**2. Consequences of hypoxia**

sion during embryonic development, by tumour growth, or by vasculature dysfunction due

Hypoxia is defined by a reduced oxygen tension relative to those normally extant within a particular tissue. It has multiple impacts on the vascular system and cell function [3]. The

to the pathophysiology of tumour progression and cell apoptosis [4] and is a feature of conditions that include cancer, ischemic heart disease, peripheral artery disease, wound healing and neovascular retinopathy. Hypoxia promotes vessel growth by stimulating an upregulation of multiple proangiogenic pathways that mediate key aspects of endothelial, stromal and vascular support cell biology. The role of hypoxia in human disease is now becoming increasingly clear [5] including the association between hypoxia and endothelial dysfunction

Hypoxia can occur in several ways: (1) hypoxic hypoxia is caused by an insufficient oxygen concentration in the air in the lungs, which may occur during sleep apnea, when the diffusion of oxygen to the blood is reduced, or at high altitude; (2) hypoxemic hypoxia occurs when the blood has reduced transport capacity as seen in carbon monoxide poisoning when haemoglobin cannot carry oxygen at its normal concentrations; (3) stagnant hypoxia results when the cardiac output does not match the demands of the body and the flow is not sufficient to deliver enough oxygenated blood to the tissue and (4) histotoxic hypoxia occurs when cells cannot utilize the available oxygen, for example following cyanide poisoning when oxygen

Chronic tissue hypoxia (an oxygen tension of 2–3% for a prolonged period of time) may cause uncontrolled proliferation of cells. When physiological oxygen concentrations are restored, the increased blood flow supplies excessive oxygen; this may then lead to increased freeradical generation, tissue damage and concomitant activation of stress-response genes; a condition known as 'reoxygenation injury'. In these circumstances, normal cells/tissues may not survive; but tumour cells are still able to proliferate despite the hypoxic milieu, as they have

Hypoxia plays important roles in normal human physiology and development. For example, it is integral to normal embryonic development. Whatever the cause, or the severity of hypoxia, it leads to an induction of adaptive responses within the endothelial and vascular smooth muscle cells through the activation of genes that participate in angiogenesis, cell pro-

In healthy vascular tissue, vascular smooth muscle cells (SMCs) and endothelial cells (ECs) proliferate at very low levels. However, SMCs and ECs can be stimulated to re-enter the cell cycle in response to several physiological and pathological stimuli. Hypoxia is considered an important stimulus of SMC and EC proliferation and is found in atherosclerotic lesions and

The proliferation of ECs is pivotal to the formation of new micro-vessels and is important during organ development in embryogenesis and tumour growth, and also contributes to

cannot be used to produce ATP as the mitochondrial electron transport is inhibited.

developed genetic and adaptive changes leading to resistance to hypoxia [6].

liferation/survival and in glucose and iron metabolism [7].

rapidly growing tumours [4].

adaptive physiological responses in the cells. A lower oxygen tension (0–1% O<sup>2</sup>

that affects several cellular processes and signal transduction.

114 Physiologic and Pathologic Angiogenesis - Signaling Mechanisms and Targeted Therapy

) are usually reversible and are usually accompanied by

) contributes

to vessel occlusion or rupture [2].

effects of moderate hypoxia (3–5% O<sup>2</sup>

Most cells are able to survive under hypoxic conditions through the transcriptional activation of a series of genes. The oxygen-sensitive transcriptional activator, hypoxia-inducible factor-1 (HIF-1) is the key transcriptional mediator of the hypoxic response and master regulator of O2 homeostasis. It orchestrates the profound changes in cellular transcription that accompanies hypoxia by controlling the expression of numerous angiogenic, metabolic and cell cycle genes. Accordingly, the HIF pathway is currently viewed as a master regulator of angiogenesis [5].

HIF-1 is normally only found in hypoxic cells. It is a heterodimer that is composed of an O2 -regulated HIF-1α subunit and a constitutively expressed HIF-1β subunit [10]. In the α-subunit, there is an oxygen-dependent degradation (ODD) domain, where the 4-hydroxyproline formation is catalysed by proline-hydroxylase-2 (PHD-2). This leads to its ubiquitination by the von Hippel-Lindau E3 ubiquitin ligase (VHL) and subsequent proteasomal degradation under normoxic cellular conditions. This prevents the formation of a functional HIF dimer [11]. Since PHDs require oxygen for their catalytic activity, and function as cellular oxygen sensors, HIF degradation only occurs under normoxic conditions. Factor inhibiting HIF-1 (FIH) protein, which hydroxylates HIF-1, also contributes to HIF-1 inactivation in normoxic conditions, and thereby prevents the interaction of this subunit with the two transcriptional co-activators of HIF-1: p300 and CREB-binding protein (CBP) which are essential for HIF-1 transcription. Expression and stabilization of the HIF-1 complex is also regulated through feedback inhibition, as PHD-2 itself is activated by HIF-1 [12].

Under hypoxic conditions, HIF-1 protein is stable and active as the hydroxylase, VHL proteins, and FIH are all inhibited by a lack of oxygen. HIF-1 is then able to interact with its coactivators and can dimerize with its constitutively expressed β-subunit [12]. Once stabilized, the HIF-1 protein can bind to the regulatory regions of its target genes, inducing their expression; these target genes include VEGF (vascular endothelial growth factor) [13], erythropoietin [14] and nitric oxide synthase (NOS) [15, 16] and other proangiogenic factors such as PlGF (placental growth factor), or angiopoietins [12] (**Figure 1**).

It has been proposed that the induction of a pseudo-hypoxic response by inhibiting HIF prolyl 4-hydroxylases may provide a novel therapeutic target in the treatment of hypoxia-associated diseases [17].

Several small molecules, such as dimethyloxalyl glycine [18], Roxadustat (FG-4592) [19] and ZYAN1 [20], have been developed to inhibit prolyl hydroxylase domain-containing (PHD) enzymes, and cause HIF activation [21]. These agents have been applied to the treatment of renal anaemia in which there is a deficiency of erythropoietin [22, 23]. The administration of

**Figure 1.** Regulation of the hypoxia-inducible transcription factor (HIF-1α) pathway. Under normal oxygen tensions (normoxia), prolyl hydroxylase (PHD) enzymes, von Hippel-Lindau protein (pVHL), the ubiquitin ligase complex (Ub) and factor inhibiting HIF-1 (FIH) are active leading to HIF-1α proteasomal degradation. Under hypoxic conditions, PHD, pVHL and Ub are not active leading to its cytoplasmic accumulation of HIF-1α. The HIF-1α gene is transcribed in the nucleus with the help of specificity protein (Sp) 1, P300, and HIF-1β leading to transcription of HIF target genes such as EPO, NOS and VEGF.

these compounds is associated with an improved iron profile and an increase of endogenous erythropoietin production to near the physiological range. The clinical trials currently underway aim to address whether PHD enzyme inhibitors will improve clinical end-points, including cardiovascular events [24]. PHD inhibitors have been reported to reduce blood pressure [22] and plasma cholesterol concentrations [19]. Hence, there is a good reason to believe that some PHD inhibitors will reduce cardiovascular endpoints in patients with renal disease. Whether they will benefit a broader category of patients with high risk of cardiovascular disease is difficult to predict.

Hydroxylase activity can be also rescued by mutating specific regions, or by adding cobalt ions to the cell, the latter of which presumably compete for iron-binding sites. Some hydroxylases in the prolyl family can be selectively inhibited by Adriamycin *in vitro*. Cobalt (II) and nickel (II) ions increase HIF-1 activity in cells, presumably because these ions displace iron from the active sites of 2-oxo-glutarate (2OG) hydroxylases [12].

It has been shown that HIF-1α can be regulated by non-hypoxic stimuli such as lipopolysaccharides (LPS), thrombin and angiotensin II (Ang II) [25]. Hormones such as angiotensin II and platelet-derived growth factor stimulate the HIF pathway by increasing HIF-1α protein levels through production of reactive oxygen species (ROS) within the cell. Although the exact mechanism for this is unclear, it appears to be entirely distinct from the hypoxia pathways.

Thrombin and other growth factors appear to increase angiogenesis through HIF-1α protein agonist mechanisms. Insulin similarly activates HIF-1α through the action of multiple protein kinases necessary for expression and function. p53 is responsible for promoting ubiquitination of HIF-1α, and may be an another possible target for enhancing HIF-1. Homozygous deletion of the p53 gene has been found to cause HIF-1 activation [26]. Gene therapy may eventually be used to increase HIF-1 levels and relieve complications of ischemia. For example, delivery of a stabilized, recombinant form of HIF-1α through adeno-associated virus (AAV) in order to overexpress HIF-1 has been shown to result in significantly increased capillary density in skeletal muscle [27]. While gene therapy approaches aimed at the process and effects of angiogenesis continue to be developed and studied, higher levels of success in preclinical trials currently are being sought before clinical applications are pursued. Amongst the remaining obstacles in using gene therapy for this purpose is the effective mode of delivery [12]. Inhibition of PHD2 using siRNA has been shown to decrease cardiac infarction size in murine models [28, 29].

In addition to HIF-1α, there are two other members of HIF superfamily that have been described: HIF-2 and HIF-3 [30]. Both are important regulators of the hypoxia response with similar actions as HIF-1 [31] and lead to the transcriptional activation of target genes in hypoxia [32]. However, Eubank et al demonstrated opposing roles for the HIFs in tumour angiogenesis, with HIF-1 exhibiting proangiogenic properties that act through its effects on VEGF secretion, whereas HIF-2 exhibits anti-angiogenic activity by inducing the production of the endogenous angiogenesis inhibitor, sVEGFR-1 [33]. HIF-3α has complementary functions, rather than redundant to HIF-1α induction in protection against hypoxic damage in alveolar epithelial cells in protection against hypoxic damage in alveolar epithelial cells [34].

Although the oxygen-sensing mechanism involving oxygen-dependent hydroxylation of the HIF-α subunits is probably a universal mechanism in cells, and has been highly conserved during evolution, additional regulatory steps appear to determine which of the alternative subunits is induced [34]. One of the best studied hypoxic responses that will be discussed in this chapter is the induction of angiogenic factors and growth factors, which lead to the formation and growth of new blood vessels.
