**3. Hypoxia and angiogenesis**

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

**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

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

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

from the active sites of 2-oxo-glutarate (2OG) hydroxylases [12].

116 Physiologic and Pathologic Angiogenesis - Signaling Mechanisms and Targeted Therapy

disease is difficult to predict.

such as EPO, NOS and VEGF.

pathways.

Blood vessels formation occurs through two basic mechanisms: (1) vasculogenesis represents de novo formation of blood vessels, and is derived from endothelial progenitors and (2) angiogenesis and arteriogenesis (formation of blood vessels from pre-existing blood vessels).

Angiogenesis is a tightly regulated multi-step process that begins when cells within a tissue respond to hypoxia. When tissues grow beyond the physiological oxygen diffusion limit, the relative hypoxia triggers expansion of vascular beds by inducing angiogenic factors in the cells of the vascular beds, which are physiologically oxygenated by simple diffusion of oxygen. Angiogenesis may be a physiological process, as in the case in embryonic development, wound healing or vessel penetration into avascular regions. It may also be pathological, for example when it occurs during the formation of solid tumours, eye disease, chronic inflammatory disorders such as rheumatoid arthritis, psoriasis and periodontitis and atherosclerosis.

The regulation of angiogenesis (whether in physiological or pathological cases) by hypoxia is an important component of homeostatic mechanisms that link vascular oxygen supply to metabolic demand. An understanding of the processes involved in angiogenic, the role of the interacting proteins involved, and how all this is regulated by hypoxia through an everexpanding number of pathways in multiple cell types may lead to the identification of novel therapies and modalities for ischemic vascular diseases as well as diseases characterized by excessive angiogenesis, such as rheumatoid arthritis, psoriasis, tumours, ischemic brain and heart attack [5, 6].

Angiogenesis in hypoxia is regulated by several pro- and anti-angiogenic factors [1]. HIF-1 has been established as the major inducer of angiogenesis [35]. It regulates the transcription of VEGF, a major regulator of angiogenesis which promotes endothelial cell migration towards the hypoxic area. During hypoxia, HIF-1 binds to the regulatory region of the VEGF gene, inducing its transcription and initiating its expression. VEGF is then secreted and binds to cognate receptor tyrosine kinases (VEGFR1 and VEGFR2) located on the surface of vascular endothelial cells triggering a cascade of intracellular signalling pathways that initiate angiogenesis [10]. These endothelial cells are recruited to form new blood vessels which ultimately supply the given area with oxygenated blood [12]. Interestingly, recent studies have shown that hypoxia influences additional aspects of angiogenesis, including vessel patterning, maturation and function [5].

Other factors such as angiopoietin-2/angiopoietin-1 [36, 37], angiopoietin receptor (Tie2) [38], platelet-derived growth factor (PDGF) [39], basic fibroblast growth factor (bFGF) [40] and monocyte chemoattractant protein 1 (MCP-1) [41] have also been reported to be responsible not only for increasing vascular permeability, endothelial sprouting, maintenance, differentiation and remodelling but also cell proliferation, migration, enhancement of endothelial assembly and lumen formation (**Figure 2**). In hypoxia, angiogenesis is also modulated by several factors that are secreted by leucocytes, which produce a high abundance of angiogenic factors, various interleukins such as TGF-β1 and MCP-1 and proteinases [42]. Thus, hypoxia provides an important environmental stimulus not only for angiogenesis but also for related phenomena in the hypoxic or surrounding area, suggesting that hypoxia is more than simply a regulator of angiogenesis [6].

Angiogenesis may be detrimental when it is excessive. Therefore, angiogenic factors must be highly active but also be tightly regulated. Angiogenesis that is associated with pathological consequences may exhibit differences in the responsible molecular pathways in comparison to physiological angiogenesis. Mutations in oncogenes and tumour suppressor genes and disruptions in growth factor activity play an important role in tumour angiogenesis. The activation of the most prominent proangiogenic factor VEGF might be due to physiological stimuli such as hypoxia or inflammation or due to oncogene activation and tumour suppression function loss. Physiological angiogenesis that occurs during embryonic development or wound healing seems to be dependent on VEGF signalling, whereas tumour angiogenesis adopts

wound healing or vessel penetration into avascular regions. It may also be pathological, for example when it occurs during the formation of solid tumours, eye disease, chronic inflammatory disorders such as rheumatoid arthritis, psoriasis and periodontitis and atherosclerosis.

118 Physiologic and Pathologic Angiogenesis - Signaling Mechanisms and Targeted Therapy

The regulation of angiogenesis (whether in physiological or pathological cases) by hypoxia is an important component of homeostatic mechanisms that link vascular oxygen supply to metabolic demand. An understanding of the processes involved in angiogenic, the role of the interacting proteins involved, and how all this is regulated by hypoxia through an everexpanding number of pathways in multiple cell types may lead to the identification of novel therapies and modalities for ischemic vascular diseases as well as diseases characterized by excessive angiogenesis, such as rheumatoid arthritis, psoriasis, tumours, ischemic brain and

Angiogenesis in hypoxia is regulated by several pro- and anti-angiogenic factors [1]. HIF-1 has been established as the major inducer of angiogenesis [35]. It regulates the transcription of VEGF, a major regulator of angiogenesis which promotes endothelial cell migration towards the hypoxic area. During hypoxia, HIF-1 binds to the regulatory region of the VEGF gene, inducing its transcription and initiating its expression. VEGF is then secreted and binds to cognate receptor tyrosine kinases (VEGFR1 and VEGFR2) located on the surface of vascular endothelial cells triggering a cascade of intracellular signalling pathways that initiate angiogenesis [10]. These endothelial cells are recruited to form new blood vessels which ultimately supply the given area with oxygenated blood [12]. Interestingly, recent studies have shown that hypoxia influences additional aspects of angiogenesis, including vessel patterning, matu-

Other factors such as angiopoietin-2/angiopoietin-1 [36, 37], angiopoietin receptor (Tie2) [38], platelet-derived growth factor (PDGF) [39], basic fibroblast growth factor (bFGF) [40] and monocyte chemoattractant protein 1 (MCP-1) [41] have also been reported to be responsible not only for increasing vascular permeability, endothelial sprouting, maintenance, differentiation and remodelling but also cell proliferation, migration, enhancement of endothelial assembly and lumen formation (**Figure 2**). In hypoxia, angiogenesis is also modulated by several factors that are secreted by leucocytes, which produce a high abundance of angiogenic factors, various interleukins such as TGF-β1 and MCP-1 and proteinases [42]. Thus, hypoxia provides an important environmental stimulus not only for angiogenesis but also for related phenomena in the hypoxic or surrounding area, suggesting that hypoxia is more than simply

Angiogenesis may be detrimental when it is excessive. Therefore, angiogenic factors must be highly active but also be tightly regulated. Angiogenesis that is associated with pathological consequences may exhibit differences in the responsible molecular pathways in comparison to physiological angiogenesis. Mutations in oncogenes and tumour suppressor genes and disruptions in growth factor activity play an important role in tumour angiogenesis. The activation of the most prominent proangiogenic factor VEGF might be due to physiological stimuli such as hypoxia or inflammation or due to oncogene activation and tumour suppression function loss. Physiological angiogenesis that occurs during embryonic development or wound healing seems to be dependent on VEGF signalling, whereas tumour angiogenesis adopts

heart attack [5, 6].

ration and function [5].

a regulator of angiogenesis [6].

**Figure 2.** HIF-1α regulates factors involved in developmental and pathological angiogenesis. HIF-1α directly regulates genes involved in steps such as vasodilation, increased vascular permeability, extracellular matrix remodelling and proliferation.

the ability to shift its dependence from VEGF to other proangiogenic pathways, for example, through the recruitment of myeloid cells and the upregulation of alternative vascular growth factors (PlGF and FGF) [1].

The identification of alternative ways of inhibiting tumour growth by disrupting the growthtriggering mechanisms of increasing vascular supply through angiogenesis will depend on the understanding of how tumour cells develop their own vasculature. Other cofactors are essential to ensure maximum efficiency of the transcriptional machinery related to changes in oxygen availability within cells/tissues, and the roles of different HIFs in eliciting hypoxic responses seem to be more divergent as originally assumed. Chen et al. have shown new regulatory interactions of HIF-related mechanisms involving the interactions of basic HIFs, HIF-1α and HIF-2α with their regulatory binding proteins, histone deacetylase 7 (HDAC7) and translation initiation factor 6 (Int6), respectively [6]. Int6 induces HIF- 2 degradation. In addition, silencing of *Int6* produces a potent, physiological induction of angiogenesis that may be useful in the treatment of diseases related to insufficient blood supply. The newly discovered binding proteins-HDAC7 for HIF-1 and Int6 for HIF-2 support the assumption that the 2 HIF isoforms play distinct roles in eliciting hypoxia-related responses. HIF-2 may be considered as one of the master switches for inducing angiogenic factors at least in some cell types [6].

The hypoxia/reoxygenation cycle leads to the formation of reactive oxygen species (ROS) that may subsequently regulate HIF-1 but in a rather complex manner. It has been suggested that ROS promote angiogenesis, either directly through stimulation of HIF-1 genes that are involved in stimulating angiogenesis, such as NOS and NADPH oxidase orthrough the generation of active oxidation products, including lipid peroxides. ROS are associated with the development of several chronic diseases that include atherosclerosis, type 2 diabetes mellitus, and cancer [43]. Although ROS have damaging effects on tissues, causing cell death at high concentrations, lesser degrees of oxidative stress may play a positive role during angiogenesis, or other pathophysiological processes. Angiogenesis induced by oxidative stress involves vascular endothelial growth factor (VEGF) signalling, although VEGF-independent pathways have also been identified [44].

The clinical importance of this biological process has become increasingly apparent over the last decade, and angiogenesis now represents a major focus for novel therapeutic approaches to the prevention and treatment of multiple diseases, most notably ischemic cardiovascular disease and cancer [10].
