1300 genes [59]. These include genes encoding key adipokines, such as leptin, apelin, visfatin, TNF-α, IL-1, IL-6, VEGF, angiopoietin-like protein (Angptl)-4, MIF, PAI-1, and matrix metalloproteinases 2 and 9 (MMP-2, MMP-9), which are upregulated, and adiponectin, peroxisome proliferator-activated receptor (PPAR)-γ which is downregulated [3, 20, 55, 60, 61]. Hypoxia alters genes encoding key proteins for metabolic processes: glucose uptake, glycolysis, oxidative metabolism, lipolysis, and lipogenesis. Glucose uptake into adipocytes is stimulated by hypoxia because the expression of GLUT transporters is upregulated [20, 55, 62]. A switch from aerobic to anaerobic metabolism in hypoxic adipocytes is sustained by the increased activity of some glycolytic enzymes (e.g., phosphofructokinase [63]) and a net lactate release. Many studies focused on hypoxia-induced derangements of lipid metabolism reporting an increased lipolysis rather than unchanged but reduced lipogenesis in hypoxic

It seems that various degrees of adipose tissue hypoxia have different metabolic effects, and it is supposed also that subcutaneous and visceral adipocytes respond differently to factors that mediate tissue hypoxia. A recent study demonstrated that hypercaloric diet induces more severe hypoxia in mesenteric adipose tissue of mice than in the subcutaneous one [67].

Another direct effect of hypoxia is induction of insulin resistance via the upregulation of certain adipokines, the impairment of insulin-signaling pathway being a key change for white

Adipose tissue is one of the most plastic entities of an organism in terms of growth in the childhood and even in the adulthood in normal and pathological conditions, responding

angiogenesis and increase blood flow.

310 Hypoxia and Human Diseases

obese subjects compared to the leans [14, 18].

mised [58].

adipocytes [64–66].

adipocyte dysfunction in obese subjects [3, 4].

The necessary link between abdominal (visceral or central) obesity and the development of type 2 diabetes and metabolic syndrome (which includes atherosclerosis, hypertension, and hyperlipidemia) due to the expanding fat mass and adipose tissue dysfunction was first demonstrated by Spiegelman's group [73, 74]. The mild inflammation status of the adipose tissue in obese subjects is induced by the peculiar role occupied by TNF-α, a 26 kDa transmembrane protein secreted as a cytokine and acting as an endotoxin-induced factor causing necrosis of tumors in vitro and cachexia in vivo, so naturally linked to the energy homeostasis [1]. They discovered that TNF-α is an active biofactor secreted by adipocytes and stromovascular cells positively correlated with obesity and insulin resistance.

Many signaling pathways have been proposed to be involved in the pathogenesis of obesityassociated inflammation called also "metaflammation" [75] such as (i) activation of toll-like receptor 4 (TLR4) by free fatty acids released after lipolysis [76], (ii) activation of protein kinase C (PKC) by diacylglicerol and ceramide [77]), (iii) induction of ER stress [25, 78] and oxidative stress [79], and (iv) adipocyte death [39]. Recent research data suggest that adipose tissue hypoxia is one of the first pathophysiological changes and was placed as a missing link between obesity and low-grade inflammation [61, 80].

Clinical and physiological data argue that in the whole organism the oxygen level is not the same in all the tissues nor constant for the same tissue and an isolate organ or tissue may lack oxygen even if the total supply is not compromised. This seems to be the case of the hypoxia inside the WAT human depots, the expanding adipose lobules or hypertrophic adipocytes resting isolated in pockets of tissue that lack the vascular supply, while other areas could be in normoxia or even hyperoxia [20]. The lack of oxygen perfusion for the hypertrophic adipocytes made them necrotic and finally they died. Dead adipocytes and free lipid droplets liberatedly act as recruitment factors for macrophages [39]. Besides adipocytes, preadipocytes and macrophages (the main players in WAT inflammatory response stimulating the inflammatory state in adipose tissue by the release of pro-inflammatory cytokines, such as TNF-α and interleukins) also respond to hypoxia. For such controversial results regarding the hypoxia in human adipose tissue, one must consider the technique accuracy and the methodological issues, minding that the same depot could be polarized toward hypoxic areas or inflamed and hypervascularized nests. Such a clustered differentiation is not unique in the adipose tissue since data demonstrated that in obese adipose tissue the switch from M2a macrophages discriminative for lean mice to M1 inflammatory phenotype takes place in well-defined spatiotemporal areas inside the same adipose depot [81].

In order to assess the involvement of TLR signaling in inflammation in obesity-related diseases, we analyzed the expression of TLR-2, TLR-4, TNF-α, and CD-68 in subcutaneous and visceral adipose depots from lean, obese, and obese diabetic subjects. We observed that both types of depots showed an increased number of small- and medium-dilated vessels with many CD68 positive cells [82]. In the peritoneal depots, we observed leukocyte margination with CD68 positive cells, but we didn't notice the presence of macrophages crowns in none of the samples analyzed, as Cinti and coworkers found in adipose tissue with hypertrophic cells [39]. Data obtained proved that same cells from the visceral adipose depots of obese and obese-diabetic patients, mainly macrophages, intravascular leukocytes, and endothelial cells, showed a positive reaction for both TLR-4 and TNF-α [82], proving that TLR4 activation contributes to the inflammatory process in obesity and the onset of the metabolic syndrome (**Figure 3**).

**Figure 3.** Immunostaining for CD68, TLR-4, and TNF-α of visceral obese adipose depots. (a) CD68-positive leukocytes between adipocytes (ob. ×40) in adipose peritoneal depots, (b) TLR-4-positive leukocytes and endothelial cells (ob. ×40), (c) intense-positive TNF-α reaction in visceral depots (ob. ×40).

Summarizing the data linking the cellular and molecular alterations of the adipose tissue in obesity to the adipose tissue dysfunction, among the three events highlighted—oxidative stress, ER stress, and local hypoxia—hypoxia might be the first in a logical chronologically order, since it promotes oxidative and ER stress. In obesity, quick changes from normoxia/ hyperoxia to hypoxia would be needed in order to induce oxidative stress [16]. Adipose tissue hypoxia induces inflammation through activation of two main transcription factors, HIF-1α and nuclear factor (NF)-KB, each of them activating transcription of a variety of genes encoding angiogenic and/or pro-inflammatory adipocytokines [26, 83]. Available data demonstrate that in rodent, HIF-1α upregulation starts in the first 1–3 days after the administration of a highfat diet, before inflammation and insulin resistance develop [15, 19, 84].

## **5. Hypoxia: a major trigger for adipose tissue remodeling**

Adipose tissue hypoxia is a concept that can practically explain the main alterations defining the adipose tissue dysfunction due to obesity: chronic inflammation, leptin expression, adiponectin reduction, adipocyte death followed by the invasion of monocytes and activation of macrophages, elevated lipolysis and adipocyte insulin resistance, and increased activity of ROS [3, 15, 55]. This entire cellular and molecular imbalance is followed by a compulsory adipose depot remodeling. The concept of remodeling of adipose tissue refers, as in all other entities, to the turnover of the cells and of the ECM in response to the requirement for growth and expansion of the adipose depots [85]. The molecules (cytokines, adipokines, growth factors, and proteases) involved in adipose tissue remodeling are synthesized and act as a permanent result of the cross talk between adipocytes and stromal cells.

#### **5.1. Adipocyte death and inflammation**

in human adipose tissue, one must consider the technique accuracy and the methodological issues, minding that the same depot could be polarized toward hypoxic areas or inflamed and hypervascularized nests. Such a clustered differentiation is not unique in the adipose tissue since data demonstrated that in obese adipose tissue the switch from M2a macrophages discriminative for lean mice to M1 inflammatory phenotype takes place in well-defined

In order to assess the involvement of TLR signaling in inflammation in obesity-related diseases, we analyzed the expression of TLR-2, TLR-4, TNF-α, and CD-68 in subcutaneous and visceral adipose depots from lean, obese, and obese diabetic subjects. We observed that both types of depots showed an increased number of small- and medium-dilated vessels with many CD68 positive cells [82]. In the peritoneal depots, we observed leukocyte margination with CD68 positive cells, but we didn't notice the presence of macrophages crowns in none of the samples analyzed, as Cinti and coworkers found in adipose tissue with hypertrophic cells [39]. Data obtained proved that same cells from the visceral adipose depots of obese and obese-diabetic patients, mainly macrophages, intravascular leukocytes, and endothelial cells, showed a positive reaction for both TLR-4 and TNF-α [82], proving that TLR4 activation contributes to the inflammatory process in obesity and the onset of the metabolic syndrome (**Figure 3**).

**Figure 3.** Immunostaining for CD68, TLR-4, and TNF-α of visceral obese adipose depots. (a) CD68-positive leukocytes between adipocytes (ob. ×40) in adipose peritoneal depots, (b) TLR-4-positive leukocytes and endothelial cells

(ob. ×40), (c) intense-positive TNF-α reaction in visceral depots (ob. ×40).

spatiotemporal areas inside the same adipose depot [81].

312 Hypoxia and Human Diseases

Adipocyte death is accepted as the main trigger for the adipose tissue remodeling [84], but the cause of this event is not consensual: the adipocyte size or the hypoxic milieu. In mice a positive robust correlation exists between adipocyte size and adipocyte death [39]. Consecutively macrophages are accumulating in crown-like structures being a source of numerous proinflammatory cytokines. A difference in the incidence of dead adipocytes was noted, the intraabdominal cells being more susceptible than those of the inguinal depots. The clearance of the cellular detritus by the macrophages is the trigger for a homeostatic remodeling program that will allow the further expansion of the adipose depots that include matrix remodeling and vasculogenesis. Foci of adipocyte death are therefore areas where macrophages promote obesity-associated inflammation [39]. Interestingly, adipocyte loss is associated with phenotypic changes in stromal monocytic-macrophage cells. In a chronologically sequence, after the scavenging, the place occupied by the huge dead adipocytes was taken by small-size adipocytes, and the former hypertrophic adipose tissue became hyperplasic (Faust et al., 1984 cited by [84]). As a new study demonstrated that the macrophages are crowded in foci of hypoxic tissue, a second theory emphasizes that adipocyte death is caused by the hypoxia and the macrophages are trapped into the hypoxic areas by MIF [65, 86].

#### **5.2. Hypoxia underpins adipose tissue fibrosis**

There are several recent studies involving fibrosis of adipose depots in installing hypoxia and insulin resistance [27, 28, 87].

Scherer's research group proposed that in adipose obese tissues hypoxia is the most important driving force downstreaming the events associated with inflammation and fibrosis [27]. They found that in adipose tissue from the transgenic mice HIF-1α-ΔODD, in which a dominantactive deletion mutation of HIF-1α is overexpressed, fed with a hypercaloric diet, the transcription factor HIF-1α failed to promote the pro-angiogenic program by targeting genes, such as VEGF-A. Moreover, in these mice HIF-1α induces the fibrotic program by an increased synthesis of fibrotic proteins, such as lysyl oxidase (LOX), type I and type III collagens, tissue inhibitor of matrix metalloproteinases (TIMP)-1, and connective tissue growth factor (CTGF). Histology performed with trichromic staining revealed thick fibrotic streaks composed of type I collagen fibers, similar results being reported also for the adipose pads from obese human subjects [88].

LOX is a known target gene of HIF-1α, and in adipose tissue of ob/ob transgenic mouse, LOX is found in increased level compared to wild type [27]. LOX cross-links elastin and collagens in ECM and creates ECM-resistant bands of fibrosis. In adipose tissue of ob/ob mouse, these collagen bundle "streaks" are found outside the "crown-like" structures previously described [27, 39, 84]. The conclusion derived was that collagen synthesis and deposition could be anterior to the accumulation of macrophages surrounding the adipose cells because hypoxiainduced fibrotic program develops shortly after the high-fat diet is established [27]. So adipose tissue fibrosis is not necessarily induced by inflammation but could be rather an upstream phenomenon through the synthesis of HIF-1α and LOX.

From a different point of view, like in other tissues, adipose tissue fibrosis develops as a result of a persistent inflammation and a failure of the normal tissue repair with *restitutio ad integrum*.

Interestingly, fibrosis of adipose depots in obesity seems to display an otherwise intensity as the inflammation and hypoxia, those visceral seeming to be not only less fibrotic than those peripheral but also with a different distribution of collagen fibers, especially pericellular or intraparenchymatous [28]. As the visceral depots are more inflamed than the subcutaneous depots as we showed [88], this observation contradicts the accepted biological sequence that fibrosis develops as a result of an excessive and altered ECM synthesis and storage by resident cells activated in an inflammatory environment. This abnormal amount of fibrotic matrix in the subcutaneous adipose tissue could be explained if we keep in mind the histology of the host tissue where the adipose depots expand (the subcutaneous adipose tissue develops toward a much more dense tissue than the visceral one). In obese subjects fibrosis accumulates in pericellular areas—lining each adipose cell or a group of cells (interstitial fibrosis) and around the vessels (**Figure 4**).

Collagen phenotypes are also different, types I, III, and VI being present in pericellular position but only I and III form thick bundles appearing as interlobular septa surrounding more cells [28]. In visceral (omental) depots, the accumulation of fibers in pericellular position is associated with small adipocyte size and a lowest quantity of circulating triglycerides, proving that the subjects with smaller adipocytes have a less adverse metabolic profile [27, 89], so fibrosis may act as a protective reaction. In the adipose depots, the significance of type VI collagen seems to be peculiar, since its appearance changes dramatically through adipogenesis [90]. Transgenic mouse col 6KO ob/ob shows reduced necrotic cell death and consumes only a half of the amount of food that ob/ob strain [91] and type VI collagen levels correlate with hyperglycemia and insulin resistance [87, 92]. Obese humans expressed higher levels of type VI collagen and macrophage markers [92].

**Figure 4.** Pericellular and interstitial fibrosis in a visceral adipose depot from an adult obese subject (ob. **×**20).

### **5.3. Hypoxia-induced angiogenesis in white adipose tissue**

**5.2. Hypoxia underpins adipose tissue fibrosis**

phenomenon through the synthesis of HIF-1α and LOX.

insulin resistance [27, 28, 87].

314 Hypoxia and Human Diseases

around the vessels (**Figure 4**).

subjects [88].

There are several recent studies involving fibrosis of adipose depots in installing hypoxia and

Scherer's research group proposed that in adipose obese tissues hypoxia is the most important driving force downstreaming the events associated with inflammation and fibrosis [27]. They found that in adipose tissue from the transgenic mice HIF-1α-ΔODD, in which a dominantactive deletion mutation of HIF-1α is overexpressed, fed with a hypercaloric diet, the transcription factor HIF-1α failed to promote the pro-angiogenic program by targeting genes, such as VEGF-A. Moreover, in these mice HIF-1α induces the fibrotic program by an increased synthesis of fibrotic proteins, such as lysyl oxidase (LOX), type I and type III collagens, tissue inhibitor of matrix metalloproteinases (TIMP)-1, and connective tissue growth factor (CTGF). Histology performed with trichromic staining revealed thick fibrotic streaks composed of type I collagen fibers, similar results being reported also for the adipose pads from obese human

LOX is a known target gene of HIF-1α, and in adipose tissue of ob/ob transgenic mouse, LOX is found in increased level compared to wild type [27]. LOX cross-links elastin and collagens in ECM and creates ECM-resistant bands of fibrosis. In adipose tissue of ob/ob mouse, these collagen bundle "streaks" are found outside the "crown-like" structures previously described [27, 39, 84]. The conclusion derived was that collagen synthesis and deposition could be anterior to the accumulation of macrophages surrounding the adipose cells because hypoxiainduced fibrotic program develops shortly after the high-fat diet is established [27]. So adipose tissue fibrosis is not necessarily induced by inflammation but could be rather an upstream

From a different point of view, like in other tissues, adipose tissue fibrosis develops as a result of a persistent inflammation and a failure of the normal tissue repair with *restitutio ad integrum*. Interestingly, fibrosis of adipose depots in obesity seems to display an otherwise intensity as the inflammation and hypoxia, those visceral seeming to be not only less fibrotic than those peripheral but also with a different distribution of collagen fibers, especially pericellular or intraparenchymatous [28]. As the visceral depots are more inflamed than the subcutaneous depots as we showed [88], this observation contradicts the accepted biological sequence that fibrosis develops as a result of an excessive and altered ECM synthesis and storage by resident cells activated in an inflammatory environment. This abnormal amount of fibrotic matrix in the subcutaneous adipose tissue could be explained if we keep in mind the histology of the host tissue where the adipose depots expand (the subcutaneous adipose tissue develops toward a much more dense tissue than the visceral one). In obese subjects fibrosis accumulates in pericellular areas—lining each adipose cell or a group of cells (interstitial fibrosis) and

Collagen phenotypes are also different, types I, III, and VI being present in pericellular position but only I and III form thick bundles appearing as interlobular septa surrounding more cells [28]. In visceral (omental) depots, the accumulation of fibers in pericellular position is associated with small adipocyte size and a lowest quantity of circulating triglycerides, proving that Being a compulsory condition for the expansion of any tissue, angiogenesis is a very limited process in normal adulthood (in endometrial cyclic physiology or wound healing), and the endothelial cells of the adult capillary network are in a relatively quiescent state.

In adipose tissue, angiogenesis is a very complex phenomenon regulated by a lot of molecules (hormones, cytokines, and growth factors) secreted by the stromo-vascular cells, including endothelial cells, and also by the adipocytes and preadipocytes [93, 94].

During adipose depot development, adipogenesis and vasculogenesis are temporally and spatially dependent, and in an adipose depot, the vascular network seems to act as a self-stop for the adipose expansion, since the inhibition of angiogenesis reduces the adipose tissue mass [95, 96].

In hypoxia induced by a high caloric intake, the vascular network does not progress uniformly between depots, due to the differences between the initial degree of vascularization and the rate/capacity of neovascularization during adipose tissue expansion. Hypoxia is more severe in mesenteric visceral depots than in subcutaneous [67], and at the same time, human visceral depots reveal a greater capillary density and angiogenic capacity than the subcutaneous adipose tissue [97, 98]. In their study using CD31+/CD34+ immunolabeling, Villaret and coworkers revealed that in obesity the capillary network is more developed and endothelial cell number is greater in visceral than in subcutaneous adipose depots, so increased hypoxia of visceral adipose tissue is not necessarily a consequence of capillary rarefaction [98]. The proangiogenic and pro-inflammatory phenotype of visceral adipose tissue could be related to endothelial cell senescence proved by an altered expression of some senescence markers such as IGFBP3, γH2AX, and SIRT1. They postulated at least two main causes for endothelial cell senescence in visceral adipose tissue: increased cell replication and oxidative stress [98]. Hyperplasia of obese visceral adipose tissue is responsible for an increased secretion of VEGF-A2 that stimulates endothelial cell proliferation.

The compensatory angiogenesis could prevent the metabolic disturbances induced by the hypoxia. It seems that not in all conditions the expansion of adipose tissue is associated with inflammation if an appropriate capillary bed is developed. If this condition is satisfied, the obese subjects are termed "metabolically healthy obese" because they may expand their adipocyte depots without inflammation consequences. This kind of expansion is associated with an enlargement of a given fat pad through recruitment of new adipocytes along with an adequate development of the vasculature, minimal associated fibrosis, and the lack of hypoxia and inflammation [83, 99].

The effects of hypoxia for obese humans have been recently disputed, the reactions triggered by the oxygen deprivation being a matter of severity, duration, and environment, since results between in vitro experiments, cell cultures under acute hypoxia, animal models, and human obese subjects are different. In recent studies, opposite data are reported by Goossens's research group. Their experiments revealed an increased pO2 in obese insulin-resistant subjects and a positive correlation between pO2 and gene expression for pro-inflammatory markers and an inverse association between pO2 and peripheral insulin sensitivity [18]. In another experiment, after exposing mice at normoxia and hypoxia for the same duration of time, the authors reported a decrease in adipocyte size, macrophages infiltration, and inflammatory cell genes in adipose tissue from hypoxic animals [100]. The same results were reported for obese men exposed for 10 nights to hypoxia consecutively followed by increased insulin sensitivity [101]. Moreover, it was presumed that the obstructive sleep apnea could be a protective mechanism to maintain energy homeostasis in obese subjects [102, 103]. Angiogenesis in hypoxic tissues is controlled by HIF-1α, so-called the master regulatory of cellular and tissue response to hypoxic stress. In adipose hypoxic tissue, HIF-1 induces angiogenesis by upregulating VEGF gene. VEGF-A is the only endothelial growth factor that stimulates ECM degradation, proliferation, migration, and tube formation of endothelial cells [104]. VEGF secretion is regulated also by insulin stimulation, growth factor, and cytokines, such as PDGF, EGF, TNFα, TGF-α, and IL-1β [99, 104].

As we showed in a previous study, VEGF immunohistochemical expression was higher in the adipose tissue of obese and obese-diabetic patients, especially in peritoneal depots. In normal weight subjects, both peripheral and central depots were VEGF negative [88].

It was demonstrated that an overexpression of VEGF in transgenic animals increased the vascularization and reversed the metabolic dysfunctions induced by a hypercaloric diet [80]. Recently, it had been claimed that the mechanism of VEGF promoting angiogenesis in adipose tissue is controlled by HIF-1β, while HIF-1α seems to regulate vascularization in BAT but not in WAT and additionally to promote WAT inflammation [105].

Adipokines, such as leptin and adiponectin, have also angiogenic properties that are stimulated in metabolically challenging conditions: leptin stimulates the angiogenic program upregulating VEGF expression, increases the vascular permeability by the formation of fenestrations in endothelial cells, and influences microvessel density [106]. For adiponectin, the results are conflicting: one supposed to be antiangiogenic because it inhibits endothelial cell migration and proliferation in vitro and neoangiogenesis in vivo [107] and others proangiogenic [99, 108].

## **6. Hypoxia: a trigger for BAT whitening and WAT browning**

adipose tissue [97, 98]. In their study using CD31+/CD34+ immunolabeling, Villaret and coworkers revealed that in obesity the capillary network is more developed and endothelial cell number is greater in visceral than in subcutaneous adipose depots, so increased hypoxia of visceral adipose tissue is not necessarily a consequence of capillary rarefaction [98]. The proangiogenic and pro-inflammatory phenotype of visceral adipose tissue could be related to endothelial cell senescence proved by an altered expression of some senescence markers such as IGFBP3, γH2AX, and SIRT1. They postulated at least two main causes for endothelial cell senescence in visceral adipose tissue: increased cell replication and oxidative stress [98]. Hyperplasia of obese visceral adipose tissue is responsible for an increased secretion of VEGF-

The compensatory angiogenesis could prevent the metabolic disturbances induced by the hypoxia. It seems that not in all conditions the expansion of adipose tissue is associated with inflammation if an appropriate capillary bed is developed. If this condition is satisfied, the obese subjects are termed "metabolically healthy obese" because they may expand their adipocyte depots without inflammation consequences. This kind of expansion is associated with an enlargement of a given fat pad through recruitment of new adipocytes along with an adequate development of the vasculature, minimal associated fibrosis, and the lack of hypoxia

The effects of hypoxia for obese humans have been recently disputed, the reactions triggered by the oxygen deprivation being a matter of severity, duration, and environment, since results between in vitro experiments, cell cultures under acute hypoxia, animal models, and human obese subjects are different. In recent studies, opposite data are reported by Goossens's research group. Their experiments revealed an increased pO2 in obese insulin-resistant subjects and a positive correlation between pO2 and gene expression for pro-inflammatory markers and an inverse association between pO2 and peripheral insulin sensitivity [18]. In another experiment, after exposing mice at normoxia and hypoxia for the same duration of time, the authors reported a decrease in adipocyte size, macrophages infiltration, and inflammatory cell genes in adipose tissue from hypoxic animals [100]. The same results were reported for obese men exposed for 10 nights to hypoxia consecutively followed by increased insulin sensitivity [101]. Moreover, it was presumed that the obstructive sleep apnea could be a protective mechanism to maintain energy homeostasis in obese subjects [102, 103]. Angiogenesis in hypoxic tissues is controlled by HIF-1α, so-called the master regulatory of cellular and tissue response to hypoxic stress. In adipose hypoxic tissue, HIF-1 induces angiogenesis by upregulating VEGF gene. VEGF-A is the only endothelial growth factor that stimulates ECM degradation, proliferation, migration, and tube formation of endothelial cells [104]. VEGF secretion is regulated also by insulin stimulation, growth factor, and cytokines, such as PDGF, EGF, TNF-

As we showed in a previous study, VEGF immunohistochemical expression was higher in the adipose tissue of obese and obese-diabetic patients, especially in peritoneal depots. In normal

It was demonstrated that an overexpression of VEGF in transgenic animals increased the vascularization and reversed the metabolic dysfunctions induced by a hypercaloric diet [80].

weight subjects, both peripheral and central depots were VEGF negative [88].

A2 that stimulates endothelial cell proliferation.

and inflammation [83, 99].

316 Hypoxia and Human Diseases

α, TGF-α, and IL-1β [99, 104].

BAT presence has been reported once for small rodents and newborns, but recently, evidence for metabolically active BAT in adult humans has been reported [109]. BAT activation under β-adrenergic signaling is important for heat generation through uncoupled oxidative phosphorylation as a result of activation of non-shivering thermogenesis [110]. For this function, an important blood supply is required to provide the amount of oxygen and nutrients, and therefore BAT is much more vascularized than WAT. In relation to energy balance, an inverse relationship is accepted between BMI and age, BAT being less active in older subjects and in obese [111, 112]. Recent experiments highlighted the importance of hypoxia in BAT dysfunction too, the lack of an adequate BAT vascularization being involved in the overall dysfunction of the adipose organ in obesity [20]. BAT activity reduces the development of metabolic syndrome, and its activation increases insulin sensitivity and contributes to glucose homeostasis [113]. Having a high oxidative capacity, it is presumed that BAT is a contributor to systemic metabolic homeostasis, and this function was impaired in obesity, as demonstrated in the experiment performed by Shimizu and coworkers [114]. They proved in mice that obesity affects the density of the capillary network in BAT much more than in WAT and induced hypoxia in this organ. This elegant experiment shows in a very credible manner that the transition of phenotype from brown to white adipocytes is induced by the diminution of vascularization, a reverse mechanism of BAT differentiation observed in fetal development when the appearance of multilocularity is anticipated by the branching of the capillary loops (personal unpublished data). This vascular dysfunction is followed by the "whitening of the brown fat" (diminished β-adrenergic signaling, the appearance of enlarged lipid droplets in the cells and loss of mitochondria) and can impact obesity and obesity-related diseases [115]. HIF-1α increased level and suppression of UCP1 gene were observed in hypoxic BAT [114]. The same influences that hypoxia exerts on gene expression in WAT have been reported also for BAT, such as increased expression of leptin, VEGF, IL-6, and GLUT1. Due to loss of mitochondria, high glucose uptake will be accompanied by the same switch to anaerobic glycolysis as in WAT. Besides triggering inflammation in macrophages, lactate is supposed to be involved also in "browning of the white fat," recent experimental data proving that in vitro lactate induces the expression of genes encoding UCP1 and proteins involved in mitochondrial oxidation in mice and human white adipocytes [116]. Same authors demonstrated that lactate also controls the browning process in vivo because it regulates Ucp1 expression in a PPAR-δdependent manner, the combination of lactate and PPAR-δ ligand rosiglitazone constituting a strong inducer of an increased expression of some mitochondrial oxidation markers in mice white adipose depots [116]. Based on this observation, one can assume that lactate could be responsible for the recruitment of "brite" cells. In light of these results, the recruitment and activation of BAT are regarded as a potential new target for strategies to counteract obesityinduced changes.

In conclusion, hypoxia could be regarded as the leading cause of adipose tissue remodeling rather than as a consequence of the functional changes in the adipose organ. Due to the interplay between hypoxia, inflammation, and angiogenesis, targeting hypoxia pathways could be a valuable therapeutic approach to reduce the clinical consequences of the metabolic syndrome.

## **Author details**

Ana Marina Andrei, Anca Berbecaru-Iovan, Felix Rareş Ioan Din-Anghel, Camelia Elena Stănciulescu, Sorin Berbecaru-Iovan, Ileana Monica Baniţă\* and Cătălina Gabriela Pisoschi

\*Address all correspondence to: monica.banita@yahoo.com

University of Medicine and Pharmacy of Craiova, Craiova, Dolj County, Romania

## **References**


oxidation in mice and human white adipocytes [116]. Same authors demonstrated that lactate also controls the browning process in vivo because it regulates Ucp1 expression in a PPAR-δdependent manner, the combination of lactate and PPAR-δ ligand rosiglitazone constituting a strong inducer of an increased expression of some mitochondrial oxidation markers in mice white adipose depots [116]. Based on this observation, one can assume that lactate could be responsible for the recruitment of "brite" cells. In light of these results, the recruitment and activation of BAT are regarded as a potential new target for strategies to counteract obesity-

In conclusion, hypoxia could be regarded as the leading cause of adipose tissue remodeling rather than as a consequence of the functional changes in the adipose organ. Due to the interplay between hypoxia, inflammation, and angiogenesis, targeting hypoxia pathways could be a valuable therapeutic approach to reduce the clinical consequences of the metabolic

and

Ana Marina Andrei, Anca Berbecaru-Iovan, Felix Rareş Ioan Din-Anghel, Camelia Elena Stănciulescu, Sorin Berbecaru-Iovan, Ileana Monica Baniţă\*

University of Medicine and Pharmacy of Craiova, Craiova, Dolj County, Romania

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326 Hypoxia and Human Diseases


**Provisional chapter**

## **Epigenetic Programming of Cardiovascular Disease by Perinatal Hypoxia and Fetal Growth Restriction Epigenetic Programming of Cardiovascular Disease by Perinatal Hypoxia and Fetal Growth Restriction**

Paola Casanello, Emilio A. Herrera and Bernardo J. Krause Bernardo J. Krause Additional information is available at the end of the chapter

Paola Casanello, Emilio A. Herrera and

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/66740

#### **Abstract**

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[116] Carrière A, Jeanson Y, Berger-Müller S, André M, Chenouard V, Arnaud E, Barreau C, Walther R, Galinier A, Wdziekonski B, Villageois P, Louche K, Collas P, Moro C, Dani C, Villarroya F, Casteilla L: Browning of white adipose cells by intermediate metabolites: an adaptive mechanism to alleviate redox pressure. Diabetes. 2014 ;63:3253–3265.

DOI:10.2337/db13-1885

328 Hypoxia and Human Diseases

Most of the worldwide deaths in patients with non-communicable diseases are due to cardiovascular and metabolic diseases, which are determined by a mix of environmental, genetic and epigenetic factors, and by their interactions. The aetiology of most cardiovascular diseases has been partially linked with *in utero* adverse conditions that may increase the risk of developing diseases later in life, known as Developmental Origins of Health and Disease (DOHaD). Perinatal hypoxia can program the fetal and postnatal developmental patterns, resulting in permanent modifications of cells, organs and systems function. In spite of the vast evidence obtained from human and animal studies linking development under adverse intrauterine conditions with increased cardiovascular risk, still few is known about the specific effects of intrauterine oxygen deficiency and the related pathogenic mechanisms. Currently, the most accepted processes that program cellular function are epigenetic mechanisms which determine gene expression in a cell-specific fashion. In this chapter we will review the current literature regarding the perinatal exposure to chronic hypoxia and Fetal Growth Restriction (FGR) in humans and animals and how this impinges the cardiovascular physiology through epigenetic, biochemical, morphologic and pathophysiologic modifications that translate into diseases blasting at postnatal life.

**Keywords:** hypoxia, programming, vascular function, oxidative stress, epigenetics, chronic diseases

## **1. Introduction**

The worldwide prevalence of cardiovascular diseases (CVDs) and metabolic syndrome ranges between 20 and 40%. These figures are likely to rise over the next decades [1, 2]. Genetic changes associated with the traits of the metabolic syndrome and cardiovascular diseases are

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

able to explain a small proportion of cases [3], suggesting the presence of other contributory factors in these conditions. Epidemiologic studies in the late 1980s in the UK revealed a strong correlation with perinatal and fetal growth patterns. Fetal growth restriction (FGR) is thus associated with an increased risk of developing adult cardiometabolic diseases [4]. Multiple reports from across the world have documented the association between intrauterine growth mediators in early life with lifelong health. These are now recognized to be important risks in the development of non-communicable diseases in adult life. This concept so-called "Fetal Programming" has evolved into "Developmental Origins of Health and Disease" (DOHaD), which we refer as Intrauterine Programming (IUP) [5] for the purpose of this chapter. The present efforts in this field are focused on unveiling the physiological and molecular mechanisms, which drive IUP, and exploring opportunities to prevent or revert the long-term consequences. The physiologic and biochemical changes that explain IUP relate to the timing and stage of development when the insult takes place; the earlier in development, the stronger the long-term effects [5]. Conversely, the long-term consequences of IUP and reproducibility of the related phenotypes suggest that epigenetic mechanisms may underlay the altered "cell programming" [6].

## **2. Fetal growth restriction**

Fetal growth restriction (FGR) is clinically defined by a fetal weight below the 10th percentile of normal for gestational age, but in a generic manner, FGR is a condition in which the potential growth of the fetus is negatively influenced by environmental and maternal factors [7]. The short-term consequences of FGR are LBW and the corresponding phenotype, which is associated with increased perinatal morbidity and mortality [8]. The long-term effects include a two- to threefold increase in the risk of developing cardiovascular disease (hypertension and coronary heart disease) in adult life [9]. The higher CVD risk in adults resulting from FGR can be traced back to a reduced arterial compliance in pre-pubertal subjects [10] and a decreased peripheral endothelial-dependent vascular relaxation at birth [11]. Moreover, studies in human placentae show that FGR-related endothelial dysfunction can also be detected in chorionic and umbilical arteries [12, 13]. Notably, we have recently demonstrated the presence of functional and epigenetic markers of endothelial dysfunction in systemic and umbilical arteries from FGR guinea pigs. The presence of these comparable markers suggests that umbilical artery endothelial cells (ECs) may be useful to explore the endothelial function of the fetus. The etiology of FGR in humans is not fully understood; however, there are known maternal risk factors such as living at high altitude, malnutrition, smoking, stress, and vascular dysfunction [14] which induce placental dysfunction and consequently fetal growth restriction. Presently, oxygen, glucose, free radicals, amino acids, and hormones have been shown to play an important role in modulating fetal growth and development. These factors are dynamically regulated throughout gestation [15]. In the earlier stages, limitations in oxygen supply promote trophoblast proliferation; however, persistence in a hypoxic environment as occurs in FGR harms trophoblast invasion and the transformation of spiral arteries leading to a vascular dysfunction of the placenta and impaired fetal growth. Thus, chronic hypoxia and oxidative stress have an important role in the placental dysfunction observed in FGR [15]. Several studies on humans confirm the presence of molecular markers of oxidative stress in the FGR placentae, the fetus, and the mother [16–19]. Impaired placental vascular function has also been proposed to play a role in FGR, conditioned by augmented synthesis and response to vasoconstrictors [20] and limited action of vasodilators [13], as well as by an increased inhibition of endothelial-dependent relaxation mediated by prooxidants [21].

Appropriate maternal nutrient supply to the fetus is key for its development. Several approaches limiting maternal supply (i.e., diet restriction) and placental nutrient transfer have been used to alter the normal fetal growth rate and development. In order to address this issue, various animal models (sheep, rat, rabbit, and guinea pig) have been developed, where placental dysfunction is induced by a reduction in uterine blood flow [22, 23]. We have recently developed a novel model of FGR in guinea pigs, by a progressive bilateral occlusion of the uterine arteries during the second half of gestation that gradually alters placental vascular resistance [24]. Several aspects suggest that this model is relevant to human clinical significance. For instance, guinea pigs present a decreased fetal abdominal growth and impaired placental blood flow adaptation during gestation, with a preserved brain blood flow and development, translating into an asymmetric FGR. Additionally, higher resistance to blood flow in the umbilical arteries can be observed. These are relevant clinical markers of FGR. However, most of mammalian models that develop placental insufficiency present a mixed effect of undernutrition, hypoxia, and oxidative stress [22]. Therefore, complementary models on chick embryos have been used to isolate the unique fetal effects of hypoxia during development from maternal responses [22]. Interestingly, the follow-up of the chickens gestated under hypoxia has shown important insights into the pathophysiological mechanisms that impair the cardiovascular function. For instance, Tintu et al. showed that developmental hypoxia induces cardiomyopathy associated with left ventricular dilatation, reduced ventricular wall mass, and increased apoptosis [25]. These responses were coupled with pump dysfunction, decreased ejection fractions, and diastolic dysfunction, which persisted in adulthood. Further, Salinas et al. showed marked cardiovascular morphostructural changes in high-altitude chicks, which were reverted either by incubation at low altitude or by oxygen supplementation [26]. Notably, Herrera et al. followed up these chicks to adulthood describing cardiac impairment in the capacity to response to pressor challenges [27]. In addition to the cardiovascular system, several organs/functions are affected during developmental hypoxia such as central nervous system, lung, and systemic metabolism. As well as in mammalian physiology, it seems that oxidative stress might be key in establishing the impairments induced by developmental hypoxia [28].

#### **2.1. Hypoxia and oxidative stress in FGR**

able to explain a small proportion of cases [3], suggesting the presence of other contributory factors in these conditions. Epidemiologic studies in the late 1980s in the UK revealed a strong correlation with perinatal and fetal growth patterns. Fetal growth restriction (FGR) is thus associated with an increased risk of developing adult cardiometabolic diseases [4]. Multiple reports from across the world have documented the association between intrauterine growth mediators in early life with lifelong health. These are now recognized to be important risks in the development of non-communicable diseases in adult life. This concept so-called "Fetal Programming" has evolved into "Developmental Origins of Health and Disease" (DOHaD), which we refer as Intrauterine Programming (IUP) [5] for the purpose of this chapter. The present efforts in this field are focused on unveiling the physiological and molecular mechanisms, which drive IUP, and exploring opportunities to prevent or revert the long-term consequences. The physiologic and biochemical changes that explain IUP relate to the timing and stage of development when the insult takes place; the earlier in development, the stronger the long-term effects [5]. Conversely, the long-term consequences of IUP and reproducibility of the related phenotypes suggest that epigenetic mechanisms may underlay the

Fetal growth restriction (FGR) is clinically defined by a fetal weight below the 10th percentile of normal for gestational age, but in a generic manner, FGR is a condition in which the potential growth of the fetus is negatively influenced by environmental and maternal factors [7]. The short-term consequences of FGR are LBW and the corresponding phenotype, which is associated with increased perinatal morbidity and mortality [8]. The long-term effects include a two- to threefold increase in the risk of developing cardiovascular disease (hypertension and coronary heart disease) in adult life [9]. The higher CVD risk in adults resulting from FGR can be traced back to a reduced arterial compliance in pre-pubertal subjects [10] and a decreased peripheral endothelial-dependent vascular relaxation at birth [11]. Moreover, studies in human placentae show that FGR-related endothelial dysfunction can also be detected in chorionic and umbilical arteries [12, 13]. Notably, we have recently demonstrated the presence of functional and epigenetic markers of endothelial dysfunction in systemic and umbilical arteries from FGR guinea pigs. The presence of these comparable markers suggests that umbilical artery endothelial cells (ECs) may be useful to explore the endothelial function of the fetus. The etiology of FGR in humans is not fully understood; however, there are known maternal risk factors such as living at high altitude, malnutrition, smoking, stress, and vascular dysfunction [14] which induce placental dysfunction and consequently fetal growth restriction. Presently, oxygen, glucose, free radicals, amino acids, and hormones have been shown to play an important role in modulating fetal growth and development. These factors are dynamically regulated throughout gestation [15]. In the earlier stages, limitations in oxygen supply promote trophoblast proliferation; however, persistence in a hypoxic environment as occurs in FGR harms trophoblast invasion and the transformation of spiral arteries leading to a vascular dysfunction of the placenta and impaired fetal growth. Thus, chronic hypoxia and oxidative stress have an important role in the placental

altered "cell programming" [6].

330 Hypoxia and Human Diseases

**2. Fetal growth restriction**

Hypoxia is defined as a limited oxygen (O<sup>2</sup> ) supply relative to the physiological demands of a tissue, organ, or organism. This is a restrictive condition frequently seen in the hypobaric environment (hypoxia of high altitude) or by a diminished oxygen delivery. At lowlands, hypoxia is a restrictive condition often faced during fetal life, either by maternal, umbilicalplacental, or fetal conditions. Placental insufficiency leads to fetal growth restriction due to a chronic decrease in fetoplacental perfusion. This situation affects simultaneously O<sup>2</sup> and nutrient supply to the fetus [29], overlapping conditions that become difficult to isolate in order to assess the specific effect of O<sup>2</sup> deficiency in determining vascular impairment. Using avian models of FGR has served to establish that chronic hypoxia, independent of nutrition, plays a crucial role in vascular programming [30, 31]. Studies of vascular function during fetal life show remarkable similarities between the effect of hypoxia in chick embryos and placental insufficiency in mammals [26, 28]; they have also served to assess the long-term consequences [27]. In both cases (chick embryos and mammalian fetuses), the presence of endothelial dysfunction and vascular remodeling is observed mainly in peripheral arteries. The mechanism by which hypoxia induces cell damage in either case is the result of an increased generation of reactive oxygen species (ROS) due to an incomplete reduction of oxygen [15, 32].

The imbalance between endogenous antioxidant defenses and reactive oxygen species, where ROS overwhelms the antioxidant capacity, has been termed "oxidative stress" [33]. ROS includes a wide variety of highly reactive molecules, such as superoxide anion (∙O2 - ), hydrogen peroxide (H2 O2 ), ∙NO, peroxynitrite (ONOO-), organic hydroperoxide (ROOH), hypochlorous acid (HOCl), and hydroxyl (∙OH), alkoxy (RO∙), and peroxy radicals (ROO∙) [34]. Superoxide is the main ROS acting at the vascular level; it derives from the enzymatic activity of NOX (NADPH oxidases), XOR (xanthine oxidases), mitochondrial complexes I and III, uncoupled eNOS, and iNOS. In the case of NOS, ROS generation can occur because of reduced L-arginine (substrate) or BH4 (cofactor) availability [33], uncoupling eNOS enzymes. Consequently, NOS-derived ∙O2 rapidly reacts with NO generating ONOO-, which reduces NO levels and modifies the structure of proteins, lipids, and DNA, causing endothelial dysfunction. Thus, increased oxidative stress exerts a negative effect on eNOS activity and NO bioavailability at multiple levels [33].

In FGR, compelling data show that oxidative stress in parallel to chronic hypoxia contributes to vascular dysfunction in the mother, placenta, and fetus [14]. In fact, short-term hypoxia induces eNOS expression and activation in human umbilical artery endothelial cells (HUAECs) [35], while in FGR HUAEC, there is reduced eNOS activation [13]. Conversely, FGR subjects present at birth increased levels of lipid peroxidation and decreased the activity of antioxidant enzymes and circulating mediators [36]. Additionally, markers of oxidative stress have been positively associated with increased umbilical artery pulsatility index, particularly in pregnancies affected by FGR [37]. We recently addressed the role of oxidative stress in FGR by treating pregnant guinea pigs with N-acetyl cysteine, a glutathione precursor, during the second half of gestation. Our results show that maternal treatment with NAC restores fetal growth by increasing placental efficiency and reverses endothelial dysfunction in FGR guinea pigs [38]. Similarly, *in ovo* melatonin administration to chronic hypoxic chick embryos reduces the levels of oxidative stress markers (i.e., lipid peroxidation and protein nitration), by increasing the expression of glutathione peroxidase (GPx), an antioxidant enzyme [28]. This effect is associated with improved endothelial function and reversal of fetal hypoxia-induced vascular remodeling; however, melatonin does not prevent FGR. Even more, in a chronic hypoxic sheep model, melatonin decreased maternal oxidative stress but simultaneously enhanced fetal growth restriction [39]. In summary, these data suggest that hypoxia and oxidative stress participate in the genesis of FGR-induced vascular dysfunction. However, there is a need for further studies addressing the precise molecular mechanisms and effective treatments for hypoxic FGR and IUP.

At a molecular level, transcription factors nuclear factor kappa B (NFκB) [34] and nuclear factor E2-related factor 2 (Nrf2) implicated in oxidative stress [34, 40] participate in promoting and reducing cellular oxidative stress, respectively. Interestingly, Nrf2 presents the suggested properties of an oxidative stress sensor. Nrf2 is normally bound to Keap1, which targets the complex to proteasome degradation; however, a prooxidant milieu induces the oxidation of two cysteine residues in Keap1 and the release of Nrf2 that subsequently translocate to the nucleus [34]. The antioxidant response triggered by Nrf2 includes the expression of NAD(P)H dehydrogenase quinone 1 (NQO1), heme-oxygenase (HO), and other antioxidant enzymes [40]. Studies show that Nrf2-induced expression of NQO1 and HO-1 improves endothelial dysfunction increasing eNOS efficiency. However, there is no information addressing whether changes in the expression of genes involved in the antioxidant defense are present in early stages of endothelial dysfunction in FGR and whether they can be modulated during gestation.

## **3. Epigenetics and endothelial programming in FGR**

Alteration in fetal development and IUP results in permanent changes in the physiological responses to different stressors across the life course. Undoubtedly, this represents a potential "handicap" for long-term health. Growing evidence in humans from individuals with altered fetal growth, and from animal models associated with the development of later cardiometabolic alterations, confirms the presence of epigenetic markers in different cell types [41]. Epigenetics can be considered as "chromosome-based mechanisms that modify the phenotypic plasticity of a cell or organism" [6]. Development itself is controlled by epigenetic mechanisms, which regulate cell differentiation and record environmental signals under physiologic [42] and/or pathologic conditions [43]. These epigenetic mechanisms include DNA methylation, a plethora of histone posttranslational modifications (PTM) (acetylation, methylation, phosphorylation, and others), ATP-dependent chromatin modifications, and noncoding RNAs [44].

#### **3.1. DNA methylation**

nutrient supply to the fetus [29], overlapping conditions that become difficult to isolate in

avian models of FGR has served to establish that chronic hypoxia, independent of nutrition, plays a crucial role in vascular programming [30, 31]. Studies of vascular function during fetal life show remarkable similarities between the effect of hypoxia in chick embryos and placental insufficiency in mammals [26, 28]; they have also served to assess the long-term consequences [27]. In both cases (chick embryos and mammalian fetuses), the presence of endothelial dysfunction and vascular remodeling is observed mainly in peripheral arteries. The mechanism by which hypoxia induces cell damage in either case is the result of an increased generation of reactive oxygen species (ROS) due to an incomplete reduction of

The imbalance between endogenous antioxidant defenses and reactive oxygen species, where ROS overwhelms the antioxidant capacity, has been termed "oxidative stress" [33]. ROS includes a wide variety of highly reactive molecules, such as superoxide anion (∙O2

hypochlorous acid (HOCl), and hydroxyl (∙OH), alkoxy (RO∙), and peroxy radicals (ROO∙) [34]. Superoxide is the main ROS acting at the vascular level; it derives from the enzymatic activity of NOX (NADPH oxidases), XOR (xanthine oxidases), mitochondrial complexes I and III, uncoupled eNOS, and iNOS. In the case of NOS, ROS generation can occur because of reduced L-arginine (substrate) or BH4 (cofactor) availability [33], uncoupling eNOS enzymes.

NO levels and modifies the structure of proteins, lipids, and DNA, causing endothelial dysfunction. Thus, increased oxidative stress exerts a negative effect on eNOS activity and NO

In FGR, compelling data show that oxidative stress in parallel to chronic hypoxia contributes to vascular dysfunction in the mother, placenta, and fetus [14]. In fact, short-term hypoxia induces eNOS expression and activation in human umbilical artery endothelial cells (HUAECs) [35], while in FGR HUAEC, there is reduced eNOS activation [13]. Conversely, FGR subjects present at birth increased levels of lipid peroxidation and decreased the activity of antioxidant enzymes and circulating mediators [36]. Additionally, markers of oxidative stress have been positively associated with increased umbilical artery pulsatility index, particularly in pregnancies affected by FGR [37]. We recently addressed the role of oxidative stress in FGR by treating pregnant guinea pigs with N-acetyl cysteine, a glutathione precursor, during the second half of gestation. Our results show that maternal treatment with NAC restores fetal growth by increasing placental efficiency and reverses endothelial dysfunction in FGR guinea pigs [38]. Similarly, *in ovo* melatonin administration to chronic hypoxic chick embryos reduces the levels of oxidative stress markers (i.e., lipid peroxidation and protein nitration), by increasing the expression of glutathione peroxidase (GPx), an antioxidant enzyme [28]. This effect is associated with improved endothelial function and reversal of fetal hypoxia-induced vascular remodeling; however, melatonin does not prevent FGR. Even more, in a chronic hypoxic sheep model, melatonin decreased maternal oxidative stress but simultaneously enhanced fetal growth restriction [39]. In summary, these data suggest that hypoxia and oxidative stress participate in the genesis of FGR-induced vascular dysfunction.

deficiency in determining vascular impairment. Using

), ∙NO, peroxynitrite (ONOO-), organic hydroperoxide (ROOH),

rapidly reacts with NO generating ONOO-, which reduces


order to assess the specific effect of O<sup>2</sup>

oxygen [15, 32].

332 Hypoxia and Human Diseases

hydrogen peroxide (H2

Consequently, NOS-derived ∙O2

bioavailability at multiple levels [33].

O2


In higher animals, DNA is methylated via an enzymatic activity that transfers a methyl group to the 5' position of cytosine ring on CpG dinucleotide generating 5-methyl-cytosine, a reaction catalyzed by two different families of DNA methyltransferases (DNMTs), named DNMT1 and DNMT3 (DNMT3a and DNMT3b) encoded by three different genes [45]. The role of DNMT1 is to preserve the DNA methylation pattern after DNA replication during mitotic cell division as well as after fertilization [46], a process guided by the presence of hemi-methylated CpGs, which are recognized by DNMT1 in dsDNA [47]. Additionally, DNMT3a and DNMT3b catalyze *de novo* methylation allowing the establishment of new DNA methylation patterns during gametogenesis, embryonic development, and cell differentiation [46, 48]. Interestingly, the genome of different cell types from a single subject presents a high DNA methylation density; however, larger differences occur in the promoter regions of genes representing less than 5% of the total genomic DNA methylation [49]. Nonetheless, these subtle differences are likely controlling most cell-specific proteins expression at the whole organism level [50]. It is commonly accepted that DNA methylation represents a hallmark of reduced gene expression and long-term gene silencing [51, 52]; however, it is worth noting that growing evidence suggests a more dynamic role for this mechanism in the regulation of gene expression [51].

#### **3.2. Histone posttranslational modifications**

The protein structural unit of the chromosomes, the nucleosome, is formed by two copies of four histones proteins named H2A, H2B, H3, and H4. Additionally, these proteins present a globular domain to interact with other histones, and a flexible tail that participates actively in the interaction with DNA. Unlike DNA methylation, histone posttranslational modifications (PTMs) are more dynamic and do not give a straight idea regarding gene silencing or activation [52]. Moreover, histone PTMs are closely related with the context in which they take place and the presence of additional PTMs, suggesting the existence of a "histone code." Up to date, more than 50 enzymes that catalyze diverse histone modifications have been identified and classified according to the reaction they carry out [53]. **Histone acetylation** occurs in lysine residues (K) and involves the transference of an acetyl group from acetyl-CoA. In mammals, this reaction is carried out by three families of histone acetyl-transferases (HAT) named GNAT, MYST, and CBP/p300 [54]. This modification is considered an activator of gene expression, due to the fact that it stabilizes the positive charge of the lysine in the histone, reducing its affinity for DNA, avoiding the formation of highly compacted chromatin. The best characterized acetylations are those that take place in lysine 9 (K9), K14, K18, and K56 in histone 3 (H3) and K5, K8, K13, and K16 in H4 [55]. At least four types of **histone deacetylases** (HDAC I, II, III y IV) have been identified, which catalyze the reverse reaction of that done by the **histone acetyl-transferase**. This enzymatic reaction is related to gene silencing, progression of cell cycle, differentiation, and the response induced by DNA damage [56]. HDAC activity can be induced in response to DNA methylation, once repressor proteins that bind CpGs (MCP) are recruited. The latter have a site of interaction with several HDACs, suggesting that gene silencing could result from a combined action of DNA and histone modifications [51, 57].

#### **3.3. Noncoding RNAs**

The idea that noncoding RNAs could regulate the expression of genes was first proposed in the early 1960s [58], with a substantial progress in this field during the last decade. Less than 5% of the transcribed RNA encodes proteins; thus, most of them correspond to noncoding RNAs (ncRNAs) involved mainly in the regulation of gene expression [59, 60]. "Long" ncRNA (lncRNA), small interfering RNA (siRNA), and micro-RNA (miRNA) are the main regulatory ncRNAs. The lncRNA regulates the expression of a specific gene complementary either through chromatin remodeling, alternative mRNA processing (splicing), or siRNA generation [59]. Conversely, siRNA and miRNAs are interference RNA-based epigenetic mechanisms, which silence genes via noncoding RNAs of ~21 bp. To date, more than a thousand noncoding miRNAs have been reported. These are transcribed by the RNA polymerase II and encoded by specific genes (~70%) or, in lesser amounts, within the intronic regions of gene encoding proteins. Micro-RNAs are transcribed as pre-miRNA and initially processed in the nucleus by the DROSHA-DGCR8 complex. Subsequently, they are exported to the cytoplasm for miRNA maturation by the action of the complex formed by the DICER1 protein and RNase IIIa IIIb [61]. This processing leads to a single-strand RNA, which is incorporated into the "proteininduced silencing complex miRNA" (miRISC), which binds to a complementary region in a target mRNA. It has been proposed that a full complementarity between the miRNA and mRNA leads to degradation of the mRNA, while partial complementarity suppresses translation [62]. Notably, a single miRNA can regulate the expression of multiple mRNAs often associated signaling pathways or metabolic processes, while several miRNAs may converge in the regulation of a single mRNA constituting a complex mechanism for gene expression regulation [61, 62].

#### **3.4. Epigenetics in endothelial physiology**

however, larger differences occur in the promoter regions of genes representing less than 5% of the total genomic DNA methylation [49]. Nonetheless, these subtle differences are likely controlling most cell-specific proteins expression at the whole organism level [50]. It is commonly accepted that DNA methylation represents a hallmark of reduced gene expression and long-term gene silencing [51, 52]; however, it is worth noting that growing evidence suggests

The protein structural unit of the chromosomes, the nucleosome, is formed by two copies of four histones proteins named H2A, H2B, H3, and H4. Additionally, these proteins present a globular domain to interact with other histones, and a flexible tail that participates actively in the interaction with DNA. Unlike DNA methylation, histone posttranslational modifications (PTMs) are more dynamic and do not give a straight idea regarding gene silencing or activation [52]. Moreover, histone PTMs are closely related with the context in which they take place and the presence of additional PTMs, suggesting the existence of a "histone code." Up to date, more than 50 enzymes that catalyze diverse histone modifications have been identified and classified according to the reaction they carry out [53]. **Histone acetylation** occurs in lysine residues (K) and involves the transference of an acetyl group from acetyl-CoA. In mammals, this reaction is carried out by three families of histone acetyl-transferases (HAT) named GNAT, MYST, and CBP/p300 [54]. This modification is considered an activator of gene expression, due to the fact that it stabilizes the positive charge of the lysine in the histone, reducing its affinity for DNA, avoiding the formation of highly compacted chromatin. The best characterized acetylations are those that take place in lysine 9 (K9), K14, K18, and K56 in histone 3 (H3) and K5, K8, K13, and K16 in H4 [55]. At least four types of **histone deacetylases** (HDAC I, II, III y IV) have been identified, which catalyze the reverse reaction of that done by the **histone acetyl-transferase**. This enzymatic reaction is related to gene silencing, progression of cell cycle, differentiation, and the response induced by DNA damage [56]. HDAC activity can be induced in response to DNA methylation, once repressor proteins that bind CpGs (MCP) are recruited. The latter have a site of interaction with several HDACs, suggesting that gene silencing could result from a combined action of DNA and

The idea that noncoding RNAs could regulate the expression of genes was first proposed in the early 1960s [58], with a substantial progress in this field during the last decade. Less than 5% of the transcribed RNA encodes proteins; thus, most of them correspond to noncoding RNAs (ncRNAs) involved mainly in the regulation of gene expression [59, 60]. "Long" ncRNA (lncRNA), small interfering RNA (siRNA), and micro-RNA (miRNA) are the main regulatory ncRNAs. The lncRNA regulates the expression of a specific gene complementary either through chromatin remodeling, alternative mRNA processing (splicing), or siRNA generation [59]. Conversely, siRNA and miRNAs are interference RNA-based epigenetic mechanisms,

a more dynamic role for this mechanism in the regulation of gene expression [51].

**3.2. Histone posttranslational modifications**

334 Hypoxia and Human Diseases

histone modifications [51, 57].

**3.3. Noncoding RNAs**

Vascular development and endothelial differentiation and function require a fine epigenetic tuning, suggesting that epigenetic mechanisms play a key role in the IUP-associated vascular dysfunction [6]. The first stages of vascular development are determined by genetic factors, while the next processes that take place (i.e., blood vessel structure, identity, and function) are influenced/determined by hemodynamic factors, ROS, and oxygen levels [63, 64]. Considering that the effect of endothelial-specific transcription factors such as KLF2 and HoxA9 does not explain the protein expression levels present in this cell type [65], an "endothelial epigenetic code" regulating the expression of crucial genes has been suggested [52, 66]. Growing evidence shows that DNA methylation, histone PTM, and miRNAs [67] play an important role in the embryonic origins of endothelial cells (EC), as well as their homeostasis during life. The epigenetic regulation of *NOS3* gene has been extensively studied in EC and non-EC, showing that ECs have a distinctive pattern of DNA methylation and histone PTMs [65]. Conversely, the decreased expression of eNOS in HUVEC exposed to acute hypoxia is controlled by the overexpression of a natural cis-antisense noncoding RNA called sONE [68] and changes in histone PTM which occur specifically at the promoter of eNOS [69]. Similarly, in the endothelium, hypoxia and oxidative stress regulate the expression of several miRNAs that modify the expression of eNOS and other enzymes related to its short- and long-term function [70]. In support of this notion, we have recently demonstrated that eNOS-induced NO enhances arginase-2 expression by epigenetic modifications in the histones residing at *ARG2* gene promoter [71]. In summary, these data show that EC-specific eNOS expression, as well as other genes related with the L-arginine/NO pathway, is effectively controlled by multiple epigenetic mechanisms which are strongly influenced by hypoxia.

#### **3.5. Epigenetics and endothelial dysfunction**

Diverse studies show that epigenetic mechanisms can increase the risk or directly participate in the development of vascular diseases. In humans, ECs from atherosclerotic plaques have decreased levels of estrogen receptor-β along with increased DNA methylation at the promoter region of this gene, compared with nonatherosclerotic plaques cells [72]. Further studies in mice [73] and swine [74] have demonstrated that disturbed flow induces genome-wide changes in the DNA methylation of EC in vivo and in vitro, an effect that would be dependent on DNMT1 expression and that mainly affects genes related to oxidative stress. Conversely, abrogation of *Nos3* promoter DNA methylation increases basal eNOS mRNA expression in vitro and protects against hind limb ischemia injury in vivo [75]. Similarly, growing evidence suggests a central role of miRNAs in the genesis of cardiometabolic dysfunction, also proposed as sensitive molecular markers of vascular disease [76]. In fact, we recently reported that circulating levels of miRNA Let-7 and miR-126 are associated with different traits of cardiometabolic dysfunction in children as well as have a predictive value for metabolic syndrome in these subjects [77]. Comparable results in adults with type 2 diabetes have been reported, where increased levels of miR-21 and decreased levels of miR-126 correlated with cardiovascular and inflammatory complications [78].

In the context of IUP of endothelial dysfunction in rats, it has been shown that brief exposure to hypoxia at the end of gestation induces pulmonary vascular dysfunction in the newborn, which associates with increased eNOS expression accompanied by decreased DNA methylation in *Nos3* gene promoter [79]. Similarly, we reported a few years ago for the first time the presence of an altered epigenetic programming of eNOS expression in EC derived from human umbilical arteries of FGR patients [12]. Notably, the altered expression of eNOS was reversed by silencing DNMT1 expression in FGR EC, which restored the DNA methylation pattern at *NOS3* promoter, as well as the regulation of eNOS expression induced by hypoxia [12]. Furthermore, using a guinea pig model of FGR, we compared the eNOS expression and DNA methylation pattern at *Nos3* promoter to clarify whether these epigenetic changes occurring in umbilical EC would represent changes that take place in systemic arteries (i.e., aorta and femoral) [38]. We found comparable changes in eNOS expression which were associated with specific changes in DNA methylation of *Nos3* promoter in the different FGR EC studied, suggesting the presence of a common programming of endothelial dysfunction in the umbilical-placental and systemic circulation. Of note, maternal treatment with an antioxidant (NAC) prevented this epigenetic programming, restoring the eNOS mRNA levels to values observed in control fetuses. Similar studies have shown the beneficial effects of antioxidants during development, showing clear evidences that ROS have causal roles in cardiovascular programming [32]. In addition, several authors have shown that ROS may induce important epigenetic modifications that determined cardiovascular dysfunction later in life. Hypoxia and oxidative stress have been shown to be present in several conditions during pregnancy, such as preeclampsia, placental insufficiency, and high-altitude pregnancies [80]. In addition, assisted reproductive technologies induce hypoxic conditions at very early stages of development. All of the above studies have suggested epigenetic modifications of the eNOS gene [80, 81]. Conversely, the response to hypoxia and oxidative stress is primarily mediated by the hypoxia-inducible transcription factor (HIF), which is regulated by the oxygen-sensing HIF hydroxylases, members of the 2-oxoglutarate (2OG)-dependent oxygenase family. Similarly, there are demethylases from the same family modulating methylation levels. Both systems, a transcription factor and an epigenetic regulator, are being regulated by hypoxia [82]. Further, HIF-1α has been suggested as an epigenetic modulator determining chromatin remodeling of hypoxia-responsive elements (HREs) sites [83]. Interestingly, in this report, a marked hyperacetylation of histones H3 and H4 was observed in the placental growth factor (Plgf) intron in hypoxic conditions. Further studies are needed to determine the interaction of transcription factors and epigenetic regulation, which might be an efficient way of controlling gene expression.

plaques have decreased levels of estrogen receptor-β along with increased DNA methylation at the promoter region of this gene, compared with nonatherosclerotic plaques cells [72]. Further studies in mice [73] and swine [74] have demonstrated that disturbed flow induces genome-wide changes in the DNA methylation of EC in vivo and in vitro, an effect that would be dependent on DNMT1 expression and that mainly affects genes related to oxidative stress. Conversely, abrogation of *Nos3* promoter DNA methylation increases basal eNOS mRNA expression in vitro and protects against hind limb ischemia injury in vivo [75]. Similarly, growing evidence suggests a central role of miRNAs in the genesis of cardiometabolic dysfunction, also proposed as sensitive molecular markers of vascular disease [76]. In fact, we recently reported that circulating levels of miRNA Let-7 and miR-126 are associated with different traits of cardiometabolic dysfunction in children as well as have a predictive value for metabolic syndrome in these subjects [77]. Comparable results in adults with type 2 diabetes have been reported, where increased levels of miR-21 and decreased levels of miR-126 correlated with cardiovascular and

In the context of IUP of endothelial dysfunction in rats, it has been shown that brief exposure to hypoxia at the end of gestation induces pulmonary vascular dysfunction in the newborn, which associates with increased eNOS expression accompanied by decreased DNA methylation in *Nos3* gene promoter [79]. Similarly, we reported a few years ago for the first time the presence of an altered epigenetic programming of eNOS expression in EC derived from human umbilical arteries of FGR patients [12]. Notably, the altered expression of eNOS was reversed by silencing DNMT1 expression in FGR EC, which restored the DNA methylation pattern at *NOS3* promoter, as well as the regulation of eNOS expression induced by hypoxia [12]. Furthermore, using a guinea pig model of FGR, we compared the eNOS expression and DNA methylation pattern at *Nos3* promoter to clarify whether these epigenetic changes occurring in umbilical EC would represent changes that take place in systemic arteries (i.e., aorta and femoral) [38]. We found comparable changes in eNOS expression which were associated with specific changes in DNA methylation of *Nos3* promoter in the different FGR EC studied, suggesting the presence of a common programming of endothelial dysfunction in the umbilical-placental and systemic circulation. Of note, maternal treatment with an antioxidant (NAC) prevented this epigenetic programming, restoring the eNOS mRNA levels to values observed in control fetuses. Similar studies have shown the beneficial effects of antioxidants during development, showing clear evidences that ROS have causal roles in cardiovascular programming [32]. In addition, several authors have shown that ROS may induce important epigenetic modifications that determined cardiovascular dysfunction later in life. Hypoxia and oxidative stress have been shown to be present in several conditions during pregnancy, such as preeclampsia, placental insufficiency, and high-altitude pregnancies [80]. In addition, assisted reproductive technologies induce hypoxic conditions at very early stages of development. All of the above studies have suggested epigenetic modifications of the eNOS gene [80, 81]. Conversely, the response to hypoxia and oxidative stress is primarily mediated by the hypoxia-inducible transcription factor (HIF), which is regulated by the oxygen-sensing HIF hydroxylases, members of the 2-oxoglutarate (2OG)-dependent oxygen-

inflammatory complications [78].

336 Hypoxia and Human Diseases

Another epigenetic regulatory mechanism is the miRNAs in the IUP. Present evidence suggests that miRNAs could be transferred across the placenta [84] with important consequences on fetal and maternal physiology. In humans, circulating levels of miR-21 during gestation in the mother positively correlate with evidence of fetal hypoxia [85] and evidence from in vitro studies show the participation of miR-21 in the FGR placental vascular dysfunction [86, 87]. By contrast, placental miR-126 levels negatively correlate with the FGR severity [88]. Studies in umbilical endothelium from swine fetuses have shown that the expression of miRNA that targets eNOS and VEGF pathways can be modulated by maternal supplementation with an L-arginine precursor [89]. Similarly, undernutrition decreases and programs at long term the expression of an anti-remodeling miRNA and this effect is prevented by the *in utero* inhibition of corticosteroid synthesis in pregnant rats [90].

## **4. Potential role of hypoxia-induced miRNAs, miR-21 and miR-126, on the endothelial dysfunction in FGR**

As previously discussed, ncRNAs constitute an important epigenetic mechanism, which mainly regulates RNA translation; notably miR-21 and miR-126 represent two potential miR-NAs with a crucial role in the endothelium. In fact, both miRNAs are abundantly expressed in cultured endothelium [91] and respond to hypoxia with a substantial increase in miR-21 and miR-126 levels, representing ~40% of all the miRNAs present in this cell type [92]. In contrast to most miRNAs, miR-126 and miR-21 are encoded within the intronic region of genes coding for proteins. MiR-126 is encoded in the seventh intron of the gene for the endothelial-specific protein epidermal growth factor-like domain 7 (Egfl7) and its expression is partially (~30%) dependent on transcription factors that bind to the promoter region of this Egfl7 [93]. Additionally, miR-126 expression is regulated, independently of Egfl7, by the DNA methylation status of a miR-126-specific promoter located in intron 7 of Egfl7 [94], as well as the binding of Nrf2 to this region in response to oxidative stress [95]. Preliminary data from our group show that FGR human endothelial cells present increased levels of DNA methylation in miR-126 promoter, suggesting an epigenetic programming of this miRNA in FGR endothelium. Conversely, miR-21 is encoded in the 11th intron of the stress-induced protein TMEM49, but its expression is completely controlled by a specific promoter in the intron 10 of TMEM49 with predicted binding sites for transcription factors that respond to oxidative stress and inflammation [96, 97]. This suggests that the expression of miR-21 and miR-126 could be regulated by epigenetic modifications present in their specific intronic promoters.

It has been proposed that miR-126 is an endothelial-specific miRNA which promotes angiogenic activation in progenitor cells during early development, as well as vascular repair in adult subjects, while in mature endothelial cells, it has an anti-atherogenic effect maintaining endothelial quiescence and preventing inflammation [67]. In ob/ob mice, antioxidant treatment induces a miR-126-dependent anti-inflammatory and antioxidant vascular response [98], an effect also observed in HUVEC [99]. Both miRNAs, miR-21 and miR-126, are upregulated by unidirectional shear stress, protecting EC from apoptosis and increasing the activation of eNOS [100]. However, in oscillatory shear stress conditions, increased levels of miR-21 promote the expression of pro-inflammatory mediators [101]. Thus, it has been proposed that miR-21 has a dual effect on vascular function: over a short time, it protects against hypoxia and ischemia [70, 102–104], and over the longer term, leads to endothelial dysfunction, apoptosis [70, 102, 105, 106], and eNOS dysfunction. The latter would occur by targeting the expression of antioxidant enzymes [70], as well as enhancing the levels of the endogenous eNOS inhibitor asymmetric dimethyl arginine (ADMA) by downregulating the expression of the enzyme dimethyl arginine dimethylaminohydrolase 1 (DDAH1) [105, 107, 108]. These data suggest that the dynamic regulation of miR-21 and miR-126 could participate in the early defense of the endothelium to hypoxia and oxidative stress; nonetheless, they prime endothelial dysfunction over the long term. Thus, increased levels of miR-21 and decreased expression of miR-126 observed in FGR placentae at term could represent a consequence rather than a cause of the hypoxia-induced endothelial dysfunction.

## **5. Conclusions**

The programming of vascular, particularly endothelial dysfunction by hypoxia in FGR is an important issue in fetal-maternal medicine up to date. Currently, there is a serious need to undercover the real impact of hypoxia as a driving force to perinatal and postnatal cardiovascular and metabolic diseases, pointing out the main proposed mechanisms. The reviewed data support the notion that epigenetic mechanisms contribute to defining and regulating vascular responses to pathological stimuli (leading to FGR). However, evidence of how fetal exposure to hypoxia and oxidative stress lead to epigenetic modifications remains elusive.

Therefore, new knowledge on the role of epigenetic mechanisms involved in the long-term vascular function is crucial to understand and put into context adequate interventions. The timing of the vascular adaptations and epigenetic responses is one of the most relevant questions that need to be answered in order to prioritize clinical approaches to early diagnose and treat such perinatal conditions, limiting postnatal cardiometabolic risk in the progeny.

## **Author details**

stress and inflammation [96, 97]. This suggests that the expression of miR-21 and miR-126 could be regulated by epigenetic modifications present in their specific intronic promoters. It has been proposed that miR-126 is an endothelial-specific miRNA which promotes angiogenic activation in progenitor cells during early development, as well as vascular repair in adult subjects, while in mature endothelial cells, it has an anti-atherogenic effect maintaining endothelial quiescence and preventing inflammation [67]. In ob/ob mice, antioxidant treatment induces a miR-126-dependent anti-inflammatory and antioxidant vascular response [98], an effect also observed in HUVEC [99]. Both miRNAs, miR-21 and miR-126, are upregulated by unidirectional shear stress, protecting EC from apoptosis and increasing the activation of eNOS [100]. However, in oscillatory shear stress conditions, increased levels of miR-21 promote the expression of pro-inflammatory mediators [101]. Thus, it has been proposed that miR-21 has a dual effect on vascular function: over a short time, it protects against hypoxia and ischemia [70, 102–104], and over the longer term, leads to endothelial dysfunction, apoptosis [70, 102, 105, 106], and eNOS dysfunction. The latter would occur by targeting the expression of antioxidant enzymes [70], as well as enhancing the levels of the endogenous eNOS inhibitor asymmetric dimethyl arginine (ADMA) by downregulating the expression of the enzyme dimethyl arginine dimethylaminohydrolase 1 (DDAH1) [105, 107, 108]. These data suggest that the dynamic regulation of miR-21 and miR-126 could participate in the early defense of the endothelium to hypoxia and oxidative stress; nonetheless, they prime endothelial dysfunction over the long term. Thus, increased levels of miR-21 and decreased expression of miR-126 observed in FGR placentae at term could represent a consequence rather than a cause of the hypoxia-induced

The programming of vascular, particularly endothelial dysfunction by hypoxia in FGR is an important issue in fetal-maternal medicine up to date. Currently, there is a serious need to undercover the real impact of hypoxia as a driving force to perinatal and postnatal cardiovascular and metabolic diseases, pointing out the main proposed mechanisms. The reviewed data support the notion that epigenetic mechanisms contribute to defining and regulating vascular responses to pathological stimuli (leading to FGR). However, evidence of how fetal exposure to hypoxia and oxidative stress lead to epigenetic modifications

Therefore, new knowledge on the role of epigenetic mechanisms involved in the long-term vascular function is crucial to understand and put into context adequate interventions. The timing of the vascular adaptations and epigenetic responses is one of the most relevant questions that need to be answered in order to prioritize clinical approaches to early diagnose and treat such perinatal conditions, limiting postnatal cardiometabolic risk in

endothelial dysfunction.

**5. Conclusions**

338 Hypoxia and Human Diseases

remains elusive.

the progeny.

Paola Casanello1, 2\*, Emilio A. Herrera3 and Bernardo J. Krause1

\*Address all correspondence to: paolacasanello@gmail.com

1 Division of Pediatrics, Department of Neonatology, The Pontifical Catholic University of Chile, Santiago, Chile

2 Division of Obstetrics & Gynecology, School of Medicine, The Pontifical Catholic University of Chile, Santiago, Chile

3 Pathophysiology Program, Biomedical Sciences Institute (ICBM), Faculty of Medicine, University of Chile, Santiago, Chile

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#### **The Critical Role of Hypoxia in Tumor-Mediated Immunosuppression The Critical Role of Hypoxia in Tumor-Mediated Immunosuppression**

Nassera Aouali, Manon Bosseler, Delphine Sauvage, Kris Van Moer, Guy Berchem and Bassam Janji Nassera Aouali, Manon Bosseler, Delphine Sauvage, Kris Van Moer, Guy Berchem and Bassam Janji

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/65383

#### **Abstract**

Underestimated for a long time, the involvement of the microenvironment has been proven essential for a better understanding of the cancer development. In keeping with this, the tumor is not considered anymore as a mass of malignant cells, but rather as an organ composed of various malignant and nonmalignant cell populations interacting with each other to create the tumor microenvironment. The tumor immune contexture plays a critical role in shaping the tumor immune response, and it is now well supported that such an immune response is impacted by the hypoxic stress within the tumor microenvironment. Tumor hypoxia is closely linked to tumor progression, metastasis, treatment failure, and escape from immune surveillance. Thus, hypoxia seems to be a key factor involved in creating an immune-suppressive tumor by multiple overlapping mechanisms, including the impairment of the function of cytotoxic immune cells, increasing the immunosuppressive properties of immunosuppressive cells, and activating resistance mechanism in the tumor cells. In this chapter, we review some recent findings describing how hypoxic stress in the tumor microenvironment hijacks the antitumor immune response.

**Keywords:** cancer, hypoxia, immune response, tumor microenvironment, autophagy, tumor plasticity, tumor heterogeneity

### **1. Introduction**

Malignant cells are part of cellular and microenvironmental complexes which both define the initiation, progression, and maintenance of the malignant phenotype. In turn, malignant cells participate in creating a hostile microenvironment characterized by hypoxic areas within the

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

tumors. Indeed, the oxygen level in the hypoxic tumor is usually lower than that of corresponding normal tissue. The oxygenation level of tumor is likely depending on (i) the initial oxygenation of the tissue; (ii) the degree of the tumor heterogeneity; (iii) the tumor size and stage. **Table 1** summarizes the percentage of oxygen level reported as a median in some healthy organs and their corresponding tumors, as defined by several studies.


**Table 1.** Comparison of the percentage (%) of oxygen level in different healthy tissues and in their corresponding cancers.

It is now widely appreciated that hypoxia is one of the most relevant factor involved in the impairment of the antitumor immune response by damping the cytotoxic function of immune cells. There are numerous studies supporting that hypoxic stress leads to the establishment of immune tolerance of tumor cells by preventing the migration and the homing of immune effector cells into established tumors. Furthermore, hypoxia can also drive tumor cell plasticity and functional heterogeneity and, thus, favors the emergence of more aggressive tumors. Many strategies are emerging for targeting intratumor hypoxia in order to change the immunosuppressive properties of the tumor to a microenvironment able to support antitumor immunity.

## **2. Hypoxia is the major factor of the tumor microenvironment**

The long-lasting tumor immunology research has validated the concept of tumor immunosurveillance. The tumor immunosurveillance consists in the fact that cytotoxic immune cells recognize nascent transformed cells and destroy them before they become clinically apparent. Several types of immune cells are involved in the control of tumors such as immune effector and immune suppressor cells. Thus, cytotoxic T lymphocytes (CTL) belong to the adaptive immune system and they are able to recognize tumor antigens through the T-cell receptor (TCR) [1]. The antigens expressed exclusively by tumor cells are called tumor-specific antigens [2]. In addition to CTL, the tumor immune surveillance involves natural killer (NK) cells that belong to the innate immune system [1]. NK cells recognize tumor cells by mechanisms called "missing-self" and "induced-self" [3]. Briefly, NK cells are regulated by a balance of inhibitory and activating signals of surface receptors. Thus, NK cells can kill their target cell depending on the recognized ligand(s). The identification of activating or inhibitory ligands allows NK cells to distinguish between "self" versus "nonself" and "self" versus "altered self" by "missing-self" and "induced-self" recognitions. Indeed, the protection of normal cells from NK cell killing is achieved by balancing the stimulatory signals delivered by stimulatory ligands with inhibitory signals delivered by self MHC class I molecules. When the expression of self MHC class I molecules is lost following cell transformation or infection, the stimulatory signals delivered by the target cell remain unbalanced, leading to the activation of NK cells and lysis of target cells (known as missing-self recognition). Under some circumstances, transformed or infected cells overexpress stimulatory ligands that overcome the inhibitory signals leading to target cell lysis (known as induced-self recognition). It has been reported that both missing-self and induced-self recognition could operate simultaneously. In this case, NK cells display a high ability to discriminate between normal and transformed target cells [4].

tumors. Indeed, the oxygen level in the hypoxic tumor is usually lower than that of corresponding normal tissue. The oxygenation level of tumor is likely depending on (i) the initial oxygenation of the tissue; (ii) the degree of the tumor heterogeneity; (iii) the tumor size and stage. **Table 1** summarizes the percentage of oxygen level reported as a median in some healthy organs and

their corresponding tumors, as defined by several studies.

cancers.

350 Hypoxia and Human Diseases

immunity.

Brain/brain tumor 4.6/1.7 Breast/breast cancer 8.5/1.5 Cervix/cervical cancer 9.5/1.2 Kidney cortex/renal cancer 7.0/1.3 Liver/liver cancer 4.0–7.3/0.8 Lung/nonsmall cell lung carcinoma 5.6/2.2 Pancreas/pancreatic tumor 7.5/0.3 Rectal mucosa/rectal carcinoma 3.9/1.8

**Healthy tissue/corresponding cancer % of oxygen (Median)**

**Table 1.** Comparison of the percentage (%) of oxygen level in different healthy tissues and in their corresponding

**2. Hypoxia is the major factor of the tumor microenvironment**

It is now widely appreciated that hypoxia is one of the most relevant factor involved in the impairment of the antitumor immune response by damping the cytotoxic function of immune cells. There are numerous studies supporting that hypoxic stress leads to the establishment of immune tolerance of tumor cells by preventing the migration and the homing of immune effector cells into established tumors. Furthermore, hypoxia can also drive tumor cell plasticity and functional heterogeneity and, thus, favors the emergence of more aggressive tumors. Many strategies are emerging for targeting intratumor hypoxia in order to change the immunosuppressive properties of the tumor to a microenvironment able to support antitumor

The long-lasting tumor immunology research has validated the concept of tumor immunosurveillance. The tumor immunosurveillance consists in the fact that cytotoxic immune cells recognize nascent transformed cells and destroy them before they become clinically apparent. Several types of immune cells are involved in the control of tumors such as immune effector and immune suppressor cells. Thus, cytotoxic T lymphocytes (CTL) belong to the adaptive immune system and they are able to recognize tumor antigens through the T-cell receptor (TCR) [1]. The antigens expressed exclusively by tumor cells are called tumor-specific antigens [2]. In addition to CTL, the tumor immune surveillance involves natural killer (NK) cells that belong to the innate immune system [1]. NK cells recognize tumor cells by mechanisms In addition to cytotoxic immune cells, the tumor immune contexture contains immune suppressive cells such as myeloid-derived suppressor cells (MDSC) able to inhibit the function of immune effectors. Macrophages and neutrophil granulocytes are also involved in antitumor immunity [5]. These cells display tumor antigens and can stimulate other immune cells such as CTL, NK cells, or antigen-presenting cells (APC) [6]. Although both CTL and NK cells kill their target following the establishment of immunological synapse (IS) [7], the molecular mechanism by which they recognize their target tumor cells is fundamentally different. Two major pathways are used by CTL and NK cells to recognize and destroy tumor cells: (i) through the release by immune cells of cytotoxic granules containing perforin and granzymes and these cytotoxic granules are captured by tumor cells to induce cell death by apoptosis [8], and (ii) through tumor necrosis factor (TNF) superfamily-dependent mechanism [9].

It has been proposed that despite the powerful ability of the immune system to attack cancer cells, tumors can outmaneuver the immune effectors cells and escape the immune surveillance. It is now well documented that the ability of tumor cells to escape immune cell control is most likely resulted from the activation of several resistance mechanisms to evade effective and functional host immune response. Therefore, it stands to reason that established tumors, displaying multiple resistance mechanism, are likely not fully controlled by the immune system. In keeping with this, it is strongly believed that clinically detected cancers have most likely evaded effective antitumor immune responses. Recently, it has been reported that in addition to its role in protecting host against tumor development, the immune system can under certain circumstances sculpt the immunogenic phenotype of well-developed tumors. Such a mechanism favors the emergence of resistant tumor cell clones [10]. Accumulating experimental and clinical evidence suggest that the resistance mechanisms activated in tumor cells are multifactorial and that such resistance mechanisms are primarily evolved and activated in the tumor microenvironment [11]. It appears that hypoxia is the major tumor microenvironmental factor involved in the alteration of the transcriptome and the metabolome of tumor cells as well as their proliferation, survival, and invasion [12].

In this chapter, we summarize some recent findings describing how hypoxic stress in the tumor microenvironment regulates the antitumor immune response and leads to tumor escape from immunosurveillance. We focus on how hypoxia confers resistance to immune attack and impairs tumor cell killing mediated by CTL and NK cells.

#### **2.1. Hypoxia and hypoxia-inducible factors (HIF) regulation**

Tumor cells are able to adapt to hypoxic stress through the regulation of the hypoxia inducible factor family of transcription factors (HIFs) [13]. It has been reported for a large number of human cancers that HIFs were overexpressed and such overexpression is associated with poor response to treatment [14]. Moreover, evidence showed a clear positive correlation between enhanced hypoxic expression of HIFs and mortality [13]. Therefore, inhibition of HIFs could represent a novel approach to improve cancer therapies. Currently, efforts are being actively pursued to identify inhibitors of HIFs and to test their efficacy as anticancer therapeutics.

Three isoforms of HIF have been identified: HIF-1, HIF-2, and HIF-3. The hypoxia-inducible factor-1 (HIF-1) is the major factor mediating adaptive responses to changes in tissue oxygen level [15]. Indeed, HIF-1 is a heterodimer composed of a constitutively expressed HIF-1β subunit and an O2-dependent regulated HIF-1α subunit. HIF-1α is a DNA-binding basic helix-loop helix of the PAS family [Per (period circadian protein); Arnt (aryl hydrocarbon receptor nuclear translocator protein); Sim (single-minded protein)] [16]. HIF-1α contains two oxygen-dependent degradation domains (ODDD), one in the N-terminal (N-ODDD) moiety and one in the Cterminal moiety (C-ODDD) [17, 18]. It also contains two transactivation domains (TADs), one N-terminal, which overlaps with the C-ODDD, and one C-terminal [19].

#### **2.2. Regulation of HIF-1 level**

The expression level of HIF-1α is determined by the rates of protein synthesis and protein degradation. While the synthesis of HIF-1α is regulated in an O2-independent manner, its degradation is primarily regulated via an O2-dependent mechanism. Thus, normoxic cells constantly synthesize HIF-1α protein and degrade it rapidly [17]. It has been shown that under normoxic conditions HIF-1α has a short half-life of less than 5 min [20]. However, under hypoxia or low oxygen level, the degradation of HIF-1α is blocked or dramatically decreased [21]. Under normoxia, HIF-1α is hydroxylated on proline residue 402 and/or 564 in the ODDD by prolyl hydroxylase domain protein 2 (PHD2) [17, 22]. Such oxygen-dependent hydroxylation of HIF-1α results in its binding to the von Hippel-Lindau tumor suppressor protein (pVHL). pVHL is the recognition component of an E3 ubiquitin-protein ligase complex that targets HIF-1α for proteolysis by the ubiquitin-proteasome pathway [23].

Enzymes regulating HIF-1α proteasomal degradation were first identified to be related to egl-9 in caenorhabditis elegans and to termed prolyl hydroxylase domain (PHD) enzymes (PHD1, PHD-2, and PHD3) [24, 25]. PHD2 uses oxygen as a substrate, and thus, its activity is inhibited under hypoxic conditions [25]. The inhibition of PHD2 leads to the inhibition of prolyl hydroxylation of HIF-1α and subsequently to the inhibition of HIF-1α-dependent proteasomal degradation. Consequently, HIF-1α rapidly accumulates in the cytoplasm, translocates to the nucleus and dimerizes with HIF-1β. The HIF-1α/HIF-1β heteromeric dimer binds to the hypoxia responsive element (HRE) in target genes, recruits coactivators and activates transcription [14] (**Figure 1A**).

In this chapter, we summarize some recent findings describing how hypoxic stress in the tumor microenvironment regulates the antitumor immune response and leads to tumor escape from immunosurveillance. We focus on how hypoxia confers resistance to immune attack and

Tumor cells are able to adapt to hypoxic stress through the regulation of the hypoxia inducible factor family of transcription factors (HIFs) [13]. It has been reported for a large number of human cancers that HIFs were overexpressed and such overexpression is associated with poor response to treatment [14]. Moreover, evidence showed a clear positive correlation between enhanced hypoxic expression of HIFs and mortality [13]. Therefore, inhibition of HIFs could represent a novel approach to improve cancer therapies. Currently, efforts are being actively pursued to identify inhibitors of HIFs and to test their efficacy as anticancer therapeutics.

Three isoforms of HIF have been identified: HIF-1, HIF-2, and HIF-3. The hypoxia-inducible factor-1 (HIF-1) is the major factor mediating adaptive responses to changes in tissue oxygen level [15]. Indeed, HIF-1 is a heterodimer composed of a constitutively expressed HIF-1β subunit and an O2-dependent regulated HIF-1α subunit. HIF-1α is a DNA-binding basic helix-loop helix of the PAS family [Per (period circadian protein); Arnt (aryl hydrocarbon receptor nuclear translocator protein); Sim (single-minded protein)] [16]. HIF-1α contains two oxygen-dependent degradation domains (ODDD), one in the N-terminal (N-ODDD) moiety and one in the Cterminal moiety (C-ODDD) [17, 18]. It also contains two transactivation domains (TADs), one

The expression level of HIF-1α is determined by the rates of protein synthesis and protein degradation. While the synthesis of HIF-1α is regulated in an O2-independent manner, its degradation is primarily regulated via an O2-dependent mechanism. Thus, normoxic cells constantly synthesize HIF-1α protein and degrade it rapidly [17]. It has been shown that under normoxic conditions HIF-1α has a short half-life of less than 5 min [20]. However, under hypoxia or low oxygen level, the degradation of HIF-1α is blocked or dramatically decreased [21]. Under normoxia, HIF-1α is hydroxylated on proline residue 402 and/or 564 in the ODDD by prolyl hydroxylase domain protein 2 (PHD2) [17, 22]. Such oxygen-dependent hydroxylation of HIF-1α results in its binding to the von Hippel-Lindau tumor suppressor protein (pVHL). pVHL is the recognition component of an E3 ubiquitin-protein ligase complex that

Enzymes regulating HIF-1α proteasomal degradation were first identified to be related to egl-9 in caenorhabditis elegans and to termed prolyl hydroxylase domain (PHD) enzymes (PHD1, PHD-2, and PHD3) [24, 25]. PHD2 uses oxygen as a substrate, and thus, its activity is inhibited under hypoxic conditions [25]. The inhibition of PHD2 leads to the inhibition of prolyl hydroxylation of HIF-1α and subsequently to the inhibition of HIF-1α-dependent proteasomal degradation. Consequently, HIF-1α rapidly accumulates in the cytoplasm, translocates to the

impairs tumor cell killing mediated by CTL and NK cells.

**2.1. Hypoxia and hypoxia-inducible factors (HIF) regulation**

N-terminal, which overlaps with the C-ODDD, and one C-terminal [19].

targets HIF-1α for proteolysis by the ubiquitin-proteasome pathway [23].

**2.2. Regulation of HIF-1 level**

352 Hypoxia and Human Diseases

**Figure 1.** The role of hypoxic stress in the impairment CTL and NK-cell mediated lysis. (A) Under normoxia, the oxygen-sensitive prolyl hydroxylase domain protein 2 (PHD2) hydroxylates HIF-1α subunit. Hydroxylated HIF-1 interacted with Von Hippel-Lindau protein (VHL), subjected to ubiquitination and subsequently degraded by the ubiquitinproteasome system. Under hypoxic stress, the function of PHD2 protein is blocked, HIF-1α is therefore stabilized and translocated to the nucleus to form heterodimeric complex with HIF-1β to transcriptionally induce the expression of HIF-target genes involved in several pathways such as autophagy. (B) Under hypoxia, STAT3 is phosphorylated at Ser-705 residue in a HIF-dependent manner by a mechanism which is not fully understood. (C) The hypoxia-dependent induction of autophagy leads to the degradation of the adaptor protein p62/SQSTM1, involved in targeting phospho-STAT3 to the ubiquitin proteasome system for degradation. Thus, targeting autophagy accumulated p62/SQSTM1 and therefore accelerated the degradation of phospho-STAT3. The degradation of phospho-STAT3 restores CTL-mediated lysis of tumor cells. In addition, the induction of autophagy in hypoxic tumor cells leads to the selective degradation of granzyme B (GZMB), a serine protease released by natural killer (NK) cells and contained in the cytotoxic granules. Such degradation inhibits NK-mediated lysis of tumor cells.

Using genomewide chromatin immunoprecipitation combined with DNA microarray (ChIPchip) or DNA sequencing (ChIP-seq) analysis, it has been shown that more than 800 genes involved in several cell functions are direct targets of HIF [26, 27]. HIF-1 activates the

expression of these genes by binding to a 50 base pair cis-acting HRE located in their enhancer and promoter regions [28]. The HREs of all these genes contain the core sequence 5′-[A/ G]CGT-3′, which in most cases is ACGTG [29]. It has been reported that HIF transcription factors preferentially bind to specific bases in the 5′ and 3′ proximity of the core that has led to define the following HRE consensus sequence [T/G/C][A/G]CGTG[CGA][GTC][GTC] [CTG] [29].

Similar to HIF-1α, the stabilization of HIF-2α is also regulated by oxygen-dependent hydroxylation [30]. This could be related to the fact that HIF-1α and HIF-2α displayed a similar structure of their DNA binding and dimerization domains. However, the major difference between the structure of HIF-1α and HIF-2α is in their transactivation domains [31]. In terms of genes expression, both HIF-1α and HIF-2α share overlapping target genes, and each one also regulates a set of unique targets [32].

In sharp contrast with HIF-1α and HIF-2α, HIF-3α lacks the transactivation domain and could function as an inhibitor of HIF-1α and HIF-2α. It has been reported that the expression of HIF-3α is regulated by HIF-1 [33]. In addition to the regulation of the expression of a large number of genes, HIF family members regulate hypoxia-related microRNAs (HRM) [34] and some chromatin modifying enzymes [35].

## **3. Intra-tumor hypoxia: a key feature that triggers several resistance mechanisms of tumor evasion from immune surveillance**

It has been clearly established that the immune effector activity and the antitumor immune response are significantly regulated by hypoxia. Indeed, hypoxia, via HIF-1α, decreases the susceptibility of lung cancer cells to CTL-mediated killing. It appears that the resistance to CTL is related to the effect of HIF-1α to induce the phosphorylation of signal transducer and activator of transcription 3 (STAT3) in tumor cells by a mechanism involving the vascular endothelial growth factor (VEGF) secretion. These data suggest that following its translocation to the nucleus, HIF-1α cooperates with pSTAT3 to impair lung carcinoma cell susceptibility to CTL-mediated killing [36] (**Figure 1B**). More recently, it has been shown that the expression of the phosphorylated form of STAT3 at Ser-705 residue is tightly controlled by the induction of autophagy in hypoxic tumor cells as the accumulation of pSTAT3 was no longer observed when autophagy was targeted genetically in tumor cells [37]. Autophagy is a catabolic cell degradation process. Autophagy plays an essential role in preventing accumulation of altered cell components [38] and as an adaptive metabolic response to provide nutrients. Recently, an unexpected role of autophagy in shaping the antitumor immune response [39] and the acquisition of resistance to TNFα has been shown [40]. Autophagy is activated under stress conditions such as hypoxia, nutrient starvation, growth factor withdrawal, and endoplasmic reticulum stress. It has been reported that the molecular mechanism by which autophagy regulates the pSTAT3 level involves the protein p62/SQSTM1 the ubiquitin proteasome system [37, 41].

Another study showed that in addition to the mechanism described earlier, it has been shown that the stem cell self-renewal transcription factor NANOG is also involved in the regulation of CTL-mediated tumor cell lysis [42, 43]. Hypoxia regulates NANOG at both transcriptional and translational levels and targeting NANOG in hypoxic cells restored CTL-mediated tumor cell killing. Furthermore, NANOG depletion results in the inhibition of STAT3 phosphorylation and its nuclear translocation. The hypoxia-induced microRNA (miR)-210 is also involved in the regulation of CTL-mediated tumor cells lysis. In fact, HIF-1 induces the expression of miR-210 which subsequently targets nonreceptor protein tyrosine phosphatase type 1 (PTPN1), homeobox A1 (HOXA1), and tumor protein p53-inducible protein 11 (TP53I11), and thereby decreases tumor cell susceptibility to CTL [44]. In the context of NK-mediated tumor cell lysis, it has been described that hypoxia increases the shedding of the major histocompatibility complex (MHC) class I polypeptide-related sequence A (MICA), a ligand for the activating receptor natural killer group 2 member D (NKG2D), on the surface of prostate cancer cells leading to an impairment of NO signaling [45] and subsequent escape of tumor cells from NK- and CTL-mediated killing. MICA expression is also downregulated by HIF-1 in osteosarcoma cells resulting in tumor resistance to NK-mediated lysis [46]. Through the activation of autophagy, it has been recently reported that melanoma and breast tumor cells escape NKmediated lysis and that targeting autophagy in hypoxic tumor cells was sufficient to restore NK-mediated lysis. In this study, it has been shown that the activation of autophagy under hypoxia was responsible for the degradation of NK-derived granzyme B making hypoxic tumor cells less sensitive to NK-mediated killing [39, 47, 48] (**Figure 1C**). In line with the studies described earlier, it is now well admitted that hypoxic stress in the tumor microenvironment is a key factor involved in the control of antitumor immune response. Beside its role in impairing the function of cytotoxic immune cells, the immunosuppressive effect of hypoxia contributes to the emergence of resistant tumor cells that compromise the effectiveness of the anti-tumor immune response [49].

## **4. Hypoxia and tumor cell heterogeneity and plasticity**

expression of these genes by binding to a 50 base pair cis-acting HRE located in their enhancer and promoter regions [28]. The HREs of all these genes contain the core sequence 5′-[A/ G]CGT-3′, which in most cases is ACGTG [29]. It has been reported that HIF transcription factors preferentially bind to specific bases in the 5′ and 3′ proximity of the core that has led to define the following HRE consensus sequence [T/G/C][A/G]CGTG[CGA][GTC][GTC]

Similar to HIF-1α, the stabilization of HIF-2α is also regulated by oxygen-dependent hydroxylation [30]. This could be related to the fact that HIF-1α and HIF-2α displayed a similar structure of their DNA binding and dimerization domains. However, the major difference between the structure of HIF-1α and HIF-2α is in their transactivation domains [31]. In terms of genes expression, both HIF-1α and HIF-2α share overlapping target genes, and each one

In sharp contrast with HIF-1α and HIF-2α, HIF-3α lacks the transactivation domain and could function as an inhibitor of HIF-1α and HIF-2α. It has been reported that the expression of HIF-3α is regulated by HIF-1 [33]. In addition to the regulation of the expression of a large number of genes, HIF family members regulate hypoxia-related microRNAs (HRM) [34] and

**3. Intra-tumor hypoxia: a key feature that triggers several resistance**

It has been clearly established that the immune effector activity and the antitumor immune response are significantly regulated by hypoxia. Indeed, hypoxia, via HIF-1α, decreases the susceptibility of lung cancer cells to CTL-mediated killing. It appears that the resistance to CTL is related to the effect of HIF-1α to induce the phosphorylation of signal transducer and activator of transcription 3 (STAT3) in tumor cells by a mechanism involving the vascular endothelial growth factor (VEGF) secretion. These data suggest that following its translocation to the nucleus, HIF-1α cooperates with pSTAT3 to impair lung carcinoma cell susceptibility to CTL-mediated killing [36] (**Figure 1B**). More recently, it has been shown that the expression of the phosphorylated form of STAT3 at Ser-705 residue is tightly controlled by the induction of autophagy in hypoxic tumor cells as the accumulation of pSTAT3 was no longer observed when autophagy was targeted genetically in tumor cells [37]. Autophagy is a catabolic cell degradation process. Autophagy plays an essential role in preventing accumulation of altered cell components [38] and as an adaptive metabolic response to provide nutrients. Recently, an unexpected role of autophagy in shaping the antitumor immune response [39] and the acquisition of resistance to TNFα has been shown [40]. Autophagy is activated under stress conditions such as hypoxia, nutrient starvation, growth factor withdrawal, and endoplasmic reticulum stress. It has been reported that the molecular mechanism by which autophagy regulates the pSTAT3 level involves the protein p62/SQSTM1 the ubiquitin proteasome

**mechanisms of tumor evasion from immune surveillance**

[CTG] [29].

354 Hypoxia and Human Diseases

system [37, 41].

also regulates a set of unique targets [32].

some chromatin modifying enzymes [35].

Solid tumors frequently reveal pronounced tumor cell heterogeneity with regards to cell organization, cell morphology, cell size, and nuclei morphology [50]. The molecular mechanisms underlying the phenotypic heterogeneity involve genetic, epigenetic, and environmental factors. It is now well established that hypoxia is an important contributor to intra- and intertumor cell heterogeneity [15, 51] by altering the expression of specific genes involved in cellular phenotype. In this respect, it has been reported that neuroblastoma cells and breast cancer cells lose their differentiated gene expression patterns and develop stem cell-like phenotypes under hypoxic stress [52, 53]. As a low stage of differentiation in neuroblastoma and breast cancer is associated with poor prognosis, it is strongly believed that, in addition to its contribution to tumor heterogeneity, hypoxia-dependent induction of tumor cell dedifferentiation contributes to tumor cell plasticity and aggressiveness.

Several lines of evidence suggest that tumor microenvironment drives stem cell renewal and differentiation. Indeed, poorly vascularized tumors contain hypoxic regions with undifferentiated 'stem-like' tumor cells that survive under control of HIFs [54]. It has been reported that hypoxic stress in colon cancer inhibits the differentiation of tumor cells and maintains their stem-like phenotype [55]. In addition, myofibroblasts stromal cells secrete factors involved in maintaining cancer stem cells (CSC) population in colon cancer [56]. Furthermore, stromal cells drive a CSC phenotype on differentiated cancer cells, allowing a transient morphological heterogeneity observed in several cancers. In this regard, transient phenotypic changes from epithelial to mesenchymal (epithelial-mesenchymal transition (EMT)) or mesenchymal to epithelial (mesenchymal to epithelial transitions (MET)) phenotype, are initially considered as conversions facilitating cell plasticity but have recently gained appreciation as events involved in tumor heterogeneity [57]. In the context of tumor immunity, recent evidence revealed that tumor cell plasticity has serious implications in terms of immunological recognition and killing of the tumor, since such tumor cell plasticity may lead to the emergence of immunoresistant variants [58].

Although the role of the immune system in inhibiting early stages of tumor growth is well established, it is now strongly suggested that the immune system can also facilitate the advanced stages of tumor progression by sculpting the immunogenic phenotype of a developing tumor to favor the emergence of immune-resistant tumor cell variants. This has led to the concept of "immunoediting" which encompasses three phases: elimination, equilibrium, and escape. Thus, immunoediting allows tumors to evade immune destruction by becoming less immunogenic or more immunosuppressive [59]. Such adaptability, achieved through cell reprogramming, reflects an important property of tumors called immune-induced plasticity. While the molecular basis of immune-dependent induction of tumor cell plasticity and its effective contribution to the selection of tumor aggressive variants is still elusive, recent findings have revealed that activated CD8+ T cells can stimulate mammary epithelial tumor cells to undergo EMT and acquire the increased tumorigenic capability and therapy resistance of breast CSCs [60]. In this regard, it has been shown that reciprocal interactions between melanoma and immune cells enhances tumor cell plasticity and drives therapy resistance [61]. Based on these data, it is now well defined that targeting phenotypic plasticity should be considered for the development of novel therapeutic strategies with the ultimate goal to prevent the establishment of a more aggressive phenotype of cancer cells.

## **5. The clinical significance of targeting hypoxia**

For many years, the major issue in the field of cancer immunity was to understand how cancer cells manage to evade immune surveillance despite the presence of a competent immune system. To address this issue, the major focus was on the mechanisms by which tumor cells escape cytotoxic immune cell recognition without considering the impact of the tumor microenvironment. This could partially explain why despite intense investigation, the gains provided by immunotherapy until recently are relatively modest. In addition, accumulating evidence suggests that tumor cell resistance mechanisms are likely evolved in the hypoxic tumor microenvironment. In keeping with this, it is therefore more accurate to consider cancer as a disease of the microenvironment rather than a disease of cells. Although remarkable progresses have been achieved over the past two decades regarding the impact of the tumor microenvironment in cancer biology and treatment, its contribution in the development of tumor resistance to immune cell killing remains fragmented.

tiated 'stem-like' tumor cells that survive under control of HIFs [54]. It has been reported that hypoxic stress in colon cancer inhibits the differentiation of tumor cells and maintains their stem-like phenotype [55]. In addition, myofibroblasts stromal cells secrete factors involved in maintaining cancer stem cells (CSC) population in colon cancer [56]. Furthermore, stromal cells drive a CSC phenotype on differentiated cancer cells, allowing a transient morphological heterogeneity observed in several cancers. In this regard, transient phenotypic changes from epithelial to mesenchymal (epithelial-mesenchymal transition (EMT)) or mesenchymal to epithelial (mesenchymal to epithelial transitions (MET)) phenotype, are initially considered as conversions facilitating cell plasticity but have recently gained appreciation as events involved in tumor heterogeneity [57]. In the context of tumor immunity, recent evidence revealed that tumor cell plasticity has serious implications in terms of immunological recognition and killing of the tumor, since such tumor cell plasticity may lead to the emergence of immunoresistant

Although the role of the immune system in inhibiting early stages of tumor growth is well established, it is now strongly suggested that the immune system can also facilitate the advanced stages of tumor progression by sculpting the immunogenic phenotype of a developing tumor to favor the emergence of immune-resistant tumor cell variants. This has led to the concept of "immunoediting" which encompasses three phases: elimination, equilibrium, and escape. Thus, immunoediting allows tumors to evade immune destruction by becoming less immunogenic or more immunosuppressive [59]. Such adaptability, achieved through cell reprogramming, reflects an important property of tumors called immune-induced plasticity. While the molecular basis of immune-dependent induction of tumor cell plasticity and its effective contribution to the selection of tumor aggressive variants is still elusive, recent findings have revealed that activated CD8+ T cells can stimulate mammary epithelial tumor cells to undergo EMT and acquire the increased tumorigenic capability and therapy resistance of breast CSCs [60]. In this regard, it has been shown that reciprocal interactions between melanoma and immune cells enhances tumor cell plasticity and drives therapy resistance [61]. Based on these data, it is now well defined that targeting phenotypic plasticity should be considered for the development of novel therapeutic strategies with the ultimate goal to

prevent the establishment of a more aggressive phenotype of cancer cells.

For many years, the major issue in the field of cancer immunity was to understand how cancer cells manage to evade immune surveillance despite the presence of a competent immune system. To address this issue, the major focus was on the mechanisms by which tumor cells escape cytotoxic immune cell recognition without considering the impact of the tumor microenvironment. This could partially explain why despite intense investigation, the gains provided by immunotherapy until recently are relatively modest. In addition, accumulating evidence suggests that tumor cell resistance mechanisms are likely evolved in the hypoxic tumor microenvironment. In keeping with this, it is therefore more accurate to consider cancer as a disease of the microenvironment rather than a disease of cells. Although remarkable

**5. The clinical significance of targeting hypoxia**

variants [58].

356 Hypoxia and Human Diseases

Emerging data indicates that hypoxia stress within the tumor microenvironment is a key factor involved in the impairment of the antitumor immune response. [62] Therefore, a deep understanding of the molecular mechanism by which hypoxia induces tumor resistance may contribute to the development of more effective tumor immunotherapies.

Consistent with the fact that hypoxia-dependent overexpression of HIF-1α is associated with an increased patient mortality in several cancer types, it stands to reason that inhibition of HIF-1 activity in preclinical studies would have marked effects on tumor growth and survival. In keeping with this, efforts are underway to identify selective inhibitors of HIF-1 and to assess their efficacy as anticancer therapeutics. Currently, two main approaches are used to target hypoxia in tumors, namely bioreductive prodrugs, and inhibitors of molecular targets upon which hypoxic cell survival depends [63, 64]. However, several lines of evidence indicate that the HIF pathway is technically extremely challenging to target. Indeed, the first evidence is that transcription factors in general, including HIF, have long been considered "undruggable," and therefore, no specific inhibitor of HIF has been brought to the market so far. The second evidence is that multiple levels of regulation and signaling pathways converge on and emerge from HIF [65]. Nevertheless, based on the molecular mechanism of HIF-1 protein, it has been suggested that small molecules could be used to inhibit HIF-1 activity through a variety of mechanisms including inhibition of (i) HIF-1α protein synthesis; (ii) HIF-1α protein stabilization; (iii) HIF-1α/β dimerization, and (iv) HIF-1/DNA binding. Two comprehensive recent reviews summarize these mechanisms in detail and give fairly exhaustive lists of the smallmolecule inhibitors for each level [15, 66].

Using a cell-based assay, several small-molecule inhibitors of HIF-1 activity have been identified. Briefly, topoisomerase I inhibitors block the expression of HIF-1α via an undefined mechanism [67]. The small molecule YC-1 (3-(5′-hydroxy-methyl-2′-furyl)-1-benzylindazole) was also shown to reduce the level of HIF-1α by a mechanism that has not been established but at least is known to work independently from its function as a stimulator of soluble guanylate-cyclase activity [68]. YC-1 is not in clinical use. The HSP90 inhibitor 17-allylaminogeldanamycin (17-AAG) has been reported to induce the degradation of HIF-1α in a VHL-independent manner [69–71]. PX-12 (thioredoxin-1 redox inhibitor) and PX-478 are both inhibitors of HIF-1α protein expression and HIF-1-mediated transactivation [72, 73]. Finally, the disruptor of microtubule polymerization 2-methoxyoestradiol (2ME2) is able to decrease the expression of HIF-1α. Currently, only topoisomerase I inhibitors, camptothecin and topotecan, are clinically approved agents, PX-478, 2ME2, and 17-AAG are under evaluation in clinical trials, whereas YC-1 and thioredoxin-1 inhibitors are not in clinical use.

Despite of the anticancer effects of these agents could be related, in part, to their inhibition of HIF-1, it seems that none of these drugs specifically targets HIF-1. Although such lack of selectivity does not disqualify these drugs as anticancer agents, it enhances the difficulty to correlate molecular and clinical responses in patients. Therefore, the identification of more selective HIF-1 inhibitors in the near future is required and more investigation needs to be

done to identify novel potent and more specific inhibitors targeting clearly defined points in the HIF pathway.

## **Acknowledgements**

This work was supported by grants from the Luxembourg Institute of Health (LECR 2013 11 05), Fondation Cancer (FC/2016/01) and Kriibskrank Kanner Foundation, Luxembourg.

## **Author details**

Nassera Aouali1 , Manon Bosseler1 , Delphine Sauvage1 , Kris Van Moer1 , Guy Berchem1,2 and Bassam Janji1\*

\*Address all correspondence to: bassam.janji@lih.lu

1 Laboratory of Experimental Cancer Research, Department of Oncology, Luxembourg Institute of Health, Luxembourg City, Luxembourg

2 Luxembourg Hospital Center, Department of Hemato-Oncology, Luxembourg City, Luxembourg

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This work was supported by grants from the Luxembourg Institute of Health (LECR 2013 11 05), Fondation Cancer (FC/2016/01) and Kriibskrank Kanner Foundation, Luxembourg.

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1 Laboratory of Experimental Cancer Research, Department of Oncology, Luxembourg

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**Author details**

Nassera Aouali1

Bassam Janji1\*

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#### **Cross‐Talk Between Hypoxia and the Tumour via Exosomes Cross**‐**Talk Between Hypoxia and the Tumour via Exosomes**

Shayna Sharma, Mona Alharbi, Andrew Lai, Miharu Kobayashi, Richard Kline, Katrina Wade, Gregory E. Rice and Carlos Salomon Shayna Sharma, Mona Alharbi, Andrew Lai, Miharu Kobayashi, Richard Kline, Katrina Wade, Gregory E. Rice and Carlos Salomon

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/65688

#### **Abstract**

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364 Hypoxia and Human Diseases

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Cancer is one of the leading causes of death worldwide, and this is often attributed to the nonspecific symptoms. Additionally, delayed diagnosis and a lack of treatment options negatively impact prognosis. Recently, the role of extracellular vesicles in cancer progression, specifically, in metastasis and in the capacity of several tumours to invade and colonise specific organs has been established. Reduced oxygen tension due to imbalanced oxygen supply and consumption is termed hypoxia and is one of the most commonly observed features in solid tumours. This is often correlated with poor cancer prognosis. Several reports have established that low oxygen tension (i.e. hypoxia) is a common feature of the tumour microenvironment often enhancing the process of epithelial‐to‐mesenchymal transition (EMT) in cancer cells, thus promoting tumouri‐ genesis and metastasis. Furthermore, hypoxia increases the number of extracellular vesicles released from cancer cells and also modifies their bioactivity and function. The aim of this chapter is to review the association between the tumour microenvironment and extracellular vesicles (EVs), focusing on a specific subpopulation of EVs of endocytic origin, termed exosomes.

**Keywords:** exosomes, metastasis, metastatic niche, tumourigenesis, cancer

### **1. Introduction**

The global burden of cancer is on the rise and in 2012 around 14.1 million new cases were reported with 8.2 million deaths attributed to cancer [1]. Cancer can be subdivided into categories

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

dependingontheareathatisaffected,includingbutnotlimitedtolungcancer,pancreatic cancer and ovarian cancer [2, 3].

Consequently, the development of targeted treatments for a large population is difficult due to the heterogeneity of the tumours. Furthermore, in cases such as ovarian cancer, current treatments, which include the use of platinum‐based cytotoxic chemotherapy, antiangiogenic drugs and poly (ADP‐ribose) polymerase inhibitors, are only beneficial for patients with early stage disease [2]. However, in patients with more advanced stage disease, there is often recurrence of the disease after treatment due to the development of resistance [2]. Therefore, it is essential that diagnostic procedures be explored.

This paradigm shift from focusing on treatments to focusing on early diagnosis of cancer has brought exosomes to the forefront.

Exosomes are small membranous vesicles that are released following the fusion of multive‐ sicular bodies (MVBs) with the cell membrane. They have multiple characteristics including a cup or spherical shape, maximum diameter of approximately 100 nm, a buoyant density of ∼1.12 to ∼1.19 g /mL on a sucrose gradient, endosomal origin and the enrichment of late endosomal membrane markers, including TSG101 and proteins from the tetraspanin family (e.g. CD63) [3, 4]. Exosomes are covered in a variety of cell surface receptors and contain several proteins such as cytoskeletal proteins, adhesion molecules and heat‐shock proteins. Addition‐ ally, they encapsulate diverse miRNA and mRNA, which can impact the bioactivity and functionality of the target cells with which the exosomes interact.

While the role of exosomes during tumour progression remains to be fully established, we postulate that tumour cells release exosomes loaded with specific molecules in response to the microenvironment to prepare for and promote metastasis to specific organs.

## **2. Exosomes: a specific type of extracellular vesicle**

Cells secrete a multitude of EVs of different origin, size, content and function. Recent reports have recognised a specific type of extracellular vesicle termed exosomes. Exosomes are believed to be tumour 'couriers', carrying signals and relocating packages of signalling molecules to initiate processes such as metastasis by preparing the metastatic niche [5, 6].

In contrast to other EVs, which are formed by an inward budding of the plasma membrane, exosomes are secreted through the intraluminal invagination of vesicles termed early endo‐ somes [7]. This leads to the formation of multivesicular bodies (MVBs) which contain intralu‐ minal vesicles (ILVs). These ILVs are then released by the cell through the fusion of the MVB with the cellular membrane. The released ILVs are termed exosomes [4, 5, 8]. Exosomes carry a common set of molecules along with cell‐specific components. Therefore, exosomes contain proteins which are associated with the biogenesis of MVBs such as tetraspanins, Rab GTPases and Annexins [9]. The endosomal‐sorting complex required for transport (ESCRT) pathway facilitates plasma membrane remodelling and is also believed to have a role in ILV formation [10]. Research has also shown that other pathways independent of the ESCRT complex also exist, as an MVB is also formed when the ESCRT complexes are repressed [11, 12, 14, 15].

dependingontheareathatisaffected,includingbutnotlimitedtolungcancer,pancreatic cancer

Consequently, the development of targeted treatments for a large population is difficult due to the heterogeneity of the tumours. Furthermore, in cases such as ovarian cancer, current treatments, which include the use of platinum‐based cytotoxic chemotherapy, antiangiogenic drugs and poly (ADP‐ribose) polymerase inhibitors, are only beneficial for patients with early stage disease [2]. However, in patients with more advanced stage disease, there is often recurrence of the disease after treatment due to the development of resistance [2]. Therefore,

This paradigm shift from focusing on treatments to focusing on early diagnosis of cancer has

Exosomes are small membranous vesicles that are released following the fusion of multive‐ sicular bodies (MVBs) with the cell membrane. They have multiple characteristics including a cup or spherical shape, maximum diameter of approximately 100 nm, a buoyant density of ∼1.12 to ∼1.19 g /mL on a sucrose gradient, endosomal origin and the enrichment of late endosomal membrane markers, including TSG101 and proteins from the tetraspanin family (e.g. CD63) [3, 4]. Exosomes are covered in a variety of cell surface receptors and contain several proteins such as cytoskeletal proteins, adhesion molecules and heat‐shock proteins. Addition‐ ally, they encapsulate diverse miRNA and mRNA, which can impact the bioactivity and

While the role of exosomes during tumour progression remains to be fully established, we postulate that tumour cells release exosomes loaded with specific molecules in response to the

Cells secrete a multitude of EVs of different origin, size, content and function. Recent reports have recognised a specific type of extracellular vesicle termed exosomes. Exosomes are believed to be tumour 'couriers', carrying signals and relocating packages of signalling molecules to initiate processes such as metastasis by preparing the metastatic niche [5, 6].

In contrast to other EVs, which are formed by an inward budding of the plasma membrane, exosomes are secreted through the intraluminal invagination of vesicles termed early endo‐ somes [7]. This leads to the formation of multivesicular bodies (MVBs) which contain intralu‐ minal vesicles (ILVs). These ILVs are then released by the cell through the fusion of the MVB with the cellular membrane. The released ILVs are termed exosomes [4, 5, 8]. Exosomes carry a common set of molecules along with cell‐specific components. Therefore, exosomes contain proteins which are associated with the biogenesis of MVBs such as tetraspanins, Rab GTPases and Annexins [9]. The endosomal‐sorting complex required for transport (ESCRT) pathway facilitates plasma membrane remodelling and is also believed to have a role in ILV formation

and ovarian cancer [2, 3].

366 Hypoxia and Human Diseases

it is essential that diagnostic procedures be explored.

functionality of the target cells with which the exosomes interact.

**2. Exosomes: a specific type of extracellular vesicle**

microenvironment to prepare for and promote metastasis to specific organs.

brought exosomes to the forefront.

Whilst the biogenesis of exosomes has been well understood and defined in recent literature, a consensus on the method to extract exosomes is yet to be established. However, a detailed discussion of the current methodological approaches is beyond the scope of this chapter [16, 17]. A NanoSight Tracking Analysis (NTA) comparison between exosomes and microvesicles is shown in **Figure 1**.

**Figure 1.** Nanoparticle‐tracking analysis using the NanoSight. Representative image of the size distribution of 100,000 g pellet (A) and exosomes (B). The NanoSight instrument measured the rate of Brownian motion of nanoparticles and consists in a light‐scattering system that provides a reproducible platform for specific and general nanoparticle charac‐ terization (NanoSight Ltd., Amesbury, UK).

Nonetheless, the requirement for a standard isolation procedure is essential as research moves towards examining exosomes as potential therapeutic agents in the context of several diseases such as cancer. Additionally, exosomes are being used to understand the characteristics of the solid tumour, circulating tumour cells and the tumour microenvironment, especially under conditions such as hypoxia.

## **3. The tumour microenvironment and hypoxia**

Under normal conditions, the cellular microenvironment inhibits the development of cancer‐ ous cells through tumour interactions thus allowing the environment to annihilate the growth of cancerous cells. The tumour microenvironment comprises endothelial cells (ECs), fibro‐ blasts, perivascular cells and inflammatory cells. These components tend to control the tumourigenic processes—that is, angiogenesis, desmoplasia, lymphangiogenesis and inflam‐ mation. Oxygen deficiency in tumour cells, also known as hypoxia, is among the major factors that trigger tumour development and hinder clinical diagnoses [13–15].

An imbalance between oxygen supply and demand causes hypoxic or anoxic conditions. The oxygen supply rate is equivalent to that of metabolic requirements in a normal cell or a tissue. However, in developed solid tumours, the oxygen consumption rate may fluctuate to adjust for the insufficient oxygen supply, allowing the tissues to develop even in regions with low oxygen levels [15]. Accumulating evidence suggests that up to 60% of locally advanced tumours display hypoxic (≤1% O2 compared to 2–9% O2 or 40 mm Hg on average in most mammalian tissues) and/or anoxic (≤0.01% O2 or undetectable oxygen) areas distributed heterogeneously throughout the tumour and that tumour hypoxia correlates with advanced stages of malignancy [16]. In cancer cells, respective mechanisms are activated to respond to changes in the availability of oxygen. The cells are subjected to lower levels of oxygen and must therefore modify their metabolism, respectively. In such conditions, the hypoxia‐ inducible factor‐1 (HIF‐1) transcription factor programme of gene expression changes. This change in expression is assumed to enable the cell to cope with the new environment [17–19].

HIF‐1 is a heterodimer complex consisting of two bHLH transcription factors: HIF‐1α and HIF‐ 1β [20]. HIF‐1α expression is significantly overexpressed in advanced ovarian tumours. O2‐ dependent mechanisms primarily regulate HIF‐1α degradation. Under normoxic conditions, the O2‐dependent hydroxylation of proline residues in HIF‐1α by prolyl hydrolase‐domain protein is recognised by the von Hippel‐Lindau tumour‐suppressor protein and ubiquitinated to be targeted for degradation. Under hypoxic conditions, HIF‐1α stabilises and accumulates due to the inhibition of hydroxylation and von Hippel‐Lindau protein‐mediated ubiquitina‐ tion, translocates to the nucleus and forms a complex with HIF‐1β and a transcriptional co‐ activator CBP/p300 to activate the transcription of target genes by directly binding to their hypoxia‐responsive elements [21].

During hypoxia, HIF‐1 activates genes involved in proliferation, cell survival, angiogenesis, vascular tone, metal transport, glycolysis, mitochondrial function, cell growth and survival, and apoptosis and EMT, which all contribute to tumour progression. HIF‐1‐dependent expression of erythropoietin and angiogenic compounds further enhances the formation of blood vessels and thus facilitates the delivery of oxygenated blood to the hypoxic tissue through the induction of vascular endothelial growth factors (VEGFs). In vitro studies show that increasingly subjecting cancer cells to a hypoxic stimulus results in a gradual increase in VEGF mRNA levels and VEGF protein levels [22, 23].

The addition of HIF‐1‐induced glycolytic enzymes provides energy as a substrate for oxidative phosphorylation when mitochondria are starved of oxygen [17]. Moreover, due to lower levels of oxygen and nutrients, the ATP level decreases causing a deregulation in the actin cytoske‐ leton controlled by the down‐regulation of Rho proteins. Rho kinase facilitates contractile force generation mediated by actin‐myosin by phosphorylating a number of target proteins. Rho/Rho kinase plays a critical role in movement, penetration, cell‐cell adhesion, smooth muscle contraction, cytokinesis, mitosis, multiplication, variation, apoptosis and oncogenic transformation within the cell [24–28].

Tumour hypoxia and HIF‐1α overexpression have been demonstrated to induce EMT and metastatic phenotypes in cancer cells, yet the crosstalk between the HIF‐signalling pathway and EMT is not completely understood [29]. Several studies propose potential molecular mechanisms, such as HIF‐1‐promoting EMT through the up‐regulation of EMT transcriptional factors. Nonetheless, it is known that HIF‐1 regulates TWIST expression by binding to their hypoxia‐responsive elements. Thus, cells cultured under hypoxia or constitutive HIF‐1α expression promoted EMT, whereas the repression of TWIST expression abolished the effect of HIF‐1α, shifting the cells back to an epithelial phenotype from the mesenchymal phenotype [29]. HIF‐1 expression induced by hypoxia represses E‐cadherin‐coding genes through SNAI1 and SNAI2 [30–32]. Along with transcriptional factors, hypoxia and HIF‐1 activate EMT‐ associated signalling pathways. Hypoxia also activates the Wnt/β‐catenin‐signalling pathway by inhibiting GSK3β activation, preventing β‐catenin phosphorylation and destruction to increase SNAI1 expression [33]. HIF‐1 further interacts with the Notch intracellular domain to increase its transcriptional activity [34].

However, in developed solid tumours, the oxygen consumption rate may fluctuate to adjust for the insufficient oxygen supply, allowing the tissues to develop even in regions with low oxygen levels [15]. Accumulating evidence suggests that up to 60% of locally advanced tumours display hypoxic (≤1% O2 compared to 2–9% O2 or 40 mm Hg on average in most mammalian tissues) and/or anoxic (≤0.01% O2 or undetectable oxygen) areas distributed heterogeneously throughout the tumour and that tumour hypoxia correlates with advanced stages of malignancy [16]. In cancer cells, respective mechanisms are activated to respond to changes in the availability of oxygen. The cells are subjected to lower levels of oxygen and must therefore modify their metabolism, respectively. In such conditions, the hypoxia‐ inducible factor‐1 (HIF‐1) transcription factor programme of gene expression changes. This change in expression is assumed to enable the cell to cope with the new environment [17–19]. HIF‐1 is a heterodimer complex consisting of two bHLH transcription factors: HIF‐1α and HIF‐ 1β [20]. HIF‐1α expression is significantly overexpressed in advanced ovarian tumours. O2‐ dependent mechanisms primarily regulate HIF‐1α degradation. Under normoxic conditions, the O2‐dependent hydroxylation of proline residues in HIF‐1α by prolyl hydrolase‐domain protein is recognised by the von Hippel‐Lindau tumour‐suppressor protein and ubiquitinated to be targeted for degradation. Under hypoxic conditions, HIF‐1α stabilises and accumulates due to the inhibition of hydroxylation and von Hippel‐Lindau protein‐mediated ubiquitina‐ tion, translocates to the nucleus and forms a complex with HIF‐1β and a transcriptional co‐ activator CBP/p300 to activate the transcription of target genes by directly binding to their

During hypoxia, HIF‐1 activates genes involved in proliferation, cell survival, angiogenesis, vascular tone, metal transport, glycolysis, mitochondrial function, cell growth and survival, and apoptosis and EMT, which all contribute to tumour progression. HIF‐1‐dependent expression of erythropoietin and angiogenic compounds further enhances the formation of blood vessels and thus facilitates the delivery of oxygenated blood to the hypoxic tissue through the induction of vascular endothelial growth factors (VEGFs). In vitro studies show that increasingly subjecting cancer cells to a hypoxic stimulus results in a gradual increase in

The addition of HIF‐1‐induced glycolytic enzymes provides energy as a substrate for oxidative phosphorylation when mitochondria are starved of oxygen [17]. Moreover, due to lower levels of oxygen and nutrients, the ATP level decreases causing a deregulation in the actin cytoske‐ leton controlled by the down‐regulation of Rho proteins. Rho kinase facilitates contractile force generation mediated by actin‐myosin by phosphorylating a number of target proteins. Rho/Rho kinase plays a critical role in movement, penetration, cell‐cell adhesion, smooth muscle contraction, cytokinesis, mitosis, multiplication, variation, apoptosis and oncogenic

Tumour hypoxia and HIF‐1α overexpression have been demonstrated to induce EMT and metastatic phenotypes in cancer cells, yet the crosstalk between the HIF‐signalling pathway and EMT is not completely understood [29]. Several studies propose potential molecular mechanisms, such as HIF‐1‐promoting EMT through the up‐regulation of EMT transcriptional factors. Nonetheless, it is known that HIF‐1 regulates TWIST expression by binding to their

hypoxia‐responsive elements [21].

368 Hypoxia and Human Diseases

transformation within the cell [24–28].

VEGF mRNA levels and VEGF protein levels [22, 23].

The Notch‐targeted genes HES1 and HEY1 were increased under hypoxic conditions; however, a knockdown of HIF‐1α abrogated the hypoxia‐induced HES1 and HEY1 expression as well as the SNAI1 expression [35]. Furthermore, HIF‐1α targeted lysyl oxidase and lysyl oxidase‐ like 2 and 3 enzymes, which promote tumour metastasis by mediating cells to matrix adhesion and stabilising SNAI1 activity to induce EMT [36, 37]. Under hypoxia, the consumption of glucose and GLUT1 expression in cancer cells increased as well [17].

It is also well established that bidirectional communication between cancer cells and their tumour microenvironment is essential for cancer progression. For example, most ovarian cancer patients present with ascites—excess fluid in the peritoneal cavity [38]. Ovarian cancer ascites contain molecular factors, including VEGF, cytokines, chemokines and TGF‐β, to mediate cellular communication for effective tumourigenesis. Accumulating evidence suggests that cellular communication is not only limited to secretary molecules, but also includes EVs (such as exosomes) that mediate such communication [39]. The nomenclature of EVs is still a matter of debate due to the many terms used (e.g. microvesicles, nanovesicles, shedding vesicles and ectosomes), emphasising the range of EV populations secreted [9].

However, during tumourigenesis, hypoxia serves as a selective agent at various physiological levels. Under hypoxia, a number of transcriptional factors control the cell environment, including Nuclear Factor‐kappaB (NF‐*κ*B), Activating Transcription Factors (ATFs) and p53s [40–42]. In NF‐*κ*B pathways activated by HIF during irregular hypoxia and re‐oxygenation [41, 43] and ATF, anoxia drives signalling [44].

Moreover, carbonic anhydrase IX (CAIX) is among the genes in the hypoxic environment of solid tumours that increasingly express themselves. CAIX expression is perceived as causing bladder, ovarian, cervical, colorectal, oral, brain and breast cancers. It enables the balancing of intracellular pH through the extracellular hydration of CO2 and the production of bicarbonate and protons. The bicarbonate goes back into the cell through bicarbonate transporters and balances the intracellular pH as alkaline, which is favourable for the cell's survival. The protons acidify the extracellular space, thus facilitating the tumour's migratory and invasive behav‐ iour [45–47]. CAIX expression and activity also facilitates the production of Granulocyte‐ colony stimulating factor (G‐CSF),which is in turn required for the transportation of granulocytic Myeloid‐derived Suppressor Cells (MDSC) to the metastatic niche—an environ‐ ment that promotes metastasis. CAIX expression is also required to stimulate NF‐*κ*B activity and G‐CSF production mediated through hypoxia. The hypoxia‐mediated NF‐*κ*B activity is triggered by a decrease in the pH level of the culture as well as hypoxia‐induced glycolytic activity in the cancer cells [46, 48–50]. The hypoxic areas of tumours usually have lower levels of extracellular pH due to increased metabolic activity [45]. It has been proven that the cells are hampered from acidifying the medium due to a smaller production rate of CAIX in a hypoxic environment [51]. Therefore, hypoxia and the tumour microenvironment are essential factors in regulating disease progression and metastasis.

## **4. Exosomes, the tumour microenvironment and hypoxia**

An evaluation of cancer cells and their microenvironment plays a critical role in hypoxia. Tumour cells under hypoxia secrete molecules that modulate their microenvironment and facilitate tumour angiogenesis and metastasis. Hypoxia is a major hallmark of the tumour environment and is caused when there is a lack of blood supply. The lowered blood supply indicates a lower number of red blood cells being able to reach the tumour cells resulting in decreased oxygen delivery [52]. Moreover, hypoxic tumours have a greater ability to resist standard treatments and the tumour cells are often in a less differentiated or more stem cell‐like state [53]. Emerging evidence has shown that exosomes are key membrane vesicles secreted by most cell types under hypoxia. It has also been shown that they have an ability to modulate the tumourmicroenvironmenttoensureadequatenutritionandoxygensupply[54].Therehasbeen an increasing interest in the role of exosomes as a mediator of cell‐to‐cell communication and its role in ultimately aiding cancer progression.

There have been several processes proposed regarding the release of exosomes into the tumour microenvironment.Theseprocessesinvolveseveralmoleculessuchasproteinsinvolvedinfusion ofthemultivesicularbodiesaswellasplasmamembraneproteins.Additionally,ithasbeenshown that exosomes present in a cell's environment also regulate exosome release. Riches and collea‐ gues showed that when exosomes were added to the culture medium of cells, the number of exosomes released by the cells decreased evidently [55]. Other proteins that may be involved in increased exosome secretion during hypoxia include the Rab family of proteins, specifically Rab27 as they regulate exosome secretion. The Rab27 protein has two isoforms: Rab27a and Rab27b.OstrowskiandcolleaguesnotedthatinhibitingRab9a,Rab5a,Rab27a,Rab27bandRab2b led to an inhibition in exosome release [35]. Furthermore, it has been previously shown that the presence of calcium (Ca2+) ionophores can lead to an increase in the release of exosomes [56]. Therefore, although there are several hypotheses, the exact mechanism is still unclear. Thus, the mechanisms underlying exosome release under different tumour microenvironmental condi‐ tions such as hypoxia remain to be elucidated. Nonetheless, progress is being made.

The role of exosomes in tumour progression and invasion has been highlighted in literature with a clear correlation being found between the number of hypoxic exosomes released and the aggressiveness of the tumour [57, 58]. A significant increase in the number of exosomes released under hypoxia (1% oxygen) and severe anoxia (0.1% oxygen) was found in a study conducted on three breast cancer cell lines, in which the impact of hypoxia on tumour progression and the release of exosomes was investigated [58]. King and colleagues postulated that the enhancement of exosome release might be mediated by the hypoxia‐inducible factor 1 oxygen‐sensing pathway (detailed above). They tested their hypothesis by using the HIF hydroxylase inhibitor, Dimethyloxalylglycine (DMOG), to treat the breast cancer cell line, MDA‐MB‐231 [58]. The role of the DMOG was to trigger an HIF response. This led to a minor although significant rise in the number of exosomes secreted by the cells when quantified by nanoparticle‐tracking analysis (NTA). Moreover, when the HIF‐1α transcription factor was silenced using siRNA, the increase in exosomes in response to hypoxia was not seen. Therefore, it was concluded that the HIF pathway may have a significant role in the release of exosomes in response to hypoxia. Similar studies were carried out on different cell lines (e.g. leukaemia cell line, K562; human microvascular endothelial cells (HMEC‐1); A431 squamous carcinoma; A549 non‐small‐cell lung (NSCL); H1299 NSCL and HFF‐1 foreskin fibroblast cells) to investigate the level of exosomes released under hypoxia and normoxia [57]. The outcome was that the number of exosomes released under hypoxic conditions increased when compared to exosomes released by cells under normoxic conditions in the same amount of time. However, the pathways underlying the hypoxic enhancement of exosome release were unclear [15].

activity in the cancer cells [46, 48–50]. The hypoxic areas of tumours usually have lower levels of extracellular pH due to increased metabolic activity [45]. It has been proven that the cells are hampered from acidifying the medium due to a smaller production rate of CAIX in a hypoxic environment [51]. Therefore, hypoxia and the tumour microenvironment are essential

An evaluation of cancer cells and their microenvironment plays a critical role in hypoxia. Tumour cells under hypoxia secrete molecules that modulate their microenvironment and facilitate tumour angiogenesis and metastasis. Hypoxia is a major hallmark of the tumour environment and is caused when there is a lack of blood supply. The lowered blood supply indicates a lower number of red blood cells being able to reach the tumour cells resulting in decreased oxygen delivery [52]. Moreover, hypoxic tumours have a greater ability to resist standard treatments and the tumour cells are often in a less differentiated or more stem cell‐like state [53]. Emerging evidence has shown that exosomes are key membrane vesicles secreted by most cell types under hypoxia. It has also been shown that they have an ability to modulate the tumourmicroenvironmenttoensureadequatenutritionandoxygensupply[54].Therehasbeen an increasing interest in the role of exosomes as a mediator of cell‐to‐cell communication and

There have been several processes proposed regarding the release of exosomes into the tumour microenvironment.Theseprocessesinvolveseveralmoleculessuchasproteinsinvolvedinfusion ofthemultivesicularbodiesaswellasplasmamembraneproteins.Additionally,ithasbeenshown that exosomes present in a cell's environment also regulate exosome release. Riches and collea‐ gues showed that when exosomes were added to the culture medium of cells, the number of exosomes released by the cells decreased evidently [55]. Other proteins that may be involved in increased exosome secretion during hypoxia include the Rab family of proteins, specifically Rab27 as they regulate exosome secretion. The Rab27 protein has two isoforms: Rab27a and Rab27b.OstrowskiandcolleaguesnotedthatinhibitingRab9a,Rab5a,Rab27a,Rab27bandRab2b led to an inhibition in exosome release [35]. Furthermore, it has been previously shown that the presence of calcium (Ca2+) ionophores can lead to an increase in the release of exosomes [56]. Therefore, although there are several hypotheses, the exact mechanism is still unclear. Thus, the mechanisms underlying exosome release under different tumour microenvironmental condi‐

tions such as hypoxia remain to be elucidated. Nonetheless, progress is being made.

The role of exosomes in tumour progression and invasion has been highlighted in literature with a clear correlation being found between the number of hypoxic exosomes released and the aggressiveness of the tumour [57, 58]. A significant increase in the number of exosomes released under hypoxia (1% oxygen) and severe anoxia (0.1% oxygen) was found in a study conducted on three breast cancer cell lines, in which the impact of hypoxia on tumour progression and the release of exosomes was investigated [58]. King and colleagues postulated that the enhancement of exosome release might be mediated by the hypoxia‐inducible factor 1 oxygen‐sensing pathway (detailed above). They tested their hypothesis by using the HIF

factors in regulating disease progression and metastasis.

370 Hypoxia and Human Diseases

its role in ultimately aiding cancer progression.

**4. Exosomes, the tumour microenvironment and hypoxia**

In addition, oncogenic miR‐21 was identified at a significant level in exosome fractions [59, 64]. miR‐21 is known to down‐regulate programmed cell death 4 (PDCD4) expression by directly targeting its 3′‐untranslated region. Moreover, it was found that exosomes isolated from peritoneal effusions (ovarian cancer) contained low PDCD4 expression, whereas oncogenic miR‐21 was highly expressed compared to exosomes isolated from non‐neoplastic peritoneal effusions [59]. The use of exosomal miR‐21 as a biomarker for cancer diagnosis has been suggested in several studies as it exists in almost all bodily fluids, is stable and is protected from degradation [60]. Exosomal miR‐21 has an effect on a number of signalling pathways which promote metastatic capacity and proliferation. It has been found that miR‐21 suppresses phosphatase and tensin homolog expression and promotes the growth and migration of tumour cells [61]. miR‐21 also regulates cellular functions by influencing signal transduction, proliferation, carcinogenesis, differentiation and immune response [62–64]. These observa‐ tions provide key evidence that elevated exosome release under hypoxia is a critical factor affecting tumour proliferation.

Tumour‐derived exosomes have the ability to transfer oncogenic activity among tumour cells. Human glioma cells can horizontally transfer an oncogenic form of epidermal growth factor variant III (EGFRvIII) to glioma cells lacking EGFRvIII [65]. The transfer results in an increased expression of the pro‐survival gene and a reduction in the cell cycle inhibitor, increasing anchorage‐independent growth capacity [65]. An interesting possibility that exosomes are key factors that affect the neighbouring cells is provided by these studies.

Exosomes facilitate communication among tumour cells and contribute to the development of a favourable microenvironment for tumour progression by enhancing processes such as angiogenesis. Angiogenesis is promoted by the activation of endothelial cells through tumour‐ derived exosomes, and is followed up by the activation of myofibroblasts, a source of matrix‐ remodelling protein [66, 67]. Tumour‐derived exosomes trigger fibroblast to myofibroblast differentiation [68]. In addition to fibroblasts, exosomes can trigger conversion of mesenchy‐ mal stem cells from the tumour stroma and adipose tissue to myofibroblasts [69]. The exosomes also contribute to the formation of pre‐metastatic niches by educating the bone marrow‐ derived cells (BMDC). BMDCs when combined with exosomes derived from highly and poorly metastatic melanoma cells accelerated primary tumour growth and also increased the magnitude and number of metastases [6]. Additionally, evidence has shown that exosomes interact with immune cells to suppress antitumour responses and skew them towards the pro‐ tumourigenic phenotype [70]. Exosomes from hypoxic endothelial cells (EC) show up‐ regulation of collagen crosslinking activity by activation of lysyl oxidase‐like 2 [71]. Lysyl oxidase‐like 2 (LOXL2) has been linked to extracellular matrix (ECM) remodelling, angiogen‐ esis, cell proliferation, migration, transcription regulation, fibroblast activation, EMT and metastatic niche formation through a number of processes [36, 72, 73]. The tumour cells can communicate with multiple different cell types via exosomes. Therefore, it is highly likely that this leads to a complex network of interactions.

The reaction of the target cells upon treatment with exosomes depends on the exosomal composition, which has been previously described as being diverse, and the transfer of encapsulated molecules [4, 67]. This ability of exosomes to protect and transfer molecules has led to the hypothesis that they could be used as potential tumour biomarkers or as a non‐ invasive tumour biopsy.

## **5.** *In vivo* **biodistribution of exosomes**

Functional characterisation of exosomes often involves the use of an *in vivo* mouse model. Such experiments can give the biodistribution and pharmacokinetic parameters of the exosomes tested, which is important for understanding exosome trafficking and their physiological roles [74].

The starting point at which exosomes are to be isolated varies based on the experimental goals. In studies investigating the role of tumour‐derived exosomes in cancer progression, exosomes were isolated from various cancer cell lines such as breast cancer, pancreatic cancer, gastric cancer and colorectal cancer [75]. Another area of interest is the potential use of exosomes as therapeutic carriers of antitumour microRNA or chemotherapy agents [76, 77]. This may allow for improved tissue targeting, increasing the potency of the delivered drug [78]. Exosomes are often isolated from cell‐conditioned media with differential centrifugation being the most common method of enriching exosomes [75–77, 79, 80]. Most of the exosome isolation protocols involved low‐speed centrifugation steps to remove cells and cell debris followed by high‐speed centrifugation at 100,000 g and a washing step of the pellet with a final centrifugation. In a study by Alvarez‐Erviti et al. [77], exosomes were derived from cultured dendritic cells, which was chosen based on data demonstrating that dendritic cell‐derived exosomes contained immune‐stimulating components such as major histocompatibility complex (MHC) class I and class II molecules in addition to T‐cell‐stimulating molecule, CD86 [81]. Studies also showed that isolated exosomes can be loaded with exogenous RNA or chemotherapy drugs by different methods, including electroporation and sonication [77, 78].

To enable the *in vivo* tracking of exosomes, they can be labelled post isolation with a lipophilic membrane dye such as Paul Karl Horan (PKH), DiOC18 (DIR) or DiIC18 (Dil) [75, 80, 82]. An alternative method of generating labelled exosomes is by transfecting donor cells with a construct encoding for a fluorescence‐membrane fusion protein. In this approach, a mem‐ brane‐bound variant of bioluminescence reporter, Gaussia luciferase, is transfected into the donor cells, producing luciferase‐labelled exosomes [82, 83]. A major difference between the two labelling approaches is the time and expertise required. The post‐isolation membrane dye labelling is quick (∼1 h), whereas the transfection of cells requires additional time (∼2 weeks) and expertise in vector and viral cloning and transfection [79, 84]. Additionally, a study by Lai et al [83] reported quicker rates of clearance of transfected luciferase‐labelled exosomes compared to the dye‐labelled exosomes. The authors attributed this difference to the possibility of the highly stable dyes being an artefact instead of indicating intact exosomal presence.

metastatic melanoma cells accelerated primary tumour growth and also increased the magnitude and number of metastases [6]. Additionally, evidence has shown that exosomes interact with immune cells to suppress antitumour responses and skew them towards the pro‐ tumourigenic phenotype [70]. Exosomes from hypoxic endothelial cells (EC) show up‐ regulation of collagen crosslinking activity by activation of lysyl oxidase‐like 2 [71]. Lysyl oxidase‐like 2 (LOXL2) has been linked to extracellular matrix (ECM) remodelling, angiogen‐ esis, cell proliferation, migration, transcription regulation, fibroblast activation, EMT and metastatic niche formation through a number of processes [36, 72, 73]. The tumour cells can communicate with multiple different cell types via exosomes. Therefore, it is highly likely that

The reaction of the target cells upon treatment with exosomes depends on the exosomal composition, which has been previously described as being diverse, and the transfer of encapsulated molecules [4, 67]. This ability of exosomes to protect and transfer molecules has led to the hypothesis that they could be used as potential tumour biomarkers or as a non‐

Functional characterisation of exosomes often involves the use of an *in vivo* mouse model. Such experiments can give the biodistribution and pharmacokinetic parameters of the exosomes tested, which is important for understanding exosome trafficking and their physiological

The starting point at which exosomes are to be isolated varies based on the experimental goals. In studies investigating the role of tumour‐derived exosomes in cancer progression, exosomes were isolated from various cancer cell lines such as breast cancer, pancreatic cancer, gastric cancer and colorectal cancer [75]. Another area of interest is the potential use of exosomes as therapeutic carriers of antitumour microRNA or chemotherapy agents [76, 77]. This may allow for improved tissue targeting, increasing the potency of the delivered drug [78]. Exosomes are often isolated from cell‐conditioned media with differential centrifugation being the most common method of enriching exosomes [75–77, 79, 80]. Most of the exosome isolation protocols involved low‐speed centrifugation steps to remove cells and cell debris followed by high‐speed centrifugation at 100,000 g and a washing step of the pellet with a final centrifugation. In a study by Alvarez‐Erviti et al. [77], exosomes were derived from cultured dendritic cells, which was chosen based on data demonstrating that dendritic cell‐derived exosomes contained immune‐stimulating components such as major histocompatibility complex (MHC) class I and class II molecules in addition to T‐cell‐stimulating molecule, CD86 [81]. Studies also showed that isolated exosomes can be loaded with exogenous RNA or chemotherapy drugs by different

To enable the *in vivo* tracking of exosomes, they can be labelled post isolation with a lipophilic membrane dye such as Paul Karl Horan (PKH), DiOC18 (DIR) or DiIC18 (Dil) [75, 80, 82]. An alternative method of generating labelled exosomes is by transfecting donor cells with a

this leads to a complex network of interactions.

**5.** *In vivo* **biodistribution of exosomes**

methods, including electroporation and sonication [77, 78].

invasive tumour biopsy.

372 Hypoxia and Human Diseases

roles [74].

Exosomes injected into mice are commonly quantified using the Coomassie dye (Bradford)‐ based method, or copper‐based chemistry such as the Bicinchoninic Acid Assay (BCA) [75–77, 82–85]. The yield of exosomes obtained often ranges from 6 to 12 μg/106 ‐cultured dendritic cells, 69.2 μg/2–5 × 107 of HEK293 and 2–4 ug/106 HEK cells [76, 77, 79]. There is some ambiguity in the quantification of exosomal protein concentration in these studies. Presumably, the exosomes were first lysed pre‐quantification as without lysing the exosomes, only the mem‐ brane‐bound proteins would be quantified. Another method of determining the required number of exosomes is to use the number of exosomes per gram of animal weight. Techniques such as NTA are used to quantify the number of exosomal particles and their size distribution [85, 90]. Importantly, in order to translate the use of exosomes into a clinical setting, standard‐ ising the dose of exosomes injected is critical. Given that isolated exosomes from current techniques such as ultracentrifugation are heterogeneous in size when observed using NTA [85], it is likely that the difference in size translates to differences in total protein concentration. Therefore, methods that quantify the total protein content within exosomes such as the Bradford/BCA assays are a better means of measuring the protein content and thus exoso‐ mal dose.

For biodistribution and tissue‐uptake studies, the dose of injected exosomes ranged from 4 to 10 μg per mouse [75, 82]. Alternatively, a dosing range of 1.5 × 1010 particles/gram body weight (p/g), 1.0 × 1010 p/g and 0.25 × 1010 p/g was used [85]. In studies where exosomes were used as a potential therapeutic siRNA carrier, the dose of exosomes chosen was much higher, at 150 μg/mouse [77]. An explanation of a higher dose employed could be that systemically admin‐ istered exosomes are rapidly cleared from the bloodstream, with evidence to suggest that macrophages play a role in exosome clearance [80]. Therefore, the higher dose was chosen to induce a measurable response.

Once the labelled exosomes are administered, the duration of monitoring ranged from 10 min to 6 h for biodistribution studies, which met the goal of tracking the localisation of exosomes over time [82, 83]. It was demonstrated that injected exosomes localised primarily in the liver and lungs [82, 85]. Moreover, it was shown recently that particular integrin expression on tumour‐derived exosomes could be used to predict organ‐specific metastasis [75]. In particular, exosomes expressing α6β4 and α6β1 were linked with lung metastasis, while exosomal integrin αvβ5 was associated with liver metastasis. For exosomes which carried modified cargo, such as siRNA targeting the abundant GAPDH, the effect induced by the cargo was measured 3 days post injection [77]. This study showed the possibility of using exosome‐ mediated delivery of potentially therapeutic siRNA to induce a gene‐specific knockdown.

In summary, *in vivo* characterisation is an important step in gaining an understanding of the physiological pathways that exosomes are involved in. Further research will strengthen the proposal of using exosomes as a therapeutic carrier and potential diagnostic tool.

## **6. New approaches to elucidate the role of exosomes in cancer**

Identification of biomarkers to detect cancer during its early stages has the potential to improve patient outcomes significantly with exosomes currently being considered. As exosomes are released and circulate in the peripheral circulation, they can be collected from diverse bio‐ fluids through minimally invasive procedures from the blood and non‐invasive procedures from saliva and urine. Through the isolation and purification process, exosomes are separated from highly abundant proteins present in bodily fluids [56]. Furthermore, cancer‐derived exosomes can be specifically distinguished from exosomes originating from other cells by the expression of markers such as CD24 and EpCAM [86]. Storage of exosomes does not signifi‐ cantly affect their protein and miRNA contents thus highlighting their high stability [87]. Most importantly, the release and content of exosomes reflect the tumour state and their microen‐ vironment [88].

Encapsulation of cellular proteins and RNA molecules into exosomes makes exosomes an enriched source of tumour markers, which provides an insight into the originating tumour cells. miRNAs are evolutionarily conserved regulating several cellular processes such as cell differentiation, proliferation and apoptosis [89]. These cellular processes are often altered in cancer‐enhancing cellular transformation and tumourigenesis by impaired miRNA biogenesis; therefore, miRNA profiles can differentiate cancer tissues from benign tissues [90]. A complete miRNA‐profiling study in epithelial ovarian cancer (EOC) has identified aberrantly expressed miRNA in different subtypes of EOC compared to normal ovaries [91]. Ovarian tumour‐ derived exosomes isolated from patient sera exhibited similar miRNA profiles to originating tumour cells and the exosome concentration was positively correlated with the progression of disease, highlighting the diagnostic potential of exosomal miRNA [92]. High exosomal miR‐ 21, miR‐23b and miR‐29a expression of ovarian cancer patient effusion correlated with poor progression‐free survival and poor overall survival was related to high expression of miR‐21 suggesting their use as prognostic markers [93].

A recent study established the role of EOC‐derived exosomes in mediating the activation of macrophages to a tumour‐associated macrophage (TAM) state [94]. They also demonstrated that SKOV‐3 cells when grown with conditioned media from the transformed macrophages were more likely to migrate and proliferate.

Additional studies have proposed the use of exosomes as both diagnostic biomarkers and therapeutic agents [95]. It has been proposed that exosomes be used to transport antitumour complexes such as drugs to the tumour cells, thus providing a form of targeted therapy. Furthermore, it has been shown that decreasing exosome production by blocking Rab27a (responsible for exosome release) can also reduce primary tumour growth [96].

Compared to the currently available detection methods, the use of exosomes as biomarkers will involve minimally invasive procedures and as the exosomal content reflects the originating cancer cells and their microenvironment, they will have greater specificity. This will decrease the need for surgical interventions and deaths from surgical complications as a result of false‐ positive results [97].

## **Author details**

measured 3 days post injection [77]. This study showed the possibility of using exosome‐ mediated delivery of potentially therapeutic siRNA to induce a gene‐specific knockdown.

In summary, *in vivo* characterisation is an important step in gaining an understanding of the physiological pathways that exosomes are involved in. Further research will strengthen the

Identification of biomarkers to detect cancer during its early stages has the potential to improve patient outcomes significantly with exosomes currently being considered. As exosomes are released and circulate in the peripheral circulation, they can be collected from diverse bio‐ fluids through minimally invasive procedures from the blood and non‐invasive procedures from saliva and urine. Through the isolation and purification process, exosomes are separated from highly abundant proteins present in bodily fluids [56]. Furthermore, cancer‐derived exosomes can be specifically distinguished from exosomes originating from other cells by the expression of markers such as CD24 and EpCAM [86]. Storage of exosomes does not signifi‐ cantly affect their protein and miRNA contents thus highlighting their high stability [87]. Most importantly, the release and content of exosomes reflect the tumour state and their microen‐

Encapsulation of cellular proteins and RNA molecules into exosomes makes exosomes an enriched source of tumour markers, which provides an insight into the originating tumour cells. miRNAs are evolutionarily conserved regulating several cellular processes such as cell differentiation, proliferation and apoptosis [89]. These cellular processes are often altered in cancer‐enhancing cellular transformation and tumourigenesis by impaired miRNA biogenesis; therefore, miRNA profiles can differentiate cancer tissues from benign tissues [90]. A complete miRNA‐profiling study in epithelial ovarian cancer (EOC) has identified aberrantly expressed miRNA in different subtypes of EOC compared to normal ovaries [91]. Ovarian tumour‐ derived exosomes isolated from patient sera exhibited similar miRNA profiles to originating tumour cells and the exosome concentration was positively correlated with the progression of disease, highlighting the diagnostic potential of exosomal miRNA [92]. High exosomal miR‐ 21, miR‐23b and miR‐29a expression of ovarian cancer patient effusion correlated with poor progression‐free survival and poor overall survival was related to high expression of miR‐21

A recent study established the role of EOC‐derived exosomes in mediating the activation of macrophages to a tumour‐associated macrophage (TAM) state [94]. They also demonstrated that SKOV‐3 cells when grown with conditioned media from the transformed macrophages

Additional studies have proposed the use of exosomes as both diagnostic biomarkers and therapeutic agents [95]. It has been proposed that exosomes be used to transport antitumour complexes such as drugs to the tumour cells, thus providing a form of targeted therapy.

proposal of using exosomes as a therapeutic carrier and potential diagnostic tool.

**6. New approaches to elucidate the role of exosomes in cancer**

vironment [88].

374 Hypoxia and Human Diseases

suggesting their use as prognostic markers [93].

were more likely to migrate and proliferate.

Shayna Sharma1 , Mona Alharbi1 , Andrew Lai1 , Miharu Kobayashi1 , Richard Kline2 , Katrina Wade2 , Gregory E. Rice1,2 and Carlos Salomon1,2\*

\*Address all correspondence to: c.salomongallo@uq.edu.au

1 Exosome Biology Laboratory, Centre for Clinical Diagnostics, University of Queensland Centre for Clinical Research, Royal Brisbane and Women's Hospital, The University of Queensland, Brisbane, QLD, Australia

2 Department of Obstetrics and Gynecology, Maternal‐Fetal Medicine, Ochsner Clinic Foun‐ dation, New Orleans, LA, USA

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#### **A Novel Hypoxia Imaging Endoscopy System A Novel Hypoxia Imaging Endoscopy System**

Kazuhiro Kaneko, Hiroshi Yamaguchi and Kazuhiro Kaneko, Hiroshi Yamaguchi and Tomonori Yano

Tomonori Yano

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/66091

#### **Abstract**

Measurement of tumor hypoxia is required for the diagnosis of tumor and the evaluation of therapeutic outcome. Currently, invasive and noninvasive techniques being exploited for tumor hypoxia measurement include polarographic needle electrodes, immunohistochemical (IHC) staining, magnetic resonance imaging (MRI), radionuclide imaging (positron emission tomography [PET] and single-photon emission computed tomography [SPECT]), optical imaging (bioluminescence and fluorescence), and hypoxia imaging endoscopy. This review provides a summary of the modalities available for assessment of tissue oxygenation as well as a discussion of current arguments for and against each modality, with a particular focus on noninvasive hypoxia imaging with emerging agents and new imaging technologies intended to detect molecular events associated with tumor hypoxia.

**Keywords:** Hypoxia imaging endoscopy, innovation of endoscopy

## **1. Introduction**

In the 1950s, hypoxia research began, and many clinical trials have been reported. Hypoxia of tumor affects outcomes after radiotherapy. But hypoxia has also been shown to be a poor prognostic factor after chemotherapy and surgery. These findings are attributed to chronic hypoxia. Hypoxic tumors are more likely to recur loco-regionally than well-oxygenated tumors regardless of whether surgery or radiation therapy is the primary local treatment. However, the common oxygen measurement used in these reports was polarographic needle electrodes inserted directly into specific sections of tumor tissue. In this method, hypoxia was measured in only pinpointed area for the tumor. In other words, there was no modality used

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

in which the hypoxia imaging results were visible in real time and which reflected the hypoxic state in the whole tumor. Therefore, hypoxia imaging is expected to allow direct visualization of the biological and functional changes in cancer.

Hypoxia is a histopathological condition in which cells in tissues suffer from lack of oxygen for their normal metabolism. An oxygen saturation (StO2) of arterial blood is almost 100% and that of venous blood is approximately 70%. In contrast, the StO2 in half of cancers is 50–60% at the highest. Hypoxia takes hold as a tumor becomes large enough to disrupt the balance of oxygen supply and consumption in the area. Approximately, 50–60% of advanced cancer forming solid tumor may show hypoxic and/or anoxic conditions exhibiting heterogeneous distribution in the inside of tumor [1]. Hypoxia proliferates rapidly in solid tumors, and their intratumoral vessels with significantly structural abnormalities are distributed spatially with dilated, tortuous, saccular, and heterogeneous figures. As a result, this distribution leads to perfusion-limited delivery of O2 [2]. There are mainly two types of hypoxia regarding solid tumor and tissue around tumor. One is perfusion-limited O2 delivery type, the so-called acute hypoxia, which leads to ischemic condition, however, it is often transient. Another type is diffusion-limited hypoxia, the so-called chronic hypoxia, which can also be caused by an increase in diffusion distances, so that cells far away (>70 μm) from a nutritive blood vessel receive less oxygen (and nutrients) than needed [1]. Regarding hypoxia-induced proteome and/or genome changes, cell cycle arrest, differentiation, apoptosis, and necrosis are found in solid tumor. In contrast, hypoxia-induced changes of proteome may progress tumor growth because of mechanisms enabling cells to overcome nutritive deprivation, to escape from the hostile environment and to favor unrestricted growth. Furthermore, continuous hypoxia can also bring cellular changes as a more aggressive phenotype [3]. Since the presence of hypoxic status in solid tumors was first reported in 1953 to be among the factors associated with treatment failure following radiation therapy [4], tumor hypoxia has drawn attention as a pivotal event in tumor invasion, angiogenesis, apoptosis, metastasis [1], resistance to chemotherapy [5], surgery, and resistance to radiotherapy [6]. In tumor diagnosis and treatment planning, it is crucial to have a grasp of the degree and extent of tumor hypoxia involved prior to the start of treatment.

## **2. Clinical importance for measurement of tumor hypoxic state**

A variety of techniques are being proposed to assess tumor hypoxia, which can be broadly categorized into direct measurements and indirect measurements according to different principles and the ability to quantify tissue oxygenation. Direct measurements, including polarographic needle electrode, phosphorescence imaging, near-infrared spectroscopy (NIRS), blood oxygen level dependent (BOLD) and 19F magnetic resonance imaging (MRI) and electron paramagnetic resonance (EPR) imaging, can detect oxygen partial pressure (pO2), oxygen concentration, or oxygen percentage. Recently, a hypoxia imaging endoscopy that can derive the oxygen saturation (StO2) was developed in endoscopic fields. Indirect measurements, including measuring exogenous and endogenous hypoxia markers, can provide parameters related to oxygenation.

Many clinical trials have been performed using direct and indirect measurement methods. It is now known that hypoxia affects outcome after radiotherapy, with poor prognosis in hypoxic cancers. Next, hypoxia has also been shown to be a poor prognostic factor after chemotherapy and surgery. Furthermore, hypoxic tumors are more likely to recur loco-regionally than welloxygenated tumors regardless of whether surgery or radiation therapy was the primary local treatment.

## **3. Direct measurements for hypoxia**

in which the hypoxia imaging results were visible in real time and which reflected the hypoxic state in the whole tumor. Therefore, hypoxia imaging is expected to allow direct visualization

Hypoxia is a histopathological condition in which cells in tissues suffer from lack of oxygen for their normal metabolism. An oxygen saturation (StO2) of arterial blood is almost 100% and that of venous blood is approximately 70%. In contrast, the StO2 in half of cancers is 50–60% at the highest. Hypoxia takes hold as a tumor becomes large enough to disrupt the balance of oxygen supply and consumption in the area. Approximately, 50–60% of advanced cancer forming solid tumor may show hypoxic and/or anoxic conditions exhibiting heterogeneous distribution in the inside of tumor [1]. Hypoxia proliferates rapidly in solid tumors, and their intratumoral vessels with significantly structural abnormalities are distributed spatially with dilated, tortuous, saccular, and heterogeneous figures. As a result, this distribution leads to perfusion-limited delivery of O2 [2]. There are mainly two types of hypoxia regarding solid tumor and tissue around tumor. One is perfusion-limited O2 delivery type, the so-called acute hypoxia, which leads to ischemic condition, however, it is often transient. Another type is diffusion-limited hypoxia, the so-called chronic hypoxia, which can also be caused by an increase in diffusion distances, so that cells far away (>70 μm) from a nutritive blood vessel receive less oxygen (and nutrients) than needed [1]. Regarding hypoxia-induced proteome and/or genome changes, cell cycle arrest, differentiation, apoptosis, and necrosis are found in solid tumor. In contrast, hypoxia-induced changes of proteome may progress tumor growth because of mechanisms enabling cells to overcome nutritive deprivation, to escape from the hostile environment and to favor unrestricted growth. Furthermore, continuous hypoxia can also bring cellular changes as a more aggressive phenotype [3]. Since the presence of hypoxic status in solid tumors was first reported in 1953 to be among the factors associated with treatment failure following radiation therapy [4], tumor hypoxia has drawn attention as a pivotal event in tumor invasion, angiogenesis, apoptosis, metastasis [1], resistance to chemotherapy [5], surgery, and resistance to radiotherapy [6]. In tumor diagnosis and treatment planning, it is crucial to have a grasp of the degree and extent of tumor hypoxia involved prior

**2. Clinical importance for measurement of tumor hypoxic state**

A variety of techniques are being proposed to assess tumor hypoxia, which can be broadly categorized into direct measurements and indirect measurements according to different principles and the ability to quantify tissue oxygenation. Direct measurements, including polarographic needle electrode, phosphorescence imaging, near-infrared spectroscopy (NIRS), blood oxygen level dependent (BOLD) and 19F magnetic resonance imaging (MRI) and electron paramagnetic resonance (EPR) imaging, can detect oxygen partial pressure (pO2), oxygen concentration, or oxygen percentage. Recently, a hypoxia imaging endoscopy that can derive the oxygen saturation (StO2) was developed in endoscopic fields. Indirect measurements, including measuring exogenous and endogenous hypoxia markers, can provide

of the biological and functional changes in cancer.

384 Hypoxia and Human Diseases

to the start of treatment.

parameters related to oxygenation.

#### **3.1. Polarographic needle electrodes for direct tumor tissue**

The invasive polarographic needle electrodes have been widely employed since the 1990s to assess tumor oxygen status and to measure pO2 in both human and animal studies [7, 8]. As the gold standard modality, their use has been extended not only to lymph node metastases but to more accessible tumors, which include head and neck cancer, cervical cancer, soft tissue sarcomas of the extremities, astrocytic brain tumors, lung cancer, pancreatic cancer, prostate cancer, and lymph node metastases [8–12]. With the average median pO2 before treatment of 11.2 mmHg (range 0.4–60 mmHg) [7], these measured values help prediction of the tumor response to treatment [13] and tumor metastatic potential [14]. The polarographic needle electrode is currently available under CT guidance for evaluating tumor pO2 in deep-seated organs as well as for assessing overall tumor oxygen status [15] with the caveat, however, that insertion of an electrode into the tumor leads to disruption of tissues, thus rendering it difficult to distinguish the necrotic areas and to establish the patterns of hypoxia involved. Furthermore, the use of the modality not only calls for great expertise but is associated with large interobserver variability.

## **4. Noninvasive imaging of hypoxia**

While the polarographic needle electrode and immunohistochemical (IHC) staining can provide a relatively accurate estimation of tumor oxygenation, being subject to selection bias, provide only a partial, but not complete, picture of the entire tumor site [16]. This has led to an increasing interest in the use of noninvasive functional and molecular imaging modalities, which is capable of yielding a large amount of high-quality experimental data per protocol by increasing the number of quantitative data collections and by guiding tissue sampling and allowing a rapid and effective combination of analyses to be conducted [17].

Several imaging modalities have been developed, to date, to allow direct or indirect measurement of tumor oxygenation, with a few of these remaining less mature for clinical application. Of these, EPR spectroscopy, which involves the use of unpaired electron species to obtain images and spectra, is currently being explored in animals as a means to provide a quantitative measure of tissue oxygenation [18]. Although this modality has considerable potential to be developed as a tumor oximeter, i.e., in monitoring changes after tumor oxygenation [19], a suitable paramagnetic marker with low toxicity for human remains yet to become available. The need for appropriate EPR instrumentation in the clinical setting also prevents this promising modality from becoming widespread [20]. Photoacoustic tomography (PAT) is also available for imaging blood oxygenation using the differential optical contrast between O2Hb and dHb. PAT has been implemented for imaging cerebral blood oxygenation of rats *in vivo*, demonstrating that PAT is capable of capturing the changes from hyperoxia to hypoxia [21], while no study reported on its clinical application.

#### **4.1. Magnetic resonance imaging**

BOLD-MRI is shown to have potential as a diagnostic modality for tumor hypoxia [22]. Hemoglobin occurs as deoxyhemoglobin in oxygen-deficient states, where not oxyhemoglobin but paramagnetic deoxyhemoglobin can increase the transverse relaxation of the surrounding protons [23]. BOLD-MRI employs deoxyhemoglobin-derived endogenous signals as image contrast to depict changes in oxygenation in blood. Decreased oxygenation in blood results in decreased signal intensity in T2-weighted images, and this correlation between the BOLD-MRI signal and vascular oxygenation allows pO2 to be directly estimated. This has indeed led to numerous studies being conducted to investigate carbogen breathing in mice, oxygenation in tumor models [24], and kidney function in patients [22, 25, 26] using the modality as a noninvasive technique with high spatial and temporal resolution [22]. As with phosphorescence and near-infrared fluorescence imaging, the major disadvantage of BOLD-MRI is that it reflects change in oxygen tension in vasculature but not those in tissues. Again, not being a quantitative method, it may easily be affected by multiple factors such as flow effects, hematocrit, pH, and temperature [27].

19F MRI involves the use of two types of markers, i.e., perfluorocarbons (PFCs) and fluorinated nitroimidazoles as contrast agents, which are not used in conventional T1-weighted MRI. While being highly hydrophobic, PFCs are highly oxygen soluble [28]. Due to the linear relationship between the 19F spin lattice relaxation rate of PFCs and the dissolved oxygen concentration, the 19F-based oximetry allows vascular oxygenation to be measured *in vivo* [29]. PFCs investigated to date include hexafluorobenzene (HFB) [30] and perfluoro-15-crown-5 ether (PF15C5) [31, 32], which are injectable intravenously or intratumorally. 19F MRI is increasingly employed to detect changes in tumor oxygenation that occur in response to treatments that are radio-sensitizing and oxygen-augmenting [33]. The disadvantages of 19F MRI are that flow artifacts affect the measurements and that, with some contrast agents, oxygen sensitivity is easily influenced by such conditions as temperature, dilution, pH, common proteins, and blood [34]. Following intravenous injection, most PFC contrast agent is extensively ingested by the reticuloendothelial system (RES) and their slow clearance may cause adverse reactions. Their intratumoral injection may also raise concern over its associated risk, e.g., embolism associated with accidental injection of PFC emulsion into the tumoral vein [35]. One major drawback of the nitroimidazole derivatives is their central nervous system (CNS) toxicity profile, with misonidazole shown to be associated with neuropathy and acute toxicity on the CNS [33].

"Vessel architectural imaging" (VAI) has recently been proposed as a new paradigm in MRI providing a basis for vessel caliber estimation [36] by incorporating an overlooked temporal shift in the MR signal, thus generating, unlike any other noninvasive imaging modality, new information on vessel type and function. Indeed, this new modality allowed an oral panvascular endothelial growth factor (pan-VEGF) receptor kinase inhibitor to be evaluated for its therapeutic efficacy in glioblastoma patients [37], demonstrating using VAI that anti-VEGF therapy not only normalizes tumor vasculature and alleviates edema but also prolongs survival in these patients.

#### **4.2. Positron emission tomography**

suitable paramagnetic marker with low toxicity for human remains yet to become available. The need for appropriate EPR instrumentation in the clinical setting also prevents this promising modality from becoming widespread [20]. Photoacoustic tomography (PAT) is also available for imaging blood oxygenation using the differential optical contrast between O2Hb and dHb. PAT has been implemented for imaging cerebral blood oxygenation of rats *in vivo*, demonstrating that PAT is capable of capturing the changes from hyperoxia to hypoxia [21],

BOLD-MRI is shown to have potential as a diagnostic modality for tumor hypoxia [22]. Hemoglobin occurs as deoxyhemoglobin in oxygen-deficient states, where not oxyhemoglobin but paramagnetic deoxyhemoglobin can increase the transverse relaxation of the surrounding protons [23]. BOLD-MRI employs deoxyhemoglobin-derived endogenous signals as image contrast to depict changes in oxygenation in blood. Decreased oxygenation in blood results in decreased signal intensity in T2-weighted images, and this correlation between the BOLD-MRI signal and vascular oxygenation allows pO2 to be directly estimated. This has indeed led to numerous studies being conducted to investigate carbogen breathing in mice, oxygenation in tumor models [24], and kidney function in patients [22, 25, 26] using the modality as a noninvasive technique with high spatial and temporal resolution [22]. As with phosphorescence and near-infrared fluorescence imaging, the major disadvantage of BOLD-MRI is that it reflects change in oxygen tension in vasculature but not those in tissues. Again, not being a quantitative method, it may easily be affected by multiple factors such as flow effects, hema-

19F MRI involves the use of two types of markers, i.e., perfluorocarbons (PFCs) and fluorinated nitroimidazoles as contrast agents, which are not used in conventional T1-weighted MRI. While being highly hydrophobic, PFCs are highly oxygen soluble [28]. Due to the linear relationship between the 19F spin lattice relaxation rate of PFCs and the dissolved oxygen concentration, the 19F-based oximetry allows vascular oxygenation to be measured *in vivo* [29]. PFCs investigated to date include hexafluorobenzene (HFB) [30] and perfluoro-15-crown-5 ether (PF15C5) [31, 32], which are injectable intravenously or intratumorally. 19F MRI is increasingly employed to detect changes in tumor oxygenation that occur in response to treatments that are radio-sensitizing and oxygen-augmenting [33]. The disadvantages of 19F MRI are that flow artifacts affect the measurements and that, with some contrast agents, oxygen sensitivity is easily influenced by such conditions as temperature, dilution, pH, common proteins, and blood [34]. Following intravenous injection, most PFC contrast agent is extensively ingested by the reticuloendothelial system (RES) and their slow clearance may cause adverse reactions. Their intratumoral injection may also raise concern over its associated risk, e.g., embolism associated with accidental injection of PFC emulsion into the tumoral vein [35]. One major drawback of the nitroimidazole derivatives is their central nervous system (CNS) toxicity profile, with misonidazole shown to be associated with neuropathy and acute toxicity

while no study reported on its clinical application.

**4.1. Magnetic resonance imaging**

386 Hypoxia and Human Diseases

tocrit, pH, and temperature [27].

on the CNS [33].

Efforts have recently been directed toward developing contrast agents for noninvasive hypoxia imaging with positron emission tomography (PET) and single-photon emission computed tomography (SPECT). Organic molecular markers labeled with positron-emitting radioisotopes are employed in PET imaging to allow the extent of tumor hypoxia to be measured. Commonly used radioisotopes include 18F, 124I, and 60/64Cu and the molecular markers to be labeled with these isotopes include 2-nitroimidazoles, e.g., fluoromisonidazole (FMISO), EF5, and fluoroetanidazole (FETA), nucleoside conjugates, e.g., iodoazomycin arabinoside (IAZA), and Cu(II)-diacetyl-bis (N4-methylthiosemicarbazone) (Cu-ATSM) [38–40]. These markers are shown not only to bind maximally to severely hypoxic cells to form such stable adducts as are detectable with a PET scanner but to provide a clear demarcation of hypoxic cells *in vivo* through their rapid reoxidization and removal from normal cells.

Of the first-generation nitroimidazoles, 18F-labeled misonidazole (18F-FMISO) is the most commonly used as being sensitive only to the presence of hypoxia in viable cells [41]. It is reported that a hypoxic state defined as <10 mmHg is required to induce significant 18FMISO uptake [42]. 18F-FMISO uptake is shown to vary widely depending on the type of patients and tumors, whereas 18 F-FMISO is shown to allow hypoxia to be detected in various tumors such as glioma, head and neck cancer, renal tumor, and non-small cell lung cancer [42–44]. A clinical trial of glioblastoma multiforme patients [45] demonstrated increased 18F-FMISO uptake and retention on both post-treatment FMISO and FDG images, suggesting that reoxygenation did not take place. It is reported that the distribution of oxygen and hypoxia was increased and decreased, respectively, in non-small cell lung carcinomas following treatment, as assessed by sequential FMISO imaging [46]. Given that no correlation is shown between patient diagnosis and degree of decrease in FMISO uptake and retention, in selectively boosting the radiation dose to hypoxic subvolumes, there appears to be a larger role for serial imaging during treatment than for baseline volume measurement. Again, pretreatment FMISO uptake/ retention and survival has been shown to be correlated and allow treatment failure to be predicted [45, 47]. However, 18F-FMISO may not be readily available for use in other cancers [42, 48].

The second-generation nitroimidazoles include 18F-fluorerythronitroimidazole (FETNIM) [49, 50], FETA [51], and EF5 [52, 53], which are more water soluble and not readily susceptible to degradation by most oxidizing mechanisms in place in humans. 18F-EF5 was tested in clinical trials for its feasibility as an imaging agent for hypoxia [54] and was shown to be hypoxia-specific, with its increased uptake shown to be correlated with the extent of tumor and high risk of metastasis in cancer patients [52], suggesting its usefulness in identifying high-risk candidates for clinical trials evaluating the influence of early chemotherapy on the occurrence of metastasis [55]. 18F-FAZA has great promise as an imaging agent for tumor hypoxia due to its faster diffusion into cells and faster clearance from normal tissues than 18F-FMISO [56]. PET imaging using 18F-FMISO demonstrated very high tracer uptake in all seven patients with high-grade gliomas evaluated, showing the potential of 18F-FMISO as an imaging agent in assessing hypoxia in this tumor type [57].

#### **4.3. Phosphorescence imaging**

Phosphorescence imaging with injection of porphyrin complex (Oxyphor) into the vasculature also allows tumor vascular pO2 to be measured [58, 59]. Recently, a general approach has been proposed through which to construct phosphorescent nanosensors with tunable spectral characteristics, varying degrees of quenching, and a high oxygen selectivity [60]. These probes are shown to exhibit excellent performance in measuring vascular pO2 in the rat brain with *in vivo* microscopy [60]. NIRS are also available for analysis of tumor oxygenation *in vivo* based on recorded spectral changes by hemoglobin in the vasculature [61–63]. Kim and Liu [64] demonstrated in an animal study that NIRS is associated with comparable efficacy to that with electrode measurements in evaluating tumor hypoxia. They showed that either carbogen (95% CO2 and 5% O2) or 100% oxygen inhalation could improve the vascular oxygen level of rat breast tumors. However, both phosphorescence imaging and NIRS are not readily translatable into clinical applications due to their low spatial resolution, light scattering, limited path length, low sensitivity, and susceptibility to environmental influence.

#### **4.4. Visible light spectroscopy**

In the search for noninvasive, continuous modalities for monitoring ischemia, electrical bioimpedance cardiac output monitoring has been proposed but shown to be incompatible with the thermodilution methods [65, 66]. Again, while near-infrared spectroscopy (NIRS) [67] is shown to respond to both hypoxemia [68, 69] and ischemia [70–72], its clinical use has been limited to large organs, such as the brain [73, 74, 85–87] with its broad normal ranges reported to be between 48% and 88% [75, 76]. Similarly, wide normal ranges are reported for sublingual capnography [77–79]. Also available, albeit invasive are polarographic oximetry probes [80] and fiber-enabled pulmonary catheters.

Visible light spectroscopy (VLS) appears to be similar to NIRS on some counts [81] with its mean VLS StO2 shown to be not significantly different from NIRS StO2 reported in human studies [67–76]. Again, the fractional contribution of venous blood to the cerebral NIRS signal has been reported to be 0.84 ± 0.21 ranging from 0.60 to 1.00 [82–84]. Using central venous and pulse oximetry saturation as estimates for local venous and arterial saturation, it is shown to be not significantly different at 0.89 ± 0.04. It is suggested that the two modalities cover similar microvascular compartments.

At the same time, VLS is shown to be superior to NIRS in monitoring tissues that lend themselves to monitoring, thus suggesting a more versatile role for VLS in patient treatment [81]. The NIRS light sources and detectors require to be spaced 2–5 cm apart or more to illuminate and monitor a large, homogenous tissue volume (>30 ml), thus making NIRS with its long and bulky sensors unsuitable for monitoring tissue regions, e.g., thin tissues such as gastrointestinal mucosa or small tumors. In contrast, the visible light used in VLS is shown to be strongly absorbed by tissue and VLS measurement to be highly localized thus making VLS unsuitable for transcranial use or use over thick skin dominated by surface tissue properties. Using VLS, a rapid real-time drop in tumor oxygenation was detected during local ischemia following clamping or epinephrine administration [85], with the tissue oximetry performed during endoscopy demonstrating a significantly lower tissue oxygenation (StO2) in tumors (46% ± 22%) than in normal mucosa (72% ± 4%) (*P* < 0.0001). Thus, VSL tissue oximetry may be able to distinguish neoplastic tissue with a high specificity to aid in the endoscopic detection of gastrointestinal tumors. Again, of note, chronic gastrointestinal ischemia was also detected using the same method [86] (**Figure 1**).

hypoxia-specific, with its increased uptake shown to be correlated with the extent of tumor and high risk of metastasis in cancer patients [52], suggesting its usefulness in identifying high-risk candidates for clinical trials evaluating the influence of early chemotherapy on the occurrence of metastasis [55]. 18F-FAZA has great promise as an imaging agent for tumor hypoxia due to its faster diffusion into cells and faster clearance from normal tissues than 18F-FMISO [56]. PET imaging using 18F-FMISO demonstrated very high tracer uptake in all seven patients with high-grade gliomas evaluated, showing the potential of 18F-FMISO as

Phosphorescence imaging with injection of porphyrin complex (Oxyphor) into the vasculature also allows tumor vascular pO2 to be measured [58, 59]. Recently, a general approach has been proposed through which to construct phosphorescent nanosensors with tunable spectral characteristics, varying degrees of quenching, and a high oxygen selectivity [60]. These probes are shown to exhibit excellent performance in measuring vascular pO2 in the rat brain with *in vivo* microscopy [60]. NIRS are also available for analysis of tumor oxygenation *in vivo* based on recorded spectral changes by hemoglobin in the vasculature [61–63]. Kim and Liu [64] demonstrated in an animal study that NIRS is associated with comparable efficacy to that with electrode measurements in evaluating tumor hypoxia. They showed that either carbogen (95% CO2 and 5% O2) or 100% oxygen inhalation could improve the vascular oxygen level of rat breast tumors. However, both phosphorescence imaging and NIRS are not readily translatable into clinical applications due to their low spatial resolution, light scattering, limited

In the search for noninvasive, continuous modalities for monitoring ischemia, electrical bioimpedance cardiac output monitoring has been proposed but shown to be incompatible with the thermodilution methods [65, 66]. Again, while near-infrared spectroscopy (NIRS) [67] is shown to respond to both hypoxemia [68, 69] and ischemia [70–72], its clinical use has been limited to large organs, such as the brain [73, 74, 85–87] with its broad normal ranges reported to be between 48% and 88% [75, 76]. Similarly, wide normal ranges are reported for sublingual capnography [77–79]. Also available, albeit invasive are polarographic oximetry probes [80]

Visible light spectroscopy (VLS) appears to be similar to NIRS on some counts [81] with its mean VLS StO2 shown to be not significantly different from NIRS StO2 reported in human studies [67–76]. Again, the fractional contribution of venous blood to the cerebral NIRS signal has been reported to be 0.84 ± 0.21 ranging from 0.60 to 1.00 [82–84]. Using central venous and pulse oximetry saturation as estimates for local venous and arterial saturation, it is shown to be not significantly different at 0.89 ± 0.04. It is suggested that the two modalities cover similar

an imaging agent in assessing hypoxia in this tumor type [57].

path length, low sensitivity, and susceptibility to environmental influence.

**4.3. Phosphorescence imaging**

388 Hypoxia and Human Diseases

**4.4. Visible light spectroscopy**

and fiber-enabled pulmonary catheters.

microvascular compartments.

**Figure 1.** VLS measurements using a fiber-optic catheter-based VLS oximeter. The catheter is passed through the accessory channel of the endoscope and positioned about 1–5 mm above the mucosa.

## **5. Hypoxia imaging endoscopy with no phosphor**

Kaneko et al. [87] reported hypoxia imaging endoscopy equipped with a laser light source. In this system, signals from the laser light passed through the processor were calculated as StO2. The measurement range of StO2 was from 0% to 100% in contactless of tumor or normal mucosa under endoscopic observation. Display imaging was performed with the use of laser light alone without phosphor, provided a display of overlay and pseudocolor images. The laser light used was not near-infrared but ranged within visible light wavelengths. In principle, this utilized the difference in absorption coefficient between oxyhemoglobin and deoxyhemoglobin. Two challenges were identified, however, in deriving the StO2 of tissue in alimentary tracts from differences in absorption spectra between oxyhemoglobin and deoxyhemoglobin using small numbers of wavelengths. First, there is not only a small difference in optical absorption spectra in the visible light region but also a narrow bandwidth between isosbestic points. Second, the reflectance of a tissue depends on hematocrit (Hct) as well as StO2, given that light absorption increases as hemoglobin density increases.

An imaging system equipped with laser diodes of 445 and 473 nm and a white fluorescent pigment body was therefore developed. Hypoxia imaging with this system rendered visible an alimentary tract tumor in real time and allowed the whole tumor to be visualized. With the tumor surface and normal mucosa rendered visible, no heterogeneity was seen with the use of this system. In the first-in-human clinical trial, early cancers of the esophagus, stomach, and colorectum were detected as hypoxic areas (**Figure 2**). Furthermore, colorectal adenomas with histologically low-grade atypia were also detected as hypoxic areas and no complications were reported in the patients with visualization of these tumors in real-time hypoxia imaging which involved only laser light without injection or oral administration of phosphor. As mentioned above, it will be expected that the hypoxia imaging endoscopy is shown to be superior to VLS or NIRS in measuring StO2 of surface of tumor and normal mucosa.

**Figure 2.** StO2 maps obtained in human subject research. (A) White light image by endoscopic observation in rectal adenocarcinoma (left). Line (L-R) corresponds to cross section of pathological diagnosis. StO2 map visualized by laser endoscope system (middle: pseudocolor StO2 image; right: StO2 overlay image). (B) Cross-sectional appearance stained with H&E (upper) and HIF1 alpha antibody (lower) corresponding to the hypoxic area visualized with StO2 map. (C) Endoscopic images of a colorectal adenoma (upper) showing clear hypoxia: white light image (upper left), pseudocolor StO2 map (upper middle) and overlayed image (upper right). Another case of a colonic lesion (lower) consisting of an adenoma (red arrow) and a hyperplasia (blue arrow): white light image (lower left), pseudocolor StO2 map (lower middle) and overlayed image (lower right). Only the adenoma was detected as hypoxia.

## **6. Indirect hypoxia evaluation**

the difference in absorption coefficient between oxyhemoglobin and deoxyhemoglobin. Two challenges were identified, however, in deriving the StO2 of tissue in alimentary tracts from differences in absorption spectra between oxyhemoglobin and deoxyhemoglobin using small numbers of wavelengths. First, there is not only a small difference in optical absorption spectra in the visible light region but also a narrow bandwidth between isosbestic points. Second, the reflectance of a tissue depends on hematocrit (Hct) as well as StO2, given that light absorption

An imaging system equipped with laser diodes of 445 and 473 nm and a white fluorescent pigment body was therefore developed. Hypoxia imaging with this system rendered visible an alimentary tract tumor in real time and allowed the whole tumor to be visualized. With the tumor surface and normal mucosa rendered visible, no heterogeneity was seen with the use of this system. In the first-in-human clinical trial, early cancers of the esophagus, stomach, and colorectum were detected as hypoxic areas (**Figure 2**). Furthermore, colorectal adenomas with histologically low-grade atypia were also detected as hypoxic areas and no complications were reported in the patients with visualization of these tumors in real-time hypoxia imaging which involved only laser light without injection or oral administration of phosphor. As mentioned above, it will be expected that the hypoxia imaging endoscopy is shown to be superior to VLS

**Figure 2.** StO2 maps obtained in human subject research. (A) White light image by endoscopic observation in rectal adenocarcinoma (left). Line (L-R) corresponds to cross section of pathological diagnosis. StO2 map visualized by laser endoscope system (middle: pseudocolor StO2 image; right: StO2 overlay image). (B) Cross-sectional appearance stained with H&E (upper) and HIF1 alpha antibody (lower) corresponding to the hypoxic area visualized with StO2 map. (C) Endoscopic images of a colorectal adenoma (upper) showing clear hypoxia: white light image (upper left), pseudocolor StO2 map (upper middle) and overlayed image (upper right). Another case of a colonic lesion (lower) consisting of an adenoma (red arrow) and a hyperplasia (blue arrow): white light image (lower left), pseudocolor StO2 map (lower mid-

or NIRS in measuring StO2 of surface of tumor and normal mucosa.

dle) and overlayed image (lower right). Only the adenoma was detected as hypoxia.

increases as hemoglobin density increases.

390 Hypoxia and Human Diseases

Proteins and genes whose expression is associated with hypoxia have potential as endogenous molecular markers of hypoxia and have been explored over the years; meanwhile, hypoxia-specific agents have also been explored and shown to be useful in monitoring hypoxia [88]. Immunohistochemical (IHC) staining for hypoxia marker adducts in situ is also available to provide indirect quantitative information on the relative oxygenation of tissue at a cellular resolution. IHC approaches have a role to play particularly *in vitro* studies, including assays of human biopsy specimens. Given the complex biology of tumor hypoxia for which no single marker is expected to have a strong prognostic power in clinical practice, efforts have been directed toward combining various markers to create a prognostic profile of hypoxia [89].

#### **6.1. Hypoxia-inducible factor 1**

Optical imaging has had an important role to play in evaluating hypoxia, especially in biopsy specimens. With the introduction of transgenes with the hypoxia responsive element as promoter sequences coupled to reporter genes, e.g., luciferase reporter gene [90, 91] or green fluorescent protein (GFP) [92], a number of modalities have been developed to allow HIF-1 activity to be directly measured. Of these, a HIF-1-dependent promoter-regulated luciferase reporter gene, shown to produce a 100-fold increased luciferase response to hypoxia, has been used to evaluate anti-hypoxia therapy for its efficacy in animals [93]. Again, an imaging probe has been developed for HIF-1-active cells using a PTD-ODD fusion protein. given that, being involved in the same ODD control as HIF-1α, PTD-ODD fusion proteins are thought likely to be co-localized with HIF-1α [93–96]. First developed as a model probe, PTD-ODD-enhanced GFP-labeled with near-infrared fluorescent dye Cy5.5 was shown to permeate cell membrane with high efficiency, with its stability controlled in an oxygen concentration-dependent manner; to accumulate in hypoxic tumor cells with HIF-1 activity, thus allowing the hypoxic tumor cells with HIF-1 activity to be imaged in contrast to the surrounding cells under aerobic conditions [96]. Bioluminescence imaging has also been used to noninvasively depict HIF-1α as it is upregulated *in vivo* following chemotherapy, suggesting that this modality may prove useful in the evaluation of emerging anti-HIF-1 therapeutics [97]. While these imaging tools have a role to play in elucidating the biology of hypoxia and mechanisms of tumor response to therapy, heterogeneous gene responses to HIF-1 pose challenges to these HIF-1-targeted modalities. Furthermore, only weak correlation has been shown between HIF-1α expression and oxygen electrode or PET imaging measurements [98, 99], thus throwing in doubt the value of HIF-1α quantification as a measure of hypoxia.

#### **6.2. Carbonic anhydrase IX**

Downstream of HIF-1, carbonic anhydrase 9 (CA IX), a member of the CA family known to exist in cytosolic, membrane-associated, mitochondrial, and secreted carbonic anhydrases (CAs), may represent an alternative target [100]. A membrane-associated enzyme involved in the respiratory gas exchange and acid-base balance, CA IX is shown to be found less abundantly in normal tissue and only in gastric mucosa, small intestine, and muscle. Under hypoxic conditions, CA IX is shown to be overexpressed in different types of cancer [101], with the staining pattern shown to be more generalized in VHL-associated tumors and focalperinecrotic in non-VHL-associated tumors [102].

CA IX has been imaged with fluorescent-labeled sulfonamides in a tumor xenograft model to allow hypoxic and (re)-oxygenated cells to be distinguished [103], which demonstrated that CA IX required exposure to hypoxia for its binding and retention—a finding confirmed by an *in vivo* imaging study [103]. In renal-cell carcinoma xenografts, a G250 monoclonal antibody against CA IX was shown to significantly inhibit tumor growth [104]. Again, phase II clinical trials employed G250-based radioimmunoimaging to detect primary and metastatic lesions as well as to guide radioimmunotherapy after labeling G250 with therapeutic radioisotopes, which included 177 Lu, 90 Y, or 186 Re [105]. High-affinity human monoclonal antibodies (A3 and CC7) specific to human CA IX were developed using phage display technology [106] and these reagents may have a role to play in a wide range of settings, including noninvasive imaging of hypoxia and drug delivery [106]. In this regard, combining CA IX and a proliferation marker may prove helpful in identifying proliferating cells under hypoxic conditions [107, 108], while no correlation is shown between the amount of CA IX and direct oxygen measurement with a needle electrode [109].

Furthermore, hypoxia markers have been identified and shown to be induced by hypoxia and expressed in human tumors, including VEGF and GLUTs, both of which are upregulated by increased activity of HIF-1 under hypoxic conditions [110]. Imaging strategies targeting these proteins have also been explored for their ability to assess tumor vasculature and proliferation, while the relationship between pO2 values and protein expression levels remains unclear [111].

## **7. Heterogeneity of tumor**

Tissue oxygenation is shown to be highly heterogeneous due to the presence of both highly oxygenated arterial vascular regions and poorly oxygenated tissues and cells. Spatial and temporal heterogeneity also contribute to the complexity of the issue. Heterogeneity is thus a major factor in hypoxia measurement that affects our ability to stratify patients and predict outcomes using the imaging technologies available, and its biological implications need to be further explored, and effective approaches to assessing heterogeneity remain to be established. Hypoxia imaging endoscopy allowed early cancers of the pharynx, esophagus, stomach, and colorectum to be captured in whole for the first time [87], with no heterogeneity found in nearly all early cancers or colorectal neoplasia detected. Given that tissue heterogeneity may vary between early, advanced, and metastatic tumors, however, it remains crucial to elucidate tissue heterogeneity as it is associated with tumor progression.

## **8. Future of hypoxia measuring methods**

abundantly in normal tissue and only in gastric mucosa, small intestine, and muscle. Under hypoxic conditions, CA IX is shown to be overexpressed in different types of cancer [101], with the staining pattern shown to be more generalized in VHL-associated tumors and focal-

CA IX has been imaged with fluorescent-labeled sulfonamides in a tumor xenograft model to allow hypoxic and (re)-oxygenated cells to be distinguished [103], which demonstrated that CA IX required exposure to hypoxia for its binding and retention—a finding confirmed by an *in vivo* imaging study [103]. In renal-cell carcinoma xenografts, a G250 monoclonal antibody against CA IX was shown to significantly inhibit tumor growth [104]. Again, phase II clinical trials employed G250-based radioimmunoimaging to detect primary and metastatic lesions as well as to guide radioimmunotherapy after labeling G250 with therapeutic radioisotopes, which included 177 Lu, 90 Y, or 186 Re [105]. High-affinity human monoclonal antibodies (A3 and CC7) specific to human CA IX were developed using phage display technology [106] and these reagents may have a role to play in a wide range of settings, including noninvasive imaging of hypoxia and drug delivery [106]. In this regard, combining CA IX and a proliferation marker may prove helpful in identifying proliferating cells under hypoxic conditions [107, 108], while no correlation is shown between the amount of CA IX and direct oxygen measurement with a

Furthermore, hypoxia markers have been identified and shown to be induced by hypoxia and expressed in human tumors, including VEGF and GLUTs, both of which are upregulated by increased activity of HIF-1 under hypoxic conditions [110]. Imaging strategies targeting these proteins have also been explored for their ability to assess tumor vasculature and proliferation, while the relationship between pO2 values and protein expression levels

Tissue oxygenation is shown to be highly heterogeneous due to the presence of both highly oxygenated arterial vascular regions and poorly oxygenated tissues and cells. Spatial and temporal heterogeneity also contribute to the complexity of the issue. Heterogeneity is thus a major factor in hypoxia measurement that affects our ability to stratify patients and predict outcomes using the imaging technologies available, and its biological implications need to be further explored, and effective approaches to assessing heterogeneity remain to be established. Hypoxia imaging endoscopy allowed early cancers of the pharynx, esophagus, stomach, and colorectum to be captured in whole for the first time [87], with no heterogeneity found in nearly all early cancers or colorectal neoplasia detected. Given that tissue heterogeneity may vary between early, advanced, and metastatic tumors, however, it remains crucial to elucidate tissue

perinecrotic in non-VHL-associated tumors [102].

needle electrode [109].

392 Hypoxia and Human Diseases

remains unclear [111].

**7. Heterogeneity of tumor**

heterogeneity as it is associated with tumor progression.

Given the wide variety of techniques available for assessing hypoxia, e.g., polarographic needle electrodes, IHC staining, PET, MRI, optical imaging with NIR fluorescence or bioluminescence, visible light spectroscopy, and hypoxia imaging endoscopy, it remains critically important to determine their relative advantages and disadvantages for clinical application. Improvements in hypoxia measuring techniques will hinge primarily on which techniques are chosen and how these techniques are applied in the clinic. Clearly, the best of these are expected to be sensitive to the biological sequel of hypoxia, and the ideal one expected to be clinically safe, readily available, minimally invasive, and free from radiation exposure, while at the same time providing high resolution and ease of use. In addition, NIR over 1000 nm wavelength, the socalled biological window, will be promising, because this wavelength area is good for tissue permeability due to reducing both light scattering and infrared absorption [112].

In the endoscopic fields of alimentary tracts, the existing diagnosis for neoplasia is based on the morphologic features of the tumor. However, imaging of a tumor focused on its function or metabolism yields a novel set of data. Hypoxia imaging endoscope system equipped with a laser source allows oxygen saturation to be shown with two types of overlay and pseudocolor images displayed one on top of the other [87]. Available for handling similarly to conventional endoscopy, this modality is easy to treat with and completely safe without being invasive. Of the large number of patients with cancers in the alimentary tract, such as oral cavity, esophagus, stomach, and colorectum in the world, a majority with advanced cancer patients receives chemotherapy, radiotherapy, and combination therapy. In this regard, this modality is expected to allow not only hypoxic states but also hyperoxic states of tumor to be detected in these patients, thus contributing to selection of therapy or drug as well as evaluation of their therapeutic efficacy. Furthermore, this modality will serve as a screening method facilitating detection of early cancer. Advances in research into hypoxia and intratumoral microvessels of tumor with this endoscopic modality are expected and lead to development of new drugs. Thus, the proposed laser source-equipped hypoxia endoscope system appears to have the potential to redraw the endoscopic landscape.

## **9. Conclusions and perspectives**

Tumor hypoxia assessment allows cancer patients to be followed up early after treatment initiation and drug resistance and radioresistance to be predicted. Current insights into the molecular mechanisms of hypoxia have indeed led to novel probes being developed for noninvasive imaging of hypoxia. Again, real-time hypoxic imaging in digestive endoscopy was obtained using such laser light as remains within visible light wavelengths, with no use of any probes. For innovation of endoscopy, it was elucidated that most of all early cancers and precursor lesions have already been to hypoxic state. This is a cutting edge finding. This imaging technology highlights a novel aspect of cancer biology as a potential biomarker which may come to be widely used in cancer diagnosis and treatment effect prediction. These approaches appear to have great promise and further studies on the predictive value of hypoxia measurement in tumors may help identify independent predictive marker of hypoxia as well as optimal parameters for assessing hypoxia. It remains to be clarified whether these new agents may help reduce hypoxic disease or whether they are available for hypoxia imaging.

## **Author details**

Kazuhiro Kaneko1 , Hiroshi Yamaguchi2 and Tomonori Yano1\*

\*Address all correspondence to: toyano@east.ncc.go.jp

1 Division of Science and Technology for Endoscopy, National Cancer Center Hospital East, Chiba, Japan

2 Imaging Technology Center, FUJIFILM Corporation, Tokyo, Japan

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#### **Hypoxia and its Emerging Therapeutics in Neurodegenerative, Inflammatory and Renal Diseases Hypoxia and its Emerging Therapeutics in Neurodegenerative, Inflammatory and Renal Diseases**

Deepak Bhatia, Mohammad Sanaei Ardekani, Qiwen Shi and Shahrzad Movafagh Deepak Bhatia, Mohammad Sanaei Ardekani, Qiwen Shi and Shahrzad Movafagh

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/66089

#### **Abstract**

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819

402 Hypoxia and Human Diseases

Hypoxia is a common underlying condition of many disease states. Hypoxia can occur with ischemia, a lack of blood flow to tissues, or independent of ischemia as in acute lung injury, anemia, and carbon monoxide poisoning. Hypoxia may be observed in patients with diseases such as obstructive sleep apnea, cerebrovascular diseases, systemic hypertension, cardiovascular diseases, chronic obstructive pulmonary disease (COPD), pulmonary hypertension and congestive heart failure (CHF), inflammatory disease states, and acute and chronic renal diseases. In the past decade, research has shown hypoxic signaling to be involved in a range of responses from adaptation of the body to reduced oxygen to pathogenesis of disease. Hypoxic signaling intermediates orchestrate a whole host of responses from angiogenesis, glycolysis, and erythropoiesis to inflammation and remodeling, which could be beneficial or harmful to the hosting organ. The length of exposure to low oxygen pressure as well as the existing signaling pathways within different cells dictates their benefit or disadvantage from hypoxic signaling. Therefore, activation or inhibition of hypoxic intermediates could serve as novel therapeutic strategies. In this chapter, we review the role of hypoxic signaling in neurodegenerative, inflammatory, and renal disease states and the emerging therapeu‐ tic approaches involving hypoxic signaling.

**Keywords:** hypoxia, hypoxia‐inducible factor, neurodegenerative disease, Parkinson's disease, Alzheimer's disease, ischemia/reperfusion, inflammation, epigenetics, micro‐ RNA, inflammatory bowel disease, rheumatoid arthritis, acute kidney injury, chronic kidney disease, erythropoiesis, anemia, allograft rejection

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **1. Hypoxia and neurodegenerative diseases**

#### **1.1. Introduction**

Neurodegenerative diseases are defined by the progressive loss of specific neuronal cell population and protein misfolding and aggregate. Reduced oxygen supply has been detected during the aging process as well as the pathogenesis of neurodegenerative diseases. Besides, diseases associated with a lowering of systemic oxygen levels predispose individuals to neurodegenerative diseases. Although the connection between hypoxia and neurodegenera‐ tion has been well established, the exact role of hypoxia in neurodegenerative diseases has yet to be elucidated.

This section summarizes current identified clues linking hypoxia to the onset and progression of neurodegenerative diseases, including neurotoxic effects, altered signaling transduction and protein expression, and abnormal epigenetic modification. Furthermore, the following discussion emphasizes on the detrimental impacts of cerebral oxygen deficiency on three major neurodegenerative diseases: Alzheimer's disease (AD), Parkinson's disease (PD), and amyo‐ trophic lateral sclerosis (ALS).

#### *1.1.1. Hypoxia and Alzheimer's disease*

AD is characterized by progressive impairments in memory and cognitive function. The hallmark features of AD are extracellular plaques whose major components are amyloid β peptide (Aβ) and intracellular neurofibrillary tangles constituted by hyperphosphorylated tau protein. Other changes identified in AD brains are loss of synapses and neurons, proliferation of reactive astrocytes, and microglial activation. The incidence of AD in the United States is 11% among the population aged over 65 years and approximately 32% among those 85 years and older (Alzheimer's Association, 2015) [1]. Apparently, aging is the most significant risk factor for AD, since the risk of developing AD doubles every 5 years after the age of 65 years. Other factors, including environmental neurotoxins/metals, gene mutations, susceptibility polymorphisms, cardiovascular diseases, traumatic brain injury, and ischemia/hypoxia, also potentially prompt the development of AD.

Although the exact mechanisms and triggers initiating AD remain unclear, both clinical and preclinical studies suggest that hypoxia should be considered as an important risk factor in AD pathogenesis. Chronic cerebral hypoperfusion and glucose hypometabolism appearing decades before cognitive dysfunction promote the initiation and progression of cognitive decline and AD [2]. Patients after cerebral hypoxia or ischemia are more susceptible to developing dementia. Cerebral blood flow (CBF) reduction decreases the synthesis of proteins necessary for memory and learning and contributes likely to neuritic injury, neuronal death, and the onset and progression of dementia [3]. Correspondingly, significantly reduced resting CBF is distinguished in AD patients and is also present in the early stages of AD pathogenesis [4].

Generally, hypoxia modifies Aβ production and tau phosphorylation at numerous points (**Figure 1**). Aβ is a cleavage product generated through the sequential actions of β‐ and γ‐ secretases on amyloid precursor protein (APP). Hypoxia can stimulate Aβ generation and senile plaque formation in AD through increasing the expression of β‐ and γ‐secretases along with the localization of γ‐secretase from cell body to axon [5]. Furthermore, hypoxia elevates the levels of APP and presenilin‐1 (PS‐1), a main component of γ‐secretase complex, in vivo [6]. The expression of neprilysin (NEP), an enzyme responsible for Aβ degeneration, is reduced during hypoxia [7]. Rats exposed to hypoxic stress display tau hyperphosphorylation in the hippocampus as well as memory deficit, and Aβ‐induced tau phosphorylation is raised through calpain upon hypoxia exposure [8, 9]. The activity of protein phosphatase 2A (PP2A) is compromised in AD and is believed to be a cause of tau neurofibrillary. Brain hypoxia generates an acidic environment that promotes the cleavage of I2 PP2A, a potent inhibitor of PP2A, by activating asparaginyl endopeptidase, thus giving rise to tau hyperphosphorylation [10].

**Figure 1.** The molecular mechanisms of hypoxic predisposition to AD.

#### *1.1.2. Hypoxia and Parkinson's disease*

**1. Hypoxia and neurodegenerative diseases**

Neurodegenerative diseases are defined by the progressive loss of specific neuronal cell population and protein misfolding and aggregate. Reduced oxygen supply has been detected during the aging process as well as the pathogenesis of neurodegenerative diseases. Besides, diseases associated with a lowering of systemic oxygen levels predispose individuals to neurodegenerative diseases. Although the connection between hypoxia and neurodegenera‐ tion has been well established, the exact role of hypoxia in neurodegenerative diseases has yet

This section summarizes current identified clues linking hypoxia to the onset and progression of neurodegenerative diseases, including neurotoxic effects, altered signaling transduction and protein expression, and abnormal epigenetic modification. Furthermore, the following discussion emphasizes on the detrimental impacts of cerebral oxygen deficiency on three major neurodegenerative diseases: Alzheimer's disease (AD), Parkinson's disease (PD), and amyo‐

AD is characterized by progressive impairments in memory and cognitive function. The hallmark features of AD are extracellular plaques whose major components are amyloid β peptide (Aβ) and intracellular neurofibrillary tangles constituted by hyperphosphorylated tau protein. Other changes identified in AD brains are loss of synapses and neurons, proliferation of reactive astrocytes, and microglial activation. The incidence of AD in the United States is 11% among the population aged over 65 years and approximately 32% among those 85 years and older (Alzheimer's Association, 2015) [1]. Apparently, aging is the most significant risk factor for AD, since the risk of developing AD doubles every 5 years after the age of 65 years. Other factors, including environmental neurotoxins/metals, gene mutations, susceptibility polymorphisms, cardiovascular diseases, traumatic brain injury, and ischemia/hypoxia, also

Although the exact mechanisms and triggers initiating AD remain unclear, both clinical and preclinical studies suggest that hypoxia should be considered as an important risk factor in AD pathogenesis. Chronic cerebral hypoperfusion and glucose hypometabolism appearing decades before cognitive dysfunction promote the initiation and progression of cognitive decline and AD [2]. Patients after cerebral hypoxia or ischemia are more susceptible to developing dementia. Cerebral blood flow (CBF) reduction decreases the synthesis of proteins necessary for memory and learning and contributes likely to neuritic injury, neuronal death, and the onset and progression of dementia [3]. Correspondingly, significantly reduced resting CBF is distinguished in AD patients and is also present in the early stages of AD pathogenesis

Generally, hypoxia modifies Aβ production and tau phosphorylation at numerous points (**Figure 1**). Aβ is a cleavage product generated through the sequential actions of β‐ and γ‐

**1.1. Introduction**

404 Hypoxia and Human Diseases

to be elucidated.

[4].

trophic lateral sclerosis (ALS).

*1.1.1. Hypoxia and Alzheimer's disease*

potentially prompt the development of AD.

The clinical features of PD include classical motor symptoms (bradykinesia, rigidity, postural instability, resting tremor) and non‐motor symptoms (dementia, sleep disorder, depression, autonomic dysfunction), resulting from a continuous degeneration and loss of dopaminergic neurons in the substantia nigra (SN) and the presence of intracytoplasmic proteinaceous inclusions called Lewy bodies (LB) [11].

α‐Synuclein (α‐syn), a major constituent of LB, is the pathological hallmark of PD. Hypoxic brain injury is a potential cause of PD, as it enhances α‐synuclein expression and aggregation [12]. ATP13A2 (PARK9) mutations have been found in postmortem PD patients, declaring its relevance to PD pathogenesis [13]. Although the exact molecular mechanism remains un‐ known, it turns out that hypoxia upregulates ATP13A2 transcription via HIF‐1 alpha (HIF‐1α) in dopaminergic cells [14]. Hypoxia changes the localization of intracellular hemo‐ globin whose overexpression is correlated with an increased risk of PD [15]. In addition, subnormal sensitivity to hypoxia has been noticed in PD patients even at an early stage of diseases, probably leading to the exacerbation of respiratory failure in PD [16].

#### *1.1.3. Hypoxia and amyotrophic lateral sclerosis*

ALS, also known as Lou Gehrig's disease, is a progressive and fetal disease resulted from damaged motor neurons in the spinal cord, brain stem, and motor cortex. The incidence rate of ALS worldwide is estimated to be 2 in 100,000 people, and in the United States, about 5000 persons are diagnosed with ALS every year [17]. ALS risk is influenced by physical activity, smoking habit, type of diet, and exposure to agriculture chemicals and heavy metals. Occu‐ pations that may cause intermittent hypoxia, such as fire fighter, double the risk of ALS, and genetic impairment in reaction to hypoxia predisposes motor neuron to death [18].

Hypoxia is not only a causative factor of ALS but also accelerates the progression of ALS. Motor neurons under hypoxic conditions fail to survive and undergo degeneration [19]. SOD1G93A mutant mice, an ALS animal model, have experienced aggravation in motor neuronal loss, neuromuscular weakness and possibly cognitive deficiency, with higher level of oxidative stress and inflammation after chronic intermittent hypoxia [20]. Chronic sustained hypoxic condition induces the activation of apoptosis‐related genes such as caspase 3, apoptosis‐ inducing factor (AIF), and cytochrome C in motor neurons from the spinal cord of ALS mice, facilitating the progression of ALS [21].

#### **1.2. The mechanism of hypoxia-induced injury in neural cells**

Cellular and molecular pathways underlying hypoxia‐induced neurotoxicity and cell death are multifaceted and complex, including a number of cross‐talked mechanisms. Ensuing hypoxia stimulates the production and release of proteins mediating oxidative stress, inflammation, apoptosis, mitochondrial metabolism, metal homeostasis, synaptic transmis‐ sion, and autophagy, contributing to neuronal death (**Figure 2**).

**Figure 2.** Different pathogenic mechanisms linking hypoxia to neurodegenerative diseases.

#### *1.2.1. Hypoxia-promoted oxidative stress*

subnormal sensitivity to hypoxia has been noticed in PD patients even at an early stage of

ALS, also known as Lou Gehrig's disease, is a progressive and fetal disease resulted from damaged motor neurons in the spinal cord, brain stem, and motor cortex. The incidence rate of ALS worldwide is estimated to be 2 in 100,000 people, and in the United States, about 5000 persons are diagnosed with ALS every year [17]. ALS risk is influenced by physical activity, smoking habit, type of diet, and exposure to agriculture chemicals and heavy metals. Occu‐ pations that may cause intermittent hypoxia, such as fire fighter, double the risk of ALS, and

Hypoxia is not only a causative factor of ALS but also accelerates the progression of ALS. Motor neurons under hypoxic conditions fail to survive and undergo degeneration [19]. SOD1G93A mutant mice, an ALS animal model, have experienced aggravation in motor neuronal loss, neuromuscular weakness and possibly cognitive deficiency, with higher level of oxidative stress and inflammation after chronic intermittent hypoxia [20]. Chronic sustained hypoxic condition induces the activation of apoptosis‐related genes such as caspase 3, apoptosis‐ inducing factor (AIF), and cytochrome C in motor neurons from the spinal cord of ALS mice,

Cellular and molecular pathways underlying hypoxia‐induced neurotoxicity and cell death are multifaceted and complex, including a number of cross‐talked mechanisms. Ensuing hypoxia stimulates the production and release of proteins mediating oxidative stress, inflammation, apoptosis, mitochondrial metabolism, metal homeostasis, synaptic transmis‐

diseases, probably leading to the exacerbation of respiratory failure in PD [16].

genetic impairment in reaction to hypoxia predisposes motor neuron to death [18].

*1.1.3. Hypoxia and amyotrophic lateral sclerosis*

406 Hypoxia and Human Diseases

facilitating the progression of ALS [21].

**1.2. The mechanism of hypoxia-induced injury in neural cells**

sion, and autophagy, contributing to neuronal death (**Figure 2**).

**Figure 2.** Different pathogenic mechanisms linking hypoxia to neurodegenerative diseases.

Oxidative stress has been implicated in hypoxic injury and neurodegenerative diseases. It occurs due to the disruption of oxidative balance and excessive production of reactive oxy‐ gen species (ROS) and reactive nitrogen species (RNS), including hydrogen peroxide (H2O2), nitric oxide (NO), superoxide (O2 − ), and the highly reactive hydroxyl radicals (·OH) [22]. The production of ROS and RNS is increased under hypoxic condition, probably because there is no acceptor for the electrons available. During hypoxic events, high levels of free radicals are produced through mitochondrial complex III, and the antioxidant status is depleted, thus leading to oxidative damage of vital cellular components. For instance, neuroblastoma cells exposed to hypoxia have augmented production of free radicals accompanied by a con‐ comitant decrease in reduced glutathione (GSH) content, glutathione reductase (GR), gluta‐ thione peroxidase (GPx), and superoxide dismutase (SOD) activities, further inciting apoptotic death [22].

Increased oxidative stress is believed to be associated with neurological disorders and classical neuropathy. Reduced antioxidant capacity is a trait of AD. The activation of NO/NOS signaling system by cerebral ischemia in aged rats triggers hippocampal Aβ production through β‐ secretase 1 (BACE1) pathway, implying RNS is a bridge linking hypoxia to AD [23]. In retinal ganglion cells (RGEs) derived from rats, hypoxia exposure triggers Aβ formation, intracellular ROS accumulation, and following cell death, suggesting the involvement of Aβ in hypoxia‐ induced retinal degeneration in AD [24]. In PD, the promotion of ROS formation is highly correlated to mutant α‐syn phosphorylation at serine 129 (Ser129), possibly preceding cell degeneration [25]. Agents with antioxidant property ameliorate neurodegenerative situation, including natural compounds and iron chelators.

#### *1.2.2. Hypoxia-altered ionic homeostasis*

Impaired cellular homeostasis of metals can be triggered by hypoxic conditions, resulting in neurodegeneration through various mechanisms, such as oxidative stress, inflammation, and aberrant expression of metalloproteins.

Calcium dyshomeostasis is a fundamental mechanism in the pathogenesis of neurodegener‐ ative diseases. The interaction between γ‐aminobutyric acid (GABA) and calcium‐dependent neurotransmission as well as calcium‐dependent neuronal metabolism also reveals the role of Ca2+ in neuronal degeneration. Ca2+ acts as an intracellular messenger, controlling not only transsynaptic signal transmission but also cellular metabolism by reaching the mitochondria [26]. Hypoxia can disrupt Ca2+ entry and signaling in various cell types. In hypoxic human neuroblastoma cells, the storage of intracellular Ca2+, Na+ /Ca2+ exchange, and capacitative Ca2+ entry are boosted, indicating adaptive cellular remodeling in response to prolonged hypoxia [27]. Similarly, chronic hypoxia enhances capacitative Ca2+ entry and mitochondria Ca2+ content in the primary culture of rat type‐I cortical astrocytes [28]. In terms of AD, chronic hypoxia potentiates posttranscriptional trafficking of L‐type Ca2+ channels that may result from the interaction between Aβ and Ca2+ channel subunit [29].

Iron can be released from storage protein in the brain under hypoxic circumstances, and disruption of intracellular free iron homeostasis is an early event upon hypoxic stimulation in oligodendrocytes that contain enriched iron and ferritin [30]. Progressive hypoxia dramatically activates the synthesis of ferritin, a major iron‐binding protein, in oligodendrocytes, and this induction may require ROS formation as it can be enhanced by co‐treatment with H2O2 [31]. Intracellular free iron has neurotoxic effects. Iron promotes Aβ aggregation in vitro [32], and iron‐Aβ interaction exhibits toxic effects through ROS [33]. Iron also binds to tau, but inter‐ estingly, its effect on tau relies on the oxidation state. Fe3+ induces the aggregation of hyper‐ phosphorylated tau and reduces the phosphorylation of tau, whereas Fe2+ exerts an opposite action [34]. As for PD, abnormal accumulation of iron results in α‐syn aggregation by pro‐ moting its synthesis and inhibiting its degradation [35].

#### *1.2.3. Hypoxia-disrupted mitochondrial functions*

The consequences of mitochondrial dysfunction cover oxidative stress, intracellular Ca2+ dysregulation, apoptosis, and metabolic failure, aggravating the deleterious effect.

Respiratory chain reprogramming is the first stage in the development of hypoxia‐triggered mitochondrial disorders, converting complex I electron transport chain (ETC) to complex II succinate oxidation. The activation of succinate is regarded as a protective and compensatory mechanism in response to oxygen shortage and preserves the aerobic energy production [36]. Otherwise, the dysregulation of complex I during oxygen deficiency may lead neurons to acute degeneration, characterized by decreased membrane potential, loss of ATP, and respiration disorders caused by abnormal oxidation of nicotinamide adenine dinucleotide (NADH) [37]. The study of mitochondrial genes informs that hypoxia upregulates genes involved in glycolytic pathways, indicating a shift in energy production from oxidative phosphorylation to glycolysis, which converts glucose to pyruvate and eventually lactate. This shift is supported by the observation of elevated brain extracellular lactate concentration in traumatic brain injury (TBI) patients. A cerebral microdialysis study discloses that the neurons in TBI patient are unable to utilize lactate produced by astrocyte through tricarboxylic acid (TCA) cycle, leading to increased lactate/pyruvate ratio [38]. In addition, the ketogenic capacity of cultured astroglia and neurons is augmented under hypoxia, probably because of the susceptibility of pyruvate dehydrogenase to oxygen deprivation [39].

Many rare mitochondrial diseases are actually models of neurodegeneration, such as Leber's hereditary optic neuropathy (LHON) and autosomal dominant optic atrophy (ADOA), and abnormal mitochondrial function has been discovered in several age‐related neurodegenera‐ tive diseases. Suppression of complex I potentiates tau phosphorylation, pointing out the role of mitochondrial dysfunction in the formation of tangles in AD [40]. During prolonged exposure to hypoxia, ROS production, Aβ accumulation, and Ca2+ dyshomeostasis are enhanced through regulation on ETC [41]. The SN of PD patients has reduced activity of mitochondrial complex I, and inhibitors of complex I produce neurological changes similar to PD [42].

#### *1.2.4. Hypoxia-mediated apoptotic cascades*

Iron can be released from storage protein in the brain under hypoxic circumstances, and disruption of intracellular free iron homeostasis is an early event upon hypoxic stimulation in oligodendrocytes that contain enriched iron and ferritin [30]. Progressive hypoxia dramatically activates the synthesis of ferritin, a major iron‐binding protein, in oligodendrocytes, and this induction may require ROS formation as it can be enhanced by co‐treatment with H2O2 [31]. Intracellular free iron has neurotoxic effects. Iron promotes Aβ aggregation in vitro [32], and iron‐Aβ interaction exhibits toxic effects through ROS [33]. Iron also binds to tau, but inter‐ estingly, its effect on tau relies on the oxidation state. Fe3+ induces the aggregation of hyper‐ phosphorylated tau and reduces the phosphorylation of tau, whereas Fe2+ exerts an opposite action [34]. As for PD, abnormal accumulation of iron results in α‐syn aggregation by pro‐

The consequences of mitochondrial dysfunction cover oxidative stress, intracellular Ca2+

Respiratory chain reprogramming is the first stage in the development of hypoxia‐triggered mitochondrial disorders, converting complex I electron transport chain (ETC) to complex II succinate oxidation. The activation of succinate is regarded as a protective and compensatory mechanism in response to oxygen shortage and preserves the aerobic energy production [36]. Otherwise, the dysregulation of complex I during oxygen deficiency may lead neurons to acute degeneration, characterized by decreased membrane potential, loss of ATP, and respiration disorders caused by abnormal oxidation of nicotinamide adenine dinucleotide (NADH) [37]. The study of mitochondrial genes informs that hypoxia upregulates genes involved in glycolytic pathways, indicating a shift in energy production from oxidative phosphorylation to glycolysis, which converts glucose to pyruvate and eventually lactate. This shift is supported by the observation of elevated brain extracellular lactate concentration in traumatic brain injury (TBI) patients. A cerebral microdialysis study discloses that the neurons in TBI patient are unable to utilize lactate produced by astrocyte through tricarboxylic acid (TCA) cycle, leading to increased lactate/pyruvate ratio [38]. In addition, the ketogenic capacity of cultured astroglia and neurons is augmented under hypoxia, probably because of the susceptibility of pyruvate

Many rare mitochondrial diseases are actually models of neurodegeneration, such as Leber's hereditary optic neuropathy (LHON) and autosomal dominant optic atrophy (ADOA), and abnormal mitochondrial function has been discovered in several age‐related neurodegenera‐ tive diseases. Suppression of complex I potentiates tau phosphorylation, pointing out the role of mitochondrial dysfunction in the formation of tangles in AD [40]. During prolonged exposure to hypoxia, ROS production, Aβ accumulation, and Ca2+ dyshomeostasis are enhanced through regulation on ETC [41]. The SN of PD patients has reduced activity of mitochondrial complex I, and inhibitors of complex I produce neurological changes similar to

dysregulation, apoptosis, and metabolic failure, aggravating the deleterious effect.

moting its synthesis and inhibiting its degradation [35].

*1.2.3. Hypoxia-disrupted mitochondrial functions*

408 Hypoxia and Human Diseases

dehydrogenase to oxygen deprivation [39].

PD [42].

Cerebral hypoxia results in increased activities of caspase‐9, caspase‐8, and caspase‐3 in the cerebral cortex of newborn piglets and enhances cytochrome C expression and caspase‐3 activity followed by the induction of apoptosis in neuroblastoma cells. NO induced by hypoxia exerts proapoptotic property through elevating the expression of proteins such as Bax and Bad, leading to APAF‐1 activation and consequential activation of caspase‐9 and caspase‐3, and, on the other hand, through downregulating antiapoptotic proteins of the B‐cell lymphoma‐2 (Bcl‐2) family [22, 43] . Exposure of primary neuron cells from ALS mice to chronic sustained hypoxia results in enhanced cellular apoptosis, suggesting hypoxia could accelerate ALS via neuronal apoptosis [21]. Angiogenin (ANG) is a potent inducer of neovascularization and is responsive to hypoxia. Silence of ANG promotes hypoxic injury‐induced motor neuron apoptosis, while exogenous overexpression of ANG has an antiapoptotic function. Mutation of ANG has been identified in ALS patients, proposing the importance of ANG in ALS pathogenesis [44].

Blockage of apoptosis can be neuroprotective. Rasagiline and its derivatives, a group of highly potent irreversible monoamine oxidase (MAO) B inhibitor, exert their anti‐Parkinson feature by preventing apoptotic cascades. They activate Bcl‐2 and protein kinase C (PKC) and inhibit proapoptosis FAS and Bax against neuronal apoptosis [45]. Treatment of 0.5% isoflurane, an inhaled anesthetic, attenuates caspase‐3 activation, BACE upregulation, and Bcl‐2 reduction caused by hypoxia in H4 human neuroglioma cells, hinting the neuroprotective effect of isoflurane in AD [46].

#### *1.2.5. Hypoxia-modified synaptic signaling*

Synaptic transmission in the central nervous system (CNS) is extremely sensitive to hypoxia, since it requires 30–50% of cerebral oxygen. Decrease in synaptic efficacy occurs very early during hypoxia and is possibly the first response of neurons to ischemic insult.

Oxygen‐sensitive ion channels and voltage‐gated Ca2+ and K+ channel are activated in response to hypoxia, bringing about changes in excitation and inhibition of neuronal and glial cells [47]. Under hypoxic circumstance, there is an accumulation of adenosine in the extracellular space, due to the increased catabolism of adenosine triphosphate (ATP) into adenosine monophos‐ phate (AMP) [48]. Adenosine is a neurotransmitter inhibiting synaptic transmission, and its effect is mediated by adenosine A1 receptor. The mechanism is that receptor activation stimulates inwardly rectifying K+ channels, substantially inhibiting Ca2+ channels, phospholi‐ pase C activation, and the release of neurotransmitters including glutamate, dopamine, serotonin, and acetylcholine [49].

P2Y1 receptor is a G‐protein‐coupled ATP receptor activated by ATP released from neurons and astrocytes during neuronal activity or under pathophysiological conditions such as hypoxia, brain injury, and AD [50]. Emerging evidence shows that P2Y1 receptor obstructs the release of neurotransmitters and modulates synaptic plasticity in the brain, especially in the prefrontal cortex, hippocampus, and cerebellum, leading to impaired cognitive process [50]. P2Y1 receptors are localized with AD features such as neurofibrillary tangles and neuritic plaques, suggesting the altered distribution of P2Y1 in AD brains [51]. Astrocytic hyperactivity consisting of single‐cell transients and Ca2+ waves has been observed around Aβ plaques. P2Y1 receptors are strongly expressed by reactive astrocytes, and blockade of P2Y1 receptors can reduce astrocytic hyperactivity back to normal [52].

#### *1.2.6. Hypoxia and autophagy*

In general, autophagy is regarded as a survival mechanism, but under severe hypoxia/ ischemia, autophagy may cause self‐digestion and eventual cell death due to its overactiva‐ tion [53]. The morphological characteristics of autophagic‐programmed cell death have been observed in both mice and rats with cerebral ischemia [54, 55].

Enormous studies indicate autophagy dysfunction in AD. Autophagic vacuoles (AVs) are significantly accumulated in the brain of AD patients compared to normal brain, possibly leading to lysosomal enzyme dysfunction [56]. The cross talk between autophagy and tau aggregation indicates the change of autophagic function in the pathogenesis of AD. Autophagy initially degrades tau to protect neurons; however, hyperphosphorylation of tau results in autophagic dysfunction, which substantially exacerbates AD via inducing tau aggregation [57, 58]. Remarkably, hypoxia induces autophagic activation through AMPK‐mTOR signaling, resulting in more Aβ production and AD aggravation in vitro [56].

Defective autophagy has been implicated in PD [59], and several mutations in PD are strongly relevant to autophagy dysregulation, such as PTEN‐induced putative kinase 1 (PINK1) [60]. Autophagy in ALS prevents neurons from degeneration, and inhibition of autophagy aggra‐ vates motor neuron viability, since the aggregates composed of intermediate filaments and insoluble forms of proteins can be cleared by autophagy pathway [61].

#### **1.3. The role of hypoxia-sensitive transcription factors in neurodegenerative diseases**

Several transcription factors are responsive to hypoxia and subsequently alter gene expression and cellular activity. The signaling pathways relevant to these transcription factors have been indicated in the development of neurodegenerative diseases. Therefore, these transcription factors may provide a link between hypoxic environment and neurodegeneration. The following discussion will include HIF‐1, the most well‐studied hypoxia‐inducible gene, and two other redox‐sensitive transcription factors, nuclear factor‐kappa B (NF‐κB) and NF‐E2‐ related factor 2 (Nrf2).

#### *1.3.1. Hypoxia-inducible factor-1*

Hypoxia‐inducible factor‐1 (HIF‐1) is a transcriptional activator involved in oxygen hemosta‐ sis, regulating the expression of genes and the activation of signaling pathways that participate in angiogenesis, erythropoiesis, neovascularization, iron metabolism, glucose metabolism, cell proliferation, apoptosis, and cell cycle control (**Figure 3**).

**Figure 3.** The neuroprotective role of HIF‐1α activation in hypoxia.

plaques, suggesting the altered distribution of P2Y1 in AD brains [51]. Astrocytic hyperactivity consisting of single‐cell transients and Ca2+ waves has been observed around Aβ plaques. P2Y1 receptors are strongly expressed by reactive astrocytes, and blockade of P2Y1 receptors can

In general, autophagy is regarded as a survival mechanism, but under severe hypoxia/ ischemia, autophagy may cause self‐digestion and eventual cell death due to its overactiva‐ tion [53]. The morphological characteristics of autophagic‐programmed cell death have been

Enormous studies indicate autophagy dysfunction in AD. Autophagic vacuoles (AVs) are significantly accumulated in the brain of AD patients compared to normal brain, possibly leading to lysosomal enzyme dysfunction [56]. The cross talk between autophagy and tau aggregation indicates the change of autophagic function in the pathogenesis of AD. Autophagy initially degrades tau to protect neurons; however, hyperphosphorylation of tau results in autophagic dysfunction, which substantially exacerbates AD via inducing tau aggregation [57, 58]. Remarkably, hypoxia induces autophagic activation through AMPK‐mTOR signaling,

Defective autophagy has been implicated in PD [59], and several mutations in PD are strongly relevant to autophagy dysregulation, such as PTEN‐induced putative kinase 1 (PINK1) [60]. Autophagy in ALS prevents neurons from degeneration, and inhibition of autophagy aggra‐ vates motor neuron viability, since the aggregates composed of intermediate filaments and

**1.3. The role of hypoxia-sensitive transcription factors in neurodegenerative diseases**

Several transcription factors are responsive to hypoxia and subsequently alter gene expression and cellular activity. The signaling pathways relevant to these transcription factors have been indicated in the development of neurodegenerative diseases. Therefore, these transcription factors may provide a link between hypoxic environment and neurodegeneration. The following discussion will include HIF‐1, the most well‐studied hypoxia‐inducible gene, and two other redox‐sensitive transcription factors, nuclear factor‐kappa B (NF‐κB) and NF‐E2‐

Hypoxia‐inducible factor‐1 (HIF‐1) is a transcriptional activator involved in oxygen hemosta‐ sis, regulating the expression of genes and the activation of signaling pathways that participate in angiogenesis, erythropoiesis, neovascularization, iron metabolism, glucose metabolism, cell

reduce astrocytic hyperactivity back to normal [52].

observed in both mice and rats with cerebral ischemia [54, 55].

resulting in more Aβ production and AD aggravation in vitro [56].

insoluble forms of proteins can be cleared by autophagy pathway [61].

*1.2.6. Hypoxia and autophagy*

410 Hypoxia and Human Diseases

related factor 2 (Nrf2).

*1.3.1. Hypoxia-inducible factor-1*

proliferation, apoptosis, and cell cycle control (**Figure 3**).

In AD, HIF‐1α upregulates neuronal glucose transporters such as GLUT‐1 and GLUT‐3 and facilitates glucose uptake, thus providing increased oxygen supply to hypoxic tissues [62]. It also contributes to cell survival by inducing the key enzymes in pentose phosphate pathway, including glucose‐6‐phosphate dehydrogenase and 6‐phosphogluconate dehydrogenase [63]. HIF‐1α also connects hypoxia to amyloidogenic processing of APP through transcriptionally upregulating BACE1 and eventually increases Aβ formation [64].

The protective role of HIF‐1 in PD has been demonstrated by its ability to increase dopamine synthesis and dopaminergic neuron growth. Tyrosine hydroxylase (TH) is the rate‐limiting enzyme of dopamine synthesis in dopaminergic neurons, and interestingly, it contains an HRE [65]. HIF‐1 elevated in response to hypoxia increases TH expression in rat brain stem, and HIF‐1α conditional knockout mice exhibit reduced expression of TH and aldehyde dehydrogenase in SN [57]. HIF‐1 activation may defend against dysregulation of brain iron homeostasis and mitochondria in PD. Iron accumulation has been observed in the SN of PD patients and is considered as a culprit of ROS generation and intracellular α‐syn aggregation [66]. Moreover, the neurotransmitter dopamine is a metal reductant that reduces the oxidation state of metals such as Fe3+ and subsequently results in elevated oxidative stress [67]. Defer‐ oxamine (DFO), an iron chelator, prevents neurotoxicity in MPTP‐treated mice through upregulation of HIF‐1α protein expression, leading to declined expression of proteins such as α‐syn, divalent metal transporter with iron‐responsive element (DMT1 + IRE) and transferrin receptor (TFR), and elevated expression of HIF‐1 target genes, including TH, vascular endo‐ thelial growth factor (VEGF), and growth associated protein 43 (GAP43) [68].

HIF‐1 activation during hypoxia should be beneficial to ALS. HIF‐1‐VEGF pathway can induce angiogenesis and increase blood supply to motor neurons. VEGF overexpression delays motor neuron loss and impairment in SOD1G93A mutant mice and prolongs the survival of mice [69]. Deletion of HRE in VEGF promoter region abolishes hypoxia‐increased VEGF expression, causing motor neuron degeneration [70]. Additionally, HIF‐1‐erythropoietin (EPO) pathway is suggested to be a new therapeutic target for ALS. EPO treatment in SOD1G93A mice postpones the onset and progression of motor deterioration and modulates the immune-inflammatory response through reducing the levels of pro-inflammatory cytokines and enhancing the expression of anti-inflammatory cytokines [71, 72]. However, both above pathways are impaired in ALS. The level of VEGF is low in the CSF of early ALS patients, and likewise, the expression of VEGF in the CSF from hypoxemic ALS patients is lower than that in the CSF from normoxemic ALS patients [73, 74]. EPO protein level is declined in the surrounding glial cells of SOD1G93A mice, and in the anterior horn cells (AHCs) from SOD1G93A mice, impaired cytoplasmic-nuclear transport of HIF-1α has appeared since the presymptomatic stage, indicating the abnormality in HIF-1 pathway might precede motor neuron degradation [75, 76].

The well-studied group of agents targeting HIF-1 is iron chelators. The neuroprotective and neurorestorative activities of M30, an iron chelator with brain-selective monoamine oxidase (MAO) AB inhibitory function, share a same pathway, the activation of HIF-1, in different neurodegenerative diseases. M30 elevates HIF-1 to regulate neurotrophins BDNF, GDNF, VEGF, and EPO in PD, and meanwhile, it delays the onset of ALS in SOD1G93A mutant mice through HIF-1 upregulation [77, 78]. In APP/PS1 AD mice model, M30 treatment upregulates HIF-1α in the frontal cortex, resulting in the beneficial modulation of target glycolytic gene expression, such as aldolase A, enolase-1, and GLUT-1 [79].

Taken together, HIF-1 is a key player protecting neuron cells against hypoxia and oxidative stress, as well as a reasonable therapeutic target against major neurodegenerative diseases, since its participation in the pathogenesis of neurodegeneration has been well identified.

#### *1.3.2. Nuclear factor-kappa B*

Nuclear factor-kappa B (NF-κB) is analogous to HIF-1 in structure, function, and mechanism of activation and plays a critical role in inflammation, immune response, synaptic transmission, neuronal plasticity, and apoptosis [80]. In resting state, NF-κB is complexed with the inhibitory subunit I-κB; however, under physiological or pharmacological stimulus such as oxidative stress, I-kappa B (I-κB) is degraded, leading to translocation of NF-κB from cytoplasm to nucleus to modulate gene transcription. NF-κB and I-κB proteins comprise a growing family of structurally related transcription factors, and functional NF-κB complexes are present in generally all cell types in the nervous system, such as neurons, astrocytes, microglia, and oligodendrocytes [81, 82]. In neurons, the most common variants consist of p50, p65/RelA, and I-κB subunits.

As a redox-sensitive transcription factor, the mobilization and upregulation of NF-κB have been reported in hypoxia and ischemia-reperfusion damage. Hypoxic-ischemic brain damage (HIBD) upregulates the expression of NF-κB and the NO content in rat cortex cells, suggesting the involvement of NF-κB/nNOS pathway during the recovery of HIBD-induced neuron damage [83]. The role of NF-κB in neonatal HIBD depends on the duration of hypoxia. Early activation of NF-κB is detrimental, and at that time point, treatment of NF-κB inhibitor, TAT-NBD, exhibits significant therapeutic outcomes, whereas late NF-κB activation enhances antiapoptotic pathway and contributes to endogenous neuroprotection [84]. The overall effect of NF‐κB activation seems to facilitate ischemic neuronal degeneration, but still, the effect can be either neuroprotective or deleterious depending on the cell type and the strength of signal [85]. The suppression of NF‐κB or I‐κB in neuron can reduce infarct size after stroke, and the inhibition of NF‐κB caused by Ginkgolide B has protective effects on ischemic stroke [86, 87].

NF‐κB activation has been observed in neurons and astroglia of brain sections from AD patients but only in cells surrounding early plaques, suggesting that the induction of NF‐κB activity by Aβ is partially responsible for the aberrant gene expression in diseased nervous tissue [88]. In addition, intraperitoneal injection of sodium hydrosulfide (NaHS), a donor of H2S whose level is reduced in the hippocampus of Aβ‐injected rats, inhibits MAPK/NF‐κB pathway and dramatically mitigates cognitive decline and neuroinflammation [83]. Another novel drug for AD, Gx‐50, exerts anti‐inflammatory effects against Aβ‐triggered microglial overactivation in AD mice model via inhibition of NF‐κB signaling [89].

Increased NF‐κB activation has been reported in dopaminergic neurons of SN from PD patients, as well as in astrocytes of spinal cords from ALS patients [90]. Compounds inhibiting NF‐κB translocation in microglia such as vinyl sulfone compound (VSC2) downregulate the expression of inducible NOS (iNOS) and TNF‐α, leading to anti‐inflammatory and antioxidant events in PD animal model [91]. NF‐κB is also involved in microglia‐induced motor neuron death in ALS. Deletion of NF‐κB signaling in microglia rescues motor neuron from microglia‐ mediated death and extends survival in ALS mice by impairing pro‐inflammatory microglial activation [92].

Collectively, NF‐κB is responsive to the injury of nervous system in both acute and chronic neurodegenerative conditions. Agents suppressing NF‐κB activation have been tested in animal models of neurodegenerative conditions, but their usage should be considered cautiously because of the involvement of NF‐κB in learning and memory.

#### *1.3.3. NF-E2-related factor 2*

is suggested to be a new therapeutic target for ALS. EPO treatment in SOD1G93A mice postpones the onset and progression of motor deterioration and modulates the immune-inflammatory response through reducing the levels of pro-inflammatory cytokines and enhancing the expression of anti-inflammatory cytokines [71, 72]. However, both above pathways are impaired in ALS. The level of VEGF is low in the CSF of early ALS patients, and likewise, the expression of VEGF in the CSF from hypoxemic ALS patients is lower than that in the CSF from normoxemic ALS patients [73, 74]. EPO protein level is declined in the surrounding glial cells of SOD1G93A mice, and in the anterior horn cells (AHCs) from SOD1G93A mice, impaired cytoplasmic-nuclear transport of HIF-1α has appeared since the presymptomatic stage, indicating the abnormality in HIF-1 pathway might precede motor neuron degradation [75,

The well-studied group of agents targeting HIF-1 is iron chelators. The neuroprotective and neurorestorative activities of M30, an iron chelator with brain-selective monoamine oxidase (MAO) AB inhibitory function, share a same pathway, the activation of HIF-1, in different neurodegenerative diseases. M30 elevates HIF-1 to regulate neurotrophins BDNF, GDNF, VEGF, and EPO in PD, and meanwhile, it delays the onset of ALS in SOD1G93A mutant mice through HIF-1 upregulation [77, 78]. In APP/PS1 AD mice model, M30 treatment upregulates HIF-1α in the frontal cortex, resulting in the beneficial modulation of target glycolytic gene

Taken together, HIF-1 is a key player protecting neuron cells against hypoxia and oxidative stress, as well as a reasonable therapeutic target against major neurodegenerative diseases, since its participation in the pathogenesis of neurodegeneration has been well identified.

Nuclear factor-kappa B (NF-κB) is analogous to HIF-1 in structure, function, and mechanism of activation and plays a critical role in inflammation, immune response, synaptic transmission, neuronal plasticity, and apoptosis [80]. In resting state, NF-κB is complexed with the inhibitory subunit I-κB; however, under physiological or pharmacological stimulus such as oxidative stress, I-kappa B (I-κB) is degraded, leading to translocation of NF-κB from cytoplasm to nucleus to modulate gene transcription. NF-κB and I-κB proteins comprise a growing family of structurally related transcription factors, and functional NF-κB complexes are present in generally all cell types in the nervous system, such as neurons, astrocytes, microglia, and oligodendrocytes [81, 82]. In neurons, the most common variants consist of p50, p65/RelA, and

As a redox-sensitive transcription factor, the mobilization and upregulation of NF-κB have been reported in hypoxia and ischemia-reperfusion damage. Hypoxic-ischemic brain damage (HIBD) upregulates the expression of NF-κB and the NO content in rat cortex cells, suggesting the involvement of NF-κB/nNOS pathway during the recovery of HIBD-induced neuron damage [83]. The role of NF-κB in neonatal HIBD depends on the duration of hypoxia. Early activation of NF-κB is detrimental, and at that time point, treatment of NF-κB inhibitor, TAT-NBD, exhibits significant therapeutic outcomes, whereas late NF-κB activation enhances antiapoptotic pathway and contributes to endogenous neuroprotection [84]. The overall effect

expression, such as aldolase A, enolase-1, and GLUT-1 [79].

*1.3.2. Nuclear factor-kappa B*

I-κB subunits.

76].

412 Hypoxia and Human Diseases

NF‐E2‐related factor 2 (Nrf2) is a basic leucine zipper (bZIP) transcription factor that is ubiquitously expressed in a wide range of tissues and cell types. It heterodimerizes with small Maf or Jun proteins and binds to the antioxidant response element (ARE) in the promoter region of target genes in response to oxidative stress [93]. Nrf2 knockout mice are susceptible to oxidative stress and neurodegeneration without obvious phenotypic defects [94].

The upregulation of Nrf2 exerts neuroprotective action during hypoxia/ischemia. Hypoxia preconditioning on rat brain against severe hypoxia or ischemia insult is through upregulating Nrf2 and HO‐1 expression and alleviating oxidative stress damage [95]. rhEPO administration in ischemic rat activates Keap‐Nrf2/ARE pathway to decrease H2O2 concentration and to protect brain tissue [96]. Similarly, in oxygen‐deficient astrocytes, sulfiredoxin‐1, an endoge‐ nous antioxidant protein, ameliorates oxidative stress via Nrf2/ARE pathway to prevent the brain from ischemic injury [97].

The expression level of Nrf2 is significantly decreased in the hippocampal neurons from AD patients [98]. The beneficial effect of Nrf2 upregulation in AD is evidenced by the finding that Nrf2 is able to induce NDP52, an autophagy adaptor protein, which facilitates the clearance of phosphorylated tau in neurons [99]. Examination of postmortem brain samples from PD patients reveals that NQO1 and p62 whose expression is associated with Nrf2 are partly sequestered in LB, demonstrating the impaired Nrf2 signaling in PD, and pharmacological activation of Nrf2 defends PD by protecting nigral dopaminergic neurons against α‐syn toxicity and decreasing astrocytosis and microgliosis [100]. Correspondingly, in ALS mice model, WN1316, a novel acylaminoimidazole, boosts the activity of Nrf2 to protect motor neurons against oxidative injury and repress glial inflammation, microgliosis, and astrocyto‐ sis [101].

The Nrf2 signaling pathway is an attractive therapeutic target for neurodegenerative diseases, and thus, the chemopreventive agents aiming at Nrf2 might be suitable candidates against the development and progression of neurodegeneration.

#### **1.4. Epigenetic modification**

Epigenetics is the study of heritable and nonheritable changes in gene expression without changes to the underlying DNA sequence. Currently, at least three systems, DNA methylation, histone medication, and noncoding RNA (ncRNA)‐associated gene silencing, are identified in epigenetic changes. A large body of evidence documents that hypoxia triggers epigenetic alternation that contributes to the initiation and aggravation of neurodegeneration.

#### *1.4.1. Modification of DNA and histone*

DNA methylation and histone modification are two important epigenetic mechanisms altering the transcription of genes. The methylation of CpG island in the promoter region results in the silence of gene expression, whereas demethylation undergoes the opposite direction. The posttranslational modification (PTM) of histone includes acetylation, methylation, and phosphorylation that are regulated by pairs of enzymes, impacting gene expression via altering chromatin structure or recruiting histone modifiers.

Short‐term hypoxia causes long‐lasting changes in genomic DNA methylation in hippocampal neuronal cells and subsequent alternation in the expression of a number of genes participating in neural growth and development [102]. Chronic hypoxia‐mediated downregulation of NEP in mouse primary cortical and hippocampal neurons is through G9a histone methyltransferase and histone deacetylase 1 (HDAC1) other than methylation of gene promoter [103]. Cultured astrocytes under ischemia‐hypoxia (IH) condition show hypermethylation of global DNA and hypoacetylation of histone H3/H4, manifesting epigenetic reprogramming induced by hypoxia [104]. Chronic hypoxia exaggerated the neuropathology and cognitive impairment in AD mice through decreasing the expression of DNA methyltransferase 3b (DNMT3b) to prevent the methylation of γ‐secretase promoter [105].

Epigenetic modifications are reversible that make it a promising candidate for therapy. Valproic acid is a neuroprotective agent showing HDAC inhibitory property. It prevents the decrease of H3‐Ace in the NEP promoter regions in prenatal hypoxia‐induced AD neuropathology, upregulating NEP to improve learning deficits and decrease Aβ level [106].

#### **1.5. Conclusion**

of phosphorylated tau in neurons [99]. Examination of postmortem brain samples from PD patients reveals that NQO1 and p62 whose expression is associated with Nrf2 are partly sequestered in LB, demonstrating the impaired Nrf2 signaling in PD, and pharmacological activation of Nrf2 defends PD by protecting nigral dopaminergic neurons against α‐syn toxicity and decreasing astrocytosis and microgliosis [100]. Correspondingly, in ALS mice model, WN1316, a novel acylaminoimidazole, boosts the activity of Nrf2 to protect motor neurons against oxidative injury and repress glial inflammation, microgliosis, and astrocyto‐

The Nrf2 signaling pathway is an attractive therapeutic target for neurodegenerative diseases, and thus, the chemopreventive agents aiming at Nrf2 might be suitable candidates against the

Epigenetics is the study of heritable and nonheritable changes in gene expression without changes to the underlying DNA sequence. Currently, at least three systems, DNA methylation, histone medication, and noncoding RNA (ncRNA)‐associated gene silencing, are identified in epigenetic changes. A large body of evidence documents that hypoxia triggers epigenetic

DNA methylation and histone modification are two important epigenetic mechanisms altering the transcription of genes. The methylation of CpG island in the promoter region results in the silence of gene expression, whereas demethylation undergoes the opposite direction. The posttranslational modification (PTM) of histone includes acetylation, methylation, and phosphorylation that are regulated by pairs of enzymes, impacting gene expression via

Short‐term hypoxia causes long‐lasting changes in genomic DNA methylation in hippocampal neuronal cells and subsequent alternation in the expression of a number of genes participating in neural growth and development [102]. Chronic hypoxia‐mediated downregulation of NEP in mouse primary cortical and hippocampal neurons is through G9a histone methyltransferase and histone deacetylase 1 (HDAC1) other than methylation of gene promoter [103]. Cultured astrocytes under ischemia‐hypoxia (IH) condition show hypermethylation of global DNA and hypoacetylation of histone H3/H4, manifesting epigenetic reprogramming induced by hypoxia [104]. Chronic hypoxia exaggerated the neuropathology and cognitive impairment in AD mice through decreasing the expression of DNA methyltransferase 3b (DNMT3b) to

Epigenetic modifications are reversible that make it a promising candidate for therapy. Valproic acid is a neuroprotective agent showing HDAC inhibitory property. It prevents the decrease of H3‐Ace in the NEP promoter regions in prenatal hypoxia‐induced AD neuropathology, upregulating NEP to improve learning deficits and decrease Aβ level [106].

alternation that contributes to the initiation and aggravation of neurodegeneration.

development and progression of neurodegeneration.

altering chromatin structure or recruiting histone modifiers.

prevent the methylation of γ‐secretase promoter [105].

**1.4. Epigenetic modification**

*1.4.1. Modification of DNA and histone*

sis [101].

414 Hypoxia and Human Diseases

This section reviews the major consequences of hypoxia in the CNS and the contribution of individual consequence to the pathogenesis of several neurodegenerative diseases. However, the cross‐link among these consequences and how they may predispose hypoxic patients to neurodegeneration remain to be determined, as well as the communication between neurons and glia in response to hypoxic environment. Different types of hypoxia, acute, chronic, sustained, or intermittent, may vary in terms of their effects on neural cells. Therefore, further investigation is required. The prevention of hypoxic condition is clearly helpful for the reduction of neurodegeneration, and the molecules targeted by hypoxia provide therapeutic strategies and interventions against common neurodegenerative diseases.

## **2. Hypoxia and the inflammatory diseases**

#### **2.1. Introduction**

Inflammatory diseases are pathological conditions associated with local or systemic activation and persistent activity of inflammatory mediators, leading to cellular, tissue, or organ damage. The inflammatory cascade leads to increased vascular leakage, recruitment of leukocytes, increased generation and secretion of local and systemic inflammatory cytokines and chemo‐ kines, and activation and proliferation of innate and adaptive immune cell members. Ulti‐ mately, the inflammatory response leads to destruction of target molecules as well as their hosting cells and tissues, which could lead to pathological conditions such as inflammatory bowel disease and rheumatoid arthritis.

Hypoxia and inflammation have been extensively studied, and the two conditions seem to have a complex interrelated relationship. In general, hypoxia induces the inflammatory response via activation of cytokines and inflammatory cells, while inflammatory states are complemented with severe hypoxia and induction of hypoxic signaling intermediates [107, 108]. A key mediator of hypoxic signaling in inflammation is HIF‐1. Aside from low oxygen tension, recent evidence shows that various oxygen‐independent pathways regulate HIF‐1α transcription and translation under normoxia. For example, endogenous nitric oxide has been shown to stabilize HIF‐1α under normoxia [109–111]. Angiotensin II is another factor that increases HIF‐1α transcription and translation under normoxia, and angiotensin receptor blockade has shown to independently reduce HIF‐1α levels under hypoxic injury [112, 113]. Other nonhypoxic HIF‐1 regulatory molecules are via growth factors, thrombin, bacterial lipopolysaccharide (LPS), interleukins, and tumor necrosis factor‐α (TNF‐α) [114]. In general transcriptional and translational regulation of HIF‐1α occurring as a secondary mode of HIF‐1 regulation may aggravate or hinder the hypoxic response of the protein.

It has been noted that during hypoxemic states the levels of inflammatory cytokines such as IL‐1, IL6, and TNF‐α increase in serum [107, 115, 116]. Activation of macrophages and other innate and adaptive immune cell members is also shown to be induced by HIF‐1 under hypoxia via activation of Toll‐like receptor (TLR) signaling [117, 118]. Likewise, ischemia reperfusion is associated with recruitment of polymorphonuclear (PMN) leukocytes and vascular leakage [116, 119, 120]. This response is shown to be mediated via several endothelial cell surface glycoproteins and receptors and secondary activation of signaling via HIF‐1–induced adeno‐ sine generation and NF‐κB [116, 119].

It is noteworthy that ischemia and hypoxia are observed in inflamed tissues due to occlusion of blood flow via inflammatory cells [108]. As a result, signaling via inflammatory intermedi‐ ates has been shown to potentiate hypoxic signaling via HIF‐1. Macrophages in specific have been shown to release cytokines that stabilize and increase the activity of HIF‐1 [111, 121]. Ultimately, transcriptional activation of factors such as VEGF by HIF‐1 seems to increase angiogenesis and blood flow restoration to the site of inflammation.

Activation of HIF‐1 further assures energy supply and survival of myeloid cells as well as bactericidal capacity of macrophages [122, 123]. Among the signaling pathways induced by HIF‐1 in macrophages are mediators such as NF‐κB, TNF‐α, and nitric oxide that play key roles in the inflammatory capacity of the myeloid cells [111, 121, 123]. Interestingly, HIF‐1α stabilization in turn positively regulates the production of inflammatory cytokines such as TNF‐α, and therefore, through a positive feedback mechanism, inflammation and hypoxic signaling potentiate one another [123]. In the following sections, detailed mechanisms of this interaction will be discussed. Furthermore, the role of hypoxia and HIF molecules in arthritic and inflammatory bowel disease (IBD) pathophysiology and potential therapeutic targets relating to hypoxic signaling will be examined.

#### **2.2. Hypoxic signaling and key inflammatory intermediates**

#### *2.2.1. TNF-α*

TNF‐α is a key mediator of the inflammatory response. It has been shown that HIF‐1a stabi‐ lization and DNA‐binding activity are enhanced by TNF‐α [111]. Interaction of TNF‐α and HIF‐1 is rather complex. Physiologically, the stabilization of HIF‐1a by TNF‐α is thought to be mediated by activated macrophages [121]. Accumulation of HIF‐1α via the TNF‐α is via a mechanism independent from hypoxic accumulation or transcriptional activation of HIF‐1α. Several studies have investigated the mechanism of HIF‐1α stabilization via TNF‐α, and among such mechanisms, NF‐κB signaling seems to be the key mediator of this process [124, 125]. Studies by Zhou et al. have shown that TNF‐α leads to accumulation of ubiquiti‐ nated form of HIF‐1α, which is normally one of HIF‐1α degradation steps. This interaction was mediated through increased NF‐κB transcription [124]. They also noted that transfec‐ tion of cells with p50/p65 members of NF‐κB family leads to normoxic accumulation of HIF‐1α in the absence of TNF‐α [124]. Interestingly it has also been shown that reactive oxy‐ gen species (ROS) such as H2O2 or SO− interfere with TNF‐α–mediated accumulation of HIF‐1α [126]. Aside from protein accumulation, additional studies have shown increased translation of HIF‐1α via TNF‐α that is also mediated via NF‐κB through upregulation of an antiapoptotic protein Bcl‐2 [127].

#### *2.2.2. Nuclear factor-kappa β*

is associated with recruitment of polymorphonuclear (PMN) leukocytes and vascular leakage [116, 119, 120]. This response is shown to be mediated via several endothelial cell surface glycoproteins and receptors and secondary activation of signaling via HIF‐1–induced adeno‐

It is noteworthy that ischemia and hypoxia are observed in inflamed tissues due to occlusion of blood flow via inflammatory cells [108]. As a result, signaling via inflammatory intermedi‐ ates has been shown to potentiate hypoxic signaling via HIF‐1. Macrophages in specific have been shown to release cytokines that stabilize and increase the activity of HIF‐1 [111, 121]. Ultimately, transcriptional activation of factors such as VEGF by HIF‐1 seems to increase

Activation of HIF‐1 further assures energy supply and survival of myeloid cells as well as bactericidal capacity of macrophages [122, 123]. Among the signaling pathways induced by HIF‐1 in macrophages are mediators such as NF‐κB, TNF‐α, and nitric oxide that play key roles in the inflammatory capacity of the myeloid cells [111, 121, 123]. Interestingly, HIF‐1α stabilization in turn positively regulates the production of inflammatory cytokines such as TNF‐α, and therefore, through a positive feedback mechanism, inflammation and hypoxic signaling potentiate one another [123]. In the following sections, detailed mechanisms of this interaction will be discussed. Furthermore, the role of hypoxia and HIF molecules in arthritic and inflammatory bowel disease (IBD) pathophysiology and potential therapeutic targets

TNF‐α is a key mediator of the inflammatory response. It has been shown that HIF‐1a stabi‐ lization and DNA‐binding activity are enhanced by TNF‐α [111]. Interaction of TNF‐α and HIF‐1 is rather complex. Physiologically, the stabilization of HIF‐1a by TNF‐α is thought to be mediated by activated macrophages [121]. Accumulation of HIF‐1α via the TNF‐α is via a mechanism independent from hypoxic accumulation or transcriptional activation of HIF‐1α. Several studies have investigated the mechanism of HIF‐1α stabilization via TNF‐α, and among such mechanisms, NF‐κB signaling seems to be the key mediator of this process [124, 125]. Studies by Zhou et al. have shown that TNF‐α leads to accumulation of ubiquiti‐ nated form of HIF‐1α, which is normally one of HIF‐1α degradation steps. This interaction was mediated through increased NF‐κB transcription [124]. They also noted that transfec‐ tion of cells with p50/p65 members of NF‐κB family leads to normoxic accumulation of HIF‐1α in the absence of TNF‐α [124]. Interestingly it has also been shown that reactive oxy‐ gen species (ROS) such as H2O2 or SO− interfere with TNF‐α–mediated accumulation of HIF‐1α [126]. Aside from protein accumulation, additional studies have shown increased translation of HIF‐1α via TNF‐α that is also mediated via NF‐κB through upregulation of an

angiogenesis and blood flow restoration to the site of inflammation.

sine generation and NF‐κB [116, 119].

416 Hypoxia and Human Diseases

relating to hypoxic signaling will be examined.

antiapoptotic protein Bcl‐2 [127].

*2.2.1. TNF-α*

**2.2. Hypoxic signaling and key inflammatory intermediates**

NF‐κB is a family of transcription factors involved in development, proliferation, survival, and antimicrobial response of innate and adaptive immune system cells. Numerous extensive studies have been conducted to elucidate the very complex role of NF‐κB in the immune response [128]. The NF‐κB family is composed of five related transcription factors, which can form homodimers or heterodimer complexes with DNA‐binding activity. These identified members are p50, p52, RelA (p65), RelB, and c‐Rel [128]. NF‐κB complexes are inactive in the cytoplasm and are bound to an inhibitory protein called I‐κB. Once NF‐κB signaling is activated, the I‐κB proteins are degraded, which then allow the transcription factors to translocate to the nucleus [128]. In the innate immune response, NF‐κB is activated secondary to Toll‐like receptor (TLR) activation. Toll‐like receptors are pattern recognition receptors (PRR), which help immune cells recognize and combat pathogenic components. There are 11 identified mammalian TLRs with various coupled signaling pathways. TLRs are expressed in the cytosol as well as on the plasma membrane of immune cells [128]. Upon ligand binding, TLR signaling leads to recruitment of specific adaptor proteins and second messenger molecules, which in turn activate several transcription factors. Among such signaling path‐ ways are mediators that result in degradation of I‐κB proteins and activation of NF‐κB [128]. NF‐κB in turn induces gene expression of cytokines and other proteins involved in bactericidal activity against pathogens. NF‐κB activation and signaling are also involved in adaptive immunity. T‐cell and B‐cell receptor activation and signaling activate NF‐κB, which in turn activates antiapoptotic proteins and increases transcription of cytokines that ensure survival, proliferation, and differentiation of B and T cells [128].

#### *2.2.3. Hypoxia and the cross talk between HIF-1 and NF-κB*

It has been shown that NF‐κB is directly activated under hypoxic conditions [129, 130]. Although the mechanism of NF‐κB activation under hypoxia remains to be an extensive area of research, it has been shown that I‐κB tyrosine residues are phosphorylated under hypoxia [129]. More recent studies suggest phosphorylation and inactivation of I‐κB under hypoxia occur secondary to activation of transforming growth factor beta‐activated kinase‐1 (TAK1) and I‐kappa B kinase (IKK) complex, primarily responsible for in I‐κB degradation resulting in NF‐κB activation [130–133]. Additionally, it has been shown that O2‐dependent prolyl hydroxylases (PHDs) that are involved in HIF‐1 inactivation also play a role in proline hydroxylation of IKKβ and NF‐κB repression [133]. Thus, during hypoxia loss of PHD activity would activate NF‐κB.

Although hypoxic activation of NF‐κB is to be better understood, a large body of convincing evidence shows a critical role for NF‐κB in induction of HIF‐1. Activation of NF‐κB leads to induction of HIF‐1α gene expression and basal HIF‐1α mRNA, and protein levels are depend‐ ent upon NF‐κB subunit expression levels [134, 135]. Several studies have explored the mechanism of regulation of HIF‐1 by NF‐κB [124, 127, 134, 136, 137]. It has been shown that NF‐κB induces expression and increases protein levels of HIF‐1α both in hypoxia and nor‐ moxia [124, 134, 137]. Indeed, certain studies suggest that HIF‐1α gene expression under hypoxia is dependent upon intact NF‐κB signaling pathway [134, 137]. These studies also provide mechanistic evidence into the regulation of HIF‐1α gene expression via binding of several NF‐κB subunits to the HIF‐1α promoter region [134, 135]. Thus, secondary to direct activation of HIF‐1 under hypoxic conditions, interaction of NF‐κB additionally contributes to this process by increasing basal levels of HIF‐1α protein.

Respective regulation of NF‐κB by HIF‐1 has also been reported in the literature [114, 138, 139]. These studies suggest direct activation of NF‐κB via HIF‐1 signaling in inflammatory cells. Among suggested mechanisms are increased expression of TLR2 and TLR6 leading to activation of NF‐κB, hyperphosphorylation of IKKβ, and phosphorylation of serine residues of p65 subunit of NF‐κB leading to its translocation to nucleus and transcriptional activity [117, 138, 139].

Overall, hypoxia and signaling via NF‐κB and HIF‐1 are closely linked and, respectively, regulate one another to enhance the inflammatory response.

#### **2.3. Hypoxia and inflammatory bowel disease (IBD)**

IBD is associated with loss of intestinal mucosal barrier, inflammation of mucosa, and increased incidence of bacterial infections [140]. IBD is categorized as ulcerative colitis (UC) and Crohn's disease (CD). Both conditions are associated with severe inflammation and breakdown of intestinal mucosal barrier and chronic gastrointestinal discomfort. Current therapeutic approaches to IBD include anti‐inflammatory agents mostly targeted at TNF‐α and immune cell members.

Hypoxia has been shown to be a critical component of inflammation in IBD. Surgical specimens of intestinal mucosa of IBD patients show increased expression of HIF‐1 and HIF‐2 [141]. Increased vascular proliferation and density has been noted in intestines of IBD patients secondary to hypoxia‐induced VEGF activity [142]. Additionally microvascular abnormality and loss of endothelial nitric oxide production are seen in IBD mucosa [143].

The intestinal mucosa is exposed to fluctuating levels of oxygen. On the one hand, the intestinal lumen is nearly anoxic, and oxygen pressure is generally low on the luminal side of the mucosa. On the other hand, the rate of perfusion of the subendothelium is dependent upon meal intake, and PO2 changes dramatically from high to low in between meals. The shift in oxygen tension in the mucosal layer renders it resistant to hypoxic states. This could be in part due to basal activity of hypoxic signaling intermediates such as HIF‐1 in the intestinal mucosal. Indeed, HIF‐1α–null mice in the intestinal epithelium show diminished mucosal protection and increased clinical symptoms in murine model of colitis [144]. HIF‐1–induced epithelial protection is shown to be due to induction of several proteins such as mucin, p‐glycoprotein, and ecto‐5′‐nucleotidase (CD73), an enzyme that converts AMP to adenosine (A2B) receptor [140]. Adenosine production during hypoxia has shown to decrease vascular leakage and neutrophil accumulation and thus plays an anti‐inflammatory role [120]. In a case‐control cohort study, patients with polymorphisms in CD39, a vascular and immune cell ecto‐ nucleotidase that converts extracellular ATP and ADP to AMP, had increased susceptibility to Crohn's disease [145]. Therefore, HIF‐1 signaling via adenosine is a key step in protection against IBD inflammation (**Figure 4**).

Hypoxia and its Emerging Therapeutics in Neurodegenerative, Inflammatory and Renal Diseases http://dx.doi.org/10.5772/66089 419

**Figure 4.** Hypoxia and IBD pathogenesis.

provide mechanistic evidence into the regulation of HIF‐1α gene expression via binding of several NF‐κB subunits to the HIF‐1α promoter region [134, 135]. Thus, secondary to direct activation of HIF‐1 under hypoxic conditions, interaction of NF‐κB additionally contributes to

Respective regulation of NF‐κB by HIF‐1 has also been reported in the literature [114, 138, 139]. These studies suggest direct activation of NF‐κB via HIF‐1 signaling in inflammatory cells. Among suggested mechanisms are increased expression of TLR2 and TLR6 leading to activation of NF‐κB, hyperphosphorylation of IKKβ, and phosphorylation of serine residues of p65 subunit of NF‐κB leading to its translocation to nucleus and transcriptional activity [117,

Overall, hypoxia and signaling via NF‐κB and HIF‐1 are closely linked and, respectively,

IBD is associated with loss of intestinal mucosal barrier, inflammation of mucosa, and increased incidence of bacterial infections [140]. IBD is categorized as ulcerative colitis (UC) and Crohn's disease (CD). Both conditions are associated with severe inflammation and breakdown of intestinal mucosal barrier and chronic gastrointestinal discomfort. Current therapeutic approaches to IBD include anti‐inflammatory agents mostly targeted at TNF‐α and

Hypoxia has been shown to be a critical component of inflammation in IBD. Surgical specimens of intestinal mucosa of IBD patients show increased expression of HIF‐1 and HIF‐2 [141]. Increased vascular proliferation and density has been noted in intestines of IBD patients secondary to hypoxia‐induced VEGF activity [142]. Additionally microvascular abnormality

The intestinal mucosa is exposed to fluctuating levels of oxygen. On the one hand, the intestinal lumen is nearly anoxic, and oxygen pressure is generally low on the luminal side of the mucosa. On the other hand, the rate of perfusion of the subendothelium is dependent upon meal intake, and PO2 changes dramatically from high to low in between meals. The shift in oxygen tension in the mucosal layer renders it resistant to hypoxic states. This could be in part due to basal activity of hypoxic signaling intermediates such as HIF‐1 in the intestinal mucosal. Indeed, HIF‐1α–null mice in the intestinal epithelium show diminished mucosal protection and increased clinical symptoms in murine model of colitis [144]. HIF‐1–induced epithelial protection is shown to be due to induction of several proteins such as mucin, p‐glycoprotein, and ecto‐5′‐nucleotidase (CD73), an enzyme that converts AMP to adenosine (A2B) receptor [140]. Adenosine production during hypoxia has shown to decrease vascular leakage and neutrophil accumulation and thus plays an anti‐inflammatory role [120]. In a case‐control cohort study, patients with polymorphisms in CD39, a vascular and immune cell ecto‐ nucleotidase that converts extracellular ATP and ADP to AMP, had increased susceptibility to Crohn's disease [145]. Therefore, HIF‐1 signaling via adenosine is a key step in protection

and loss of endothelial nitric oxide production are seen in IBD mucosa [143].

this process by increasing basal levels of HIF‐1α protein.

regulate one another to enhance the inflammatory response.

**2.3. Hypoxia and inflammatory bowel disease (IBD)**

138, 139].

418 Hypoxia and Human Diseases

immune cell members.

against IBD inflammation (**Figure 4**).

Aside from HIF‐1, NF‐κB is also involved in inflammatory events of IBD [146, 147]. Nuclear levels of NF‐κB p65 have long been seen in lamina propria biopsies of patients with Crohn's disease [148]. Activation of NF‐κB in mucosal macrophages leads to induction of pro‐inflam‐ matory cytokines such as TNF‐α, IL‐1, and IL‐6, which mediate mucosal tissue damage [149]. NF‐κB activation in intestinal mucosa also plays a role in differentiation of T‐helper cells, which also play a role in IBD inflammation (**Figure 4**) [149]. In addition to pro‐inflammatory activity, some studies have shown a protective role for NF‐κB [146]. Loss of β or γ subunits of the IKK complex leads to colitis and apoptosis of intestinal mucosa [150, 151]. Additionally, polymor‐ phisms of TLR4 and TLR5, which are involved in NF‐κB activation, have been strongly associated with IBD in canines [152]. The protective role of NF‐κB in IBD is thought to be in terms of maintaining mucosal barrier and integrity. Overall, NF‐κB seems to play a dual role in IBD.

Due to the protective role of HIF‐1 in models of colitis, it has been proposed that induction of HIF‐1 could serve as a potential therapeutic target for treatment of IBD. The common phar‐ macological method of HIF‐1 induction is via inhibition of PHD enzymes, which break down the HIF‐1α subunit in the presence of oxygen. In vitro pharmacological inhibition of PHD using 2‐oxoglutarate analogs as co‐substrates of PHDs or dimethyloxaloglycine, has shown to stabilize HIF‐1α [153–155]. In these studies PHD inhibitors decreased clinical symptoms in murine models of colitis and thus present promising therapeutic targets for IBD [153, 155, 156]. As mentioned previously blockade of PHDs can also lead to NF‐κB activation. Using PHD inhibitors has thus been suggested to have dual benefits in treatment of IBD.

NF‐κB activity, however, is associated with increased inflammation, and therefore, inhibition of NF‐κB has also been examined and shows promise in treatment of IBD [149]. Selective NF‐ κB inhibitors, antisense oligonucleotides against NF‐κB, and targeting DNA‐binding activity of NF‐κB using decoy oligodeoxynucleotides have been among the strategies tested that have produced promising results in murine models of colitis and IBD [157, 158].

#### **2.4. Hypoxia and rheumatoid arthritis (RA)**

 Rheumatoid arthritis is the most common type of inflammatory arthritis. As an autoimmune disorder, RA is characterized as inflammation of the synovium, loss of cartilage, and bone erosion leading to joint pain and dysfunction [159]. The synovial fluid in RA is infiltrated with fibroblasts, immune cells, and angiogenesis of new vasculature [159, 160]. Additionally, a key feature of synovial fluid in RA is hypoxia. It has been shown that the synovium of knee joints of RA patients has significantly less O2 7 pressure than that of osteoarthritis (OA) patients [161]. Immunohistochemical analysis of synovial stromal cells and macrophages of RA‐ and OA‐ affected joints show significant increases in HIF1α and HIF2α expression compared to normal. Additionally, the levels of HIFs were directly correlated with VEGF expression in the stromal cell lining in these specimens [162]. Other studies have identified HIF‐2α significantly upre‐ gulated in fibroblast‐like synoviocytes of RA and associated IL‐6 upregulation in these cells [163]. These and other similar studies imply HIF signaling as the orchestrator of synovial inflammation and secondary joint damage [159, 164, 165]. A large number of HIF‐activated inflamamtory mediators have been identified in RA synovial fluid including but not limited to stromal cell–derived factor 1 (SDF‐1), VEGF, TNF‐α, IL‐1β, and IL‐8 [166]. Various TLRs are also expressed in synovial tissue and macrophages, which further activate NF‐κB pathway and increase expression of other inflammatory proteins [167]. Not surprisingly, HIF‐dependent pathways have also been implicated in TLR expressions in many tissues including synovial cells [117, 118]. Finally, recruitment of CXCR4+ lymphocytes and matrix metalloproteinases (MMPs) in the synovial fibroblasts involved in cartilage destruction has also shown to be HIF‐1 mediated and NF‐κB mediated [168, 169]. Overall, a large body of evidence implicates hypoxia and HIF signaling as a key underlying mechanism in pathogenesis of RA (**Figure 5**).

**Figure 5.** Hypoxia and pathogenesis of rheumatoid arthritis.

As discussed above, hypoxia‐ and HIF‐mediated signaling is highly pro‐inflammatory and destructive in RA. The key approach to treatment of RA is thus inhibition of HIF signaling. Many HIF inhibitors have been tested in cancer that may show promise in treatment of RA [170]. The limiting factor in administering HIF inhibitors is pharmacokinetic availability of these compounds in the synovial space as well as specific targetting of joints rather than systemic therapy. Gene targetting of HIF molecules using antisense oligonucleotides targetting HIF‐1α mRNA has also been tested, which may show efficacy in RA treatment [159]. Addi‐ tional approaches including anti‐VEGF antibodies or anti‐VEGF receptor molecules have been tested in models of arthritis and have shown efficacy in delaying onset and severity of RA in animal models [159, 171]. These strategies remain to be clinically tested yet show great promise in novel therapeutics of RA.

### **2.5. Conclusion**

1 **2.4. Hypoxia and rheumatoid arthritis (RA)**

18 Hypoxia and Human Diseases 420 Hypoxia and Human Diseases

24

25 **Figure 5.** Hypoxia and pathogenesis of rheumatoid arthritis.

 Rheumatoid arthritis is the most common type of inflammatory arthritis. As an autoimmune disorder, RA is characterized as inflammation of the synovium, loss of cartilage, and bone erosion leading to joint pain and dysfunction [159]. The synovial fluid in RA is infiltrated with fibroblasts, immune cells, and angiogenesis of new vasculature [159, 160]. Additionally, a key feature of synovial fluid in RA is hypoxia. It has been shown that the synovium of knee joints of RA patients has significantly less O2 7 pressure than that of osteoarthritis (OA) patients [161]. Immunohistochemical analysis of synovial stromal cells and macrophages of RA‐ and OA‐ affected joints show significant increases in HIF1α and HIF2α expression compared to normal. Additionally, the levels of HIFs were directly correlated with VEGF expression in the stromal cell lining in these specimens [162]. Other studies have identified HIF‐2α significantly upre‐ gulated in fibroblast‐like synoviocytes of RA and associated IL‐6 upregulation in these cells [163]. These and other similar studies imply HIF signaling as the orchestrator of synovial inflammation and secondary joint damage [159, 164, 165]. A large number of HIF‐activated inflamamtory mediators have been identified in RA synovial fluid including but not limited to stromal cell–derived factor 1 (SDF‐1), VEGF, TNF‐α, IL‐1β, and IL‐8 [166]. Various TLRs are also expressed in synovial tissue and macrophages, which further activate NF‐κB pathway and increase expression of other inflammatory proteins [167]. Not surprisingly, HIF‐dependent pathways have also been implicated in TLR expressions in many tissues including synovial cells [117, 118]. Finally, recruitment of CXCR4+ lymphocytes and matrix metalloproteinases (MMPs) in the synovial fibroblasts involved in cartilage destruction has also shown to be HIF‐1 mediated and NF‐κB mediated [168, 169]. Overall, a large body of evidence implicates hypoxia

23 and HIF signaling as a key underlying mechanism in pathogenesis of RA (**Figure 5**).

Section 2 discussed the complex relationship between hypoxia and inflammatory process and highlighted the key intermediates and pathways involved in this relationship. The discovery of hypoxic‐inflammatory pathways has led to a greater understanding of inflammatory and autoimmune diseases such as IBD and RA and the use of novel pharmacological approaches targetting HIF and hypoxic signaling intermediates in these conditions. So far, these agents have been mostly studied in cancer clinical trials. Additional clinical studies are needed to examine the safety and efficacy of new HIF‐modulating agents in treatment of inflammatory disease states.

## **3. Hypoxia and renal diseases**

#### **3.1. Introduction**

Approximately 26 million Americans have some evidence of chronic kidney disease (CKD) and are at risk to develop kidney failure. The number of Americans with end‐stage renal diseases (ESRD) is expected to grow to 785,000 by 2020 (currently 485,000). The annual cost of treating ESRD is currently over \$32 billion. It is estimated that healthcare system can save up to \$18.5 to \$60.6 billion by reducing rate of progression of chronic kidney disease (CKD) by 10–30% over the next decade.

In acute setting acute kidney injury (AKI) has been shown to be associated with bad outcome, for instance, mortality rate of hospitalized patients with AKI increases more than fourfold [172]. Due to high medical and economic impact of AKI and CKD, finding new therapeutic tools in treatment of CKD is becoming of an increasing importance.

Hypoxia‐inducible factor (HIF) has become the focus of medical community as a putative target because its augmentation is likely to ease the burden of kidney disease. The following sections discuss the evidence regarding the role of HIF molecules in various kidney pathologies and potential therapeutic approaches with respect to the HIF system.

#### *3.1.1. Pathophysiology*

Kidneys have a rich blood supply. In fact human kidneys receive 20% of cardiac output, while they weigh less than 1% of the total body weight. However, renal medulla, physiologically, has low oxygen tension and hence is very sensitive to hypoxia.

Hypoxia is the final common pathway to irreversible renal damage and eventually ESRD [173]. Since Fine et al. introduced chronic hypoxia hypothesis for the first time, it has been confirmed in several studies [174]. Also, hypoxia plays a role in pathogenesis of AKI as well as transfor‐ mation of AKI to CKD.

Three phases of cell damage have been recognized following hypoxic insult to kidneys (by ligation of a branch of renal artery) [175]:


In order to survive hypoxemia or regional hypoxia, the kidneys adopt a set of sophisticated defense mechanisms, which include expression of HIF. HIF is the cornerstone of adaptation to hypoxia. This master regulator of the cellular response to hypoxia orchestrates several hundred target genes affecting metabolism, the cell cycle, and inflammation [176]. The hypoxia‐ inducible transcription factors have been extensively studied in the kidneys [177]. HIF‐1α is mainly expressed in tubular cells, while HIF‐2α is found in peritubular, interstitial, endothelial, and glomerular regions [178]. Likewise, PHD1 and PHD3 are mostly present in glomeruli, and PHD1, PHD2, and PHD3 express more in the distal tubules than in the proximal tubules [179].

Numerous studies have found critical roles for HIF molecules in hypoxic adaptation of the kidneys as well as pathophysiology of various kidney diseases [177]. Given the fact HIF is the key step in renal response to hypoxia targeting HIF and its regulatory mechanisms is a plausible approach to prevent and treat hypoxic insults to kidney. In quest for novel therapeutic tools for treatment and prevention of kidney diseases, HIF‐related pathways have shown promising results.

#### **3.2. HIF in acute kidney injury**

AKI is defined by rapid decline in renal function. AKI has multitude of causes. One of the most common causes of AKI is ischemia as a result of decreased renal perfusion, which leads to acute tubular necrosis (ATN) [180]. With renal ischemia several mechanisms in small arterioles will perpetuate regional hypoxia (**Figure 6**); these mechanisms include:


Hypoxia and its Emerging Therapeutics in Neurodegenerative, Inflammatory and Renal Diseases http://dx.doi.org/10.5772/66089 423

**Figure 6.** Diagram summarizing the interrelation between different factors causing hypoxia and CKD.

It has been shown that after renal ischemic attack, the number of capillaries in the medulla will decrease, which in turn leads to chronic ischemia, fibrosis, and progression to CKD [184]. Therefore, AKI is a risk factor for development of CKD. At the same time, patients with CKD have more incidence of AKI. In fact the most important risk factor of AKI is CKD [185]. AKI carries high risk of mortality; among patients older than 66 years with a first AKI hospitaliza‐ tion, the in‐hospital mortality rate in 2013 was up to 14.4% (2015 USRDS Annual Data Report). Mortality rate in patients with AKI admitted to intensive care unit may surpass 50%. These data obviated the need for finding new therapies in AKI focused on renal hypoxia.

The key hypoxic intermediates mostly studied in animal models of AKI are HIF‐1 and HIF‐2. Rosenberger et al. showed that upregulation of HIF‐1α occurs up to 7 days following ligation of a branch of the renal artery. HIF‐2α expression has also been noted but to a lesser degree than HIF‐1α and was confined to resident and infiltrating peritubular cells in the cortex [186]. Numerous studies have shown the involvement of HIF proteins in protection against acute renal injury [177]. Induction of HIF‐1 or its target genes have shown to reduce injury secondary to various types of acute renal insult [187, 188].

#### *3.2.1. HIF in contrast-induced nephropathy*

*3.1.1. Pathophysiology*

422 Hypoxia and Human Diseases

mation of AKI to CKD.

completed.

promising results.

**3.2. HIF in acute kidney injury**

endothelium and leukocytes [183]

ligation of a branch of renal artery) [175]:

Kidneys have a rich blood supply. In fact human kidneys receive 20% of cardiac output, while they weigh less than 1% of the total body weight. However, renal medulla, physiologically, has

Hypoxia is the final common pathway to irreversible renal damage and eventually ESRD [173]. Since Fine et al. introduced chronic hypoxia hypothesis for the first time, it has been confirmed in several studies [174]. Also, hypoxia plays a role in pathogenesis of AKI as well as transfor‐

Three phases of cell damage have been recognized following hypoxic insult to kidneys (by

**•** Phase II or intermediate phase: 1–3 days following insult; in this phase tissue damage is

In order to survive hypoxemia or regional hypoxia, the kidneys adopt a set of sophisticated defense mechanisms, which include expression of HIF. HIF is the cornerstone of adaptation to hypoxia. This master regulator of the cellular response to hypoxia orchestrates several hundred target genes affecting metabolism, the cell cycle, and inflammation [176]. The hypoxia‐ inducible transcription factors have been extensively studied in the kidneys [177]. HIF‐1α is mainly expressed in tubular cells, while HIF‐2α is found in peritubular, interstitial, endothelial, and glomerular regions [178]. Likewise, PHD1 and PHD3 are mostly present in glomeruli, and PHD1, PHD2, and PHD3 express more in the distal tubules than in the proximal tubules [179]. Numerous studies have found critical roles for HIF molecules in hypoxic adaptation of the kidneys as well as pathophysiology of various kidney diseases [177]. Given the fact HIF is the key step in renal response to hypoxia targeting HIF and its regulatory mechanisms is a plausible approach to prevent and treat hypoxic insults to kidney. In quest for novel therapeutic tools for treatment and prevention of kidney diseases, HIF‐related pathways have shown

AKI is defined by rapid decline in renal function. AKI has multitude of causes. One of the most common causes of AKI is ischemia as a result of decreased renal perfusion, which leads to acute tubular necrosis (ATN) [180]. With renal ischemia several mechanisms in small arterioles

**c.** Small vessel occlusion due to activation of coagulation system interaction between the

will perpetuate regional hypoxia (**Figure 6**); these mechanisms include:

**a.** Decreased generation of nitric oxide (vasodilator) by endothelial cells [181]

**b.** Enhanced reactivity to endogenous mediators of vasoconstriction [182]

**•** Phase I: 1–6 h post hypoxic damage; in this phase parenchymal cells still appear viable.

**•** Phase III or late phase: after 3 days; when tissue repair and remodeling are initiated.

low oxygen tension and hence is very sensitive to hypoxia.

The exact mechanism of contrast‐induced nephropathy (CIN) remains elusive. Among possible mechanisms are renal vasoconstriction and decreased vasodilatation, which leads to tubular hypoxemia, decreased mitochondrial function and generation of reactive oxygen species (ROS), increased prostaglandins, decreased NO levels, and increased oxygen con‐ sumption due to osmotic demand of contrast media on tubular Na/K ATPase, all of which lead to medullary cell damage [189, 190]. Clearly, a direct link with hypoxia and CIN exists. Reversible renal vasoconstriction has been demonstrated in animal studies [191]. In an animal study, Rosenberger et al. induced renal hypoxia by a combination of COX inhibition, radio‐ contrast material, and blockade of nitric oxide synthase. In this study generalized HIF induction (tubules, interstitium, and endothelial cells) initiated within minutes of regional renal hypoxia. They showed medullary thick ascending limb (TAL) of Henle had less HIF expression, which may explain the higher susceptibility of this region to hypoxia [175]. These findings render regional hypoxia a plausible cause for CIN pathophysiology and a potential role for preventative HIF induction therapy in this condition.

#### *3.2.2. Ischemic acute kidney injury*

Ischemic injury in thick ascending limb of Henle is believed to play a pivotal role in patho‐ genesis of AKI due to regional renal low oxygen tension. Activation of HIF‐1 has shown to be protective in models of ischemia‐reperfusion injury. Schley and his colleagues showed that deletion of the von Hippel‐Lindau (*VHL*) protein in thick ascending limb (TAL) of Henle preserved its function following ischemia‐reperfusion. Notably, this study demonstrated better recovery in *VHL*‐knocked‐out animals by showing higher number of proliferating cells [192]. Furthermore, preconditional activation of HIF‐1 via carbon monoxide or PHD1 inhibitor has shown to ameliorate the degree of renal ischemic damage in rat models of ischemia‐ reperfusion injury [188]. Others have shown activation of HIF‐1 via cobalt chloride leads to reduction of tubulointerstitial damage secondary to acute renal injury in rats [187].

#### **3.3. HIF in chronic kidney disease (CKD)**

Chronic renal hypoxia causes apoptosis and also differentiation of tubular cells to myofibro‐ blasts. Under hypoxic condition renal fibroblasts will also get activated. These together will lead to progressive renal failure and eventually ESRD. Glomerulosclerosis as a result of chronic high blood pressure or high blood sugar can also cause tubular ischemia by impairing tubular perfusion.

Several pharmacological means of reducing renal hypoxia are already widely available for use in daily clinical practice. Treatment with erythropoietin (EPO)‐stimulating agents has been shown to slow down the progression of CKD [193]. Renin‐angiotensin system (RAS) blockade can also be protective against hypoxia. RAS blockade will improve perfusion of peritubular capillaries by decreasing tone of efferent arteriols in parent glomerulus [194]. Yu et al. studied the effect of HIF activation (via a nonselective PHD inhibitor, l‐mimosine) in rats with CKD. Animals underwent subtotal nephrectomy. In this study they demonstrated HIF activation can have different (beneficial or deleterious) effects on renal tissue. It was also shown that function of remnant kidney is also dependent upon the timing of HIF activation. Early activation of HIF in CKD caused increased fibrosis (rise in mRNA of collagen type III) and inflammation, while late activation of HIF showed anti‐fibrotic effects [195].

#### *3.3.1. HIF in diabetic nephropathy*

species (ROS), increased prostaglandins, decreased NO levels, and increased oxygen con‐ sumption due to osmotic demand of contrast media on tubular Na/K ATPase, all of which lead to medullary cell damage [189, 190]. Clearly, a direct link with hypoxia and CIN exists. Reversible renal vasoconstriction has been demonstrated in animal studies [191]. In an animal study, Rosenberger et al. induced renal hypoxia by a combination of COX inhibition, radio‐ contrast material, and blockade of nitric oxide synthase. In this study generalized HIF induction (tubules, interstitium, and endothelial cells) initiated within minutes of regional renal hypoxia. They showed medullary thick ascending limb (TAL) of Henle had less HIF expression, which may explain the higher susceptibility of this region to hypoxia [175]. These findings render regional hypoxia a plausible cause for CIN pathophysiology and a potential

Ischemic injury in thick ascending limb of Henle is believed to play a pivotal role in patho‐ genesis of AKI due to regional renal low oxygen tension. Activation of HIF‐1 has shown to be protective in models of ischemia‐reperfusion injury. Schley and his colleagues showed that deletion of the von Hippel‐Lindau (*VHL*) protein in thick ascending limb (TAL) of Henle preserved its function following ischemia‐reperfusion. Notably, this study demonstrated better recovery in *VHL*‐knocked‐out animals by showing higher number of proliferating cells [192]. Furthermore, preconditional activation of HIF‐1 via carbon monoxide or PHD1 inhibitor has shown to ameliorate the degree of renal ischemic damage in rat models of ischemia‐ reperfusion injury [188]. Others have shown activation of HIF‐1 via cobalt chloride leads to

reduction of tubulointerstitial damage secondary to acute renal injury in rats [187].

Chronic renal hypoxia causes apoptosis and also differentiation of tubular cells to myofibro‐ blasts. Under hypoxic condition renal fibroblasts will also get activated. These together will lead to progressive renal failure and eventually ESRD. Glomerulosclerosis as a result of chronic high blood pressure or high blood sugar can also cause tubular ischemia by impairing tubular

Several pharmacological means of reducing renal hypoxia are already widely available for use in daily clinical practice. Treatment with erythropoietin (EPO)‐stimulating agents has been shown to slow down the progression of CKD [193]. Renin‐angiotensin system (RAS) blockade can also be protective against hypoxia. RAS blockade will improve perfusion of peritubular capillaries by decreasing tone of efferent arteriols in parent glomerulus [194]. Yu et al. studied the effect of HIF activation (via a nonselective PHD inhibitor, l‐mimosine) in rats with CKD. Animals underwent subtotal nephrectomy. In this study they demonstrated HIF activation can have different (beneficial or deleterious) effects on renal tissue. It was also shown that function of remnant kidney is also dependent upon the timing of HIF activation. Early activation of HIF in CKD caused increased fibrosis (rise in mRNA of collagen type III) and inflammation, while

role for preventative HIF induction therapy in this condition.

*3.2.2. Ischemic acute kidney injury*

424 Hypoxia and Human Diseases

**3.3. HIF in chronic kidney disease (CKD)**

late activation of HIF showed anti‐fibrotic effects [195].

perfusion.

Diabetic kidney disease (diabetic nephropathy (DN)) is the leading cause of ESRD. Hypergly‐ cemia and resultant hyperfiltration will increase renal oxygen consumption. Eighty percent of the total renal oxygen consumption is related to sodium‐potassium pump in cortical proximal tubule. Diabetes causes decreased renal oxygen tension by increasing oxygen consumption. Inoue et al. by using diffusion‐weighted (DW) and blood oxygen level‐dependent (BOLD) MRI showed tissue hypoxia in diabetic kidneys [196]. Palm et al. also demonstrated lower paren‐ chymal oxygen tension along with higher oxygen consumption in diabetic rats [197]. In the setting of hypoxia, paradoxically, the activity of HIF‐1α seems to be decreased or altered in diabetic rat kidneys [198, 199]. Polymorphism of pro582ser in HIF‐1α gene, which results in altered response of HIF‐1α to low oxygen, is associated with increased incidence of diabetic nephropathy in diabetic patients [199]. It appears from this evidence that HIF‐1α‐protective activity in the kidney is compromised in the setting of diabetes. This is further supported by the finding that pharmacologic activation of HIF pathway decreases renal damage in diabetic rats by decreasing proteinuria, improving tubulointerstitial damage and normalizing glomer‐ ular hyperfiltration [200]. There is thus indication for the use of HIF‐1–activating approaches in prevention of diabetic nephropathy.

#### **3.4. HIF in anemia of kidney disease**

HIF plays a role in anemia of CKD and ESRD. Erythropoietin is secreted from human kidneys after birth. The kidney accounts for ∼90% of the total EPO production in the adult human [201]. Renal erythropoietin‐producing cells are fibroblasts in peritubular capillaries in the cortex and outer medulla [202].

Kidneys are the perfect choice to be responsible for erythropoietin secretion due to their regional low oxygen tension. Any minute changes in renal oxygen tension will lead to adjustments of serum hematocrit. In subcellular level HIF binds to the EPO enhancer, the hypoxia‐responsive element, and activates the transcription of EPO. Renal EPO synthesis is regulated by HIF‐2 [203]. HIF‐2 exerts its multipronged effect by increasing EPO production, increasing iron absorption, and also increasing maturation of erythroid progenitors in the bone marrow. Studies indicate that in ESRD patients erythropoietin concentration is below normal due to dysfunctional EPO‐producing cells (not due to cell death) [204]. Erythropoietin‐ producing cells in renal fibrosis remain alive and preserve their plasticity: although the exact mechanism of erythropoietin production in ESRD remains elusive, it is possible plasticity of erythropoietin‐producing cells plays a role when signals for HIF pathway are augmented. Pathways to stabilize or even augment HIF response will mimic the state of hypoxia, which will lead to erythropoietin production; this is considered a novel therapeutic tool in our armamentarium to treat anemia of CKD. HIF stabilizers inhibit PHDs, which will subsequently cause accumulation of HIF, and as a result erythropoietin production ensues.

In 2010 a phase 1 clinical trial revealed PHD inhibitor (FG‐2216) led to increased EPO produc‐ tion and plasma EPO levels in patients with ESRD [205]. In a phase 2‐b study of nondialysis‐ dependent patients with chronic kidney disease, related anemia treatment with an oral PHD inhibitor (Roxadustat) was shown to increase EPO level and correct anemia. Clinical response was independent of iron intake (oral or IV) [206].

#### **3.5. HIF in renal transplant**

As of January 2016, there are 100,791 people waiting for renal transplants in the United States. Every 14 min a patient is added to the kidney transplant waiting list. In 2012, the probability of 1‐year graft survival was 92% and 97% for deceased and living donor kidney transplant recipients, respectively. The estimated US average charges for a kidney transplant in 2011 is \$262,900. This data emphasizes on the need for exploring new ways to save and preserve more allografts.

In the process of renal transplantation, harvested organ is subjected to hypoxia. Hypoxia‐ reperfusion occurs during organ procurement, preservation, and after implantation. Ischemia‐ Reperfusion injury (IRI) has prognostic implications for the allograft and kidney recipient. As mentioned before HIF has been shown to be a renoprotective agent and may alter transplan‐ tation outcome.

Conde et al. found HIF‐1α increases in human proximal tubular cells (in vitro) after hypo‐ xia and also during reoxygenation period. A similar biphasic pattern was observed in IRI model in SD rats (en vivo). The en vivo part of the study proved that HIF‐1α induction during reperfusion phase was required for survival of proximal tubule cells and expedited recovery. Conde and his colleagues also studied human allograft biopsies (7–15 days post‐ transplant): HIF‐1α expression was more robust in proximal tubule cells with minimal is‐ chemic damage. Again, this finding indicate a protective role of HIF in IRI. AN interesting finding in this study was demonstration of the role of Akt/mTOR signaling in HIF‐1α in‐ duction. Using rapamycin (mTOR inhibitor) during reoxygenation period prevented HIF‐1α stabilization [207].

Renal ischemia‐reperfusion injury is an important factor in determination of the fate of a renal allograft. Immunological response is potentiated under ischemia‐reperfusion. CD4+ T cells play the main role in pathogenesis of IRI and natural killer (NK) cells are part of the immediate response to IRI. Regulatory family differentially affect the immune response to the of HIF affect allograft's during ischemia‐reperfusion. While HIF‐1α plays a crucial role in T‐cell survival and function , HIF‐2α has a protective function in T‐cell mediated renal IRI [208]. In an animal study, Zhang et al. showed the role of HIF‐2α in mitigating NK T‐cell–mediated cytotoxicity in IRI. In this study HIF‐2α and adenosine A2A receptor (adora2a) worked in concert with each other (so‐called hypoxia‐adenosinergic immunosuppression) to restrict NK T‐cell activation [209]. This finding is of clinical importance as pharmacologic activation of HIF‐2α can potentially limit allograft IRI and subsequently improve the outcome of renal transplan‐ tation.

#### **3.6. Conclusion**

The overwhelming clinical and economical impact of renal disease and the limited thera‐ peutic options available have placed a great demand on finding additional therapeutic ap‐ proaches. The evidence discussed in this section suggests a widespread role of hypoxia and HIF signaling in a range of acute and chronic renal diseases and a clear indication for HIF‐ targeted therapies. It appears that HIF‐1 activity is protective in acute renal injury, while prolonged activity of HIF‐1 may lead to worsened outcomes in CKD. The protective versus deleterious roles of HIF‐1 thus complicate the use of HIF‐1–targeted approaches. On the other hand, HIF‐2 therapies may be more promising especially in terms of anemia of kidney disease and renal allograft rejection. In either case, additional clinical research is needed in the use and efficacy of both HIF‐1 and HIF‐2 therapies in prevention or treatment of various renal diseases.

## **Author details**

inhibitor (Roxadustat) was shown to increase EPO level and correct anemia. Clinical response

As of January 2016, there are 100,791 people waiting for renal transplants in the United States. Every 14 min a patient is added to the kidney transplant waiting list. In 2012, the probability of 1‐year graft survival was 92% and 97% for deceased and living donor kidney transplant recipients, respectively. The estimated US average charges for a kidney transplant in 2011 is \$262,900. This data emphasizes on the need for exploring new ways to save and preserve more

In the process of renal transplantation, harvested organ is subjected to hypoxia. Hypoxia‐ reperfusion occurs during organ procurement, preservation, and after implantation. Ischemia‐ Reperfusion injury (IRI) has prognostic implications for the allograft and kidney recipient. As mentioned before HIF has been shown to be a renoprotective agent and may alter transplan‐

Conde et al. found HIF‐1α increases in human proximal tubular cells (in vitro) after hypo‐ xia and also during reoxygenation period. A similar biphasic pattern was observed in IRI model in SD rats (en vivo). The en vivo part of the study proved that HIF‐1α induction during reperfusion phase was required for survival of proximal tubule cells and expedited recovery. Conde and his colleagues also studied human allograft biopsies (7–15 days post‐ transplant): HIF‐1α expression was more robust in proximal tubule cells with minimal is‐ chemic damage. Again, this finding indicate a protective role of HIF in IRI. AN interesting finding in this study was demonstration of the role of Akt/mTOR signaling in HIF‐1α in‐ duction. Using rapamycin (mTOR inhibitor) during reoxygenation period prevented

Renal ischemia‐reperfusion injury is an important factor in determination of the fate of a renal allograft. Immunological response is potentiated under ischemia‐reperfusion. CD4+ T cells play the main role in pathogenesis of IRI and natural killer (NK) cells are part of the immediate response to IRI. Regulatory family differentially affect the immune response to the of HIF affect allograft's during ischemia‐reperfusion. While HIF‐1α plays a crucial role in T‐cell survival and function , HIF‐2α has a protective function in T‐cell mediated renal IRI [208]. In an animal study, Zhang et al. showed the role of HIF‐2α in mitigating NK T‐cell–mediated cytotoxicity in IRI. In this study HIF‐2α and adenosine A2A receptor (adora2a) worked in concert with each other (so‐called hypoxia‐adenosinergic immunosuppression) to restrict NK T‐cell activation [209]. This finding is of clinical importance as pharmacologic activation of HIF‐2α can potentially limit allograft IRI and subsequently improve the outcome of renal transplan‐

The overwhelming clinical and economical impact of renal disease and the limited thera‐ peutic options available have placed a great demand on finding additional therapeutic ap‐

was independent of iron intake (oral or IV) [206].

**3.5. HIF in renal transplant**

426 Hypoxia and Human Diseases

allografts.

tation outcome.

HIF‐1α stabilization [207].

tation.

**3.6. Conclusion**

Deepak Bhatia1 , Mohammad Sanaei Ardekani2 , Qiwen Shi3 and Shahrzad Movafagh1\*

\*Address all correspondence to: smovafag@su.edu

1 Bernard J Dunn School of Pharmacy, Shenandoah University, VA, USA

2 Kidney and Hypertension Specialists, VA, USA

3 Collaborative Innovation Center of Yangtza River Delta Region Green Pharmaceuticals, Zhejiang University of Technology, Hangzhou, Zhejiang

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2016.

## *Edited by Jing Zheng and Chi Zhou*

This book contains a total of 21 chapters, each of which was written by experts in the corresponding field. The objective of this book is to provide a comprehensive and updated overview of cellular and molecular mechanisms underlying hypoxia's impacts on human health, as well as current advances and future directions in the detection, recognition, and management of hypoxia-related disorders. This collection of articles provides a clear update in the area of hypoxia research for biomedical researchers, medical students, nurse practitioners, and practicing clinicians in the fields of high altitude biology, cardiovascular biology and medicine, tumor oncology, obstetrics, pediatrics, and orthodontics and for others who may be interested in hypoxia.

Hypoxia and Human Diseases

Hypoxia and Human Diseases

*Edited by Jing Zheng and Chi Zhou*

Photo by porpeller / iStock