**Meet the editors**

Dr. Zheng is a professor in the Department of Obstetrics and Gynecology at the University of Wisconsin-Madison. He received his PhD in Reproductive Physiology. Over the last two decades, his research interests are in the cellular and molecular mechanisms governing endothelial functions. Dr. Zheng's laboratory has been continuously funded by AHA, NIH, and private foun-

dations, and he has served as a regular and ad hoc member of several NIH and AHA study sections. He has also been actively involved in training students and other young scientists.

Dr. Zhou is a research associate in the Department of Obstetrics and Gynecology at the University of Wisconsin-Madison. She obtained her PhD in Reproductive Physiology, primarily in functional genomics and embryonic development. Currently, her research focuses on exploring mechanisms regulating human placental/ fetal vascular growth and development relevant to

human pregnancy complications using functional genomics and bioinformatics approaches.

## Contents

### **Preface XIII**


Contents **VII**

Chapter 9 **Adaptations to Chronic Hypoxia Combined with Erythropoietin Deficiency in Cerebral and Cardiac Tissues 161**

> Mitchell Huber, Hong Lian Duan, Ankush Chandra, Fengwu Li, Longfei Wu, Longfei Guan, Xiaokun Geng and Yuchuan Ding

Chapter 10 **Hypothermia in Stroke Therapy: Systemic versus Local**

Nicoletta Charolidi and Veronica A. Carroll

Sandeep Artham and Payaningal R. Somanath

Chapter 13 **Hypoxia Modulates the Adenosinergic Neural Network 247**

Chapter 14 **The HIF System Response to ESA Therapy in CKD‐Anemia 267** Sandra Ribeiro, Luís Belo, Flávio Reis and Alice Santos‐Silva

Xiao Xiao Wang, Yu Chen and Wai Keung Leung

Chapter 16 **Interplay between Hypoxia, Inflammation and Adipocyte Remodeling in the Metabolic Syndrome 303**

Monica Baniţă and Cătălina Gabriela Pisoschi

**Hypoxia and Fetal Growth Restriction 329**

Chapter 18 **The Critical Role of Hypoxia in Tumor-Mediated**

**Immunosuppression 349**

Guy Berchem and Bassam Janji

Chapter 17 **Epigenetic Programming of Cardiovascular Disease by Perinatal**

Paola Casanello, Emilio A. Herrera and Bernardo J. Krause

Nassera Aouali, Manon Bosseler, Delphine Sauvage, Kris Van Moer,

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

Raja El Hasnaoui-Saadani

Chapter 11 **Hypoxia and Pulmonary Hypertension 211**

Chapter 12 **Stage-Specific Effects of Hypoxia on Interstitial**

Susana P. Gaytán and Rosario Pasaro

Chapter 15 **Role of the Hypoxia-Inducible Factor in Periodontal**

**Application 179**

**Lung Disease 227**

**Inflammation 285**

#### Chapter 19 **Cross‐Talk Between Hypoxia and the Tumour via Exosomes 365** Shayna Sharma, Mona Alharbi, Andrew Lai, Miharu Kobayashi,

Richard Kline, Katrina Wade, Gregory E. Rice and Carlos Salomon

#### Chapter 20 **A Novel Hypoxia Imaging Endoscopy System 383** Kazuhiro Kaneko, Hiroshi Yamaguchi and Tomonori Yano

Chapter 21 **Hypoxia and its Emerging Therapeutics in Neurodegenerative, Inflammatory and Renal Diseases 403** Deepak Bhatia, Mohammad Sanaei Ardekani, Qiwen Shi and Shahrzad Movafagh

## Preface

Hypoxia refers to a state in which oxygen supply to the whole body or a region of the body is inadequate. To date, after extensive and systemic research, it is clear that chronic and se‐ vere hypoxia could be detrimental to human health and is related to many pathological con‐ ditions such as cardiovascular disorders and cancers. Additionally, we should also recognize that under physiological conditions, most cells within the tissue actually reside in low O2 environments (~3–16% O2) relative to ambient O2 (~ 21% O2). This physiological low O2 is critical to many essential cellular functions.

This book aims to provide a comprehensive and most updated overview of our current un‐ derstanding of physiological (i.e., at high altitude) and pathological hypoxia's roles in vari‐ ous aspects of human diseases. It also concludes with current advances and future directions of therapeutics of human hypoxic diseases. We hope that this book will become useful and attractive to medical students, practicing clinicians, and biomedical researchers who are working or are interested in the biology of hypoxia.

It has been an extraordinarily exciting and rewarding experience to put this book together. We wish to express our deep gratitude to all contributors for their hard work and scholarly efforts in preparation of each individual chapter. We also would like to thank our publishing manag‐ ers, Ms. Dajana Pemac and Ms. Maja Bozicevic at InTech, for making this book available.

> **Chi Zhou, PhD and Jing Zheng, PhD** Department of Obstetrics and Gynecology, University of Wisconsin-Madison, Madison, WI, USA

#### **The Multifaceted Role of Hypoxia‐Inducible Factor 1 (HIF1) in Lipid Metabolism The Multifaceted Role of Hypoxia**‐**Inducible Factor 1 (HIF1) in Lipid Metabolism**

Guomin Shen and Xiaobo Li Guomin Shen and Xiaobo Li

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

http://dx.doi.org/10.5772/65340

#### **Abstract**

Hypoxia‐inducible factor 1 (HIF1) is a master transcription factor and regulates expression of a large number of genes involving many aspects of biology. In addition to HIF1's roles in glucose metabolism and angiogenesis, numerous studies have revealed an emerging role of HIF1 in controlling lipid homeostasis. In this chapter, we discuss that lipid accumulation is related to HIF1's activity in several diseases and the growing evidence demonstrating the functional importance of HIF1 in controlling lipid metabolism. The functions include lipid uptake and trafficking, fatty acid metabolism, sterol metabolism, triacylglycerol synthesis, phospholipid metabolism, lipid droplet biogenesis, and lipid signaling. Defining the role of HIF1 in lipid metabolism is crucial to understand the pathophysiology of lipid in disease and may help us to identify additional target sites for drug development. This review would shed light on our understanding of the critical role of HIF1 in lipid metabolism.

**Keywords:** hypoxia‐inducible factor 1, lipid accumulation, lipid metabolism

## **1. Introduction**

Hypoxia has been identified as a common symptom in many diseases, such as cancer [1, 2], obesity [3], atherosclerosis [4], and ischemic heart disease (IHD) [5]. Adaptation to hypoxia involves hypoxia‐inducible factor 1 (HIF1) and requires reprogramming of essential elements of cellular metabolism [6]. HIF1 was described about 20 years ago [7]. It is a heterodimeric transcription factor that is composed of an oxygen‐regulated HIF1α subunit and a constitu‐ tively expressed HIF1β subunit [7, 8]. HIF1α is mainly regulated by protein degradation. Under normoxic conditions, HIF1α is subjected to oxygen‐dependent hydroxylation by three

© 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.

prolyl hydroxylase domain proteins (PHD1–3) on two proline residues in the oxygen‐ dependent degradation (ODD) domain [9]. The prolyl‐hydroxylated HIF1α is targeted for degradation by the tumor suppressor protein von Hippel‐Lindau (VHL), an E3 ubiquitin‐ protein ligase [10, 11]. HIF1α is also regulated in an oxygen‐dependent manner by factor inhibiting HIF1 (FIH1) [12, 13]. In this case, FIH1 mediates the hydroxylation of an asparagine residue in the C‐terminal trans‐activation domain, which prevents the binding of HIF1α with coactivators p300 and CBP [13–15]. Hydroxylation of proline and asparagine is inhibited under hypoxic conditions causing HIF1α to rapidly accumulate [12, 13]. HIF1α subsequently heterodimerizes with HIF1β, and the complex binds to hypoxic responsive elements (HREs) within the promoter regions of target genes, and allows for recruitment of coactivators and activation of transcription [16]. In addition to hypoxia, HIF1 accumulation can also be induced by growth‐factor stimulation, gene mutations, and intermediate metabolites [17] (**Figure 1**).

**Figure 1.** Regulation of HIF1 and its downstream roles related to lipid metabolism. HIF1 accumulation can be induced by hypoxia, gene mutations, intermediate metabolites, and growth factors. HIF1 plays a pivotal role in lipid metabo‐ lism. It can increase lipid uptake and trafficking, fatty acid synthesis, sterol synthesis, TAG synthesis, lipid droplet bio‐ genesis, and lipid signal production, and suppress fatty acid β‐oxidation. Lipid droplet accumulation may be the final result of HIF1 in lipid metabolism. It is unclear about its role in phospholipids metabolism.

It has been reported that HIF1 regulates the transcription of hundreds of genes involving many aspect of biology, especially energy metabolism and vascularization [16]. The role of HIF1 in glucose metabolism had been well established [17]. Most of genes involving glucose uptake and glycolysis are directly regulated by HIF1 [17]. Recent studies demonstrated that HIF1 also plays an important role in lipid metabolism [1, 2, 18–21]. Currently, our understanding of HIF1 in regulating lipid metabolism has lagged behind that of glucose metabolism. Lipids, struc‐ turally and functionally important in all organisms, are not only one of the major components of cellular membrane systems, but also the source of energy storage. Moreover, signal molecules, such as prostaglandin E2 (PGE2), hydroxyeicosatetraenoic acid (HETE), and steroid hormones, are derived from lipids. This review would focus on the HIF1's activity related to dysregulation of lipid metabolism in several diseases, including atherosclerosis [4], fatty liver disease (FLD) [19], heart failure diseases [5], obesity [3], and cancer [1, 2] as well as the involvement of HIF1 in lipid metabolism, including lipid uptake and trafficking, fatty acid metabolism, sterol metabolism, triacylglycerol (TAG) synthesis, phospholipid metabolism, lipid droplet (LD) biogenesis, and lipid signaling.

## **2. Lipid accumulation is associated with HIF1's activity in diseases**

Most of the studies have demonstrated that HIF1's activity is associated with lipid accumula‐ tion positively [3, 18, 20–27], while few researches have indicated the opposite effect [28–31]. PHD2 inhibition or deletion, increasing HIF1's activity (**Figure 1**), decreased lipid accumula‐ tion in different animal models [28, 30, 31]. It indicated that the role of HIF1 in lipid metabolism may be different in different animal models. Details were described and discussed in the following sections.

#### **2.1. Atherosclerosis**

prolyl hydroxylase domain proteins (PHD1–3) on two proline residues in the oxygen‐ dependent degradation (ODD) domain [9]. The prolyl‐hydroxylated HIF1α is targeted for degradation by the tumor suppressor protein von Hippel‐Lindau (VHL), an E3 ubiquitin‐ protein ligase [10, 11]. HIF1α is also regulated in an oxygen‐dependent manner by factor inhibiting HIF1 (FIH1) [12, 13]. In this case, FIH1 mediates the hydroxylation of an asparagine residue in the C‐terminal trans‐activation domain, which prevents the binding of HIF1α with coactivators p300 and CBP [13–15]. Hydroxylation of proline and asparagine is inhibited under hypoxic conditions causing HIF1α to rapidly accumulate [12, 13]. HIF1α subsequently heterodimerizes with HIF1β, and the complex binds to hypoxic responsive elements (HREs) within the promoter regions of target genes, and allows for recruitment of coactivators and activation of transcription [16]. In addition to hypoxia, HIF1 accumulation can also be induced by growth‐factor stimulation, gene mutations, and intermediate metabolites [17] (**Figure 1**).

2 Hypoxia and Human Diseases

**Figure 1.** Regulation of HIF1 and its downstream roles related to lipid metabolism. HIF1 accumulation can be induced by hypoxia, gene mutations, intermediate metabolites, and growth factors. HIF1 plays a pivotal role in lipid metabo‐ lism. It can increase lipid uptake and trafficking, fatty acid synthesis, sterol synthesis, TAG synthesis, lipid droplet bio‐ genesis, and lipid signal production, and suppress fatty acid β‐oxidation. Lipid droplet accumulation may be the final

It has been reported that HIF1 regulates the transcription of hundreds of genes involving many aspect of biology, especially energy metabolism and vascularization [16]. The role of HIF1 in glucose metabolism had been well established [17]. Most of genes involving glucose uptake and glycolysis are directly regulated by HIF1 [17]. Recent studies demonstrated that HIF1 also

result of HIF1 in lipid metabolism. It is unclear about its role in phospholipids metabolism.

Hypoxia has been demonstrated in atherosclerotic plaques [4]. Arterial wall hypoxia exists in a rabbit atherosclerosis injury model [32–34], confirmed in rabbit atherosclerotic plaques [35, 36] as well as in several mouse models [23, 37, 38]. Recently, in vivo studies have demonstrated hypoxia in human atherosclerotic plaques [39]. Macrophages are the major cell types in human plaques that display signs of hypoxia. TAG‐loaded foam cells derived from macrophages are characteristic of both early and late atherosclerotic plaques [40, 41]. Exposure of human macrophages to hypoxia causes an accumulation of TAG‐containing lipid droplets [42]. HIF1α is expressed in various cell types of atherosclerotic lesions and is associated with lesional inflammation [43]. Knockdown of HIF1α with small interfering RNAs inhibits TAG‐loaded foam cell formation in the human monoblastic cell line U937 [22]. Dyslipidemia are regarded as the key risk factors for the development of atherosclerosis, and HIF1 has been suggested to have both detrimental and beneficial roles in atherosclerosis [28, 44, 45]. In murine atheroscle‐ rosis, the hypoxia‐induced accumulation of cholesterol was substantially reversed in vitro by reducing the expression of the HIF1α [23]. While in another model, PHD2 inhibition stabilized HIF1α and reduced serum cholesterol levels in low‐density lipoprotein receptor‐deficient mice that were fed a high‐fat diet (HFD) [28]. So the role of HIF1 should be further studied in atherosclerosis lipid metabolism.

#### **2.2. Heart failure diseases**

Ischemic heart disease, systemic hypertension, and pathological cardiac hypertrophy eventu‐ ally result in heart failure. Myocardial hypoxia has been associated with these clinical condi‐ tions [25, 46]. Several studies showed a correlation between TAG accumulation and heart failure [26, 47–49]. Hypoxia promotes TAG accumulation in cardiomyocytes [48, 50]. Overex‐ pression of the constitutive active form of HIF1α in cardiomyocytes promotes intracellular lipid accumulation under normoxia [24]. The specific deletion of VHL in mice cardiac myocytes results in lipid accumulation [25, 26]. In a pathological cardiac hypertrophy mouse model, cardiac TAG accumulation in ventricles was abolished in HIF1α knockout mice [26].

#### **2.3. Fatty liver disease (FLD)**

Lipid accumulation is a common feature of fatty liver disease, whether it is alcoholic (AFLD) or nonalcoholic (NAFLD) [19]. FLD initially begins with simple hepatic steatosis, but can irreversibly progress to steatohepatitis, fibrosis, cirrhosis, or hepatocellular carcinoma [19]. Hypoxia in liver has been documented in vivo in rats on a continuous ethanol diet at a constant rate for prolonged periods [51–54]. Recent studies have demonstrated that hypoxia is also observed in NAFLD [55]. Indeed, HIF1 expression is increased in fatty liver diseases [19]. Nath and his colleagues found that ethanol feeding resulted in liver steatosis in wild‐type mice compared with isocaloric diet‐fed controls [27]. Constitutive activation of HIF1α in hepatocytes accelerates lipid accumulation with chronic ethanol feeding compared with wild‐type mice [27]. In contrast, hepatocyte‐specific deletion of HIF1α protected mice from alcohol‐induced liver lipid accumulation [27]. However, another group reported that hepatocyte‐specific HIF1α‐null mice developed severe hyper‐triglyceridemia with enhanced lipid accumulation in the liver of mice after 4 weeks of exposure to a 6% ethanol‐containing liquid diet [29]. Different genetic techniques used to create specific gene expression or knockout mice in each of these studies may offer some explanation of the different results each described. The other possible explanation is that the presence of inflammation may rewire the HIF‐1 pathway, which leads to a different gene expression profile compared to that observed in simple steatosis [19].

#### **2.4. Obesity**

Hypoxia has been directly demonstrated in adipose tissue of several obese mouse models, such as ob/ob mice [56, 57], KKAy obese mice [58], and high‐fat diet‐induced obese mice [56–58]. In HFD‐induced obese mice, HIF1 activation in visceral white adipocytes is critical to maintain dietary obesity [3] and adipocyte‐specific HIF1β or HIF1α knockout mice exhibit reduced fat formation compared with wild‐type controls [59]. Conversely, another group, using transgenic mice with adipose tissue selective expression of a dominant negative version of HIF1, found that mice with inhibition of HIF1's activity developed a more severe obesity in HFD‐induced obese mice [60]. Inactivation of PHD2 resulted in the activation of HIF1. Transgenic mice with PHD2‐specific deletion in adipocyte were resistant to HFD‐induced obesity and decreased lipid accumulation [30]. In another PHD2‐deficient mice model, they also had improved glucose tolerance and insulin sensitivity. Whether fed normal chow or HFD, PHD2 inhibition had less adipose tissue, smaller adipocytes, and less adipose tissue inflammation than their littermates. In addition, serum cholesterol level and de novo lipid synthesis were decreased, and the mice were protected against hepatic steatosis in PHD2‐deficient mice [31]. It seems that HIF1 in adipocyte of obesity had different effect on lipid metabolism compared with other models. Thus, the effect of HIF1 in lipid metabolism of obesity has yet to be defined.

#### **2.5. Cancer**

**2.2. Heart failure diseases**

4 Hypoxia and Human Diseases

**2.3. Fatty liver disease (FLD)**

**2.4. Obesity**

Ischemic heart disease, systemic hypertension, and pathological cardiac hypertrophy eventu‐ ally result in heart failure. Myocardial hypoxia has been associated with these clinical condi‐ tions [25, 46]. Several studies showed a correlation between TAG accumulation and heart failure [26, 47–49]. Hypoxia promotes TAG accumulation in cardiomyocytes [48, 50]. Overex‐ pression of the constitutive active form of HIF1α in cardiomyocytes promotes intracellular lipid accumulation under normoxia [24]. The specific deletion of VHL in mice cardiac myocytes results in lipid accumulation [25, 26]. In a pathological cardiac hypertrophy mouse model,

Lipid accumulation is a common feature of fatty liver disease, whether it is alcoholic (AFLD) or nonalcoholic (NAFLD) [19]. FLD initially begins with simple hepatic steatosis, but can irreversibly progress to steatohepatitis, fibrosis, cirrhosis, or hepatocellular carcinoma [19]. Hypoxia in liver has been documented in vivo in rats on a continuous ethanol diet at a constant rate for prolonged periods [51–54]. Recent studies have demonstrated that hypoxia is also observed in NAFLD [55]. Indeed, HIF1 expression is increased in fatty liver diseases [19]. Nath and his colleagues found that ethanol feeding resulted in liver steatosis in wild‐type mice compared with isocaloric diet‐fed controls [27]. Constitutive activation of HIF1α in hepatocytes accelerates lipid accumulation with chronic ethanol feeding compared with wild‐type mice [27]. In contrast, hepatocyte‐specific deletion of HIF1α protected mice from alcohol‐induced liver lipid accumulation [27]. However, another group reported that hepatocyte‐specific HIF1α‐null mice developed severe hyper‐triglyceridemia with enhanced lipid accumulation in the liver of mice after 4 weeks of exposure to a 6% ethanol‐containing liquid diet [29]. Different genetic techniques used to create specific gene expression or knockout mice in each of these studies may offer some explanation of the different results each described. The other possible explanation is that the presence of inflammation may rewire the HIF‐1 pathway, which leads to a different gene expression profile compared to that observed in simple steatosis [19].

Hypoxia has been directly demonstrated in adipose tissue of several obese mouse models, such as ob/ob mice [56, 57], KKAy obese mice [58], and high‐fat diet‐induced obese mice [56–58]. In HFD‐induced obese mice, HIF1 activation in visceral white adipocytes is critical to maintain dietary obesity [3] and adipocyte‐specific HIF1β or HIF1α knockout mice exhibit reduced fat formation compared with wild‐type controls [59]. Conversely, another group, using transgenic mice with adipose tissue selective expression of a dominant negative version of HIF1, found that mice with inhibition of HIF1's activity developed a more severe obesity in HFD‐induced obese mice [60]. Inactivation of PHD2 resulted in the activation of HIF1. Transgenic mice with PHD2‐specific deletion in adipocyte were resistant to HFD‐induced obesity and decreased lipid accumulation [30]. In another PHD2‐deficient mice model, they also had improved glucose tolerance and insulin sensitivity. Whether fed normal chow or HFD, PHD2 inhibition had less adipose tissue, smaller adipocytes, and less adipose tissue inflammation than their

cardiac TAG accumulation in ventricles was abolished in HIF1α knockout mice [26].

Hypoxia in the tumor microenvironment leads to the metabolic changes in cancer cells. Over 50% cellular energy is produced by glycolysis and HIF1 plays a central role in the changes [16, 61]. Recently disorders of lipid metabolism had been demonstrated in solid tumors [62, 63], such as pancreatic cancer [64], liver cancer [1], breast cancer [65], colon cancer [66], and ovarian cancer [67]. Lipid accumulation is observed in human tumor tissue [66, 68]. Accumulation of cholesterol also has been reported in prostate cancer [69]. Indeed, recent researches had demonstrated that HIF1's activity is really involved abnormal lipid metabolism of cancer cells. Hypoxia‐induced lipid accumulation depends on HIF1's activity in cancer cells [18, 20, 21]. Under hypoxic condition, the flux from glutamine into fatty acid is mediated by reductive carboxylation, and HIF1α plays an important role in this metabolic shift in tumor cells [70]. HIF1α also inhibits fatty acid β‐oxidation to promote lipid accumulation in human hepato‐ cellular carcinoma [1]. Valli and his colleagues revealed that hypoxia induced many changes in lipid metabolites. Enzymatic steps in fatty acid synthesis and the Kennedy pathway were modified in an HIF1α‐dependent fashion in HCT116 cell line [2]. However, the role of HIF1 in cancer lipid metabolism has not been well addressed, so more researches should be further studied.

## **3. The role of HIF1 in lipid metabolism**

Lipid metabolism is more complicated than glucose metabolism. Besides as major components of membrane, lipids are also a source of energy storage and signal molecules. HIF1‐induced genes involving lipid metabolism are listed in **Table 1**. We would discuss the role of HIF1 in lipid metabolism from the following linked aspects: lipid uptake and trafficking, fatty acid metabolism, sterol metabolism, TAG synthesis, phospholipids metabolism, lipid droplets biogenesis, and lipid signaling (**Figure 1**).

#### **3.1. Lipid uptake and trafficking**

#### *3.1.1. Free fatty acid (FFA) uptake*

At the plasma membrane, uptake of fatty acid is mainly regulated by the fatty acid transport protein family, such as CD36 [89–91], and plasma membrane‐associated fatty‐acid‐binding proteins (FABPs). Fatty acid transporter CD36 transports long chain fatty acid (LCFA) across plasma membrane. In cardiac myocytes, acute hypoxia (15 min) induced the redistribution of CD36 from an intracellular pool to the plasma membrane [92]. Similarly, in intact Langendorff‐ perfused heart, a similar effect was demonstrated [92]. Thus, indicating the increased intra‐ cellular lipid accumulation in hypoxic hearts is attributable to accumulation of fatty acid in the heart [92]. CD36 also can be regulated at the transcriptional level. In neonatal mouse cardiac myocytes, phenyl‐epinephrine (PE) induced free fatty acid uptake in an HIF1α ‐dependent fashion while inhibition of CD36 led to decreased TAG accumulation upon PE stimulation [26]. In this model, CD36 was induced through HIF1‐PPARγ axis [26]. In human retinal pigment epithelial cells, CD36 is mediated by HIF1 binding on its promoter region [71]. Hypoxia also markedly induced CD36 mRNA in corneal and retinal tissue in in vivo [71].


PPARγ, peroxisome proliferator‐activated receptor gamma; VLDLR, very‐low‐density lipoprotein receptor; LRP1, low‐ density lipoprotein receptor‐related protein 1; CAV1, caveolin 1; PPARα, peroxisome proliferator‐activated receptor alpha; TWIST1, twist family bHLH transcription factor 1; SIRT2, sirtuin 2; DEC1, deleted in esophageal cancer 1; ABCA1, ATP‐binding cassette subfamily A member 1; LPIN1, lipin 1; HIG2, hypoxia inducible gene 2; CHKA, choline kinase alpha; COX2, cyclooxygenase 2; PTGES, prostaglandin E synthase 1.

"\*" genes suppressed by HIF1.

**Table 1.** HIF1 targets genes that regulate lipid metabolism.

FABPs are part of a larger family of cytoplasmic proteins comprising nine members (FABP1– FABP9) [93], and are involved in reversibly binding intracellular hydrophobic ligands and trafficking them throughout cellular compartments [89]. Some evidence suggested that FABPs could interact directly with CD36 [94]. In *in vitro*, FABP3 and FABP7 were induced by hypoxia in a HIF1‐dependent manner, and both are involved in fatty acid uptake [21]. Knockdown of endogenous expression of FABP3 or FABP7 significantly impaired lipids droplets formation under hypoxia [21]. More specifically, the role of FABP3 is evident from the phenotype of FABP3 knockout mice, which show a rate of palmitate uptake reduced by 50% in cardiac myocytes [95, 96]. FABP7 binds long‐chain polyunsaturated FA (PUFA), allowing uptake and intracellular trafficking [97], and is involved in proliferation and invasion of melanoma cells [98] and glioblastoma cells [21]. High expression of FABP7 in glioblastomas is associated with poor prognosis and more invasive tumors [99].

#### *3.1.2. LDL and VLDL uptake*

cellular lipid accumulation in hypoxic hearts is attributable to accumulation of fatty acid in the heart [92]. CD36 also can be regulated at the transcriptional level. In neonatal mouse cardiac myocytes, phenyl‐epinephrine (PE) induced free fatty acid uptake in an HIF1α ‐dependent fashion while inhibition of CD36 led to decreased TAG accumulation upon PE stimulation [26]. In this model, CD36 was induced through HIF1‐PPARγ axis [26]. In human retinal pigment epithelial cells, CD36 is mediated by HIF1 binding on its promoter region [71]. Hypoxia also

markedly induced CD36 mRNA in corneal and retinal tissue in in vivo [71].

**Products of HIF1's target genes Functions in lipid metabolism References** CD36, PPARγ, FABP3, FABP7 Fatty acid uptake [21, 26, 71] VLDLR, LRP1 LDL and VLDL uptake [18, 48, 72, 73] CAV1, RAB20 Endocytosis and lipid trafficking [74, 75] PPARα\*, TWIST1, Sirt2\* Fatty acid β‐oxidation [3, 76, 77] DEC1 Fatty acid synthesis [30, 78] ABCA1\* Cholesterol efflux [79] PPARγ, Lipin1 TAG synthesis [20, 26] CHKA Phospholipids synthesis [80, 81] ADRP, HIG2, CAV1 Lipid droplet biogenesis [42, 74, 82–85] COX2, PTGES1 Lipid signaling [86–88]

PPARγ, peroxisome proliferator‐activated receptor gamma; VLDLR, very‐low‐density lipoprotein receptor; LRP1, low‐ density lipoprotein receptor‐related protein 1; CAV1, caveolin 1; PPARα, peroxisome proliferator‐activated receptor alpha; TWIST1, twist family bHLH transcription factor 1; SIRT2, sirtuin 2; DEC1, deleted in esophageal cancer 1; ABCA1, ATP‐binding cassette subfamily A member 1; LPIN1, lipin 1; HIG2, hypoxia inducible gene 2; CHKA, choline kinase

FABPs are part of a larger family of cytoplasmic proteins comprising nine members (FABP1– FABP9) [93], and are involved in reversibly binding intracellular hydrophobic ligands and trafficking them throughout cellular compartments [89]. Some evidence suggested that FABPs could interact directly with CD36 [94]. In *in vitro*, FABP3 and FABP7 were induced by hypoxia in a HIF1‐dependent manner, and both are involved in fatty acid uptake [21]. Knockdown of endogenous expression of FABP3 or FABP7 significantly impaired lipids droplets formation under hypoxia [21]. More specifically, the role of FABP3 is evident from the phenotype of FABP3 knockout mice, which show a rate of palmitate uptake reduced by 50% in cardiac myocytes [95, 96]. FABP7 binds long‐chain polyunsaturated FA (PUFA), allowing uptake and intracellular trafficking [97], and is involved in proliferation and invasion of melanoma cells [98] and glioblastoma cells [21]. High expression of FABP7 in glioblastomas is associated with

alpha; COX2, cyclooxygenase 2; PTGES, prostaglandin E synthase 1.

**Table 1.** HIF1 targets genes that regulate lipid metabolism.

poor prognosis and more invasive tumors [99].

"\*" genes suppressed by HIF1.

6 Hypoxia and Human Diseases

LDL and VLDL are major source of extracellular lipid, and HIF1 has been implicated in the transport of LDL and VLDL into cells. LDL receptor (LDLR) and VLDL receptor (VLDLR) are major receptors that are responsible for LDL and VLDL uptake. It had been reported that hypoxia significantly increased LDL uptake and enhances lipid accumulation in arterial smooth muscle cells (SMCs), exclusive LDLR activity [100]. In addition, hypoxia increased VLDL uptake in cardiac myocytes, which might be partially dependent on up‐regulating VLDLR expression [101]. Some studies had also reported that VLDLR could be induced un‐ der hypoxia [102]. In human cancer cell lines, we had demonstrated that HIF1‐mediated VLDLR induction influenced intracellular lipid accumulation through regulating LDL and VLDL uptake under hypoxia [18]. In hepatocellular carcinoma, expression of VLDR was as‐ sociated positively with HIF1 [18]. In mice, hypoxia‐induced VLDLR expression in HL‐1 cells was dependent on HIF1α through its interaction with an HRE in the *VLDLR* promoter. VLDLR promoted the endocytosis of lipoproteins, and causes lipid accumulation in cardio‐ myocytes [48].

Low‐density lipoprotein receptor related protein 1 (LRP1) belongs to LDL receptor superfam‐ ily, and is a key receptor for selective cholesterol uptake in human vascular smooth muscle cells (VSMCs). Hypoxia increased LRP1 expression through HIF1α, and overexpression of LRP1 mediated hypoxia‐induced aggregated LDL (agLDL) uptake in human VSMCs [72] as well as VLDL‐cholesteryl ester (VLDL‐CE) uptake in neonatal rat ventricular myocytes (NRVMs) [73]. In contrast to the strong impact of LRP1 inhibition on VLDL‐CE uptake in hypoxic cardiomyocytes, LRP1 deficiency did not exert any significant effect on VLDL‐TG uptake or VLDL‐TG accumulation [73]. This indicated that VLDLR might be a key receptor for VLDL‐TG uptake. Therefore, more experiments should be done to value the precise contribution of VLDLR and LRP1 in myocardial VLDL‐CE and VLDL‐TG uptake in patho‐ physiological situation in the heart.

LDL and VLDL uptake are through vesicular transport pathways [103]. The LDL receptor superfamily has NPXY motif in cytoplasmic domain that interacts with the endocytotic machinery to mediate rapid clathrin‐dependent endocytosis of the receptor‐ligand complex [104, 105]. Caveolaes are formed in the process of receptor‐mediated endocytosis. Numerous proteins are involved in caveolae formation, including caveolins, Rabs, VAT‐1, SNAP, and VAMP [106]. Caveolin‐1 (CAV1) is an essential structural constituent of caveolae that is involved in constitutive endocytic vesicular trafficking. Loss of VHL function, an E3 ligase involving HIF1α degradation, was associated with increased caveolae formation [74]. CAV1, as a direct target of HIF1, accentuated the formation of caveolae [74]. Knockdown expression of CAV1 inhibited uptake of oxidized LDL (oxLDL) without changing its binding to the plasma membrane [107]. These results indicated that CAV1 was part of the pathway that allowed cells to take up oxLDL [107]. Rab20, a member of the Rab family of small GTP‐binding proteins, regulating intracellular trafficking and vesicle formation, had also been characterized as an HIF‐1 target [75]. Although there was no direct evidence of the involvement of CAV1 and Rab20 in hypoxia‐induced LDL and VLDL uptake, we hypothesized that they might play role in hypoxia‐induced LDL and VLDL uptake and/or intracellular lipid trafficking.

Taken together, HIF1 promoting lipid accumulation may increase lipid uptake and intracel‐ lular lipid trafficking by inducing related genes directly. It should be further studied if there are more genes targeted by HIF1 in the process.

#### **3.2. Fatty acid metabolism**

## *3.2.1. Fatty acid β‐oxidation*

Hypoxia increased intracellular lipid accumulation through suppression of fatty acid β‐ oxidation (FAO) in several models, and the molecular mechanism involvement of HIF1 in the process had been demonstrated (**Figure 2**). Under hypoxic condition, human macrophages showed in an increased TAG accumulation that was associated with a decreasing rate of FAO. The decreasing rate of FAO was shown to be partly dependent on the reduced expression of enzymes involved in FAO [42]. Peroxisome proliferator‐activated receptors (PPARs), including α, γ, and β/δ, belong to the nuclear receptor family of ligand‐activated transcription factors that were originally described as gene regulators of various metabolic pathways. PPARα and PPARβ/δ control expression of genes implicated in FAO. PPARγ, in contrast, is a key regulator of glucose homeostasis and adipogenesis [108].

Muscle carnitine palmitoyltransferase 1 (M‐CPT1), a known PPARα target gene, catalyzes the rate‐limiting step in the mitochondrial import of fatty acids for the FAO cycle [109]. In cardiomyocytes, hypoxia and adenovirus‐mediated expression of a constitutively active form of HIF1α reduced the mRNA and protein levels of PPARα and M‐CPT1 [24, 50, 110] as well as the DNA binding activity of PPARα [24, 50]. CoCl2 treatment also decreased PPARα and M‐ CPT1 mRNA levels [110]. In intestinal epithelial cells, hypoxia rapidly down‐regulated PPARα mRNA and protein in an HIF1‐dependent manner in vitro and in vivo [76]. HIF1 could down‐regulate PPARα directly through binding a functional HRE in the promoter region [76]. These results suggested that the mechanism of HIF‐1 suppression of FAO involved the partial reduction of the expression of PPARα and M‐CPT1.

**Figure 2.** The molecular mechanism involving HIF1 repression of fatty acid β‐oxidation. HIF1 targets PPARα, PPARδ, and Sirt2 directly and thereby suppresses the genetic expression of fatty acid β‐oxidation.

HIF1 also suppressed FAO by inhibition of PPARδ's activity. In a pathological cardiac hyper‐ trophy mouse model, myocardial hypoxia provoked Dnm3os activation and concomitantly mir‐199a and mir‐214 expression through the HIF1‐TWIST1 axis [49]. TWIST1 is a direct target gene of HIF1 [77]. DNM3os is a noncoding RNA transcript that harbors the mi‐RNA cluster mir‐199a∼214, for which PPARδ is a target. Increased expression of mir‐199a and mir‐214 decreased cardiac PPARδ expression and mitochondrial fatty acid oxidative capacity. Reduced expression of enzymes involved in FAO, for example long‐chain acyl‐CoA dehydrogenase (LCAD) and medium‐chain acyl‐CoA dehydrogenase (MCAD), was also observed. Converse‐ ly, antagomir‐based silencing of miR‐199a∼214 in mice subjected to pressure overload de‐ repressed cardiac PPARδ, LCAD and MCAD levels, and restored mitochondrial FAO [49].

PPARγ coactivator 1α (PGC‐1α) has been prominently associated with the expression of the genes involving FAO and energy expenditure [111]. In obese mouse model, HIF1α suppressed FAO in visceral white adipocytes, in part, through transcriptional repression of sirtuin 2 (Sirt2), an NAD+ ‐dependent deacetylase [3]. Reduced Sirt2 function directly translated into dimin‐ ished deacetylation of PGC1α and the expression of FAO genes. HIF1α negated adipocyte‐ intrinsic pathway of fatty acid catabolism by negatively regulating the Sirt2‐PGC1α regulatory axis [3].

PPARγ coactivator 1β (PGC‐1β) is a transcription factor that also plays critical roles in regulating mitochondrial function and lipid metabolism [112, 113]. PGC‐1β could regulate FAO through activating medium‐chain acyl‐CoA dehydrogenase (MCAD) and long‐chain acyl‐CoA dehydrogenase (LCAD), which catalyzes the first step of FAO in mitochondria [1, 112]. It had been documented previously that hypoxia inhibited PGC‐1β activity through HIF1‐ dependent c‐Myc suppression in VHL‐null RCC4 renal carcinoma cells [114]. Under hypoxic condition in Hep3B and HepG2 cells, and also in PC3 prostate cancer cells, Huang and his colleagues revealed a role of the HIF1/C‐MYC/PGC‐1β regulatory axis in hypoxia‐mediated regulation of MCAD and LCAD by which HIF1 suppressed FAO [1]. This study confirmed that hypoxia inhibited FAO in an HIF1‐dependent mechanism in cancer cells [1].

In summary, it had been confirmed by different models that hypoxia inhibits FAO depending on HIF1's activity (**Figure 2**). However, HIF1 did not target FAO‐related genes directly, and it was always cross‐talk with other pathway to suppress FAO indirectly. It should be further studied if HIF1 could involve cross‐talk with more pathways to suppress FAO.

#### *3.2.2. Fatty acid synthesis*

Taken together, HIF1 promoting lipid accumulation may increase lipid uptake and intracel‐ lular lipid trafficking by inducing related genes directly. It should be further studied if there

Hypoxia increased intracellular lipid accumulation through suppression of fatty acid β‐ oxidation (FAO) in several models, and the molecular mechanism involvement of HIF1 in the process had been demonstrated (**Figure 2**). Under hypoxic condition, human macrophages showed in an increased TAG accumulation that was associated with a decreasing rate of FAO. The decreasing rate of FAO was shown to be partly dependent on the reduced expression of enzymes involved in FAO [42]. Peroxisome proliferator‐activated receptors (PPARs), including α, γ, and β/δ, belong to the nuclear receptor family of ligand‐activated transcription factors that were originally described as gene regulators of various metabolic pathways. PPARα and PPARβ/δ control expression of genes implicated in FAO. PPARγ, in contrast, is a key regulator

Muscle carnitine palmitoyltransferase 1 (M‐CPT1), a known PPARα target gene, catalyzes the rate‐limiting step in the mitochondrial import of fatty acids for the FAO cycle [109]. In cardiomyocytes, hypoxia and adenovirus‐mediated expression of a constitutively active form of HIF1α reduced the mRNA and protein levels of PPARα and M‐CPT1 [24, 50, 110] as well as the DNA binding activity of PPARα [24, 50]. CoCl2 treatment also decreased PPARα and M‐ CPT1 mRNA levels [110]. In intestinal epithelial cells, hypoxia rapidly down‐regulated PPARα mRNA and protein in an HIF1‐dependent manner in vitro and in vivo [76]. HIF1 could down‐regulate PPARα directly through binding a functional HRE in the promoter region [76]. These results suggested that the mechanism of HIF‐1 suppression of FAO involved the partial

**Figure 2.** The molecular mechanism involving HIF1 repression of fatty acid β‐oxidation. HIF1 targets PPARα, PPARδ,

and Sirt2 directly and thereby suppresses the genetic expression of fatty acid β‐oxidation.

are more genes targeted by HIF1 in the process.

of glucose homeostasis and adipogenesis [108].

reduction of the expression of PPARα and M‐CPT1.

**3.2. Fatty acid metabolism**

8 Hypoxia and Human Diseases

*3.2.1. Fatty acid β‐oxidation*

De novo fatty acid synthesis begins with acetyl coenzyme A (Ac‐CoA). Ac‐CoA is primarily generated from glucose through tri‐carboxylic acid (TCA) cycle in the mitochondrion, the citrate shuttle and ATP citrate lyase in the cytosol. Under hypoxic condition, cells converted glucose to lactate and the TCA cycle is largely disconnected from glycolysis [70, 115–117], thereby directing glucose carbon away from fatty acid synthesis. Recently, several groups had found that hypoxic tumor cells maintain proliferation by running the TCA cycle in reverse [70, 115–117]. In these cells, the source of carbon for Ac‐CoA and fatty acid switched from glucose to glutamine. This hypoxic flux from glutamine to fatty acid was mediated by the reductive carboxylation of glutamine‐derived α‐ketoglutarate.

The reductive carboxylation of glutamine was part of the metabolic reprogramming associated with HIF1. Glutamine‐derived α‐ketoglutarate is reductively carboxylated by the cytosolic isocitrate dehydrogenase 1 (IDH1) [70, 115] and the mitochondrial isocitrate dehydrogenase 2 (IDH2) to form isocitrate [70, 115, 116], which could then be isomerized to citrate. The combined action of IDH1 and IDH2 was necessary and sufficient to affect the reverse TCA flux [115]. Citrate was converted into Ac‐CoA by ATP citrate lyase in the cytosol. Renal cell lines deficient in the VHL preferentially used reductive glutamine metabolism for lipid biosynthesis even at normal oxygen levels [70]. Constitutive activation of HIF1 recapitulated the preferential reductive metabolism of glutamine‐derived α‐ketoglutarate even in normoxic condition [116]. This regulation by HIF1 of the reverse TCA cycle occurred partly through HIF1‐inducing PDK1. Knocking down PDK1 suppressed reductive carboxylation [70, 118]. However, more details should be studied about the role of HIF1 in TCA cycle reverse.

The first step of fatty acid synthesis is catalyzed by AcCoA carboxylase (ACC) which converts Ac‐CoA to malonyl‐CoA. Then fatty‐acid synthase (FASN) catalyzes acetyl‐CoA and malonyl‐ CoA to palmitate. Further elongation and de‐saturation of newly synthesized fatty acid takes place at the cytoplasmic face of the endoplasmic reticulum membrane. It had been reported that hypoxia regulated FASN expression [78, 119, 120]. However, different conclusions on hypoxia regulation of FASN had been reported. One group using human breast cancer cell lines found that FASN was significantly up‐regulated by hypoxia via activation of the Akt and HIF1 followed by the induction of the SREBP1 gene [119]. Another group, using several cell lines other than breast cancer cell lines, found that hypoxia suppressed FASN expression through HIF1‐DEC1 and/or DEC2‐SREBP1 axis. They found that HIF1 repressed the SREBP1 gene by inducing DEC1 and DEC2, and further repressing FASN expression [78]. These results might indicate that HIF1 regulated FASN in a cell‐type specific manner. In addition, it had been reported that hypoxia could induce the expression of SCD1 which introduces a double bond in the Δ9 position of palmitic acid and stearic acid to produce mono‐unsaturated fatty acid [42, 121]. It is unknown if HIF1 is involved in hypoxic‐induced SCD1.

Taken together, the role of HIF1 in de novo fatty acid synthesis may depend on different models and conditions, and more researches should be done in the direction.

#### **3.3. Cholesterol metabolism**

Cholesterol is an essential structural component of membrane. It modulates membrane permeability and fluidity and also forms microdomains named lipid rafts that integrate the activation of some signal transduction pathways [14]. Intermediates generated by the choles‐ terol biosynthesis pathway were required for the posttranslational modification of small GTPases, such as the farnesylation of Ras and the geranyl‐geranylation of Rho [15]. Finally, cholesterol also serves as a precursor for the biosynthesis of steroid hormones, bile acids, and vitamin D.

Cellular cholesterol level can be modulated by three processes: cholesterol uptake, synthesis, and efflux [122]. In the preceding paragraph, we had discussed the role of HIF1 in LDL and VLDL uptake that are main source of extracellular cholesterol. Here, we discuss the cholesterol synthesis and efflux. Cholesterol biosynthesis begins with the condensation of AcCoA with acetoacetyl‐CoA to form 3‐hydroxy‐3‐methylglutaryl (HMG)‐CoA. Then HMG‐CoA reduc‐ tase (HMGCR) reduces of HMG‐CoA to mevalonate. Early research found that Hypoxia also suppressed cholesterol synthesis in cultured rabbit skin fibroblasts [123]. However, recently research indicated that hypoxia increased sterol synthesis depending on HIF1's activity [23, 124]. In hypoxic macrophages, the increase of intracellular cholesterol content was correlated with elevated HMGCR's activity and mRNA levels [23]. In HepG2 cells, HIF1α accumulation was able to increase the level and activity of HMGCR by stimulating its transcription [124]. But it was unclear if HIF1 regulated HMGCR directly.

Hypoxia suppressed the efflux of cholesterol, and this efflux was substantially reversed in vitro by reducing the expression of HIF1 [23, 123]. ATP‐binding cassette transporter A1 (ABCA1) plays a major role in cholesterol efflux. Hypoxia severely reduced ABCA1‐mediated choles‐ terol efflux, which could be explained by subcellular redistribution of ABCA1 protein under acute hypoxia and decreased protein level under prolonged hypoxia [23]. One group reported that HIF1 could repress the transcription of ABCA1 directly [79]. Hypoxia, partly mediated by HIF1α, increased intracellular cholesterol content due to the induction of cholesterol synthesis and the suppression of cholesterol efflux [23]. In addition, accumulation of cholesterol in hypoxic cells was in esterified form [23, 100]. At 2% O2 tension, twice the total cholesteryl ester was observed compared with that at 21% O2. At the same time, no significant difference was found in the concentration of cellular‐free cholesterol [100]. Accumulation of cholesteryl ester in hypoxic cells might depend on the increased activity of AcCoA:cholesterol acyltransferases (ACATs) [123], which are important enzymes for the esterification of cholesterol. Therefore, more studies should be done to define the role of HIF1 involving the cholesterol metabolism in detail.

### **3.4. TAG synthesis and phospholipids metabolism**

### *3.4.1. TAG synthesis*

The reductive carboxylation of glutamine was part of the metabolic reprogramming associated with HIF1. Glutamine‐derived α‐ketoglutarate is reductively carboxylated by the cytosolic isocitrate dehydrogenase 1 (IDH1) [70, 115] and the mitochondrial isocitrate dehydrogenase 2 (IDH2) to form isocitrate [70, 115, 116], which could then be isomerized to citrate. The combined action of IDH1 and IDH2 was necessary and sufficient to affect the reverse TCA flux [115]. Citrate was converted into Ac‐CoA by ATP citrate lyase in the cytosol. Renal cell lines deficient in the VHL preferentially used reductive glutamine metabolism for lipid biosynthesis even at normal oxygen levels [70]. Constitutive activation of HIF1 recapitulated the preferential reductive metabolism of glutamine‐derived α‐ketoglutarate even in normoxic condition [116]. This regulation by HIF1 of the reverse TCA cycle occurred partly through HIF1‐inducing PDK1. Knocking down PDK1 suppressed reductive carboxylation [70, 118]. However, more

The first step of fatty acid synthesis is catalyzed by AcCoA carboxylase (ACC) which converts Ac‐CoA to malonyl‐CoA. Then fatty‐acid synthase (FASN) catalyzes acetyl‐CoA and malonyl‐ CoA to palmitate. Further elongation and de‐saturation of newly synthesized fatty acid takes place at the cytoplasmic face of the endoplasmic reticulum membrane. It had been reported that hypoxia regulated FASN expression [78, 119, 120]. However, different conclusions on hypoxia regulation of FASN had been reported. One group using human breast cancer cell lines found that FASN was significantly up‐regulated by hypoxia via activation of the Akt and HIF1 followed by the induction of the SREBP1 gene [119]. Another group, using several cell lines other than breast cancer cell lines, found that hypoxia suppressed FASN expression through HIF1‐DEC1 and/or DEC2‐SREBP1 axis. They found that HIF1 repressed the SREBP1 gene by inducing DEC1 and DEC2, and further repressing FASN expression [78]. These results might indicate that HIF1 regulated FASN in a cell‐type specific manner. In addition, it had been reported that hypoxia could induce the expression of SCD1 which introduces a double bond in the Δ9 position of palmitic acid and stearic acid to produce mono‐unsaturated fatty

Taken together, the role of HIF1 in de novo fatty acid synthesis may depend on different models

Cholesterol is an essential structural component of membrane. It modulates membrane permeability and fluidity and also forms microdomains named lipid rafts that integrate the activation of some signal transduction pathways [14]. Intermediates generated by the choles‐ terol biosynthesis pathway were required for the posttranslational modification of small GTPases, such as the farnesylation of Ras and the geranyl‐geranylation of Rho [15]. Finally, cholesterol also serves as a precursor for the biosynthesis of steroid hormones, bile acids, and

Cellular cholesterol level can be modulated by three processes: cholesterol uptake, synthesis, and efflux [122]. In the preceding paragraph, we had discussed the role of HIF1 in LDL and VLDL uptake that are main source of extracellular cholesterol. Here, we discuss the cholesterol synthesis and efflux. Cholesterol biosynthesis begins with the condensation of AcCoA with

details should be studied about the role of HIF1 in TCA cycle reverse.

acid [42, 121]. It is unknown if HIF1 is involved in hypoxic‐induced SCD1.

and conditions, and more researches should be done in the direction.

**3.3. Cholesterol metabolism**

10 Hypoxia and Human Diseases

vitamin D.

TAG is formed by the addition of three molecules of fatty acid to glycerol. There are two major pathways for TAG biosynthesis in mammalian cell: the glycerol phosphate pathway and the mono‐acylglycerol (MG) pathway. In the glycerol phosphate pathway, two molecules of fatty acyl‐CoA are esterified to glycerol‐3‐phosphate to yield 1,2‐diacylglycerol (DAG) phosphate (commonly identified as phosphatidic acid). The phosphate is then removed to yield 1,2‐ diacylglycerol, which is followed by addition of the third fatty acid to form TAG. TAG accumulation under hypoxia could be mediated by HIF1‐inducing Lipin1 [20], a phosphatidate phosphatase isoform that catalyzes the penultimate step in TAG biosynthesis, the removal of phosphate from diacylglycerol phosphate to yield DAG. It also had been reported that hypoxia produced a marked intracellular accumulation of diacylglycerol in different cell types [125]. DAG may also serve a feedback role regulating HIF1's activity [125]. In a mouse model of pathological hypertrophy, HIF1α promoted TAG accumulation in cardiomyocytes via the regulation of PPARγ expression. PPARγ was the principal mediator of TAG anabolism through its transcriptional regulation glycerol‐3‐phosphate generation (via GPD1), and downstream esterification processes (via GPAT) [26].

#### *3.4.2. Phospholipids metabolism*

Phospholipids are indispensable for cell growth. Phospholipids synthesis and TAG synthesis share similar steps. DAG is a precursor for phosphatidylcholine and phosphatidylethanola‐ mine. Phosphatidic acid utilizes cytidine triphosphate (CTP) as an energy source to produce a CDP‐DAG intermediate followed by conversion to phosphatidylcholine. It had been reported that the intracellular level of phosphatidic acid (PA) and DAG rose in response to hypoxia [125, 126]. However, PA accumulation in response to hypoxia was both HIF1 and VHL‐independ‐ ent [127]. Choline kinase α (ChKα) catalyzes the phosphorylation of choline, the first step of phosphatidylcholine synthesis. In cancer cells, one group had shown that hypoxia increased ChKα expression and this was driven by HIF1 [80]. Conversely, another group had shown that choline kinase activity and choline phosphorylation were decreased, that might be mediated via HIF1α binding to the promoter of ChKα gene [81]. Thus, further studies should be done to address the role of HIF1 in phospholipids metabolism.

#### **3.5. Lipid droplet (LD) biogenesis and lipid signaling**

Lipid droplet, also named lipid body, has been largely associated with neutral lipid storage and transport in cells [106]. The internal core of the LD is rich in neutral lipids, predominantly TAGs or cholesteryl esters, that are surrounded by an outer monolayer of phospholipids and associated proteins [128]. LD was considered to be highly regulated, dynamic and functionally active organelle [106]. Proteins on the surface of lipid droplets are crucial to the droplet structure and dynamics. Currently, the complete protein composition of LD has not been defined. The best characterized LD' proteins are the perilipin/ADRP/TIP47 (PAT) domain family. Apart from the PAT domain proteins, there are other lipid droplets associated proteins which involve the catabolism of lipids, vesicular transport, eicosanoid‐forming enzymes, protein kinases, etc. [106]. Hypoxia increased LD number and size [42, 129]. Several LD‐ associated proteins were induced by HIF1 and might also involve HIF1‐induced LD biogenesis and lipid signaling (**Figure 3**).

#### *3.5.1. Lipid droplet biogenesis*

Adipose differentiation‐related protein (ADRP), a PAT domain protein, is a structural com‐ ponent of LD and had been reported by several groups to be inducible by HIF1 [42, 82–84]. Lipid accumulation was associated with high expression level of ADRP in solid tumors [68, 130], especially in clear cell lesions [131]. During the process of carcinogenesis, the ADRP expression was increased during early tumorigenesis and was associated with the proliferation rate [68]. The expression of ADRP was also correlated with atherosclerosis [132]. In mouse macrophages in vitro, ADRP expression facilitated foam cell formation induced by modified lipoproteins [132]. In apolipoprotein E‐deficient mice, ADRP inactivation reduced the number of LD in foam cells in atherosclerotic lesions [132]. Under hypoxia, knockdown of ADRP in U87 and T98G or in MCF‐7 and MDA‐MB‐231 cells significantly decreased the formation of LD, and resulted in decreased fatty acid uptake [21]. It indicated that ADRP promoted LD formation mainly through increasing FA uptake under hypoxic condition. It had been reported that ADRP can also stimulate LCFA uptake [133]. While another research reported that ADRP did not involve LDL‐ and VLDL‐induced LD formation under hypoxia [84].

**Figure 3.** A hypothetical representation of molecular mechanism involving hypoxia‐induced lipid droplet biogenesis and function. HIF1‐induced structural proteins of the LD, such as ADRP, HIG2, combine with HIF1‐increased lipids to form the LD. Enzymes involving eicosanoid production are also induced by HIF1, and are recruited to the LD. These proteins can increase lipid signaling that can involve many aspects of biology, such as HIF1α's stability, angiogenesis, inflammation, cell proliferation and survival.

Hypoxia‐inducible protein 2 (HIG2), a newly identified protein associated with LD, was up‐ regulated by hypoxia and was a direct and specific target gene of HIF1 [85]. Overexpression of HIG2 under normoxic condition was sufficient to increase LD in HeLa cells. HIG2‐driven LD might contribute to an inflammatory response. Overexpression of HIG2 stimulated cytokine expression of vascular endothelial growth factor‐A (VEGFA), macrophage migration inhibitory factor (MIF), and interleukin‐6 (IL‐6). Increasing expression of HIG2 was also detected under several conditions of pathological lipid accumulation, such as atherosclerotic arteries and fatty liver disease [85]. We had mentioned that CAV1 was a target of HIF1. CAV1 could distribute to LD under several conditions [134–137] and the association with LD was reversible [134]. However, It is unknown if hypoxia can redistribute CAV1 to LD and CAV1 involves LD biogenesis under hypoxia.

#### *3.5.2. Lipid signaling*

*3.4.2. Phospholipids metabolism*

12 Hypoxia and Human Diseases

and lipid signaling (**Figure 3**).

*3.5.1. Lipid droplet biogenesis*

to address the role of HIF1 in phospholipids metabolism.

**3.5. Lipid droplet (LD) biogenesis and lipid signaling**

Phospholipids are indispensable for cell growth. Phospholipids synthesis and TAG synthesis share similar steps. DAG is a precursor for phosphatidylcholine and phosphatidylethanola‐ mine. Phosphatidic acid utilizes cytidine triphosphate (CTP) as an energy source to produce a CDP‐DAG intermediate followed by conversion to phosphatidylcholine. It had been reported that the intracellular level of phosphatidic acid (PA) and DAG rose in response to hypoxia [125, 126]. However, PA accumulation in response to hypoxia was both HIF1 and VHL‐independ‐ ent [127]. Choline kinase α (ChKα) catalyzes the phosphorylation of choline, the first step of phosphatidylcholine synthesis. In cancer cells, one group had shown that hypoxia increased ChKα expression and this was driven by HIF1 [80]. Conversely, another group had shown that choline kinase activity and choline phosphorylation were decreased, that might be mediated via HIF1α binding to the promoter of ChKα gene [81]. Thus, further studies should be done

Lipid droplet, also named lipid body, has been largely associated with neutral lipid storage and transport in cells [106]. The internal core of the LD is rich in neutral lipids, predominantly TAGs or cholesteryl esters, that are surrounded by an outer monolayer of phospholipids and associated proteins [128]. LD was considered to be highly regulated, dynamic and functionally active organelle [106]. Proteins on the surface of lipid droplets are crucial to the droplet structure and dynamics. Currently, the complete protein composition of LD has not been defined. The best characterized LD' proteins are the perilipin/ADRP/TIP47 (PAT) domain family. Apart from the PAT domain proteins, there are other lipid droplets associated proteins which involve the catabolism of lipids, vesicular transport, eicosanoid‐forming enzymes, protein kinases, etc. [106]. Hypoxia increased LD number and size [42, 129]. Several LD‐ associated proteins were induced by HIF1 and might also involve HIF1‐induced LD biogenesis

Adipose differentiation‐related protein (ADRP), a PAT domain protein, is a structural com‐ ponent of LD and had been reported by several groups to be inducible by HIF1 [42, 82–84]. Lipid accumulation was associated with high expression level of ADRP in solid tumors [68, 130], especially in clear cell lesions [131]. During the process of carcinogenesis, the ADRP expression was increased during early tumorigenesis and was associated with the proliferation rate [68]. The expression of ADRP was also correlated with atherosclerosis [132]. In mouse macrophages in vitro, ADRP expression facilitated foam cell formation induced by modified lipoproteins [132]. In apolipoprotein E‐deficient mice, ADRP inactivation reduced the number of LD in foam cells in atherosclerotic lesions [132]. Under hypoxia, knockdown of ADRP in U87 and T98G or in MCF‐7 and MDA‐MB‐231 cells significantly decreased the formation of LD, and resulted in decreased fatty acid uptake [21]. It indicated that ADRP promoted LD formation mainly through increasing FA uptake under hypoxic condition. It had been reported

Eicosanoids are signaling molecules made by oxidation of 20‐carbon fatty acids, mainly from arachidonic acid. Cyclooxygenases and Lipoxygenases are two families of enzymes catalyzing fatty acid oxygenation to produce the eicosanoids. There are multiple subfamilies of eicosa‐ noids, including prostaglandins, prostacyclins, thromboxanes, lipoxins, and leukotrienes. Prostaglandins, such as PGI2 and PGE2, are synthesized via cyclooxygenase (COX) by oxidation of arachidonic acid. PGE2 is synthesized in three steps catalyzed by phospholipase (PL) A2, COX, and terminal prostaglandin E synthase (PTGES), where each catalytic activity is repre‐ sented by multiple enzymes and/or isoenzymes. It had been reported that hypoxia could increase prostaglandins (PGI2 and PGE2) synthesis [138]. Hypoxia‐induced synthesis of PGE2 was accompanied by up‐regulation of COX2, which is a direct target gene of HIF1 [86]. Several studies had indicated that LD was reservoirs of COX2 and sites of PGE2 synthesis [66, 139, 140]. PTGES1 could also be regulated by HIF1 directly [87, 88]; however, it is unknown if PTGES1 localizes to hypoxia‐induced LD.

Lipoxygenases are a family of nonheme iron‐containing enzymes which dioxygenate polyun‐ saturated fatty acid to hydroperoxyl metabolite, and mainly include 5‐lipoxygenase (5‐LO), 12‐lipoxygenase (12‐LO), and 15‐lipoxygenase (15‐LO). 5‐LO and 15‐LO were shown by immuno‐cytochemistry, immuno‐fluorescence, ultrastructural postembedding immuno‐gold EM and/or western blotting from subcellular fractions to localize within lipid droplets stimulated in vitro [141–144]. Increasing level of 5‐LO was detected in lung tissue of rodent model of hypoxia‐induced pulmonary hypertension [145]. Hypoxia increased 12‐LO in rat lung and in in vitro cultured rat pulmonary artery smooth muscle cell (PASMC) and may contribute to the production of 12(S)‐hydroxyeicosatetraenoic acid (12(S)‐HETE) [146]. Increasing 12(S)‐HETE had also been demonstrated in hypoxic macrophage cells [147]. Under hypoxia, increased levels of 15‐LO had been demonstrated by different groups [147, 148] and its product, 15‐hydroperoxyeicosatetraenoic acid (15‐HETE), was up‐regulated [147]. Up‐ regulation of 15‐LO/15‐HETE in response to hypoxia might be partially mediated by HIF1α [149]. In addition, HIF1α was shown to be regulated by 15‐HETE in a positive feedback manner [149]. However, it is unknown if lipoxygenases are regulated by HIF1 directly.

## **4. Conclusions and perspectives**

HIF1 plays an important role in lipid metabolism and a number of studies support the findings that HIF1 promotes lipid accumulation. Nevertheless, many questions remain. HIF1, as a master transcriptional factor, may target many genes directly or indirectly involved in lipid metabolism. HIF1 plays a pivotal role in glucose metabolism. Inhibition of GULT3, an HIF1 target gene, could significantly reduce both glucose uptake and hypoxia‐induced de novo lipid synthesis in human monocyte‐derived macrophages [150]. PGAM1, induced by hypoxia [151], catalyzes the reversible reaction of 3‐phosphoglycerate (3‐PGA) to 2‐phosphoglycerate (2‐ PGA) in the glycolytic pathway. Inhibition of PGAM1 led to significantly decreased glycolysis and de novo lipid synthesis in cancer cells [152]. Thus, it is possible that glucose metabolism might couple with lipid metabolism under hypoxia. The source of carbon for fatty acid switched from glucose to glutamine under hypoxia [70]. The question thus arises. Does HIF1 induce lipid accumulation through targeting genes involving glucose metabolism, and how does glucose metabolism affect lipid metabolism under hypoxia?

HIF1 could interact with other pathways to regulate lipid metabolism besides PPARα, PPARγ, PPARδ, PGC1α, and SREBP1. There might be a pivotal role for mTOR in controlling lipid homeostasis in many settings, both physiological and pathological [153]. AMPK is a cellular energy sensor that normalizes lipid, glucose, and energy imbalances [154]. Inhibition of cMYC was accompanied by accumulation of intracellular LD in tumor cells as a direct consequence of mitochondrial dysfunction [155]. Recently, p53 had also been shown to regulate lipid metabolism [156]. The role of HIF1 in these pathways and the molecular mechanism will require further investigation.

Lipid accumulation in diseases, including obesity, atherosclerosis, ALD, heart failure disease and cancer, had been associated with HIF1's activity. There may be additional pathologies with lipid metabolism disorder associated with HIF1. HIF1 is an attractive target candidate for therapeutic intervention in diseases with disorder of lipid metabolism including cancer. Its involvement in the etiology of a number of diseases and its interaction with a number of regulatory genes make it an important area for further study.

## **Acknowledgements**

of arachidonic acid. PGE2 is synthesized in three steps catalyzed by phospholipase (PL) A2, COX, and terminal prostaglandin E synthase (PTGES), where each catalytic activity is repre‐ sented by multiple enzymes and/or isoenzymes. It had been reported that hypoxia could increase prostaglandins (PGI2 and PGE2) synthesis [138]. Hypoxia‐induced synthesis of PGE2 was accompanied by up‐regulation of COX2, which is a direct target gene of HIF1 [86]. Several studies had indicated that LD was reservoirs of COX2 and sites of PGE2 synthesis [66, 139, 140]. PTGES1 could also be regulated by HIF1 directly [87, 88]; however, it is unknown if

Lipoxygenases are a family of nonheme iron‐containing enzymes which dioxygenate polyun‐ saturated fatty acid to hydroperoxyl metabolite, and mainly include 5‐lipoxygenase (5‐LO), 12‐lipoxygenase (12‐LO), and 15‐lipoxygenase (15‐LO). 5‐LO and 15‐LO were shown by immuno‐cytochemistry, immuno‐fluorescence, ultrastructural postembedding immuno‐gold EM and/or western blotting from subcellular fractions to localize within lipid droplets stimulated in vitro [141–144]. Increasing level of 5‐LO was detected in lung tissue of rodent model of hypoxia‐induced pulmonary hypertension [145]. Hypoxia increased 12‐LO in rat lung and in in vitro cultured rat pulmonary artery smooth muscle cell (PASMC) and may contribute to the production of 12(S)‐hydroxyeicosatetraenoic acid (12(S)‐HETE) [146]. Increasing 12(S)‐HETE had also been demonstrated in hypoxic macrophage cells [147]. Under hypoxia, increased levels of 15‐LO had been demonstrated by different groups [147, 148] and its product, 15‐hydroperoxyeicosatetraenoic acid (15‐HETE), was up‐regulated [147]. Up‐ regulation of 15‐LO/15‐HETE in response to hypoxia might be partially mediated by HIF1α [149]. In addition, HIF1α was shown to be regulated by 15‐HETE in a positive feedback manner [149]. However, it is unknown if lipoxygenases are regulated by HIF1 directly.

HIF1 plays an important role in lipid metabolism and a number of studies support the findings that HIF1 promotes lipid accumulation. Nevertheless, many questions remain. HIF1, as a master transcriptional factor, may target many genes directly or indirectly involved in lipid metabolism. HIF1 plays a pivotal role in glucose metabolism. Inhibition of GULT3, an HIF1 target gene, could significantly reduce both glucose uptake and hypoxia‐induced de novo lipid synthesis in human monocyte‐derived macrophages [150]. PGAM1, induced by hypoxia [151], catalyzes the reversible reaction of 3‐phosphoglycerate (3‐PGA) to 2‐phosphoglycerate (2‐ PGA) in the glycolytic pathway. Inhibition of PGAM1 led to significantly decreased glycolysis and de novo lipid synthesis in cancer cells [152]. Thus, it is possible that glucose metabolism might couple with lipid metabolism under hypoxia. The source of carbon for fatty acid switched from glucose to glutamine under hypoxia [70]. The question thus arises. Does HIF1 induce lipid accumulation through targeting genes involving glucose metabolism, and how

HIF1 could interact with other pathways to regulate lipid metabolism besides PPARα, PPARγ, PPARδ, PGC1α, and SREBP1. There might be a pivotal role for mTOR in controlling

PTGES1 localizes to hypoxia‐induced LD.

14 Hypoxia and Human Diseases

**4. Conclusions and perspectives**

does glucose metabolism affect lipid metabolism under hypoxia?

This review is supported by a National Natural Science Foundation of China (Grant No. 31301076 to G. S; Grant No. 81401961 to X. L.), We thank Gerard Moskowitz, Ph. D. (Washington University in St. Louis, St. Louis, MO) for critical reading of the manuscript. We sincerely apologize to the colleagues whose works are not covered in this review due to limitations of time and space.

## **Author details**

Guomin Shen1\* and Xiaobo Li2

\*Address all correspondence to: shenba433@163.com

1 Department of Medical Genetics, Medical College, Henan University of Science and Technology, Luoyang, Henan Province, China

2 Department of Pathology & Translational Medicine Center, Harbin Medical University, Harbin, Heilongjiang Province, China

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#### **Hypoxic Upregulation of ARNT (HIF-1β): A Cell-Specific Attribute with Clinical Implications Hypoxic Upregulation of ARNT (HIF-1**b**): A Cell-Specific Attribute with Clinical Implications**

Markus Mandl and Reinhard Depping Markus Mandl and Reinhard Depping

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

http://dx.doi.org/10.5772/67171

#### **Abstract**

According to the current point of view described in the literature, the transcription fac‐ tor aryl hydrocarbon receptor nuclear translocator (ARNT), also designated as hypoxia‐ inducible factor (HIF)‐1β, is constitutively expressed and not influenced by oxygen tension. However, a study published two decades ago provided early evidence regarding a hypoxia‐dependent ARNT upregulation. This finding was subsequently challenged and neglected. Until now, only a limited number of publications focus on the regulation of ARNT in hypoxia. Therefore, appropriate studies and the putative mechanism mediating this cellular attribute are discussed. The advantages of an elevated ARNT expression level in tumour cells are delineated. This chapter provides an overview of hypoxia‐inducible ARNT as an emerging concept in HIF biology.

**Keywords:** aryl hydrocarbon receptor nuclear translocator, ARNT, HIF‐1β, crosstalk, cancer

## **1. Introduction**

The name aryl hydrocarbon receptor nuclear translocator (ARNT) designates a transcrip‐ tion factor of the Per‐ARNT‐Sim family which is ubiquitously expressed. This protein is also known as hypoxia‐inducible factor (HIF)‐1β. The use of these two equal synonyms for the same transcription factor throughout the literature already implies its role in various signal‐ ling pathways [1]. Unfortunately, the term "hypoxia‐inducible" might be misleading in this context. According to the current point of view described in the literature, ARNT expression is not affected by environmental conditions such as hypoxia. Therefore, ARNT is considered to be a constitutively expressed gene [1]. Although this notion might be true for the majority of cells/tissues investigated, numerous studies reported the capability of tumour cells to elevate

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© 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.

ARNT expression in response to hypoxia [1–7]. This cellular attribute was found in cells of different tumour types of both human and murine origin. These key findings clearly suggest that hypoxia‐dependent ARNT upregulation might provide a certain benefit for appropriate cells [1].

ARNT and its paralogue ARNT2 [1] share a 90% identical amino acid sequence [8]. In contrast to ARNT, ARNT2 is mainly expressed in the central nervous system [8, 9]. ARNT2 expression was shown to be positively correlated with breast cancer prognosis [8]. In addition, high‐ ARNT2 levels in hepatocellular carcinomas are associated with a prolonged overall survival of cancer patients [8]. However, many functions of this transcription factor are still unknown [1, 8]. Moreover, the regulation of ARNT and ARNT2 varied in human hepatocellular carci‐ noma Hep3B cells. ARNT was elevated in hypoxia, whereas ARNT2 was not affected in this model [3].

This chapter describes the current knowledge regarding hypoxic upregulation of ARNT, which appears to be beneficial for certain types of tumour cells. Therefore, the aim of this section is to emphasise this unique cellular attribute. A potential altered ARNT expression level due to hypoxic exposure of cells should be considered and not generally excluded. In this context, the use of ARNT as loading control or as a reference gene is basically not recom‐ mended [1].

## **2. Regulation of ARNT**

### **2.1. Upregulation of ARNT in response to hypoxia**

First evidence for a hypoxia‐dependent regulation of ARNT was provided by Wang et al. [5]. Herein, Hep3B cells were used to study the effects of HIF‐1α and ARNT under hypoxic condi‐ tions. ARNT was elevated on mRNA level in this cell line due to hypoxic exposure (1% v/v O2 ). In addition, treatment with hypoxia mimetics such as cobalt chloride and desferrioxamine had similar effects. Nuclear extracts prepared from Hep3B and HeLa cells were used to investigate the response of both HIF‐1 subunits to oxygen deprivation. Re‐oxygenation experiments were also included into the study [5]. The data revealed that both transcription factors HIF‐1α and ARNT were inducible in hypoxic cells on mRNA as well as protein levels [5]. Huang et al. [10] reported that ARNT protein levels remained constant regardless of cellular oxygen tension. In this study, Hep3B, HeLa and HEK293 cells were used. Unfortunately, not all experiments were conducted with all cell lines [10], thereby making a direct comparison with the study of Wang et al. [5] complicated. Nevertheless, the latter report [10] challenged the results of the previous one [5] due to signal variations of Northern blots and discontinuities of time‐course experiments [10].

However, Huang et al. [10] proposed a very graphic working model including a specific sensor for hypoxia located in the cell membrane [10]. The depicted mechanism is similar to our nowa‐ days HIF scheme. These days the prolylhydroxylase domain enzymes (PHDs), which require O2 as a substrate, are known to act as cellular oxygen sensors [11]. The comparison of both seminal studies conducted by Wang et al. [5] and Huang et al. [10] also requires a glance on citation frequencies of both reports. Noteworthily, the report of Wang et al. [5], which describes the upregulation of ARNT for the first time, was cited approximately four times more often as compared to Huang et al. [10]. Despite this clear distinction of citation frequencies, the opinion that ARNT is unaffected by cellular oxygen tension became a guideline in HIF biology [1].

ARNT expression in response to hypoxia [1–7]. This cellular attribute was found in cells of different tumour types of both human and murine origin. These key findings clearly suggest that hypoxia‐dependent ARNT upregulation might provide a certain benefit for appropriate

ARNT and its paralogue ARNT2 [1] share a 90% identical amino acid sequence [8]. In contrast to ARNT, ARNT2 is mainly expressed in the central nervous system [8, 9]. ARNT2 expression was shown to be positively correlated with breast cancer prognosis [8]. In addition, high‐ ARNT2 levels in hepatocellular carcinomas are associated with a prolonged overall survival of cancer patients [8]. However, many functions of this transcription factor are still unknown [1, 8]. Moreover, the regulation of ARNT and ARNT2 varied in human hepatocellular carci‐ noma Hep3B cells. ARNT was elevated in hypoxia, whereas ARNT2 was not affected in this

This chapter describes the current knowledge regarding hypoxic upregulation of ARNT, which appears to be beneficial for certain types of tumour cells. Therefore, the aim of this section is to emphasise this unique cellular attribute. A potential altered ARNT expression level due to hypoxic exposure of cells should be considered and not generally excluded. In this context, the use of ARNT as loading control or as a reference gene is basically not recom‐

First evidence for a hypoxia‐dependent regulation of ARNT was provided by Wang et al. [5]. Herein, Hep3B cells were used to study the effects of HIF‐1α and ARNT under hypoxic condi‐ tions. ARNT was elevated on mRNA level in this cell line due to hypoxic exposure (1% v/v O2

In addition, treatment with hypoxia mimetics such as cobalt chloride and desferrioxamine had similar effects. Nuclear extracts prepared from Hep3B and HeLa cells were used to investigate the response of both HIF‐1 subunits to oxygen deprivation. Re‐oxygenation experiments were also included into the study [5]. The data revealed that both transcription factors HIF‐1α and ARNT were inducible in hypoxic cells on mRNA as well as protein levels [5]. Huang et al. [10] reported that ARNT protein levels remained constant regardless of cellular oxygen tension. In this study, Hep3B, HeLa and HEK293 cells were used. Unfortunately, not all experiments were conducted with all cell lines [10], thereby making a direct comparison with the study of Wang et al. [5] complicated. Nevertheless, the latter report [10] challenged the results of the previous one [5] due to signal variations of Northern blots and discontinuities of time‐course

However, Huang et al. [10] proposed a very graphic working model including a specific sensor for hypoxia located in the cell membrane [10]. The depicted mechanism is similar to our nowa‐ days HIF scheme. These days the prolylhydroxylase domain enzymes (PHDs), which require

 as a substrate, are known to act as cellular oxygen sensors [11]. The comparison of both seminal studies conducted by Wang et al. [5] and Huang et al. [10] also requires a glance on

).

cells [1].

32 Hypoxia and Human Diseases

model [3].

mended [1].

experiments [10].

O2

**2. Regulation of ARNT**

**2.1. Upregulation of ARNT in response to hypoxia**

The capability to elevate ARNT expression in hypoxia was also found in murine L929 and Hepa1 cells. Interestingly, human Hep3B cells were also used in this study conducted by Chilov et al. [7], but ARNT was unaffected by oxygen deprivation [7]. This seemingly con‐ flicting observation compared to a previous report [5] is likely due to different experimental conditions. Obviously, a short‐term exposure of Hep3B cells to hypoxia (4 h in Ref. [7]) is not sufficient to induce ARNT protein expression in this model. However, other studies clearly confirmed the hypoxia‐dependent upregulation of ARNT in Hep3B cells [3, 4].

First mechanistic clues regarding the induction of ARNT under oxygen deprivation were provided by Zhong et al. [6]. Herein, the authors tested the hypothesis whether HIF‐1α and ARNT are regulated by similar signalling pathways in human prostate cancer cells. Indeed, an elevated ARNT protein level was observed in hypoxic PC‐3 cells. Interestingly, this effect was attenuated by inhibition of the phosphatidylinositol‐3 kinase (PI3K)/AKT‐pathway by Wortmannin [6]. Another hint regarding the ARNT expression pattern in cancer cells came from Skinner et al. [12]. Herein, the authors investigated the transcriptional regulation of VEGF in ovarian cancer cell lines in response to PI3K/Akt signalling. Blocking of this path‐ way using the compound LY294002 specifically inhibited HIF‐1α expression but had no effect on ARNT. Unfortunately, the inducibility of ARNT in hypoxia was not tested in this study [12]. Most important, the observation that PI3K/Akt inhibition decreased HIF‐1α but not ARNT in one model [12] whereas both transcription factors were reduced on protein level in another model [6] might suggest a HIF‐dependent regulation of ARNT in certain cell lines.

The studies discussed so far clearly show that certain cell lines are capable to induce ARNT in hypoxia and that this effect is dependent on the experimental conditions (i.e. time points). Thus, one might assume that scientists became more aware of this phenomenon over time. However, ARNT was also used as a loading control in Western blot analysis [12, 13]. This application clearly demonstrates the major opinion of a complete non‐hypoxic regulation of ARNT. Of note, effects of hypoxia and hypoxia mimetics on ARNT expression should be taken into consideration when studying the HIF pathway as previously proposed [1]. Such an approach will help to identify new cell types harbouring the hypoxia‐inducible ARNT attribute and might provide novel mechanistic insights. The current proposed mechanism is discussed later in the chapter.

The seminal study conducted by Choi et al. shed light on ARNT expression and turnover [14]. The authors assumed that the regulation of ARNT expression or activity might significantly affect cell metabolism. Thus, ARNT should be regarded as a drug target and appropriate compounds need to be investigated. De novo synthesis of ARNT, enhanced stability of the protein and dimerisation with HIF‐1α, represents three ways how this transcription factor can be controlled. Curcumin, the major component of the spice turmeric, was tested in this study for potential inhibitory effects on HIF‐1. Interestingly, curcumin facilitated the degradation of ARNT and blocked HIF signalling [14]. Similar effects were reported by Ströfer et al. [15]. Curcumin‐mediated ARNT depletion was observed in human HepG2, Hep3B and MCF‐7 cells [15]. The half‐life of ARNT was determined in Hep3B cells after cycloheximide treat‐ ment and calculated with approximately 5 h [14]. In contrast, curcumin exposure decreased ARNT half‐life to roughly 2 h. It turned out that the curcumin‐dependent degradation of ARNT was redox sensitive and could be reversed by antioxidants and the proteasome inhibi‐ tor MG‐132 [14]. Remarkably, MG‐132 did not affect ARNT protein level in the absence of cur‐ cumin. Therefore, the authors proposed the existence of two different mechanisms mediating ARNT turnover: a proteasome‐independent mechanism under physiological conditions and a proteasome‐dependent degradation in response to stress [14].

The elevation of ARNT protein expression under oxygen deprivation might not be an exclu‐ sive trait of cell lines. Exposure of primary mouse keratinocytes to acute hypoxia (1% O<sup>2</sup> ) resulted in an upregulation of ARNT after 4 and 5 h, respectively [16]. However, this effect was not statistically significant. Putative alterations on ARNT mRNA expression were also evalu‐ ated in this cell model. In contrast, time‐course experiments revealed no apparent changes on mRNA level in murine keratinocytes cultured in hypoxia up to 48 h [16]. The selection of an inappropriate internal control in qPCR analysis can also lead to different expression lev‐ els in normoxia and hypoxia [17]. Therefore, Vavilala et al. determined the expression level of three housekeeping genes (ribosomal protein L32, β‐actin and GAPDH) in normoxic and hypoxic cells, respectively [17]. The authors observed no significant changes on mRNA levels between both experimental settings. The aim of this study was to investigate inhibitory effects of Honokiol, a biphenolic phytochemical compound, on HIF signalling in several cell lines. The results presented in this report consist solely of gene expression data. Among them, the HIF‐1α, HIF‐2α and ARNT mRNA level were compared under normoxic and hypoxic condi‐ tions [17]. Remarkably, an approximately 7‐fold increase in ARNT mRNA was observed in D407 human retinal pigment epithelial cells. In addition, a 2‐fold upregulation was detected in HT‐29 cells and a slight increase in the HEK293 cell line. MCF‐7 cells showed no increase in ARNT mRNA due to hypoxic exposure. Unfortunately, the comparison of these effects among the cell lines tested in this study is limited because of different time points used (12 versus 24 h in D407 cells; 1% O2 ) [17]. Nevertheless, the study provides clear evidence of a cell‐specific transcriptional ARNT upregulation in hypoxia although these findings were not confirmed by Western blotting.

Further mechanistic insights into this cellular trait were provided by a research project inves‐ tigating the regulation of ARNT in human melanoma cells [2]. Among a panel of five different cell lines, ARNT was rapidly elevated on protein level in 518A2 cells after treatment with the hypoxia mimetic cobalt chloride (CoCl2 ). Interestingly, knockdown of HIF‐1α in CoCl<sup>2</sup> stimulated and hypoxic 518A2 cells abolished the hypoxia‐dependent upregulation of ARNT. Overexpression of a dominant‐negative HIF mutant in this cell model indicated that ARNT expression is dependent on the HIF pathway itself. In agreement with these findings, overex‐ pression of HIF‐1α caused an elevation of ARNT protein in CoCl<sup>2</sup> treated 518A2 cells. Taken together, this study demonstrated a regulatory relationship between HIF‐1α and its binding partner ARNT for the first time. In addition, it was concluded that this capability might pre‐ vent ARNT to become a limiting factor in hypoxia [2].

The first comprehensive study aiming to re‐evaluate the regulation of ARNT was conducted by Wolff et al. [4]. Herein, numerous cell lines were exposed to 1 and 3% O2 for different time points. In addition, hypoxia mimetics such as CoCl2 and dimethyloxalylglycine (DMOG) were used and the quantity of ARNT protein determined by Western blotting. The authors found out that ARNT expression was induced in MCF‐7, HeLa and Hep3B cells. Interestingly, the ARNT level was also dependent on the hypoxic environment used. A concentration of 1% O2 led to a faster increase in ARNT protein but also to an earlier decline to basal levels as compared to 3% hypoxia. Moreover, the appropriate mRNA levels did not correlate with the amount of protein detected. In particular, in MCF‐7 and Hep3B cells, a downregulation of ARNT mRNA was observed due to hypoxia. Therefore, the authors hypothesised the existence of a reciprocal feedback regulation between ARNT protein stability and de novo synthesis. This study provides convincing evidence that the predominant point of view that ARNT is unaffected by hypoxia and hypoxia mimetics cannot be applied to all cell lines in general [4].

The first review highlighting the topic of hypoxia‐inducible ARNT was published by Mandl and Depping [1]. Herein, two major questions were raised: (1) How can cells acquire this attri‐ bute? and (2) What is the benefit for these cells? [1] Both issues will be discussed below. An updated list of cell lines capable to elevate ARNT in response to hypoxia is presented in **Table 1**. Among them, the human Hep3B cell line is obviously the best studied model in this context.


**Table 1.** Cell lines with hypoxia‐inducible ARNT expression.

#### *2.1.1. Purpose of hypoxia‐inducible ARNT*

of ARNT and blocked HIF signalling [14]. Similar effects were reported by Ströfer et al. [15]. Curcumin‐mediated ARNT depletion was observed in human HepG2, Hep3B and MCF‐7 cells [15]. The half‐life of ARNT was determined in Hep3B cells after cycloheximide treat‐ ment and calculated with approximately 5 h [14]. In contrast, curcumin exposure decreased ARNT half‐life to roughly 2 h. It turned out that the curcumin‐dependent degradation of ARNT was redox sensitive and could be reversed by antioxidants and the proteasome inhibi‐ tor MG‐132 [14]. Remarkably, MG‐132 did not affect ARNT protein level in the absence of cur‐ cumin. Therefore, the authors proposed the existence of two different mechanisms mediating ARNT turnover: a proteasome‐independent mechanism under physiological conditions and

The elevation of ARNT protein expression under oxygen deprivation might not be an exclu‐ sive trait of cell lines. Exposure of primary mouse keratinocytes to acute hypoxia (1% O<sup>2</sup>

resulted in an upregulation of ARNT after 4 and 5 h, respectively [16]. However, this effect was not statistically significant. Putative alterations on ARNT mRNA expression were also evalu‐ ated in this cell model. In contrast, time‐course experiments revealed no apparent changes on mRNA level in murine keratinocytes cultured in hypoxia up to 48 h [16]. The selection of an inappropriate internal control in qPCR analysis can also lead to different expression lev‐ els in normoxia and hypoxia [17]. Therefore, Vavilala et al. determined the expression level of three housekeeping genes (ribosomal protein L32, β‐actin and GAPDH) in normoxic and hypoxic cells, respectively [17]. The authors observed no significant changes on mRNA levels between both experimental settings. The aim of this study was to investigate inhibitory effects of Honokiol, a biphenolic phytochemical compound, on HIF signalling in several cell lines. The results presented in this report consist solely of gene expression data. Among them, the HIF‐1α, HIF‐2α and ARNT mRNA level were compared under normoxic and hypoxic condi‐ tions [17]. Remarkably, an approximately 7‐fold increase in ARNT mRNA was observed in D407 human retinal pigment epithelial cells. In addition, a 2‐fold upregulation was detected in HT‐29 cells and a slight increase in the HEK293 cell line. MCF‐7 cells showed no increase in ARNT mRNA due to hypoxic exposure. Unfortunately, the comparison of these effects among the cell lines tested in this study is limited because of different time points used (12 versus 24 h

) [17]. Nevertheless, the study provides clear evidence of a cell‐specific

). Interestingly, knockdown of HIF‐1α in CoCl<sup>2</sup>

treated 518A2 cells. Taken

transcriptional ARNT upregulation in hypoxia although these findings were not confirmed

Further mechanistic insights into this cellular trait were provided by a research project inves‐ tigating the regulation of ARNT in human melanoma cells [2]. Among a panel of five different cell lines, ARNT was rapidly elevated on protein level in 518A2 cells after treatment with

stimulated and hypoxic 518A2 cells abolished the hypoxia‐dependent upregulation of ARNT. Overexpression of a dominant‐negative HIF mutant in this cell model indicated that ARNT expression is dependent on the HIF pathway itself. In agreement with these findings, overex‐

together, this study demonstrated a regulatory relationship between HIF‐1α and its binding partner ARNT for the first time. In addition, it was concluded that this capability might pre‐

)

a proteasome‐dependent degradation in response to stress [14].

in D407 cells; 1% O2

34 Hypoxia and Human Diseases

by Western blotting.

the hypoxia mimetic cobalt chloride (CoCl2

pression of HIF‐1α caused an elevation of ARNT protein in CoCl<sup>2</sup>

vent ARNT to become a limiting factor in hypoxia [2].

The capability of certain tumour cells to upregulate ARNT under hypoxic conditions might provide a specific survival advantage as previously proposed [1]. Indeed, we recently discov‐ ered a relationship between ARNT and the cellular response to radiation [18]. Tumour hypoxia is associated with radioresistance and poor patient prognosis. Therefore, we investigated the effects of an altered expression of ARNT on radioresistance and performed clonogenic survival assays. As expected, silencing of ARNT in Hep3B and MCF‐7 cells by siRNA ren‐ dered these models susceptible to radiation. Interestingly, overexpression of ARNT in these cell lines promoted radioresistance. Therefore, it was hypothesised that radiation treatment might provide a selection pressure and lead to an enrichment of high‐ARNT expressing cells. Taken together, these findings provide evidence to consider ARNT as a drug target in order to increase radiosensitivity in tumour cells and as a predictive marker in this context [18].

As outlined above, there is evidence that HIF‐1α mediates the elevation of ARNT under hypoxic conditions in certain cell lines. This regulatory relationship is the prerequisite of a feed‐forward loop (FFL) as demonstrated recently in Hep3B cells. In such a network motif, one transcription factor regulates the other and both controls the expression of a target gene cooperatively. Given the fact that HIF‐1α and ARNT form the transcriptional active complex HIF‐1, which regulates a plethora of target genes, the FFL definition is fulfilled. By using reporter gene assays, we were able to demonstrate that overexpression of ARNT in Hep3B cells increased the luciferase signal in hypoxia. Therefore, it was concluded that augmented HIF signalling in terms of elevated target gene expression might be beneficial for tumour cells. These findings support the concept of ARNT being a limiting factor in at least certain cell models [3].

Moreover, general considerations regarding inducible gene expression are in line with the studies discussed above. In order to respond rapidly to micro‐environmental alterations required genes need to be specifically activated. Inducible genes are highly regulated and must be quickly shut down to basal expression levels once the stimulus disappeared [19].

#### *2.1.2. Mechanism of hypoxia‐dependent ARNT upregulation*

The mechanism(s) underlying this unique cellular attribute is (are) unclear. There is mount‐ ing evidence indicating a pivotal role of HIF‐1α [2–4]. It was demonstrated that ARNT was increased in 518A2 human melanoma cells in a HIF‐1α‐dependent manner under hypoxic conditions [2]. A very similar mechanism was revealed in Hep3B cells [3]. Knockdown and overexpression of HIF‐1α affected the ARNT protein level accordingly. Moreover, a clear tran‐ scriptional relationship between HIF‐1α and its binding partner ARNT was established in this model system. Treatment with actinomycin D, an inhibitor of RNA synthesis, diminished the induction of ARNT under oxygen deprivation. In addition, appropriate gene‐silencing experi‐ ments and qRT‐PCR analysis confirmed this finding [3]. Another important observation might designate HIF‐1α as a mediator of this cellular attribute. The PI3K/Akt inhibitor LY294002 was shown to inhibit HIF‐1α expression in ovarian cancer cell lines but had no effect on ARNT protein [12]. In contrast, several independent studies have shown that the hypoxia‐dependent increase in ARNT was abolished by blocking the PI3K/Akt pathway with LY294002 or similar compounds [2, 6, 20]. This finding—the susceptibility of ARNT to PI3K/Akt inhibition in certain models—might be characteristic for cells capable to induce ARNT in hypoxia. Taken together, this suggests a linear model and might imply ARNT to be a downstream target of HIF‐1α.

The cellular cause of the regulatory relationship between HIF‐1α and ARNT is not known. HIF‐1α can act independent of its binding partner ARNT and regulate gene expression [1]. It was shown that HIF‐1α can act as a co‐activator or co‐repressor on certain genes. In addi‐ tion, an indirect regulatory connection between both transcription factors might exist [1]. HIF‐regulated genes encode for growth factors, glucose transporters, glycolytic enzymes but also other transcription factors and miRNAs. Therefore, HIF‐controlled transcription fac‐ tors and miRNAs might influence ARNT expression [1, 3]. A general working concept is discussed below.

#### *2.1.2.1. Working concept of hypoxia‐inducible ARNT*

is associated with radioresistance and poor patient prognosis. Therefore, we investigated the effects of an altered expression of ARNT on radioresistance and performed clonogenic survival assays. As expected, silencing of ARNT in Hep3B and MCF‐7 cells by siRNA ren‐ dered these models susceptible to radiation. Interestingly, overexpression of ARNT in these cell lines promoted radioresistance. Therefore, it was hypothesised that radiation treatment might provide a selection pressure and lead to an enrichment of high‐ARNT expressing cells. Taken together, these findings provide evidence to consider ARNT as a drug target in order to increase radiosensitivity in tumour cells and as a predictive marker in this context [18].

As outlined above, there is evidence that HIF‐1α mediates the elevation of ARNT under hypoxic conditions in certain cell lines. This regulatory relationship is the prerequisite of a feed‐forward loop (FFL) as demonstrated recently in Hep3B cells. In such a network motif, one transcription factor regulates the other and both controls the expression of a target gene cooperatively. Given the fact that HIF‐1α and ARNT form the transcriptional active complex HIF‐1, which regulates a plethora of target genes, the FFL definition is fulfilled. By using reporter gene assays, we were able to demonstrate that overexpression of ARNT in Hep3B cells increased the luciferase signal in hypoxia. Therefore, it was concluded that augmented HIF signalling in terms of elevated target gene expression might be beneficial for tumour cells. These findings support the concept

Moreover, general considerations regarding inducible gene expression are in line with the studies discussed above. In order to respond rapidly to micro‐environmental alterations required genes need to be specifically activated. Inducible genes are highly regulated and must be quickly shut down to basal expression levels once the stimulus disappeared [19].

The mechanism(s) underlying this unique cellular attribute is (are) unclear. There is mount‐ ing evidence indicating a pivotal role of HIF‐1α [2–4]. It was demonstrated that ARNT was increased in 518A2 human melanoma cells in a HIF‐1α‐dependent manner under hypoxic conditions [2]. A very similar mechanism was revealed in Hep3B cells [3]. Knockdown and overexpression of HIF‐1α affected the ARNT protein level accordingly. Moreover, a clear tran‐ scriptional relationship between HIF‐1α and its binding partner ARNT was established in this model system. Treatment with actinomycin D, an inhibitor of RNA synthesis, diminished the induction of ARNT under oxygen deprivation. In addition, appropriate gene‐silencing experi‐ ments and qRT‐PCR analysis confirmed this finding [3]. Another important observation might designate HIF‐1α as a mediator of this cellular attribute. The PI3K/Akt inhibitor LY294002 was shown to inhibit HIF‐1α expression in ovarian cancer cell lines but had no effect on ARNT protein [12]. In contrast, several independent studies have shown that the hypoxia‐dependent increase in ARNT was abolished by blocking the PI3K/Akt pathway with LY294002 or similar compounds [2, 6, 20]. This finding—the susceptibility of ARNT to PI3K/Akt inhibition in certain models—might be characteristic for cells capable to induce ARNT in hypoxia. Taken together, this suggests a linear model and might imply ARNT to be a downstream target of HIF‐1α.

The cellular cause of the regulatory relationship between HIF‐1α and ARNT is not known. HIF‐1α can act independent of its binding partner ARNT and regulate gene expression [1].

of ARNT being a limiting factor in at least certain cell models [3].

*2.1.2. Mechanism of hypoxia‐dependent ARNT upregulation*

36 Hypoxia and Human Diseases

Based on the studies mentioned above, a general working concept can be deduced (**Figure 1**). In addition to its oxygen regulation, the HIF pathway, that is, HIF‐1α, is also controlled by growth factors via the PI3K/Akt signalling cascade leading to elevated translation [21, 22]. Upon activation HIF‐1α induces the upregulation of its binding partner ARNT either on mRNA and/or protein level in appropriate cell lines. For instance, it was shown that hypoxic induction of ARNT in Hep3B cells is mediated by de novo synthesis [3]. This effect can be achieved either directly or indirectly. A direct mechanism might involve the recruitment of HIF‐1α to the ARNT promoter, whereas an indirect mechanism might be mediated by other HIF‐regulated transcription factors or miRNAs [1]. Indeed, a complex mutual regulatory rela‐ tionship between miRNAs and PAS proteins exists. However, the physiological and patho‐ physiological mechanisms behind are unclear [23].

**Figure 1.** General working concept of hypoxia‐inducible ARNT. See text for details.

Our recent experiments revealed that HIF‐1α and ARNT are recruited to the ARNT gene promoter in hypoxic Hep3B cells. Deployment of CRISPR/Cas9 gene editing technology con‐ firmed the importance of a unique genomic sequence for hypoxia‐dependent ARNT upreg‐ ulation. Therefore, these findings suggest a direct mechanism and render ARNT a putative HIF‐1 target gene in Hep3B cells (unpublished observations; manuscript in preparation).

The regulatory relationship between HIF‐1α and ARNT is part of a feed‐forward loop (FFL; **Figure 1**: red arrows) as already demonstrated in Hep3B cells [3]. Subsequently, HIF‐1α and its binding partner ARNT form the transcriptional active heterodimer HIF‐1 and initiate the expression of various target genes. Therefore, an increased target gene expression seems to be beneficial for tumour cells [3].

## *2.1.3. Experimental conditions*

Although the hypoxic inducibility of ARNT is described in specific cell lines by convincing data, not every study could confirm this circumstance. This obvious conflict depends mainly on the experimental conditions used. For instance, it was demonstrated that in Hep3B cells, 3% O2 for 8 h was sufficient to elevate ARNT on protein level [3]. In contrast, a peak induction on mRNA level was observed after 5 h in the same setting [3]. Of note, these conditions need not to be appropriate in other cells. Until now, a few studies reported that ARNT mRNA and protein levels do not correlate in a number of cell lines [4, 18].

#### **2.2. Regulation of ARNT by other factors**

The regulation of ARNT or whether it responds to stimulation is poorly understood. There is evidence that ARNT expression is controlled by the NF‐κB pathway in different models. It was demonstrated that ARNT mRNA was induced in HEK293 cells due to TNF‐α stimula‐ tion. This effect was abrogated by pharmacological blocking or silencing of the NF‐κB cascade [24]. Moreover, Per‐ARNT‐Sim (PAS) transcription factors belonging to different signalling circuits can compete for common binding partners such as ARNT (discussed below). Thus, misregulation of these proteins might contribute to tumour survival [9]. Noteworthily, the mutual regulation of PAS transcription factors on mRNA level was also mentioned in the literature, but appropriate citations are missing [22].

## **3. Crosstalk between Per‐ARNT‐Sim transcription factors**

The HIF, AhR and BMAL1/Clock pathways respond to a decline in cellular oxygen concentra‐ tion, environmental xenobiotics or govern circadian rhythms, respectively. All of these tran‐ scription factors are related. They belong to the group of Per‐ARNT‐Sim (PAS) transcription factors which are characterised by the presence of a PAS domain (composed of PAS‐A and PAS‐B subdomains) required for protein‐protein interactions. Therefore, all family members are able to form homo‐ and heterodimers among the group [9, 25].

The transcription factor ARNT plays a pivotal role within the HIF and AhR pathways. It serves as the common binding partner for HIF‐α subunits and ligands activated AhR proteins [1]. Therefore, a competition between both signalling cascades regarding the recruitment of ARNT might be obvious. Indeed, early evidence for such an antagonism was provided by Gradin et al. [26]. By using luciferase reporter gene constructs under the control of xenobi‐ otic‐responsive elements (XRE), the effect of HIF and AhR activation was studied in HepG2 cells. As expected, stimulation of cells with an appropriate AhR ligand leads to a pronounced induction of reporter gene expression. This effect was suppressed by co‐treatment with the hypoxia mimetic cobalt chloride. Co‐immunoprecipitation experiments clearly indicated a competition between the HIF and AhR pathway relating to ARNT binding. In addition, it was shown that HIF‐1α could efficiently compete with the AhR for dimerisation with ARNT. This study provided evidence for a HIF‐1α‐mediated inhibition of AhR signalling by sequestra‐ tion of ARNT [26]. Vorrink et al. [27] observed similar effects again in human hepatocellular carcinoma HepG2 cells and in the human keratinocyte HaCaT cell line. One major advantage of this study was the genuine hypoxic exposure of cells instead of stimulation with hypoxia mimetics such as cobalt chloride. AhR signalling was triggered by treatment with the dioxin‐ like compound PCB126. Again, hypoxia inhibited CYP1A1 reporter gene activity in PCB126 stimulated HepG2 cells. Importantly, ARNT overexpression caused an elevated luminescence signal under normoxic and hypoxic conditions. Moreover, forced ARNT expression was suf‐ ficient to overcome the inhibitory effect of hypoxia on AhR signalling. The authors concluded that ARNT is sequestered by HIF‐1α in hypoxia thus limiting the availability of this transcrip‐ tion factor for AhR heterodimerisation [27]. Noteworthily, another report published nearly two decades ago claims the complete opposite [28]. This study might provide evidence for a lack of competition between HIF and AhR signalling on ARNT recruitment. Unfortunately, the presented arguments and data are not convincing at many points [28].

Furthermore, a crosstalk between AhR and BMAL1/Clock exists. Lipophilic AhR ligands such as dioxin or dietary polyphenols bind within the AhR PAS‐B domain and trigger nuclear trans‐ location. Within the nucleus activated AhR can dimerise with BMAL1 thus disrupting the auto‐ regulatory loop of BMAL1/Clock genes. Therefore, AhR activation leads to a suppression of circadian rhythms, whereas AhR inhibition strengthens rhythm amplitude [25]. Interestingly, there is evidence that both AhR and ARNT are expressed in an oscillatory pattern in vivo [29].

## **4. Subcellular dynamics of ARNT and turnover**

Our recent experiments revealed that HIF‐1α and ARNT are recruited to the ARNT gene promoter in hypoxic Hep3B cells. Deployment of CRISPR/Cas9 gene editing technology con‐ firmed the importance of a unique genomic sequence for hypoxia‐dependent ARNT upreg‐ ulation. Therefore, these findings suggest a direct mechanism and render ARNT a putative HIF‐1 target gene in Hep3B cells (unpublished observations; manuscript in preparation).

The regulatory relationship between HIF‐1α and ARNT is part of a feed‐forward loop (FFL; **Figure 1**: red arrows) as already demonstrated in Hep3B cells [3]. Subsequently, HIF‐1α and its binding partner ARNT form the transcriptional active heterodimer HIF‐1 and initiate the expression of various target genes. Therefore, an increased target gene expression seems

Although the hypoxic inducibility of ARNT is described in specific cell lines by convincing data, not every study could confirm this circumstance. This obvious conflict depends mainly on the experimental conditions used. For instance, it was demonstrated that in Hep3B cells,

The regulation of ARNT or whether it responds to stimulation is poorly understood. There is evidence that ARNT expression is controlled by the NF‐κB pathway in different models. It was demonstrated that ARNT mRNA was induced in HEK293 cells due to TNF‐α stimula‐ tion. This effect was abrogated by pharmacological blocking or silencing of the NF‐κB cascade [24]. Moreover, Per‐ARNT‐Sim (PAS) transcription factors belonging to different signalling circuits can compete for common binding partners such as ARNT (discussed below). Thus, misregulation of these proteins might contribute to tumour survival [9]. Noteworthily, the mutual regulation of PAS transcription factors on mRNA level was also mentioned in the

The HIF, AhR and BMAL1/Clock pathways respond to a decline in cellular oxygen concentra‐ tion, environmental xenobiotics or govern circadian rhythms, respectively. All of these tran‐ scription factors are related. They belong to the group of Per‐ARNT‐Sim (PAS) transcription factors which are characterised by the presence of a PAS domain (composed of PAS‐A and PAS‐B subdomains) required for protein‐protein interactions. Therefore, all family members

The transcription factor ARNT plays a pivotal role within the HIF and AhR pathways. It serves as the common binding partner for HIF‐α subunits and ligands activated AhR proteins [1].

 for 8 h was sufficient to elevate ARNT on protein level [3]. In contrast, a peak induction on mRNA level was observed after 5 h in the same setting [3]. Of note, these conditions need not to be appropriate in other cells. Until now, a few studies reported that ARNT mRNA and

to be beneficial for tumour cells [3].

**2.2. Regulation of ARNT by other factors**

literature, but appropriate citations are missing [22].

**3. Crosstalk between Per‐ARNT‐Sim transcription factors**

are able to form homo‐ and heterodimers among the group [9, 25].

protein levels do not correlate in a number of cell lines [4, 18].

*2.1.3. Experimental conditions*

38 Hypoxia and Human Diseases

3% O2

Translocation of ARNT from the cytoplasm into the nucleus is mediated by importins as also demonstrated for other HIF family members [30, 31]. Blocking of this specific process was proposed as a novel way to suppress HIF signalling [30]. Whether ARNT shuttles, back into the cytoplasm is unknown. Under these circumstances, inhibition of the putative nuclear export might prolong HIF activity. Moreover, whether ARNT is degraded, within the nucleus is not investigated in greater depth (depicted in **Figure 2**).

In general, there is evidence for two different mechanisms leading to ARNT degradation. It was found out that ARNT was not affected by the proteasome inhibitor MG‐132 under physiological conditions. In contrast, proteasomal degradation of ARNT might be triggered by reactive oxygen species [3, 14].

**Figure 2.** Subcellular logistics of ARNT. See text for details.

## **5. Clinical aspects**

Inhibition of the HIF pathway is proposed as a treatment strategy in oncology. Several appro‐ priate compounds have been identified and confirmed in xenograft models. These drugs are able to block different processes of the HIF signalling pathway. For instance, HIF‐1α protein synthesis is diminished by rapamycin which is an inhibitor of mTOR. The antibiotic acrifla‐ vine prevents heterodimerisation of HIF‐1α and ARNT subunits [32]. Moreover, a plethora of other HIF inhibitors was discovered which comprise of different chemical entities. HIF is con‐ sidered as an attractive drug target, and blocking of its activity might lead to cytostatic anti‐ tumour effects. A synergistic outcome with radiotherapy is also expected [33, 34]. However, such drugs might be useful in multidrug regimes only in a subset of cancer patients. Cancers in which HIF is a strong driving force for disease progression are assumed to be susceptible for anti‐HIF treatment [32].

The temporal importance of ARNT during tumour growth was investigated by Shi et al. [35]. Herein, the authors used murine hepatoma Hepa‐1 cells transduced with a Tet‐Off mArnt construct. Xenograft experiments conducted with these cells indicated that ARNT is particularly required during the early stage of tumour growth. The authors proposed that a profound inhibition of the HIF pathway might be achieved only by suppressing both HIF‐1α and HIF‐2α proteins. Therefore, it was concluded that the binding partner ARNT might represent a preferable therapeutic target rather than HIF‐α subunits [35]. More convincing evidence regarding the role of ARNT in this malignancy was provided by a study using human tissue samples and cell lines [36]. ARNT expression was analysed by immunohistochemistry in hepatocellular carcinoma (HCC) and liver tissues. ARNT was found primarily in the nucleus but also in the cytoplasm in a minor fraction of cells. Interestingly, ARNT expression was significantly higher in normal liver samples as com‐ pared to appropriate HCC tissues. In addition, the impact of ARNT expression on overall survival (OS) of HCC patients was evaluated [36]. Surprisingly, a high intra‐tumour ARNT level was associated with a prolonged OS. In agreement with this observation, stably len‐ tiviral transduced ARNT‐knockdown HCCLM6 cells showed a high proliferation rate, whereas overexpression of ARNT had the opposite effect. In addition, ARNT‐suppressed cells formed smaller tumours in a murine xenograft model as compared to appropriate ARNT‐overexpressing counterparts. This finding is in line with clinical data indicating a smaller tumour size in high‐ARNT expressing hepatocellular carcinoma [36]. Moreover, the incidence of recurrence after surgery was significantly lower when a high intra‐tumour ARNT level was detected. Taken together, this study describes an inhibitory role of ARNT in HCC progression. It was concluded that ARNT is a central regulator in HCC progres‐ sion and a useful predictive marker regarding curative resection. The authors proposed that the relative balance of ARNT and its binding partners might be an important determi‐ nant in HCC [36]. In addition, another study demonstrated an important role of ARNT in this malignancy. Choi et al. [37] silenced ARNT expression in several human hepatoma cell lines by using siRNA and evaluated the effects on cell growth. It was shown that knock‐ down of this transcription factor inhibited proliferation and sensitised cells to apoptosis [37]. An elegant approach to target ARNT by small molecule inhibitors was conducted by Guo et al. [38]. Herein, nuclear magnetic resonance and biochemical screens were used in order to identify molecules selectively binding to the PAS domain of ARNT. The com‐ pound KG‐548 was discovered to compete with the co‐activator TACC3 for ARNT binding. The specific blocking of protein‐protein interactions among transcription factors repre‐ sents a novel technique to inhibit HIF signalling. Due to the shared use of ARNT among alpha subunits, targeting this protein was proposed to be more efficient as compared to its counterparts [38]. Evidence highlighting the importance of ARNT as a drug target was also provided by another study. Chan and colleagues [39] described that ARNT expression enhances cisplatin resistance in cancer cells. This phenotype was mediated by upregula‐ tion of MDR1, a multidrug efflux pump of the ABC superfamily, by a direct mechanism. Accordingly, knockdown of ARNT by siRNA transfection reduced cisplatin resistance in human cancer cells. Moreover, ARNT silencing increased the therapeutic efficacy of this cytotoxic drug in a murine xenograft model [39].

**5. Clinical aspects**

40 Hypoxia and Human Diseases

**Figure 2.** Subcellular logistics of ARNT. See text for details.

for anti‐HIF treatment [32].

Inhibition of the HIF pathway is proposed as a treatment strategy in oncology. Several appro‐ priate compounds have been identified and confirmed in xenograft models. These drugs are able to block different processes of the HIF signalling pathway. For instance, HIF‐1α protein synthesis is diminished by rapamycin which is an inhibitor of mTOR. The antibiotic acrifla‐ vine prevents heterodimerisation of HIF‐1α and ARNT subunits [32]. Moreover, a plethora of other HIF inhibitors was discovered which comprise of different chemical entities. HIF is con‐ sidered as an attractive drug target, and blocking of its activity might lead to cytostatic anti‐ tumour effects. A synergistic outcome with radiotherapy is also expected [33, 34]. However, such drugs might be useful in multidrug regimes only in a subset of cancer patients. Cancers in which HIF is a strong driving force for disease progression are assumed to be susceptible

The temporal importance of ARNT during tumour growth was investigated by Shi et al. [35]. Herein, the authors used murine hepatoma Hepa‐1 cells transduced with a Tet‐Off mArnt construct. Xenograft experiments conducted with these cells indicated that ARNT is particularly required during the early stage of tumour growth. The authors proposed that a profound inhibition of the HIF pathway might be achieved only by suppressing both HIF‐1α and HIF‐2α proteins. Therefore, it was concluded that the binding partner ARNT might represent a preferable therapeutic target rather than HIF‐α subunits [35]. More convincing evidence regarding the role of ARNT in this malignancy was provided by a study using human tissue samples and cell lines [36]. ARNT expression was analysed by immunohistochemistry in hepatocellular carcinoma (HCC) and liver tissues. ARNT Targeting the HIF pathway in cancer therapy in order to achieve tumour control has been pro‐ posed by several reports [40–42]. Remarkably, the majority of HIF inhibitors described until now lack specificity. For instance, the drug topotecan blocks topoisomerase I activity but also diminished HIF signalling in preclinical models. This inhibitory effect on HIF was accom‐ plished by preventing the accumulation of HIF‐1α. In multihistology target‐driven clinical trial, Kummar et al. [43] evaluated the oral use of this compound in a small number of cancer patients. Different tumour entities were diagnosed in these patients including ovarian cancer, sarcoma and melanoma among others. A complete inhibition of HIF‐1α was detected in biop‐ sies of a few patients, but inherent sampling and heterogeneous HIF‐1α expression might limit this finding [43]. In contrast, despite the clear role of ARNT in tumour progression, its drug‐ability and appropriate treatment effects need to be evaluated in a clinical setting.

## **6. Concluding remarks**

The attribute of certain tumour cells to elevate the transcription factor ARNT in hypoxia was shown decades ago but since neglected in HIF biology. Only a small number of studies focus on the regulation of ARNT, especially under hypoxic conditions. Therefore, hypoxia‐induc‐ ible ARNT is an emerging concept in this field. According to the major opinion, ARNT is a constitutively expressed gene. This means that ARNT expression is not effected by environ‐ mental factors such as hypoxia. Due to the fact that there are exceptions from this dogma, the statement of a constitutive ARNT expression should be revised and not used in general terms. Thus, ARNT should be regarded as a "cell‐specific facultative gene" in tumour cells which indicates an expression as needed.

## **Author details**

Markus Mandl and Reinhard Depping\*

\*Address all correspondence to: reinhard.depping@uni‐luebeck.de

Institute of Physiology, Center for Structural and Cell Biology in Medicine, University of Luebeck, Luebeck, Germany

## **References**


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**6. Concluding remarks**

42 Hypoxia and Human Diseases

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\*Address all correspondence to: reinhard.depping@uni‐luebeck.de

**Author details**

**References**

The attribute of certain tumour cells to elevate the transcription factor ARNT in hypoxia was shown decades ago but since neglected in HIF biology. Only a small number of studies focus on the regulation of ARNT, especially under hypoxic conditions. Therefore, hypoxia‐induc‐ ible ARNT is an emerging concept in this field. According to the major opinion, ARNT is a constitutively expressed gene. This means that ARNT expression is not effected by environ‐ mental factors such as hypoxia. Due to the fact that there are exceptions from this dogma, the statement of a constitutive ARNT expression should be revised and not used in general terms. Thus, ARNT should be regarded as a "cell‐specific facultative gene" in tumour cells which

Institute of Physiology, Center for Structural and Cell Biology in Medicine, University of

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#### **The Hypoxia-Reoxygenation Injury Model** The Hypoxia-Reoxygenation Injury Model

#### Domokos Gerő Domokos Gerő

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

http://dx.doi.org/10.5772/65339

#### Abstract

Hypoxia-reoxygenation injury is a commonly used in vitro model of ischemia, which is useful to study the recovery processes following the hypoxic period. Hypoxia can be rapidly induced in vitro by replacing the culture atmosphere with hypoxic or anoxic gas mixture. Cellular injury mostly occurs as a result of energetic failure in this model: the lack of oxygen blocks the mitochondrial respiration and anaerobic metabolism becomes the major source of high-energy molecules in the cells. In the absence of glucose, glycolysis and pentose phosphate pathway fail to suffice the cellular energy prerequisite and longer periods of oxygen-glucose deprivation (OGD) can completely deplete the cellular NAD<sup>+</sup> and ATP pools. The lack of NAD<sup>+</sup> results in severe metabolic suppression and predisposes the cells to other injury types. This includes oxidant-induced damage, since oxidative stress activates poly(ADP-ribose) polymerase (PARP) that further depletes the cellular NAD<sup>+</sup> pool and leads to excessive cell death. The impaired mitochondrial respiration also leads to an increase in the mitochondrial membrane potential and augments the mitochondrial superoxide generation leading to oxidative stress. The above processes ultimately lead to necrotic cell death, but in certain cell types, mitochondrial damage can also trigger apoptosis.

Keywords: hypoxia-reoxygenation injury, poly(ADP-ribose) polymerase, energetic failure, mitochondrial dysfunction, oxidative stress

## 1. Introduction

This chapter gives an overview of the hypoxia-reoxygenation model, provides guidance to perform hypoxia-reoxygenation or oxygen-glucose deprivation (OGD) experiments and discusses the mechanism of cellular damage in this model.

In vivo ischemia-reperfusion models are technically simple and reproduce many aspects of ischemic diseases, but in vitro models are equally important, because they allow detailed study of the mechanism of cellular damage and make it possible to test large chemical libraries or sets of human small interfering RNAs (siRNAs) that are essential for early phase drug discovery [1–5]. Chemical hypoxia models that use mitochondrial uncoupling agents or respiration

© The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons

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

blockers reproduce the rapid onset of ischemia but there is no way to study the recovery processes that occur during reperfusion [6–9]. The significance of true hypoxia-reoxygenation models is in the capacity for recovery from the hypoxic phase, which makes these models especially useful for ischemia-related experiments. Oxygen-glucose deprivation is a variation of the hypoxia model to mimic the shortage of nutrients in ischemia.

## 2. Hypoxia-reoxygenation induction

The hypoxia/OGD models are simple experimental models that do not require expensive laboratory instruments. Regular cell culture plasticware can be placed in a gas-tight chamber and the culture atmosphere replaced with oxygen-free gas mixture using an inexpensive flow meter. In addition, OGD can be induced by replacement of the culture medium with glucosefree medium. The reoxygenation period is initiated by glucose supplementation and by returning the culture vessels to regular atmosphere. The severity of the injury can be adjusted to specific needs by varying the length of the hypoxic/OGD period. Therapeutic interventions may be delivered prior to hypoxia induction or immediately following the reoxygenation modelling preventive or reperfusion therapies.

In most hypoxia experiments, above the hypoxic and OGD groups it is essential to use normoxia controls with normal glucose concentration or to expose normoxic controls to glucose deprivation (GD). Since the normoxic and hypoxic cells must be physically separated during the hypoxic period, identical cell plates must be prepared for the hypoxia and simultaneous normoxia exposures. Culture medium is replaced with fresh medium either containing glucose or without glucose prior to the induction of hypoxia. Serum deprivation may be necessary for complete removal of glucose in OGD injury. To induce hypoxia, the culture plates are placed in gas-tight incubation chambers (Billups-Rothenberg Inc., Del Mar, CA) and the chamber is flushed with oxygen-free gas mixture at 25–30 L/min flow rate for 5–10 min to completely remove oxygen [1–5, 7, 10]. Hypoxia is maintained by clamping and incubating the chambers at 37°C for the requested period. The composition of the gas mixture may vary depending on the bicarbonate content of the culture medium and the required level of acidity change (pH level), since hypercapnia can mimic the rapid development of acidic pH of ischemic tissues [11]. The CO2 content is typically between 5 and 20% with 80–95% N2. This procedure removes oxygen from the atmosphere but dissolved oxygen remains in all fluids in the chamber including the culture medium and additionally in water used for humidification, thus complete anoxia is reached with a delay, following depletion of the remaining oxygen. Following the hypoxic exposure, restoration of the normal culture conditions is achieved by supplementing the culture medium with glucose and foetal bovine serum (FBS) and by reoxygenating the culture vessels in regular culture atmosphere. In most cells, the cellular ATP level is recovered during a recovery period of 16–24 hours that might be the period of interest in most experiments.

Drug treatments may be administered before the hypoxia induction to test preventive effects or following the hypoxic period to test the therapeutic potential in ischemic diseases [1–3]. For gene silencing small interfering RNAs may be added 48 hours prior to the hypoxia exposure to effectively reduce RNA and protein levels of the gene of interest at the time of the hypoxia experiment [4, 5]. Unfortunately, gene silencing cannot be selectively used to study the hypoxic or the reoxygenation phase. Pharmacological treatments using small compounds allow specific post-hypoxic treatments that permit the specific study of the recovery phase.

## 3. Mechanism of cellular damage in hypoxia-reoxygenation injury

#### 3.1. Cellular energy depletion

blockers reproduce the rapid onset of ischemia but there is no way to study the recovery processes that occur during reperfusion [6–9]. The significance of true hypoxia-reoxygenation models is in the capacity for recovery from the hypoxic phase, which makes these models especially useful for ischemia-related experiments. Oxygen-glucose deprivation is a variation

The hypoxia/OGD models are simple experimental models that do not require expensive laboratory instruments. Regular cell culture plasticware can be placed in a gas-tight chamber and the culture atmosphere replaced with oxygen-free gas mixture using an inexpensive flow meter. In addition, OGD can be induced by replacement of the culture medium with glucosefree medium. The reoxygenation period is initiated by glucose supplementation and by returning the culture vessels to regular atmosphere. The severity of the injury can be adjusted to specific needs by varying the length of the hypoxic/OGD period. Therapeutic interventions may be delivered prior to hypoxia induction or immediately following the reoxygenation

In most hypoxia experiments, above the hypoxic and OGD groups it is essential to use normoxia controls with normal glucose concentration or to expose normoxic controls to glucose deprivation (GD). Since the normoxic and hypoxic cells must be physically separated during the hypoxic period, identical cell plates must be prepared for the hypoxia and simultaneous normoxia exposures. Culture medium is replaced with fresh medium either containing glucose or without glucose prior to the induction of hypoxia. Serum deprivation may be necessary for complete removal of glucose in OGD injury. To induce hypoxia, the culture plates are placed in gas-tight incubation chambers (Billups-Rothenberg Inc., Del Mar, CA) and the chamber is flushed with oxygen-free gas mixture at 25–30 L/min flow rate for 5–10 min to completely remove oxygen [1–5, 7, 10]. Hypoxia is maintained by clamping and incubating the chambers at 37°C for the requested period. The composition of the gas mixture may vary depending on the bicarbonate content of the culture medium and the required level of acidity change (pH level), since hypercapnia can mimic the rapid development of acidic pH of ischemic tissues [11]. The CO2 content is typically between 5 and 20% with 80–95% N2. This procedure removes oxygen from the atmosphere but dissolved oxygen remains in all fluids in the chamber including the culture medium and additionally in water used for humidification, thus complete anoxia is reached with a delay, following depletion of the remaining oxygen. Following the hypoxic exposure, restoration of the normal culture conditions is achieved by supplementing the culture medium with glucose and foetal bovine serum (FBS) and by reoxygenating the culture vessels in regular culture atmosphere. In most cells, the cellular ATP level is recovered during a recovery period of 16–24 hours that might be the period of

Drug treatments may be administered before the hypoxia induction to test preventive effects or following the hypoxic period to test the therapeutic potential in ischemic diseases [1–3]. For gene silencing small interfering RNAs may be added 48 hours prior to the hypoxia exposure to

of the hypoxia model to mimic the shortage of nutrients in ischemia.

2. Hypoxia-reoxygenation induction

48 Hypoxia and Human Diseases

modelling preventive or reperfusion therapies.

interest in most experiments.

Hypoxia and glucose deprivation cause energy depletion in the cells and may be directly responsible for the viability reduction caused by the injury. Since the lack of oxygen blocks aerobic metabolism, which is responsible for the larger part of ATP production in the cells, the cells need to use other pathways to produce sufficient ATP for survival. Most cells can adapt to low oxygen conditions in cell culture, producing ATP solely by anaerobic metabolism if adequate glucose supply is present. However, the anaerobic pathways, glycolysis and pentose phosphate pathway need to use high amounts of glucose to produce comparable output. Glycolysis produces only two ATP molecules, but oxidative phosphorylation is capable to produce ~30 ATPs per glucose molecule oxidized [12]. The typical mitochondrial ATP production is lower than the theoretical maximum, since up to 20% of the basal metabolic rate may be used to drive the proton leak [13], but it is still more than 10 times higher than the anaerobic ATP production. The compensatory increase in anaerobic metabolism would be stopped by the limited availability of NAD<sup>+</sup> , since protons are transferred to NAD<sup>+</sup> by glyceraldehyde phosphate dehydrogenase to produce NADH during glycolysis, if lactate dehydrogenase (LDH) did not recycle NAD<sup>+</sup> . This step helps maintain the higher anaerobic metabolic rate, but at the expense of metabolic acidosis (lactic acidosis).

However, in the absence of glucose, the ATP production will drop rapidly as the cellular energy storage is depleted and cell death will be induced. Most cells can survive in culture if the cellular ATP concentration will be reduced by less than 75–80% the normal ATP level [1–3, 5]. Following an OGD injury that does not reduce the cellular ATP concentration below 20% of the initial baseline value full recovery is expectable if optimal culture conditions are provided. Since the cells try to maintain normal ATP level and use all resources that can be utilized for energy production during the OGD phase, the recovery process is time-consuming: all precursor molecules need to be resynthesized in the cells. A more robust injury that decreases the cellular ATP concentration below 20% will initiate severe viability loss in the cell population [2] (Figure 1).

The cellular energy production remains impaired following an OGD injury: the cellular ATP production is slow even if the energy sources are provided in liberal amounts. The loss of all high-energy molecules is responsible for the diminished ATP synthesis following OGD. Not only ATP, but also adenosine diphosphate (ADP) and NAD<sup>+</sup> are greatly reduced in the cells to minimize the ATP loss that will sustain the metabolic suppression [5]. ATP is the central coenzyme in the cells that functions as universal energy currency to transfer chemical energy. ATP molecules are generated in large quantities by constant recycling of ADP to ATP; the daily

estimated ATP synthesis is around 1000 g/kg bodyweight [14]. Organic compounds are catabolized via a series of redox reactions in the cells and ultimately generate carbon dioxide and water. During these reactions, energy is collected via transferring electrons from organic donors to the acceptor molecule NAD<sup>+</sup> and reducing it to NADH. Energy is retrieved from NADH in the mitochondria as the electrons are gradually transferred to oxygen through the electron transport chain and ATP is produced in the coupled oxidative phosphorylation reaction. Thus, the energy stored by NAD<sup>+</sup> molecules is interconvertible to ATP molecules and the lack of NAD<sup>+</sup> can severely limit the ATP generation.

Figure 1. Post-hypoxic recovery of the cellular ATP content. (A and B) LLC-PK1 cells were subjected to hypoxia in the absence (OGD) or presence of 300 µM adenosine (ADE), inosine (INO) or glucose (GLC) to reduce the cellular ATP content to 5, 10 or 20% of normoxic controls, and ATP concentration was measured during the 24-hour-long recovery period. (A) ATP content gradually increased proportional to the hypoxic ATP depletion. (B) ATP resynthesis requires both adenosine deaminase (ADA) and adenosine kinase (AK) activity in the cells. Blockage of ADA by EHNA (10 µM) and/or AK by ABT 702 (ABT, 30µM) blocks the recovery of the cellular ATP content. (Data are shown as mean ± SD values. \*p < 0.05 compared to OGD, #p < 0.05 compared to adenosine, †p < 0.05 compared to inosine, &p < 0.05 compared to glucose.) From Ref. [2].

NAD+ biosynthesis occurs either via the de novo (kynurenine) pathway from tryptophan or via the salvage pathway using nicotinamide as substrate [15–17]. NAD+ is not only used in cellular energy production reactions catalyzed by dehydrogenases, but it is also utilized by poly(ADPribose) polymerases (PARPs) in ADP-ribosylation reactions and by sirtuins in deacetylation reactions that produce nicotinamide [18, 19]. Nicotinamide can be reused for NAD+ synthesis via the salvage pathway: an energy-requiring (endothermic) two-step process that uses ATP. The salvage pathway is considered as the main NAD+ biosynthesis pathway in humans and the major substrate is nicotinamide, since nicotinamide deamidase, the enzyme that catalyzes the conversion of nicotinamide to nicotinic acid, is missing in humans [20]. In the first step, nicotinamide is converted to nicotinamide mononucleotide (NMN) by nicotinamide phosphoribosyltransferase (NamPRT) using phosphoribosyl pyrophosphate (PRPP) as cosubstrate and one ATP molecule (Figure 2). The second step is the conversion of NMN to NAD+ by nicotinamide mononucleotide adenylyl transferases (NMNATs) that also requires one ATP molecule. This step is catalyzed by NMNAT-1 in the nucleus, NMNAT-2 in the Golgi and NMNAT-3 in the mitochondria [21, 22]. Since the conversion of ribose 5-pyrophosphate (coming from the degradation of ADP-ribose polymers) to PRPP requires a third ATP molecule, altogether three ATP molecules are necessary for the resynthesis of one NAD+ molecule [15, 16, 20]. NamPRT is recognized as the rate-limiting enzyme in NAD+ salvage, partly because this step requires more energy, if PRPP synthesis is also considered, and because it relies on a single enzyme, while the cells contain multiple NMNAT isoenzymes. NAD+ biosynthesis is an energy-requiring process, and it is further complicated by sequestered localization of NAD+ in the cells: there are separate mitochondrial, cytoplasmic and nuclear NAD+ pools and they are not completely exchangeable [16]. NAD+ biosynthesis is estimated to occur at 5g/kg tissue/day [16] suggesting that nicotinamide may be recycled several times a day. Still, the recovery occurs at a slower rate following a severe OGD injury, because the lack of ATP limits the NAD+ turnover and the low NAD+ availability blocks the ATP generation from metabolic sources.

estimated ATP synthesis is around 1000 g/kg bodyweight [14]. Organic compounds are catabolized via a series of redox reactions in the cells and ultimately generate carbon dioxide and water. During these reactions, energy is collected via transferring electrons from organic donors to the acceptor molecule NAD<sup>+</sup> and reducing it to NADH. Energy is retrieved from NADH in the mitochondria as the electrons are gradually transferred to oxygen through the electron transport chain and ATP is produced in the coupled oxidative phosphorylation reaction. Thus, the energy stored by NAD<sup>+</sup> molecules is interconvertible to ATP molecules and the

NAD+ biosynthesis occurs either via the de novo (kynurenine) pathway from tryptophan or via the salvage pathway using nicotinamide as substrate [15–17]. NAD+ is not only used in cellular energy production reactions catalyzed by dehydrogenases, but it is also utilized by poly(ADPribose) polymerases (PARPs) in ADP-ribosylation reactions and by sirtuins in deacetylation reactions that produce nicotinamide [18, 19]. Nicotinamide can be reused for NAD+ synthesis via the salvage pathway: an energy-requiring (endothermic) two-step process that uses ATP. The salvage pathway is considered as the main NAD+ biosynthesis pathway in humans and the major substrate is nicotinamide, since nicotinamide deamidase, the enzyme that catalyzes the conversion of nicotinamide to nicotinic acid, is missing in humans [20]. In the first step, nicotinamide is converted to nicotinamide mononucleotide (NMN) by nicotinamide phosphoribosyltransferase (NamPRT) using phosphoribosyl pyrophosphate (PRPP) as cosubstrate and one ATP molecule (Figure 2). The second step is the conversion of NMN to NAD+ by nicotinamide mononucleotide adenylyl transferases (NMNATs) that also requires one ATP molecule. This step is catalyzed by NMNAT-1 in the nucleus, NMNAT-2 in the Golgi and NMNAT-3 in the mitochondria [21, 22]. Since the conversion of ribose 5-pyrophosphate (coming from the degradation of ADP-ribose polymers) to PRPP requires a third ATP molecule, altogether three ATP molecules are necessary

Figure 1. Post-hypoxic recovery of the cellular ATP content. (A and B) LLC-PK1 cells were subjected to hypoxia in the absence (OGD) or presence of 300 µM adenosine (ADE), inosine (INO) or glucose (GLC) to reduce the cellular ATP content to 5, 10 or 20% of normoxic controls, and ATP concentration was measured during the 24-hour-long recovery period. (A) ATP content gradually increased proportional to the hypoxic ATP depletion. (B) ATP resynthesis requires both adenosine deaminase (ADA) and adenosine kinase (AK) activity in the cells. Blockage of ADA by EHNA (10 µM) and/or AK by ABT 702 (ABT, 30µM) blocks the recovery of the cellular ATP content. (Data are shown as mean ± SD values. \*p < 0.05 compared to OGD, #p < 0.05 compared to adenosine, †p < 0.05 compared to inosine, &p < 0.05 compared to glucose.)

lack of NAD<sup>+</sup> can severely limit the ATP generation.

From Ref. [2].

50 Hypoxia and Human Diseases

Figure 2. Compartmentalization of NAD<sup>+</sup> biosynthesis. The 'de novo' synthesis of NAD<sup>+</sup> starts from tryptophan and produces the precursor quinolinic acid (QA), while the 'salvage' pathway utilizes the NAD<sup>+</sup> break-down products nicotinamide (Nam) and nicotinic acid (NA). QA, NA and Nam are converted to nicotinic acid mononucleotide (NAMN) and nicotinamide mononucleotide (NMN) by the respective phosphoribosyltransferases (QAPRT: quinolinic acid phosphoribosyltransferase, NAPRT: nicotinic acid phosphoribosyltransferase, NamPRT: nicotinamide phosphoribosyltransferase). NAMN and NMN are converted to nicotinic acid adenine dinucleotide (NAAD) and nicotinamide adenine dinucleotide (NAD<sup>+</sup> ) by transferring the adenylate moiety of ATP to the mononucleotides by compartment-specific NMN adenylyl transferase (NMNAT) enzymes. NAAD is amidated by NAD synthetase (NADS) using glutamine as an ammonium donor. There are three NMNAT isoforms: NMNAT1 is ubiquitous and is localized to the nucleus, NMNAT2 is cytoplasmic and is predominantly expressed in the brain and NMNAT3 is present in the mitochondria. PARP-1 utilizes NAD<sup>+</sup> as a substrate to produce ADP-ribose polymers and nicotinamide.

The lack of NAD<sup>+</sup> affects both mitochondrial respiration and anaerobic metabolism following the OGD injury [5]. Severe metabolic suppression is detectable following the OGD injury if the resynthesis of NAD<sup>+</sup> is prevented by NamPRT inhibition: the mitochondrial oxygen consumption of the cells is severely reduced in the cells (Figure 3). The respiratory capacity of the cells is suppressed following OGD and while normal cells typically use no more than ~50–60% of their

Figure 3. Suppressed cellular metabolism following oxygen-glucose deprivation (OGD). (A–F) H9c2 cells were transfected with PARP-1 (siPARP-1) or CTL siRNA and 48 hours later the cells were exposed to hypoxia or oxygenglucose deprivation for 8 hours. Following the hypoxic phase, glucose and serum concentrations were normalized and the cells were treated with NamPRT inhibitor FK866 (10 μM) to block NAD<sup>+</sup> resynthesis (or vehicle) at normal oxygen tension for 16 hours. The metabolic profile of the cells was determined by extracellular flux analysis. (A and D) The oxygen consumption rate (OCR) and (C and F) the extracellular acidification rate (ECAR) were monitored using Oligomycin (1 μg/mL), FCCP (0.3 μM) and antimycin A (2 μg/mL) injections. (B and E) Basal oxygen consumption and total respiratory capacity were determined following the addition of FCCP. NamPRT inhibition severely blocks the recovery of the respiratory capacity and prevents the anaerobic metabolic compensation. PARP-1 silencing increases the respiratory capacity in cells with diminished NAD<sup>+</sup> content. (n = 3, \*p < 0.05 compared to CTL, #p < 0.05 PARP-1 silenced cells compared to respective CTL siRNA treated cells.) From Ref. [5].

respiratory capacity under baseline conditions, the cells use their full respiratory capacity following hypoxia of OGD injury. While the basal anaerobic metabolism is less affected by the lack of NAD<sup>+</sup> the anaerobic compensation is reduced by 70%, which makes the cells extremely sensitive to other injuries that require excess energy. At this stage, NAD<sup>+</sup> is functionally shared between the mitochondrial and cytoplasmic pools, as the blockage of mitochondrial NAD<sup>+</sup> recycling by inhibition of ATP synthase immediately draws a halt to anaerobic metabolism. This phenomenon can help explain the vulnerability of the cells: any injury that causes mitochondrial impairment can simultaneously block the anaerobic metabolism in the cells.

#### 3.2. Oxidative stress during reoxygenation

resynthesis of NAD<sup>+</sup> is prevented by NamPRT inhibition: the mitochondrial oxygen consumption of the cells is severely reduced in the cells (Figure 3). The respiratory capacity of the cells is suppressed following OGD and while normal cells typically use no more than ~50–60% of their

52 Hypoxia and Human Diseases

Figure 3. Suppressed cellular metabolism following oxygen-glucose deprivation (OGD). (A–F) H9c2 cells were transfected with PARP-1 (siPARP-1) or CTL siRNA and 48 hours later the cells were exposed to hypoxia or oxygenglucose deprivation for 8 hours. Following the hypoxic phase, glucose and serum concentrations were normalized and the cells were treated with NamPRT inhibitor FK866 (10 μM) to block NAD<sup>+</sup> resynthesis (or vehicle) at normal oxygen tension for 16 hours. The metabolic profile of the cells was determined by extracellular flux analysis. (A and D) The oxygen consumption rate (OCR) and (C and F) the extracellular acidification rate (ECAR) were monitored using Oligomycin (1 μg/mL), FCCP (0.3 μM) and antimycin A (2 μg/mL) injections. (B and E) Basal oxygen consumption and total respiratory capacity were determined following the addition of FCCP. NamPRT inhibition severely blocks the recovery of the respiratory capacity and prevents the anaerobic metabolic compensation. PARP-1 silencing increases the respiratory capacity in cells with diminished NAD<sup>+</sup> content. (n = 3, \*p < 0.05 compared to CTL, #p < 0.05 PARP-1 silenced cells

compared to respective CTL siRNA treated cells.) From Ref. [5].

Oxidative stress is an important contributor to cellular damage in hypoxia- or OGDreoxygenation injury. While it is recognized as the major cause of cellular damage in ischemia-reperfusion injury in vivo [23–25], reoxygenation does not induce severe oxidative stress in the in vitro injury. Mitochondria are the major sources of oxidants in vitro following OGD or hypoxia. The mitochondrial respiratory chain is turned off by the lack of oxygen during hypoxia or OGD, but the electrons and protons are fed to the mitochondria as long as possible. As a result, the protons pumped from the matrix to the intermembrane space may increase the transmembrane gradient [5]. Mitochondrial uncoupling proteins are responsible for maintaining the physiological mitochondrial membrane potential [26]. They allow reverse transfer of protons from the intermembrane space to the matrix without coupled ATP synthesis. This proton leak may reduce the efficiency of ATP production, but it also helps against mitochondrial hyperpolarization [27–29].

Superoxide is produced by the mitochondrial electron transport chain itself, most importantly at complex III: a low percentage of electrons from quinone molecules are transferred to oxygen instead of complex III even in healthy mitochondria [30–34]. The amount of ROS generation is relatively low, approximately 0.2–2% of the oxygen consumed by the mitochondria is reduced to superoxide [28]. However, this process would leave behind excess protons in the intermembrane space and increase the mitochondrial membrane potential, if mitochondria did not possess a safety mechanism against it. Uncoupling proteins and especially UCP2 are responsible for protecting against hyperpolarization. The elevated mitochondrial membrane potential directly increases the mitochondrial superoxide generation [35, 36]. This action is reversible: if the mitochondrial membrane potential is normalized, the superoxide generation will decrease to normal levels [27, 34, 37]. However, the action of UCP2 and UCP3 is regulated by reactive oxygen species (ROS) generation as their activity is affected by glutathionylation: increase in ROS production prompts the deglutathionylation and activation of proton conductivity via UCP2 and UCP3, while at low ROS levels the uncoupling proteins are glutathionylated that effectively deactivates the proton conductance process [28, 38]. During hypoxia or OGD, the absence of oxygen completely deactivates UCPs in the cell and it excludes the compensation for the hyperpolarization in the beginning of the reoxygenation phase. While an increase is detectable in the mitochondrial membrane potential, the amount of superoxide generation hardly exceeds the normal levels immediately following hypoxia or OGD due to the suppressed mitochondrial activity [5], but increased ROS production can be detected in the cells even after full recovery of the cellular ATP and NAD<sup>+</sup> contents [5] (Figure 4).

Figure 4. Mitochondrial oxidant production in hypoxia-reoxygenation injury. (A–F) H9c2 cardiomyocytes were exposed to hypoxia or oxygen-glucose deprivation (OGD) for 8 hours, followed by 16-hour-long recovery. Cells were simultaneously maintained at normoxia in glucose-containing culture medium as controls (CTL) or subjected to glucose deprivation (GD). (A and B) ATP and (C and D) NAD+ contents were determined both at the end of the hypoxia (A and C) and following the recovery (B and D). (E and F) The mitochondrial potential and (G and H) superoxide production were measured by JC-1 and MSOX Red (MSOX) at the end of the hypoxia (E and G) and following the recovery (F and H). (n = 4, \*p < 0.05 compared to CTL.) From Ref. [5].

Oxidative stress damages the DNA and RNA molecules causing modified bases and strand breaks and also induces oxidative protein damage. To minimize further dysfunction caused by impaired molecules, repair processes are promptly activated in the cells and PARP-1 is the key enzyme that orchestrates this process. The activation of PARP-1 is an easily detectable sign of oxidative stress in the cells and tissues [39–41].

#### 3.3. The function of PARP-1 and its role in oxidative stress-induced cell death

OGD due to the suppressed mitochondrial activity [5], but increased ROS production can be detected in the cells even after full recovery of the cellular ATP and NAD<sup>+</sup> contents [5]

Figure 4. Mitochondrial oxidant production in hypoxia-reoxygenation injury. (A–F) H9c2 cardiomyocytes were exposed to hypoxia or oxygen-glucose deprivation (OGD) for 8 hours, followed by 16-hour-long recovery. Cells were simultaneously maintained at normoxia in glucose-containing culture medium as controls (CTL) or subjected to glucose deprivation (GD). (A and B) ATP and (C and D) NAD+ contents were determined both at the end of the hypoxia (A and C) and following the recovery (B and D). (E and F) The mitochondrial potential and (G and H) superoxide production were measured by JC-1 and MSOX Red (MSOX) at the end of the hypoxia (E and G) and following the recovery (F and H). (n =

4, \*p < 0.05 compared to CTL.) From Ref. [5].

(Figure 4).

54 Hypoxia and Human Diseases

PARP-1 is the major isoform of poly(ADP-ribose) polymerases in the cells that mainly resides in the nucleus. It detects DNA strand breaks and plays a role in base excision repair by adding multiple ADP-ribose units to the DNA associated histone proteins using NAD<sup>+</sup> as a substrate. It promotes DNA repair by recruiting components of the repair machinery and also by providing sequestered energy source for the repair in the form of ADP-ribose. Poly(ADP-ribose) (PAR) induces conformation changes in the DNA due to its negative charge, which may serve as a surface for interaction in DNA repair. The removal of PAR is catalyzed by poly(ADPribose) glycohydrolase (PARG), an enzyme that is mainly localized to the cytoplasm and needs to translocate to the nucleus to counteract PARP.

While the far-reaching activity of PARP-1 in DNA repair suggests that it is essential for DNA integrity and cell survival, PARP-1 knockout mice are viable and do not exhibit high susceptibility for spontaneous tumour development [42]. There is no human 'PARP-1 deficiency syndrome'. Single nucleotide polymorphisms of the PARP gene have been identified, but only few studies found association with functional changes and increased risk of cancer, nephritis or arthritis [43–46]. DNA repair processes possibly rely on redundant actions of many other components or PARP-1 is substituted by other PARP isoforms [47, 48]. On the other hand, the principal role of PARP-1 is indisputable in cell metabolism and oxidative stress-induced cell death.

In oxidative stress, the enzyme is capable of over-activation by creating huge branching PAR polymers within minutes, thereby depleting the available NAD<sup>+</sup> pool of the cells and causing energetic failure [40, 49, 50]. Inhibition of PARP activity prevents necrotic cell death in oxidative stress and promotes cell survival and apoptosis, a favourable cell death form. During apoptosis PARP is inactivated by caspase cleavage that dissociates the DNA binding and catalytic domains of PARP and prevents PARP activation by DNA strand breaks. Apart from caspases, various proteases (cathepsin, calpain, granzyme B) may inactivate PARP by proteolytic cleavage following OGD or hypoxia injury [2]. PARP also catalyzes its self-PARylation and this auto-modification reduces the catalytic activity of the enzyme, thus, it also serves as a control of its activity. It was suggested that other post-translational modifications of the enzyme (phosphorylation, acetylation) are also implicated in the regulation of PARP activity [49, 51].

PARP also regulates gene transcription via interacting with other transcription factors or by directly binding to promoter regions to control cellular metabolism [52, 53]. Among others, the PAR-degrading enzyme PARG, the nuclear NAD<sup>+</sup> synthesis enzyme NMNAT-1 and Nuclear Respiratory Factor 1, which activates the expression of metabolic genes regulating cellular growth and mitochondrial respiration, were identified as PARP interactors [53–56]. The interplay between PARP-1 and the NAD<sup>+</sup> biosynthesis enzyme NMNAT-1 is particularly interesting because it suggests that under baseline conditions the nuclear NAD<sup>+</sup> utilization and recycling are fully coupled processes [54, 57].

PARP activation may cause mitochondrial dysfunction in cells exposed to oxidative stress that is best characterized by reduced mitochondrial reserve capacity [58]. The cellular NAD<sup>+</sup> pool is compartmentalized within the cells and since the NAD<sup>+</sup> pools are non-exchangeable between the nucleus and the mitochondria [22], the PARP-mediated nuclear NAD<sup>+</sup> depletion may develop mitochondrial failure via prior depletion of the cytoplasmic NAD<sup>+</sup> pool and inhibition of glycolysis. There seems to be a competition for substrate between PARP-1 and other NAD<sup>+</sup> consuming enzymes including the sirtuins [40, 59]. The sirtuin family members use NAD<sup>+</sup> for their deacetylation function and are mainly implicated in the regulation of glucose and lipid metabolism [59, 60]. The various sirtuins show distinct intracellular localization profile, Sirt1, Sirt6 and Sirt7 are predominantly nuclear proteins [59]. While PARP and sirtuins share their common substrate, the NAD<sup>+</sup> consumption by sirtuins is hardly comparable to that of PARP, thus competition for substrate has little impact on PARP activity. Still Sirt1 may affect the action of PARP-1 via direct interaction of the two proteins and by modulating PARP activity via deacetylation [61]. On the other hand, the nuclear sirtuins are possibly affected by PARP1 mediated NAD<sup>+</sup> consumption under oxidative stress, since the lack of PARP-1 increases Sirt-1 activity and stimulates the mitochondrial metabolism [62]. Thus, it suggests that sirtuins and especially Sirt-1 may play a role in PARP-mediated mitochondrial suppression, as PARPmediated NAD+ consumption decreases Sirt-1 activity and mitochondrial metabolism.

PARP-1 activation is generally associated with necrotic cell death, but PARP-1 may be involved in other cell death forms. The obligatory trigger of PARP activation is DNA single strand break, which can be induced by a variety of oxidants. In pathophysiological conditions, reactive species capable of inducing DNA strand breakage, and thereby PARP activation, include hydroxyl radical, nitroxyl radical, as well as peroxynitrite (a reactive oxidant produced from the reaction of nitric oxide and superoxide) [63–65]. In response to DNA damage, PARP becomes activated and, using NAD<sup>+</sup> as a substrate, catalyzes the building of homopolymers of adenosine diphosphate ribose units. Depending of the severity of DNA damage this process can be overwhelming and it may deplete the cellular NAD<sup>+</sup> and ATP pools and can eventually lead to cell death via the necrotic route [39]. Hypoxia- or OGD-reoxygenation injury predisposes the cells to PARP-1 mediated NAD<sup>+</sup> depletion: lower level of oxidative stress and PARP-1 activity can exhaust the cellular NAD<sup>+</sup> pool and lead to necrosis (Figure 5).

The activation of PARP-1 is a regulated process and the enzyme also plays an important role in programmed cell death forms [66, 67]. PARP-1 activity level depends on the severity of oxidative stress, and its high catalytic activity is necessary to promote immediate DNA repair. This protective mechanism helps maintain genome integrity: the ADP-ribose units provide energy source for base excision repair and the negatively charged polymer recruits other repair proteins to the site of the damage [68]. Low level of PARP activity is always detectable, and it is associated with normal gene expression and physiological maintenance of DNA integrity. Severe DNA damage that occurs under pathological conditions induces excessive activation of the enzyme that can rapidly deplete the cellular NAD<sup>+</sup> content. Less severe oxidative damage can induce moderate PARP activation to restore the DNA integrity and if the repair process is unsuccessful, apoptosis may be induced [39, 40, 66]. The apoptotic process follows the intrinsic or mitochondrial pathway in this case [69], and it requires a nuclear-to-mitochondrial signal for initiation. The signalling molecules have not been unequivocally identified, but PARP-1 and the PAR polymer might be directly involved in this process [70]. PARP-1 can generate large PAR polymers that may escape from the nucleus. The PAR polymer itself can induce membrane damage, mitochondrial depolarization and apoptosis-inducing factor (AIF) release [70]. AIF released from the mitochondria translocates to the nucleus and plays a role in cell death progression [71]. This PAR-mediated cell death program is occasionally discriminated from necrosis and apoptosis as parthanatos, a distinct cell death form [70]. Triggering of the mitochondrial apoptotic signal leads to caspase activation, which becomes detectable 1 hour following the start of reoxygenation and remains elevated for several hours in hypoxiareoxygenation injury [2]. During apoptosis caspase cleavage inactivates PARP-1 by removing the catalytic region of the protein from the DNA binding region to avoid unnecessary NAD<sup>+</sup> consumption caused by the fragmented DNA [72].

growth and mitochondrial respiration, were identified as PARP interactors [53–56]. The interplay between PARP-1 and the NAD<sup>+</sup> biosynthesis enzyme NMNAT-1 is particularly interesting because it suggests that under baseline conditions the nuclear NAD<sup>+</sup> utilization and

PARP activation may cause mitochondrial dysfunction in cells exposed to oxidative stress that is best characterized by reduced mitochondrial reserve capacity [58]. The cellular NAD<sup>+</sup> pool is compartmentalized within the cells and since the NAD<sup>+</sup> pools are non-exchangeable between the nucleus and the mitochondria [22], the PARP-mediated nuclear NAD<sup>+</sup> depletion may develop mitochondrial failure via prior depletion of the cytoplasmic NAD<sup>+</sup> pool and inhibition of glycolysis. There seems to be a competition for substrate between PARP-1 and other NAD<sup>+</sup> consuming enzymes including the sirtuins [40, 59]. The sirtuin family members use NAD<sup>+</sup> for their deacetylation function and are mainly implicated in the regulation of glucose and lipid metabolism [59, 60]. The various sirtuins show distinct intracellular localization profile, Sirt1, Sirt6 and Sirt7 are predominantly nuclear proteins [59]. While PARP and sirtuins share their common substrate, the NAD<sup>+</sup> consumption by sirtuins is hardly comparable to that of PARP, thus competition for substrate has little impact on PARP activity. Still Sirt1 may affect the action of PARP-1 via direct interaction of the two proteins and by modulating PARP activity via deacetylation [61]. On the other hand, the nuclear sirtuins are possibly affected by PARP1 mediated NAD<sup>+</sup> consumption under oxidative stress, since the lack of PARP-1 increases Sirt-1 activity and stimulates the mitochondrial metabolism [62]. Thus, it suggests that sirtuins and especially Sirt-1 may play a role in PARP-mediated mitochondrial suppression, as PARP-

mediated NAD+ consumption decreases Sirt-1 activity and mitochondrial metabolism.

1 activity can exhaust the cellular NAD<sup>+</sup> pool and lead to necrosis (Figure 5).

PARP-1 activation is generally associated with necrotic cell death, but PARP-1 may be involved in other cell death forms. The obligatory trigger of PARP activation is DNA single strand break, which can be induced by a variety of oxidants. In pathophysiological conditions, reactive species capable of inducing DNA strand breakage, and thereby PARP activation, include hydroxyl radical, nitroxyl radical, as well as peroxynitrite (a reactive oxidant produced from the reaction of nitric oxide and superoxide) [63–65]. In response to DNA damage, PARP becomes activated and, using NAD<sup>+</sup> as a substrate, catalyzes the building of homopolymers of adenosine diphosphate ribose units. Depending of the severity of DNA damage this process can be overwhelming and it may deplete the cellular NAD<sup>+</sup> and ATP pools and can eventually lead to cell death via the necrotic route [39]. Hypoxia- or OGD-reoxygenation injury predisposes the cells to PARP-1 mediated NAD<sup>+</sup> depletion: lower level of oxidative stress and PARP-

The activation of PARP-1 is a regulated process and the enzyme also plays an important role in programmed cell death forms [66, 67]. PARP-1 activity level depends on the severity of oxidative stress, and its high catalytic activity is necessary to promote immediate DNA repair. This protective mechanism helps maintain genome integrity: the ADP-ribose units provide energy source for base excision repair and the negatively charged polymer recruits other repair proteins to the site of the damage [68]. Low level of PARP activity is always detectable, and it is associated with normal gene expression and physiological maintenance of DNA integrity. Severe DNA damage that occurs under pathological conditions induces excessive activation

recycling are fully coupled processes [54, 57].

56 Hypoxia and Human Diseases

Figure 5. The mechanism of energetic failure in hypoxia-reoxygenation injury. The events of hypoxia/OGDreoxygenation injury leading to ATP depletion with the contribution of PARP labelled in red.

PARP-1 itself can exit the nucleus in oxidative stress and interact with cytoplasmic or mitochondrial proteins [4]. Thereby, PARP-1 can have direct access to the cytoplasmic or mitochondrial NAD<sup>+</sup> pools and can PARylate cytoplasmic and mitochondrial proteins [73]. In this process, the PAR-binding E3 ubiquitin ligase RNF146 (ring finger protein 146, dactylidin also named Iduna) is involved [74–76], which can capture the PARP-1 protein and promote its ubiquitination and proteasomal degradation [4]. RNF146 was discovered as a neuroprotective gene product that when over-expressed exerted protection against NMDA excitotoxicity and MNNG-induced PARP-1 dependent cell death in vitro [77] and protects against oxidative stress-mediated neural injury in transgenic mice [78]. RNF146 is a 359 amino acid long, cytoplasmic protein that contains conserved Really Interesting New Gene (RING) and WWE domains. The special zinc finger domain (RING domain) between amino acids 38–75 is responsible for the E3 ubiquitin-protein ligase activity [79]. The WWE domain at 92–168 mediates specific interaction with ADP-ribosylated proteins (PAR-recognition sequence) and the carboxy-terminal half of the protein, which shows similarity to nucleoporin 155, a component of the nuclear pore complex, possibly plays a role in bidirectional trafficking of molecules between the nucleus and the cytoplasm [78, 79]. RNF146 can bind to the PAR polymer, thus it can recognize the auto-PARylated PARP-1 and other PARylated proteins, but their distinct subcellular localization (PARP-1 is present in the nucleus and RNF146 in the cytoplasm) prevents their direct association under physiological conditions. However, when the nuclear membrane integrity is disrupted, RNF146 can translocate to the nucleus, directly interact with PARP-1 and both proteins are rapidly degraded by the proteasome [4]. This interaction affects PARP-1 removal during cell division: PARP-1 is sequestered and degraded during the mitotic phase, and also results in rapid PARP-1 removal in oxidative stress. In the latter case, not only PARP-1 but also its targets, the PARylated proteins are affected, including the NAD<sup>+</sup> biosynthesis enzymes NamPRT and nicotinamide N-methyltransferase and various metabolic enzymes, e.g. lactate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), pyruvate dehydrogenase and succinate dehydrogenase [80] that can slow down the recovery following OGD or hypoxic injury. A small fraction of PARP-1 is localized to the mitochondria and plays a role in mitochondrial DNA repair, but it may also be involved in degradation of interacting mitochondrial proteins [73, 81, 82]. Inhibition of PARP-1 activity renders protection against reoxygenation-induced oxidative damage and cell death in OGD injury but this effect is limited by the low concentration of cellular ATP [2, 5] and may not be comparable to the beneficial effects of PARP inhibitors observed in ischemia-reperfusion injury in vivo [83, 84].

#### 3.4. Increased sensitivity to oxidative damage

Post-hypoxic cells show increased sensitivity to oxidant-induced cellular injury due to (1) diminished ATP and NAD<sup>+</sup> pools, (2) low mitochondrial metabolic output and (3) reduced antioxidant capacity. Hypoxia and glucose deprivation decrease the intracellular concentrations of ATP and NAD<sup>+</sup> that greatly reduce the tolerance to cytotoxic injuries since they are associated with enhanced energy consumption. Oxidant-induced cellular damage is further aggravated by the diminished NAD<sup>+</sup> and ATP synthesis due to mitochondrial dysfunction and restricted glycolytic capacity. The exposure to low oxygen atmosphere induces down-regulation of antioxidant genes that reduces the buffering capacity during the reoxygenation phase [85, 86]. Changes in oxygen supply are detected via reduced levels of oxidants and hypoxiainducible factor-α (HIF-1α) is responsible for transcriptional regulation of the antioxidant enzymes [87, 88]. The diminished scavenging capability and the higher oxidant generation during the recovery period greatly reduce the tolerance to oxidants. Overall, these factors increase the vulnerability of the cells and oxidants can induce devastating damage during the reoxygenation period.

named Iduna) is involved [74–76], which can capture the PARP-1 protein and promote its ubiquitination and proteasomal degradation [4]. RNF146 was discovered as a neuroprotective gene product that when over-expressed exerted protection against NMDA excitotoxicity and MNNG-induced PARP-1 dependent cell death in vitro [77] and protects against oxidative stress-mediated neural injury in transgenic mice [78]. RNF146 is a 359 amino acid long, cytoplasmic protein that contains conserved Really Interesting New Gene (RING) and WWE domains. The special zinc finger domain (RING domain) between amino acids 38–75 is responsible for the E3 ubiquitin-protein ligase activity [79]. The WWE domain at 92–168 mediates specific interaction with ADP-ribosylated proteins (PAR-recognition sequence) and the carboxy-terminal half of the protein, which shows similarity to nucleoporin 155, a component of the nuclear pore complex, possibly plays a role in bidirectional trafficking of molecules between the nucleus and the cytoplasm [78, 79]. RNF146 can bind to the PAR polymer, thus it can recognize the auto-PARylated PARP-1 and other PARylated proteins, but their distinct subcellular localization (PARP-1 is present in the nucleus and RNF146 in the cytoplasm) prevents their direct association under physiological conditions. However, when the nuclear membrane integrity is disrupted, RNF146 can translocate to the nucleus, directly interact with PARP-1 and both proteins are rapidly degraded by the proteasome [4]. This interaction affects PARP-1 removal during cell division: PARP-1 is sequestered and degraded during the mitotic phase, and also results in rapid PARP-1 removal in oxidative stress. In the latter case, not only PARP-1 but also its targets, the PARylated proteins are affected, including the NAD<sup>+</sup> biosynthesis enzymes NamPRT and nicotinamide N-methyltransferase and various metabolic enzymes, e.g. lactate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), pyruvate dehydrogenase and succinate dehydrogenase [80] that can slow down the recovery following OGD or hypoxic injury. A small fraction of PARP-1 is localized to the mitochondria and plays a role in mitochondrial DNA repair, but it may also be involved in degradation of interacting mitochondrial proteins [73, 81, 82]. Inhibition of PARP-1 activity renders protection against reoxygenation-induced oxidative damage and cell death in OGD injury but this effect is limited by the low concentration of cellular ATP [2, 5] and may not be comparable to the beneficial effects of PARP inhibitors observed in ischemia-reperfusion injury in vivo [83, 84].

Post-hypoxic cells show increased sensitivity to oxidant-induced cellular injury due to (1) diminished ATP and NAD<sup>+</sup> pools, (2) low mitochondrial metabolic output and (3) reduced antioxidant capacity. Hypoxia and glucose deprivation decrease the intracellular concentrations of ATP and NAD<sup>+</sup> that greatly reduce the tolerance to cytotoxic injuries since they are associated with enhanced energy consumption. Oxidant-induced cellular damage is further aggravated by the diminished NAD<sup>+</sup> and ATP synthesis due to mitochondrial dysfunction and restricted glycolytic capacity. The exposure to low oxygen atmosphere induces down-regulation of antioxidant genes that reduces the buffering capacity during the reoxygenation phase [85, 86]. Changes in oxygen supply are detected via reduced levels of oxidants and hypoxiainducible factor-α (HIF-1α) is responsible for transcriptional regulation of the antioxidant enzymes [87, 88]. The diminished scavenging capability and the higher oxidant generation

3.4. Increased sensitivity to oxidative damage

58 Hypoxia and Human Diseases

The cells may be treated with exogenous oxidants following the in vitro hypoxic or OGD injury to better mimic tissue reperfusion, since (1) the infiltration and ROS production of circulating leukocytes is missing and (2) the ratio of culture volume/packed cell volume is a couple of orders of magnitude higher than the ratio of extracellular/intracellular space, thus oxidants produced by the cells are instantaneously diluted in in vitro hypoxia-reoxygenation injury. Oxidants induce more severe cell damage in post-OGD cells than in normal cells, since the cellular NAD<sup>+</sup> content is much lower following OGD exposure (Figure 6). The cellular NAD<sup>+</sup>

Figure 6. Hypoxia and OGD increases the sensitivity to exogenous oxidants. H9c2 cells were subjected to 8-hour-long hypoxia/OGD or GD, and then following the normalization of glucose concentration and oxygen tension the cells were exposed to various concentrations of H2O2 for 3 hours. (A and B) The viability of the cells was evaluated by the MTT assay. (C and D) LDH activity was measure in the supernatant. Non-linear curve-fitting was applied to the raw data (A and C) and the concentration of H2O2 that caused 50% reduction in the viability (B) or 50% increase in the LDH release (D) are shown. GD and OGD resulted in narrower range of H2O2 tolerance (steeper curves). From Ref. [5].

pools may be completely depleted by a moderate oxidative damage that hardly induces viability reduction in normal cells [5]. Cells undergoing OGD injury are less tolerant to oxidants and can survive oxidant-exposure in a narrow concentration range. PARP inhibition reduces the NAD<sup>+</sup> consumption and has protective effect against oxidant-induced cell damage in post-hypoxic or post-OGD cells that is in line with in vivo ischemia-reperfusion data [23, 67, 89–91].

## 4. Interventions to increase the recovery following hypoxia-reoxygenation or OGD-reoxygenation injury

In drug discovery, the ultimate goal of using in vitro models, like hypoxia-reoxygenation or the OGD-reoxygenation is to find novel drugs that show efficacy against ischemic diseases in vivo. The relative contribution of various pathways leading to cellular damage in hypoxia or OGDinjury has not been definitely established, thus it is unclear which pathways can serve as drug targets in this injury. Furthermore, there are notable differences between the hypoxiareoxygenation model and ischemia-reperfusion injury that may result in discrepancy between the in vitro and in vivo drug efficacy [92, 93]. There are numerous factors that may account for this difference and their significance should be individually evaluated depending on the target disease for each organ or tissue type. While the organs consist of various cell types and the extracellular matrix, usually a single cell type is used in the in vitro model. This excludes the cells that build up the blood vessels and the circulating blood cells, and the secreted proinflammatory mediators and ROS produced by leukocytes are similarly absent. There are functional differences between tissues and cultured cells including muscle contraction, absorption of nutrients in the digestive system, kidney filtration, excretion and reabsorption and the detoxification function of the liver that all require lot of metabolic energy. Cultured cells may show slightly different expression profiles than their in vivo counterparts that may affect the expression level of drug targets and can change the observed responses. There are deficiencies of the in vitro model, which are associated with the differences in extracellular volume: the hypoxia induction is slower, the energy resources are more abundant and the dilutions of secreted cytotoxic or cytoprotective agents are greater than in vivo. On the other hand, there are no drug absorption, solubility and metabolism issues in vitro that may reduce the drug effects in vivo.

Interventions that reduce the cellular damage in hypoxia-reoxygenation injury and enhance recovery following hypoxia or OGD exposure may target (1) the metabolism and energy resources, (2) the oxidative stress pathways and antioxidant responses or (3) the proteasome and proteolytic activity. Apart from these universal cellular targets, some tissue-specific receptors were also found to have beneficial effects in some models. Energy replenishment using adenosine or inosine is effective in various cell types exposed to OGD injury since the pentose part of these nucleosides can be anaerobically metabolized through the pentose phosphate pathway [1–3]. Purine nucleosides are preferable to glucose in hypoxia since their metabolism can produce more ATP molecules than glycolysis and their utilization is more effective at low concentrations. Furthermore, they possess anti-inflammatory and weak PARP inhibitor activity that supports their activity in vivo [94]. Various ROS scavengers and antioxidants also exert cytoprotective effect in hypoxiareoxygenation models [95] and inhibition of the NAD<sup>+</sup> consumer PARP-1 that recognizes the oxidative DNA damage is also beneficial [96, 97]. Not only the necrosis-associated PARP-1 blockage is effective in OGD-reoxygenation injury, but also caspase inhibition has protective effect in select cell types, confirming that the cell death features both apoptotic and necrotic elements in this injury [2]. Proteasome inhibition that possibly prevents the degradation of key signalling proteins and metabolic enzymes is also beneficial in hypoxia-reoxygenation injury [98, 99].

## 5. Conclusion

pools may be completely depleted by a moderate oxidative damage that hardly induces viability reduction in normal cells [5]. Cells undergoing OGD injury are less tolerant to oxidants and can survive oxidant-exposure in a narrow concentration range. PARP inhibition reduces the NAD<sup>+</sup> consumption and has protective effect against oxidant-induced cell damage in post-hypoxic or post-OGD cells that is in line with in vivo ischemia-reperfusion data [23, 67,

4. Interventions to increase the recovery following hypoxia-reoxygenation

In drug discovery, the ultimate goal of using in vitro models, like hypoxia-reoxygenation or the OGD-reoxygenation is to find novel drugs that show efficacy against ischemic diseases in vivo. The relative contribution of various pathways leading to cellular damage in hypoxia or OGDinjury has not been definitely established, thus it is unclear which pathways can serve as drug targets in this injury. Furthermore, there are notable differences between the hypoxiareoxygenation model and ischemia-reperfusion injury that may result in discrepancy between the in vitro and in vivo drug efficacy [92, 93]. There are numerous factors that may account for this difference and their significance should be individually evaluated depending on the target disease for each organ or tissue type. While the organs consist of various cell types and the extracellular matrix, usually a single cell type is used in the in vitro model. This excludes the cells that build up the blood vessels and the circulating blood cells, and the secreted proinflammatory mediators and ROS produced by leukocytes are similarly absent. There are functional differences between tissues and cultured cells including muscle contraction, absorption of nutrients in the digestive system, kidney filtration, excretion and reabsorption and the detoxification function of the liver that all require lot of metabolic energy. Cultured cells may show slightly different expression profiles than their in vivo counterparts that may affect the expression level of drug targets and can change the observed responses. There are deficiencies of the in vitro model, which are associated with the differences in extracellular volume: the hypoxia induction is slower, the energy resources are more abundant and the dilutions of secreted cytotoxic or cytoprotective agents are greater than in vivo. On the other hand, there are no drug absorption, solubility and metabolism issues in vitro that may reduce the drug

Interventions that reduce the cellular damage in hypoxia-reoxygenation injury and enhance recovery following hypoxia or OGD exposure may target (1) the metabolism and energy resources, (2) the oxidative stress pathways and antioxidant responses or (3) the proteasome and proteolytic activity. Apart from these universal cellular targets, some tissue-specific receptors were also found to have beneficial effects in some models. Energy replenishment using adenosine or inosine is effective in various cell types exposed to OGD injury since the pentose part of these nucleosides can be anaerobically metabolized through the pentose phosphate pathway [1–3]. Purine nucleosides are preferable to glucose in hypoxia since their metabolism can produce more ATP molecules than glycolysis and their utilization is more effective at low concentrations. Furthermore, they possess

89–91].

60 Hypoxia and Human Diseases

effects in vivo.

or OGD-reoxygenation injury

The hypoxia-reoxygenation model is a valuable tool in hypoxia and ischemia research that may be combined with other injury models to fully reproduce features of inflammatory and vascular diseases. This low-cost model does not require advanced research skills and may be optimized within a short time in the laboratory. The cellular damage mostly occurs as a consequence of energetic failure and shows necrotic characteristics in this model. Both the hypoxic phase and the post-hypoxic recovery period involve massive changes in the cellular metabolism: a characteristic suppression of mitochondrial energy production is caused by the lack of oxygen and later by the shortage of NAD<sup>+</sup> supply. The recovery from this state is a delicate process that recreates the balance in cellular energetics.

## Acknowledgement

D.G. received funding from the People Programme (Marie Curie Actions) of the European Union's Seventh Framework Programme under the grant agreement number 628100.

## Author details

Domokos Gerő

Address all correspondence to: gerodomokos@yahoo.com

University of Exeter Medical School, Exeter, UK

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#### **Vascular Smooth Muscle as an Oxygen Sensor: Role of Elevation of the [Na+]i /[K+]i Vascular Smooth Muscle as an Oxygen Sensor: Role of Elevation of the [Na+]i /[K+]i**

Sergei N. Orlov , Yulia G. Birulina , Liudmila V. Smaglii and Svetlana V. Gusakova Sergei N. Orlov, Yulia G. Birulina, Liudmila V. Smaglii and Svetlana V. Gusakova

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

http://dx.doi.org/10.5772/65384

#### **Abstract**

The article presents a review of data from our own research and data obtained by other authors about the role of intracellular sodium (Na <sup>+</sup>) and potassium (Ki +) in transcrip‐ tomic changes in vascular smooth muscle cells (VSMC) during hypoxia. It was found that acute hypoxia suppressed [K+ ]o and phenylephrine‐induced contractions of aortic rings through voltage‐gated as well as by Cai 2+‐ and ATP‐sensitive K+ channels; 24‐h incubation of VSMC in ischemic conditions resulted in attenuation of ATP content, elevation of [Na+ ]i and loss of [K+ ]i . Dissipation of Na+ and K+ gradients in low‐Na+ , high‐ K+ medium completely eliminated increment in Fos, Atf3, Ptgs2 and Per2 mRNAs and sharply diminished augmentation of Klf10, Edn1, Nr4a1 and Hes1 expression evoked by hypoxia. All these data suggest that Nai +/Ki +‐mediated signaling contribute to transcriptomic changes in VSMC subjected to sustained hypoxia.

**Keywords:** smooth muscle cells, hypoxia, intracellular [Na+ ]/[K+ ] ratio, transcription, contraction

## **1. Introduction**

Maintaining optimal oxygen tension level in cells promotes the metabolic and plastic processes that ensure their functional stability. To date, there are a lot of reports showing the high sensitivity of endothelium‐denuded blood vessels to oxygen deficiency (hypoxia) [1–5]. These data allow considering vascular smooth muscle cells (VSMC) as an oxygen sensor involved in modulation of blood vessel tone and gene expression. Previously, using global gene expression profiling, we found that in several cell types including rat aortic VSMC Na+ , K+ ‐ATPase inhibition by

© 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.

ouabain or K+ ‐free medium led to the differential expression of dozens of genes whose altered expression was previously detected in cells subjected to hypoxia and ischemia/reperfusion [6, 7]. In view of this finding, we examined the relative impact of canonical hypoxia‐inducible factor 1alpha (HIF‐1α)‐ and Nai +/Ki +‐mediated signaling on transcriptomic changes evoked by hypoxia and glucose deprivation as well as its possible involvement in regulation of VSMC contraction.

## **2. Hypoxia affects excitation-contraction and excitation-transcription coupling: role of HIF-1α-mediated signaling**

Blood vessels play a key role in the maintenance of a balanced supply of oxygen and nutrition in target tissues under acute and chronic hypoxic conditions. In systemic circulation, acute hypoxic conditions resulted in dilatation of vascular beds via direct actions of attenuated partial oxygen pressure (pO2) on vascular smooth muscle cells (VSMC) as well as by ATP release from erythrocytes that, in turn, leads to activation of purinergic P2Y receptors and augmented production of nitric oxide by endothelial cells (for comprehensive reviews, see [1– 3]).

**Figure 1A** shows that in the absence of erythrocytes, hypoxia attenuated by 20–30% the contraction of rat aortic strips triggered by agonist of α1‐adrenergic receptors phenylephrine. We found that inhibitory action of hypoxia was partially abolished by 4‐aminopyridine (**Figure 1B**) and glibenclamide (**Figure 1C**), thus indicating activation of voltage‐gated and ATP‐ sensitive K+ channels, respectively. Recently, Gun et al. reported that hypoxic relaxation of mesenteric arteries is suppressed by a selective inhibitor of the large conductance Ca2+‐ activated K+ channels (BKCa) iberiotoxin [4].

Unlike systemic circulation, hypoxia results in augmented contraction of pulmonary arterial smooth muscle cells via inhibition of voltage‐gated K+ channels Kv1.5 and Kv2.1 and activation of nonselective cation channels TRPC1 (for reviews, see [5, 8]). It was shown that ATP release from erythrocytes triggered by shear stress and activation of cAMP‐mediated signaling is sharply decreased in human with primary pulmonary hypertension [9]. To the best of our knowledge, the comparative analysis of hypoxia‐induced ATP release from erythrocytes of normotensive and hypertensive patients and implication of purinergic receptors in regulation of vascular tone in systemic and pulmonary circulations have not yet been performed.

In addition, the regulation of vascular tone hypoxia leads to cell type‐specific differential expression of hundreds of genes documented in global gene profiling studies [10–16]. It is generally accepted that these transcriptomic changes are mediated by hypoxia‐inducible factor 1alpha (HIF‐1α) involved in regulation of gene expression via interaction of HIF‐1α/HIF‐1β heterodimer with hypoxia‐response elements (HREs) in promoter/enhancer regions of the target gene's DNA. In normoxia, oxygen‐dependent prolyl hydroxylase hydroxylates HIF‐1α and induces its proteasomal degradation. In contrast, under hypoxic conditions, HIF‐1α is translocated to the nucleus, where it forms HIF‐1α/HIF‐1β complex [17–20]. The list of HIF‐1‐ sensitive genes includes Hif‐1α per se and others related to angiogenesis (vascular endothelial growth factor (Vegf) and its receptor Flt1), vasomotor control (endothelin‐1, adrenomedullin, nitric oxide synthase‐2), erythropoiesis and iron metabolism (transferrin, transferrin receptor, erythropoietin, ceruloplasmin), energy metabolism (phosphoenolpyruvate carboxylase, aldose, endolase, phosphoglucokinase‐1, ‐L and ‐C, lactate dehydrogenase A, tyrosine hydroxylase and plasminogen activator inhibitor‐1, glucose transporters Glut1‐Glut3), and cell proliferation (Tgfb, Igf1, Igfbp1) [21]. Shimoda and coworkers reported that reduction in voltage‐gated K+ currents following hypoxia was absent in pulmonary arterial smooth muscle cells from heterozygous HIF‐1α mice, thus suggesting and implicating this oxygen‐sensing machinery in vascular bed‐specific contractile responses [22].

ouabain or K+

74 Hypoxia and Human Diseases

3]).

sensitive K+

activated K+

1alpha (HIF‐1α)‐ and Nai

+/Ki

**coupling: role of HIF-1α-mediated signaling**

channels (BKCa) iberiotoxin [4].

smooth muscle cells via inhibition of voltage‐gated K+

‐free medium led to the differential expression of dozens of genes whose altered

+‐mediated signaling on transcriptomic changes evoked by hypoxia

expression was previously detected in cells subjected to hypoxia and ischemia/reperfusion [6, 7]. In view of this finding, we examined the relative impact of canonical hypoxia‐inducible factor

and glucose deprivation as well as its possible involvement in regulation of VSMC contraction.

Blood vessels play a key role in the maintenance of a balanced supply of oxygen and nutrition in target tissues under acute and chronic hypoxic conditions. In systemic circulation, acute hypoxic conditions resulted in dilatation of vascular beds via direct actions of attenuated partial oxygen pressure (pO2) on vascular smooth muscle cells (VSMC) as well as by ATP release from erythrocytes that, in turn, leads to activation of purinergic P2Y receptors and augmented production of nitric oxide by endothelial cells (for comprehensive reviews, see [1–

**Figure 1A** shows that in the absence of erythrocytes, hypoxia attenuated by 20–30% the contraction of rat aortic strips triggered by agonist of α1‐adrenergic receptors phenylephrine. We found that inhibitory action of hypoxia was partially abolished by 4‐aminopyridine (**Figure 1B**) and glibenclamide (**Figure 1C**), thus indicating activation of voltage‐gated and ATP‐

mesenteric arteries is suppressed by a selective inhibitor of the large conductance Ca2+‐

Unlike systemic circulation, hypoxia results in augmented contraction of pulmonary arterial

of nonselective cation channels TRPC1 (for reviews, see [5, 8]). It was shown that ATP release from erythrocytes triggered by shear stress and activation of cAMP‐mediated signaling is sharply decreased in human with primary pulmonary hypertension [9]. To the best of our knowledge, the comparative analysis of hypoxia‐induced ATP release from erythrocytes of normotensive and hypertensive patients and implication of purinergic receptors in regulation of vascular tone in systemic and pulmonary circulations have not yet been performed.

In addition, the regulation of vascular tone hypoxia leads to cell type‐specific differential expression of hundreds of genes documented in global gene profiling studies [10–16]. It is generally accepted that these transcriptomic changes are mediated by hypoxia‐inducible factor 1alpha (HIF‐1α) involved in regulation of gene expression via interaction of HIF‐1α/HIF‐1β heterodimer with hypoxia‐response elements (HREs) in promoter/enhancer regions of the target gene's DNA. In normoxia, oxygen‐dependent prolyl hydroxylase hydroxylates HIF‐1α and induces its proteasomal degradation. In contrast, under hypoxic conditions, HIF‐1α is translocated to the nucleus, where it forms HIF‐1α/HIF‐1β complex [17–20]. The list of HIF‐1‐ sensitive genes includes Hif‐1α per se and others related to angiogenesis (vascular endothelial growth factor (Vegf) and its receptor Flt1), vasomotor control (endothelin‐1, adrenomedullin,

channels, respectively. Recently, Gun et al. reported that hypoxic relaxation of

channels Kv1.5 and Kv2.1 and activation

**2. Hypoxia affects excitation-contraction and excitation-transcription**

**Figure 1.** Hypoxia influences on phenylephrine (PE)‐induced contraction of ring aortic segments from male Wistar rats. Aortic segments were incubated for 60 min in hypoxic Krebs solution (pO2 ~ 30 mmHg) and then contacted with phenylephrine (1 µM). Registration of constrictive responses was performed by Myobath‐2 Multi‐Channel Tissue Bath System. Incubation in hypoxic solution decreased the amplitude of PE‐induced constriction in comparison with con‐ traction in normoxic solution (A). Both blocker of voltage‐dependent potassium channel 4‐aminopyridine (1 mM) (B) and blocker of ATP‐dependent potassium channels glibenclamide (10 µM) (C) significantly decreased mechanical ten‐ sion of aortic segments in comparison with PE‐induced contraction in hypoxic solution (*p* < 0.05). *X* axis—time (h), *Y* axis—mechanical tension (mN). The arrows indicate the addition and removal of the respective solutions.

It should be noted that side‐by‐side with activation of HIF‐1α‐mediated signaling, attenuation of pO2 and delivery of cell fuels resulted in decreased intracellular ATP content that, in turn, led to activation of AMP‐sensitive protein kinase (AMPK) [23, 24], decline of ion transport ATPase activities and dissipation of electrochemical gradients of K+ , Na+ , Cl− and Ca2+ [25]. Numerous research teams reported that [Ca2+]i elevation triggers transcriptomic alterations via Cai 2+‐sensitive transcriptional elements [26]. Importantly, along with the increment in [Ca2+]i , even transient ischemia increases [Na+ ]i from 5–8 to 25–40 mM and causes reciprocal changes in [K+ ]i [27]. These data motivate us to propose that Nai +/Ki +‐sensitive signaling pathways contribute to cellular responses triggered by sustained hypoxia [6, 28]. Investigations exam‐ ining this hypothesis are considered below.

## **3. Intracellular monovalent cations as regulators of gene transcription**

In the late 1990s, we observed that elevation of the [Na+ ]i /[K+ ]i ratio protects rat aortic VSMC against apoptosis triggered by serum deprivation and staurosporine addition [29]. To further explore this antiapoptotic pathway, we treated cells with actinomycin D or cycloheximide. Both macromolecular synthesis inhibitors abolished protection against apoptosis by ouabain [30]. Later we employed proteomic technology and detected hundreds of differentially expressed protein spots in VSMC subjected to Na+ , K+ ‐ATPase inhibition by ouabain and other cardiotonic steroids (CTS) [30]. These data, together with augmented RNA synthesis observed in ouabain‐ treated VSMC [31], suggest that sharp transcriptomic changes seen in ouabain‐treated cells are mediated by immediate response genes (IRG). Indeed, in both RASMC and HeLa cells, ouabain treatment resulted in augmentation of immunoreactive c‐Fos and c‐Jun by 10‐fold and fourfold, respectively [32, 33]. Addition of ouabain induced a fourfold c‐Fos mRNA increment accompanied by fivefold increment in [Na+ ]i within 30 min. At the same time, we observed only 10–15% decrease in [K+ ]i [32, 33]. Thus, we can assume that c‐Fos expression is more sensitive to increase in [Na+ ]i rather than [K+ ]i .

Recent studies have revealed that CTS may affect cells independently of suppression of Na+ , K+ ‐ATPase. Thus, ouabain induced interaction of α‐subunit of the Na+ , K+ ‐ATPase with the membrane‐associated nonreceptor tyrosine kinase Src, activation of Ras/Raf/ERK1,2, phos‐ phatidylinositol 3‐kinase (PI(3)K), PI(3)K‐dependent protein kinase B, phospholipase C, [Ca2+]i oscillations and increased production of the reactive oxygen species (for review, see [34– 36]). Considering this, we employed K+ ‐free medium as an alternative approach for Na+ , K+ ‐ ATPase inhibition. To identify Nai +,Ki +‐sensitive transcriptomes, both ubiquitous and cell type‐ specific, we compared the effect of ouabain and K+ ‐free medium on profiles of gene expression in rat VSMC, human umbilical vein endothelial cells (HUVEC) and the human carcinoma HeLa cell line [26]. Using Affymetrix‐based technology, we found that expression of 684, 737 and 1839 transcripts in HeLa, HUVEC and RASMC, respectively, changes up to 60‐fold. It is worth noting that there was a strong correlation in cells pretreated with ouabain or K+ ‐free medium for 3 h. We also found that 80 transcripts of examined Nai +/Ki +‐sensitive genes were common for all examined types of cells [26].

We found that genes involved in the regulation of transcription represents a half of ubiquitous Nai +,Ki +‐sensitive transcriptome. This amount was ~sevenfold higher than in the total human genome [37]. The group of ubiquitous Nai +/Ki +‐sensitive genes, whose expression was in‐ creased by more than threefold, included the transcription factor of the steroid‐thyroid hormone‐retinoid receptor superfamily Nr4a2, transcriptional regulator of C2H2‐type zinc finger protein Egr‐1, the basic helix‐loop‐helix transcription regulator Hes1, members of the superfamily of b‐zip transcriptional factors possessing leucine‐zipper dimerization motif and basic DNA‐binding domain and forming heterodimeric activating protein AP‐1 (Fos, FosB, Jun, JunB, Atf3) [26].

#### **4. Evidence for** Nai +/Ki <sup>+</sup>**-mediated,** Cai 2+**-independent excitationtranscription coupling**

in [K+ ]i

76 Hypoxia and Human Diseases

[27]. These data motivate us to propose that Nai

In the late 1990s, we observed that elevation of the [Na+

ining this hypothesis are considered below.

protein spots in VSMC subjected to Na+

accompanied by fivefold increment in [Na+

36]). Considering this, we employed K+

specific, we compared the effect of ouabain and K+

for 3 h. We also found that 80 transcripts of examined Nai

ATPase inhibition. To identify Nai

for all examined types of cells [26].

genome [37]. The group of ubiquitous Nai

]i

rather than [K+

‐ATPase. Thus, ouabain induced interaction of α‐subunit of the Na+

+,Ki

noting that there was a strong correlation in cells pretreated with ouabain or K+

]i

only 10–15% decrease in [K+

sensitive to increase in [Na+

K+

[Ca2+]i

Nai +,Ki +/Ki

]i /[K+ ]i

contribute to cellular responses triggered by sustained hypoxia [6, 28]. Investigations exam‐

**3. Intracellular monovalent cations as regulators of gene transcription**

, K+

against apoptosis triggered by serum deprivation and staurosporine addition [29]. To further explore this antiapoptotic pathway, we treated cells with actinomycin D or cycloheximide. Both macromolecular synthesis inhibitors abolished protection against apoptosis by ouabain [30]. Later we employed proteomic technology and detected hundreds of differentially expressed

steroids (CTS) [30]. These data, together with augmented RNA synthesis observed in ouabain‐ treated VSMC [31], suggest that sharp transcriptomic changes seen in ouabain‐treated cells are mediated by immediate response genes (IRG). Indeed, in both RASMC and HeLa cells, ouabain treatment resulted in augmentation of immunoreactive c‐Fos and c‐Jun by 10‐fold and fourfold, respectively [32, 33]. Addition of ouabain induced a fourfold c‐Fos mRNA increment

]i

]i .

Recent studies have revealed that CTS may affect cells independently of suppression of Na+

membrane‐associated nonreceptor tyrosine kinase Src, activation of Ras/Raf/ERK1,2, phos‐ phatidylinositol 3‐kinase (PI(3)K), PI(3)K‐dependent protein kinase B, phospholipase C,

in rat VSMC, human umbilical vein endothelial cells (HUVEC) and the human carcinoma HeLa cell line [26]. Using Affymetrix‐based technology, we found that expression of 684, 737 and 1839 transcripts in HeLa, HUVEC and RASMC, respectively, changes up to 60‐fold. It is worth

We found that genes involved in the regulation of transcription represents a half of ubiquitous

+/Ki

creased by more than threefold, included the transcription factor of the steroid‐thyroid

+‐sensitive transcriptome. This amount was ~sevenfold higher than in the total human

oscillations and increased production of the reactive oxygen species (for review, see [34–

+‐sensitive signaling pathways

ratio protects rat aortic VSMC

,

, K+ ‐

‐free medium

‐ATPase with the

‐ATPase inhibition by ouabain and other cardiotonic

within 30 min. At the same time, we observed

, K+

‐free medium on profiles of gene expression

+‐sensitive genes, whose expression was in‐

+‐sensitive genes were common

[32, 33]. Thus, we can assume that c‐Fos expression is more

‐free medium as an alternative approach for Na+

+‐sensitive transcriptomes, both ubiquitous and cell type‐

+/Ki

Because of the high electrochemical gradient, the opening of calcium channels resulted in rapid elevation of [Ca2+]i from ~0.1 to 1 µM, its interaction with calmodulin and other [Ca2+]i sensors, in turn, affects the expression of hundreds of genes, i.e., phenomenon termed excitation‐ transcription coupling [38]. Increase in [Ca2+]i affects transcription via several signaling pathways. Thus, [Ca2+]i elevation induces translocation of kappa‐light‐chain enhancer of nuclear factor (NFκB) of activated B cells from the cytosol to the nucleus. This process is triggered by activation of Ca2+/calmodulin‐sensitive protein kinase (CaMKI, II or III) and phosphorylated IkB kinase that phosphorylates the inhibitor of kB (IkB) [38]. [Ca2+]i elevation also promotes translocation from cytosol to the nucleus; nuclear factor of activated T cells (NFAT) is evoked by its dephosphorylation by the (Ca2+/calmodulin)‐dependent phosphatase calcineurin [39]. In addition, increased cytosolic and nucleoplasmic Ca2+ concentrations lead to phosphorylation of cAMP response element‐binding protein (CREB) by CaMKII and CaMKIV, respectively. Phosphorylated CREB regulates transcription via their binding to the (Ca2++cAMP)‐response element (CRE) sequences of DNA [40].

Because the c‐Fos promoter contains CRE, its augmented expression might be mediated by depolarization of ouabain‐treated VSMC and the opening of voltage‐gated Ca2+ channels. However, unlike high‐K+ medium, c‐Fos expression in ouabain‐treated cells was not affected by inhibition of L‐type Ca2+ channels with nicardipine [41]. In additional experiments, we found that augmented c‐Fos expression evoked by ouabain was preserved in Ca2+‐free medium and in the presence of extracellular (EGTA) and intracellular (BAPTA) Ca2+ chelators [30]. To study the role of Cai 2+‐mediated and Nai +/Ki +‐independent signaling, we compared transcrip‐ tomic changes triggered by elevation of the [Na+ ]i /[K+ ]i ratio in control and Ca2+‐depleted cells. Depletion of Ca2+ led to prevalent increase in Nai +/Ki +‐sensitive genes, both ubiquitous and cell‐type specific [26]. For further investigation, we examined ubiquitous Cai 2+‐sensitive genes whose expression is regulated by more than threefold independently of the presence of Ca2+ chelators and selected several transcription factors (Fos, Hes1, Nfkbia, Jun), protein phospha‐ tase 1, dual specificity phosphatase Dusp8, interleukin‐6, regulatory subunit, type 2 cycloox‐ ygenase COX‐2, cyclin L1 [41].

Considering these data, it is important to underline that Ca2+ chelators may affect cellular functions independently of Ca2+ depletion. Thus, we observed that the addition of EGTA increases permeability of VSMC for Na+ [41]. It is also known that the affinity of EGTA for Mn2+, Zn2+, Cu2+, Co2+, Fe2+/3+ is 10‐fold to 107 ‐fold higher than for Ca2+ [42–44]. These polyva‐ lent cations are important in regulation of metaloenzymes activity and participate in pro‐ tein‐DNA and protein‐protein interactions. Moreover, EGTA causes irreversible conformational transition and inactivation of transcriptional adaptor Zn2+‐binding domain that affects gene expression [45]. It is worth noting that in the human genome, the C2H2 zinc finger superfamily includes about half of all annotated transcription factors [46]. This implies that this and other chelators have Cai 2+‐independent action on transcriptomic changes evoked by diverse stimuli. Keeping these data in mind, we compared the actions of Ca2+ chelators and Na+ , K+ ‐ATPase inhibitors on transcriptomic changes and concentration of monovalent cations in VSMC [47]. Our results show that transcriptomic changes seen in Ca2+‐depleted VSMC are at least partially caused by elevation of the [Na+ ]i /[K+ ]i ratio and activation of Nai +/Ki +‐independent signaling pathways. This conclusion is supported by sev‐ eral observations. First, Ca2+ depletion led to a ~threefold elevation of [Na+ ]i and a twofold attenuation of [K+ ]i . An increment in the [Na+ ]i /[K+ ]i ratio seen in Ca2+‐depleted cells was caused by elevation of plasma membrane permeability for monovalent cations. Indeed, Ca2+ depletion resulted in almost threefold elevation of the rate of 22Na and 86Rb influx measured in the presence of inhibitors of Na+ , K+ ‐ATPase and Na+ , K+ , 2Cl− cotransport. Second, the list of genes whose mRNA content was increased in Ca2+‐depleted cells by more than fourfold includes a large number of genes whose expression was also attenuated by the Na+ , K+ ‐AT‐ Pase inhibition in K+ ‐free medium. Third, there was a strong positive correlation in mRNA content of 2071 genes whose expression was changed by more than 1.2‐fold in cells subject‐ ed to Na+ , K+ ‐ATPase inhibition in K+ ‐free medium as well as in Ca2+‐depleted cells. Fourth, dissipation of transmembrane gradients of Na+ and K+ in high‐K+ , low‐Na+ medium abolish‐ ed the increment in the [Na+ ]i /[K+ ]i ratio as well as sharp elevation of Atf3, Nr4a1 and Erg3 mRNA content triggered by 3‐h incubation of VSMC in Ca2+‐free, EGTA‐containing medium [47]. Thus, novel molecular biological and pharmacological approaches should be devel‐ oped for precise identification of the relative impact of Ca2+‐mediated and Ca2+‐independent pathways on transcriptomic changes evoked by elevation of the [Na+ ]i /[K+ ]i ratio.

#### **5. Evidence for implication of [Na+ ]i /[K+ ]i -sensitive pathways in transcriptomic changes evoked by hypoxia**

The crosstalk between transcriptomic changes and monovalent ion handling was initially supported by comparative analysis of Nai +/Ki +‐sensitive genes documented in our investiga‐ tions [26] and data on genes whose expression in hypoxic conditions was changed in studies performed by other research groups [9, 11, 48–56]. Indeed, among genes whose augmented expression was detected both in vivo and in vitro models of ischemia/reperfusion, we found several ubiquitous Nai +/Ki +‐sensitive genes, including transcription factors EGR1, ATF3, NFKBIZ, HES1 as well as type 2 cyclooxygenase, IL6, thioredoxin‐interacting protein TXNIP.

Moreover, using IPA‐knowledge base data, we observed that ubiquitous Na+ i ,K+ i ‐sensitive transcriptomes are highly significantly correlated with differential expression of genes in disorders triggered by kidney, liver and heart ischemia (**Figure 2**). These data allowed us to propose that transcriptomic changes in ischemic tissues are at least partially mediated by a novel Nai +, Ki +‐mediated excitation‐transcription coupling [26, 27].

increases permeability of VSMC for Na+

Mn2+, Zn2+, Cu2+, Co2+, Fe2+/3+ is 10‐fold to 107

implies that this and other chelators have Cai

, K+

‐ATPase inhibition in K+

]i /[K+ ]i

pathways on transcriptomic changes evoked by elevation of the [Na+

dissipation of transmembrane gradients of Na+

**5. Evidence for implication of [Na+**

supported by comparative analysis of Nai

**transcriptomic changes evoked by hypoxia**

+/Ki

+/Ki

]i

in the presence of inhibitors of Na+

Ca2+ chelators and Na+

78 Hypoxia and Human Diseases

activation of Nai

attenuation of [K+

Pase inhibition in K+

, K+

several ubiquitous Nai

ed the increment in the [Na+

ed to Na+

[41]. It is also known that the affinity of EGTA for

lent cations are important in regulation of metaloenzymes activity and participate in pro‐ tein‐DNA and protein‐protein interactions. Moreover, EGTA causes irreversible conformational transition and inactivation of transcriptional adaptor Zn2+‐binding domain that affects gene expression [45]. It is worth noting that in the human genome, the C2H2 zinc finger superfamily includes about half of all annotated transcription factors [46]. This

changes evoked by diverse stimuli. Keeping these data in mind, we compared the actions of

monovalent cations in VSMC [47]. Our results show that transcriptomic changes seen in

]i /[K+ ]i

caused by elevation of plasma membrane permeability for monovalent cations. Indeed, Ca2+ depletion resulted in almost threefold elevation of the rate of 22Na and 86Rb influx measured

‐ATPase and Na+

of genes whose mRNA content was increased in Ca2+‐depleted cells by more than fourfold

content of 2071 genes whose expression was changed by more than 1.2‐fold in cells subject‐

mRNA content triggered by 3‐h incubation of VSMC in Ca2+‐free, EGTA‐containing medium [47]. Thus, novel molecular biological and pharmacological approaches should be devel‐ oped for precise identification of the relative impact of Ca2+‐mediated and Ca2+‐independent

> **]i /[K+ ]i**

+/Ki

The crosstalk between transcriptomic changes and monovalent ion handling was initially

tions [26] and data on genes whose expression in hypoxic conditions was changed in studies performed by other research groups [9, 11, 48–56]. Indeed, among genes whose augmented expression was detected both in vivo and in vitro models of ischemia/reperfusion, we found

NFKBIZ, HES1 as well as type 2 cyclooxygenase, IL6, thioredoxin‐interacting protein TXNIP.

and K+

includes a large number of genes whose expression was also attenuated by the Na+

Ca2+‐depleted VSMC are at least partially caused by elevation of the [Na+

eral observations. First, Ca2+ depletion led to a ~threefold elevation of [Na+

, K+

. An increment in the [Na+

‐fold higher than for Ca2+ [42–44]. These polyva‐

2+‐independent action on transcriptomic

]i /[K+ ]i

]i

cotransport. Second, the list

ratio seen in Ca2+‐depleted cells was

ratio and

and a twofold

, K+ ‐AT‐

medium abolish‐

‐ATPase inhibitors on transcriptomic changes and concentration of

+‐independent signaling pathways. This conclusion is supported by sev‐

, K+ , 2Cl−

‐free medium. Third, there was a strong positive correlation in mRNA

in high‐K+

+‐sensitive genes, including transcription factors EGR1, ATF3,

‐free medium as well as in Ca2+‐depleted cells. Fourth,

**-sensitive pathways in**

ratio as well as sharp elevation of Atf3, Nr4a1 and Erg3

, low‐Na+

]i /[K+ ]i ratio.

+‐sensitive genes documented in our investiga‐

To examine this hypothesis, we compared the effect of ouabain and hypoxia on the content of monovalent ions and ATP in VSMC from the rat aorta. We observed that 24‐h incubation of VSMC in hypoxia and glucose starvation decreased intracellular ATP content by ~three‐ fold, whereas ouabain attenuated this parameter by <20% (**Figure 3**). Ouabain led to almost 10‐fold increase in [Na+ ]i and similar decrease in [K+ ]i . Hypoxia also caused threefold in‐ crease in [Na+ ]i and twofold decrease in [K+ ]i . At the same time, reduction in monovalent cations transmembrane gradients in low‐Na+ , high‐K+ medium almost completely eliminat‐ ed the actions of ouabain and hypoxia on the [Na+ ]i /]K+ ]i ratio [57].

**Figure 2.** Disorders significantly associated with differential expression of genes whose expression was ubiquitously changed in VSMC from rat aorta, human umbilical vein endothelial cells and HeLa cell line subjected to Na+ ,K+ ‐ATPase inhibition by both ouabain and K+ ‐free medium. The criteria with a threshold for significance of *p* = 0.05 (or 1.3 when expressed as −log(*p*‐value) are shown as straight line. Adopted with permission from [26].

We then identified the [Na+ ]i /]K+ ]i ‐sensitive transcriptome in rat VSMC. We found that 6‐h inhibition of the Na+ , K+ ‐ATPase with ouabain or in K+ ‐free medium resulted in differential expression of 6412 transcripts exhibit highly significant (*p* < 4 × 10−9) and positive (*R*<sup>2</sup> > 0.80) correlation and classified as Cai 2+‐sensitive genes [57]. To continue our studies, we selected genes whose participation in the pathogenesis of hypoxia was shown in previous studies combined with the property of the highest expression increments under sustained Na+ , K+ ‐ ATPase inhibition. These genes include Fos, Cyp1a1, Klf10, Atf3, Nr4a1, Hes1, Ptgs2 and Per2. Among these genes, Fos, Atf3 and JUN together form dimeric transcription factor AP‐1 whose expression increased in all types of cells subjected to hypoxia [58]. Klf10 is a Kruppel‐like zinc finger transcription factor family member involved in hypoxia‐dependent angiogenesis via COX‐1 activation [59]. Ptgs2 encodes an inducible isoform of cyclooxygenase‐2 (COX‐2) whose role in the pathophysiology of hypoxia is well documented [60]. Nur77 or Nr4a1, also known as nerve growth factor IB, is the nuclear receptor of transcription factors stabilizing HIF‐1α which increases its transcriptional activity [61]. Hes1 is the main helix‐loop‐helix transcription factor that enhances the expression after ischemic renal failure [52]. Clock, Bmal1, Per1, Per2, Cry1 and Cry2 are the positive (Clock and Bmal1) and negative (others) regulators of a transcription‐translation feedback loop forming the core circadian oscillator [62]. Cyp1a1 encodes a cytochrome P450 family member and its expression is mediated by HIF‐1β [63, 64]. Per2 promotes circadian stabilization of HIF‐1α activity that is critical for myocardial adapta‐ tion to ischemia. The positive controls for canonical HIF‐1α‐sensitive genes are endothelin (Edn1) and vascular endothelial growth factor (Vegfa).

**Figure 3.** Effect of ouabain and hypoxia on intracellular Na+ , K+ and ATP concentrations in VSMC from the rat aorta. Cells were exposed to normal oxygen partial pressure (5% CO2/air—control) ±3 µM ouabain or exposure to hypoxia (5% CO2/95% N2)/glucose deprivation for 24 h in normal high‐Na+ , low‐K+ ([Na+ ]/[K+ ] = 140/5) or in low‐Na+ , high‐K+ DMEM‐like medium ([Na+ ]/[K+ ] = 131/115). Intracellular K+ and Na+ Cl− content was measured as the steady‐state dis‐ tribution of extra‐ and intracellular 86Rb and 22Na, respectively. Intracellular ATP content was measured by assaying luciferase‐dependent luminescence with ATP bioluminescent assay kit. Means ± S.E. from three independent experi‐ ments performed in quadruplicate are shown. \**p* < 0.05 compared to the controls. Adopted with permission from [57].

To assess the role of [Na+ ]i /[K+ ]i ‐dependent and HIF‐1α‐mediated signaling, we compared expression of the above‐listed selected genes in hypoxic conditions and under the action of ouabain in control high‐Na+ , low‐K+ medium and in high‐K+ , low‐Na+ medium with dissipated transmembrane gradients of monovalent cations and after cells transfection with Hif‐1a siRNA

[57]. As demonstrated in other cell types [65, 66], hypoxia slightly enhanced Hif‐1a mRNA (**Figure 4**) but increased immunoreactive HIF‐1α protein content by ~fivefold (**Figure 5**).

ATPase inhibition. These genes include Fos, Cyp1a1, Klf10, Atf3, Nr4a1, Hes1, Ptgs2 and Per2. Among these genes, Fos, Atf3 and JUN together form dimeric transcription factor AP‐1 whose expression increased in all types of cells subjected to hypoxia [58]. Klf10 is a Kruppel‐like zinc finger transcription factor family member involved in hypoxia‐dependent angiogenesis via COX‐1 activation [59]. Ptgs2 encodes an inducible isoform of cyclooxygenase‐2 (COX‐2) whose role in the pathophysiology of hypoxia is well documented [60]. Nur77 or Nr4a1, also known as nerve growth factor IB, is the nuclear receptor of transcription factors stabilizing HIF‐1α which increases its transcriptional activity [61]. Hes1 is the main helix‐loop‐helix transcription factor that enhances the expression after ischemic renal failure [52]. Clock, Bmal1, Per1, Per2, Cry1 and Cry2 are the positive (Clock and Bmal1) and negative (others) regulators of a transcription‐translation feedback loop forming the core circadian oscillator [62]. Cyp1a1 encodes a cytochrome P450 family member and its expression is mediated by HIF‐1β [63, 64]. Per2 promotes circadian stabilization of HIF‐1α activity that is critical for myocardial adapta‐ tion to ischemia. The positive controls for canonical HIF‐1α‐sensitive genes are endothelin

, K+

and Na+

, low‐K+

Cl−

 ([Na+ ]/[K+

‐dependent and HIF‐1α‐mediated signaling, we compared

, low‐Na+

Cells were exposed to normal oxygen partial pressure (5% CO2/air—control) ±3 µM ouabain or exposure to hypoxia

tribution of extra‐ and intracellular 86Rb and 22Na, respectively. Intracellular ATP content was measured by assaying luciferase‐dependent luminescence with ATP bioluminescent assay kit. Means ± S.E. from three independent experi‐ ments performed in quadruplicate are shown. \**p* < 0.05 compared to the controls. Adopted with permission from [57].

expression of the above‐listed selected genes in hypoxic conditions and under the action of

transmembrane gradients of monovalent cations and after cells transfection with Hif‐1a siRNA

medium and in high‐K+

and ATP concentrations in VSMC from the rat aorta.

] = 140/5) or in low‐Na+

medium with dissipated

content was measured as the steady‐state dis‐

, high‐K+

(Edn1) and vascular endothelial growth factor (Vegfa).

**Figure 3.** Effect of ouabain and hypoxia on intracellular Na+

]/[K+

DMEM‐like medium ([Na+

80 Hypoxia and Human Diseases

To assess the role of [Na+

ouabain in control high‐Na+

(5% CO2/95% N2)/glucose deprivation for 24 h in normal high‐Na+

]i /[K+ ]i

, low‐K+

] = 131/115). Intracellular K+

**Figure 4.** Effect of hypoxia and ouabain on gene expression in VSMC from the rat aorta. Cells were incubated for 24 h under normoxia, hypoxia/glucose deprivation or 3 mM ouabain in control high‐Na+ , low‐K+ medium (A, C), or high‐ K+ , low‐Na+ medium (B). In some experiments, RASMC were transfected with Hif‐1α siRNA (C). The content of mRNA in normoxia was taken as 1.00 and shown as broken lines. Adopted with permission from [57].

**Figure 5.** (A). Representative Western blots of GAPDH and HIF‐1α in VSMC incubated for 24 h under control condi‐ tions (normoxia), hypoxia/glucose deprivation, 3 mM ouabain or hypoxia/glucose deprivation in cells transfected with Hif‐1α siRNA. (B). Hypoxia/glucose deprivation and ouabain influence on HIF‐1α protein relative content in RASMC. The HIF‐1α/GAPDH ratio in control conditions was taken as 1.00. Data obtained in three independent experiments are reported as means ± S.E. Adopted with permission from [57].

Transfection of rat VSMC with Hif‐1α siRNA but not with scrambled siRNA led to ~threefold expression reduction in Hif‐1a and lowered hypoxia‐induced HIF‐1a protein gain (**Figure 5**). Pretreatment with ouabain slightly changed HIF‐1α protein content (**Figure 5**) and amplified baseline Hif‐1a mRNA by ~50% (**Figure 4**). Hypoxia causes fourfold and 12‐fold increase in Edn1 and Vegfa mRNA content, respectively, (**Figure 4**), which is consistent with earlier observations [19]. Hypoxia‐dependent increase in Edn1 and Vegfa mRNA was attenuated after transfection with Hif‐1a siRNA by ~twofold and fourfold, respectively. At the same time, ouabain augmented Edn1 mRNA by 2.5‐fold but did not significantly impair Vegfa. Similarly, low‐Na+ , high‐K+ medium that is characterized with dissipation of the transmembrane gradients of monovalent cations also did not affect hypoxia‐induced expression of Vegfa and reduced Edn1 mRNA by twofold. All these data strongly support the efficacy of Hif1α‐siRNA function [57].

In hypoxic conditions, dissipation of monovalent cations transmembrane gradients completely suppressed increments in Fos, Atf3, Ptgs2 and Per2 mRNA and diminished increase in Klf10, Edn1, Nr4a1 and Hes1 expression (**Figure 4**). Hypoxia caused from twofold to sixfold aug‐ mentation of Atf3, Fos, Ptgs2, Klf10, Nr4a2, Hes1 and Per2 expression (**Figure 4**). These data 17 are consistent with the observations obtained in other cell types, including human VSMC [67, 68]. Transfection with Hif‐1a siRNA led to twofold attenuation of hypoxia‐induced increase in Nr4a and Klf10 mRNA without significant influence on expression of Fos, Atf3, Ptgs2 and Per2 evoked by hypoxia. At the same time, hypoxic conditions led to twofold decrease in Cyp1a1 mRNA and attenuated expression of Cyp1a1 obtained from human microvasculature [69]. Ouabain enhanced the expression of all eight tested genes from threefold to 10‐fold that were completely abolished in low‐Na+ , high‐K+ medium characterized with dissipation of the transmembrane gradients of monovalent cations [57]. However, in ouabain‐treated RASMC, the expression of these genes was not affected by transfection with Hif‐1a siRNA, but decrease in monovalent cations transmembrane gradient sharply decreased elevation of Edn1, Klf10, Hes1 and Nr4a1 expression seen in hypoxic conditions and com‐ pletely abolished increase in Atf3, Fos, Ptgs2 and Per2 mRNA (**Figure 4**).

#### **6. Unresolved issues and future directions**

Viewed collectively, our results demonstrate a key role of [Na+ ]i /[K+ ]i ‐mediated excitation‐ transcription coupling in overall transcriptomic changes triggered by sustained ischemia. The molecular organization of sensors for monovalent cation is still unclear in contrast to rapid progress in the identification of Cai 2+sensors. Initially, such sensors were identified in parval‐ bumin and calmodulin. These high‐affinity binding sites (the so‐called EF‐hand domains) are formed by a highly conservative linear amino acid sequence consisting of 14 amino acid residues. Further screening of cDNA libraries allowed to identify more than 30 other Cai 2+ [70]. Moreover, high‐affinity sensors for Nai <sup>+</sup> are almost completely saturated at [Ca2+]i of 1 µM. This allows identifying amino acid residues using 45Ca binding assay. In contrast, molecular sensors for monovalent ion may be presented by 3D protein structures formed with space‐separated amino acid residues [27, 71]. Besides this, cellular functions are affected by monovalent cations when they act in the millimolar concentrations that make their detection with radioisotopes more complicate. As it was shown by Ono and coworkers, Na+ may interact with calpain Ca2+‐ binding sites at the baseline level of [Ca2+]i (~100 nM). Thus, calpain functions as Cai 2+‐ dependent protease with K0.5 of 15 mM for Na+ [72]. Additional experiments should be performed to examine the role of Ca2+‐binding proteins as [Na+ ]i sensors involved in cellular responses evoked by hypoxia.

Transfection of rat VSMC with Hif‐1α siRNA but not with scrambled siRNA led to ~threefold expression reduction in Hif‐1a and lowered hypoxia‐induced HIF‐1a protein gain (**Figure 5**). Pretreatment with ouabain slightly changed HIF‐1α protein content (**Figure 5**) and amplified baseline Hif‐1a mRNA by ~50% (**Figure 4**). Hypoxia causes fourfold and 12‐fold increase in Edn1 and Vegfa mRNA content, respectively, (**Figure 4**), which is consistent with earlier observations [19]. Hypoxia‐dependent increase in Edn1 and Vegfa mRNA was attenuated after transfection with Hif‐1a siRNA by ~twofold and fourfold, respectively. At the same time, ouabain augmented Edn1 mRNA by 2.5‐fold but did not significantly impair Vegfa. Similarly,

gradients of monovalent cations also did not affect hypoxia‐induced expression of Vegfa and reduced Edn1 mRNA by twofold. All these data strongly support the efficacy of Hif1α‐siRNA

In hypoxic conditions, dissipation of monovalent cations transmembrane gradients completely suppressed increments in Fos, Atf3, Ptgs2 and Per2 mRNA and diminished increase in Klf10, Edn1, Nr4a1 and Hes1 expression (**Figure 4**). Hypoxia caused from twofold to sixfold aug‐ mentation of Atf3, Fos, Ptgs2, Klf10, Nr4a2, Hes1 and Per2 expression (**Figure 4**). These data 17 are consistent with the observations obtained in other cell types, including human VSMC [67, 68]. Transfection with Hif‐1a siRNA led to twofold attenuation of hypoxia‐induced increase in Nr4a and Klf10 mRNA without significant influence on expression of Fos, Atf3, Ptgs2 and Per2 evoked by hypoxia. At the same time, hypoxic conditions led to twofold decrease in Cyp1a1 mRNA and attenuated expression of Cyp1a1 obtained from human microvasculature [69]. Ouabain enhanced the expression of all eight tested genes from

with dissipation of the transmembrane gradients of monovalent cations [57]. However, in ouabain‐treated RASMC, the expression of these genes was not affected by transfection with Hif‐1a siRNA, but decrease in monovalent cations transmembrane gradient sharply decreased elevation of Edn1, Klf10, Hes1 and Nr4a1 expression seen in hypoxic conditions and com‐

transcription coupling in overall transcriptomic changes triggered by sustained ischemia. The molecular organization of sensors for monovalent cation is still unclear in contrast to rapid

bumin and calmodulin. These high‐affinity binding sites (the so‐called EF‐hand domains) are formed by a highly conservative linear amino acid sequence consisting of 14 amino acid

allows identifying amino acid residues using 45Ca binding assay. In contrast, molecular sensors

residues. Further screening of cDNA libraries allowed to identify more than 30 other Cai

threefold to 10‐fold that were completely abolished in low‐Na+

**6. Unresolved issues and future directions**

progress in the identification of Cai

Moreover, high‐affinity sensors for Nai

pletely abolished increase in Atf3, Fos, Ptgs2 and Per2 mRNA (**Figure 4**).

Viewed collectively, our results demonstrate a key role of [Na+

medium that is characterized with dissipation of the transmembrane

, high‐K+

]i /[K+ ]i

2+sensors. Initially, such sensors were identified in parval‐

<sup>+</sup> are almost completely saturated at [Ca2+]i

medium characterized

‐mediated excitation‐

2+ [70].

of 1 µM. This

low‐Na+

function [57].

, high‐K+

82 Hypoxia and Human Diseases

It is generally accepted that transcription is under the control of proteins interacting with specific response elements within 5'‐ and 3'‐untranslated region (UTR). Considering this, we tried to find Na+ response element (NaRE) within c‐Fos promoter. With the CRE and all other known c‐Fos promoter transcription elements, we observed massive accumulation of endog‐ enous c‐Fos mRNA and immunoreactive protein in HeLa cells subjected to 6‐h inhibition of Na+ , K+ ‐ATPase, but we did not find any significant increase in luciferase expression in ouabain‐ treated HeLa cells [33]. Negative results obtained in this study may be explained by the following hypotheses: (i) NaRE is located within the c‐Fos 3'‐UTR and/or introns. (ii) Elevation of [Na+ ]i /[K+ ]i ratio influences on gene expression through epigenetic modification of regula‐ tory mechanism having a significant impact on various cellular functions, such the DNA, histones or nucleosome remodeling [73]. Importantly, the epigenetic mechanism of gene expression does not contribute to the regulation of L‐luc transcription in the plasmid employed in our experiments [33]. (iii) More evidence indicates that gene activation or silencing is under the complex control of three‐dimensional (3D) positioning of genetic materials and chromatin in the nuclear space (for review, see [74]). It may be proposed that gene transcription is affected by increased [Na+ ]i /[K+ ]i ratio through changing of the 3D organization of DNA‐chromatin complex. These hypotheses will be verified in forthcoming studies.

Some studies have shown that epigenetic modulatory mechanism of histone methylation is a key process that helps cells to adapt to hypoxia [75]. Growing evidence shows that along with the 5'‐UTR regulation by transcription factors, gene activation or silencing is controlled by 3D positioning of genetic materials and chromatin in nuclear spaces [74, 76]. The epigenetic regulation of 3D genome organization with considering the [Na+ ]i /[K+ ]i ratio and its role in gene silencing and activation is currently being examined in our laboratory.

Matrix metalloproteinases play an important role in pathophysiology of hypoxic chronic ve‐ nous disease via their implication in the regulation of migration, proliferation and endothe‐ lium‐dependent VSMC contraction [77]. We found that sustained elevation of the [Na+ ]i /[K+ ]i ratio resulted in ~fivefold elevation of Mmp28 metalloproteinase expression in rat VSMC [57]. The same procedure resulted in sevenfold elevation of the content of Nccp mRNA en‐ coding natriuretic peptide precursor C [57]. NCCP is proteolytically processed to C‐type na‐ triuretic peptide (CNP), i.e., a selective agonist for the B‐type natriuretic receptor whose role in cGMP‐mediated vasorelaxation is well documented. We noted that in endothelial cells, modest long‐term inhibition of the Na+ ,K+ ‐ATPse causes ~sevenfold attenuation of expres‐ sion of Edn encoding preproendothelin‐1 that is proteolytically processed to the most pow‐ erful endothelium‐derived vasoconstrictor endothelin‐1. We also observed ~10‐fold elevation of the content of mRNA encoding ubiquitously derived vasodilator adrenomedul‐ lin (unpublished results). Do these [Na+ ]i /[K+ ]i ‐mediated transcriptomic changes contribute to the pathophysiology of hypoxic vascular disorders? Does partial dissipation of electro‐ chemical gradients of monovalent cations seen in VSMC subjected to ischemia and glucose deprivation have an impact on the distinct regulation of systemic and pulmonary circulation under hypoxic conditions? We will address these questions to forthcoming studies.

## **Acknowledgements**

This work was supported by Grants from the Russian Foundation for Basic Research #15‐04‐00101 and the Russian Science Foundation #14‐15‐00006 and #16‐15‐10026.

## **Author details**

Sergei N. Orlov1,2\*, Yulia G. Birulina1 , Liudmila V. Smaglii1 and Svetlana V. Gusakova1

\*Address all correspondence to: sergei.n.orlov@yandex.ru

1 Siberian State Medical University, Tomsk, Russia

2 MV Lomonosov Moscow State University, Moscow, Russia

## **References**


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This work was supported by Grants from the Russian Foundation for Basic Research

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1 Siberian State Medical University, Tomsk, Russia


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**Chapter 5 Provisional chapter**

#### **Hypoxia in Mesenchymal Stem Cell Hypoxia in Mesenchymal Stem Cell**

Wahyu Widowati, Dwi Davidson Rihibiha, Khie Khiong, M. Aris Widodo, Sutiman B. Sumitro and Indra Bachtiar Wahyu Widowati, Dwi Davidson Rihibiha, Khie Khiong, M. Aris Widodo, Sutiman B. Sumitro and Indra Bachtiar

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

http://dx.doi.org/10.5772/65510

#### **Abstract**

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[77] MacColl E, Khalil RA. Matrix metalloproteinases as regulator of vein structure and function: implications in chronic venous disease. J Pharmacol Exp Ther. 2015; 355: 410–

[76] Gibcus JH, Dekker J. The hierarchy of the 3D genome. Mol Cell. 2013; 49: 773–782.


2006; 210: 161–172.

90 Hypoxia and Human Diseases

calpain is an intracellular Na+

Genet. 2007; 8: 104–115.

420.

Mesenchymal stem cells (MSCs) are non-hematopoietic multipotent stem cells with selfrenewal properties and ability to differentiate into a variety of mesenchymal tissues. This chapter overviews effects of hypoxia on MSCs, makes it promising therapy to various diseases. Cultivation of MSCs under hypoxic condition results in variety of outcome that is important to be noted in clinical use. In most studies, hypoxic condition appears to increase proliferation, differentiation, and immune regulatory performance of MSCs without affecting its characteristic. Those benefits are therefore utilized in clinical application. However, there are also studies that report on negative effects of hypoxia in MSCs such as chromosomal instability. Molecular mechanism of MSCs in hypoxic condition is provided for better understanding, which is crucial for further development with better outcome.

**Keywords:** mesenchymal stem cells, hypoxia

## **1. Introduction**

In these days, stem cell therapy is becoming more believable in treating degenerative diseases compared to conventional medicine. Various diseases such as diabetes, myocardial infarction, spinal cord injury, stroke, and Parkinson's and Alzheimer's diseases have become more prevalent with increasing life expectancy. It has been estimated that in the United States alone, ~128 million individuals would benefit from regenerative stem cell therapy during their lifetime. Mesenchymal stem cells (MSCs) have been highly utilized to treat degenerative diseases among other stem cells. These cells are found in tissues such as bone marrow, adipose tissue, umbilical cord,

© 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.

and dental pulp. Self-renewal and multipotency are the key features of MSCs that make it promising tool. These properties have raised interest on researchers for finding appropriate method to optimize the genetic and environmental factors, which later enhance the biological activities of MSCs.

Many researches have been conducted in the last two decades to study the complex processes in stem cell maintenance. The role of hypoxic conditions (usually 2–9% O2 concentration) on stem cell biology is very interesting subject due to its beneficial effects. Thus, cultivation of MSCs under hypoxia is currently studied to obtain better understanding, as well as further development to generate better outcome.

## **2. Mesenchymal stem cell**

About 130 years ago, German pathologist Conheim proposed the presence of non-hematopoietic stem cells in the bone marrow that contributes to wound healing in numerous peripheral tissues. Later in the early 1970s, Friedenstein and colleagues demonstrated that the rodent bone marrow had fibroblastoid cells with clonogenic potential *in vitro* [1, 2]. In the study, after the non-adherent cells were removed a few hours later, spindle-like cells, which were morphologically heterogeneous, appeared to attach to the plastic, capable of forming colonies. These cells could also make bone and reconstitute a hematopoietic microenvironment in subcutaneous transplants. Moreover, they could regenerate heterotopic bone tissue in serial transplants, thus indicated their self-renewal potential. Over the years, many studies have investigated these findings and found that these cells were also present in the human bone marrow and could be sub-passaged and differentiated *in vitro* into a variety of the mesenchymal lineages such as osteoblasts, chondrocytes, adipocytes, and myoblasts [3–7]. It has been further renamed as "mesenchymal stem cell" or MSC [4].

MSCs or MSC-like cells are also found in fat, umbilical cord blood, amniotic fluid, placenta, dental pulp, tendons, synovial membrane, and skeletal muscle, yet the complete equivalency of such populations remains unclear [8–16]. Characteristic of MSCs according to The International Society for Cell Therapy [17] consists of (1) adherence to plastic in standard culture conditions; (2) expression of the surface molecules CD73, CD90, and CD105 in the absence of CD34, CD45, HLA-DR, CD14 or CD11b, CD79a, or CD19 surface molecules; and (3) a capacity for differentiation to osteoblasts, adipocytes, and chondroblasts *in vitro*. These criteria were established to standardize human MSC isolation but may not apply uniformly to other species. For instance, marker expression and behavior in murine MSCs were different compared to human MSCs [18]. Certain *in vivo* surface markers may no longer be expressed after Transplantation, although new markers are obtained during expansion. In study done by Jones et al., MSC uniformly expressed HLA-DR (a marker that should not be expressed on MSCs by the above definition) while also expressing CD90 and CD105, adhering to plastic in culture, and differentiating into osteoblasts, adipocytes, and chondroblasts [19]. Indeed, clear definition of MSC-specific characteristics is difficult to apply in both human and animal models.

## **3. Hypoxia in mesenchymal stem cell**

and dental pulp. Self-renewal and multipotency are the key features of MSCs that make it promising tool. These properties have raised interest on researchers for finding appropriate method to optimize the genetic and environmental factors, which later enhance the biological

Many researches have been conducted in the last two decades to study the complex processes in stem cell maintenance. The role of hypoxic conditions (usually 2–9% O2 concentration) on stem cell biology is very interesting subject due to its beneficial effects. Thus, cultivation of MSCs under hypoxia is currently studied to obtain better understanding, as well as further

About 130 years ago, German pathologist Conheim proposed the presence of non-hematopoietic stem cells in the bone marrow that contributes to wound healing in numerous peripheral tissues. Later in the early 1970s, Friedenstein and colleagues demonstrated that the rodent bone marrow had fibroblastoid cells with clonogenic potential *in vitro* [1, 2]. In the study, after the non-adherent cells were removed a few hours later, spindle-like cells, which were morphologically heterogeneous, appeared to attach to the plastic, capable of forming colonies. These cells could also make bone and reconstitute a hematopoietic microenvironment in subcutaneous transplants. Moreover, they could regenerate heterotopic bone tissue in serial transplants, thus indicated their self-renewal potential. Over the years, many studies have investigated these findings and found that these cells were also present in the human bone marrow and could be sub-passaged and differentiated *in vitro* into a variety of the mesenchymal lineages such as osteoblasts, chondrocytes, adipocytes, and myoblasts [3–7]. It has been

MSCs or MSC-like cells are also found in fat, umbilical cord blood, amniotic fluid, placenta, dental pulp, tendons, synovial membrane, and skeletal muscle, yet the complete equivalency of such populations remains unclear [8–16]. Characteristic of MSCs according to The International Society for Cell Therapy [17] consists of (1) adherence to plastic in standard culture conditions; (2) expression of the surface molecules CD73, CD90, and CD105 in the absence of CD34, CD45, HLA-DR, CD14 or CD11b, CD79a, or CD19 surface molecules; and (3) a capacity for differentiation to osteoblasts, adipocytes, and chondroblasts *in vitro*. These criteria were established to standardize human MSC isolation but may not apply uniformly to other species. For instance, marker expression and behavior in murine MSCs were different compared to human MSCs [18]. Certain *in vivo* surface markers may no longer be expressed after Transplantation, although new markers are obtained during expansion. In study done by Jones et al., MSC uniformly expressed HLA-DR (a marker that should not be expressed on MSCs by the above definition) while also expressing CD90 and CD105, adhering to plastic in culture, and differentiating into osteoblasts, adipocytes, and chondroblasts [19]. Indeed, clear definition of MSC-specific characteristics is difficult to apply in both human and animal models.

activities of MSCs.

92 Hypoxia and Human Diseases

development to generate better outcome.

further renamed as "mesenchymal stem cell" or MSC [4].

**2. Mesenchymal stem cell**

Numerous *in vitro* studies have been conducted in the last two decades to observe the complex processes in stem cell maintenance. However, the role of physiologically hypoxic conditions (usually 2–9% O2 concentration) on stem cell biology received very little attention. O2 concentration is an environmental factor that plays a vital role on stem cell fate and function [20]. Stem cells are typically cultured under the ambient O2 concentration without paying attention to the metabolic milieu of the niche in which they normally grow [21]. The effects of different O2 levels in MSC culture were first studied in 1958, when Cooper et al. and Zwartouw and Westwood observed that some cells proliferated more rapidly under low O2 tension levels compared to normal atmospheric levels [22, 23]. MSCs are present in perivascular niches in close association with blood vessels in virtually all tissues [11, 16, 24] and have been compared to pericytes [25]. Even though MSCs are located close to vascular structures, the different tissues where these stem cells are found exhibit low oxygen tensions [26–29]. Therefore, it is possible that maintaining MSCs in an undifferentiated state may require a hypoxic environment, in addition to other factors.

The higher O2 concentration might cause environmental stress to the *in vitro* cultured MSCs. Recent studies have presented significant evidences regarding the negative outcome under ambient O2 concentration on MSCs, including early senescence, longer population doubling (PD) time, DNA damage [30, 31], and poor engraftment following transplantation [32, 33]. These have shown the influential effect of O2 concentration on MSCs biology and raised serious concern over its therapeutic efficiency and biosafety. Thus, the effect of different O2 concentration on MSCs biology is further discussed based on recent research outcomes.

## **4. Characteristic of MSCs in hypoxic condition**

As described above, MSC immunophenotype is characterized by the expression of CD73, CD90, CD105, CD106, CD146, and MHC class I molecules, and the absence of markers such as CD45 and CD34 or MHC class II molecules [17]. Many studies suggest that hypoxia has no effect on MSCs characteristic, indicated by surface markers. According to one study by Holzwarth et al., there were no significant differences in the expression of cell surface markers after 14 days of culture at 1% when compared to 20% of O2 [34]. Referring to study carried out by Nekanti et al., WJ-MSCs cultured under both hypoxia and normoxia for 10 passages were positive for CD44, CD73, CD90, CD105, and CD166 and negative for CD34, CD45, and HLA-DR, and there was no significant difference between the two populations [35]. These results are also supported by study carried out by Widowati et al. The surface marker of WJ-MSCs of P4 and P8 both normoxic and hypoxic 5% O2 were not significantly different. WJ-MSCs were positive for CD105, CD73, and CD90 and negative for CD34, CD45, CD14, CD19, and HLA-II [36].

Morphology changes are also documented in MSCs under hypoxia. Referring to Nekanti et al., WJ-MSC cultured under hypoxia showed a higher amount of large, flattened cells both at early and late passages, compared to normoxic cultures. The enlargement in cell size under hypoxia might be due to a natural response to low oxygen, in which increased surface area would allow for an increase in oxygen diffusion rate [35].

## **5. MSCs proliferation in hypoxic condition**

Capability for self-renewal is a key feature of stem cells. An increased proliferation rate is necessary for more efficient use of stem cells in regenerative therapies. Fehrer et al. demonstrated that bone marrow-derived MSCs (BM-MSCs) cultured in 3% O2 concentration showed significant increased *in vitro* proliferative lifespan, with ~10 additional population doublings (PDs) (28.5 ± 3.8 PD in 20% O2 and 37.5 ± 3.4 PD in 3% O2) before reaching senescence compared to cells cultured in the ambient O2 environment [31]. In addition, early passaged MSCs cultured in hypoxic conditions also exhibit increased proliferative lifespan along with significant difference in population doubling [30]. Furthermore, it is possible to harvest more than 1 × 109 MSCs from the first five passages cultured in 3% O2, whereas in ambient condition only 2 × 107 cells can be obtained [30]. Higher *in vitro* expansion rate in hypoxic conditions has also been reported by other researchers [37–39]. Such *in vitro* culture environment also allows to maintain a higher proportion of rapidly self-renewing MSCs for a longer period of time [40]. Other study showed that the increased hypoxic (O2 2.5%) condition was the best microenvironment for stem cell proliferation compared to normoxic and hypoxic (O2 5%) for cells at a high passage (P7, P8) [41].

However, various responses of stem cells under hypoxia have been reported [42]. Those differences in cellular responses on hypoxia might be associated with degrees and durations of hypoxia, as well as other cell conditions. Oxygen tension in the stem cell niche for MSCs is suggested to be various from 1 to 7% [43]. A study by Holzwarth et al. showed that rates of MSCs proliferation were reduced after 7 days of culture under hypoxia at 21, 5, 3, and 1% O2. In their study, only 1.37% of the cells entered the G2/M phase in hypoxic cultures (1% O2) after 7 days, compared to 2.50% at hyperoxic culture (21% O2). Reduced O2 concentrations were therefore confirmed to inhibit cell proliferation as indicated by reduced number of cells in the G2/M phase [34].

## **6. Chromosomal stability of MSCs in hypoxic condition**

Some recent studies have found that human mesenchymal stem cells (hMSCs) retained chromosomal stability following long-term culture *in vitro* [44–46]. Hypoxic environments have shown to increase mutation frequencies in cancer cell lines and trigger genomic rearrangements [47, 48]. It is suggested that oxygen concentration has a major impact on karyotypic aberration. Referring to study of Ueyama et al., chromosomal instability is associated with repeated cell division. A high frequency of chromosomal abnormality breakpoints in common fragile sites (CFSs) was detected by karyotypic analysis (e.g., 2q33, 7q11, 7q36, 8q22.1, 8q24.1, 11p15.1, 19q13) [49]. Generally, chromosomes have fragile sites that are prone to exhibiting gaps and breaks during metaphase [50], in which chromosome rearrangement occurs in cultured cells. Fragile sites are categorized into two main classes, common and rare, according to their frequency in the population [51]. In Ueyama study, several genes involved in regulation of the cell cycle, transcription and cell adhesion, are located in that region with a frequency of 6, 5, and 2%, respectively. In particular, the 11p15.5 domain known as an important tumor suppressor gene region such as tetraspanin 32 (TSPAN32) and tumor-suppressing subtransferable candidate 4 (TSSC4) is present in this region. Alterations in this region have been associated with some neoplasia. It is suggested that the deletion of contiguous genes may induce a multisystem developmental disorder and that these alterations might influence normal functioning and cell survival.

Sex chromosome aneuploidy was also one of the most observed aberrational karyotypes. Frequency of sex chromosome in cultured lymphocytes was significantly higher in females than in males, and that loss of Y chromosomes correlated with age in human bone marrow cells [52, 53]. There are several factors influencing karyotypic stability such as hypoxic culture conditions, donor age, and multiple passages. Karyotypic aberrations increased with passage number and hMSCs undergo spontaneous transformation with tumorigenic potential, especially in later passages under hypoxic culture conditions in hMSCs of elderly donors [49]. Shortly, monitoring of chromosomal stability in culture expanded hMSCs is required prior to exposure to human beings, in order to detect mutations and potentially immortalized clones and to prevent transplant-associated tumor formation.

## **7. MSCs plasticity in hypoxic condition**

early and late passages, compared to normoxic cultures. The enlargement in cell size under hypoxia might be due to a natural response to low oxygen, in which increased surface area

Capability for self-renewal is a key feature of stem cells. An increased proliferation rate is necessary for more efficient use of stem cells in regenerative therapies. Fehrer et al. demonstrated that bone marrow-derived MSCs (BM-MSCs) cultured in 3% O2 concentration showed significant increased *in vitro* proliferative lifespan, with ~10 additional population doublings (PDs) (28.5 ± 3.8 PD in 20% O2 and 37.5 ± 3.4 PD in 3% O2) before reaching senescence compared to cells cultured in the ambient O2 environment [31]. In addition, early passaged MSCs cultured in hypoxic conditions also exhibit increased proliferative lifespan along with significant difference in population doubling [30]. Furthermore, it is possible to harvest more than

MSCs from the first five passages cultured in 3% O2, whereas in ambient condition only

 cells can be obtained [30]. Higher *in vitro* expansion rate in hypoxic conditions has also been reported by other researchers [37–39]. Such *in vitro* culture environment also allows to maintain a higher proportion of rapidly self-renewing MSCs for a longer period of time [40]. Other study showed that the increased hypoxic (O2 2.5%) condition was the best microenvironment for stem cell proliferation compared to normoxic and hypoxic (O2 5%) for cells at a

However, various responses of stem cells under hypoxia have been reported [42]. Those differences in cellular responses on hypoxia might be associated with degrees and durations of hypoxia, as well as other cell conditions. Oxygen tension in the stem cell niche for MSCs is suggested to be various from 1 to 7% [43]. A study by Holzwarth et al. showed that rates of MSCs proliferation were reduced after 7 days of culture under hypoxia at 21, 5, 3, and 1% O2. In their study, only 1.37% of the cells entered the G2/M phase in hypoxic cultures (1% O2) after 7 days, compared to 2.50% at hyperoxic culture (21% O2). Reduced O2 concentrations were therefore confirmed to inhibit cell proliferation as indicated by reduced number of cells in the

Some recent studies have found that human mesenchymal stem cells (hMSCs) retained chromosomal stability following long-term culture *in vitro* [44–46]. Hypoxic environments have shown to increase mutation frequencies in cancer cell lines and trigger genomic rearrangements [47, 48]. It is suggested that oxygen concentration has a major impact on karyotypic aberration. Referring to study of Ueyama et al., chromosomal instability is associated with repeated cell division. A high frequency of chromosomal abnormality breakpoints in common fragile sites (CFSs) was detected by karyotypic analysis (e.g., 2q33, 7q11, 7q36, 8q22.1, 8q24.1,

**6. Chromosomal stability of MSCs in hypoxic condition**

would allow for an increase in oxygen diffusion rate [35].

**5. MSCs proliferation in hypoxic condition**

1 × 109

94 Hypoxia and Human Diseases

2 × 107

high passage (P7, P8) [41].

G2/M phase [34].

The multilineage potential of MSCs is one of the reasons underlying their use in regenerative medicine [54]. Results of MSC differentiation into other lineages diverse according to several studies [34, 55, 56]. Some *in vitro* studies have shown that cultures with low O2 concentrations stimulated cells to differentiate into adipogenic, osteogenic, or chondrogenic cells. Previous study showed that Rat mesenchymal stem cells (rMSCs) cultured in 5% oxygen produced more bone than cells cultured in 20% O2 throughout their cultivation time, as indicated by increased markers of osteogenesis, including alkaline phosphatase activity, calcium content, and von Kossa staining. These markers were usually elevated above basal levels when cells were switched from control to low oxygen at first passage and decreased for cells switched from low to control oxygen [57]. Hypoxia appears to exert a potent lipogenic effect independent of PPAR-γ2 maturation pathway [58]. The level of differentiated antigen H-2Dd and the number of G2/S/M phase cells increased evidently under 8% O2 condition. Also, the proportion of wide, flattened, and epithelial-like cells increased significantly in MSCs. When cultured in adipogenic medium, there was a fivefold to sixfold increase in the number of lipid droplets under hypoxic conditions compared with that in normoxic culture. Oct4 was downregulated under 8% O2 condition but still expressed after adipocyte differentiation in normoxic culture and treated with hypoxia-mimicking agents, cobalt chloride (CoCl2) and deferoxamine mesylate (DFX). These findings indicate that hypoxia enhances MSC differentiation and hypoxia and hypoxia-mimicking agents generate different effects on MSC differentiation [59].

Conversely, some others have reported suppressive effects of low O2 tension levels on the plasticity of MSCs. Differentiation capacity into adipogenic progeny was diminished, and no osteogenic differentiation was detected at 3% oxygen. In turn, MSC that had previously been cultured at 3% oxygen could subsequently be stimulated to successfully differentiate at 20% oxygen [31]. Temporary exposure of MSCs to hypoxia resulted in (i) persistent (up to 14 days postexposure) downregulation of cbfa-1/Runx2, osteocalcin, and type I collagen and (ii) permanent (up to 28 days postexposure) upregulation of osteopontin mRNA expressions [60]. Another study by Widowati et al. showed both nor-WJ-MSCs and hypo-WJ-MSCs differentiated to osteocytes, chondrocytes, and adipocytes, although there was no significant difference among treatments [36]. Study conducted by Georgi et al. showed that molecular fingerprints of human MSCs, primary chondrocytes, and MSC/primary chondrocytes coculture differ when cultured in either normoxic (21% O2) or hypoxic (2.5% O2) conditions [61]. In the study, cartilage formation increased in cocultures of MSCs and primary chondrocytes was lost when the cells were cultured under hypoxia which was associated with a decrease in the mRNA expression of the chondrogenic marker SOX9 and FGF-1. This coincided with a significant decrease in lipids. Lipid profiles of normoxic and hypoxic cultures are different. The improved cartilage formation in cocultures of MSCs and chondrocytes may employ soluble factors, including small molecules, lipids, or proteins [62]. Lipids such as phospholipids, cholesterol, and diacylglycerols play significant roles in cellular signaling, membrane integrity, and metabolism [63]. Recent study described that short-term changes in sphingolipid metabolism resulted in long-term effects on the chondrogenic phenotype, and the stimulation of chondrocytes with acylceramidase improves cartilage repair and MSC differentiation [64].

## **8. Immunomodulatory effects of MSCs in hypoxic condition**

One of the key factors of MSC in therapeutics development is their known anti-inflammatory/ immunomodulatory properties. Clinical studies showed efficacy of MSC at inhibiting lethal, immune-based condition of graft versus host disease [65–70]. It has been reported that MSCs derived from adipose, bone marrow, and placenta have the capability to recover ischemic injury by increasing vascularization and reducing inflammation in ischemia-injured hindlimb, lung, heart, and brain [71–73]. Thus, these cells have been used in clinical trials to treat ischemic disease [74]. MSCs produce a broad variety of cytokines, chemokines, and growth factors that may potentially be involved in tissue repair. Hypoxia increases the production of several of these factors, although different responses are also noted in few studies. Referring to Chang et al., hypoxic preconditioning enhances the capacity of the secretome obtained from cultured human MSCs to release several of these factors and the therapeutic potential of the cultured MSC secretome in experimental TBI [75].

One of the most studied mechanisms of inflammation-induced MSC activity is treatment with interferon gamma (IFN-γ). This cytokine is usually secreted during inflammatory Th1 immune responses that are associated with autoimmunity mediated by cellular means, such as CD8 T cells and NK cells, which commonly occur in multiple sclerosis, diabetes type 1, and rheumatoid arthritis [76]. Treatment of IFN-γ in MSC has been reported to enhance the immunosuppressive activity through stimulation of the enzyme IDO [77–80]. MSC expression of the tryptophan-catabolizing enzyme indolamine 2,3 deoxygenase (IDO) was markedly upregulated under hypoxia [81]. IDO is critical in immune regulation by MSC through induction of T cell anergy [82] and stimulation of T regulatory cells (T-regs) [83, 84].

(DFX). These findings indicate that hypoxia enhances MSC differentiation and hypoxia and

Conversely, some others have reported suppressive effects of low O2 tension levels on the plasticity of MSCs. Differentiation capacity into adipogenic progeny was diminished, and no osteogenic differentiation was detected at 3% oxygen. In turn, MSC that had previously been cultured at 3% oxygen could subsequently be stimulated to successfully differentiate at 20% oxygen [31]. Temporary exposure of MSCs to hypoxia resulted in (i) persistent (up to 14 days postexposure) downregulation of cbfa-1/Runx2, osteocalcin, and type I collagen and (ii) permanent (up to 28 days postexposure) upregulation of osteopontin mRNA expressions [60]. Another study by Widowati et al. showed both nor-WJ-MSCs and hypo-WJ-MSCs differentiated to osteocytes, chondrocytes, and adipocytes, although there was no significant difference among treatments [36]. Study conducted by Georgi et al. showed that molecular fingerprints of human MSCs, primary chondrocytes, and MSC/primary chondrocytes coculture differ when cultured in either normoxic (21% O2) or hypoxic (2.5% O2) conditions [61]. In the study, cartilage formation increased in cocultures of MSCs and primary chondrocytes was lost when the cells were cultured under hypoxia which was associated with a decrease in the mRNA expression of the chondrogenic marker SOX9 and FGF-1. This coincided with a significant decrease in lipids. Lipid profiles of normoxic and hypoxic cultures are different. The improved cartilage formation in cocultures of MSCs and chondrocytes may employ soluble factors, including small molecules, lipids, or proteins [62]. Lipids such as phospholipids, cholesterol, and diacylglycerols play significant roles in cellular signaling, membrane integrity, and metabolism [63]. Recent study described that short-term changes in sphingolipid metabolism resulted in long-term effects on the chondrogenic phenotype, and the stimulation of chondro-

hypoxia-mimicking agents generate different effects on MSC differentiation [59].

96 Hypoxia and Human Diseases

cytes with acylceramidase improves cartilage repair and MSC differentiation [64].

One of the key factors of MSC in therapeutics development is their known anti-inflammatory/ immunomodulatory properties. Clinical studies showed efficacy of MSC at inhibiting lethal, immune-based condition of graft versus host disease [65–70]. It has been reported that MSCs derived from adipose, bone marrow, and placenta have the capability to recover ischemic injury by increasing vascularization and reducing inflammation in ischemia-injured hindlimb, lung, heart, and brain [71–73]. Thus, these cells have been used in clinical trials to treat ischemic disease [74]. MSCs produce a broad variety of cytokines, chemokines, and growth factors that may potentially be involved in tissue repair. Hypoxia increases the production of several of these factors, although different responses are also noted in few studies. Referring to Chang et al., hypoxic preconditioning enhances the capacity of the secretome obtained from cultured human MSCs to release several of these factors and the therapeutic potential of the cultured

One of the most studied mechanisms of inflammation-induced MSC activity is treatment with interferon gamma (IFN-γ). This cytokine is usually secreted during inflammatory Th1 immune

**8. Immunomodulatory effects of MSCs in hypoxic condition**

MSC secretome in experimental TBI [75].

Moreover, IFN-γ induced secretion of other inhibitors of inflammation by MSCs, including the complement inhibitor factor H [85], as well as the immunomodulatory molecules TGF-β and HGF [86]. At a functional level, Noone et al. demonstrated that IFN-γ pretreatment of MSC resulted in protection of MSCs from NK-mediated killing via upregulation of prostaglandin E (PGE)-2 synthesis [87]. IFN-γ, along with necrosis factor-alpha (TNF-α), IL-1α, and IL-1β, induces Gal-9 in MSC [88].

Another inflammatory mediator known to induce regenerative activities in MSC is the macrophage-derived cytokine TNF-α. Pretreatment of TNF-α in MSCs provided superior angiogenic activity *in vitro*, as indicated by expression of VEGF, as well as *in vivo* in an animal model of critical limb ischemia, as compared to untreated MSCs [89]. In other study, TNF-α preconditioning increased proliferation, mobilization, and osteogenic differentiation of MSCs and upregulated bone morphogenetic protein-2 (BMP-2) protein level. Osteogenic differentiation of MSC induced by TNF-α was partially inhibited after BMP-2 knockdown by siRNA [90]. Lipopolysaccharide and toll-like receptor (TLR) agonists, as activators of innate immunity, are also responsible for regenerative activity of MSCs by inducing paracrine factors secretion such as VEGF [91]. IFN-γ and TLR also upregulate the glucocorticoids production, which decreases T cells stimulated by radiotherapy in colonic mucosa [92].

Akiyama et al. reported that MSCs induced T cell apoptosis via the Fas/FasL pathway [93]. Telomerase improved immunomodulatory properties of MSCs by upregulating FasL expression [94]. Dental follicle cells and cementoblasts have been reported to trigger apoptosis of ameloblast-lineage cells, as well as Hertwig's epithelial root sheath (HERS)/epithelial rests of Malassez (ERM) cells, via the Fas/FasL pathway during tooth development [95]. FasL regulated the immunomodulatory properties of Human gingiva-derived mesenchymal stem cells (hGMSCs), which is promoted by hypoxia. However, the underlying pathways of such event remain unclear. Further studies regarding the pathways involved in hGMSC-mediated immunomodulation are encouraged.

## **9. Molecular mechanism of MSCs in hypoxic condition**

O2 concentration in the stem cell niche (usually 2–9% O2) is considered a driver of cell function [20]. Hypoxia plays a vital role in maintaining homeostasis within the body from the early stage of embryonic development. It facilitates proper embryonic development, maintains stem cell pluripotency, induces differentiation, and regulates the signaling of multiple cascades, including angiogenesis [96]. In hypoxic conditions, these functions are regulated by several transcription factors such as hypoxia-inducible factors (HIFs), prolyl hydroxylases (PHDs), factor-inhibiting HIF-1 (FIH-1), activator protein 1 (AP-1), nuclear factor (NF)-*κ*B, p53, and c-Myc [97]. Although interaction among all of the transcription factors is required for cellular response, HIFs (especially HIF-1) are the key regulators of cellular response to hypoxia [98]. The discovery of HIF-1a by Greg Semenza provided profound insight into the cellular mechanisms that control hypoxic adaptation [99–101].

Generally, under hypoxic conditions, low O2 level suppresses the prolyl hydroxylation that leads to HIF-1*α* accumulation and nuclear translocation [102]. After nuclear translocation, it binds with HIF-1*β* to form the heterodimer. Then, the HIF-1 heterodimer binds to a hypoxiaresponse element (HRE) in the target genes, associated with coactivators such as CBP/p300, and regulates the transcription (**Figure 1**) of as many as 70 genes involved in metabolism, angiogenesis, invasion/metastasis, and cell fate [103].

**Figure 1.** Regulation of hypoxia in MSCs. HIF, hypoxia-inducible factor; HPH, HIF-prolyl hydroxylases; HPE, HIFprolyl hydroxylases; HRE, hypoxia-response element; CDK, cyclin-dependent kinase; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor; IGF, insulin-like growth factor; TNF, tumor necrosis factor; PGE-2, prostaglandin E-2; Gal9, galectin-9. The prolyl hydroxylation process is suppressed due to lack of O2 that leads HIF-1α accumulation and nuclear translocation after nuclear translocation, and it binds HIF-1β to form the heterodimer. Then, the HIF-1 heterodimer binds HRE in the genes target, associated with coactivators such as CBP/ p300, and regulates the transcription of as many as 70 genes involved in proliferation, differentiation, and immunomodulatory.

In 2007, iPSCs were discovered by Shinya Yamanaka and colleagues, and the subsequent identification of the necessary transcriptional programs was required to maintain stem cells in a pluripotent state [104, 105]. The measurement of low partial pressures of oxygen in various stem cell niches raises question whether HIF-1a and iPSCs pathways were converged. It was described initially in Embryonic stem cells (ESCs) [106], hematopoietic stem cells (HSCs) [107], neural stem cells (NSCs) [108], and cancer stem cells (CSCs) [109], which now further expanded to include iPSCs [110]. Remarkably, Yamanaka first reprogrammed fibroblasts to iPSCs using only four transcription factors (Oct4, Sox2, c-Myc, and Klf4) [105] in the same year that Oct4 was shown to be a specific target gene of HIF-2a [111]. The correlation between HIF-2a and Oct4 has been proposed as underlying mechanism of stem cells response to hypoxic conditions in their niche and direct modification of stem cell function by low O2. HIF-2a expression has recently been investigated in several stem cell lineages, and Oct4 expression is tightly regulated throughout embryogenesis. Loss or even decrease in Oct4 expression leads to differentiation [112]. Oct4 works in concert with Nanog and Sox2 to maintain stem cell identity and repress genes that promote differentiation [113]. The recent identification of HIF-2a upregulation by Oct4 in CSCs and ESCs underscores the importance of this axis in maintaining stemness in both development and disease.

transcription factors such as hypoxia-inducible factors (HIFs), prolyl hydroxylases (PHDs), factor-inhibiting HIF-1 (FIH-1), activator protein 1 (AP-1), nuclear factor (NF)-*κ*B, p53, and c-Myc [97]. Although interaction among all of the transcription factors is required for cellular response, HIFs (especially HIF-1) are the key regulators of cellular response to hypoxia [98]. The discovery of HIF-1a by Greg Semenza provided profound insight into the cellular

Generally, under hypoxic conditions, low O2 level suppresses the prolyl hydroxylation that leads to HIF-1*α* accumulation and nuclear translocation [102]. After nuclear translocation, it binds with HIF-1*β* to form the heterodimer. Then, the HIF-1 heterodimer binds to a hypoxiaresponse element (HRE) in the target genes, associated with coactivators such as CBP/p300, and regulates the transcription (**Figure 1**) of as many as 70 genes involved in metabolism,

**Figure 1.** Regulation of hypoxia in MSCs. HIF, hypoxia-inducible factor; HPH, HIF-prolyl hydroxylases; HPE, HIFprolyl hydroxylases; HRE, hypoxia-response element; CDK, cyclin-dependent kinase; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor; IGF, insulin-like growth factor; TNF, tumor necrosis factor; PGE-2, prostaglandin E-2; Gal9, galectin-9. The prolyl hydroxylation process is suppressed due to lack of O2 that leads HIF-1α accumulation and nuclear translocation after nuclear translocation, and it binds HIF-1β to form the heterodimer. Then, the HIF-1 heterodimer binds HRE in the genes target, associated with coactivators such as CBP/ p300, and regulates the transcription of as many as 70 genes involved in proliferation, differentiation, and immunomo-

mechanisms that control hypoxic adaptation [99–101].

98 Hypoxia and Human Diseases

angiogenesis, invasion/metastasis, and cell fate [103].

dulatory.

It is known that phosphorylation of protein kinase B (Akt), a downstream gene of phosphatidylinositol 3-kinase (PI3K) signaling pathway, is an important step in signaling pathways that mediate cell proliferation [114, 115]. In PI3K/Akt pathway, a large number of substrates are phosphorylated, including HIF-1 [116]. Referring to study done by Rosová et al., the preculture of MSCs in hypoxia prior to injection activated the PI3K/Akt signaling pathway while maintaining their viability and cell cycle rates [117].

Hypoxia-mediated MSC differentiation by reducing apoptosis via activating the PI3K/Akt/ FoxO pathway. Referring to Wang et al., MSCs underwent apoptosis upon induction for chondrogenic differentiation [118]. Apoptosis has been demonstrated as a general phenomenon that occurs during endochondral differentiation of chondrocytes [119]. One study demonstrated that chondrocytes progression to endochondral ossification employed higher FAS receptor and caspase protein as indicators of apoptosis [120]. Other studies showed that both the Wnt/beta-catenin and Indian hedgehog (Ihh) signaling pathways play important roles in endochondral ossification. Beta-catenin is needed at upstream of Ihh signaling for chondrocyte survival and inhibition of apoptosis [121]. The expression of Sox9, col2a1 and aggrecan in prechondrogenic cells 30 and chondrocytes 14 is regulated by PI3K/Akt pathway. It has also been demonstrated that PI3K/Akt regulated col2a1 and aggrecan by modulating Sox9 expression and transcriptional activity in nucleus pulposus cells 31.

Lee et al. [122] showed novel pathway for hypoxia-induced proliferation and migration in human mesenchymal stem cells that employ HIF-1α, FASN, and mTORC1O. Hypoxia treatment stimulates UCB-hMSC proliferation, along with the expression of two lipogenic enzymes: fatty acid synthase (FASN) and stearoyl-CoA desaturase-1 (SCD1). FASN is a key enzyme in UCB-hMSC proliferation and migration. Hypoxia-induced FASN expression was regulated by the HIF-1α/SCAP/SREBP1 pathway. Mammalian target of rapamycin (mTOR) was phosphorylated by hypoxia, whereas inhibition of FASN by cerulenin suppressed hypoxia-induced mTOR phosphorylation, as well as UCB-hMSC proliferation and migration. Hypoxia-induced proliferation and migration are significantly inhibited by raptor small interfering RNA. Hypoxia-induced mTOR also regulates CDK2, CDK4, cyclin D1, cyclin E, and F-actin expression as well as c-myc, p-cofilin, profilin, and Rho GTPase. Moreover, hypoxia-induced FASN stimulates FFA production as well as proliferation and migration. Several studies reported that FAS and FA derivatives inhibited and uncoupled oxidative phosphorylation of various cells [123–125]. Palmitic acid treatment rescues inhibition of mTOR phosphorylation as well as restriction of UCB-hMSC proliferation and migration. Change in cellular metabolite ratios may be another pathway, in addition to the HIF1a/SCAP/SREBP1 pathway, involved in the regulation of lipid metabolism in UCB-hMSCs. Some studies reported that alteration of cellular metabolite ratios, such as NADP/NADPH, by hypoxia has also an important role in the regulation of various stem cell functions such as cell cycle and selfrenewal activities [126, 127].

## **10. Hypoxic MSCs in clinical application**

MSCs possess anti-inflammatory/immunomodulatory properties, which are utilized in therapeutics development. Clinical studies on efficacy of MSCs have been shown to inhibit lethal, immune-based condition of graft versus host disease [65–70]. Moreover, MSCs derived from adipose, bone marrow, and placenta have the capability to recover ischemic injury by increasing vascularization and reducing inflammation in ischemia-injured hindlimb, lung, heart, and brain [71–73, 128]. These cells have been used in clinical trials to treat ischemic disease [74], and the safety of MSCs has been evaluated [129, 130]. There are several modified approaches, which have been proposed to improve the effect of MSCs on ischemia-related disease, such as over expression of angiogenesis-related genes such as bFGF on MSCs [131], combination with other cells such as endothelial cells [84], antioxidants such as melatonin [132], serum deprivation [72], and cell spheroids [133].

From isolation to engraftment, the MSCs usually pass through two different phases, consisting of *in vitro* culture condition (from isolation to transplantation) and *in vivo* or physiological condition (before isolation and after transplantation). At present, most of the expansion procedures of MSCs are performed under ambient O2 concentration, where cells are exposed to 20% O2, which is ~4–10 times more than the concentration of O2 in their natural niches [134, 135]. Maintaining genetic stability has been a challenge during *in vitro* expansion of MSCs. Increased rates of aneuploidy, double-stranded DNA breakdown, and faster telomere shortening have been reported for MSCs cultured in ambient condition [30]. According to recent review, major causes behind aneuploidy were defective spindle assembly checkpoint, centrosome amplification, and merotelic attachments [136], which are caused by ROS [137]. ROS also acts in acceleration of telomere shortening and DNA breakdown [138, 139]. Correlation between telomere shortening and aneuploidy in embryonic and hepatocellular carcinoma cells has also been reported in recent studies [140, 141]. The higher ROS production due to the increased mitochondrial respiration during expansion of MSCs in ambient O2 concentration might be the cause behind genetic instability in them. However, cells undergo anaerobic respiration during hypoxia, which lowers the ROS concentration within the cells. This might reduce the DNA damage, telomere shortening, and aneuploidy which in return may increase the biosafety of stem cell-based therapy.

hypoxia-induced mTOR phosphorylation, as well as UCB-hMSC proliferation and migration. Hypoxia-induced proliferation and migration are significantly inhibited by raptor small interfering RNA. Hypoxia-induced mTOR also regulates CDK2, CDK4, cyclin D1, cyclin E, and F-actin expression as well as c-myc, p-cofilin, profilin, and Rho GTPase. Moreover, hypoxia-induced FASN stimulates FFA production as well as proliferation and migration. Several studies reported that FAS and FA derivatives inhibited and uncoupled oxidative phosphorylation of various cells [123–125]. Palmitic acid treatment rescues inhibition of mTOR phosphorylation as well as restriction of UCB-hMSC proliferation and migration. Change in cellular metabolite ratios may be another pathway, in addition to the HIF1a/SCAP/SREBP1 pathway, involved in the regulation of lipid metabolism in UCB-hMSCs. Some studies reported that alteration of cellular metabolite ratios, such as NADP/NADPH, by hypoxia has also an important role in the regulation of various stem cell functions such as cell cycle and self-

MSCs possess anti-inflammatory/immunomodulatory properties, which are utilized in therapeutics development. Clinical studies on efficacy of MSCs have been shown to inhibit lethal, immune-based condition of graft versus host disease [65–70]. Moreover, MSCs derived from adipose, bone marrow, and placenta have the capability to recover ischemic injury by increasing vascularization and reducing inflammation in ischemia-injured hindlimb, lung, heart, and brain [71–73, 128]. These cells have been used in clinical trials to treat ischemic disease [74], and the safety of MSCs has been evaluated [129, 130]. There are several modified approaches, which have been proposed to improve the effect of MSCs on ischemia-related disease, such as over expression of angiogenesis-related genes such as bFGF on MSCs [131], combination with other cells such as endothelial cells [84], antioxidants such as melatonin

From isolation to engraftment, the MSCs usually pass through two different phases, consisting of *in vitro* culture condition (from isolation to transplantation) and *in vivo* or physiological condition (before isolation and after transplantation). At present, most of the expansion procedures of MSCs are performed under ambient O2 concentration, where cells are exposed to 20% O2, which is ~4–10 times more than the concentration of O2 in their natural niches [134, 135]. Maintaining genetic stability has been a challenge during *in vitro* expansion of MSCs. Increased rates of aneuploidy, double-stranded DNA breakdown, and faster telomere shortening have been reported for MSCs cultured in ambient condition [30]. According to recent review, major causes behind aneuploidy were defective spindle assembly checkpoint, centrosome amplification, and merotelic attachments [136], which are caused by ROS [137]. ROS also acts in acceleration of telomere shortening and DNA breakdown [138, 139]. Correlation between telomere shortening and aneuploidy in embryonic and hepatocellular carcinoma cells has also been reported in recent studies [140, 141]. The higher ROS production due to the increased mitochondrial respiration during expansion of MSCs in ambient O2 concentration might be the cause behind genetic instability in them. However, cells undergo anaerobic

renewal activities [126, 127].

100 Hypoxia and Human Diseases

**10. Hypoxic MSCs in clinical application**

[132], serum deprivation [72], and cell spheroids [133].

The ability of stem or progenitor cells to home and engraft into target tissues after transplantation is the key to succeed in clinical application. The degree of homing and engraftment of MSCs in adult recipients is very low [142–144]. Hypoxic culture conditions may also provide a solution for more efficient engraftment. Recently, early passaged mouse BM-MSCs showed better engraftment than late passaged mouse BM-MSCs in *in vivo* model [145]. In other study, hypoxic preconditioned murine MSCs also enhanced skeletal muscle regeneration and improved blood flow and vascular formation compared to normoxic condition [146]. Furthermore, hypoxic conditions cause MSCs to grow faster [30] while maintaining a higher number of rapidly self-renewing cells [40]. Hypoxic environment also upregulated chemokine receptors CXCR4, CXCR7 and CX3CR1 [147, 148], and they may facilitate tissue-specific trafficking of MSCs. Thus, sufficient numbers of MSCs with a higher fraction of rapidly selfrenewing cells are suggested, and highly expressed chemokine receptors on their surface can be obtained from the early passages of hypoxic cultures, which could increase the efficiency of damaged tissue-specific migration and engraftment following transplantation.

MSCs cultured under hypoxic conditions also increased in vascular endothelial growth factor receptor 1 (VEGFR1) expression and VEGF- or placental growth factor (PLGF)-dependent migration(Okuyamaetal.,2006).Preconditioningwithoxygenandcombinedglucosedepletion also increased the survival of stem cell antigen (Sca)-1þ cells via PI3K/Akt-dependent caspase-3 downregulation and thereby increased the engraftment rate [149]. In addition to the increase in migration and survival, MSCs with hypoxic preconditioning have also been shown to enhance revascularization after transplantation for hindlimb ischemia [117]. Therefore, culturing MSCs in hypoxic conditions can also be considered as a solution for tissue-specific engraftment.

Hypoxia-stimulated immune regulation of MSCs has been observed in the situation of allogeneic use of BM-MSCs for stimulation of therapeutic angiogenesis. Recent study showed hypoxia-conditioned BM-MSCs from B6 mice repair limb of Balb/c mice compared to normoxic MSCs. Engraftment in allogeneic recipients increased by decreasing NK cells cytotoxicity and the accumulation of host-derived NK cells when transplanted *in vivo*. These allogeneic hypoxia-treated BM-MSCs increased CD31+ endothelial cells and αSMA+ and desmin + muscle cells, thereby enhancing angiogenesis and restoring muscle structure. Moreover, anti-NK antibodies along with normoxic MSCs enhanced angiogenesis and prevented limb amputation in allogeneic recipients with limb ischemia [150].

Some studies have shown that MSC transplantation contributes to tumor formation *in vivo* [24, 151, 152], whereas Furlani et al. reported that cultured MSCs with spontaneous transformations had no functional effects after intracardiac transplantation [153]. Further studies regarding tumorigenicity and safety of the stem cell-based products are encouraged. However, complexity of cell therapy requires more standards for advanced medicinal products [154]. Thus, especially in the field of regenerative medicine, concrete and specific standards and governmental support systems are necessary to promote their production [154].

## **11. Perspective of hypoxic MSCs**

Hypoxic condition has been confirmed to enhance MSCs proliferation, differentiation, and immune regulatory performance. However, some studies have also reported opposite and negative effects. Different outcomes in each study raise interest in availability of more appropriate methods for cell cultures, which require further study in standardizing the culture of MSCs for use in cell therapy. Optimal conditions for the culture of MSCs have not yet been clearly defined, and it is very crucial to precisely determine the effects of hypoxia on MSCs differentiation, proliferation, and morphology, among other aspects. Moreover, hypoxic MSCbased therapies require a complete understanding of stem cell molecular mechanism. The clarity in stem cell regulation is important for further development such as periodic monitoring of chromosomal stability in culture prior to exposure to human to detect mutations and to prevent transplant-associated tumor formation, and also genetic engineering of physiology of MSCs to acquire better outcome.

## **12. Conclusion**

The growing interest in the potential application of MSCs in regenerative medicine was followed by the several studies measuring the effects of low O2 levels on the behavior and function of MSCs. Hypoxic condition appears to enhance MSCs proliferation, differentiation, and immune regulatory performance in damaged tissues without affecting its characteristic. However, there are also studies that report on negative effects of hypoxia in MSCs.

## **Author details**

Wahyu Widowati1\*, Dwi Davidson Rihibiha2 , Khie Khiong2 , M. Aris Widodo3 , Sutiman B. Sumitro4 and Indra Bachtiar5

\*Address all correspondence to: wahyu\_w60@yahoo.com

1 Faculty of Medicine, Maranatha Christian University, Bandung, Indonesia

2 Biomolecular and Biomedical Research Center, Aretha Medika Utama, Bandung, Indonesia

3 Laboratory of Pharmacology, Faculty of Medicine, Brawijaya University, Malang, Indonesia

4 Department of Biology, Faculty of Mathematics and Natural Sciences, Brawijaya University, Malang, Indonesia

5 Stem Cell and Cancer Institute, Jakarta, Indonesia

## **References**

**11. Perspective of hypoxic MSCs**

MSCs to acquire better outcome.

**12. Conclusion**

102 Hypoxia and Human Diseases

**Author details**

Sutiman B. Sumitro4

Indonesia

Indonesia

ty, Malang, Indonesia

Wahyu Widowati1\*, Dwi Davidson Rihibiha2

and Indra Bachtiar5

\*Address all correspondence to: wahyu\_w60@yahoo.com

5 Stem Cell and Cancer Institute, Jakarta, Indonesia

Hypoxic condition has been confirmed to enhance MSCs proliferation, differentiation, and immune regulatory performance. However, some studies have also reported opposite and negative effects. Different outcomes in each study raise interest in availability of more appropriate methods for cell cultures, which require further study in standardizing the culture of MSCs for use in cell therapy. Optimal conditions for the culture of MSCs have not yet been clearly defined, and it is very crucial to precisely determine the effects of hypoxia on MSCs differentiation, proliferation, and morphology, among other aspects. Moreover, hypoxic MSCbased therapies require a complete understanding of stem cell molecular mechanism. The clarity in stem cell regulation is important for further development such as periodic monitoring of chromosomal stability in culture prior to exposure to human to detect mutations and to prevent transplant-associated tumor formation, and also genetic engineering of physiology of

The growing interest in the potential application of MSCs in regenerative medicine was followed by the several studies measuring the effects of low O2 levels on the behavior and function of MSCs. Hypoxic condition appears to enhance MSCs proliferation, differentiation, and immune regulatory performance in damaged tissues without affecting its characteristic.

, Khie Khiong2

, M. Aris Widodo3

,

However, there are also studies that report on negative effects of hypoxia in MSCs.

1 Faculty of Medicine, Maranatha Christian University, Bandung, Indonesia

2 Biomolecular and Biomedical Research Center, Aretha Medika Utama, Bandung,

3 Laboratory of Pharmacology, Faculty of Medicine, Brawijaya University, Malang,

4 Department of Biology, Faculty of Mathematics and Natural Sciences, Brawijaya Universi-


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114 Hypoxia and Human Diseases

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#### **Cardiovascular Adaptation to High-Altitude Hypoxia** Cardiovascular Adaptation to High-Altitude Hypoxia

Jun Ke, Lei Wang and Daliao Xiao Jun Ke, Lei Wang and Daliao Xiao

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

http://dx.doi.org/10.5772/65354

#### Abstract

High-altitude exposure has been well recognized as a hypoxia exposure that significantly affects cardiovascular function. However, the pathophysiologic adaptation of cardiovascular system to high-altitude hypoxia (HAH) varies remarkably. It may depend on the exposed time and oxygen partial pressure in the altitude place. In short-term HAH, cardiovascular adaptation is mainly characterized by functional alteration, including cardiac functional adjustments, pulmonary vascular constriction, transient pulmonary hypertension, and changes in cerebral blood flow (CBF). These changes may be explained mainly by ventilatory acclimatization and variation of autonomic nervous activity. In long-term HAH, cardiovascular adaptation is mainly characterized by both functional and structural alterations. These changes include right ventricle (RV) hypertrophy, persistent pulmonary hypertension, lower CBF and reduced uteroplacental and fetal volumetric blood flows.

Keywords: high altitude, hypoxia, cardiovascular adaptation, compensatory and pathologic adaptation

### 1. Introduction

High-altitude environment exerts a unique challenge to human life, which is chiefly characterized by lower partial pressure of O2 (PO2) relative to sea level at the same latitudes. The conventional definition of high-altitude hypoxia (HAH) is that arterial blood O2 saturation (SaO2) in body measurably begins to fall at altitudes >2500 m [1]. It is one of the hypoxemic types, which is due to a decrease in the amount of breathable oxygen caused by the low atmospheric pressure of high altitudes, and in turn low maximal oxygen uptake (VO2 max), and the arterial partial pressure of O2 (PaO2) in the body [2]. Reduced oxygen availability at high altitude is associated with significant changes in cardiovascular function and increased the risk of cardiovascular disease. Human body has both short-term and long-term adaptations to altitude that allow it to partially compensate for the lowered amount of oxygen in the atmosphere [3]. In this chapter we present the physiologic and pathologic adaptation of cardiovascular system to short-term and long-term HAH and its underlying mechanisms.

© 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, © 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.

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

## 2. Cardiovascular adaptation to high-altitude hypoxia

### 2.1. Cardiovascular adaptation to short-term high-altitude hypoxia and the underlying mechanisms

With ascent to high altitude, there is a nonlinear decrease in barometric pressure and a reduction in ambient partial pressure of oxygen (PO2), and, subsequently a decrease in the PO2 at every point along the oxygen transport cascade from inspired air to the alveolar space, arterial blood, the tissues, and venous blood. The higher the elevation attained, the greater the drop in PO2 in the human body. These declines in oxygen tensions trigger a variety of physiologic responses in the cardiovascular system over a period of minutes to weeks after the initial altitude hypoxia exposure that enable the individual adapt to or compensate for the hypoxic environment.

At high altitude, in the short term, the low PO2 of inspired air will typically concomitantly reduce SaO2, so a compensatory adjustment may immediately take place to meet the large and consistent O2 demand of the aerobic metabolism of tissues and cells. Initial lack of oxygen is sensed by the carotid bodies, which causes an increase in the breathing rate. Then the cardiovascular functions are changed in response to the short-term HAH.

#### 2.1.1. Cardiac system

The main cardiac response to short-term HAH is the adjustment of cardiac function, which includes the changes in heart rate (HR) and cardiac output, left ventricular ejection fraction (LVEF), both ventricular systolic and diastolic function, and arterial blood pressure (ABP). At high altitude, an initial response is that the heart beats faster. Cardiac contractility and submaximal cardiac output also increase acutely during the first few days at altitude. This acute increase in cardiac output may largely be explained by the increased heart rate and may be offset by reduced stroke volume. For example, an earlier study demonstrated that acutely breathing an inspired fraction of O2 of 0.12 caused 22% increase in cardiac output accompanied with 18% increase in heart rate and unchanged stroke volume, so the oxygen delivery to the tissues remained unchanged [4]. In addition, an evaluation of ventricular functions by Doppler echocardiography with tissue Doppler imaging (TDI) in volunteers exposed to acutely short-term (90 min) HAH reported that short-term HAH significantly increased HR, LVEF, isovolumic contraction wave velocity (ICV), acceleration (ICA), and systolic ejection wave velocity at the mitral annulus, indicating enhanced left ventricular systolic function. However, there was no change in right ventricular area shortening fraction, tricuspid annular plane systolic excursion (TAPSE), ICV, and ICA at the tricuspid annulus, demonstrating preserved right ventricular systolic function. Furthermore, increase in isovolumic relaxation time (IRT) at both annuli indicated altered diastolic function of both ventricles [5].

In response to a short-term high-altitude exposure, blood pressure is likely increased to a variable extent in many individuals. The changes in blood pressure may be dependent on individual conditions, the absolute altitude of exposure, and the duration of stay at altitude. A recent study has reported that after acute exposure to 3700 m, diastolic blood pressure and mean arterial blood pressure rose gradually and continually in healthy male young adults [6]. Further analysis showed that higher blood pressure accompanied poor sleeping quality and higher incidence of acute mountain sickness. In addition, systolic blood pressure also significantly increased after high-altitude exercise [6]. Significant rise in systolic and diastolic blood pressure in the initial phase of exposure to altitude was also reported in other studies [7, 8].

There are several possible mechanisms involved in short-term HAH-mediated cardiac dysfunction. One of the important mechanisms is the changes of autonomic nervous system including parasympathetic nervous system and sympathetic nervous system (SNS). A functional approach to assess the role of parasympathetic nerves by muscarinic blockade reported that tachycardia after 8 hours of exposure to hypoxia can be prevented by muscarinic blockade, which indicated that a muscarinic effect was involved in the tachycardia after short-term HAH [9]. Acute exposure to high altitude induced a statistically significant increase in heart rate associated with a shift of sympathovagal balance towards more sympathetic and less parasympathetic activity, which suggests a depression of autonomic functions and a relative increase in sympathetic activity at higher hypoxic levels [10]. These adaptations consist of significantly increased sympathetic activation as evidenced by heightened circulating catecholamine levels (such as norepinephrine) [11–14]. Mazzeo et al. reported a different catecholamine response between acute and chronic high-altitude exposure [13]. In response to acute exposure to 4300 m (4 hours), the arterial plasma epinephrine levels but not norepinephrine levels were significantly increased. However, both epinephrine and norepinephrine concentrations were increased after 21 days of chronic exposure [13]. These findings provide evidence for a differential adaptive response between sympathetic neural activity and that of the adrenal medulla during high-altitude exposure.

The increase in sympathetic tone may be a natural response by acute nonadapted subjects to counteract the effects of hypoxia. Indeed, short-term altitude exposure can directly or indirectly affect the vascular tone of systemic resistance vessels and enhances ventilation and sympathetic activity through the activation of peripheral chemoreceptors [15]. The peripheral chemoreceptors mainly include carotid bodies and aortic chemoreceptor, which are served as hypoxia sensors in the arterial walls. Carotid bodies act as sensitive monitors of arterial O2 tension (PaO2), whereas aortic chemoreceptors mainly monitor arterial O2 content (CaO2). So, carotid bodies evoke stronger respiratory responses than aortic chemoreceptors [16]. A study in humans exposed to hypoxia demonstrated that carotid bodies are chiefly responsible for ventilatory and vascular response, whereas aortic chemoreceptors mainly mediated the tachycardic response [17]. Another study indicated that hyperventilation induced by hypoxic stimulation of carotid bodies decreased vagal traffic to the heart through Hering-Breuer reflex, which plays an important indirect role in tachycardic response to hypoxia [18]. Meanwhile, hypoxic stimulation of carotid bodies also directly activated SNS to accelerate HR through increasing circulating catecholamine [19]. In addition, hypoxic activation of peripheral chemoreceptors was addressed to reset the baroreflex control of both HR and sympathetic nervous system (SNS) activity to higher levels, so that HR and sympathetic vasoconstriction were increased, which were independent of breathing rate and tidal volume [20].

#### 2.1.2. Pulmonary vascular system

2. Cardiovascular adaptation to high-altitude hypoxia

2.1. Cardiovascular adaptation to short-term high-altitude hypoxia

vascular functions are changed in response to the short-term HAH.

With ascent to high altitude, there is a nonlinear decrease in barometric pressure and a reduction in ambient partial pressure of oxygen (PO2), and, subsequently a decrease in the PO2 at every point along the oxygen transport cascade from inspired air to the alveolar space, arterial blood, the tissues, and venous blood. The higher the elevation attained, the greater the drop in PO2 in the human body. These declines in oxygen tensions trigger a variety of physiologic responses in the cardiovascular system over a period of minutes to weeks after the initial altitude hypoxia exposure that enable the individual adapt to or compensate for the hypoxic environment.

At high altitude, in the short term, the low PO2 of inspired air will typically concomitantly reduce SaO2, so a compensatory adjustment may immediately take place to meet the large and consistent O2 demand of the aerobic metabolism of tissues and cells. Initial lack of oxygen is sensed by the carotid bodies, which causes an increase in the breathing rate. Then the cardio-

The main cardiac response to short-term HAH is the adjustment of cardiac function, which includes the changes in heart rate (HR) and cardiac output, left ventricular ejection fraction (LVEF), both ventricular systolic and diastolic function, and arterial blood pressure (ABP). At high altitude, an initial response is that the heart beats faster. Cardiac contractility and submaximal cardiac output also increase acutely during the first few days at altitude. This acute increase in cardiac output may largely be explained by the increased heart rate and may be offset by reduced stroke volume. For example, an earlier study demonstrated that acutely breathing an inspired fraction of O2 of 0.12 caused 22% increase in cardiac output accompanied with 18% increase in heart rate and unchanged stroke volume, so the oxygen delivery to the tissues remained unchanged [4]. In addition, an evaluation of ventricular functions by Doppler echocardiography with tissue Doppler imaging (TDI) in volunteers exposed to acutely short-term (90 min) HAH reported that short-term HAH significantly increased HR, LVEF, isovolumic contraction wave velocity (ICV), acceleration (ICA), and systolic ejection wave velocity at the mitral annulus, indicating enhanced left ventricular systolic function. However, there was no change in right ventricular area shortening fraction, tricuspid annular plane systolic excursion (TAPSE), ICV, and ICA at the tricuspid annulus, demonstrating preserved right ventricular systolic function. Furthermore, increase in isovolumic relax-

ation time (IRT) at both annuli indicated altered diastolic function of both ventricles [5].

In response to a short-term high-altitude exposure, blood pressure is likely increased to a variable extent in many individuals. The changes in blood pressure may be dependent on individual conditions, the absolute altitude of exposure, and the duration of stay at altitude. A recent study has reported that after acute exposure to 3700 m, diastolic blood pressure and mean arterial blood pressure rose gradually and continually in healthy male young adults [6]. Further analysis showed that higher blood pressure accompanied poor sleeping quality and higher incidence of acute mountain sickness. In addition, systolic blood pressure also

and the underlying mechanisms

118 Hypoxia and Human Diseases

2.1.1. Cardiac system

Pulmonary circulation is the important portion of the cardiovascular system responsible for the gas exchange. Short-term HAH can immediately trigger hypoxic pulmonary vasoconstriction (HPV), which, in conjunction with increased cardiac output, leads to an enhanced pulmonary vascular resistance and a rise in pulmonary artery pressure. An investigation reported that human pulmonary vascular tone rose rapidly to reach a maximum within 5 min and was then maintained for the duration of the altitude exposure. This acute hypoxic pulmonary vasoconstriction (HPV) was reversed to baseline values within 5 min after breathing oxygen [21]. HPV is intrinsic to the pulmonary vascular smooth cells and independent of the endothelium, as demonstrated in experiments with endothelium-denuded pulmonary arteries (PAs) [22]. In short-term HAH, it was confirmed that small resistance pulmonary arteries (<200 μm) were highly sensitive to the alveolar O2 tension. There is a functional "O2-sensing unit" in the pulmonary artery smooth muscle cell (PASMC) mitochondria, which can detect falls in alveolar O2, leading to produce a mediator to modulate the function of effector proteins. During hypoxia, the production of the mediator is low, which causes the inhibition of specific O2-sensitive K+ channels resulting in depolarization of PASMCs and activation of voltage-gated L-type Ca2+ channels. Ca2+ influx is thereby increased and cytosolic Ca2+ elevated, resulting in activation of the PASMCs' contractile machinery and development of HPV [23]. Given the fact that there is a lack of voltage-gated Ltype Ca2+ channels in endothelium, it is most likely that HPV is endothelium-independent and intrinsic to pulmonary smooth muscle cells. However, endothelium-dependent and -independent mechanisms could modulate this response. Hypoxia may enhance pulmonary artery resistance through endothelin (ET) and sympathetic stimulation, whereas HPV may be attenuated by increased release of NO, hyperventilation improving alveolar PO2, and respiratory alkalosis [24].

High-altitude pulmonary edema (HAPE) is not an uncommon form of acute altitude illness that occurs in otherwise healthy mountaineers at altitudes typically above 2500 m. The initial cause of HAPE is a shortage of oxygen caused by the lower air pressure at high altitudes. The mechanisms underlying this oxygen shortage-induced HAPE are poorly understood, but one of the critical mechanisms is an excessive rise in pulmonary vascular resistance or hypoxic pulmonary vasoconstriction leading to increased microvascular pressures. This enhanced hydrostatic stress causes dynamic changes in the permeability of the alveolar capillary barrier and induces a highpermeability noninflammatory lung edema. Previous report indicated that decreased nitric oxide release and enhanced endothelin levels following acute high-altitude exposure may be the major determinants of exaggerated hypoxic pulmonary vasoconstriction in HAPE-susceptible individuals [25]. In addition, other hypoxia-mediated changes of sympathetic nervous activity, endothelial function, and altered levels of other vasoactive mediators such as endothelin and angiotensin II may also contribute additionally to HAPE susceptibility. Although higher pulmonary arterial pressure is associated with the development of HAPE, pulmonary hypertension may not in itself be sufficient to explain the development of high-altitude pulmonary edema. Development of pulmonary hypertension can occur in the absence of HAPE in humans at high altitude.

#### 2.1.3. Cerebrovascular system

The brain is the most oxygen-dependent organ in the body. In response to acute exposure to high altitude, cerebral blood flow (CBF) rises significantly to ensure an adequate supply of O2 to meet the brain tissues' large and consistent demand [26–28]. The mechanisms underlying the regulation of CBF during short-term HAH are complex and depend partly on the degree of hypoxia per se and on the partial pressures of arterial oxygen (PaO2) and arterial carbon

dioxide (PaCO2) [29]. Upon ascent to high altitude, a severe drop in PaO2 (to <40–45 mmHg) induces a cerebral vasodilation, which suggests that altitude-mediated reduced PaO2 may act as a cerebral vasodilator. However, the fall in PaCO2 following hyperventilation caused by hypoxic-induced activation of peripheral chemoreceptor also produces cerebral vasoconstriction. Therefore, the changes in CBF at high altitude are highly related to the balance of PaO2/ PaCO2 in the circulation. Indeed, it has been demonstrated that the low PaO2-to-PaCO2 ratio explains 40% of the increase in brain blood flow upon arrival at high altitude (5050 m) [26]. The increased CBF is mainly due to heightened hypoxic-induced dilatation in the cerebral circulation prior to ventilatory adjustments [26]. A number of mechanisms are proposed to contribute to the cerebral vasodilation. One of the mechanisms is that hypoxia may increase adenosine and nitric oxide level, which causes an increase in arterial diameter [27]. In addition, the cerebral dilatation can be also regulated through other factors (such as hypoxia inducible factor). Furthermore, the fact that acute altitude exposure-mediated increased CBF and cerebral vasodilation can be reversed by supplemental oxygen suggests a direct hypoxic effect. With increasing altitude, increased CBF is believed to be one compensatory mechanism serving to maintain normal oxygen flux to the brain in the face of arterial hypoxemia. However, the profound hypoxemia experienced by climbers at extreme altitude (>5500 m) is known to be related with cerebral dysfunction. Previous investigation has shown that hypoxia-mediated cerebral vascular dysfunction and cerebral edema is one of the major cause of deaths over 8000 m on Everest [30].

(HPV), which, in conjunction with increased cardiac output, leads to an enhanced pulmonary vascular resistance and a rise in pulmonary artery pressure. An investigation reported that human pulmonary vascular tone rose rapidly to reach a maximum within 5 min and was then maintained for the duration of the altitude exposure. This acute hypoxic pulmonary vasoconstriction (HPV) was reversed to baseline values within 5 min after breathing oxygen [21]. HPV is intrinsic to the pulmonary vascular smooth cells and independent of the endothelium, as demonstrated in experiments with endothelium-denuded pulmonary arteries (PAs) [22]. In short-term HAH, it was confirmed that small resistance pulmonary arteries (<200 μm) were highly sensitive to the alveolar O2 tension. There is a functional "O2-sensing unit" in the pulmonary artery smooth muscle cell (PASMC) mitochondria, which can detect falls in alveolar O2, leading to produce a mediator to modulate the function of effector proteins. During hypoxia, the production of the mediator is low, which causes the inhibition of specific O2-sensitive K+ channels resulting in depolarization of PASMCs and activation of voltage-gated L-type Ca2+ channels. Ca2+ influx is thereby increased and cytosolic Ca2+ elevated, resulting in activation of the PASMCs' contractile machinery and development of HPV [23]. Given the fact that there is a lack of voltage-gated Ltype Ca2+ channels in endothelium, it is most likely that HPV is endothelium-independent and intrinsic to pulmonary smooth muscle cells. However, endothelium-dependent and -independent mechanisms could modulate this response. Hypoxia may enhance pulmonary artery resistance through endothelin (ET) and sympathetic stimulation, whereas HPV may be attenuated by increased release of NO, hyperventilation improving alveolar PO2, and respiratory alkalosis [24]. High-altitude pulmonary edema (HAPE) is not an uncommon form of acute altitude illness that occurs in otherwise healthy mountaineers at altitudes typically above 2500 m. The initial cause of HAPE is a shortage of oxygen caused by the lower air pressure at high altitudes. The mechanisms underlying this oxygen shortage-induced HAPE are poorly understood, but one of the critical mechanisms is an excessive rise in pulmonary vascular resistance or hypoxic pulmonary vasoconstriction leading to increased microvascular pressures. This enhanced hydrostatic stress causes dynamic changes in the permeability of the alveolar capillary barrier and induces a highpermeability noninflammatory lung edema. Previous report indicated that decreased nitric oxide release and enhanced endothelin levels following acute high-altitude exposure may be the major determinants of exaggerated hypoxic pulmonary vasoconstriction in HAPE-susceptible individuals [25]. In addition, other hypoxia-mediated changes of sympathetic nervous activity, endothelial function, and altered levels of other vasoactive mediators such as endothelin and angiotensin II may also contribute additionally to HAPE susceptibility. Although higher pulmonary arterial pressure is associated with the development of HAPE, pulmonary hypertension may not in itself be sufficient to explain the development of high-altitude pulmonary edema. Development of

pulmonary hypertension can occur in the absence of HAPE in humans at high altitude.

The brain is the most oxygen-dependent organ in the body. In response to acute exposure to high altitude, cerebral blood flow (CBF) rises significantly to ensure an adequate supply of O2 to meet the brain tissues' large and consistent demand [26–28]. The mechanisms underlying the regulation of CBF during short-term HAH are complex and depend partly on the degree of hypoxia per se and on the partial pressures of arterial oxygen (PaO2) and arterial carbon

2.1.3. Cerebrovascular system

120 Hypoxia and Human Diseases

High-altitude cerebral edema (HACE) is a medical condition in which the brain swells with fluid because of ascending to a high altitude. It occurs when the body fails to acclimatize while ascending to a high altitude. HACE can be prevented by ascending to heights slowly to allow the body more time to acclimatize. The major cause of HACE is oxygen deprivation. It is most often a complication of acute mountain sickness or high-altitude pulmonary edema. The current leading theory of its pathophysiology is that HACE is likely a result of vasogenic edema [31]. High-altitude hypoxia increases vascular permeability, which passes through the vasogenic endothelium in the brain. The leaking may be caused by increased pressure, or it may be caused by inflammation that makes the endothelium vulnerable to leaking. It has been reported that activation of vascular endothelial growth factor (VEGF) by hypoxia-inducible factor may be one of the major causes leading to overperfusion of microvascular beds, endothelial leakage, and hence edema [31]. In addition, high-altitude hypoxia can alter cerebral vasodilation coupled with a possible impairment of the autoregulation of cerebral blood flow and disruption of the integrity of the blood brain barrier possibly by hypoxia-mediated release of certain neuromodulators such as VEGF and calcitonin gene-related peptide (CGRP). Furthermore, the increased sympathetic nervous activity at high altitude may also play a role in the development of cerebral edema.

#### 2.2. Cardiovascular adaptation to long-term HAH and the underlying mechanisms

Acute short-term exposure to high altitude has been recognized as a type of cardiovascular stress, and results in an immediate increase in heart rate, cardiac output, and a transient rise in the blood pressure but without significant changes in the ejection fraction. However, long-term exposure to high altitude or people who reside at high altitude show compensate change in cardiovascular system that has allowed them to adapt to high-altitude chronic hypoxia.

#### 2.2.1. Cardiac system

In long-term HAH, the changes in cardiac function are different from those in acute short-term HAH. Similar to the short-term HAH, heart rate and arterial blood pressure may remain increased, but stroke volume is decreased and the cardiac output returns to baseline after a longer hypoxic exposure [4]. In long-term HAH, the heart must preserve adequate contractile function in spite of lowered oxygen tension in the cardiac circulation. Suarez et al. had conducted studies in young men during acclimation to a simulated altitude in a chamber for 40 days and their results showed that left ventricular systolic function indices including ejection fraction, ratio of peak systolic pressure to end systolic volume, and mean normalized systolic ejection rate at rest and exercise, were sustained in all subjects at high altitude despite reduced preload, pulmonary hypertension and severe hypoxemia, which means remarkably preserved contractility and excellent tolerance of the normal myocardium to long-term HAH [32]. Another study also demonstrated that cardiac contractility remained normal during exposure to altitude-induced hypoxia with preservation of LV ejection fraction and LV percent fractional shortening [33].

Cardiac adaptation to long-term HAH is characterized by a variety of functional adjustments to maintain homeostasis with minimum expenditure of energy. Such adjustments may help to protect the heart from development of ischemic heart disease. An epidemiological study reported that men residing at high altitude resulted in protection against death from ischemic heart disease [34]. The epidemiological observations on the cardioprotective effect of high altitude were confirmed in various experimental models [35–37]. It has been reported that the hearts of animals adapted to long-term HAH develop better functional recovery following ischemia and produce smaller cardiac infarction. In addition, it has also been reported that adaptation to HAH could protect the heart against ischemia-induced arrhythmias [38]. However, the cardioprotective effect of adaptation to HAH is age-dependent. For example, a recent experiment with rats at stimulated altitude of 5000 m from 7-week-old to their entire lifetime [39] showed that cardiac tolerance to acute hypoxia was significantly increased in up to 18 months-old rats, but it was lost in senescent rats (25 months-old). Similarly, people living at high altitude in the Andeans lost their adaptation and have higher incidence of pulmonary hypertension in their aged life.

The mechanisms underlying cardiac functional changes in response to long-term HAH remain far from being understood. However, recent studies in animals and man have highlighted the role of both sympathetic and parasympathetic nervous system in cardiac adaptation following long-term HAH. The role of the parasympathetic system in regulation of heart rate has been examined in humans from the response to muscarinic blockade. A study in human after exposing to an altitude of 5260 m for 9 weeks found that muscarinic blockade increased HR both at rest and during exercise, which suggested that enhanced parasympathetic activity involves in the altered HR during long-term HAH [40]. Meanwhile, another study in animals reported that the muscarinic receptor density in animals native to high altitude was significantly higher than in those living at low altitude. After 5 weeks of relocation to sea level the muscarinic receptor density concomitantly declined to the level in sea level animals [41]. In addition, similar to the short-term HAH, the sympathetic nervous system also plays a key role in the regulation of HR and cardiac function during long-term HAH, but the pattern is different from those in short-term HAH. Previous studies in long-term HAH subjects reported that plasma norepinephrine level increased more significantly than epinephrine levels [13, 14, 42, 43]. Although the resting heart rate remains increased, the maximal heart rate (the heart rate at maximal exercise) is reduced at long-term HAH. In view of the evidence of elevated systemic catecholamine levels after long-term HAH, the lower maximal heart rate suggests a change in adrenergic receptor density. Indeed, several studies in animals have shown the change in adrenergic receptor density in response to long-term HAH. For example, a study in rats exposed to 21 days of hypobaric hypoxia found that there was a significant reduction in βadrenergic receptor density [44]. Another study in rats following 21 days of exposure to a simulated altitude of 5500 m also reported a downregulation of α- and β-adrenergic receptor density in ventricular tissues [45]. Furthermore, studies in humans using isoprenaline as an indirect measure of density of β-adrenergic receptors demonstrated a downregulation of βadrenergic receptors at high altitude [46]. It has been reported that prolonged HAH exposure could also alter peripheral and central adrenergic receptor expression, leading to changes in cardiac function [47, 48]. Taken together, sustained long-term HAH exposure causes progressive enhancement of both sympathetic and parasympathetic activity, resulting in alteration of cardiovascular function.

In addition to cardiac functional adaptation, cardiac structural adaptation also occurs following long-term HAH exposure. One of the changes in response to sustained HAH is development of right ventricle (RV) hypertrophy. Long-term high altitude-induced RV hypertrophy is a beneficial adaptation that helps to counteract the increased afterload caused by persistent pulmonary hypertension and maintain a normal cardiac output [49]. During the compensated phase of hypertrophy, a study using an isolated preparation of the RV working heart demonstrated that mechanical performance was almost doubled compared with the control group, while the index of contractility remained unchanged, which means that the elevated ventricular performance is merely the result of the increased muscle mass. Meantime, the markedly improved ability of the RV maintaining cardiac output against increased pulmonary resistance was observed [49]. Hypertrophic RV is associated with significant changes of cardiac protein profiling [50]. Experimental results in rats exposed to intermittent high-altitude hypoxia have shown that the concentration of collagenous and noncollagenous proteins was significantly increased both in hypertrophic RV and nonhypertrophic LV [51]. Cardiac enlargement may be the result of both an increase in the number of individual cell elements (hyperplasia) and an increase in their volume (hypertrophy).

#### 2.2.2. Pulmonary vascular system

exposure to high altitude or people who reside at high altitude show compensate change in cardiovascular system that has allowed them to adapt to high-altitude chronic hypoxia.

In long-term HAH, the changes in cardiac function are different from those in acute short-term HAH. Similar to the short-term HAH, heart rate and arterial blood pressure may remain increased, but stroke volume is decreased and the cardiac output returns to baseline after a longer hypoxic exposure [4]. In long-term HAH, the heart must preserve adequate contractile function in spite of lowered oxygen tension in the cardiac circulation. Suarez et al. had conducted studies in young men during acclimation to a simulated altitude in a chamber for 40 days and their results showed that left ventricular systolic function indices including ejection fraction, ratio of peak systolic pressure to end systolic volume, and mean normalized systolic ejection rate at rest and exercise, were sustained in all subjects at high altitude despite reduced preload, pulmonary hypertension and severe hypoxemia, which means remarkably preserved contractility and excellent tolerance of the normal myocardium to long-term HAH [32]. Another study also demonstrated that cardiac contractility remained normal during exposure to altitude-induced hypoxia with preservation of LV ejection fraction and LV percent fractional shortening [33].

Cardiac adaptation to long-term HAH is characterized by a variety of functional adjustments to maintain homeostasis with minimum expenditure of energy. Such adjustments may help to protect the heart from development of ischemic heart disease. An epidemiological study reported that men residing at high altitude resulted in protection against death from ischemic heart disease [34]. The epidemiological observations on the cardioprotective effect of high altitude were confirmed in various experimental models [35–37]. It has been reported that the hearts of animals adapted to long-term HAH develop better functional recovery following ischemia and produce smaller cardiac infarction. In addition, it has also been reported that adaptation to HAH could protect the heart against ischemia-induced arrhythmias [38]. However, the cardioprotective effect of adaptation to HAH is age-dependent. For example, a recent experiment with rats at stimulated altitude of 5000 m from 7-week-old to their entire lifetime [39] showed that cardiac tolerance to acute hypoxia was significantly increased in up to 18 months-old rats, but it was lost in senescent rats (25 months-old). Similarly, people living at high altitude in the Andeans lost their adaptation and have higher incidence of pulmonary

The mechanisms underlying cardiac functional changes in response to long-term HAH remain far from being understood. However, recent studies in animals and man have highlighted the role of both sympathetic and parasympathetic nervous system in cardiac adaptation following long-term HAH. The role of the parasympathetic system in regulation of heart rate has been examined in humans from the response to muscarinic blockade. A study in human after exposing to an altitude of 5260 m for 9 weeks found that muscarinic blockade increased HR both at rest and during exercise, which suggested that enhanced parasympathetic activity involves in the altered HR during long-term HAH [40]. Meanwhile, another study in animals reported that the muscarinic receptor density in animals native to high altitude was significantly higher than in those living at low altitude. After 5 weeks of relocation to sea level the

2.2.1. Cardiac system

122 Hypoxia and Human Diseases

hypertension in their aged life.

The most common effect of long-term sustained HAH exposure is the development of pulmonary hypertension. Previous studies have reported a prevalence of high-altitude pulmonary hypertension between 5% and 18% of the population living at high altitude [52]. High-altitude pulmonary hypertension is characterized by increased pulmonary vascular resistance secondary to hypoxia-induced pulmonary vasoconstriction and vascular remodeling. The pulmonary vascular adaptations involve all elements of the vessel wall and include endothelial dysfunction, extension of smooth muscle into previously nonmuscular vessels and adventitial thickening. Long-term high-altitude-induced pulmonary hypertension was not completely reversed by oxygen breathing, suggesting that pulmonary arteries structural remodeling plays a pivotal role in pulmonary hypertension during long-term HAH [53]. Pulmonary arteries (PAs) remodeling involves cellular hypertrophy and hyperplasia in all three structural layers of PAs, namely adventitia, media, and intima. In addition, long-term HAH also causes other structural changes, such as the migration of medial smooth muscle cells (SMCs) into the intima, fibroblast proliferation and increased collagen deposition in the adventitia, more extracellular matrix proteins secreted by endothelial cells, and the appearance of SM-like cells in previously nonmuscularized vessels of the alveolar wall. All these changes eventually result in a reduction of the vascular lumen diameter and an increase in pulmonary vascular resistance [54].

The molecular mechanisms underlying the pathogenesis of high altitude-induced pulmonary hypertension are not fully understood, but several hypoxia-mediated signaling pathways are thought to play a key role. In the pulmonary vasculature, some membrane-bound receptors and signaling proteins are sensitive to hypoxia and play important role in the vascular medial proliferation. For example, a recent study in a sheep model of in utero high-altitude long-term hypoxia exposure demonstrated pulmonary vascular remodeling similar to that seen in other animal models of pulmonary hypertension [55]. The results indicated that pulmonary arteries of long-term HAH-exposed fetuses exhibited medial wall thickening and distal muscularization associated with an increased epidermal growth factor receptor (EGFR) protein expression in the pulmonary arteries. Furthermore, it has been demonstrated that the proliferation of fetal ovine pulmonary vascular smooth muscle cell was attenuated by inhibition of EGFR with a specific EGFR protein tyrosine kinase inhibitor [55]. These findings suggest that EGFR plays a role in fetal ovine pulmonary vascular remodeling following long-term HAH and that inhibition of EGFR signaling may reverse high altitude-induced pulmonary vascular remodeling. Similar to EGFR, platelet-activating factor (PAF) and PAF receptor have also been implicated in the pathogenesis of long-term HAH-induced pulmonary remodeling and hypertension in different animal models [56, 57]. In those studies, high PAF and PAF receptor expression levels in the pulmonary arteries have been reported in the long-term hypoxia-exposed animals [56, 57]. Furthermore, PAF receptor antagonists attenuated hypoxia-induced pulmonary hypertension and pulmonary vascular remodeling [56], suggesting that PAF receptor-mediated signaling also plays a key role in pulmonary vascular remodeling.

Accumulating evidence indicates that intrinsic changes in the ionic balance and calcium homeostasis of pulmonary arterial smooth muscle cells (PASMCs) caused by long-term hypoxia have a profound effect on PA remodeling. The membrane depolarization of PASMCs following the hypoxic inhibition of O2 sensitive K+ channels activated Ca2+ influx and elevated cytoplasmic ionized Ca2+ via voltage-gated Ca2+ channels. Changes in the transport of K+ and Ca2+ through their respective ion channels modulate these processes by affecting cell volume, membrane potential, gene transcription, apoptosis, and cell-cycle progression. The adaptation of these ion channels at high altitude appears to involve in pulmonary arteries remodeling [58].

Although PASMCs are the major components of arteries that actively involve long-term HAHmediated sustained vasoconstriction and enhanced medial hypertrophy, endothelial cells, on the other hand, can sense humoral and hemodynamic changes incurred by high-altitude hypoxia, triggering their production of vasoactive and mitogenic factors that then affect PASMCs' function and growth [54, 59, 60]. Endothelin (ET)-1 is an important mediator of hypoxia-induced pulmonary vasoconstriction and vascular remodeling [61]. Chronic hypoxia increases ET-1 gene transcription and peptide synthesis in cultured endothelial cells. ET-1 and its receptors are selectively upregulated in patients with primary pulmonary hypertension and in humans exposed to high altitude [61]. Rats exposed to chronic hypoxia exhibit increased pulmonary artery pressure associated with an increase in ET-1 peptide levels. Moreover, hypoxic pulmonary vascular remodeling can be prevented and reversed by administration of ET receptor antagonist [61], suggesting a key role of ET-1 and its receptor-mediated signaling in chronic hypoxia-induced pulmonary hypertension and vascular remodeling.

#### 2.2.3. Cerebrovascular system

vascular adaptations involve all elements of the vessel wall and include endothelial dysfunction, extension of smooth muscle into previously nonmuscular vessels and adventitial thickening. Long-term high-altitude-induced pulmonary hypertension was not completely reversed by oxygen breathing, suggesting that pulmonary arteries structural remodeling plays a pivotal role in pulmonary hypertension during long-term HAH [53]. Pulmonary arteries (PAs) remodeling involves cellular hypertrophy and hyperplasia in all three structural layers of PAs, namely adventitia, media, and intima. In addition, long-term HAH also causes other structural changes, such as the migration of medial smooth muscle cells (SMCs) into the intima, fibroblast proliferation and increased collagen deposition in the adventitia, more extracellular matrix proteins secreted by endothelial cells, and the appearance of SM-like cells in previously nonmuscularized vessels of the alveolar wall. All these changes eventually result in a reduction of the vascular

The molecular mechanisms underlying the pathogenesis of high altitude-induced pulmonary hypertension are not fully understood, but several hypoxia-mediated signaling pathways are thought to play a key role. In the pulmonary vasculature, some membrane-bound receptors and signaling proteins are sensitive to hypoxia and play important role in the vascular medial proliferation. For example, a recent study in a sheep model of in utero high-altitude long-term hypoxia exposure demonstrated pulmonary vascular remodeling similar to that seen in other animal models of pulmonary hypertension [55]. The results indicated that pulmonary arteries of long-term HAH-exposed fetuses exhibited medial wall thickening and distal muscularization associated with an increased epidermal growth factor receptor (EGFR) protein expression in the pulmonary arteries. Furthermore, it has been demonstrated that the proliferation of fetal ovine pulmonary vascular smooth muscle cell was attenuated by inhibition of EGFR with a specific EGFR protein tyrosine kinase inhibitor [55]. These findings suggest that EGFR plays a role in fetal ovine pulmonary vascular remodeling following long-term HAH and that inhibition of EGFR signaling may reverse high altitude-induced pulmonary vascular remodeling. Similar to EGFR, platelet-activating factor (PAF) and PAF receptor have also been implicated in the pathogenesis of long-term HAH-induced pulmonary remodeling and hypertension in different animal models [56, 57]. In those studies, high PAF and PAF receptor expression levels in the pulmonary arteries have been reported in the long-term hypoxia-exposed animals [56, 57]. Furthermore, PAF receptor antagonists attenuated hypoxia-induced pulmonary hypertension and pulmonary vascular remodeling [56], suggesting that PAF receptor-mediated signaling also

Accumulating evidence indicates that intrinsic changes in the ionic balance and calcium homeostasis of pulmonary arterial smooth muscle cells (PASMCs) caused by long-term hypoxia have a profound effect on PA remodeling. The membrane depolarization of PASMCs following the hypoxic inhibition of O2 sensitive K+ channels activated Ca2+ influx and elevated cytoplasmic ionized Ca2+ via voltage-gated Ca2+ channels. Changes in the transport of K+ and Ca2+ through their respective ion channels modulate these processes by affecting cell volume, membrane potential, gene transcription, apoptosis, and cell-cycle progression. The adaptation of these ion channels at high altitude appears to involve in pulmonary arteries remodeling [58].

Although PASMCs are the major components of arteries that actively involve long-term HAHmediated sustained vasoconstriction and enhanced medial hypertrophy, endothelial cells, on

lumen diameter and an increase in pulmonary vascular resistance [54].

124 Hypoxia and Human Diseases

plays a key role in pulmonary vascular remodeling.

Upon ascent to high altitude, cerebral blood flow (CBF) rises substantially. However, as HAHexposed time is increased, the increased CBF will return to near sea level values within 1–3 weeks, which displays clear time-dependent changes during acclimatization. In general, highaltitude native residents have lower CBF values compared to sea level natives. The major mechanism underlying the reduction in CBF of high-altitude residents is the reported elevation in hematocrit and consequently increased arterial oxygen content (CaO2), suggesting an inverse relationship between CBF and CaO2. There are at least four reflex mechanisms that regulate CBF: (1) hypoxic ventilator response; (2) hypercapnic ventilatory response; (3) hypoxic cerebral vasodilation; and (4) hypocapnic cerebral vasoconstriction [62]. On initial arrival at high altitude, hypobaric hypoxia changes the mediators of CBF because of a decrease in arterial oxygen tension, which is an independent mediator of cerebral arteriolar dilatation. In addition, hypoxemia can trigger hyperventilation associated decrease in arterial carbon dioxide tension, which will cause cerebral arterial constriction because of an associated increase in periarteriolar pH. Therefore, over a few days period at a constant altitude, the influence of the arterial oxygen tension-induced threshold for cerebral vasodilation is attenuated and the degree of hypocapnia is enhanced. Furthermore, during a prolonged stay at altitude, the hematocrit also increases, resulting in an increased arterial oxygen content at an unchanged oxygen tension. This change will tend to decrease CBF. Therefore, cerebral hemodynamics during acclimatization to altitude is the result of these homeostatic mechanisms. In addition to these reflex responses, CBF is also regulated by some other hypoxia-induced changes. For example, high-altitude hypoxia-induced changes of cerebral capillary density, hypoxiainduced factor (HIF), nitric oxide, endothelin-1, reactive oxygen species (ROS), and neurotransmitters may be responsible for the falling CBF during long-term HAH [29].

#### 2.2.4. Uteroplacental vascular system

Pregnancy is associated with a significant increase in uterine blood flow that optimizes the delivery of oxygen and nutrients to the developing fetus. The greater fall in uteroplacental vascular resistance preferentially directs blood flow to this vascular bed, raising the uterine blood flow from 20–50 ml/min in the nonpregnant state to 450–800 ml/min in the near-term pregnant stage [63]. The adaptations in the uterine circulation to pregnancy are complex and are mainly achieved through the remodeling of uterine vasculature, enhanced vasodilator response, blunted vasoconstrictor response, and reduced pressure-dependent myogenic reactivity. At sea level pregnant uterine artery diameter doubles due to the vascular growth and remodeling as well as due to alterations in vasoreactivity, and changes in the active and passive properties of the uterine artery vascular wall. The molecular mechanisms prompting uterine vascular growth and enlargement of the vascular diameter are not fully understood. However, one of the major mechanisms underlying pregnancy-mediated decreased uterine vascular resistance may be regulated through hormonal stimuli. It has been reported that estradiol is likely a key player because of its angiogenic properties and stimulatory effects on nitric oxide-mediated vasodilation [64]. Estrogen receptors (ERs) have been identified in uterine artery vascular smooth muscle and their expressions are significantly increased in pregnant uterine arteries as compared with nonpregnant uterine arteries [65]. The pregnancy-associated increased ER expression may directly upregulate vascular endothelial growth factor (VEGF), MAP kinase, and eNOS expression and their activities, leading to promote uterine vascular growth and vasodilation [65]. The decreased uterine vascular resistance can also be regulated by contractile agonists or related proteins. For example, pregnancy decreases PKC activity but increases ERK kinase activity in uterine arteries, leading to decrease in uterine artery contractility [66, 67]. In addition, myogenic tone and distensibility are additional factors that can alter uterine arterial intraluminal diameter and uterine vascular resistance. It has been reported that pregnancy significantly downregulates pressure-dependent myogenic tone and increases the pressure-dependent passive uterine arterial diameter. The reduced myogenic tone is mediated by an increase in the inhibitory effect of ERK and a decrease in the PKC signal pathway [68].

High-altitude hypoxia has profound effects on uteroplacental circulation including altered uteroplacental and fetal volumetric blood flows, resulting in fetal intrauterine growth restriction. It has been demonstrated that high-altitude hypoxia decreases the pregnancy-associated rise in uterine blood flow [69]. Reduced uterine blood flow and inadequate perfusion of the placenta have been attributed to the increased incidence of preeclampsia and fetal intrauterine growth restriction [1]. One of the mechanisms that contributes to the decreased uterine blood flow may be a significant inhibition of pregnancy-associated increase in uterine vascular growth. It has been reported that there is only half as much pregnancy-mediated increase in uterine arterial DNA synthesis in chronic hypoxic vs. normoxic animals [70]. The proliferative response to serum stimulation in cultured uterine arterial smooth muscle cells is also attenuated by hypoxia exposure [70]. In addition, high-altitude hypoxia also can alter pregnancy-associated responses to contractile proteins and vasodilator-mediated signaling pathways. Experimental studies in sheep, that experienced long-term high-altitude exposure during pregnancy, showed significant increase in the pressure-dependent myogenic tone of resistance-sized uterine arteries by suppressing the ERK1/2 activity and increasing the PKC signaling pathway [65]. Furthermore, high-altitude hypoxia exposure selectively downregulated estrogen-α receptor expression in uterine arteries of pregnant animals and inhibited the steroid hormone-mediated adaptation of ERK1/2 and PKC signaling pathways to cause an increase in the myogenic tone of uterine arteries in pregnancy [65]. These observations provide a novel molecular mechanism underlying high altitude-induced decrease in uterine blood flow by inhibition of estrogen/receptor-mediated signaling in pregnancy. The large-conductance Ca2+-activated K+ (BKca) is abundantly expressed in vascular smooth muscle cells. Previous studies have suggested that BKca channel is involved in the regulation of uterine circulation and the increase in uterine blood flow during pregnancy [71]. The BKca channel in vascular smooth muscle is a major effector in response to hypoxia. Studies in pregnant sheep model of long-term high-altitude (3801 m) exposure provide novel evidence that long-term high-altitude hypoxia during pregnancy adversely affects the uterine circulation via downregulating BKca channel function in uterine vasculatures [72]. High-altitude hypoxia during gestation significantly inhibited pregnancy-associated upregulation of BKca channel activity and attenuated BKca channel current density in pregnant uterine arteries [72]. This was mediated by a selective downregulation of BKca channel β1 subunit expression in the uterine arteries. In accordance, high-altitude hypoxia impaired the role of the BKca channel in regulating pressure-induced myogenic tone of uterine arteries that was enhanced in pregnant animals acclimatized to high altitude. These results suggest that selectively targeting BKca channel may be another key mechanism in the maladaptation of uteroplacental circulation caused by high-altitude hypoxia, which may contribute to the decreased uterine blood flow and fetal intrauterine growth restriction associated with maternal hypoxia. The molecular mechanisms underlying high-altitude hypoxia-mediated alteration of targeting gene expression in pregnant uterine arteries are not completely understood. However, recent studies suggest that epigenetic mechanism plays an important role in regulation of gene expression in adaptation to high altitude [73]. The results showed that chronic hypoxia increased estrogen receptor α subunit (ER-α) promoter DNA methylation at both specific protein-1 and upstream stimulatory factor binding sites, decreased specificity protein-1 and upstream stimulatory factor binding to the promoter, and suppressed ER-α expression in uterine arteries of pregnant animals [73]. Furthermore, the studies provide novel evidence that hypoxia-mediated DNA methylation plays a causal role in ER-α gene repression and ablation of estrogen-mediated adaptation of uterine arterial BKca channel activity, resulting in increased uterine arterial myogenic tone in pregnancy [73].

are mainly achieved through the remodeling of uterine vasculature, enhanced vasodilator response, blunted vasoconstrictor response, and reduced pressure-dependent myogenic reactivity. At sea level pregnant uterine artery diameter doubles due to the vascular growth and remodeling as well as due to alterations in vasoreactivity, and changes in the active and passive properties of the uterine artery vascular wall. The molecular mechanisms prompting uterine vascular growth and enlargement of the vascular diameter are not fully understood. However, one of the major mechanisms underlying pregnancy-mediated decreased uterine vascular resistance may be regulated through hormonal stimuli. It has been reported that estradiol is likely a key player because of its angiogenic properties and stimulatory effects on nitric oxide-mediated vasodilation [64]. Estrogen receptors (ERs) have been identified in uterine artery vascular smooth muscle and their expressions are significantly increased in pregnant uterine arteries as compared with nonpregnant uterine arteries [65]. The pregnancy-associated increased ER expression may directly upregulate vascular endothelial growth factor (VEGF), MAP kinase, and eNOS expression and their activities, leading to promote uterine vascular growth and vasodilation [65]. The decreased uterine vascular resistance can also be regulated by contractile agonists or related proteins. For example, pregnancy decreases PKC activity but increases ERK kinase activity in uterine arteries, leading to decrease in uterine artery contractility [66, 67]. In addition, myogenic tone and distensibility are additional factors that can alter uterine arterial intraluminal diameter and uterine vascular resistance. It has been reported that pregnancy significantly downregulates pressure-dependent myogenic tone and increases the pressure-dependent passive uterine arterial diameter. The reduced myogenic tone is mediated by an increase in the inhibitory

High-altitude hypoxia has profound effects on uteroplacental circulation including altered uteroplacental and fetal volumetric blood flows, resulting in fetal intrauterine growth restriction. It has been demonstrated that high-altitude hypoxia decreases the pregnancy-associated rise in uterine blood flow [69]. Reduced uterine blood flow and inadequate perfusion of the placenta have been attributed to the increased incidence of preeclampsia and fetal intrauterine growth restriction [1]. One of the mechanisms that contributes to the decreased uterine blood flow may be a significant inhibition of pregnancy-associated increase in uterine vascular growth. It has been reported that there is only half as much pregnancy-mediated increase in uterine arterial DNA synthesis in chronic hypoxic vs. normoxic animals [70]. The proliferative response to serum stimulation in cultured uterine arterial smooth muscle cells is also attenuated by hypoxia exposure [70]. In addition, high-altitude hypoxia also can alter pregnancy-associated responses to contractile proteins and vasodilator-mediated signaling pathways. Experimental studies in sheep, that experienced long-term high-altitude exposure during pregnancy, showed significant increase in the pressure-dependent myogenic tone of resistance-sized uterine arteries by suppressing the ERK1/2 activity and increasing the PKC signaling pathway [65]. Furthermore, high-altitude hypoxia exposure selectively downregulated estrogen-α receptor expression in uterine arteries of pregnant animals and inhibited the steroid hormone-mediated adaptation of ERK1/2 and PKC signaling pathways to cause an increase in the myogenic tone of uterine arteries in pregnancy [65]. These observations provide a novel molecular mechanism underlying high altitude-induced decrease in

effect of ERK and a decrease in the PKC signal pathway [68].

126 Hypoxia and Human Diseases

There are significant differences in uterine arterial adaptation to pregnancy between the long- and short-resident high-altitude populations. The weight of the babies born to Tibetan residents at high altitude is more than that of those born to Han women living at the same altitude, which is associated with a higher uterine flow velocity and larger uterine arterial diameters [1]. The uterine arterial diameters in Andean pregnant women are also most doubling increased at high altitude whereas there are about half as much increase in European pregnant women [74]. As a result, Andean pregnant women have much higher uterine blood flows and birth weights of their babies than Europeans at high altitude. However, the values are the same at low altitude in both Andean and European women, which suggests a much higher protective effect of Andean ancestry at high altitude. The questions why longresident high-altitude populations (such as Tibetan and Andean women) have higher resistant to the adverse effects of high-altitude hypoxia than the short-resident populations (such as Han and European women) are not fully understood. However, recent reports suggest that genetic background may play a key role in the altitude-related changes in birth weight and uterine blood flow [1].

## 3. Conclusion

The adaptation of the cardiovascular system to altitude is variable, depending on individual predisposition, the actual elevation, the rate of ascent, and the duration of exposure. In acute short-term exposure to HAH, the initial response is increasing sympathetic activity and hyperventilation resulting in increases in systemic vascular resistance, blood pressure, heart rate, and cardiac output. Pulmonary vasoconstriction leads to pulmonary hypertension. However, in response to acute HAH, cerebral blood flow (CBF) rises significantly to ensure an adequate supply of O2 to meet the brain tissues' large and consistent demand. The sympathetic excitation results from acute HAH, partly through chemoreceptor reflexes and partly through altered baroreceptor function. In long-term HAH exposure or resident at high altitude, cardiovascular system progresses a compensatory adaptation. The cardiovascular system may promote adaptational changes in cardiovascular structure, remodeling, and functional proteins through different molecular mechanisms including epigenetic regulatory and/or genetic factormediated mechanisms. However, cardiovasculatures may progress a pathologic adaptation and develop a maladaptation syndrome known as high-altitude pulmonary edema, cerebral edema, chronic mountain sickness, pulmonary hypertension, heart failure, and fetal intrauterine growth restriction. In conclusion, cardiovascular system progresses a compensatory and pathologic adaptation to HAH. Understanding those adaptation processes will help us to reduce the development of adverse changes and simultaneously preserve the beneficial signs of the process of adaptation.

## Author details

Jun Ke1,2, Lei Wang1,3 and Daliao Xiao1 \*

\*Address all correspondence to: Dxiao@llu.edu

1 Center for Perinatal Biology, Department of Basic Science, Loma Linda University School of Medicine, Loma Linda, California, USA

2 Guangdong Cardiovascular Institute, Guangdong General Hospital, Guangdong Academy of Medical Science, Guangzhou, Guangdong, China

3 Department of Traditional Chinese Medicine, People's Hospital of Shanghai Putuo District, Shanghai, China

## References

[1] Moore LG, Charles SM, Julian CG. Humans at high altitude: hypoxia and fetal growth. Respir Physiol Neurobiol 2011;178:181–190. DOI: 10.1016/j.resp.2011.04.017.

[2] Naeije R. Physiological adaptation of the cardiovascular system to high altitude. Prog Cardiovasc Dis 2010;52:456–466. DOI: 10.1016/j.pcad.2010.03.004.

3. Conclusion

128 Hypoxia and Human Diseases

of the process of adaptation.

Jun Ke1,2, Lei Wang1,3 and Daliao Xiao1

Medicine, Loma Linda, California, USA

\*Address all correspondence to: Dxiao@llu.edu

of Medical Science, Guangzhou, Guangdong, China

\*

1 Center for Perinatal Biology, Department of Basic Science, Loma Linda University School of

2 Guangdong Cardiovascular Institute, Guangdong General Hospital, Guangdong Academy

3 Department of Traditional Chinese Medicine, People's Hospital of Shanghai Putuo District,

[1] Moore LG, Charles SM, Julian CG. Humans at high altitude: hypoxia and fetal growth.

Respir Physiol Neurobiol 2011;178:181–190. DOI: 10.1016/j.resp.2011.04.017.

Author details

Shanghai, China

References

The adaptation of the cardiovascular system to altitude is variable, depending on individual predisposition, the actual elevation, the rate of ascent, and the duration of exposure. In acute short-term exposure to HAH, the initial response is increasing sympathetic activity and hyperventilation resulting in increases in systemic vascular resistance, blood pressure, heart rate, and cardiac output. Pulmonary vasoconstriction leads to pulmonary hypertension. However, in response to acute HAH, cerebral blood flow (CBF) rises significantly to ensure an adequate supply of O2 to meet the brain tissues' large and consistent demand. The sympathetic excitation results from acute HAH, partly through chemoreceptor reflexes and partly through altered baroreceptor function. In long-term HAH exposure or resident at high altitude, cardiovascular system progresses a compensatory adaptation. The cardiovascular system may promote adaptational changes in cardiovascular structure, remodeling, and functional proteins through different molecular mechanisms including epigenetic regulatory and/or genetic factormediated mechanisms. However, cardiovasculatures may progress a pathologic adaptation and develop a maladaptation syndrome known as high-altitude pulmonary edema, cerebral edema, chronic mountain sickness, pulmonary hypertension, heart failure, and fetal intrauterine growth restriction. In conclusion, cardiovascular system progresses a compensatory and pathologic adaptation to HAH. Understanding those adaptation processes will help us to reduce the development of adverse changes and simultaneously preserve the beneficial signs


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#### **Arterial Oxygen Saturation During Ascent to 5010 m: Heart Rate and AMS Scores Arterial Oxygen Saturation During Ascent to 5010 m: Heart Rate and AMS Scores**

Christopher B. Wolff, Annabel H. Nickol and David J. Collier Christopher B. Wolff, Annabel H. Nickol and David J. Collier

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

http://dx.doi.org/10.5772/66088

#### **Abstract**

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ajpregu.00806.2006.

134 Hypoxia and Human Diseases

The hypothesis here is that tissues exposed to the hypoxia of altitude have increased blood flow so that the rate of arrival of oxygen is as rapid as normal. If the ascent is too rapid, the system starts to fail. The study involves an ascent to high altitude (5010 m) during which 59 subjects recorded their resting arterial oxygen saturation (SaO2), heart rate (HR) and Lake Louise acute mountain sickness (AMS) scores, twice daily. During the major ascent SaO2 fell progressively. In 42 subjects, HR increased in a highly significant, negative, relationship to SaO2. In 10 subjects heart rate (HR) remained unchanged. Three subjects showed extreme HR variability. Data were incomplete in four subjects. For nine of the subjects, showing the progressive HR versus SaO2 correlation during ascent, the sequence terminated with a lower HR than would be expected from the correlation so far. Individual AMS scores showed no correlation with SaO2 but averaged values from 19 of the subjects from each 'one night' stopover; showed a strong, negative, correlation. Average stopover HR values correlated negatively with the average SaO2 values. Cardiac output (CO) is likely to have increased during ascent as HR increased, since there is a progressive relationship between HR and cardiac output (CO). Hence, despite the progressive fall in SaO2, tissue oxygen delivery (DO2) would have remained close to normal in the 42 subjects who showed the significant HR: SaO2 relationship.

**Keywords:** high altitude, SaO2, heart rate, acute mountain sickness, oxygen delivery

© 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**

During ascent to high altitude, the fractional concentration of oxygen in the atmospheric gas is unchanged but overall barometric pressure falls. This means that there are less oxygen molecules per unit volume, so the activity, or oxygen partial pressure, falls. Breathing will only compensate for this, with a closer approach to normal concentration of oxygen in the blood (known as 'content,' CaO2), if it is increased. Initially, lowered oxygen in the lung and hence in the blood causes blood flow to the brain to increase, washing out carbon dioxide (CO2) from the brain environment. The increase in blood flow allows the rate of arrival of oxygen at the brain to normalize, the higher flow compensating for the lower CaO2. So the rate of arrival of oxygen (oxygen delivery, DO2) is sustained at or near the normal rate—three times the rate of cerebral oxygen consumption cerebral metabolic rate for oxygen, CMRO2 [1]. However, the lower CO2 in the brain, and resulting alkalinity, inhibits breathing via the central chemoreceptor, counter‐ acting the stimulating effect of low oxygen at the peripheral arterial chemoreceptor. There is therefore an initial pause in ventilatory stimulation. Over 2–5 days, for a subject remaining at the same altitude, brain inhibition is removed as acidity is corrected. This restores the central chemoreceptor level of respiratory stimulus (removal of inhibition). So now the ventilatory stimulus from the peripheral arterial chemoreceptor activity stimulates ventilation, with improvement in the arterial oxygen level [1]. Since these effects are operating at the same time as subjects ascend, with environmental oxygen falling progressively, arterial oxygen content (CaO2) and oxygen saturation (SaO2) usually fall progressively during ascent.

Since individual breathing responses (known as ventilatory responses) vary between individ‐ uals the progressive drop in CaO2 also varies between individuals. Most of the oxygen in the blood is carried on hemoglobin in the red cells and low CaO2 is paralleled by lower arterial oxygen saturation of the hemoglobin (SaO2). With little change in hemoglobin concentration (Hb) during ascent, SaO2 therefore provides a guide to CaO2 change. If there was no change in cardiac output (CO) the rate of oxygen delivery to the tissues (DO2 = CO × CaO2) would fall in proportion to the fall in SaO2. Here we are interested in the possibility that there may be compensatory increases in CO helping to sustain a more normal DO2. During ascent, the use of a pulse oximeter provides SaO2 and also gives the heart rate (HR). Cardiac output is equal to stroke volume (SV) times HR, that is, CO = HR × SV. With only modest changes in SV during ascent HR changes can therefore act as a surrogate for CO changes. Increases in HR as SaO2 falls during ascent would suggest that CO increases too. Increases in CO as SaO2/CaO2 fall will mean that there is compensatory response helping to maintain DO2. It is therefore important to see whether there is a significant HR increase in relation to falling SaO2 and to see whether this occurs in all or some subjects. Increases in heart rate can therefore be used as an indirect indicator of any cardiac output increase mitigating DO2 reduction.

In a recent high‐altitude study [2], undertaken in South America, eight normal subjects acclimatized to moderately high altitude during an initial 5‐day sojourn at Cusco (3324 m). This was followed by two brief ascents to around 5000 m over a 4‐week period. The preliminary acclimatization and brief ascents meant the subjects were likely to be less stressed by their hypoxic exposure than occurs with progressive ascents from sea level. Total time at high altitude was 28 days, the largest proportion at Cusco. SaO2 and HR were recorded twice daily. For seven of the subjects, there was a highly significant relationship between HR and SaO2, with the highest HR accompanying the lowest SaO2 value. The remaining subject sustained near normal SaO2 and HR. HR × SaO2 (assumed to give a value changing in relation to DO2) remained near constant throughout the trek in all subjects. Since HR × SaO2 will have a similar trend to CO × CaO2, DO2 will have been well sustained despite the varying degrees of hypoxia. Despite apparently relatively good DO2 maintenance, for these subjects, individual mean acute mountain sickness (AMS) scores correlated significantly with mean SaO2 values both at rest and with mild exercise (30 cm step up over 2 min).

In a second high‐altitude study, undertaken by 14 schoolboys and their teacher, an ascent was made to Annapurna base camp (4130 m) [3]. There was no preliminary acclimatization and, due to shortage of stopover sites, the final two ascent stages were large. The subjects each recorded SaO2 and HR soon after arrival at each new altitude both at rest and with mild exercise. Not all subjects showed individually significant HR versus SaO2 changes, but the overview across subjects (mean values at each altitude) was highly significant both for rest and exercise. Of interest was the fact that, for exercise, HR × SaO2, otherwise constant, showed a large fall for the last ascent stage and base camp. Anecdotally, most subjects suffered consid‐ erable AMS symptoms during the 1 day stop at base camp. A plot of the mean values of HR versus SaO2 for exercise shows a highly significant trend (**Figure 1**), but the HR value for base camp gives a point lower than expected from the rest of the values trend for HR versus SaO2. This may well represent a failure of DO2 compensation.

**Figure 1.** Data from this subject (15) illustrates rising HR during ascent. On the left HR (open squares), SaO2 (filled circles) and AMS × 10 scores (mini flags) are shown (left hand axis) with altitude (right hand axis, continuous line). The middle plot shows the ascending values of HR as filled squares and omits the AMS scores. The plot on the right shows the ascending HR values plotted against SaO2—slope significant at the *p* < 0.001 level.

#### **1.1. Reasons why DO2 control is important**

**1. Introduction**

136 Hypoxia and Human Diseases

During ascent to high altitude, the fractional concentration of oxygen in the atmospheric gas is unchanged but overall barometric pressure falls. This means that there are less oxygen molecules per unit volume, so the activity, or oxygen partial pressure, falls. Breathing will only compensate for this, with a closer approach to normal concentration of oxygen in the blood (known as 'content,' CaO2), if it is increased. Initially, lowered oxygen in the lung and hence in the blood causes blood flow to the brain to increase, washing out carbon dioxide (CO2) from the brain environment. The increase in blood flow allows the rate of arrival of oxygen at the brain to normalize, the higher flow compensating for the lower CaO2. So the rate of arrival of oxygen (oxygen delivery, DO2) is sustained at or near the normal rate—three times the rate of cerebral oxygen consumption cerebral metabolic rate for oxygen, CMRO2 [1]. However, the lower CO2 in the brain, and resulting alkalinity, inhibits breathing via the central chemoreceptor, counter‐ acting the stimulating effect of low oxygen at the peripheral arterial chemoreceptor. There is therefore an initial pause in ventilatory stimulation. Over 2–5 days, for a subject remaining at the same altitude, brain inhibition is removed as acidity is corrected. This restores the central chemoreceptor level of respiratory stimulus (removal of inhibition). So now the ventilatory stimulus from the peripheral arterial chemoreceptor activity stimulates ventilation, with improvement in the arterial oxygen level [1]. Since these effects are operating at the same time as subjects ascend, with environmental oxygen falling progressively, arterial oxygen content

(CaO2) and oxygen saturation (SaO2) usually fall progressively during ascent.

indicator of any cardiac output increase mitigating DO2 reduction.

Since individual breathing responses (known as ventilatory responses) vary between individ‐ uals the progressive drop in CaO2 also varies between individuals. Most of the oxygen in the blood is carried on hemoglobin in the red cells and low CaO2 is paralleled by lower arterial oxygen saturation of the hemoglobin (SaO2). With little change in hemoglobin concentration (Hb) during ascent, SaO2 therefore provides a guide to CaO2 change. If there was no change in cardiac output (CO) the rate of oxygen delivery to the tissues (DO2 = CO × CaO2) would fall in proportion to the fall in SaO2. Here we are interested in the possibility that there may be compensatory increases in CO helping to sustain a more normal DO2. During ascent, the use of a pulse oximeter provides SaO2 and also gives the heart rate (HR). Cardiac output is equal to stroke volume (SV) times HR, that is, CO = HR × SV. With only modest changes in SV during ascent HR changes can therefore act as a surrogate for CO changes. Increases in HR as SaO2 falls during ascent would suggest that CO increases too. Increases in CO as SaO2/CaO2 fall will mean that there is compensatory response helping to maintain DO2. It is therefore important to see whether there is a significant HR increase in relation to falling SaO2 and to see whether this occurs in all or some subjects. Increases in heart rate can therefore be used as an indirect

In a recent high‐altitude study [2], undertaken in South America, eight normal subjects acclimatized to moderately high altitude during an initial 5‐day sojourn at Cusco (3324 m). This was followed by two brief ascents to around 5000 m over a 4‐week period. The preliminary acclimatization and brief ascents meant the subjects were likely to be less stressed by their hypoxic exposure than occurs with progressive ascents from sea level. Total time at high

Surprisingly, oxygen combines extreme toxicity with capability to provide energy more efficiently than other biochemical means utilized by other species. The ready conversion of oxygen to dangerous free radicals is largely prevented in our cells by the specific energy‐ generating biochemical sequences, especially featuring the Krebs cycle. There is an optimum rate at which oxygen flows to a particular tissue and this bears a constant relationship to the rate at which oxygen is utilized (VO2). Hence, we have values for DO2/VO2 which are normally sustained at values specific to the tissue: for the brain 3:1, for heart 1.6:1 and at exercise rates below competitive levels skeletal muscle sustains a ratio 1.5:1 [4]. Maintaining these DO2 values not only provides sufficient oxygen but also avoids an excess, which would endanger toxicity. Inadequacy of oxygen supply can endanger life following surgery partly from a tendency for an oxygen debt to develop during an operation. This results from reduced arterial pressure since some of the arterial volume escapes into veins relaxed by the anesthesia. The reduced pressure lowers cardiac output so that DO2 falls, hence development of an oxygen debt [5].

Cerebral arterial blood flow and metabolic rate obtained by Severinghaus et al. [6] were further examined and showed that cerebral oxygen delivery was sustained after ascent to 3100 m, over the next 5 days. There was an initial fall in SaO2 and compensatory rise in cerebral blood flow. The acclimatization process allowed the initially lowest SaO2 to improve over the 5 days at altitude [7, 8].

## **2. Methods**

Fifty‐nine normal subjects undertook the trek to Kanchenjunga base camp (altitude 5010 m) from Kathmandu (1345 m). The first leg by plane took them to Tumlingtar (470 m) and then they made an initial partial ascent to 2900 m. There was then a descent to 675 m by the 7th day. From there, the rest of the trek (the main ascent) took around 14 days (a conservative ascent profile of just over 300 m per day). Parties of 7–11 subjects set off from Kathmandu separated by a few days. Each group was accompanied by porters and cooks who went ahead each day to prepare the next stopover site. Porters and yaks carried most of the research equipment for other studies and much of the individual luggage, largely housed in individual 60 L barrels. This meant that each subject carried a low‐weight 'day pack.' Each subject measured their arterial saturation (SaO2) and heart rate (HR) using a (Nonin, model 9500, Nonin Medical Inc. MIN, USA) oximeter. The evening measurement was made after at least 5 min rest, seated in the mess tent prior to supper. Readings were made after around 1 min to allow stability. A second measurement of SaO2 and HR was made in the morning at each stopover site. Again, each measurement was made after at least 5 min rest in the mess tent prior to breakfast. At both morning and evening sessions, each subject filled in individual 'altitude sickness scores' for each of five symptom complexes (head, guts, tired, dizzy, sleep). Each category belongs to the mode of acute mountain sickness (AMS) assessment known as the Lake Louise consensus AMS scoring system [9]. The numerical system requires scores of 0–3 for each category. The AMS score is the total of the values entered in each category. For each category, zero represents no effect and for three, the symptom in the category is severe. Hence, the total possible range, theoretically, for a given score is from 0 to 15. A subject is deemed to be sick, however, with AMS with scores above 3.

## **3. Results**

rate at which oxygen flows to a particular tissue and this bears a constant relationship to the rate at which oxygen is utilized (VO2). Hence, we have values for DO2/VO2 which are normally sustained at values specific to the tissue: for the brain 3:1, for heart 1.6:1 and at exercise rates below competitive levels skeletal muscle sustains a ratio 1.5:1 [4]. Maintaining these DO2 values not only provides sufficient oxygen but also avoids an excess, which would endanger toxicity. Inadequacy of oxygen supply can endanger life following surgery partly from a tendency for an oxygen debt to develop during an operation. This results from reduced arterial pressure since some of the arterial volume escapes into veins relaxed by the anesthesia. The reduced pressure lowers cardiac output so that DO2 falls, hence development of an oxygen debt [5].

Cerebral arterial blood flow and metabolic rate obtained by Severinghaus et al. [6] were further examined and showed that cerebral oxygen delivery was sustained after ascent to 3100 m, over the next 5 days. There was an initial fall in SaO2 and compensatory rise in cerebral blood flow. The acclimatization process allowed the initially lowest SaO2 to improve over the 5 days at

Fifty‐nine normal subjects undertook the trek to Kanchenjunga base camp (altitude 5010 m) from Kathmandu (1345 m). The first leg by plane took them to Tumlingtar (470 m) and then they made an initial partial ascent to 2900 m. There was then a descent to 675 m by the 7th day. From there, the rest of the trek (the main ascent) took around 14 days (a conservative ascent profile of just over 300 m per day). Parties of 7–11 subjects set off from Kathmandu separated by a few days. Each group was accompanied by porters and cooks who went ahead each day to prepare the next stopover site. Porters and yaks carried most of the research equipment for other studies and much of the individual luggage, largely housed in individual 60 L barrels. This meant that each subject carried a low‐weight 'day pack.' Each subject measured their arterial saturation (SaO2) and heart rate (HR) using a (Nonin, model 9500, Nonin Medical Inc. MIN, USA) oximeter. The evening measurement was made after at least 5 min rest, seated in the mess tent prior to supper. Readings were made after around 1 min to allow stability. A second measurement of SaO2 and HR was made in the morning at each stopover site. Again, each measurement was made after at least 5 min rest in the mess tent prior to breakfast. At both morning and evening sessions, each subject filled in individual 'altitude sickness scores' for each of five symptom complexes (head, guts, tired, dizzy, sleep). Each category belongs to the mode of acute mountain sickness (AMS) assessment known as the Lake Louise consensus AMS scoring system [9]. The numerical system requires scores of 0–3 for each category. The AMS score is the total of the values entered in each category. For each category, zero represents no effect and for three, the symptom in the category is severe. Hence, the total possible range, theoretically, for a given score is from 0 to 15. A subject is deemed to be sick, however, with

altitude [7, 8].

138 Hypoxia and Human Diseases

**2. Methods**

AMS with scores above 3.

AMS scores showed no obvious trends during the major ascent for any individual, whereas SaO2 fell during ascent in all cases (where the data had been recorded—a few subjects omitted variable amounts of data). The heart rate, however, showed an obvious progressively increas‐ ing trend in 42 individuals. For 10 subjects, there was no HR change during ascent.

**Figure 1** shows a particularly clear example of increasing HR and decreasing SaO2 for one individual during the major ascent. The AMS score is included in the left‐hand panel and omitted in the middle panel, where the square HR symbol during ascent is filled in, to emphasis the progressively increasing HR. In the right‐hand panel, HR from the ascent is plotted against SaO2, showing the highly significant trend in this subject. For the 42 subjects showing increasing HR during ascent and falling SaO2, individual least squares regression plots (HR versus SaO2) give significance levels (*p* values). The degree of the HR versus SaO2 relationship significance is shown for each subject in **Table 1**, according to the subject's identification number. Nineteen subjects (32%) showed significance at the 0.001 level; 17 (29%) at the 0.01 level and for 6 subjects (10%) significance level was only 0.05. The status for each of the remaining subjects is also shown: four subjects with poor data (very few recorded points or none at all), three subjects with apparently random data (labeled 'variability') and the ten subjects in whom HR was effectively unchanged. **Figure 2** shows HR, SaO2, AMS score and altitude against time for three of the subjects in whom there was an unchanging HR. The first stopped recording prior to reaching base camp, the second stopped recording at base camp and the third continued, at least HR recording, even during descent.


**Table 1.** Listing of all subjects according to (a) whether they showed a significant relationship between HR and SaO2 during ascent in the upper part of the table (*p* < 0.001, *p* < 0.01 and *p* < 0.05) and (b) those without a significant relationship (poor data, variability or a constant HR).

In the face of any clear individual indication of progress of AMS scores during ascent, it was important to see whether the expected general tendency held good. Mean values for AMS scores and for HR and SaO2 were calculated for 19 subjects (numbers 1–19) at each altitude. Mean AMS increased progressively with increasing altitude and was significantly related to SaO2 (**Figure 3**, middle panel). This simply confirms the trend expected with ascent to altitude. Mean arterial oxygen saturation at each one‐night stopover fell progressively during ascent (**Figure 3**, left panel, SaO2% = −0.0043 × altitude (m) + 102.24; *R*<sup>2</sup> = 0.972) though there was considerable variation for the mean values at each stopover site. The error bars show the maximum and minimum individual values.

**Figure 2.** Plots against time of HR (open squares), SaO2 (filled circles), AMS score (mini‐flag) and altitude (continuous line) against time. Each is an example of data from a subject in whom HR remained near constant (subjects, 15, 44 and 38).

**Figure 3.** Mean values at each altitude of, SaO2%, AMS score and HR. The left panel shows SaO2 against altitude with maximum and minimum values as 'error bars.' The middle panel shows mean AMS score against SaO2 for one‐night stopovers during the major ascent. The right‐hand panel shows mean (HR ‐ 72) plotted against SaO2% above the first two stopovers of the major ascent. The points are from the lowest altitudes were probably from subjects who were not fully rested).

For mean HR (minus 72 as an assumed normal at sea level), values at each stopover site show a smooth relationship to SaO2 for altitudes above 2400 m (**Figure 3**, right‐hand panel). The higher mean HR at lower SaO2 is consistent with the tendency for an increasing heart rate during ascent, found for most individuals.

Most subjects in the present study (*n* = 42, 71%) showed the significant HR: SaO2 relationship. The trend relative to SaO2 was sustained in 33 subjects; however, for nine subjects (numbered: 20, 25, 48, 50, 52, 53, 55, 56 and 57), the trend ended at the lowest SaO2 with a lower HR than predicted by the trend to date. **Figure 4** shows an example (subject 25). HR (open rectangles) changes in the figure during base camp to larger open circles, where the points are obviously lower than expected from the trend during ascent. Although anecdotal, it may be no coinci‐ dence that the AMS scores are at their maximum at the same time.

Most subjects in the present study (*n* = 42, 71%) showed the significant HR: SaO2 relationship. The trend relative to SaO2 was sustained in 33 subjects; however, for nine subjects (numbered: 20, 25, 48, 50, 52, 53, 55, 56 and 57), the trend ended at the lowest SaO2 with a lower HR than predicted by the trend to date. **Figure 4** shows an example (subject 25). HR (open rectangles) change in the figure during base camp to larger open circles, where the points are obviously lower than expected from the trend during ascent. Although anecdotal, it may be no coinci‐ dence that the AMS scores are at their maximum at the same time.

**Figure 4.** Example of late fall in HR. For subject 25, there was a steady rise in HR (left figure, open squares) during ascent (the period 9–18 days—from 1235 m to base camp at 5100 m). The next two values of HR after reaching base camp (large open circles) were significantly reduced. SaO2 (filled diamonds) had fallen as usual during ascent. On the left, altitude is shown as a continuous line and AMS score (×10) shows as a thin jagged line. The plot on the right shows HR during the ascent plotted against SaO2. The open circle represents the one of the two lower HR values for which SaO2 was recorded. The lowered HR is thought to reflect impaired compensation for hypoxia.

## **4. Discussion**

Mean arterial oxygen saturation at each one‐night stopover fell progressively during ascent

considerable variation for the mean values at each stopover site. The error bars show the

**Figure 2.** Plots against time of HR (open squares), SaO2 (filled circles), AMS score (mini‐flag) and altitude (continuous line) against time. Each is an example of data from a subject in whom HR remained near constant (subjects, 15, 44 and

**Figure 3.** Mean values at each altitude of, SaO2%, AMS score and HR. The left panel shows SaO2 against altitude with maximum and minimum values as 'error bars.' The middle panel shows mean AMS score against SaO2 for one‐night stopovers during the major ascent. The right‐hand panel shows mean (HR ‐ 72) plotted against SaO2% above the first two stopovers of the major ascent. The points are from the lowest altitudes were probably from subjects who were not

For mean HR (minus 72 as an assumed normal at sea level), values at each stopover site show a smooth relationship to SaO2 for altitudes above 2400 m (**Figure 3**, right‐hand panel). The higher mean HR at lower SaO2 is consistent with the tendency for an increasing heart rate

Most subjects in the present study (*n* = 42, 71%) showed the significant HR: SaO2 relationship. The trend relative to SaO2 was sustained in 33 subjects; however, for nine subjects (numbered: 20, 25, 48, 50, 52, 53, 55, 56 and 57), the trend ended at the lowest SaO2 with a lower HR than predicted by the trend to date. **Figure 4** shows an example (subject 25). HR (open rectangles) changes in the figure during base camp to larger open circles, where the points are obviously lower than expected from the trend during ascent. Although anecdotal, it may be no coinci‐

= 0.972) though there was

(**Figure 3**, left panel, SaO2% = −0.0043 × altitude (m) + 102.24; *R*<sup>2</sup>

maximum and minimum individual values.

140 Hypoxia and Human Diseases

38).

fully rested).

during ascent, found for most individuals.

dence that the AMS scores are at their maximum at the same time.

This HR, SaO2 and AMS data, collected during a high‐altitude trek to Kanchenjunga base camp in 1998, has shown important variations in individual responses. These differences raise questions concerning physiological mechanisms, such as the reason why a significant propor‐ tion of subjects (10 out of a total of 59 or 52 if we exclude 3 with inadequate data and variability —see **Table 1**) maintained a constant resting HR, despite falling SaO2 during ascent. In contrast, in most subjects (42), HR increased in relation to falling SaO2. Despite inspection of the individual time‐based plots of AMS scores, there seemed no help there with its prediction, though there was, of course, the overall upward trend in the average AMS scores during ascent, accompanying lowered SaO2 (**Figure 3**, middle panel).

A highly significant difference in vulnerability to acute mountain sickness (AMS) has been shown to be related to differences in the type of ACE gene (angiotensin‐converting enzyme) carried by the subject. It is part of the renin‐angiotensin system, which regulates blood pressure and the balance of fluids and salts in the body. Those with so‐called 'double insertion' of the ACE gene experience far less trouble with high‐altitude ascent than do those with 'double deletion.' Subjects with the mixed 'insertion/deletion' properties are intermediate [10]. It seems likely that good compensation with maintained DO2 will be the background reason for the altitude advantage of those with double insertion ACE gene but this, of course, would need detailed examination in a major study. It is also possible that the subjects who sustained a constant heart rate with progressive lowering of SaO2 belong to the double deletion group. Specific measurement would be required in the study to answer this question. There seems no known difference in responses to hypoxic exposure between men and women.

The significant inverse correlation between HR and SaO2 in 42 subjects is consistent with the DO2 priority of body tissues, illustrated for skeletal muscle [11], the brain [7, 8] and heart [4] and demonstrated for the whole body [12] in subjects breathing 12% oxygen—resting DO2 for each individual was the same on 12% oxygen as on air, despite variations in SaO2 in each individual on 12% oxygen.

It has been pointed out that HR usually increases as CO increases, but there may not have been complete DO2 compensation in those who increased their HR during ascent. It is possible that the CO increase falls short of sustaining normal DO2. Again, it would be useful to know whether the compensation indicated by the HR increase with falling SaO2 is actually complete.

The depression of HR below that expected from the trend, usually found when it occurs, close to or actually at base camp was most likely to reflect inadequate DO2. If this did represent a significant fall in DO2 such subjects could be more vulnerable to AMS. For the subject, illustrated AMS scores were already increasing. It would be helpful in confirmation or refutation of assertions about HR if CO (in preference to HR) could be measured during ascent. Suitable portable equipment is awaited though none is at present on the horizon.

#### **4.1. The value of the study**

It is hoped that the illustration here of a variety of different features of the responses of individuals to the hypoxic environment of altitude will help guide future investigation and throw light on mechanisms responsible for AMS.

The study reported here is consistent with the ability of the body to sustain normal and adequate rates of oxygen delivery to the tissues. This has been shown to be limited by the severity of the hypoxic exposure with variation between subjects as to whether the limit is reached, the level of SaO2 at which it happens, and the fact that a significant proportion of subjects do not make the compensatory adjustments seen in the majority.

The novelty of this study is the new insight that the heart rate increase is a reflection of increased cardiac output sustaining a compensatory rate of oxygen delivery to the tissues. When the heart rate fails to increase as expected from results to date it may be a clue that compensation is failing.

## **Acknowledgements**

Thanks are due to Medical Expeditions (Altitude Education and Research Charity). This work was facilitated by the NIHRB Biomedical Research Unit in Cardiovascular Disease at Barts.

## **Author details**

altitude advantage of those with double insertion ACE gene but this, of course, would need detailed examination in a major study. It is also possible that the subjects who sustained a constant heart rate with progressive lowering of SaO2 belong to the double deletion group. Specific measurement would be required in the study to answer this question. There seems no

The significant inverse correlation between HR and SaO2 in 42 subjects is consistent with the DO2 priority of body tissues, illustrated for skeletal muscle [11], the brain [7, 8] and heart [4] and demonstrated for the whole body [12] in subjects breathing 12% oxygen—resting DO2 for each individual was the same on 12% oxygen as on air, despite variations in SaO2 in each

It has been pointed out that HR usually increases as CO increases, but there may not have been complete DO2 compensation in those who increased their HR during ascent. It is possible that the CO increase falls short of sustaining normal DO2. Again, it would be useful to know whether the compensation indicated by the HR increase with falling SaO2 is actually complete. The depression of HR below that expected from the trend, usually found when it occurs, close to or actually at base camp was most likely to reflect inadequate DO2. If this did represent a significant fall in DO2 such subjects could be more vulnerable to AMS. For the subject, illustrated AMS scores were already increasing. It would be helpful in confirmation or refutation of assertions about HR if CO (in preference to HR) could be measured during ascent.

It is hoped that the illustration here of a variety of different features of the responses of individuals to the hypoxic environment of altitude will help guide future investigation and

The study reported here is consistent with the ability of the body to sustain normal and adequate rates of oxygen delivery to the tissues. This has been shown to be limited by the severity of the hypoxic exposure with variation between subjects as to whether the limit is reached, the level of SaO2 at which it happens, and the fact that a significant proportion of

The novelty of this study is the new insight that the heart rate increase is a reflection of increased cardiac output sustaining a compensatory rate of oxygen delivery to the tissues. When the heart rate fails to increase as expected from results to date it may be a clue that compensation

Thanks are due to Medical Expeditions (Altitude Education and Research Charity). This work was facilitated by the NIHRB Biomedical Research Unit in Cardiovascular Disease at Barts.

known difference in responses to hypoxic exposure between men and women.

Suitable portable equipment is awaited though none is at present on the horizon.

subjects do not make the compensatory adjustments seen in the majority.

individual on 12% oxygen.

142 Hypoxia and Human Diseases

**4.1. The value of the study**

is failing.

**Acknowledgements**

throw light on mechanisms responsible for AMS.

Christopher B. Wolff1\*, Annabel H. Nickol2 and David J. Collier1

\*Address all correspondence to: chriswolff@doctors.org.uk

1 William Harvey Heart Centre, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, UK

2 Oxford Centre, for Respiratory Medicine, Churchill Hospital, Oxford, UK

## **References**


#### **Hypoxia-Induced Molecular and Cellular Changes in the Congenitally Diseased Heart: Mechanisms and Strategies of Intervention Hypoxia-Induced Molecular and Cellular Changes in the Congenitally Diseased Heart: Mechanisms and Strategies of Intervention**

Dominga Iacobazzi, Massimo Caputo and Mohamed T Ghorbel Dominga Iacobazzi , Massimo Caputo and Mohamed T Ghorbel

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

http://dx.doi.org/10.5772/66038

#### **Abstract**

[11] Wolff CB. Cardiac output, oxygen consumption and muscle oxygen delivery in submaximal exercise: normal and low O2 states. Adv Exp Med Biol. 2003;510:279–284.

[12] Bell M, Thake CD, Wolff CB. Effect of Inspiration of 12% O2 (balance N2) on cardiac output, respiration, oxygen saturation and oxygen delivery. Adv Exp Med Biol.

2011;915:327–332.

144 Hypoxia and Human Diseases

Tissue hypoxia plays a critical role in the pathobiology of congenital heart diseases, especially with regard to cyanotic patients. Here, we describe the cellular and molecular mechanisms induced by hypoxia in the diseased heart, with particular attention to the metabolic and functional changes that underlie the hypoxia-induced right ventricle remodelling. The role of reactive oxygen species in transcriptomic changes, DNA damage, contractile dysfunction and extracellular matrix remodelling will be addressed. Furthermore, the reoxygenation injury, which occurs when oxygen is reintroduced upon initiation of cardiopulmonary bypass, will be discussed. This allows a better understanding of the risks associated with the reoxygenation injury in children undergoing openheart surgery and helps to improve strategies of intervention for myocardial protection.

**Keywords:** hypoxia, congenital heart disease, cyanosis, reoxygenation injury, cardiovascular disease

## **1. Hypoxia in cardiovascular disease and congenital heart disease**

The term hypoxia refers to a condition where the tissues are not adequately oxygenated, usually due to interrupted coronary blood flow or a reduction in arterial blood oxygen partial pressure [1]. With the heart being a highly oxidative organ, relying on high oxygen consumption for the work of its contractile machinery, it appears obvious that cardiac cells are very sensitive to oxygen deprivation [2]. Heart hypoxia, which originates as a

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result of disproportion between the amount of oxygen supplied to the cardiac cell and the amount required by the cell, plays a critical role in the pathobiology of several cardiovascular diseases [3]. These include myocardial infarction, coronary artery diseases, heart failure secondary to pulmonary disease and congenital heart diseases [1, 4, 5]. In patients with coronary artery diseases and myocardial infarction, hypoxia is usually due to the formation of an atherosclerotic plaque in the wall of coronary arteries, which reduces the perfusion of myocardial tissue [6]. In addition, a rupture of the plaque might result in complete arterial occlusion, leading to the death of the ischemic tissue [6]. The increased O2 consumption caused by pressure overload and reduced O2 delivery, due to impaired coronary blood flow, are the main causes of hypoxia in patients suffering from heart failure secondary to pulmonary hypertension [7].

The scenario looks different when shifting the focus to myocardial hypoxia in paediatric patients with congenital heart diseases (CHDs). Diseases affecting the heart, in fact, have usually a different pathophysiology in children compared to adult population [8]. Furthermore, as a result of the different pathophysiological function of the defective heart, the paediatric and adult patients are differently susceptible to stress insults, although there is still disagreement on whether the vulnerability of immature heart is less or more than for adult heart [9–12].

Congenital heart diseases include a wide spectrum of anomalies of the cardiac architecture, and they are usually classified based on the anatomical and pathophysiological nature of the defect. The main anomalies involve atrioventricular junctions and valves [i.e. atrial septal defect (ASD), ventricular septal defect (VSD), atrioventricular septal defect (AVSD)], the ventricular outflow tracts [like in tetralogy of Fallot (TOF)] or can consist of univentricular hearts [like single ventricle (SV)] [13].

More often, congenital heart defects are simply classified as cyanotic and acyanotic, depending on whether or not the defect affects the amount of oxygen in the body. In cyanotic heart defects, as consequence of a mixture between oxygenated and de-oxygenated blood, less oxygen-rich blood reaches the different tissues of the body, resulting in a bluish skin, lips and nail bed colour. This category includes defects such as TOF, transposition of the great vessels or truncus arteriosus. On the other hand, non-cyanotic CHD patients do not experience a lack in blood oxygen supply; therefore, they rarely develop the bluish colour, except for few occasion, when the baby needs more oxygen, such as when crying and feeding. Atrial and/or ventricular septal defects or coarctation of the aorta are examples of acyanotic CHDs [14, 15].

Several studies have shown that, among CHDs, cyanotic patients are much more prone to develop a severe chronic hypoxia state, compared to the acyanotic ones, as the lack of oxygen exposes the cardiac tissue to an increase in free oxygen radicals [16, 17]. Therefore, when considering the treatment of these patients, the oxidative stress problem has to be taken into account, in addition to the other anomalies that characterize these defects. Nevertheless, care must be taken also for the treatment of acyanotic patients, to prevent the hypoxia that might develop in a later stage.

## **2. Mechanism underlying the hypoxia response in Congenital Heart Disease**

#### **2.1. Depletion of antioxidant defences**

result of disproportion between the amount of oxygen supplied to the cardiac cell and the amount required by the cell, plays a critical role in the pathobiology of several cardiovascular diseases [3]. These include myocardial infarction, coronary artery diseases, heart failure secondary to pulmonary disease and congenital heart diseases [1, 4, 5]. In patients with coronary artery diseases and myocardial infarction, hypoxia is usually due to the formation of an atherosclerotic plaque in the wall of coronary arteries, which reduces the perfusion of myocardial tissue [6]. In addition, a rupture of the plaque might result in complete arte-

tion caused by pressure overload and reduced O2 delivery, due to impaired coronary blood flow, are the main causes of hypoxia in patients suffering from heart failure secondary to

The scenario looks different when shifting the focus to myocardial hypoxia in paediatric patients with congenital heart diseases (CHDs). Diseases affecting the heart, in fact, have usually a different pathophysiology in children compared to adult population [8]. Furthermore, as a result of the different pathophysiological function of the defective heart, the paediatric and adult patients are differently susceptible to stress insults, although there is still disagreement on whether the vulnerability of immature heart is less or more than for adult heart

Congenital heart diseases include a wide spectrum of anomalies of the cardiac architecture, and they are usually classified based on the anatomical and pathophysiological nature of the defect. The main anomalies involve atrioventricular junctions and valves [i.e. atrial septal defect (ASD), ventricular septal defect (VSD), atrioventricular septal defect (AVSD)], the ventricular outflow tracts [like in tetralogy of Fallot (TOF)] or can consist of univentricular hearts

More often, congenital heart defects are simply classified as cyanotic and acyanotic, depending on whether or not the defect affects the amount of oxygen in the body. In cyanotic heart defects, as consequence of a mixture between oxygenated and de-oxygenated blood, less oxygen-rich blood reaches the different tissues of the body, resulting in a bluish skin, lips and nail bed colour. This category includes defects such as TOF, transposition of the great vessels or truncus arteriosus. On the other hand, non-cyanotic CHD patients do not experience a lack in blood oxygen supply; therefore, they rarely develop the bluish colour, except for few occasion, when the baby needs more oxygen, such as when crying and feeding. Atrial and/or ventricular septal defects or coarctation of the aorta are examples of acyanotic

Several studies have shown that, among CHDs, cyanotic patients are much more prone to develop a severe chronic hypoxia state, compared to the acyanotic ones, as the lack of oxygen exposes the cardiac tissue to an increase in free oxygen radicals [16, 17]. Therefore, when considering the treatment of these patients, the oxidative stress problem has to be taken into account, in addition to the other anomalies that characterize these defects. Nevertheless, care must be taken also for the treatment of acyanotic patients, to prevent the hypoxia that might

consump-

rial occlusion, leading to the death of the ischemic tissue [6]. The increased O2

pulmonary hypertension [7].

146 Hypoxia and Human Diseases

[like single ventricle (SV)] [13].

CHDs [14, 15].

develop in a later stage.

[9–12].

The exposure of a defective heart to chronic hypoxia induces molecular and cellular changes that affect the myocardial function and metabolism. One of the most typical sign of a heartdeveloping chronic hypoxia is the unbalance between the level of reactive oxygen species (ROS) and the antioxidant defence system. ROS are physiologically produced during cell metabolic and energetic reactions [18]. Nevertheless, the body is endowed with antioxidant enzymes such as catalase, glutathione peroxidase, and superoxide dismutase and vitamins (retinoic acid, alpha-tocopherol, ascorbic acid) that can counteract this physiological production [19]. Even in case of excessive free radical production, the body responds to restore harmony balance [20]. However, under chronic hypoxia, a downregulation of antioxidant defences occurs, making the cells vulnerable to oxidant damage. Two different studies analysing the oxidant status of paediatric patients with CHDs revealed that the oxidative stress index, given by the ratio between pro-oxidants and antioxidants factors, was higher in the plasma of cyanotic children compared to the controls [16, 17]. No difference was found between acyanotic and control groups, thus confirming that the anatomical defect dictates the hypoxic level and the oxidative status [16, 17].

#### **2.2. Hypoxia-induced metabolic and functional changes: the basis of right ventricle remodelling**

Metabolic markers of oxidative stress, such as 8-isoprostane, were shown to be high in cyanotic patients' heart as revealed by our study evaluating the transcriptomic analysis of patients with tetralogy of Fallot (TOF) [21]. In a different study, we performed a genome-wide investigation to determine the global gene expression profiles associated with chronic hypoxia in the heart of patients with TOF, undergoing corrective cardiac surgery. The data revealed that 795 genes were differently expressed in cyanotic versus acyanotic hearts. In particular, genes associated with the contractility machinery function and MAPK signalling, involved in cell survival and antioxidant defence, were downregulated, whereas growth, remodelling and apoptosisrelated genes were upregulated in the cyanotic group compared to the acyanotic one [22].

The altered gene expression triggered by the rise in reactive oxygen species is mostly responsible for the cellular and molecular changes that affect the myocardial function and metabolism, thus predisposing the heart to hypertrophy and failure. The hypoxia-induced downregulation of the sodium-calcium (Na+ –Ca2+) exchanger (NCX1) in cyanotic patients decreases myocyte calcium handling capacity, leading to mechanical dysfunction [22]. In addition, ROS can induce oxidative modification of the sarcoplasmic membrane channels: the ryanodine receptor2 (RyR2) becomes abnormally activated while sarcoplasmic reticulum Ca2+-ATPase (SERCA) is inhibited, causing an abnormal Ca2+ transient between cytosol and sarcoplasmic reticulum that contributes to the cardiomyocytes contractile dysfunction [23, 24]. ROS accumulation has also a detrimental effect on mitochondrial function by sustaining mitochondrial permeability transition pore (mPTP) opening and mitochondrial membrane depolarization. As a result, mitochondrial respiration is inhibited with less ATP production. The insufficient energy production also arises from the switch from an aerobic metabolism to a high glycolytic metabolic profile. Protein kinase D (PDK), which inhibits pyruvate dehydrogenase during glucose oxidation, is a key factor in the deficient energy supply [25, 26]. Another aspect of redox imbalance is the extracellular matrix (ECM) modification deriving from the matrix metalloproteinases (MMP) activation, which leads to heart remodelling and fibrosis [27].

Within the complex architecture of the heart, the right ventricle (RV) seems to be the most susceptible structure to be affected by the above-mentioned hypoxia-induced changes. The different morphology and metabolism between the left and the right ventricle can in part explain the different susceptibility [28]. Furthermore, the anatomy of most of the CHD exposes the right ventricle to higher stresses, making it more prone to fail than the left counterpart [24]. One of the main insults to which the RV is subjected is the pressure overload that can derive from pulmonary artery hypertension (PAH) or RV outflow obstruction, with both events leading to right ventricular hypertrophy (RVH) and eventually to right ventricular failure.

#### **2.3. HIF-1alpha mediated angiogenic response**

One of the key features of chronic hypoxia is the activation of the HIF-1alpha (HIF-1α) signalling, an essential regulator of the angiogenic response. The mechanisms by which HIF-1α is triggered are relatively well-understood: under hypoxia, HIF1-alpha degradation is prevented by the hydroxylation of specific protein residues, and therefore, its translocation to the nucleus promotes the transcriptions of pro-angiogenic genes like vascular endothelial growth factor (VEGF), platelet-derived endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP) and erythropoietin (EPO) [29–31].

The role of HIF-1α in adult ischemic heart disease and pressure overload heart failure has been widely demonstrated by different research groups [6, 30, 32]. However, only few studies have investigated its involvement in the pathogenesis of congenital heart disease [6, 33, 34].

An important increase in HIF-1α and related pro-angiogenic genes and proteins have been reported in ventricular biopsies from children with cyanotic congenital heart disease, compared with acyanotic or control groups [22, 35]. In addition, mRNA level of HIF-1α as well as that of two of its representative target genes, VEGF and EPO, were found to be upregulated in blood samples of newborns with cyanosis and persistent pulmonary hypertension, therefore representing early markers of generalized hypoxia [36]. If the HIF-1α/VEGF-induced collateral vessel formation in hypoxemic myocardium is essential to compensate the lack of oxygen supply in cyanotic hearts, especially in cases of coarctation of aorta, an abnormal vessel formation can become a source of morbidity, due to arteriovenous malformations [34]. However, a correlation between VEGF increase and abnormal vessel formation has not yet been found [37]. Nevertheless, increased activation of HIF-1α/VEGF signalling might be detrimental in newborn with persistent pulmonary hypertension, as these patients normally present an overexpression of VEGF receptor 1 (VEGFR1), which accounts for the vasoconstrictor effect of VEGF [38].

Further mechanisms, independent from HIF-1α might account for the hypoxia-induced VEGF production in CHDs. As hypoxia is often associated with tissue damage and apoptosis, cytokines or other mediators (IL-10, TNF-α, TGF-β, etc.) might as well initiate the cascade that leads to VEGF production [31].

#### **2.4. Hypoxia-induced DNA damage**

mitochondrial permeability transition pore (mPTP) opening and mitochondrial membrane depolarization. As a result, mitochondrial respiration is inhibited with less ATP production. The insufficient energy production also arises from the switch from an aerobic metabolism to a high glycolytic metabolic profile. Protein kinase D (PDK), which inhibits pyruvate dehydrogenase during glucose oxidation, is a key factor in the deficient energy supply [25, 26]. Another aspect of redox imbalance is the extracellular matrix (ECM) modification deriving from the matrix metalloproteinases (MMP) activation, which leads to heart remodelling and

Within the complex architecture of the heart, the right ventricle (RV) seems to be the most susceptible structure to be affected by the above-mentioned hypoxia-induced changes. The different morphology and metabolism between the left and the right ventricle can in part explain the different susceptibility [28]. Furthermore, the anatomy of most of the CHD exposes the right ventricle to higher stresses, making it more prone to fail than the left counterpart [24]. One of the main insults to which the RV is subjected is the pressure overload that can derive from pulmonary artery hypertension (PAH) or RV outflow obstruction, with both events leading to right ventricular hypertrophy (RVH) and eventually to right ventricular failure.

One of the key features of chronic hypoxia is the activation of the HIF-1alpha (HIF-1α) signalling, an essential regulator of the angiogenic response. The mechanisms by which HIF-1α is triggered are relatively well-understood: under hypoxia, HIF1-alpha degradation is prevented by the hydroxylation of specific protein residues, and therefore, its translocation to the nucleus promotes the transcriptions of pro-angiogenic genes like vascular endothelial growth factor (VEGF), platelet-derived endothelial cell growth factor/thymidine phosphory-

The role of HIF-1α in adult ischemic heart disease and pressure overload heart failure has been widely demonstrated by different research groups [6, 30, 32]. However, only few studies have investigated its involvement in the pathogenesis of congenital heart disease [6, 33, 34]. An important increase in HIF-1α and related pro-angiogenic genes and proteins have been reported in ventricular biopsies from children with cyanotic congenital heart disease, compared with acyanotic or control groups [22, 35]. In addition, mRNA level of HIF-1α as well as that of two of its representative target genes, VEGF and EPO, were found to be upregulated in blood samples of newborns with cyanosis and persistent pulmonary hypertension, therefore representing early markers of generalized hypoxia [36]. If the HIF-1α/VEGF-induced collateral vessel formation in hypoxemic myocardium is essential to compensate the lack of oxygen supply in cyanotic hearts, especially in cases of coarctation of aorta, an abnormal vessel formation can become a source of morbidity, due to arteriovenous malformations [34]. However, a correlation between VEGF increase and abnormal vessel formation has not yet been found [37]. Nevertheless, increased activation of HIF-1α/VEGF signalling might be detrimental in newborn with persistent pulmonary hypertension, as these patients normally present an overexpression of VEGF receptor 1 (VEGFR1), which accounts for the vasoconstrictor effect

fibrosis [27].

148 Hypoxia and Human Diseases

of VEGF [38].

**2.3. HIF-1alpha mediated angiogenic response**

lase (PD-ECGF/TP) and erythropoietin (EPO) [29–31].

The induction of p53 pathway, as a result of the ROS accumulation triggered by hypoxia, is one of the primary event that initiates the apoptotic cascade that occurs in hypoxic states. The activation of p53 leads to an altered expression of the pro-apoptotic gene Bcl-2, which, in turn, causes the DNA damage [39]. It has been shown that the extent of DNA damage depends on the anatomical anomaly and to the grade of cyanosis, with persistent cyanotic patients being more prone to DNA damage. In particular, children with TOF and with septal defects associated with great vessel anomaly displayed a significantly increased DNA damage compared to the ones with isolated septal defects [39, 40]. These data support the evidence that DNA damage can represent a marker of oxidative stress in CHDs as well as the common biochemical modifications and the oxidant status index.

#### **2.5. miRNA involvement in myocardial adaptation to chronic hypoxia**

Among the tissue and circulating biomarkers, microRNAs (miRNAs) have emerged as important tools to assess the hypoxic status of a variety of organs. Briefly, miRNAs are small (19–24 nucleotides) non-coding single-stranded RNAs that form complementary pair with specific target mRNAs to negatively regulate these mRNAs' expression via translational repression or degradation [41]. It has been documented that a hypoxic environment can alter the miRNA profile and their regulation of related pathways, especially with regard to apoptosis/proliferation functions [42]. Furthermore, intensive studies in cardiovascular field have shown how the heart pathophysiology is tightly regulated by miRNAs expression and function [43]. Several miRNAs (i.e. miR-208a, miR21, mi-R29) are involved in myocardial development, and their dysregulation has been linked to cardiac remodelling and hypertrophy; miR-145 upregulation was found in smooth muscle cells of vessels from both a murine model and patients with pulmonary arterial hypertension, whereas plasma upregulation of a huge number of miRNAs (miR-1, miR-133a, miT-499, miR-208) has been reported in patients with acute ischemia and, therefore, hypoxic myocardium [44–48]. Experimental studies performed on cardiac cells further validate the finding that miRNAs expression is modulated with hypoxic stimuli: 145 microRNAs were found to be differently expressed in a study conducted on the human cardiac cell cultured under hypoxia compared the normoxia [49]. Among these, miR-146b was shown to play an important role in the adaptation of cardiomyocytes to chronic hypoxia and its inhibition augmented hypoxia-induced cardiomyocyte apoptosis [50].

A wide array of miRNAs have been reported to be dysregulated in children with CHDs, most of which are crucial in RV development and are specifically linked to a particular defect [24]. In addition, the hypoxic state of some CHDs further affects the miRNA profile of the heart. A recent study by Huang and colleagues shed a light on miRNA-184 as a possible player involved in the mechanism leading to cyanotic CHDs [51]. miRNA-184 expression was, in fact, markedly decreased in myocardial samples from cyanotic CHDs patients, compared to controls and its suppression *in vitro* was also associated with decreased proliferation and induction of apoptosis, through a mechanism that likely involves the activation of Caspase-3 and -9 by the oxidised miRNA-184 [51]. In another study aimed to evaluate the involvement of miRNAs in the hypoxic response of cardiomyocytes, the expression of miR-138 in myocardial samples of cyanotic patients with TOF was almost twofold miR-138 expression in acyanotic group (VSD) patients [52]. This finding suggests that miR-138 might be used to discriminate TOF from other subtypes of CHDs and further supports the evidence that miRNAs can shed a light on the knowledge of the aetiology of different CHDs and be predictive of the clinical outcome/management of these diseases.

## **3. Reoxygenation and reperfusion injuries**

After a hypoxic event or status of the heart, it is crucial to intervene to re-establish a normal oxygen level. In most cases, the intervention involves heart surgery with cardiopulmonary bypass (CPB) and cardioplegic arrest (CA). During such heart surgery, the standard protocol involves the administration of high level of oxygen upon initiation of CPB and before CA. This causes what is commonly referred to as reoxygenation injury [53]. Following the establishment of CPB, the heart is stopped (ischemic period) to carry out the corrective surgery. When the ischemic heart is reperfused at the end of intervention, a reperfusion injury occurs. The severity of this reperfusion injury depends on the severity of the ischemic period and may be linked to delayed post-operative recovery [54].

It has been widely reported that free oxygen radical formation plays an important role in the development of ischemia-reperfusion injury in the heart as well as in various organs. In the reperfused heart, this oxidant formation derives form a series of interacting pathways in cardiac myocytes and endothelial cells, which involve also leukocyte chemotaxis and inflammation. The white blood cells are, in fact, another great source of ROS: when activated by the binding to the hypoxic endothelium, they produce chemotactic substances and oxygen radicals, which are the main responsible for cellular damage [55]. In addition, nitric oxide (NO) production is greatly increased in post-ischemic hearts, thus impairing vascular reactivity [56].

It has been demonstrated that the damage resulting from the reperfusion event is more severe in hypoxic (low oxygen supply), compared to ischemic (low coronary flow) hearts [57]. When comparing the effects of reperfusion, respectively, in ischemic and hypoxic hearts, Samaja et al. found that the myocardial depression, the energy demand, and the associated O2 free radicals were higher in the hypoxic rat hearts than the ischemic ones. Furthermore, the hearts subjected to chronic hypoxia are even more prone to the reoxygenation injury than the hearts that have experienced acute hypoxic events. The compensatory changes that occur in chronic lack of oxygen may account for the higher predisposition to generate larger amounts of oxygen radicals with the reintroduction of high levels of oxygen [58].

With many CHDs being characterized by a chronic hypoxic status, the subset of cyanotic children is obviously at a higher risk than the acyanotic CHDs population [59]. Clinical studies have shown that, despite similar cross-clamp times during open heart surgery, cyanotic children have worse clinical outcome and more reoxygenation injury, measured by troponin I release, compared with acyanotic children [11]. The major problem arises from the oxygen reintroduction during the cardiopulmonary bypass (CPB), which is a necessary procedure for the surgical management of CHDs [55]. As the chronic hypoxia produces long-term changes in the myocardial metabolism and function, the sudden oxygen reintroduction further exacerbates these effects. The impaired contractility due to hypoxia-induced calcium overload and the loss of high energy phosphates are examples of the pathological events amplified by the reoxygenation [59, 60]. In addition, the depletion of endogenous antioxidants that characterize chronic cyanosis cannot counteract the oxygen radical-mediated injury when oxygen is reintroduced [61]. On the contrary, minimal changes in the antioxidant reserve capacity were reported before and after the CPB in acyanotic infants, suggesting that, in the absence of hypoxia, a small amount of oxygen free radicals are produced [62].

fact, markedly decreased in myocardial samples from cyanotic CHDs patients, compared to controls and its suppression *in vitro* was also associated with decreased proliferation and induction of apoptosis, through a mechanism that likely involves the activation of Caspase-3 and -9 by the oxidised miRNA-184 [51]. In another study aimed to evaluate the involvement of miRNAs in the hypoxic response of cardiomyocytes, the expression of miR-138 in myocardial samples of cyanotic patients with TOF was almost twofold miR-138 expression in acyanotic group (VSD) patients [52]. This finding suggests that miR-138 might be used to discriminate TOF from other subtypes of CHDs and further supports the evidence that miRNAs can shed a light on the knowledge of the aetiology of different CHDs and be predictive of the

After a hypoxic event or status of the heart, it is crucial to intervene to re-establish a normal oxygen level. In most cases, the intervention involves heart surgery with cardiopulmonary bypass (CPB) and cardioplegic arrest (CA). During such heart surgery, the standard protocol involves the administration of high level of oxygen upon initiation of CPB and before CA. This causes what is commonly referred to as reoxygenation injury [53]. Following the establishment of CPB, the heart is stopped (ischemic period) to carry out the corrective surgery. When the ischemic heart is reperfused at the end of intervention, a reperfusion injury occurs. The severity of this reperfusion injury depends on the severity of the ischemic period and may

It has been widely reported that free oxygen radical formation plays an important role in the development of ischemia-reperfusion injury in the heart as well as in various organs. In the reperfused heart, this oxidant formation derives form a series of interacting pathways in cardiac myocytes and endothelial cells, which involve also leukocyte chemotaxis and inflammation. The white blood cells are, in fact, another great source of ROS: when activated by the binding to the hypoxic endothelium, they produce chemotactic substances and oxygen radicals, which are the main responsible for cellular damage [55]. In addition, nitric oxide (NO) production is greatly increased in post-ischemic hearts, thus impairing

It has been demonstrated that the damage resulting from the reperfusion event is more severe in hypoxic (low oxygen supply), compared to ischemic (low coronary flow) hearts [57]. When comparing the effects of reperfusion, respectively, in ischemic and hypoxic hearts, Samaja et al. found that the myocardial depression, the energy demand, and the

Furthermore, the hearts subjected to chronic hypoxia are even more prone to the reoxygenation injury than the hearts that have experienced acute hypoxic events. The compensatory changes that occur in chronic lack of oxygen may account for the higher predisposition to generate larger amounts of oxygen radicals with the reintroduction of high levels of

free radicals were higher in the hypoxic rat hearts than the ischemic ones.

clinical outcome/management of these diseases.

150 Hypoxia and Human Diseases

**3. Reoxygenation and reperfusion injuries**

be linked to delayed post-operative recovery [54].

vascular reactivity [56].

associated O2

oxygen [58].

The effect of reoxygenation injury due to CPB in corrective heart surgery in cyanotic children has further been proven by a significant change in the myocardial gene expression profile [21]. In particular, a wide genome expression array study found 32 significantly downregulated and three upregulated genes in cyanotic heart biopsies taken before and after hyperoxic CPB. Among the upregulated genes after reoxygenation, MOSC1, a factor involved in superoxide generation [63], showed a great increase at a mRNA level, thus suggesting its possible involvement in the increase in CPB-induced oxidative stress. On the other hand, the downregulation of the taurine transporter (TAUT) and the consequent depletion of the documented cardioprotective taurine [64] may in part explain some aspects of the myocardial injury, such as the mitochondrial and myofibers dysfunction. In addition, 8-isoprostane, a reliable marker of oxidative stress, was increased after CPB, and this correlated with the downregulation of keys genetic pathways related to myocardial function and to the reduction in antioxidant defenses [21]. It, therefore, appears obvious that the maintenance of endogenous antioxidants during hypoxia is a crucial determinant of tissue recovery on reoxygenation.

It has been suggested that HIF-α might as well stand as target for cardioprotection upon reoxygenation, by inhibiting mitochondrial oxidative metabolism and therefore reducing the generation of ROS under hypoxia-reoxygenation [6].

MicroRNA expression also appears to be affected by the reoxygenation event. In a study by Bolkier et al., the plasma levels of some cardiac-associated miRNA were dramatically increased after surgery of children undergoing open-heart surgery for CHDs. The increase in the selected miRNAs (microRNA-208a, -208b and -499) correlated with higher troponin levels and delayed hospital discharge [65]. This evidence further justifies the use of circulating miRNAs as biomarkers not only for the diagnosis but also for prognosis and prediction of surgical clinical outcome. In addition, through two different approaches—overexpression and inhibition—miRNAs might represent a suitable target to therapeutically treat those defect characterized by an altered expression of their level.

## **4. Strategies of surgical intervention**

In order to reduce the risk associated with reoxygenation injury in children undergoing openheart surgery, different interventional strategies have been explored. One of the strategies proposed to avoid this injury is the "controlled reoxygenation", achieved by using a partial pressure of oxygen in arterial blood (PaO2 ) similar to the patient's preoperative oxygen saturation when starting CPB [66].

Before its adoption in current clinical practices, several experimental studies on animal models have provided the evidence that the biochemical and the functional status of the cyanotic heart are improved by delaying reoxygenation upon cardiopulmonary bypass. Morita and colleagues set up an *in vivo* experimental animal model where immature piglet hearts were subjected to hypoxemia followed by uncontrolled reoxygenation at high oxygen tension (400 mmHg) or controlled oxygenation at ambient tension (40 mmHg) followed by a raising in the tension to 100 mmHg first and 400 mmHg later. The authors found that lipid peroxidation was reduced while antioxidant reserve capacity preserved in the controlledreoxygenation group, with this outcome correlating with improved ventricular contractility and functional recovery [67]. In addition, using a modified cold blood cardioplegia, enriched with potassium, the calcium influx was limited and the impaired contractility restored upon reoxygenation [66]. Similar results were obtained in another animal study where controlled normoxic reoxygenation showed a better outcome than abrupt oxygen reintroduction at high pressure. Furthermore, the effect of leukodepletion was examined in this study, in order to verify whether the removal of an important source of ROS, the white blood cells, would minimize the reoxygenation injury. The depletion of leukocytes from the blood-reduced oxygen free radical formation and preserved ventricular contractility at similar extent to the one achieved by controlled reoxygenation [55].

The beneficial effect of controlling the rate of re-introduction of molecular oxygen was also evident in adult patients. Lower lipid peroxidation and preserved antioxidant levels were observed in patients receiving normoxic reoxygenation, compared to the hyperoxic ones, although no significant difference between the two groups was found in the cardiac performance after CBP, likely because this was measured at one low time point of the Starling fraction curve [68]. The controlled-reoxygenation procedure has subsequently been adopted in the operations of cyanotic infants undergoing cardiac surgery, obtaining similar results to the ones seen in the acute experimental model [58].

Subsequent studies have further confirmed these findings and stressed the importance of controlled reoxygenation on starting CPB in cyanotic patients. In two randomized controlled trials including cyanotic children receiving CBP, we showed that the reduced myocardial injury in the controlled normoxic group was accompanied by a reduction in cerebral and hepatic injury, assessed by S100 and άGT measurement, which are markers of neuron and hepatocytes damage, respectively [69, 70]. In a different study, we have also analysed the effect of the two reoxygenation approaches on the myocardial gene expression profile of cyanotic paediatric patients undergoing corrective heart surgery. Results showed that the controlled reoxygenation reduced the transcriptomic alteration observed following hyperoxic CPB. The most differentially expressed genes, mainly downregulated, were related to remodelling and metabolic processes, suggesting that the hearts subjected to hyperoxic reoxygenation had lower adaptation and remodelling capacity than the ones with controlled reoxygenation CBP [21].

**4. Strategies of surgical intervention**

pressure of oxygen in arterial blood (PaO2

achieved by controlled reoxygenation [55].

ones seen in the acute experimental model [58].

ration when starting CPB [66].

152 Hypoxia and Human Diseases

In order to reduce the risk associated with reoxygenation injury in children undergoing openheart surgery, different interventional strategies have been explored. One of the strategies proposed to avoid this injury is the "controlled reoxygenation", achieved by using a partial

Before its adoption in current clinical practices, several experimental studies on animal models have provided the evidence that the biochemical and the functional status of the cyanotic heart are improved by delaying reoxygenation upon cardiopulmonary bypass. Morita and colleagues set up an *in vivo* experimental animal model where immature piglet hearts were subjected to hypoxemia followed by uncontrolled reoxygenation at high oxygen tension (400 mmHg) or controlled oxygenation at ambient tension (40 mmHg) followed by a raising in the tension to 100 mmHg first and 400 mmHg later. The authors found that lipid peroxidation was reduced while antioxidant reserve capacity preserved in the controlledreoxygenation group, with this outcome correlating with improved ventricular contractility and functional recovery [67]. In addition, using a modified cold blood cardioplegia, enriched with potassium, the calcium influx was limited and the impaired contractility restored upon reoxygenation [66]. Similar results were obtained in another animal study where controlled normoxic reoxygenation showed a better outcome than abrupt oxygen reintroduction at high pressure. Furthermore, the effect of leukodepletion was examined in this study, in order to verify whether the removal of an important source of ROS, the white blood cells, would minimize the reoxygenation injury. The depletion of leukocytes from the blood-reduced oxygen free radical formation and preserved ventricular contractility at similar extent to the one

The beneficial effect of controlling the rate of re-introduction of molecular oxygen was also evident in adult patients. Lower lipid peroxidation and preserved antioxidant levels were observed in patients receiving normoxic reoxygenation, compared to the hyperoxic ones, although no significant difference between the two groups was found in the cardiac performance after CBP, likely because this was measured at one low time point of the Starling fraction curve [68]. The controlled-reoxygenation procedure has subsequently been adopted in the operations of cyanotic infants undergoing cardiac surgery, obtaining similar results to the

Subsequent studies have further confirmed these findings and stressed the importance of controlled reoxygenation on starting CPB in cyanotic patients. In two randomized controlled trials including cyanotic children receiving CBP, we showed that the reduced myocardial injury in the controlled normoxic group was accompanied by a reduction in cerebral and hepatic injury, assessed by S100 and άGT measurement, which are markers of neuron and hepatocytes damage, respectively [69, 70]. In a different study, we have also analysed the effect of the two reoxygenation approaches on the myocardial gene expression profile of cyanotic paediatric patients undergoing corrective heart surgery. Results showed that the controlled reoxygenation reduced the transcriptomic alteration observed following hyperoxic CPB. The most differentially expressed genes, mainly downregulated, were related

) similar to the patient's preoperative oxygen satu-

Another approach of intervention, in the management of CPB, has involved the effect of whole body temperature during the paediatric cardiac surgery.

Although standard CPB procedures have always been conducted by cooling down the body temperature to 28° (hypothermic CPB), in order to reduce the metabolic rate and oxygen consumption, and therefore to protect organs from ischemic injury, recent evidences have demonstrated that normothermic (35°–37°) CPB is associated with lower inflammatory response and organ injury, both in adult and children [71–73].

In addition, we have shown that normothermic CPB in paediatric patients is also associated with reduced oxidative stress, assessed by troponin I and Isoprostane-8 release, compared with hypothermic CPB, while the inflammatory response has similar levels in the two groups [74].

Other researches have also investigated the effect of the temperature of cardioplegia during paediatric CPB. Warm blood cardioplegia, for long time adopted only in adult heart surgery, has proved to be safe and effective compared to standard cold CPB, with even better hydric balance and hemodynamic stability [75]. Once again, the pre-existent hypoxic status affects the biochemical and clinical outcome of the cardioplegic technique used. We have also shown that while for acyanotic patients the cardioplegic technique is not critical, for cyanotic patients, the use of cold blood cardioplegia with terminal warm blood cardioplegic reperfusion ("hot shot") improves the metabolic and functional recovery. The hot shot cardioplegia resulted in higher reperfusion ATP, ATP/ADP and glutamate levels than acyanotic patients, suggesting that this technique is advantageous only in stressed hearts [76]. Furthermore, the study shows that even if the blood cardioplegia is kept at cold temperature, this still offers a higher myocardial protection, compared to the crystalloid cardioplegia, confirming previous experimental and clinical results [77–79].

Besides CPB strategies, a pharmacological approach could be used as an interventional strategy for perioperative cardioprotection of hypoxic hearts. Experimental studies have shown that the selective inhibition of the enzyme phosphodiesterase-5 (PDE-5) can offer myocardial protection in infant hearts by improving myocardial function and reducing infarct size during reperfusion. However, no direct evidence between this protective effect and the hypoxiainduced injury was shown [80].

As for its established role in hypoxia, HIF-1α has also been investigated as a target for hypoxia-induced myocardial injury in reperfusion. By stabilizing its active form, through the compound dimethyloxyglycine (DMOG), a novel HIF-1α stabilizer, Zhang et al. showed that the progression of hypoxia-induced right ventricle remodelling was significantly reduced in a murine model of chronic hypoxia, most likely as a result of the induction of genes related to adaptive processes [81].

Furthermore, as previously mentioned, miRNAs are being extensively investigated as potential therapeutic tools in the management of CHDs. However, despite the fact that the road ahead looks promising and appealing, some obstacles, like the stability, the off-target effects and the immunogenicity of the delivery vehicles, still need to be overcome before getting miRNA-based therapeutics into clinical practice.

## **5. Conclusion**

In conclusion, important steps ahead have been made in the knowledge of the mechanisms by which hypoxia takes part to the onset of congenital heart diseases, especially with regard to cyanotic patients. Likewise, significant advances have been made in the strategies of intervention involving open-heart surgery of children with these defects; in order to reduce the injury induced by CPB reoxygenation. Hopefully, the further understanding of the signalling pathways and the mechanism underlying the pathophysiology of hypoxia and hypoxiainduced reoxygenation injury in each kind of defect will result in the development of even better therapeutic strategies and in the design of specific interventions, particularly for the high-risk population.

## **Author details**

Dominga Iacobazzi, Massimo Caputo and Mohamed T Ghorbel\*

\*Address all correspondence to: m.ghorbel@bristol.ac.uk

University of Bristol, School of Clinical Sciences, Bristol Heart Institute, Bristol, UK

## **References**


[8] Roche SL, Redington AN. The failing right ventricle in congenital heart disease. Can J Cardiol. 2013;29(7):768–78.

and the immunogenicity of the delivery vehicles, still need to be overcome before getting

In conclusion, important steps ahead have been made in the knowledge of the mechanisms by which hypoxia takes part to the onset of congenital heart diseases, especially with regard to cyanotic patients. Likewise, significant advances have been made in the strategies of intervention involving open-heart surgery of children with these defects; in order to reduce the injury induced by CPB reoxygenation. Hopefully, the further understanding of the signalling pathways and the mechanism underlying the pathophysiology of hypoxia and hypoxiainduced reoxygenation injury in each kind of defect will result in the development of even better therapeutic strategies and in the design of specific interventions, particularly for the

miRNA-based therapeutics into clinical practice.

Dominga Iacobazzi, Massimo Caputo and Mohamed T Ghorbel\*

University of Bristol, School of Clinical Sciences, Bristol Heart Institute, Bristol, UK

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#### **Adaptations to Chronic Hypoxia Combined with Erythropoietin Deficiency in Cerebral and Cardiac Tissues Adaptations to Chronic Hypoxia Combined with Erythropoietin Deficiency in Cerebral and Cardiac tissues**

Raja El Hasnaoui-Saadani Raja El Hasnaoui-Saadani

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160 Hypoxia and Human Diseases

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

http://dx.doi.org/10.5772/66974

#### **Abstract**

Chronic anemia-induced hypoxia triggers regulatory pathways that mediate long-term adaptive cardiac and cerebral changes, particularly at the transcriptional level. These adaptative mechanisms include a regulated cerebral blood flow and cardiac output, angiogenesis and cytoprotection triggered by hypoxia-inducible factor 1 alpha (HIF-1α), vascular endothelial growth factor (VEGF), neuronal nitric oxide synthase (nNOS) and Epo pathways. All these compensatory mechanisms aim to optimize oxygen delivery and to protect the brain and heart from hypoxic injury. We reviewed the effects of chronic hypobaric hypoxia as well as chronic anemia in the heart and brain, and we compared for the first time the effects of chronic hypobaric hypoxia combined with a severe lack of Epo (chronic anemia) in these vital organs. Functional cardiac adaptations such as cardiac hypertrophy, increased cardiac output as well as angiogenesis occurred along with the activation of HIF1α/VEGF and Epo/EpoR pathways under chronic anemia or hypoxia. Similarly, cerebrovascular adaptations take place through the same molecular mechanisms under chronic hypoxia or anemia. However, when both arterial pressure and content of oxygen are decreased, the cerebral and cardiac adaptative mechanisms showed their limitations. In addition, cerebral and cardiac cell injuries may have occurred following the combined effect of chronic anemia and hypoxia. By emphasizing the anemia and hypoxia-induced cerebral and myocardial adaptations, this review highlighted the crucial role of Epo in its non-erythropoietic functions such as angiogenesis and neuroprotection. Indeed, a better understanding of these protective mechanisms is of great clinical importance to the development of new therapeutic strategies for the management of ischemic heart and brain.

**Keywords:** chronic hypoxia, chronic anemia, angiogenesis, cardiac function, cerebral blood flow, oxygen homeostasis, neuroprotection, HIF-1-VEGF-Epo

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 © 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,

## **1. Introduction**

Inadequate level of oxygen like in chronic hypoxia or anemia is especially detrimental for cerebral and heart tissues. Indeed, hypoxia plays an important role in the pathogenesis of cerebral and myocardial ischemia, and chronic heart and lung diseases [1]. That is why specific mechanisms at a systemic, cellular and molecular level take place to maintain the oxygen homeostasis. It is important to clearly distinguish the differences between hypoxia and anemia. Hypoxia is a reduction of arterial pressure of oxygen (PaO<sup>2</sup> ), while anemia is a reduction of arterial content of oxygen (CaO<sup>2</sup> ) as it occurs during a decrease in hemoglobin concentration. This review focus mainly on chronic hypobaric hypoxia that occurs at high altitude and chronic anemia is referred to our model of transgenic mice that presents a constitutive erythropoietin deficiency. Furthermore, emphasis is placed on effects of chronic hypoxia and anemia in the cerebral and cardiac tissues. The discussion is mostly based on animal studies unless otherwise indicated, even though similarities of adaptative mechanisms are shown by human studies [2, 3].

Chronic hypoxia promotes angiogenesis by modulating the transcriptional regulator hypoxiainducible factor 1 alpha (HIF-1α), which in turn triggers the upregulation of the erythropoietin [4], a major factor of acclimatization to hypoxia. HIF-1α is a master regulator of the hypoxic response and its proangiogenic activities include regulation of vascular endothelial growth factor (VEGF), but also Epo and its receptors (EpoR) [1, 5]. Erythropoietin primarily regulates red blood cell formation, and Epo serum levels are increased under hypoxic stress (e.g., anemia and altitude) [6]. However, several non-hematopoietic functions of Epo and its receptors have been exposed by experimental studies using genetically modified mice [7]. It is of great clinical importance that Epo has been shown to have protective functions in many different tissues. Indeed, these studies using recombinant human EPO (rHuEPO) suggested new therapeutic indications of Epo for the management of ischemic injuries of several tissues such as myocardium and brain [8–10].

Epo-induced angiogenesis may lead to an improvement in brain perfusion since Epo protects vascular bed integrity and stimulates angiogenesis [11–14] by acting indirectly on endothelial cells via activation of VEGF/VEGF receptor system, which is the most important regulator of endothelial growth and angiogenesis. Furthermore, Epo may have a positive effect on cerebral vasculature in addition to the cerebral blood flow (CBF) through alteration in nitric oxide (NO) production, which mainly derived from arginine and catalyzed by endothelial nitric oxide synthase (eNOS) [15]. Therefore, it seems that cellular protection and angiogenesis in heart and brain tissues are the dual role of erythropoietin and VEGF. These both cytokines are triggered by HIF-1α to maintain an adequate cellular oxygen supply and protect the brain and heart against hypoxic and or anemic injuries.

Our model of erythropoietin-SV40 T antigen (Epo-TAgh) transgenic mice has a targeted disruption in the 5′ untranslated region of the Epo gene that dramatically reduces its expression. The homozygous animals are thus severely and chronically anemic [16, 17]. Therefore, these transgenic anemic mice provided us an interesting model to study the adaptive mechanisms to chronic anemia and hypoxia, especially in vital organs, such as brain and heart. The present review aims to give a brief synthesis of adaptations to chronic hypoxia in the brain and heart tissues, in absence of Epo. The first part will briefly describe the similarities of the signaling process of hypoxia-induced angiogenesis, as well as the other mechanisms that take place to protect the brain and heart against anemic and hypoxic injuries. The second part will focus on these adaptations in response to chronic anemia due to Epo deficiency (in comparison with adaptations induced by hypoxia). The third part will mainly describe the effect of both constraints (anemia and hypoxia) in cerebral and cardiac tissues.

## **2. Adaptations to chronic hypoxia in cerebral and cardiac tissues**

#### **2.1. Brain under chronic hypoxia**

**1. Introduction**

162 Hypoxia and Human Diseases

human studies [2, 3].

of arterial content of oxygen (CaO<sup>2</sup>

such as myocardium and brain [8–10].

and heart against hypoxic and or anemic injuries.

Inadequate level of oxygen like in chronic hypoxia or anemia is especially detrimental for cerebral and heart tissues. Indeed, hypoxia plays an important role in the pathogenesis of cerebral and myocardial ischemia, and chronic heart and lung diseases [1]. That is why specific mechanisms at a systemic, cellular and molecular level take place to maintain the oxygen homeostasis. It is important to clearly distinguish the differences between hypoxia and ane-

tration. This review focus mainly on chronic hypobaric hypoxia that occurs at high altitude and chronic anemia is referred to our model of transgenic mice that presents a constitutive erythropoietin deficiency. Furthermore, emphasis is placed on effects of chronic hypoxia and anemia in the cerebral and cardiac tissues. The discussion is mostly based on animal studies unless otherwise indicated, even though similarities of adaptative mechanisms are shown by

Chronic hypoxia promotes angiogenesis by modulating the transcriptional regulator hypoxiainducible factor 1 alpha (HIF-1α), which in turn triggers the upregulation of the erythropoietin [4], a major factor of acclimatization to hypoxia. HIF-1α is a master regulator of the hypoxic response and its proangiogenic activities include regulation of vascular endothelial growth factor (VEGF), but also Epo and its receptors (EpoR) [1, 5]. Erythropoietin primarily regulates red blood cell formation, and Epo serum levels are increased under hypoxic stress (e.g., anemia and altitude) [6]. However, several non-hematopoietic functions of Epo and its receptors have been exposed by experimental studies using genetically modified mice [7]. It is of great clinical importance that Epo has been shown to have protective functions in many different tissues. Indeed, these studies using recombinant human EPO (rHuEPO) suggested new therapeutic indications of Epo for the management of ischemic injuries of several tissues

Epo-induced angiogenesis may lead to an improvement in brain perfusion since Epo protects vascular bed integrity and stimulates angiogenesis [11–14] by acting indirectly on endothelial cells via activation of VEGF/VEGF receptor system, which is the most important regulator of endothelial growth and angiogenesis. Furthermore, Epo may have a positive effect on cerebral vasculature in addition to the cerebral blood flow (CBF) through alteration in nitric oxide (NO) production, which mainly derived from arginine and catalyzed by endothelial nitric oxide synthase (eNOS) [15]. Therefore, it seems that cellular protection and angiogenesis in heart and brain tissues are the dual role of erythropoietin and VEGF. These both cytokines are triggered by HIF-1α to maintain an adequate cellular oxygen supply and protect the brain

Our model of erythropoietin-SV40 T antigen (Epo-TAgh) transgenic mice has a targeted disruption in the 5′ untranslated region of the Epo gene that dramatically reduces its expression. The homozygous animals are thus severely and chronically anemic [16, 17]. Therefore, these transgenic anemic mice provided us an interesting model to study the adaptive mechanisms to chronic anemia and hypoxia, especially in vital organs, such as brain and heart. The present

), while anemia is a reduction

) as it occurs during a decrease in hemoglobin concen-

mia. Hypoxia is a reduction of arterial pressure of oxygen (PaO<sup>2</sup>

In the central nervous system, cerebrovascular and energy metabolism adaptations occur under hypoxic conditions in order to preserve an adequate tissue oxygen supply needed to support an optimal neuronal function. An acute hypoxic exposure triggers both a CBF and glucose consumption increases [18]. The stabilization of HIF-1α rapidly up regulates the vasodilatory enzyme inducible nitric oxide synthase (iNOS) [19]. NO, the enzymatic product of iNOS, relaxes vascular smooth muscle cells, providing a short-term increase in blood flow. Thus, an increase in cerebral NO following a rise in NOS isoforms expression is most probably responsible for the rise in CBF [20–23]. With longer hypoxic exposure, polycythemia and cerebral angiogenesis take place to enhance cerebral oxygenation, while cerebral NO level returns to basal value [23, 24]. Indeed, in a chronic hypoxic challenge to the central nervous system, the cerebral cortex is known to undergo a significant cerebrovascular remodeling, in order to preserve tissue oxygen and energy supply [24–29]. These microvascular changes occur relatively late compared to the physiological adaptations [30]. In the rat brain, the capillary density almost doubles, and the average intercapillary distance decreases from about 50 to about 40 μm [31]. Also, by 3 weeks of adaptation, the initial hypoxic-induced increased flow returns to baseline by 5 days [22], concomitantly there is an increasing hematocrit, glucose consumption is slightly elevated by about 15% [32–34] and tissue oxygen tension is restored [35, 36]. Finally, it is well accepted that blood flow alterations serve acute changes in oxygen delivery, while persistent changes are due to capillary density adjustments.

Molecular mechanisms underlying hypoxia-induced capillary increases are now well documented and involve specifically HIF-1α/VEGF pathway [23, 26, 33, 37, 38]. The HIF pathway regulates a host of pro-angiogenic genes, including VEGF, angiopoietin-1, angiopoietin-2 (Ang-2) and many others [39, 40]. HIF-regulated pro-angiogenic factors execute the HIF-specific angiogenic program by increasing vascular permeability (most probably through interaction with NO [41]), endothelial cell proliferation, sprouting, migration, adhesion and tube formation. In rats, HIF-1α is detected in the brain, in all cell types, shortly after the onset of hypoxia and persists for at least 2 weeks, until cerebral angiogenesis is completed within 3 weeks of exposure to hypoxia [23, 29, 35]. Brain angiogenesis also requires additional pro-angiogenic factors such as Ang-2. Ang-2, which is not constitutively expressed under normoxic conditions, is upregulated in rat and mouse endothelial cells following hypoxia [28, 38]. Ang-2 induction during hypoxia is known to occur independently of HIF-1 and is due to cyclooxygenase-2 (COX-2) enzyme activity [42, 43]. More recent results also demonstrated that the hypoxic capillary response in aged mice was preserved after 3 weeks of hypoxia despite a significant delay in the response during the first week of exposure to hypoxia [25].

#### **2.2. Heart under chronic hypoxia**

In humans, the most characteristic and important cardiovascular response to hypoxia is pulmonary vasoconstriction, which reduces the caliber of pulmonary vessels and raises vascular resistance in a region of low alveolar PO<sup>2</sup> . However, severe hypoxia has a direct deleterious effect on cardiac function. Hypoxic pulmonary vasoconstriction can cause chronic pulmonary hypertension. Myocardial contractility and maximum output are diminished during conditions of reduced oxygen supply. While maximum oxygen consumption is reduced in chronic hypoxia, cardiac output (CO) remains normal at rest, owing primarily to an increased red blood cell mass [44, 45].

In our animal model, we showed that all parameters of cardiac function were preserved when comparing wild-type (WT) mice under normoxic and chronic hypoxic conditions. Indeed, systolic blood pressure was not affected by 14-day hypoxia at 4500 m, and hypoxic wild-type mice did not develop pulmonary hypertension. Moreover, there was no cardiac hypertrophy at variance with what was shown in rats or humans in similar hypoxic conditions [46]. Moreover, cardiac output was not affected by chronic hypoxia alone and oxygen delivery was maintained. In addition, hypoxic wild-type mice responded by increasing plasma Epo and blood hemoglobin, resulting in a rise in oxygen-carrying capacity [47].

Furthermore, many reports now stated that heart could be an additional Epo productive tissue [48, 49]. Hoch et al. first showed an Epo gene and protein expression in cardiac progenitor cells [50]. Through specific binding to its receptor EpoR, Epo triggers intracellular signaling events that depend on the activation of Jak2 tyrosine kinase [51]. The exploration of these pathways revealed that Epo is also an angiogenic as well as an anti-apoptotic factor as described respectively in the brain [52] and the heart [47, 50, 53, 54]. As previously described [55], chronic hypoxia led to the activation of HIF-1α/VEGF pathway in the heart of adult wild-type mice most probably responsible for their enhanced myocardial angiogenesis. Also we demonstrated the activation of cardioprotective pathways, involving HIF-1α and Epo, as suggested by the increase of EpoR expression and P-STAT-5/STAT-5 ratio [47]. Furthermore, we could not exclude a cardiac metabolic gene remodeling since temporal changes in glucose metabolic genes in response to moderate hypobaric hypoxia [56] have been demonstrated.

However, these adaptive responses contribute to maintain an adequate tissue oxygen supply for the preservation of cardiac function and to protect the heart against hypoxic insults.

## **3. Adaptations to chronic Epo deficiency in cerebral and cardiac tissues**

Anemia is defined as a lack of oxygen-carrying red blood cells which also results in a lack of oxygen delivery to tissue. The physiological and molecular responses to tissue hypoxia are increasingly understood while the effects of anemia are still poorly documented [57]. Our model of erythropoietin-SV40 T antigen (Epo-TAgh) transgenic mice presents a severe reduction of Epo expression that induces chronic anemia [16, 17]. The first studies demonstrated that Epo-Tagh mice could survive in chronic hypoxia (14 days at 4500 m), in part through an increase in ventilation and probably a higher cardiac output as suggested by a significant cardiac hypertrophy [58–60]. Hence, it was of interest of our team to compare the physiological and cellular responses to chronic anemia (low Hb) and chronic hypoxia (low P<sup>a</sup> O2 ) in cerebral and cardiac tissues. Indeed, the objectives of our studies were to determine if chronic anemic mice developed compensatory mechanisms in the brain and heart (vascular remodeling, adaptative function, pathways involving HIF-1α) to offset the decrease in hemoglobin concentration.

#### **3.1. Brain under chronic anemia**

during hypoxia is known to occur independently of HIF-1 and is due to cyclooxygenase-2 (COX-2) enzyme activity [42, 43]. More recent results also demonstrated that the hypoxic capillary response in aged mice was preserved after 3 weeks of hypoxia despite a significant delay

In humans, the most characteristic and important cardiovascular response to hypoxia is pulmonary vasoconstriction, which reduces the caliber of pulmonary vessels and raises vascular

effect on cardiac function. Hypoxic pulmonary vasoconstriction can cause chronic pulmonary hypertension. Myocardial contractility and maximum output are diminished during conditions of reduced oxygen supply. While maximum oxygen consumption is reduced in chronic hypoxia, cardiac output (CO) remains normal at rest, owing primarily to an increased red

In our animal model, we showed that all parameters of cardiac function were preserved when comparing wild-type (WT) mice under normoxic and chronic hypoxic conditions. Indeed, systolic blood pressure was not affected by 14-day hypoxia at 4500 m, and hypoxic wild-type mice did not develop pulmonary hypertension. Moreover, there was no cardiac hypertrophy at variance with what was shown in rats or humans in similar hypoxic conditions [46]. Moreover, cardiac output was not affected by chronic hypoxia alone and oxygen delivery was maintained. In addition, hypoxic wild-type mice responded by increasing plasma Epo and

Furthermore, many reports now stated that heart could be an additional Epo productive tissue [48, 49]. Hoch et al. first showed an Epo gene and protein expression in cardiac progenitor cells [50]. Through specific binding to its receptor EpoR, Epo triggers intracellular signaling events that depend on the activation of Jak2 tyrosine kinase [51]. The exploration of these pathways revealed that Epo is also an angiogenic as well as an anti-apoptotic factor as described respectively in the brain [52] and the heart [47, 50, 53, 54]. As previously described [55], chronic hypoxia led to the activation of HIF-1α/VEGF pathway in the heart of adult wild-type mice most probably responsible for their enhanced myocardial angiogenesis. Also we demonstrated the activation of cardioprotective pathways, involving HIF-1α and Epo, as suggested by the increase of EpoR expression and P-STAT-5/STAT-5 ratio [47]. Furthermore, we could not exclude a cardiac metabolic gene remodeling since temporal changes in glucose metabolic genes in response to moderate hypobaric hypoxia [56] have been demonstrated.

However, these adaptive responses contribute to maintain an adequate tissue oxygen supply for the preservation of cardiac function and to protect the heart against hypoxic insults.

**3. Adaptations to chronic Epo deficiency in cerebral and cardiac tissues**

Anemia is defined as a lack of oxygen-carrying red blood cells which also results in a lack of oxygen delivery to tissue. The physiological and molecular responses to tissue hypoxia are

. However, severe hypoxia has a direct deleterious

in the response during the first week of exposure to hypoxia [25].

blood hemoglobin, resulting in a rise in oxygen-carrying capacity [47].

**2.2. Heart under chronic hypoxia**

164 Hypoxia and Human Diseases

blood cell mass [44, 45].

resistance in a region of low alveolar PO<sup>2</sup>

Low O2 environment is the principal regulator of HIF activity. The HIF pathway mediates the primary cellular responses to low O<sup>2</sup> , which promotes both short- and long-term adaptation to hypoxia as already described in the previous paragraph. In this regard, we considered that anemia-induced cerebral hypoxia involved the same hypoxic molecules. It has already been described that anemia increases cerebral hypoxic genes expression such as HIF, VEGF and nNOS which are involved in O<sup>2</sup> homeostasis [61]. Indeed, studies on severe hemodilution using NOS-deficient mice showed an increased expression of HIF and nNOS proteins in the brain as well as an increased whole body HIF activity [57, 61–64]. Many other molecules, including Epo, VEGF and iNOS have also been shown to be upregulated in the anemic brain [61, 65]. Thus, it seems that during anemia, HIF-1α has the potential to regulate cerebral cellular responses under both hypoxic and normoxic conditions [23, 47, 57, 64, 65].

Then, we focused on potential mediators of the increase in CBF associated with both hypoxia and anemia. Indeed, local production of NO by endothelial NOS (eNOS), and nNOS mediates CBF under a number of physiological conditions, including anemia and hypoxia [24, 61, 63, 64]. Relatively specific inhibition of nNOS has been demonstrated to impair the increase in CBF associated with acute anemia [61] and hypoxia [66–68], implicating nNOS as an important mediator of CBF in both cases [63, 64]. In our studies, we also found an increase in nNOS, while iNOS and eNOS were unchanged but no corresponding change in cerebral NO concentration. The stabilization of HIF-1α, as already described in the brain in acute [61, 69] and chronic anemia [23], promote VEGF-induced angiogenesis as shown in normoxic Epo-TAgh mice with a rise in the capillary/fiber ratio, thus optimizing oxygen diffusion as previously described in the brain [24]. Erythropoietin also plays an important role in angiogenesis through upregulation of VEGF in ischemic rats [11]. Indeed, Wang et al. showed that neural progenitor cells treated with Epo were able to produce VEGF and consequently to promote angiogenesis through the upregulation of VEGFR2 expression in cerebral endothelial cells [70].

Our work provided novel physiological data about cerebral adaptations to chronic anemia. Indeed, we evidenced that Epo deficiency activated cerebral hypoxic mechanisms through HIF activation that promote angiogenesis [23]. In addition, the JAK/STAT signaling pathway mediated by the Epo/EpoR complex seems to be activated by chronic anemia [23, 47] and could promote neuroprotection and cell proliferation [71]. Furthermore, more recent results showed that nNOS is specifically protective during anemia [57]. All these responses were probably able to minimize brain damage that could be induced by chronic anemia. Finally, the mechanisms responsible for matching capillary density to tissue oxygen levels are not unique to environmental hypoxic stimuli. Rather, these processes appear to be responsible for maintaining the oxygen availability through local blood flow in order to optimize the neuronal function.

#### **3.2. Heart under chronic anemia**

The classic physiological cardiovascular responses to anemia include an increased CO, a redistribution of blood flow and a decrease of hemoglobin-oxygen affinity. Two mechanisms are most probably responsible for the increased CO during anemia: reduced blood viscosity and increased sympathetic stimulation of the cardiovascular effectors. Blood viscosity affects both preload and afterload, two of the major determinants of the CO, whereas sympathetic stimulation primarily increases heart rate and contractility [72]. If cardiac function is normal, the increase in venous return or left ventricular preload will be the most important determinant of the increased CO during normovolemic anemia [72]. It is also known that anemia induces right and left ventricular hypertrophy [59, 73, 74] and increases CO, offsetting the fall in arterial oxygen content to maintain oxygen delivery. Our data confirmed the increase in CO by an increase in the stroke volume associated with a left ventricular dilatation as expected by Olivetti et al. [74]. Taken together, these data suggest that the enhancement in CO could be explained by both an increase in preload and autonomous nervous system stimulation. Indeed, our data showed an increase in myocardial function parameters in normoxic anemic mice. However, although the CO was increased in Epo-Tagh mice, oxygen delivery remained lower than in controls. This could induce the stabilization of the transcription factor HIF-1α as already described in the brain in both models of acute and chronic anemia [61, 69]. This stabilization promotes VEGF-induced angiogenesis as shown in normoxic Epo-TAgh mice with a rise in the capillaries/fibers ratio, thus optimizing oxygen diffusion as described in the brain [24]. This increase in capillary density could allow the development of cardiac hypertrophy without myocardial dysfunction, as previously described in rats in a model of anemia induced by iron-deficient diet [74]. Furthermore, we could not exclude that increased expression of nNOS also contributed to these adaptive cardiovascular responses in chronic anemic mice. Indeed, acute anemia resulted in an increase in CO and a reduced stroke volume in WT anemic mice while in contrast, CO and stroke volume responses were severely attenuated in anemic nNOS−/− mice [57]. In addition, a model of *Hif1a*+/− hemizigous mice revealed impaired increases in hematocrit, right ventricular mass and right ventricular pressure, allowing us to speculate that increased HIF-1α may have participated in these physiological responses to anemia in our model [75].

### **4. Effects of chronic anemia and hypoxia on cerebral and cardiac tissues**

As previously explained, plethora of studies are available to describe cerebral and cardiac adaptations and their underlying molecular mechanisms in response to chronic hypoxia or anemia separately. Our group investigated for the first time, the effects of chronic hypobaric hypoxia combined with chronic anemia in the heart and brain of the transgenic Epo-TAgh mice. So far, the few studies from other groups that also use transgenic mice overexpressing Epo (Tg6 and Tg21) display results that are complementary to our data but also more detailed. Indeed, these studies also describe the pathways involved in the ventilatory responses to hypoxia and aim to clarify the role of Epo in respiratory acclimatization to hypoxia at physiological, cellular and molecular levels. Even though, we were not able to find other animal studies combining the effects of both chronic hypoxia and anemia on cardiac or cerebral tissues, comparing studies at a multidisciplinary level may provide new approaches and therapies for diseases associated with hypoxia.

#### **4.1. Brain under chronic hypoxia and anemia**

could promote neuroprotection and cell proliferation [71]. Furthermore, more recent results showed that nNOS is specifically protective during anemia [57]. All these responses were probably able to minimize brain damage that could be induced by chronic anemia. Finally, the mechanisms responsible for matching capillary density to tissue oxygen levels are not unique to environmental hypoxic stimuli. Rather, these processes appear to be responsible for maintaining the oxygen availability through local blood flow in order to optimize the

The classic physiological cardiovascular responses to anemia include an increased CO, a redistribution of blood flow and a decrease of hemoglobin-oxygen affinity. Two mechanisms are most probably responsible for the increased CO during anemia: reduced blood viscosity and increased sympathetic stimulation of the cardiovascular effectors. Blood viscosity affects both preload and afterload, two of the major determinants of the CO, whereas sympathetic stimulation primarily increases heart rate and contractility [72]. If cardiac function is normal, the increase in venous return or left ventricular preload will be the most important determinant of the increased CO during normovolemic anemia [72]. It is also known that anemia induces right and left ventricular hypertrophy [59, 73, 74] and increases CO, offsetting the fall in arterial oxygen content to maintain oxygen delivery. Our data confirmed the increase in CO by an increase in the stroke volume associated with a left ventricular dilatation as expected by Olivetti et al. [74]. Taken together, these data suggest that the enhancement in CO could be explained by both an increase in preload and autonomous nervous system stimulation. Indeed, our data showed an increase in myocardial function parameters in normoxic anemic mice. However, although the CO was increased in Epo-Tagh mice, oxygen delivery remained lower than in controls. This could induce the stabilization of the transcription factor HIF-1α as already described in the brain in both models of acute and chronic anemia [61, 69]. This stabilization promotes VEGF-induced angiogenesis as shown in normoxic Epo-TAgh mice with a rise in the capillaries/fibers ratio, thus optimizing oxygen diffusion as described in the brain [24]. This increase in capillary density could allow the development of cardiac hypertrophy without myocardial dysfunction, as previously described in rats in a model of anemia induced by iron-deficient diet [74]. Furthermore, we could not exclude that increased expression of nNOS also contributed to these adaptive cardiovascular responses in chronic anemic mice. Indeed, acute anemia resulted in an increase in CO and a reduced stroke volume in WT anemic mice while in contrast, CO and stroke volume responses were severely attenuated in anemic nNOS−/− mice [57]. In addition, a model of *Hif1a*+/− hemizigous mice revealed impaired increases in hematocrit, right ventricular mass and right ventricular pressure, allowing us to speculate that increased HIF-1α may have participated in these physiological responses to anemia in our model [75].

**4. Effects of chronic anemia and hypoxia on cerebral and cardiac tissues**

As previously explained, plethora of studies are available to describe cerebral and cardiac adaptations and their underlying molecular mechanisms in response to chronic hypoxia or

neuronal function.

166 Hypoxia and Human Diseases

**3.2. Heart under chronic anemia**

In the brain, both Epo and its receptor are upregulated during ischaemia/hypoxia [76, 77] and Epo administration considerably inhibits apoptosis after middle cerebral artery occlusion [78]. Apart from its positive effects in acute ischaemic brain damage, Epo is a potent stimulator of the hypoxic ventilatory response (HVR) by interacting with respiratory centers in the brainstem [79]. Indeed, the blockade of Epo's activity in the brainstem of adult C57Bl6 mice by intracisternal injections of the soluble Epo receptor (sEpoR) induced a reduction of the basal minute ventilation, but it did not affect the central chemosensitivity [80, 81]. In contrast, recent study using transgenic mice Tg6 (that present a human Epo gene overexpression in brain and circulation; Tg21: Epo overexpression in brain) suggested that Epo blunts the HVR through an interaction with central and peripheral respiratory chemocenters [81]. In our model, acute hypoxic ventilatory response was increased after chronic hypoxia in wild-type mice but remained unchanged in Epo-TAgh mice, confirming that adequate erythropoietin level is necessary to obtain an appropriate HVR and a significant ventilatory acclimatization to hypoxia. Surprisingly, both constraints (chronic hypoxia and anemia) did not trigger a synergic effect in any studied parameters except a high cerebral NO level that could suggest an improved brain perfusion. Finally, the response to chronic hypoxia was divergent in the brain of wild-type and anemic mice. Indeed, these adaptation processes including angiogenesis and neuroprotection were globally altered in Epo-TAgh mice exposed to chronic hypoxia.

Taken together, all these data suggest that Epo/EpoR pathways activation is necessary to initiate neuroprotection mechanisms as well as cerebral angiogenesis under hypoxia but also might help to better understand respiratory disorders at high altitude.

#### **4.2. Heart under chronic hypoxia and anemia**

Independently, chronic anemia and chronic hypoxia increased the expression of HIF-1α, VEGF and Epo, cytokines that are involved in both angiogenesis and cardioprotection through specific signaling pathways acting to compensate oxygen transport deficiency. Recent studies also involved these same cytokines in the cardiovascular responses as well as increased cardiac output observed in acute anemia [57, 75]. Our data showed a decrease in left ventricular hypertrophy and functional left ventricular adaptation as well as a reduced oxygen delivery in the heart of hypoxic Epo-TAgh mice. Results from other groups showed that Tg6 mice did not develop pulmonary hypertension in normoxia or after exposure to chronic hypoxia (10% O2 for 3 weeks) [82] suggesting an important role of Epo in functional adaptation of the heart to chronic hypoxia. Similarly to what occurred in the brain, we did not observe a synergic effect of these combined constraints on the expression of the hypoxic genes in the heart of chronically hypoxic Epo-TAgh mice suggesting that adaptive responses to both constraints are already maximal. However, the increased P-STAT-5/STAT-5 ratio is concordant with a direct protective effect of Epo on cardiomyocytes and endothelial cells as well as stimulation of angiogenesis in the ischaemic heart [83]. Capillary density was unchanged in spite of the fall in HIF-1α/VEGF pathway probably because the initiation of the capillarization with acute hypoxia necessitates VEGF, while its maintenance in chronic hypoxia involves other factors such as angiopoietins [38, 43].

Taken together, our results suggest that adaptative mechanisms that take place with chronic anemia are somewhat similar to those in response to 14 days of hypoxia. However, when both constraints are applied, these mechanisms failed to maintain an adequate cardiac adaptation with a secondary decrease in body oxygen supply, despite the activation of cardioprotective pathways.

## **5. Perspectives and significance**

In this review, a proposal is made that chronic anemia-induced hypoxia triggers regulatory pathways that mediate long-term adaptive cardiac and cerebral changes, particularly at the transcriptional level. These adaptative mechanisms include a regulated increase in cerebral blood flow, cardiac output, angiogenesis and cytoprotection triggered by HIF-1α, VEGF and Epo pathways. All these compensatory mechanisms aim to optimize oxygen delivery and to protect the brain and heart from hypoxic injury to allow acclimatization. However, when both arterial pressure and content of oxygen are decreased, the cerebral and cardiac adaptative mechanisms showed their limitations. We could not exclude that cerebral and cardiac cell injuries occurred following the combined effect of chronic anemia and hypoxia as well as of the NO toxicity. **Figure 1** summarizes the cerebral and cardiac plasticity induced by chronic anemia and/or hypoxia. Data shown in this figure are all based on animal studies. Moreover, a recent review of our group includes also ventilatory [60], muscular [84, 85] and rheologic [86] adaptations in this model of mice. Finally, investigating the molecular mechanisms of O2 homeostasis represents a mean of gaining new insights to the hypoxia-induced cerebral and myocardial injuries. But it is of great clinical importance to study extensively these nonerythropoietic functions of Epo to contribute to the development of new therapeutic strategies for the management of brain and heart ischemia.

**Figure 1** summarizes the physiological adaptations to chronic hypoxia and anemia in the heart and brain of our model of Epo-TAgh mice. The green color represents the responses of normoxic anemic mice. The blue color represents the responses of hypoxic control mice. The red color represents the responses of hypoxic anemic mice. The arrows represent an increase or decrease of the response, while the **'='** symbol means no change between normoxia and hypoxia. PaO<sup>2</sup> is arterial pressure of oxygen, CaO<sup>2</sup> is arterial content of oxygen, PiO<sup>2</sup> is inspired pressure of oxygen and TO<sup>2</sup> is transport of oxygen.

Adaptations to Chronic Hypoxia Combined with Erythropoietin Deficiency in Cerebral and Cardiac Tissues http://dx.doi.org/10.5772/66974 169

**Figure 1.** Cerebral and cardiac plasticity induced by chronic anemia and/or hypoxia in Epo-Tagh mice.

## **Grants**

not develop pulmonary hypertension in normoxia or after exposure to chronic hypoxia (10%

Taken together, our results suggest that adaptative mechanisms that take place with chronic anemia are somewhat similar to those in response to 14 days of hypoxia. However, when both constraints are applied, these mechanisms failed to maintain an adequate cardiac adaptation with a secondary decrease in body oxygen supply, despite the activation of cardioprotective

In this review, a proposal is made that chronic anemia-induced hypoxia triggers regulatory pathways that mediate long-term adaptive cardiac and cerebral changes, particularly at the transcriptional level. These adaptative mechanisms include a regulated increase in cerebral blood flow, cardiac output, angiogenesis and cytoprotection triggered by HIF-1α, VEGF and Epo pathways. All these compensatory mechanisms aim to optimize oxygen delivery and to protect the brain and heart from hypoxic injury to allow acclimatization. However, when both arterial pressure and content of oxygen are decreased, the cerebral and cardiac adaptative mechanisms showed their limitations. We could not exclude that cerebral and cardiac cell injuries occurred following the combined effect of chronic anemia and hypoxia as well as of the NO toxicity. **Figure 1** summarizes the cerebral and cardiac plasticity induced by chronic anemia and/or hypoxia. Data shown in this figure are all based on animal studies. Moreover, a recent review of our group includes also ventilatory [60], muscular [84, 85] and rheologic [86] adaptations in this model of mice. Finally, investigating the molecular mechanisms of

 homeostasis represents a mean of gaining new insights to the hypoxia-induced cerebral and myocardial injuries. But it is of great clinical importance to study extensively these nonerythropoietic functions of Epo to contribute to the development of new therapeutic strategies

**Figure 1** summarizes the physiological adaptations to chronic hypoxia and anemia in the heart and brain of our model of Epo-TAgh mice. The green color represents the responses of normoxic anemic mice. The blue color represents the responses of hypoxic control mice. The red color represents the responses of hypoxic anemic mice. The arrows represent an increase or decrease of the response, while the **'='** symbol means no change between normoxia

is transport of oxygen.

is arterial content of oxygen, PiO<sup>2</sup>

is

is arterial pressure of oxygen, CaO<sup>2</sup>

 for 3 weeks) [82] suggesting an important role of Epo in functional adaptation of the heart to chronic hypoxia. Similarly to what occurred in the brain, we did not observe a synergic effect of these combined constraints on the expression of the hypoxic genes in the heart of chronically hypoxic Epo-TAgh mice suggesting that adaptive responses to both constraints are already maximal. However, the increased P-STAT-5/STAT-5 ratio is concordant with a direct protective effect of Epo on cardiomyocytes and endothelial cells as well as stimulation of angiogenesis in the ischaemic heart [83]. Capillary density was unchanged in spite of the fall in HIF-1α/VEGF pathway probably because the initiation of the capillarization with acute hypoxia necessitates VEGF, while its maintenance in chronic hypoxia involves other factors

O2

168 Hypoxia and Human Diseases

pathways.

O2

and hypoxia. PaO<sup>2</sup>

inspired pressure of oxygen and TO<sup>2</sup>

such as angiopoietins [38, 43].

**5. Perspectives and significance**

for the management of brain and heart ischemia.

All the original articles of our group cited in this review were supported by "Agence Nationale de la Recherche" n°ANR-08-GENOPAT-029.

## **Author details**

Raja El Hasnaoui-Saadani

Address all correspondence to: rajaelhasnaoui@hotmail.com

Research center-College of Medicine- Princess Nourah bint Abdulrahmane University, Riyadh, Saudi Arabia

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176 Hypoxia and Human Diseases


**Provisional chapter**

## **Hypothermia in Stroke Therapy: Systemic versus Local Application Hypothermia in Stroke Therapy: Systemic versus Local Application**

Mitchell Huber, Hong Lian Duan, Ankush Chandra, Fengwu Li, Longfei Wu, Longfei Guan, Xiaokun Geng and Yuchuan Ding Chandra, Fengwu Li, Longfei Wu, Longfei Guan, Xiaokun Geng and Yuchuan Ding Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

Mitchell Huber, Hong Lian Duan, Ankush

http://dx.doi.org/10.5772/65899

#### **Abstract**

Presently, there are no effective, widely applicable therapies for ischemic stroke. There is strong clinical evidence for the neuroprotective benefits of hypothermia, and surfacecooling methods have been utilized for decades in the treatment of cerebral ischemia during cardiac arrest, but complications with hypothermia induction have hindered its clinical acceptance in ischemic stroke therapy. Recently, the microcatheter-based local endovascular infusion (LEVI) of cold saline directly to the infarct site has been proposed as a solution to the drawbacks of surface cooling. The safety and efficacy of LEVI in rat models have been established, and implementation in larger animals has been similarly encouraging. A recent pilot study even established the safety of LEVI in humans. This review seeks to outline the major research on LEVI, discusses the mechanisms that mediate its superior neuroprotection over surface and systemic cooling, and identifies areas that warrant further investigation. While LEVI features improvements on surface cooling, its core mechanisms of neuroprotection are still largely shared with therapeutic hypothermia in general. As such, the mechanisms of hypothermia-based neuroprotection are discussed as well.

**Keywords:** local endovascular infusion, therapeutic hypothermia, ischemic stroke therapy, neuroprotection, microcatheter

#### **1. Introduction**

Ischemic stroke is the leading cause of death and disability worldwide, yet effective treatment is limited. Despite considerable research efforts, intravenous (IV) thrombolysis with recombinant tissue plasminogen activator (rt-PA) within the first 4.5 h of symptom onset remains

© 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.

the only proven acute therapy for ischemic stroke [1]. Outside of the treatment window, rt-PA fails to be an option, and given that only 25.4% of stroke patients arrive to the hospital within 3 h of symptom onset, a significant minority of patients are even eligible to receive rt-PA [2]. Thus, alternative treatment strategies for ischemic stroke are urgently needed. Although over a thousand drugs and nonpharmacological strategies have been tested for neuroprotective ability in acute stroke as of 2003, none have proven effective and applicable enough for widespread clinical acceptance [3]. However, hypothermia has prevailed as a promising therapeutic option for stroke patients. In fact, hypothermia is the only neuroprotective approach found thus far whose efficacy has been experimentally demonstrated in a randomized controlled clinical trial [4]. The neuroprotective benefits of hypothermia have been utilized for decades in the treatment of global cerebral ischemia following cardiac arrest and for hypoxic-ischemic encephalopathy in newborns, but its use in stroke therapy has garnered attention only in recent years [4, 5].

Hypothermia has been consistently shown to reduce infarct volumes and improve functional outcomes in animal models of focal cerebral ischemia. In a meta-analysis, the use of hypothermia in animal models of ischemic stroke was shown to reduce infarct volumes by 44% on average [6]. Given the robust neuroprotective effects of therapeutic hypothermia (TH) in animal models of temporary artery occlusion, studies are being conducted at an increasing rate to empirically establish hypothermia as a high-yield front-line stroke therapy.

#### **1.1. Degrees of hypothermia**

Therapeutic hypothermia is defined as the deliberate reduction of core body temperature for therapeutic benefit [7]. While there is no exact consensus on the optimal degree of cooling, several studies have found cooling at 33°C to be most effective [7–10]. The vast majority of investigations on the topic feature a mild to moderate degree of cooling, with very few venturing into moderate-deep to deep hypothermia (**Table 1**). In fact, temperature depressions to such an extent have been reported to primarily provide negative consequences [9]. For this review, therapeutic hypothermia refers to mild-moderate hypothermia, unless otherwise specified.


**Table 1.** Terms for degrees of hypothermia by body temperature.

## **2. Systemic hypothermia**

The majority of studies on the induction of TH in acute ischemic stroke therapy have applied whole-body cooling. Therapeutic cerebral hypothermia can be most easily established by either surface cooling or systemic endovascular infusion cold saline [11, 12]. In clinical stroke cases, surface and endovascular cooling have both been used for successful whole-body hypothermia induction and maintenance.

The sentinel study on therapeutic hypothermia exclusively considered "surface cooling," cooling with ice packs or air-circulating cooling blankets/mattresses. This study demonstrated improved survival outcomes in cardiac arrest patients with therapeutic hypothermia [5, 12]. The Cooling for Acute Ischemic Brain Damage (COOL AID) study additionally showed that moderate therapeutic hypothermia (target temperature 32°C by surface cooling) in patients with acute ischemic stroke is feasible and can be accomplished safely by surface cooling [13]. Surface-cooling methods are easy to use and permit early treatment initiation, which makes them an attractive option. However, there are numerous logistical problems associated with surface cooling that outweigh its benefit.

the only proven acute therapy for ischemic stroke [1]. Outside of the treatment window, rt-PA fails to be an option, and given that only 25.4% of stroke patients arrive to the hospital within 3 h of symptom onset, a significant minority of patients are even eligible to receive rt-PA [2]. Thus, alternative treatment strategies for ischemic stroke are urgently needed. Although over a thousand drugs and nonpharmacological strategies have been tested for neuroprotective ability in acute stroke as of 2003, none have proven effective and applicable enough for widespread clinical acceptance [3]. However, hypothermia has prevailed as a promising therapeutic option for stroke patients. In fact, hypothermia is the only neuroprotective approach found thus far whose efficacy has been experimentally demonstrated in a randomized controlled clinical trial [4]. The neuroprotective benefits of hypothermia have been utilized for decades in the treatment of global cerebral ischemia following cardiac arrest and for hypoxic-ischemic encephalopathy in newborns, but its use in stroke therapy has garnered attention only in

Hypothermia has been consistently shown to reduce infarct volumes and improve functional outcomes in animal models of focal cerebral ischemia. In a meta-analysis, the use of hypothermia in animal models of ischemic stroke was shown to reduce infarct volumes by 44% on average [6]. Given the robust neuroprotective effects of therapeutic hypothermia (TH) in animal models of temporary artery occlusion, studies are being conducted at an increasing

Therapeutic hypothermia is defined as the deliberate reduction of core body temperature for therapeutic benefit [7]. While there is no exact consensus on the optimal degree of cooling, several studies have found cooling at 33°C to be most effective [7–10]. The vast majority of investigations on the topic feature a mild to moderate degree of cooling, with very few venturing into moderate-deep to deep hypothermia (**Table 1**). In fact, temperature depressions to such an extent have been reported to primarily provide negative consequences [9]. For this review, therapeutic hypothermia refers to mild-moderate hypothermia, unless otherwise

The majority of studies on the induction of TH in acute ischemic stroke therapy have applied whole-body cooling. Therapeutic cerebral hypothermia can be most easily established by either surface cooling or systemic endovascular infusion cold saline [11, 12]. In clinical stroke cases, surface and endovascular cooling have both been used for successful whole-body

rate to empirically establish hypothermia as a high-yield front-line stroke therapy.

**Degree of hypothermia Mild Moderate Moderate-deep Deep** Body temperature (°C) 35.9–34 33.9–32 31.9–30 <30

recent years [4, 5].

180 Hypoxia and Human Diseases

specified.

**1.1. Degrees of hypothermia**

**2. Systemic hypothermia**

hypothermia induction and maintenance.

**Table 1.** Terms for degrees of hypothermia by body temperature.

Systemic endovascular infusion methods reduce body temperature invasively using intravenously placed cooling catheters or intravenous cold infusions of isotonic saline into a major systemic blood vessel. The safety of endovascular cooling in patients with acute ischemic stroke was assessed in both the COOL AID II study [14] and the Intravascular Cooling in the Treatment of Stroke (ICTuS) study [15]. The approach was shown in both cases to reduce body temperature more rapidly than surface cooling could accomplish, and since a temperature probe is embedded in the catheter, precise temperature monitoring and regulation was far superior to surface-cooling methods. The disadvantages of systemic endovascular hypothermia induction stem from its invasive nature; the method carries a much higher risk of deep venous thrombosis (DVT), bacteremia, and sepsis than surface cooling [16, 17]. Additionally, the Intravascular Cooling in the Treatment of Stroke-Longer tPA Window (ICTuS-L) study results showed a statistically significant increase in the occurrence of pneumonias in patients receiving systemic endovascular TH [18].

Unfortunately, whole-body cooling by either method creates a number of serious complications. Chiefly, whole-body cooling frequently causes shivering and dermal vasoconstriction, which can complicate effective progression to optimal cooling ranges; whole-body cooling frequently requires 3–7 h to reach target temperatures [19]. Shivering also raises intracranial pressure (ICP) and requires the use of several pharmacological agents to inhibit these effects along with skin warming to address physical discomfort [20, 21]. Another side effect of wholebody cooling is the risk of shear-induced platelet aggregation, which can develop as blood viscosity increases at low temperatures [22]. Even a minor amount of coagulation can cause a blockage of the microcirculation of the brain and heart, which ironically creates the exact problem that hypothermia attempts to treat [21]. Furthermore, whole-body cooling increases the likelihood of ventricular fibrillations, bradycardia, reduced cardiac output, hemostatic or hemorrhagic changes, decreased urine output, and metabolic dysfunction [14, 23, 24]. With this extensive list of severe complications, a more graceful therapeutic modality is urgently needed.

## **3. Hypothermia via local endovascular infusion**

Recently, the selective induction of hypothermia into the ischemic region using an endovascular microcatheter has garnered attention as a novel strategy to optimize the neuroprotective benefits of therapeutic hypothermia with the myriad of comorbidities accompanying full-body cooling. In contrast to other cooling methods, which require hypothermia to slowly spread into the ischemic region, local endovascular infusion (LEVI) reduces infarct temperatures effectively by perfusing ice-cold saline directly to the ischemic region. This allows for more rapid achievement of target temperatures and permits greater specificity of hypothermia while avoiding the side effects of systemic cooling. During these procedures, an infusion microcatheter, guided to the site of the lesion via the guide catheter over a microguidewire, is advanced distally to the site of occlusion, and cold saline is perfused [25] for a variable length of time, usually from 5 to 30 min. The logistics of actually performing LEVI in humans are relatively simple, as this is a normal part of performing endovascular interventions for many neuroendovascular surgeons [26]. Therefore, it is expected that this new therapy could easily be added to an angiography suite [27].

LEVI has been tested in animal models of stroke both before and after reperfusion. Prereperfusion flushing was first proposed by Ding et al. [28], when the technique was used in a transient middle cerebral artery occlusion (MCAO) rat model. The study produced a 65% reduction in infarct volumes and 61% reduction in leukocyte infiltration when resolution of a 2-h middle cerebral artery occlusion was preceded by LEVI (23°C saline infused at 2 mL/min for 3–4 min) [28]. Pre-reperfusion LEVI has since been shown to reduce infarct volumes by 75 [29] to 90% [30] and significantly conserve motor function both hours and weeks after stroke [29, 30]. Post-reperfusion LEVI has also been considered in some studies, in which a catheter was introduced into the internal carotid artery after blood flow to the ischemic territory had been reestablished [31, 32]. Significant improvements in both infarct volume and functional recovery were observed in every post-reperfusion LEVI trial tested, but these improvements were not as pronounced as those from pre-reperfusion LEVI.

Although the majority of current experimental data on LEVI in stroke are based on rat models, a few large animal studies that have been conducted are equally encouraging. A recent investigation using swine showed that LEVI significantly reduced infarct volumes following 4–4.5-h MCAO (the longest delay of hypothermia in any LEVI large animal study) [33]. The credibility [34], safety, and efficacy of LEVI in Rhesus monkeys were also confirmed, as infusion of cold-lactated Ringer's solution was used to achieve statistically significant degrees of peri-infarct cooling without apparent vasogenic edema or other comorbidities [35]. Additionally, the safety and feasibility of LEVI was recently verified in humans [36]. In nine human patients with partially or completely treated cerebrovascular diseases undergoing diagnostic cerebral angiogram, 7°C LEVI at ~33 mL/min for 10–13 min was able to reduce jugular venous blood temperature (a proxy for brain temperature) by 0.84°C while reducing rectal temperature by 0.15°C and having no significant effects on vital signs. LEVI was also recently implemented in patients actively undergoing ischemic stroke (within 8 h of symptom onset), which confirmed the safety and feasibility of the procedure [25]. The neuroprotective efficacy of LEVI, however, remains to be established in a clinical setting.

Despite recent milestones in LEVI testing, several systematic obstacles have hindered widespread acceptance. Chief among these obstacles is heterogeneity of experimental designs. Since TH is only widely used for cardiac arrest, the majority of studies utilize a global ischemia model, which has been found to unfaithfully simulate the physiological conditions of focal ischemia [37]. Hypothermia-based investigations also vary in animal model, animal age, duration of ischemia, duration of hypothermia, depth of hypothermia, method of hypothermia induction, and rate of cooling, all of which have consistently been shown to play critical roles in the efficacy of TH treatments. Additionally, current animal models have failed to adequately simulate the reaction of a human to such an intervention. While the majority of LEVI studies have used rat models, rats have been widely criticized for their poor translatability to clinical practice [38]. There even exists heterogeneity among the species; rats of similar strains from different suppliers have been found to show variations in response to ischemia [12]. Given that stroke accounts for 9% of deaths worldwide and ~25% of stroke survivors are permanently disabled [39], such a promising therapy is in serious need of further exploration.

#### **3.1. Benefits of LEVI over systemic infusion**

LEVI is an optimized version of general TH. As such, its mechanisms of neuroprotection are predominately the same as those of full-body cooling. However, LEVI retains a few unique features that make it considerably more effective than global cooling. These features are summarized in the present section.

#### *3.1.1. Maximized rate of cooling*

full-body cooling. In contrast to other cooling methods, which require hypothermia to slowly spread into the ischemic region, local endovascular infusion (LEVI) reduces infarct temperatures effectively by perfusing ice-cold saline directly to the ischemic region. This allows for more rapid achievement of target temperatures and permits greater specificity of hypothermia while avoiding the side effects of systemic cooling. During these procedures, an infusion microcatheter, guided to the site of the lesion via the guide catheter over a microguidewire, is advanced distally to the site of occlusion, and cold saline is perfused [25] for a variable length of time, usually from 5 to 30 min. The logistics of actually performing LEVI in humans are relatively simple, as this is a normal part of performing endovascular interventions for many neuroendovascular surgeons [26]. Therefore, it is expected that this new therapy could easily

LEVI has been tested in animal models of stroke both before and after reperfusion. Prereperfusion flushing was first proposed by Ding et al. [28], when the technique was used in a transient middle cerebral artery occlusion (MCAO) rat model. The study produced a 65% reduction in infarct volumes and 61% reduction in leukocyte infiltration when resolution of a 2-h middle cerebral artery occlusion was preceded by LEVI (23°C saline infused at 2 mL/min for 3–4 min) [28]. Pre-reperfusion LEVI has since been shown to reduce infarct volumes by 75 [29] to 90% [30] and significantly conserve motor function both hours and weeks after stroke [29, 30]. Post-reperfusion LEVI has also been considered in some studies, in which a catheter was introduced into the internal carotid artery after blood flow to the ischemic territory had been reestablished [31, 32]. Significant improvements in both infarct volume and functional recovery were observed in every post-reperfusion LEVI trial tested, but these improvements

Although the majority of current experimental data on LEVI in stroke are based on rat models, a few large animal studies that have been conducted are equally encouraging. A recent investigation using swine showed that LEVI significantly reduced infarct volumes following 4–4.5-h MCAO (the longest delay of hypothermia in any LEVI large animal study) [33]. The credibility [34], safety, and efficacy of LEVI in Rhesus monkeys were also confirmed, as infusion of cold-lactated Ringer's solution was used to achieve statistically significant degrees of peri-infarct cooling without apparent vasogenic edema or other comorbidities [35]. Additionally, the safety and feasibility of LEVI was recently verified in humans [36]. In nine human patients with partially or completely treated cerebrovascular diseases undergoing diagnostic cerebral angiogram, 7°C LEVI at ~33 mL/min for 10–13 min was able to reduce jugular venous blood temperature (a proxy for brain temperature) by 0.84°C while reducing rectal temperature by 0.15°C and having no significant effects on vital signs. LEVI was also recently implemented in patients actively undergoing ischemic stroke (within 8 h of symptom onset), which confirmed the safety and feasibility of the procedure [25]. The neuroprotective

Despite recent milestones in LEVI testing, several systematic obstacles have hindered widespread acceptance. Chief among these obstacles is heterogeneity of experimental designs. Since TH is only widely used for cardiac arrest, the majority of studies utilize a global ischemia model, which has been found to unfaithfully simulate the physiological conditions of

be added to an angiography suite [27].

182 Hypoxia and Human Diseases

were not as pronounced as those from pre-reperfusion LEVI.

efficacy of LEVI, however, remains to be established in a clinical setting.

Although there is no consensus on the exact treatment window for therapeutic hypothermia, it would be difficult to dispute the time-sensitive nature of hypothermia induction [31, 40]. While one author found that TH is ineffective after 45 min of ischemia [41] and most others have found neuroprotective efficacy when induction follows 2–3 h of ischemia [30, 42], there is a strong consensus that this efficacy diminishes over the course of hours. Considering that surface-cooling methods frequently take 3–7 h to reach target temperatures [19], it would be impossible for any stroke patient to fall within an optimal treatment window. By contrast, LEVI can establish target temperatures in a matter of minutes [36]; in a 300-g localized cerebral infarct, LEVI attained target temperatures 30 times faster than classic surface cooling and 10–20 times faster than systemic infusion of cold saline into the inferior vena cava [27]. The time saved by using LEVI translates to superior degrees of neuroprotection and an improved quality of life for ischemic stroke patients.

#### *3.1.2. Metabolite washout and attenuated hyper- or hypoperfusion*

One mechanism by which ischemic stroke damages the brain is through postischemic hyperperfusion. Under ischemic conditions, brain cells are forced to conduct anaerobic respiration, the byproducts of which (lactate, prostaglandins, and carbon dioxide) are vasodilatory at elevated levels [43]. In the absence of adequate perfusion, these vasodilatory metabolites accumulate in the ischemic region and trigger an excessive vasodilation once perfusion is restored. Literature on postischemic hyperperfusion has been discrepant, but suggests that the phenomenon is associated with larger infarcts and early death [44, 45]. This "luxury reperfusion" has been implicated in post-reperfusion edema formation, the primary cause of death within 1 month of ischemic stroke [45, 46]. While hypothermia prevents intracranial-pressure elevations by itself, LEVI provides an additional protective mechanism by washing out vasodilatory metabolites built up during the ischemic period, which minimizes the extent of hyperperfusion-related injury [29, 47]. As evidence of this mechanism, fast warm (37°C)-saline LEVI has been shown to significantly reduce infarct volumes and improve functional recoveries compared to systemic infusion of warm saline [28].

Pre-reperfusion flushing also significantly reduces leukocyte infiltration and ICAM-1 expression in the peri-infarct vasculature [28, 48], leading to improved postischemic perfusion. Luan et al. showed that LEVI was able to reduce cerebral poststroke ICAM-1 expression and leukocyte infiltration to a significantly greater degree than that of local warm-saline infusion or systemic cold-saline infusion were able to [48]. Other studies have reported similar reductions in ICAM-1 expression and infiltration/activation of PMN leukocytes and microglia [49]. These data imply that the neuroprotective advantages of LEVI over systemic infusion rely partially on its metabolite-washout ability and subsequent improved perfusion.

#### *3.1.3. Drug delivery into ischemic territory through LEVI*

In addition to the hypothermia-associated benefits of cold-saline infusion, LEVI allows for coadministration of neuroprotective drugs directly into the ischemic region along with hypothermic fluids, which maximizes local drug concentrations while minimizing systemic drug concentrations, thereby circumventing dose-dependent systemic side effects [50]. Preliminary studies using LEVI with neuroprotective drugs have shown exceptional promise; a 2012 study by Song et al. found that LEVI of a magnesium sulfate solution at 15°C caused a 65% reduction in infarct volumes compared to a 48% reduction from LEVI alone [51]. Similar results were found following LEVI of a 20% human albumin solution cooled to 0°C [52]. Normothermic local infusion of drugs has shown potential as well, as LEVI of erythropoietin at room temperature reduced infarct volumes by 21% (significant compared to control), decreased apoptosis in the ischemic core and penumbra, and significantly preserved neurological scores [53].

LEVI can also aid in drug permeation into the brain parenchyma. Blood-brain barrier (BBB) impermeability has been described as the most important factor limiting the growth of neurotherapeutic drugs [54] and remains a challenging issue today. However, BBB breakdown is a natural product of cerebral ischemia, which allows for the perfusion of drugs into the brain parenchyma that would otherwise be prevented from reaching their target [50]. When coupled with LEVI-based drug administration, BBB breakdown can be capitalized on to provide benefits for stroke therapy. This hypothesis was confirmed experimentally in a 2007 study by Woitzik et al. in which microcatheter-based infusion of MK-801 (an NMDA receptor antagonist) into the ischemic region resulted in 30% smaller infarct volumes at 24 h after infusion than when MK-801 was infused systemically [50]. MK-801 has shown significant neuroprotective potential, but has not attained clinical acceptance due to significant side effects when administered at high enough doses to be effective when infused systemically [55], a problem nullified by LEVI-based administration. While LEVI with neuroprotective drugs has never been tested in a clinical setting, it is possible that the combination could open the door for the use of neuroprotective pharmacotherapies that would otherwise be prohibited from reaching target tissues [50, 56].

## **4. Mechanisms underlying hypothermia-induced neuroprotection**

In addition to LEVI-specific neuroprotective mechanisms, LEVI benefits from neuroprotective mechanisms of therapeutic hypothermia in general. These mechanisms exhibit significant redundancy, as they affect multiple steps in several parallel pathways of hypoxia-induced brain injury. Hypothermia primarily exerts its neuroprotective effects by slowing essential metabolic processes while preserving life, which subsequently attenuates pathways involved in excitotoxicity, free radical production, inflammation, edema, and apoptosis [12, 37, 57, 58]. However, a common theme in literature on the topic is consensus on effects and uncertainty of mechanisms. While virtually every paper finds TH administration to be neuroprotective, there is very little agreement on how this works. This is due, in part, to the correlative goal of most studies. The majority of work on the topic identifies alterations in the levels of one indicator or another when TH is implemented, but fails to elucidate exactly where TH exerts its neuroprotective effects. While this is valuable information, without a causative component, these studies always leave the door open for the participation of a third variable. In light of frequently conflicting findings, this section features few concrete lessons from the literature. Rather, we attempt to discuss the pathways that TH acts on, and consider the most likely points at which TH exerts its neuroprotective effects.

#### **4.1. Metabolic crisis**

vasodilatory metabolites built up during the ischemic period, which minimizes the extent of hyperperfusion-related injury [29, 47]. As evidence of this mechanism, fast warm (37°C)-saline LEVI has been shown to significantly reduce infarct volumes and improve functional recover-

Pre-reperfusion flushing also significantly reduces leukocyte infiltration and ICAM-1 expression in the peri-infarct vasculature [28, 48], leading to improved postischemic perfusion. Luan et al. showed that LEVI was able to reduce cerebral poststroke ICAM-1 expression and leukocyte infiltration to a significantly greater degree than that of local warm-saline infusion or systemic cold-saline infusion were able to [48]. Other studies have reported similar reductions in ICAM-1 expression and infiltration/activation of PMN leukocytes and microglia [49]. These data imply that the neuroprotective advantages of LEVI over systemic infusion rely partially on its metabolite-washout ability and subsequent improved

In addition to the hypothermia-associated benefits of cold-saline infusion, LEVI allows for coadministration of neuroprotective drugs directly into the ischemic region along with hypothermic fluids, which maximizes local drug concentrations while minimizing systemic drug concentrations, thereby circumventing dose-dependent systemic side effects [50]. Preliminary studies using LEVI with neuroprotective drugs have shown exceptional promise; a 2012 study by Song et al. found that LEVI of a magnesium sulfate solution at 15°C caused a 65% reduction in infarct volumes compared to a 48% reduction from LEVI alone [51]. Similar results were found following LEVI of a 20% human albumin solution cooled to 0°C [52]. Normothermic local infusion of drugs has shown potential as well, as LEVI of erythropoietin at room temperature reduced infarct volumes by 21% (significant compared to control), decreased apoptosis in the ischemic core and penumbra, and significantly preserved neuro-

LEVI can also aid in drug permeation into the brain parenchyma. Blood-brain barrier (BBB) impermeability has been described as the most important factor limiting the growth of neurotherapeutic drugs [54] and remains a challenging issue today. However, BBB breakdown is a natural product of cerebral ischemia, which allows for the perfusion of drugs into the brain parenchyma that would otherwise be prevented from reaching their target [50]. When coupled with LEVI-based drug administration, BBB breakdown can be capitalized on to provide benefits for stroke therapy. This hypothesis was confirmed experimentally in a 2007 study by Woitzik et al. in which microcatheter-based infusion of MK-801 (an NMDA receptor antagonist) into the ischemic region resulted in 30% smaller infarct volumes at 24 h after infusion than when MK-801 was infused systemically [50]. MK-801 has shown significant neuroprotective potential, but has not attained clinical acceptance due to significant side effects when administered at high enough doses to be effective when infused systemically [55], a problem nullified by LEVI-based administration. While LEVI with neuroprotective drugs has never been tested in a clinical setting, it is possible that the combination could open the door for the use of neuroprotective pharmacotherapies that would otherwise be prohibited from reaching

ies compared to systemic infusion of warm saline [28].

*3.1.3. Drug delivery into ischemic territory through LEVI*

perfusion.

184 Hypoxia and Human Diseases

logical scores [53].

target tissues [50, 56].

The primary culprit of ischemia-induced brain damage is oxygen-supply cessation, which initiates a cascade of secondary problems. In the absence of oxygen, neurons are unable to generate high-energy metabolites, which prohibit effective maintenance of ion gradients. Ion-gradient breakdown leads to involuntary depolarization, which allows for excessive glutamate release. This wave of glutamate then stimulates NMDA and AMPA receptors, which results in increased intracellular calcium levels and ultimately leads to excitotoxicity, a phenomenon characterized by mitochondrial membrane depolarization, caspase activation, production of reactive oxygen and nitrogen species, and apoptosis [37, 59]. In addition to excitotoxicity, ion-gradient breakdown causes Na<sup>+</sup> to build up in brain cells and in particular astrocytes. This establishes an osmotic gradient favoring the movement of water into astrocytes (and to a lesser extent, all other brain cells), thereby creating cytotoxic edema [60]. The edema increases intracranial pressure and ultimately exacerbates brain damage (**Figure 1**).

Hypothermia combats this cascade at several points (**Figure 1**). Reduced brain temperatures have been shown to lower cerebral metabolic rate by 5% for every 1°C reduction in body temperature, allowing for prolonged maintenance of ion gradients (preventing excitotoxicity) and minimized need to conduct anaerobic respiration, thereby diminishing the extent of reperfusion injury [58]. In patients with traumatic brain injury who received therapeutic hypothermia to 32–33°C, cerebral oxygen consumption was reduced to 27% after 24 h of hypothermia [61]. Hypothermia has also been shown to reduce the production of glycolytic intermediates by an average of 30% and tricarboxylic acid (TCA) cycle intermediates by 30–70% [62]. Alternatively, ratios of phosphocreatinine:inorganic phosphate and adenosine triphosphate (ATP):inorganic phosphate seem to increase slightly during transient hypothermia, implying that the real energy conservation mechanism at play is one of the slowing energy-consuming reactions, rather than slowing glycolytic flux [62]. Hypothermia has also been routinely reported to improve ATP recovery after reperfusion [37]; mild hypothermia has led to a 10–20% increase in the rate of metabolic recovery in the first 10–25 min after reperfusion compared to normothermic animals [63], a finding echoed in other studies [62, 64]. It is possible, then, that the primary energy conservation mechanism that underlies TH is that of accelerated energy recovery after reperfusion rather than energy preservation during hypoxia. However, while metabolic depression during hypothermia has been well documented as a phenomenon, its underlying mechanism is still poorly understood. Thus, the points at which hypothermia exerts its neuroprotective effects are unclear, and whether its main mechanism of neuroprotection involves cellular respiration at all remains to be elucidated.

**Figure 1.** The figure describes the pathogenesis of stroke as it relates to ischemia-induced metabolic crisis. The points at which hypothermia exerts its neuroprotective effects remain largely unclear. Studies have shown that TH attenuates a multitude of steps in the cascade of ischemia-induced brain damage compared to stroke without hypothermia, but whether the observed attenuations are direct effects of TH or byproducts of upstream attenuations has yet to be elucidated. As such, blue font indicates steps discussed in the present review that hypothermia has been shown to attenuate. Black font indicates steps that we have not discussed in the present review, but does not necessarily indicate that these steps are unaffected by hypothermia.

Hypothermia has also been shown to prevent anoxic depolarization. In an aged rat model, mild hypothermia was shown to completely inhibit the efflux of excitatory amino acids (glutamate and aspartate) while significantly increasing the release of the inhibitory amino acid taurine [65]. While no mechanism has been firmly tied to this phenomenon, several have been speculated. TH has been reported to prevent activation of protein kinase C (PKC) and calcium-calmodulin kinase II during ischemia, both of which are associated with neurotransmitter release [65]. Therapeutic cooling also attenuates ischemia-induced downregulation of the GluR2 (glutamate receptor 2) CA1 subunit, which is responsible for limiting Ca2+ influx through AMPA receptors in a global cerebral ischemia model [66]. It is also possible that this facet of hypothermic neuroprotection is accomplished by the preservation of ion gradients due to metabolic downregulation. However, some reports have suggested that hypothermia simply delays anoxic depolarization rather than preventing it [67]. In light of conflicting research on the topic, it is likely that multiple mechanisms are at play, culminating in the robust excitation prevention associated with therapeutic hypothermia.

transient hypothermia, implying that the real energy conservation mechanism at play is one of the slowing energy-consuming reactions, rather than slowing glycolytic flux [62]. Hypothermia has also been routinely reported to improve ATP recovery after reperfusion [37]; mild hypothermia has led to a 10–20% increase in the rate of metabolic recovery in the first 10–25 min after reperfusion compared to normothermic animals [63], a finding echoed in other studies [62, 64]. It is possible, then, that the primary energy conservation mechanism that underlies TH is that of accelerated energy recovery after reperfusion rather than energy preservation during hypoxia. However, while metabolic depression during hypothermia has been well documented as a phenomenon, its underlying mechanism is still poorly understood. Thus, the points at which hypothermia exerts its neuroprotective effects are unclear, and whether its main mechanism of neuroprotection involves cellular

Hypothermia has also been shown to prevent anoxic depolarization. In an aged rat model, mild hypothermia was shown to completely inhibit the efflux of excitatory amino acids (glutamate and aspartate) while significantly increasing the release of the inhibitory amino acid taurine [65]. While no mechanism has been firmly tied to this phenomenon, several have

**Figure 1.** The figure describes the pathogenesis of stroke as it relates to ischemia-induced metabolic crisis. The points at which hypothermia exerts its neuroprotective effects remain largely unclear. Studies have shown that TH attenuates a multitude of steps in the cascade of ischemia-induced brain damage compared to stroke without hypothermia, but whether the observed attenuations are direct effects of TH or byproducts of upstream attenuations has yet to be elucidated. As such, blue font indicates steps discussed in the present review that hypothermia has been shown to attenuate. Black font indicates steps that we have not discussed in the present review, but does not necessarily indicate

respiration at all remains to be elucidated.

186 Hypoxia and Human Diseases

that these steps are unaffected by hypothermia.

Hypothermia has also been found to combat cytotoxic edema after ischemic stroke (**Figure 1**). This edema is largely mediated by aquaporin 4 (AQP4), which is expressed in the glial-limiting membranes, ependyma, and pericapillary foot processes of astrocytes [68]. In mice, AQP4 knockout has been associated with reduced infarct sizes, decreased brain water content, and improved neurological and survival outcomes [60]. While the brain naturally downregulates AQP4 expression following hypoxia [60], hypothermia has been shown to augment this downregulation [60, 69, 70]. It is possible that this downregulation is a downstream effect of TH. In experimental models, AQP4 levels in astrocyte cell membranes were increased by increased lactic acid concentration, but AQP4 mRNA levels were unchanged, which implies that the observed increases in membrane-bound AQP4 came as the result of redistribution or posttranslational modification, rather than increased expression [71]. Several other mechanisms have also been proposed for this upregulation [72]; thus, the specifics of ischemia-induced aquaporin modulation have still yet to be fully elucidated.

At the molecular level, several studies have implicated immediate induction of early gene expression (miRNAs) and cellular stress response (heat-shock proteins, HSPs) activation in hypothermia-induced neuroprotection. Hypothermia has been shown to suppress transcription of some pro-inflammatory molecules (interleukin (IL)-1β and osteopontin) and enhance transcription of anti-inflammatory substances (HSP70) [73]. The duration of postreperfusion hypothermia seems to play a role in the modulation of transcriptional rate, as the expression of numerous genes differs when hypothermia is sustained for 8 h compared to 4 h. One such gene is early growth response-1 (Egr-1), which is an early regulator of other pro-inflammatory mediators (IL-1β MCK-1, and MIP-2) [73]. This is consistent with other reports on the topic, which suggests that Egr-1 is the key component modulated by TH. However, information regarding early cellular response to ischemia and hypothermia has largely been conflicting, leaving the specifics of its involvement unclear [38] and inconsistent [31, 32].

While there exists a general consensus that TH is neuroprotective, the precise mechanisms of this effect are still very much theoretical. Additionally, if TH attenuated metabolic crisis alone, it would not be able to accomplish such a robust degree of neuroprotection [74]. It comes as no surprise, then, that suspended animation is just the appetizer in the multicourse meal that is TH-mediated neuroprotection.

#### **4.2. Inflammation and blood-brain barrier breakdown**

In stroke therapy, the restoration of blood flow is of chief concern. Surprisingly, however, recanalization is not exclusively beneficial. Reperfusion often initiates a detrimental cascade, collectively termed ischemia/reperfusion injury, which can be disastrous; in some animal models, reperfusion after an extended period of ischemia caused larger infarct volumes than if the occlusion had been left permanently [45]. Reperfusion injury is a complex, multifaceted injury cascade initiated by sterile inflammation from anoxic tissue damage, and propagated by both the innate and adaptive immune systems and complement system [75, 76].

Mechanistically, ischemia/reperfusion injury is initiated by the aberrant Ca2+ influx characteristic of ischemic stroke, which activates phospholipases and eventually results in the production of pro-inflammatory mediators from microglia, including proteases, leukotrienes, IL-1β IL-6, NO, and tumor necrosis factor (TNF)-a [77]. These mediators contribute to post-reperfusion insult directly, by increasing vascular permeability, and indirectly, by increasing endothelial ICAM-1 expression and serving as potent chemotactic agents for polymorphonuclear leukocytes, both of which increase leukocyte extravasation into the brain parenchyma [46, 78]. The pro-inflammatory transcription factor nuclear factor kappa B (NF-κB) is likely the cause of this upregulation, as it is responsible for inducing the expression of IL-1β IL-6, TNF-α, and ICAM-1 [78]. In addition to recruiting leukocytes to the infarct site, IL-1β and TNF-α have also been found to increase the production of matrix metalloproteinases (MMPs) [79]. MMP-2 and MMP-9 have been shown to contribute to vasogenic edema by degrading extracellular matrix components during ischemic stroke, and MMP-9 knockout mice experience reduced infarct volumes and less severe motor deficits than wild-type mice [79]. The effect of MMPs ultimately perpetuates the development of inflammation and edema, which further encourages leukocyte extravasation. Once leukocytes enter the brain tissue, they produce ROS and pro-inflammatory factors of their own, thereby creating a viscous cycle of brain injury, inflammation, and blood-brain barrier (BBB) breakdown (**Figure 2**).

A common effect of reperfusion injury mechanisms is BBB disruption. Reperfusion activates matrix-degrading proteases within hours, which makes the vessels particularly leaky and allows for migration of albumin and other blood proteins into the brain parenchyma within 4–6 h of BBB disruption [72]. Water osmotically follows these proteins, thereby creating vasogenic edema, which may increase brain water content by more than 100% in poorly perfused regions [72, 80]. Vasogenic edema is the primary cause of death within the first month of an ischemic stroke [46], as it increases intracranial pressure (ICP) and compresses cerebrovasculature within the inflexible confines of the skull, causing further ischemia and eventually brain herniation [56].

Therapeutic hypothermia is able to confer anti-inflammatory neuroprotection by reducing the secretion of pro-inflammatory cytokines (IL-1β TNF-α, and IL-6) and inflammatory mediators (reactive oxygen and nitrogen species, E-selectin, and HMGB1) [81]. TH can also prevent leukocyte extravasation into neural tissue directly by reducing the endothelial expression of ICAM-1 [27, 48]. ICAM-1 is constitutively expressed by endothelial cells at very low levels, but the expression is precipitously increased following endothelial damage when it functions as an attachment point for the CD11/CD18 integrin of leukocytes (preceding extravasation

**4.2. Inflammation and blood-brain barrier breakdown**

188 Hypoxia and Human Diseases

mation, and blood-brain barrier (BBB) breakdown (**Figure 2**).

brain herniation [56].

In stroke therapy, the restoration of blood flow is of chief concern. Surprisingly, however, recanalization is not exclusively beneficial. Reperfusion often initiates a detrimental cascade, collectively termed ischemia/reperfusion injury, which can be disastrous; in some animal models, reperfusion after an extended period of ischemia caused larger infarct volumes than if the occlusion had been left permanently [45]. Reperfusion injury is a complex, multifaceted injury cascade initiated by sterile inflammation from anoxic tissue damage, and propagated

Mechanistically, ischemia/reperfusion injury is initiated by the aberrant Ca2+ influx characteristic of ischemic stroke, which activates phospholipases and eventually results in the production of pro-inflammatory mediators from microglia, including proteases, leukotrienes, IL-1β IL-6, NO, and tumor necrosis factor (TNF)-a [77]. These mediators contribute to post-reperfusion insult directly, by increasing vascular permeability, and indirectly, by increasing endothelial ICAM-1 expression and serving as potent chemotactic agents for polymorphonuclear leukocytes, both of which increase leukocyte extravasation into the brain parenchyma [46, 78]. The pro-inflammatory transcription factor nuclear factor kappa B (NF-κB) is likely the cause of this upregulation, as it is responsible for inducing the expression of IL-1β IL-6, TNF-α, and ICAM-1 [78]. In addition to recruiting leukocytes to the infarct site, IL-1β and TNF-α have also been found to increase the production of matrix metalloproteinases (MMPs) [79]. MMP-2 and MMP-9 have been shown to contribute to vasogenic edema by degrading extracellular matrix components during ischemic stroke, and MMP-9 knockout mice experience reduced infarct volumes and less severe motor deficits than wild-type mice [79]. The effect of MMPs ultimately perpetuates the development of inflammation and edema, which further encourages leukocyte extravasation. Once leukocytes enter the brain tissue, they produce ROS and pro-inflammatory factors of their own, thereby creating a viscous cycle of brain injury, inflam-

A common effect of reperfusion injury mechanisms is BBB disruption. Reperfusion activates matrix-degrading proteases within hours, which makes the vessels particularly leaky and allows for migration of albumin and other blood proteins into the brain parenchyma within 4–6 h of BBB disruption [72]. Water osmotically follows these proteins, thereby creating vasogenic edema, which may increase brain water content by more than 100% in poorly perfused regions [72, 80]. Vasogenic edema is the primary cause of death within the first month of an ischemic stroke [46], as it increases intracranial pressure (ICP) and compresses cerebrovasculature within the inflexible confines of the skull, causing further ischemia and eventually

Therapeutic hypothermia is able to confer anti-inflammatory neuroprotection by reducing the secretion of pro-inflammatory cytokines (IL-1β TNF-α, and IL-6) and inflammatory mediators (reactive oxygen and nitrogen species, E-selectin, and HMGB1) [81]. TH can also prevent leukocyte extravasation into neural tissue directly by reducing the endothelial expression of ICAM-1 [27, 48]. ICAM-1 is constitutively expressed by endothelial cells at very low levels, but the expression is precipitously increased following endothelial damage when it functions as an attachment point for the CD11/CD18 integrin of leukocytes (preceding extravasation

by both the innate and adaptive immune systems and complement system [75, 76].

**Figure 2.** The figure describes the pathogenesis of stroke as it relates to ischemia-induced oxidative stress, inflammation, and edema. It is not known exactly where hypothermia exerts its neuroprotective effects. Studies have shown that TH attenuates a multitude of steps in the cascade of ischemia-induced brain damage compared to stroke without hypothermia, but whether the observed attenuations are direct effects of TH or byproducts of upstream attenuations has yet to be elucidated. As such, blue font indicates steps discussed in the present review that hypothermia has been shown to attenuate. Black font indicates steps that we have not discussed in the present review, but does not necessarily indicate that these steps are unaffected by hypothermia.

into damaged tissue) [82]. ICAM-1 knockout mice are resistant to cerebral ischemic injury [83], and antagonization of CD11/CD18 has been shown to substantially reduce leukocyte infiltration and subsequent cerebral edema (**Figure 2**) [82].

These effects seem to be associated with the inhibition of the extracellular signal-regulated kinase (ERK) pathway (**Figure 2**). ERK plays a significant role in the regulation of cell survival signals, and in the brain it is involved in responses to stress stimuli, including glutamate receptor stimulation and oxidative stress [84, 85]. ERK has been shown to contribute to NO and TNF-α secretion, and inhibition of the pathway prevents the release of excitotoxic amino acids following focal ischemia [86]. Transient hypothermia has been shown to reduce microglial activation, which translated to reduced phosphorylation (activation) of ERK and decreased IL-6 and TNF-α secretion [84]. However, induction of hypothermia in conjunction with U0216 (an ERK inhibitor) provided equal functional recovery to rats that did not receive U0216, implying that poststroke functional recovery progresses independently of ERK signaling [87]. Hypothermia has also been shown to reduce ICAM-1 expression in microglia in correlation with the ERK pathway, as administration of TH led to decreases in the activation of ERK as well as the inhibition of ICAM-1 expression [84].

Hypothermia-associated decreases in the expression of ICAM-1, IL-1β and TNF-α may also be due to attenuation of the NF-κB pathway (**Figure 2**). Therapeutic hypothermia has been shown to increase the expression of HSP70 in ischemic brains (but not in non-ischemic brains), and reports have suggested that HSP70 stabilizes NF-κB, thereby preventing its phosphorylation (activation) [73]. However, other NF-κB-associated proteins contribute as well. This pathway is puzzling, as the mechanism of NF-κB suppression varies depending on the type of ischemia. In models of focal ischemia, hypothermia suppresses NF-κB activity by inhibiting the activity of NF-κB kinase (IKK), a protein essential for degradation of the NF-κB inhibitor (IκB). In models of global ischemia, nuclear NF-κB levels in hypothermic subjects were still below normothermic levels, but IKK and IκB levels were unchanged [78]. These results are surprising, but emphasize the complexity of stroke pathogenesis and TH-associated neuroprotection. Moreover, regardless of the precise mechanism, therapeutic hypothermia seems to serve a beneficial role in NF-κB-associated neuroprotection.

Hypothermia can also prevent BBB breakdown directly. Nagel et al. recently found that TH increased functional recovery and reduced MMP-2 and -9 activities to the same degree as normothermic application of the MMP inhibitor minocycline, and that the application of TH in conjunction with minocycline was only marginally more effective than either by itself [88]. In addition to decreasing MMP activity, minocycline has been shown to decrease MMP production at the transcriptional level, and this report suggested that TH functions in the same way [88]. Other groups have found similar results, and this TH-induced MMP downregulation indeed translated to smaller infarct volumes and improved functional recovery [79, 89]. These data consistently show that TH is a powerful downregulator of MMP expression and activity, and that the modulation of MMP function leads to marked improvements in bigpicture end goals of stroke therapy (decreased infarct volume, increased functional recovery, etc.) (**Figure 2**).

#### **4.3. Apoptosis**

Following the initial ischemia-induced insults (hours to days), long-term brain damage (days to weeks) is greatly influenced by cellular proapoptotic mechanisms. Hypothermia has been shown to affect several aspects of apoptotic cell death in both the intrinsic (intracellular-mediated) and extrinsic (receptor-mediated) cell death pathways, and ultimately prevent apoptosis after experimental stroke (**Figure 3**) [37].

receptor stimulation and oxidative stress [84, 85]. ERK has been shown to contribute to NO and TNF-α secretion, and inhibition of the pathway prevents the release of excitotoxic amino acids following focal ischemia [86]. Transient hypothermia has been shown to reduce microglial activation, which translated to reduced phosphorylation (activation) of ERK and decreased IL-6 and TNF-α secretion [84]. However, induction of hypothermia in conjunction with U0216 (an ERK inhibitor) provided equal functional recovery to rats that did not receive U0216, implying that poststroke functional recovery progresses independently of ERK signaling [87]. Hypothermia has also been shown to reduce ICAM-1 expression in microglia in correlation with the ERK pathway, as administration of TH led to decreases in the activation of ERK as well

Hypothermia-associated decreases in the expression of ICAM-1, IL-1β and TNF-α may also be due to attenuation of the NF-κB pathway (**Figure 2**). Therapeutic hypothermia has been shown to increase the expression of HSP70 in ischemic brains (but not in non-ischemic brains), and reports have suggested that HSP70 stabilizes NF-κB, thereby preventing its phosphorylation (activation) [73]. However, other NF-κB-associated proteins contribute as well. This pathway is puzzling, as the mechanism of NF-κB suppression varies depending on the type of ischemia. In models of focal ischemia, hypothermia suppresses NF-κB activity by inhibiting the activity of NF-κB kinase (IKK), a protein essential for degradation of the NF-κB inhibitor (IκB). In models of global ischemia, nuclear NF-κB levels in hypothermic subjects were still below normothermic levels, but IKK and IκB levels were unchanged [78]. These results are surprising, but emphasize the complexity of stroke pathogenesis and TH-associated neuroprotection. Moreover, regardless of the precise mechanism, therapeutic hypothermia seems

Hypothermia can also prevent BBB breakdown directly. Nagel et al. recently found that TH increased functional recovery and reduced MMP-2 and -9 activities to the same degree as normothermic application of the MMP inhibitor minocycline, and that the application of TH in conjunction with minocycline was only marginally more effective than either by itself [88]. In addition to decreasing MMP activity, minocycline has been shown to decrease MMP production at the transcriptional level, and this report suggested that TH functions in the same way [88]. Other groups have found similar results, and this TH-induced MMP downregulation indeed translated to smaller infarct volumes and improved functional recovery [79, 89]. These data consistently show that TH is a powerful downregulator of MMP expression and activity, and that the modulation of MMP function leads to marked improvements in bigpicture end goals of stroke therapy (decreased infarct volume, increased functional recovery,

Following the initial ischemia-induced insults (hours to days), long-term brain damage (days to weeks) is greatly influenced by cellular proapoptotic mechanisms. Hypothermia has been shown to affect several aspects of apoptotic cell death in both the intrinsic (intracellular-mediated) and extrinsic (receptor-mediated) cell death pathways, and ultimately prevent apopto-

as the inhibition of ICAM-1 expression [84].

190 Hypoxia and Human Diseases

etc.) (**Figure 2**).

**4.3. Apoptosis**

sis after experimental stroke (**Figure 3**) [37].

to serve a beneficial role in NF-κB-associated neuroprotection.

**Figure 3.** The figure describes the pathogenesis of stroke as it pertains to apoptotic pathways. It is not known exactly where hypothermia exerts its neuroprotective effects. Studies have shown that TH attenuates a multitude of steps in the cascade of ischemia-induced brain damage compared to stroke without hypothermia, but whether the observed attenuations are direct effects of TH or byproducts of upstream attenuations has yet to be elucidated. As such, blue font indicates steps discussed in the present review that hypothermia has been shown to attenuate. Black font indicates steps that we have not discussed in the present review, but does not necessarily indicate that these steps are unaffected by hypothermia. BDNF, brain-derived neurotropic factor; MMP, matrix metalloproteinase; RKT, receptor tyrosine kinase; PI3K, phosphoinositide-3 kinase; PTEN, phosphatase and tensin homologue; FKHR, forkhead transcription factor; APAF1, apoptotic protease-activating factor 1.

The extrinsic apoptotic pathway is initiated by ligand binding to cell death receptors; the best studied being the FAS-ligand (FASL) and its receptor, FAS. When FASL interacts with FAS, it triggers the intercellular assembly of death-induced-signaling complexes (DISCs), which leads to caspase 8 activation. Activated caspase 8 then triggers a caspase activation cascade resulting in the stimulation of apoptosis-inducing proteins such as caspase 3, thereby mediating cell death (**Figure 3**).

Hypothermia affects this pathway at multiple levels (**Figure 3**). Cooling has been shown to suppress the expression of caspase 8, caspase 3, FAS, and FASL [90]. Additionally, there is evidence that the FAS-FASL complex must be cleaved from the cell membrane by MMPs before becoming active [91]. TH has been shown to reduce levels of both MMPs and soluble FASL in cooled rat brains [91], so is possible that the reduction in levels of these downstream effectors is simply the byproduct of inhibiting the FAS-FASL cleavage. While there are little available data to this end, the fact remains that, by one mechanism or another, hypothermia significantly reduces the production of a number of extrinsic apoptotic pathway intermediates, which translates to the preservation of penumbral tissue.

The intrinsic apoptotic pathway is triggered by intracellular cell stress signals including hypoxia, DNA damage, and cellular detachment from the extracellular matrix. These signals initiate apoptosis by disrupting the balance between proapoptotic Bcl-2 family members (BID, BAX, BAD, etc.) and anti-apoptotic Bcl-2 members (Bcl-2, Bcl-x, etc.) by a variety of mechanisms. Bcl-2 and Bcl-xL have both been found to be upregulated in neurons surviving hypoxia, while proapoptotic Bcl-2 members are highly expressed in neurons that will eventually die from hypoxic damage [37]. The imbalance between pro- and anti-apoptotic Bcl-2 members leads to the liberation of cytochrome C from the mitochondrial intermembrane space into the cytosol where it couples with APAF1 to form an apoptosome. The apoptosome activates caspase 9, which triggers a caspase activation cascade resulting in the activation of caspase 3 and apoptosis (**Figure 3**).

Hypothermia exerts its neuroprotective effects at several points along the intrinsic apoptotic pathway (**Figure 3**). TH has been found to inhibit BAX overexpression 4 h after 30 min of partial ischemia while having no effect on Bcl-2 expression [92]. TH has also been shown to diminish cytochrome C release without modifying BAX or Bcl-2 expression. This study did not observe caspase activity, which implied that TH endowed neuroprotection functions independently of caspases [93]. Interestingly, the same group found that hypothermia increased Bcl-2 expression in a global ischemia model [94], which underlines the importance of designing studies specific to local cooling in focal ischemia models. Additionally, mild hypothermia has been found to decrease cytochrome C translocation 5 h after reperfusion while leaving levels of caspase 9 and caspase 3 unchanged [74]. The conflicting nature of these findings leaves the point at which TH exerts its protective effects in question, but emphasizes the intricacy of TH-mediated neuroprotection.

Some of the anti-apoptotic effects of TH are mediated through the anti-apoptotic factor Akt/protein kinase B (**Figure 3**). Hypothermia attenuates decreases in Akt dephosphorylation (inactivation) after hypoxia [90]. In response to growth factors including BDNF (brainderived neurotropic factor), membrane receptor tyrosine kinases activate PI3 kinase, which activates Akt via phosphorylation (p-Akt), thereby allowing it to phosphorylate (inhibit) numerous proapoptotic factors, including BAD, caspase 9, and forkhead transcription factor (FKHR) [90, 95]. Under normal physiological conditions, these proteins are phosphorylated by Akt, and their dephosphorylation can have severe repercussions. Dephosphorylation of BAD allows it to migrate into the mitochondria where it triggers the release of cytochrome C [91]. Dephosphorylated FKHR functions as a transcription factor to encourage overexpression of FASL and BIM [90]. Activation of caspase 9 activates a caspase cascade that results in apoptosis.

In normothermia, poststroke p-Akt levels fluctuate constantly; Zhao et al. found that, in normothermic rats, p-Akt levels decreased 30 min after stroke, increased at 1.5 and 5h, decreased at 9 and 24h, and increased again at 48h. Moderate hypothermia was found to stabilize these fluctuations at every time point except 24h, which translated to reduced infarct volumes and improved functional recovery up to 2 months after hypoxia. Interestingly, the reduction in infarct volumes was considerably less pronounced when TH was administered in conjunction with the PI3K inhibitor LY294002, although infarcts were still substantially smaller than in control animals [90]. It is very likely that this pathway provides a significant portion of TH-mediated neuroprotection. In line with this premise, mild hypothermia has also been found to inhibit the expression of caspase-3 and Fas after resolution of focal ischemia, which also translated to significantly decreased infarct volumes [96]. Hypothermia has also been shown to augment BDNF expression during cerebral ischemia [97], as well as attenuate the decrease in the Akt activity after stroke [90], so it is also possible that the effects of hypothermia on Akt activity are mediated at the level of BDNF.

While direct enhancement of the Akt pathway likely constitutes a portion of TH-mediated neuroprotection, cerebral cooling intervenes at other steps in the pathway as well. The PI3K/Akt pathway is inhibited by phosphatase and tensin homologue (PTEN), which de-phosphorylates upstream activators of Akt. PTEN is inhibited by phosphorylation (p-PTEN), and p-PTEN levels seem to play a crucial role in TH-mediated neuroprotection. Hypothermia has been found to stabilize p-PTEN levels more effectively than levels of p-Akt and other PI3K/Akt pathway participants (p-PDK1, p-GSK3β p-FKHR) [37]. A recent investigation from Lee et al. found that TH administered 15 min before reperfusion led to massive decreases in infarct volume while TH administered 15 min after reperfusion only had modest infarct reductions. Interestingly, while early and late TH had nearly identical effects on levels of p-Akt and other proteins, only early TH maintained high levels of p-PTEN [98]. Additionally, independent of hypothermia, PTEN inhibition was recently shown to confer a 75% reduction in infarct volume in rat models [95]. PTEN clearly plays a critical part in the story of neuroprotection, and should not be neglected in future investigations on the topic.

#### **4.4. Long-term neuroprotection**

FASL in cooled rat brains [91], so is possible that the reduction in levels of these downstream effectors is simply the byproduct of inhibiting the FAS-FASL cleavage. While there are little available data to this end, the fact remains that, by one mechanism or another, hypothermia significantly reduces the production of a number of extrinsic apoptotic pathway intermedi-

The intrinsic apoptotic pathway is triggered by intracellular cell stress signals including hypoxia, DNA damage, and cellular detachment from the extracellular matrix. These signals initiate apoptosis by disrupting the balance between proapoptotic Bcl-2 family members (BID, BAX, BAD, etc.) and anti-apoptotic Bcl-2 members (Bcl-2, Bcl-x, etc.) by a variety of mechanisms. Bcl-2 and Bcl-xL have both been found to be upregulated in neurons surviving hypoxia, while proapoptotic Bcl-2 members are highly expressed in neurons that will eventually die from hypoxic damage [37]. The imbalance between pro- and anti-apoptotic Bcl-2 members leads to the liberation of cytochrome C from the mitochondrial intermembrane space into the cytosol where it couples with APAF1 to form an apoptosome. The apoptosome activates caspase 9, which triggers a caspase activation cascade resulting in the activation of

Hypothermia exerts its neuroprotective effects at several points along the intrinsic apoptotic pathway (**Figure 3**). TH has been found to inhibit BAX overexpression 4 h after 30 min of partial ischemia while having no effect on Bcl-2 expression [92]. TH has also been shown to diminish cytochrome C release without modifying BAX or Bcl-2 expression. This study did not observe caspase activity, which implied that TH endowed neuroprotection functions independently of caspases [93]. Interestingly, the same group found that hypothermia increased Bcl-2 expression in a global ischemia model [94], which underlines the importance of designing studies specific to local cooling in focal ischemia models. Additionally, mild hypothermia has been found to decrease cytochrome C translocation 5 h after reperfusion while leaving levels of caspase 9 and caspase 3 unchanged [74]. The conflicting nature of these findings leaves the point at which TH exerts its protective effects in question, but emphasizes

Some of the anti-apoptotic effects of TH are mediated through the anti-apoptotic factor Akt/protein kinase B (**Figure 3**). Hypothermia attenuates decreases in Akt dephosphorylation (inactivation) after hypoxia [90]. In response to growth factors including BDNF (brainderived neurotropic factor), membrane receptor tyrosine kinases activate PI3 kinase, which activates Akt via phosphorylation (p-Akt), thereby allowing it to phosphorylate (inhibit) numerous proapoptotic factors, including BAD, caspase 9, and forkhead transcription factor (FKHR) [90, 95]. Under normal physiological conditions, these proteins are phosphorylated by Akt, and their dephosphorylation can have severe repercussions. Dephosphorylation of BAD allows it to migrate into the mitochondria where it triggers the release of cytochrome C [91]. Dephosphorylated FKHR functions as a transcription factor to encourage overexpression of FASL and BIM [90]. Activation of caspase 9 activates a caspase cascade that results in

In normothermia, poststroke p-Akt levels fluctuate constantly; Zhao et al. found that, in normothermic rats, p-Akt levels decreased 30 min after stroke, increased at 1.5 and 5h, decreased

ates, which translates to the preservation of penumbral tissue.

caspase 3 and apoptosis (**Figure 3**).

192 Hypoxia and Human Diseases

the intricacy of TH-mediated neuroprotection.

apoptosis.

There is compelling clinical evidence of neuroprotection with prolonged moderate cerebral hypothermia initiated within a few hours after hypoxia-ischemia and continued through the resolution of ischemia in term infants and adults [99–101]. The mechanisms underlying the neuroprotection are currently under investigation. Volser et al. showed that during the postischemic phase, the brain naturally activates restorative mechanisms to counteract the effects of the ischemic insult even without the induction of hypothermia [102]. This study, among others put forth the idea of long-term neuroprotection following an ischemic event in the brain. A study by Feng et al. went a step further and found that acute brain insult led to stimulation of neural stem cell proliferation, particularly in the subventricular and hippocampal subgranular zone, corroborating long-term neuroprotection [103]. However, evident from the lasting symptoms of acute ischemia, the brain is unable to completely regenerate and recover from the injury on its own. Thus, there is a dire need for the development of effective regenerative techniques and therapies to maximize patient recovery. This is where LEVI and hypothermia can be used to further the recovery of the brain.

Over the past decade, researchers have proposed the following mechanisms of long-term neuroprotection: neurogenesis, angiogenesis, gliogenesis, preservation of the integrity of neural networks, and inhibition of apoptosis [55]. These mechanisms will be discussed in detail below.

#### *4.4.1. Neurogenesis*

Contrary to prior belief, neurogenesis is a common event observed in the brain and while it is primarily limited to two neurogenic areas of the brain, the dentate gyrus of the hippocampus and the subventricular zone of the lateral ventricles, this process plays an important role in maintaining normal brain function [104, 105]. Two potential mechanisms can be attributed to neurogenesis: enhanced differentiation of neuroprogenitor cells into neurons and preferential differentiation of neuroprogenitor cells toward neurogenesis over gliogenesis.

The formation of new neural cells from neural progenitor cells has been identified as a major contributor to new populations of neurons, and TH seems to encourage this formation. An *in vivo* study by Silasi et al. found that when forebrain ischemia was induced in adult rodents, mild hypothermia following the ischemic event led to significantly increased neurogenesis in the dentate gyrus when compared to control groups with no hypothermia induction following an ischemic event [106]. Moreover, a very recent study in a neonatal hypoxic-ischemic injury mouse model showed that hypothermia provided partial protection for neural stem and progenitor cells (NSPCs) in the dentate gyrus subgranular zone, which may facilitate the recovery of function after injury and does not impair the proliferation of NSPCs during recovery [107]. This TH-mediated neurogenesis is thought to confer a more robust, long-term conservation of brain function than would be seen in normoxic stroke patients.

Preferential differentiation of neuroprogenitor cells into neurons also plays a major role in neuroprotection. Interestingly, an *in vivo* study found that cooling of rat brains to 33°C under hypoxic conditions led to an inhibition of hypoxia-induced apoptosis of proliferating neural stem cells and an increase in preferential maturation of neural progenitor cells into neural cells in the striatum [108]. Moreover, an *in vitro* study by Saito et al. found that moderate hypothermia to 32°C prevented apoptosis, preserved the naivety of neural stem cells, and led to lower expression of GFAP in neural stem cell culture, indicating less glial differentiation [109].

On the other hand, a study from early 2016 found that in aged rats, hypothermia induced by H2 S gas for 24 h after resolution of an MCAO only provided temporary therapeutic benefit and did not correlate with enhanced neurogenesis in the subventricular zone or infarcted area [110]. However, the duration of hypothermia induction in this study was shorter than the duration of hypothermia used in most clinical trials (24–48 h) and thus led to suboptimal hypothermia which is reflected in the temporary therapeutic effects [110]. Additionally, the use of hydrogen sulfide to induce hypothermia may not be representative of the conventional hypothermia-inducing agents used in other animal studies. H2 S is a weak and reversible inhibitor of oxidative phosphorylation, thus causing a suspended animation state with hypothermia [111]. It is quite possible that the mechanism of induction of hypothermia by H2 S may have interfered with various long-term protective mechanisms observed in other studies and in clinic using conventional hypothermia techniques.

#### *4.4.2. Angiogenesis*

Over the past decade, researchers have proposed the following mechanisms of long-term neuroprotection: neurogenesis, angiogenesis, gliogenesis, preservation of the integrity of neural networks, and inhibition of apoptosis [55]. These mechanisms will be discussed in detail

Contrary to prior belief, neurogenesis is a common event observed in the brain and while it is primarily limited to two neurogenic areas of the brain, the dentate gyrus of the hippocampus and the subventricular zone of the lateral ventricles, this process plays an important role in maintaining normal brain function [104, 105]. Two potential mechanisms can be attributed to neurogenesis: enhanced differentiation of neuroprogenitor cells into neurons and preferential

The formation of new neural cells from neural progenitor cells has been identified as a major contributor to new populations of neurons, and TH seems to encourage this formation. An *in vivo* study by Silasi et al. found that when forebrain ischemia was induced in adult rodents, mild hypothermia following the ischemic event led to significantly increased neurogenesis in the dentate gyrus when compared to control groups with no hypothermia induction following an ischemic event [106]. Moreover, a very recent study in a neonatal hypoxic-ischemic injury mouse model showed that hypothermia provided partial protection for neural stem and progenitor cells (NSPCs) in the dentate gyrus subgranular zone, which may facilitate the recovery of function after injury and does not impair the proliferation of NSPCs during recovery [107]. This TH-mediated neurogenesis is thought to confer a more robust, long-term

Preferential differentiation of neuroprogenitor cells into neurons also plays a major role in neuroprotection. Interestingly, an *in vivo* study found that cooling of rat brains to 33°C under hypoxic conditions led to an inhibition of hypoxia-induced apoptosis of proliferating neural stem cells and an increase in preferential maturation of neural progenitor cells into neural cells in the striatum [108]. Moreover, an *in vitro* study by Saito et al. found that moderate hypothermia to 32°C prevented apoptosis, preserved the naivety of neural stem cells, and led to lower expression of GFAP in neural stem cell culture, indicating less glial differentiation [109].

On the other hand, a study from early 2016 found that in aged rats, hypothermia induced by

ible inhibitor of oxidative phosphorylation, thus causing a suspended animation state with hypothermia [111]. It is quite possible that the mechanism of induction of hypothermia by H2

may have interfered with various long-term protective mechanisms observed in other studies

S gas for 24 h after resolution of an MCAO only provided temporary therapeutic benefit and did not correlate with enhanced neurogenesis in the subventricular zone or infarcted area [110]. However, the duration of hypothermia induction in this study was shorter than the duration of hypothermia used in most clinical trials (24–48 h) and thus led to suboptimal hypothermia which is reflected in the temporary therapeutic effects [110]. Additionally, the use of hydrogen sulfide to induce hypothermia may not be representative of the conven-

S is a weak and revers-

S

differentiation of neuroprogenitor cells toward neurogenesis over gliogenesis.

conservation of brain function than would be seen in normoxic stroke patients.

tional hypothermia-inducing agents used in other animal studies. H2

and in clinic using conventional hypothermia techniques.

below.

H2

*4.4.1. Neurogenesis*

194 Hypoxia and Human Diseases

Angiogenesis is a normal yet important biological process that is highly regulated and leads to the formation of new blood vessels during development, wound repair, and reproduction [111]. A study on rats by Xie et al. found that mild hypothermia enhanced angiogenesis in focal cerebral ischemia by increasing microvessel diameter, number of vascular branch points, and overall vessel surface area [112]. This was found to be a brain-derived neurotrophic factor (BDNF)-dependent process. Moreover, another study using a rat MCAO model showed that the injection of BDNF fused with a collagen-binding domain (CBD-BDNF) into the lateral ventricle specifically bound to collagen of the ventricular ependyma and consequently led to neural regeneration, angiogenesis, and reduced cell death [113]. This study further confirms the pro-angiogenic activity of BDNF in ischemic conditions. Vascular endothelial growth factor (VEGF) upregulation has also been found to correlate with acute cerebral ischemia [114, 115]. A very recent prospective cohort study observed increased brain perfusion over the first month in term-asphyxiated newborn babies treated with hypothermia during the first few days of life. This increase in brain perfusion came as a result of increased angiogenesis, which was found to be associated with VEGF expression in the injured brain of asphyxiated newborns treated with hypothermia [116]. VEGF has been consistently shown to increase angiogenesis, which translates to increased functional recovery in the months following an ischemic stroke [117–119].

#### *4.4.3. Gliogenesis*

While gliogenesis refers to the development of microglia, oligodendrocytes, and astrocytes in the brain, intriguingly, oligodendrocytes have been found to have a similar susceptibility to neurons for cell death. Early studies found that combined deprivation of oxygen and glucose led to selective death of mature oligodendrocytes over other glial cells *in vitro* [120–122]. *In vivo* studies have shown that cerebral white matter, specifically oligodendrocytes and astrocytes, are highly vulnerable to focal ischemia [123]. However, *in vitro* studies have shown that hypothermia increases the number of oligodendrocyte precursors in primary neural and glial cultures from mouse brains and maintains a cell population of oligodendrocyte progenitors in a less well-differentiated state [124]. Recent studies have found that susceptible oligodendrocyte progenitors and mature oligodendrocytes exposed to hypoxia could be protected by deep hypothermia [125]. Another study demonstrated that hypothermia promoted the differentiation and maturation of oligodendrocyte precursor cells (OPCs), and indicated that OPC death was significantly suppressed by hypothermia *in vitro*, alluding to the fact that hypothermia is protective of oligodendrogliogenesis [126]. More recent studies in fetal sheep have shown that cerebral ischemia is associated with significant loss in total numbers of oligodendrocytes, decreased myelin basic protein expression, and increased microglial activation [127, 128]. However, another study in fetal sheep countered these results by showing that delayed cerebral hypothermia partially protects white matter after global cerebral ischemia by stimulating oligodendrocyte proliferation, reducing microglial induction, and restoring the amount and pattern of expression of myelin basic protein, once again confirming the neuroprotective role of hypothermia toward oligodendrogenesis [129, 130]. Moreover, researchers have found that hypothermia attenuates demyelination, trauma-induced oligodendrocyte cell death, and overall circuit dysfunction [131, 132]. While a study in preterm fetal sheep found that TH was correlated with an overall reduction in the hypoxia-induced death of immature oligodendrocytes, hypothermia did not prevent the hypoxia-induced inhibition of oligodendrocyte proliferation in the periventricular white matter zone [133, 134]. Most importantly, a recent study in rats found that hypothermia reduced the extent of hypoxia-ischemia damage in axons and increased oligodendrocyte lineage proliferation, which was reflected in the increase in myelination of axons and decreases apoptosis and pre-oligodendrocyte lineage accumulation [134]. While an ischemic environment has been shown to be detrimental to oligodendrogenesis and oligodendrocyte survival, hypothermia has been shown to rescue these processes *in vivo* and *in vitro*, as discussed above.

Since astrocytes are the largest population of cells present in the ischemic core during the subacute to chronic period of stroke, astrogliogenesis is often considered to be therapeutic following insult to the brain [131, 135, 136]. However, we still lack much information and need more investigation in this area. Most of the current literature suggests astrogliogenesis as detrimental to the brain rather than neuroprotective. As we know, activated astrocytes form the glial scar in the brain following insult or injury [112, 137]. This brings about doubt on whether astrogliogenesis is therapeutic and may actually impede the postischemic healing process by forming a glial scar that could hinder neurite growth and synaptogenesis, and lead to leakage of proapoptotic factors from astrocyte gap junctions within the glial scar [138]. Moreover, a very recent study found that in mice, hypoxia diminished the protective function of astrocytes and activated them to initiate astrogliosis in the ischemic region [139]. In fact, many studies have shown that decreased astrogliosis correlates with decreased infarct size [140]. Intriguingly, a study conducted by Xiong et al. showed that postischemic hypothermia in rats for 24 h rescued hippocampal neurons by decreasing astrocyte activation and inflammatory cytokine release [141]. Such studies truly call into question the role of astrogliogenesis in neuroprotection. More investigation needs to be done in this area to better understand the role of astrogliogenesis in neuroprotection under hypothermic conditions.

#### *4.4.4. Preservation of the integrity of neural networks*

Neural networks are functional units representing the high complexity and processivity of the brain and thus repair and preservation of this circuitry is the key for recovery from brain injury. Some of the key processes involved in neural network maintenance are axonal and neurite growth, synaptogenesis, and maintenance of neuronal architecture. Studies have found that hypothermia of the brain by 17°C enhanced neurite and axonal outgrowth in brain slices [142, 143]. A recent study on spinal cord injury rat models found that regional hypothermia promoted neurite, axonal, and nerve fiber growth to the point that hind limb function was recovered in these rats, which emphasizes the plasticity and extent of recovery via hypothermia that the central nervous system is capable of [144]. However, deep hypothermia (20°C) followed by subsequent rewarming did not change the stability of dendritic spines or the presynaptic boutons in mouse somatosensory cortex [145]. Moreover, a gene profiling study on rat model of traumatic brain injury found that mild hypothermia had significant effects on gene expression for synapse organization and biogenesis; an analysis of the hippocampal gene expression profiles of these rats found that 133 genes showed statistically significant changes in expression compared to injured rat in normoxic conditions. Of the 133, 57 genes were upregulated and were responsible for synaptic organization and biogenesis [146]. An *in vitro* study showed that hypothermia to 33°C following *in* vitro ischemia decreased the neuronal actin polymerization that reduced spine calcium kinetics, disrupted detrimental cell signaling, and protected the neurons against damage [147]. While hypoxic conditions caused changes in F-actin architecture of dendritic spines, hypothermia decreased the actin modifications in dendritic spines preventing the neuronal death [148]. All of these studies support the notion of spine and synaptogenesis preservation by hypothermia treatment.

In a functional study on ischemic gerbils treated with moderate postischemic hypothermia, the untreated (normothermic) groups experienced a 95% reduction in CA1 cells, while cell counts in the TH group were equivalent to that of sham animals. Additionally, postischemic hypothermia preserved the electrophysiological properties of CA1 neurons, which reflects the functional preservation of neural networks [149]. Moreover, mice subjected to ischemia followed by hypothermia treatment showed neuroprotection against ischemia-induced long-term potentiation (LTP) impairment as well as synaptic plasticity [150]. While there are encouraging studies on mechanisms of neural network preservation by hypothermia treatment, further research is needed to better understand how neuronal networks are preserved in the ischemic and penumbra regions in response to hypothermia.

## **5. Future research directions**

overall circuit dysfunction [131, 132]. While a study in preterm fetal sheep found that TH was correlated with an overall reduction in the hypoxia-induced death of immature oligodendrocytes, hypothermia did not prevent the hypoxia-induced inhibition of oligodendrocyte proliferation in the periventricular white matter zone [133, 134]. Most importantly, a recent study in rats found that hypothermia reduced the extent of hypoxia-ischemia damage in axons and increased oligodendrocyte lineage proliferation, which was reflected in the increase in myelination of axons and decreases apoptosis and pre-oligodendrocyte lineage accumulation [134]. While an ischemic environment has been shown to be detrimental to oligodendrogenesis and oligodendrocyte survival, hypothermia has been shown to rescue these processes *in* 

Since astrocytes are the largest population of cells present in the ischemic core during the subacute to chronic period of stroke, astrogliogenesis is often considered to be therapeutic following insult to the brain [131, 135, 136]. However, we still lack much information and need more investigation in this area. Most of the current literature suggests astrogliogenesis as detrimental to the brain rather than neuroprotective. As we know, activated astrocytes form the glial scar in the brain following insult or injury [112, 137]. This brings about doubt on whether astrogliogenesis is therapeutic and may actually impede the postischemic healing process by forming a glial scar that could hinder neurite growth and synaptogenesis, and lead to leakage of proapoptotic factors from astrocyte gap junctions within the glial scar [138]. Moreover, a very recent study found that in mice, hypoxia diminished the protective function of astrocytes and activated them to initiate astrogliosis in the ischemic region [139]. In fact, many studies have shown that decreased astrogliosis correlates with decreased infarct size [140]. Intriguingly, a study conducted by Xiong et al. showed that postischemic hypothermia in rats for 24 h rescued hippocampal neurons by decreasing astrocyte activation and inflammatory cytokine release [141]. Such studies truly call into question the role of astrogliogenesis in neuroprotection. More investigation needs to be done in this area to better understand the role of astrogliogenesis in neuroprotection under hypothermic

Neural networks are functional units representing the high complexity and processivity of the brain and thus repair and preservation of this circuitry is the key for recovery from brain injury. Some of the key processes involved in neural network maintenance are axonal and neurite growth, synaptogenesis, and maintenance of neuronal architecture. Studies have found that hypothermia of the brain by 17°C enhanced neurite and axonal outgrowth in brain slices [142, 143]. A recent study on spinal cord injury rat models found that regional hypothermia promoted neurite, axonal, and nerve fiber growth to the point that hind limb function was recovered in these rats, which emphasizes the plasticity and extent of recovery via hypothermia that the central nervous system is capable of [144]. However, deep hypothermia (20°C) followed by subsequent rewarming did not change the stability of dendritic spines or the presynaptic boutons in mouse somatosensory cortex [145]. Moreover, a gene profiling study on rat model of traumatic brain injury found that mild hypothermia had significant effects on gene expression for synapse organization and biogenesis; an analysis

*vivo* and *in vitro*, as discussed above.

196 Hypoxia and Human Diseases

*4.4.4. Preservation of the integrity of neural networks*

conditions.

Between 1935 and 2010, cancer, heart disease, and stroke have consistently been in the top five causes of death in the United States [151]. While all three are complex, multifaceted diseases, stroke differs from cancer and heart disease in one critical way; a highly effective, easily administered, cost-effective therapy has already been devised. The main factor hindering significant progress on stroke therapy is not a lack of ideas, but rather a lack of research moving hypothermia toward clinical acceptance. Since TH is still predominately discussed in the context of cardiac arrest, the majority of studies on TH feature a global ischemia (cardiac arrest) model, which cannot always be extrapolated to studies on focal cerebral ischemia. Several papers in the present review alone have arrived at a finding using a global ischemia model that is directly opposed by results from a model of focal ischemia or vice versa [37, 93, 94]. In focal ischemia models, there is significant heterogeneity in experimental methods. Studies on TH in focal cerebral ischemia frequently differ in animal model, animal age, duration of ischemia, duration of hypothermia, depth of hypothermia, method of hypothermia induction, and rate of cooling, all of which have consistently been shown to play critical roles in the efficacy of TH treatments. It is also important to note that the vast majority of investigations on neuroprotective efficacy have used transient occlusion models, which produce much more uniform and encouraging results than those using a permanent occlusion model [37]. This is problematic, considering that an estimated 50% of ischemic stroke patients display vessel occlusion 3–4 days after symptom onset, which is considered a relatively permanent occlusion [152]. This heterogeneity is likely a large source of conflicting findings, and surely prevents investigators from coming to an agreement on TH mechanisms. Another issue with present research is the goal of hypotheses. While there have been innumerable studies on the mechanisms of hypothermia-mediated neuroprotection, these reports are usually correlative rather than causative, which makes it difficult to derive any concrete, widely applicable mechanisms from the literature. This overall lack of research has hindered publicization of the procedure; given that LEVI was only developed in 2002, many groups are simply unaware that such a procedure has been proposed. For instance, a highly cited 2012 review on the topic discussed numerous problems with global cooling, but failed to mention LEVI to any extent despite the fact that the procedure remedies every problem highlighted in the paper [58]. However, as the body of research on LEVI grows, so too will its clinical acceptance.

Overall, the picture of therapeutic hypothermia-mediated neuroprotection is favorable and encouraging. TH consistently decreases infarct volumes and facilitates short- and long-term preservation of function to an unprecedented degree. Although there is little widespread consensus as to how this is accomplished, a review of the literature is scarce with detrimental effects of TH. While many questions remain to be answered before TH can be consistently implemented in humans, such a promising therapy to such a ubiquitously disastrous disease warrants a significant time investment going forward.

## **Acknowledgements**

This work was partially supported by the American Heart Association Grant-in-Aid (14GRNT20460246), Merit Review Award (I01RX-001964-01) from the US Department of Veterans Affairs Rehabilitation R&D Service, the National Natural Science Foundation of China (81501141), and the Beijing New-Star Plan of Science and Technology (xx2016061).

## **Author details**

Mitchell Huber1#, Hong Lian Duan2#, Ankush Chandra<sup>1</sup> , Fengwu Li2 , Longfei Wu<sup>3</sup> , Longfei Guan2 , Xiaokun Geng1,2\* and Yuchuan Ding1,2

\*Address all correspondence to: xgeng@ccmu.edu.cn

1 Department of Neurological Surgery, Wayne State University School of Medicine, Detroit, MI, USA

2 Institute of Neuroscience, Beijing Luhe Hospital, Capital Medical University, Beijing, China

3 China-America Institute of Neuroscience, Xuanwu Hospital, Capital Medical University, Beijing, China
