**4. Atherosclerosis and plaque angiogenesis**

Considering the important contributions of HIF-1 in angiogenesis, it may also be a promising target for treating ischaemic disease [1] and pressure-overload heart failure [45].

Atherosclerosis causes clinical disease through the occlusion of the arteries as a result of excessive build-up of plaque within the artery wall resulting from the accumulation of cholesterol, fatty material and extracellular matrix. This causes obstruction in the blood flow to the myocardium (coronary heart disease), brain (ischemic stroke) or lower extremities (peripheral vascular). The most common of these manifestations is coronary heart disease that includes stable angina pectoris and the acute coronary syndromes [46].

Coronary heart disease (CHD) is a major cause of mortality globally (1 in every 6 deaths annually). An estimated £2bn per annum is used to treat CHD and its co-morbidities [47]. Arterial injury plays a key role in the initiation and progression of CHD [48]. Treatments for CHD range from lifestyle changes and non-invasive medical therapies to pharmacological therapies and open surgical interventions. Despite the widespread use of drugs such as statins, there remains a significant proportion of individuals for whom response to therapy is sub-optimal, and who develop atherosclerosis [49, 50].

Atherosclerosis is a lipoprotein-driven disease affecting medium and large arteries that leads to plaque formation at specific sites of the arterial tree through intimal inflammation, necrosis, fibrosis and calcification. It is a chronic inflammatory process that involves increased oxidative stress, endothelial damage, and smooth muscle cell proliferation and migration. It is associated with several established risk factors, including hypertension, hyperglycaemia, ageing and dyslipidaemia [51]. It is important to control the factors involved in the progression of atherosclerosis because advanced atherosclerotic lesions are prone to rupture, leading to disability or death. Plaque at risk of rupture has been a major focus of research [52]. There is an emerging need for new therapies to stabilize atherosclerotic lesions. Further understanding of the effects of hypoxia in atherosclerotic lesions could indicate potential therapeutic targets [53, 54]. The presence of hypoxia in human carotid atherosclerotic lesions correlates with angiogenesis. Hypoxia plays a key role in the progression and development of advanced lesions by promoting lipid accumulation, increased inflammation, ATP depletion and angiogenesis. A recent study has convincingly demonstrated the presence of hypoxia in macrophage-rich regions of advanced human carotid atherosclerotic lesions [53].

## **4.1. Evidence for hypoxia within atherosclerotic plaque**

sis, or other pathophysiological processes. Angiogenesis induced by oxidative stress involves vascular endothelial growth factor (VEGF) signalling, although VEGF-independent pathways

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

Considering the important contributions of HIF-1 in angiogenesis, it may also be a promising

Atherosclerosis causes clinical disease through the occlusion of the arteries as a result of excessive build-up of plaque within the artery wall resulting from the accumulation of cholesterol, fatty material and extracellular matrix. This causes obstruction in the blood flow to the myocardium (coronary heart disease), brain (ischemic stroke) or lower extremities (peripheral vascular). The most common of these manifestations is coronary heart disease that includes

Coronary heart disease (CHD) is a major cause of mortality globally (1 in every 6 deaths annually). An estimated £2bn per annum is used to treat CHD and its co-morbidities [47]. Arterial injury plays a key role in the initiation and progression of CHD [48]. Treatments for CHD range from lifestyle changes and non-invasive medical therapies to pharmacological therapies and open surgical interventions. Despite the widespread use of drugs such as statins, there remains a significant proportion of individuals for whom response to therapy is

Atherosclerosis is a lipoprotein-driven disease affecting medium and large arteries that leads to plaque formation at specific sites of the arterial tree through intimal inflammation, necrosis, fibrosis and calcification. It is a chronic inflammatory process that involves increased oxidative stress, endothelial damage, and smooth muscle cell proliferation and migration. It is associated with several established risk factors, including hypertension, hyperglycaemia, ageing and dyslipidaemia [51]. It is important to control the factors involved in the progression of atherosclerosis because advanced atherosclerotic lesions are prone to rupture, leading to disability or death. Plaque at risk of rupture has been a major focus of research [52]. There is an emerging need for new therapies to stabilize atherosclerotic lesions. Further understanding of the effects of hypoxia in atherosclerotic lesions could indicate potential therapeutic targets [53, 54]. The presence of hypoxia in human carotid atherosclerotic lesions correlates with angiogenesis. Hypoxia plays a key role in the progression and development of advanced lesions by promoting lipid accumulation, increased inflammation, ATP depletion and angiogenesis. A recent study has convincingly demonstrated the presence of hypoxia in macrophage-rich regions of advanced human

target for treating ischaemic disease [1] and pressure-overload heart failure [45].

have also been identified [44].

**4. Atherosclerosis and plaque angiogenesis**

120 Physiologic and Pathologic Angiogenesis - Signaling Mechanisms and Targeted Therapy

stable angina pectoris and the acute coronary syndromes [46].

sub-optimal, and who develop atherosclerosis [49, 50].

carotid atherosclerotic lesions [53].

disease and cancer [10].

Hypoxia in atherosclerotic plaques is now widely recognized, because of the use of specific probes in imaging studies [4]. Imaging plaque hypoxia could provide a means of assessing putative culprit lesions that are at risk of rupture, and are consequentially liable to adverse outcomes.

Hypoxia has been consistently found in atherosclerotic plaques *in vivo* in humans and animal models using different biomarkers [55]. The immunologically identifiable hypoxia marker, 7-(4˝-(2-nitroimidazole-1-yl)-butyl)-theophylline (NITP), has been used to assess hypoxia in three murine models *in vivo*. NITP can bind to cells under low-oxygen conditions [56, 57].

Other non-invasive imaging techniques have also been applied, which directly target plaque hypoxia, and these techniques are now being further validated in human studies. The metabolic marker F-fluorodeoxyglucose (FDG) has been used to detect human atherosclerosis *in vivo* and may also serve as an indirect marker of plaque hypoxia as the enhanced glucose uptake in anaerobic metabolism results in an increased uptake of the labelled FDG [58]. F-18-fluoromisonidazole positron emission tomographic (PET) has been used for the *in vivo* assessment of hypoxia in advanced aortic atherosclerosis in rabbits where hypoxia has been found to be predominantly confined to the macrophage-rich regions within the atheromatous core, whereas the macrophages close to the lumen were hypoxia negative [47]. This was then related to hypoxia assessed by *ex vivo* tissue staining using pimonidazole, and immuno-staining for macrophages (RAM-11), new vessels (CD31) and hypoxia-inducible factor-1 α. 18F-fluoromisonidazole (18F-FMISO), a cell permeable 2-nitroimidazole derivative that is reduced i*n vivo* by nitroreductases, regardless of the intracellular oxygen concentration, has been one of the leading radiotracers for imaging hypoxia [47]. In human studies, this imaging approach has been coupled with quantitative polymerase chain reaction (qPCR) and immune-staining of plaques tissues recovered by carotid endarterectomy to determine the gene expression of HIF-1α and cluster of differentiation 68 (CD68, a marker of inflammation). HIF-1α and CD68 expression were both found to be significantly correlated with F-FDG-uptake, indicating an association between the presence of hypoxia, inflammation and increased glucose metabolism *in vivo* [59].

Imaging plaque biomarkers such as CRP, interleukins 6, 10 and 18, soluble CD40 ligand, Pand E-selectin, NT-proBNP, fibrinogen and cystatin C show great potential in the prediction and improvement for vascular patients [60].

## **4.2. The development of a hypoxic environment within the atherosclerotic plaque**

Hypoxia has been identified as a potential factor in the formation of vulnerable plaque, and it is clear that decreased oxygen plays a role in the development of plaque angiogenesis leading to plaque destabilization [61]. There have been a number of hypotheses of atherogenesis (plaque angiogenesis) proposing that an imbalance between the demand for and supply of oxygen in the arterial wall is a key factor in the development of atherosclerosis [2, 62].

During atherogenesis, the intima (the innermost layer of the artery wall) may thicken by the accumulation of cells and matrix, and the diffusion of oxygen can then become impaired. The vasa vasorum, forming the network of small blood vessels, are vulnerable to hypoxia especially at the site of arterial branching as they are end arteries and the blood flow is reduced in this region. It has been hypothesized that hypoxia within the vasa vasorum is due to reduced blood flow and consequent endothelial dysfunction, local inflammation and permeation of large particles such as microbes, LDL-lipoprotein and fatty acids which are transformed by macrophages into foam cells [63, 64], which may be an initiating factor in atherosclerosis [65]. Therefore, the micro-environment within the atherosclerotic plaque is thought to be an important determinant of whether a plaque progresses, and the likelihood of clinical complications. Recent reports provide substantial evidence that there are regions within the plaque in which hypoxia can be identified [46].

In addition to being a marker of hypoxia, HIF-1α may directly enhance atherogenesis through several mechanisms, including smooth muscle cell proliferation and migration, new vessel formation (angiogenesis) and altered lipid metabolism [66]. The effects of HIF-1α on macrophage biology and subsequent promotion of atherogenesis has been studied in mice. HIF-1α expression in macrophages affects their intrinsic inflammatory profile and promotes the development of atherosclerosis [67]. Hence, HIF-1α may play a key role in the progression of atherosclerosis by initiating and promoting the formation of foam cells, endothelial cell dysfunction, apoptosis, increasing inflammation and angiogenesis [68].

It has been also proposed that the state of hypoxia, present in the atherosclerotic plaques of mice deficient in apolipoprotein E (ApoE−/− mice), may promote lipid synthesis, and reduce cholesterol efflux through the ATP-binding cassette transporter (ABCA1) pathway: processes that are known to be mediated by HIF-1α [55]. Hypoxia has also been reported to increase the formation of lipid droplets in macrophages to promote the secretion of inflammatory mediators, and atherosclerotic lesion progression by exacerbating ATP depletion and lactate accumulation in this model of atherosclerosis [53].

Several HIF-responsive genes have been found to be upregulated in atherosclerosis, such as VEGF, endothelin-1 and matrix-metalloproteinase-2 [69]. Hypoxia has the potential to fundamentally change the function, metabolism and responses of many of the cell types found within the developing atherosclerotic plaque, and this may in turn determine whether the plaque evolves into a stable or unstable phenotype. It is likely that this is mediated through effects on angiogenesis, extracellular matrix elaboration and lipoprotein metabolism. The hypoxic milieu in the atherosclerotic plaque may therefore also have implications for the putative therapeutic interventions for atherosclerosis. However, most *in vitro* studies have been conducted under normoxic conditions. The effects observed under these conditions may not accurately reflect those extant within the plaque [69].

The role of HIF-1 in atherosclerosis is not univocal. Silencing of HIF-1α in macrophages reduces proinflammatory factors and increases macrophage apoptosis. Hyperlipidaemia impairs angiogenesis in an HIF-1b and nuclear factor (NF)-κB-dependent manner. Specific knockdown of HIF-1α in endothelial cells reduces atherosclerosis through reduced monocyte recruitment [26], whereas knockdown in antigen-presenting cells results in aggravation of atherosclerosis through T-cell polarization [70]. There is another non-lipid-driven mechanism by which alternative macrophages present in human atherosclerosis M(Hb) promote plaque neoangiogenesis and microvessel incompetence throughan HIF-1α/VEGF-A-dependent pathway [71].

HIF-1α has also been also implicated in the pathogenesis of in-stent restenosis following coronary revascularisation, stroke, peripheral artery disease, aortic aneurysm formation and pulmonary artery hypertension [72], and also appears to be involved in the calcification of blood vessels, which often accompanies atherosclerosis [73]. Despite being an intracellular transcription factor, HIF-1 could be possible released into the circulation from damaged cells, similar to other transcriptional factors such as NF-κB and p53 [73-75].

## **4.3. Other atherogenic mechanisms of hypoxia**

cially at the site of arterial branching as they are end arteries and the blood flow is reduced in this region. It has been hypothesized that hypoxia within the vasa vasorum is due to reduced blood flow and consequent endothelial dysfunction, local inflammation and permeation of large particles such as microbes, LDL-lipoprotein and fatty acids which are transformed by macrophages into foam cells [63, 64], which may be an initiating factor in atherosclerosis [65]. Therefore, the micro-environment within the atherosclerotic plaque is thought to be an important determinant of whether a plaque progresses, and the likelihood of clinical complications. Recent reports provide substantial evidence that there are regions within the plaque

In addition to being a marker of hypoxia, HIF-1α may directly enhance atherogenesis through several mechanisms, including smooth muscle cell proliferation and migration, new vessel formation (angiogenesis) and altered lipid metabolism [66]. The effects of HIF-1α on macrophage biology and subsequent promotion of atherogenesis has been studied in mice. HIF-1α expression in macrophages affects their intrinsic inflammatory profile and promotes the development of atherosclerosis [67]. Hence, HIF-1α may play a key role in the progression of atherosclerosis by initiating and promoting the formation of foam cells, endothelial cell

It has been also proposed that the state of hypoxia, present in the atherosclerotic plaques of mice deficient in apolipoprotein E (ApoE−/− mice), may promote lipid synthesis, and reduce cholesterol efflux through the ATP-binding cassette transporter (ABCA1) pathway: processes that are known to be mediated by HIF-1α [55]. Hypoxia has also been reported to increase the formation of lipid droplets in macrophages to promote the secretion of inflammatory mediators, and atherosclerotic lesion progression by exacerbating ATP depletion and lactate

Several HIF-responsive genes have been found to be upregulated in atherosclerosis, such as VEGF, endothelin-1 and matrix-metalloproteinase-2 [69]. Hypoxia has the potential to fundamentally change the function, metabolism and responses of many of the cell types found within the developing atherosclerotic plaque, and this may in turn determine whether the plaque evolves into a stable or unstable phenotype. It is likely that this is mediated through effects on angiogenesis, extracellular matrix elaboration and lipoprotein metabolism. The hypoxic milieu in the atherosclerotic plaque may therefore also have implications for the putative therapeutic interventions for atherosclerosis. However, most *in vitro* studies have been conducted under normoxic conditions. The effects observed under these conditions may

The role of HIF-1 in atherosclerosis is not univocal. Silencing of HIF-1α in macrophages reduces proinflammatory factors and increases macrophage apoptosis. Hyperlipidaemia impairs angiogenesis in an HIF-1b and nuclear factor (NF)-κB-dependent manner. Specific knockdown of HIF-1α in endothelial cells reduces atherosclerosis through reduced monocyte recruitment [26], whereas knockdown in antigen-presenting cells results in aggravation of atherosclerosis through T-cell polarization [70]. There is another non-lipid-driven mechanism by which alternative macrophages present in human atherosclerosis M(Hb) promote plaque neoangiogenesis

and microvessel incompetence throughan HIF-1α/VEGF-A-dependent pathway [71].

dysfunction, apoptosis, increasing inflammation and angiogenesis [68].

122 Physiologic and Pathologic Angiogenesis - Signaling Mechanisms and Targeted Therapy

accumulation in this model of atherosclerosis [53].

not accurately reflect those extant within the plaque [69].

in which hypoxia can be identified [46].

Although plaque angiogenesis is a physiological response that facilitates the increased oxygen demand in the plaque, it can have adverse effects by facilitating intra-plaque haemorrhage (IPH) and the influx of inflammatory mediators. IPH as a result of immature plaque neovessels is associated with subsequent ischemic events. Inflammatory cell, endothelial cell and pericyte interactions can provide insight into the biological mechanisms of plaque angiogenesis [70].

The recruitment of T lymphocytes and proliferation and migration of smooth muscle and endothelial cells are essential for atherosclerotic plaque formation and development. During this process, a number of pro-inflammatory factors and cytokines, leukotrienes and chemokines are increased in expression, especially in lipid-loaded foam cells, such as IL8, tumour necrosis factor α (TNFα), interleukin (IL)-1,vascular cell adhesion molecule 1 (VCAM-1) and 15-lipoxygenase-2 (15-LOX-2). Moreover, macrophages are trapped in hypoxic areas of the lesion; however, the exact mechanisms have yet to be determined.

The majority of inflammatory cells contributing to early atherosclerosis probably enter the artery wall from the lumen [76, 77]. However, the vasa vasorum and associated microvessels may provide an alternate route by which leucocytes can enter the vascular wall [78]. As atherosclerosis progresses, angiogenic factors within the micro-environment of the plaque may stimulate new vessel formation. This combination of delicate new vessel network and inflammatory cells, that elaborate proteolytic enzymes, may contribute to intra-plaque haemorrhage and subsequent plaque rupture [79]. The involvement of vasa vasorum and intimal hyperplasia in the pathophysiology of atherosclerosis is supported by several experimental animal studies [80, 81].

Hypoxia may also induce macrophage migration inhibitory factor (MIF). MIF plays a critical role in the progression of atherosclerosis by several different mechanisms. These include the MIF-triggered arrest and chemotaxis of monocytes and T cells through its receptors CXCR2/4. Further, *in vivo* studies have shown that the blockade of MIF in mice with advanced atherosclerosis leads to plaque regression and reduced monocyte and T-cell content. Additionally, the neuronal signalling molecule Netrin-1 was recently shown to play an important role in macrophage retention in atherosclerotic plaques. Notably, netrin-1 expression has been shown to be regulated by hypoxia, but this may be tissue or disease specific [55].

Atherosclerotic lesion formation is associated with vessel wall thickening resulting in regional limited oxygen exchange. Vascular cells respond to hypoxic conditions with changes in cell metabolism, angiogenesis, apoptosis and inflammatory responses comparable to cells in tumours. Local hypoxic regions and hypoxic cells have been identified in human atherosclerotic lesions and in experimental models. Increased oxygen consumption by cells with a high metabolic activity, such as macrophages, further depletes the oxygen availability, creating a hypoxic environment in the atherosclerotic lesion. In macrophages, hypoxia not only affects the metabolism and lipid uptake but also results in an increased inflammatory response characterized by increased IL-1β and caspase-1 activation. Hypoxia also augments the thrombogenic potential of atherosclerotic plaques through upregulation of tissue factor.

The identification of specific inflammatory markers pertaining to the arterial wall in atherosclerosis may be useful for both diagnosis and treatment. These include macrophage inhibiting factor (MIF), leucocytes and P-selectin. Purinergic signalling is involved in the control of vascular tone and remodelling. Endothelial cells release purines and pyrimidines in response to changes in blood flow (evoking shear stress) and hypoxia. They then act on P2Y, P2X and P1 receptors on endothelial cells leading to release of EDRF mediated by nitric oxide and prostaglandins and EDHF, resulting in vasodilatation. The therapeutic potential of purinergic compounds for the treatment of vascular diseases, including hypertension, ischaemia, atherosclerosis, migraine and coronary artery and diabetic vascular disease as well as vasospasm is discussed [82]. Modern therapeutic modalities involving endothelial progenitor cells therapy, angiotensin II type-2 (AT2R) and ATP-activated purinergic receptor therapy are notable to mention. Future drugs may be designed to target three signalling mechanisms of AT2R which are (a) activation of protein phosphatases resulting in protein dephosphorylation, (b) activation of bradykinin/nitric oxide/ cyclic guanosine 3˝,5˝-monophosphate pathway by vasodilation and (c) stimulation of phospholipase A(2) and release of arachidonic acid. Drugs may also be designed to act on ATP-activated purinergic receptor channel type P2X7 molecules which acts on cardiovascular system. Better understanding of the vascular inflammatory processes and the cells involved in the formation of plaques may prove to be beneficial for future diagnosis, clinical treatment and planning innovative novel anti-atherosclerotic drugs [83].

Systemic hypoxia that is, for example, associated with obstructive sleep apnoea (OSA) also promotes atherosclerosis. The processes by which it may do this include effects on lipid metabolism and efflux, inflammation, altered macrophage polarization and glucose metabolism [84].

## **5. Conclusion**

Hypoxia is involved in several pathophysiological processes, including embryogenesis, angiogenesis and atherogenesis. HIF-1 appears to be an important mediator controlling cellular response to hypoxia. It also appears to be related to atherosclerotic progression and rupture. A better understanding of the mechanism involved in these processes may provide some novel therapeutic approaches to the treatment of cardiovascular disease.
