**5. The role of HDACs in angiogenesis: HDAC-regulated ECs functions in vitro**

During development of the embryo and the physiological repairs of any tissue damages, the formation of new blood vessels plays a major role. The process can either involve vasculogenesis where ECs may be derived from the differentiation of different kinds of stem cells such as embryonic stem (ES) cells, while angiogenesis requires the proliferation, migration, and sprouting of ECs. Some of these new blood vessel formations are normal and beneficial as seen in wound healing after trauma and ischemic tissue restoration. However, pathological neovascularisation leads to many diseases such as diabetic retinopathy, tumour, and inflammation. Over the past decade, investigations into the role of HDACs in the regulations of these processes have gained some tractions. We have previously shown that the stabilisation of the class I HDAC3 plays an essential role in VEGF receptor 2 (VEGFR2)-mediated endothelial differentiation of mouse ES cells (mESC)-derived Sca-1<sup>+</sup> progenitors [31], while these events can also signal through VEGR2-HDAC3 stabilisation in a ligand-independent manner through exposure to laminar shear flow [32]. These derived EC-like cells display increased angiogenic potential by significantly enhancing re-endothelialisation with the host vessels upon their transplantation into a mouse wire injury model and substantially attenuated the injury-induced neointimal hyperplasia [32]. In addition, HDAC3 is also essential for the survival of ECs under atherogenic disturbed flow, and knock-down of HDAC3 increases neointima formation in the atheroprone ApoE−/− mice [33]. Overall, these show HDAC3 plays a role in the angiogenic processes.

Angiogenic activation of ECs to migrate and to form sprouts is associated with characteristic changes in gene expression profiles [34], which can be modulated by the inhibition of HDAC. HDAC inhibitions by pan-HDAC inhibitors such as suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA) can suppress VEGF-induced capillary-like structures formation in human umbilical vein endothelial cells (HUVEC) by suppressing angiogenic factors such as hypoxia-inducible factor 1 alpha (HIF-1α), VEGF, and eNOS while both HDACi also prevent the sprouting of capillaries from rat aorta [35]. Discrepant reports exist however, as another study has shown that treatment with SAHA or a more class I selective inhibitor valporic acid (VPA) in combination with VEGF indeed resulted in enhanced EC sprouting [36]. These discrepancies could be due to the contrasting roles that other HDACs within the same class and/ or from the other classes might play in regulating angiogenesis.

been extensively examined. However, the functions of HDACs in cardiovascular diseases and

There has been a breakthrough in the development of HDACi. These HDACi induce acetylation of histone proteins, as well as non-histone proteins, which leads to the alteration and regulation of biological events including angiogenesis, apoptosis/autophagy, cell cycle, fibrogenesis, immune response, inflammation, and metabolism (**Figure 1**). As a result, HDAC inhibitor-based therapies have gained substantial attention as treatments for cardiovascular

In the following sections, we will describe the different exerted functions of HDACi in differ-

During development of the embryo and the physiological repairs of any tissue damages, the formation of new blood vessels plays a major role. The process can either involve vasculogenesis where ECs may be derived from the differentiation of different kinds of stem cells such as embryonic stem (ES) cells, while angiogenesis requires the proliferation, migration, and sprouting of ECs. Some of these new blood vessel formations are normal and beneficial as seen in wound healing after trauma and ischemic tissue restoration. However, pathological neovascularisation leads to many diseases such as diabetic retinopathy, tumour, and inflammation. Over the past decade, investigations into the role of HDACs in the regulations of these processes have gained some tractions. We have previously shown that the stabilisation of the class I HDAC3 plays an essential role in VEGF receptor 2 (VEGFR2)-mediated endo-

events can also signal through VEGR2-HDAC3 stabilisation in a ligand-independent manner through exposure to laminar shear flow [32]. These derived EC-like cells display increased angiogenic potential by significantly enhancing re-endothelialisation with the host vessels upon their transplantation into a mouse wire injury model and substantially attenuated the injury-induced neointimal hyperplasia [32]. In addition, HDAC3 is also essential for the survival of ECs under atherogenic disturbed flow, and knock-down of HDAC3 increases neointima formation in the atheroprone ApoE−/− mice [33]. Overall, these show HDAC3 plays a

Angiogenic activation of ECs to migrate and to form sprouts is associated with characteristic changes in gene expression profiles [34], which can be modulated by the inhibition of HDAC. HDAC inhibitions by pan-HDAC inhibitors such as suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA) can suppress VEGF-induced capillary-like structures formation in human umbilical vein endothelial cells (HUVEC) by suppressing angiogenic factors such as hypoxia-inducible factor 1 alpha (HIF-1α), VEGF, and eNOS while both HDACi also prevent

progenitors [31], while these

**5. The role of HDACs in angiogenesis: HDAC-regulated ECs** 

thelial differentiation of mouse ES cells (mESC)-derived Sca-1<sup>+</sup>

arteriosclerosis are less explored [23].

156 Physiologic and Pathologic Angiogenesis - Signaling Mechanisms and Targeted Therapy

ent physiological and pathological conditions.

**4.2. HDAC inhibitors**

diseases and cancer.

**functions in vitro**

role in the angiogenic processes.

In fact, it is common that completely opposite role has been reported for other HDACs in the regulation of angiogenesis. HDAC4, a class IIa HDAC, was reported to negatively regulate angiogenesis by reducing VEGF expression [37], while others have reported HDAC4 induces angiogenesis through an increase in stability of HIF-1γ [38]. In addition, Zhang et al. showed that HDAC4 inhibition facilitated c-kit+ cardiac stem cells (CSCs) into the differentiation of cardiac lineage commitments with EC potential in vitro [4].

Diverse role has also been reported in another class IIa HDAC HDAC5. On the one hand, HDAC5 has been shown to repress KLF2 an important regulator of EC homeostasis that is normally expressed in the laminar flow-exposed (therefore atheroprotective) segments of the vessels, which results in repressed eNOS expression in ECs [39]. This anti-angiogenic role of HDAC5 was validated by siRNA-mediated knock-down of HDAC5 that promoted migration and sprouting of ECs [40]. Conversely, phosphorylation-dependent nuclear exports of HDAC5 [40] and HDAC7 [41], thereby the de-repression of target genes, are crucial for the expression of VEGF or metalloproteinase-10 in ECs that lead to increased angiogenesis. Moreover, blockade of HDAC7 phosphorylation with a signal-resistant HDAC7 mutant represses EC proliferation and migration in response to VEGF, confirming the important role of both class IIa HDACs plays in VEGF-mediated angiogenesis [42]. In addition, HDAC7 has also been identified as a key modulator of EC migration at least in part by regulating PDGF-B/PDGF-beta gene expression [43].

Evidence from our laboratory demonstrated that mouse HDAC7 undergoes alternative translation during mouse ESCs differentiation, resulting in the production of a 7-amino acid peptide (*Data not publish yet*). This peptide was shown to enhance mouse vascular progenitor cell migration and VEGF-induced differentiation towards the EC lineage in vitro. Overall, HDAC7 appears to be pro-angiogenic, while the mediating role of HDAC5 in angiogenesis could be largely dependent on its translocation within the nucleus.

There is a limited research into the role of class IIb HDAC in angiogenesis, but nevertheless HDAC6 can be classified as a pro-angiogenic factor as it induces cell migration by the deacetylation of cytoskeletal proteins [44, 45]. Class III HDAC SIRT1 is also highly expressed in the vasculature during blood vessel growth where it controls the angiogenic activity of ECs. Loss of SIRT1 function leads to blockage of sprouting angiogenesis [46]. Furthermore, SIRT1 associates with and deacetylates transcription factor Foxo1 and hence restricts its antiangiogenic activity [46].

## **6. The role of HDAC in therapeutic angiogenesis**

Different strategies for therapeutic angiogenesis, including the direct delivery of angiogenic growth factors and the delivery of cells to ischemic tissues, have been developed. Moreover, there is a recent progress on therapeutic angiogenesis by utilising polymeric biomaterials, combined with stem cell and gene therapy as well as stimulation of endogenous stem cell homing (see Ref. [47] for a more comprehensive review).

Owing to the disadvantage of invasiveness, limited drug diffusion, and lack of selectivity towards targeted tissues, treatments with traditional drugs and surgery are becoming less commonly used [48]. An emerging technique, ultrasound-targeted microbubble destruction (UTMD), has been proposed as a non-invasive and specific targeting approach in angiogenic therapy of CVDs. UTMD might create a series of biological effects, including improving recovery of local tissue damages, improving transient membrane permeability, and extravasation to facilitate the entering of targeted genes or drugs into the tissues or cells of interest.

There are several approaches indicating that inhibition of HDAC protects the heart against injury in different cardiovascular-related diseases, including myocardial infarction, myocardial hypertrophy, and diabetic cardiomyopathy. In addition, HDACi also seem to play a therapeutic role in other CVDs with vascular remodelling as one of their main manifestations. Here, we review the role of HDACs in these diseases one by one in order to better understand the context-dependent effects of HDACs in angiogenesis regulation in these diseases.

#### **6.1. Atherosclerosis**

Atherosclerosis of the arteries is a main causative pathogenesis of various CVDs including coronary artery disease (CAD), peripheral vascular disease (PVD), and stroke. It is a chronic pathological condition of the arteries that is characterised by the accumulation of lipids, chronic inflammation, generation of a fibrous cap, proliferation of SMCs, calcification in vascular smooth muscle layer, with the resultant loss of elasticity of arteries. In addition, disturbed shear stress (the tangential force of the flowing blood on the endothelial surface of the blood vessel) contributes to several elements of atherosclerotic disease. As a result of the growth of atheroma, the lumen of the artery is gradually narrowed, which changes the local environment. Activated ECs within the injury lesions produce adhesion molecules [intercellular adhesion molecule (ICAM) and vascular cell adhesion molecule-1 (VCAM-1)], chemotactic proteins [monocyte chemotactic protein-1 (MCP-1)], E- and P-selectin, and growth factors [macrophage colony-stimulating factor (M-CSF)] that create a pro-inflammatory environment. The inflammatory molecules then recruit monocytes to the vessel wall and promote their transmigration across the endothelial monolayer into the intima, where they proliferate, differentiate into macrophages, and foam cells by taking up the lipoproteins, leading to neointimal formation. With time, the foam cells and macrophages die and release lipid-filled contents and tissue factors, contributing to the formation of the lipid-rich necrotic core, which is a key component of unstable plaques. Meanwhile, SMCs migrate from the medial layer and accumulate within the intima, where they synthesise and secrete interstitial collagen and elastin and form the fibrous cap over the lesion. Ultimately, the thin fibrous caps rupture, then expose, and release procoagulant materials into the blood, triggering the thrombosis that impedes blood flow and results in acute stenosis of the arteries, leading to clinical manifestations [49–51].

Because many patients are not candidates for the standard treatments such as angioplasty or bypass surgery, a great enthusiasm has emerged for the utilisation of angiogenesis as a therapeutic modality for atherosclerotic arterial disease. It must be taken into account that angiogenesis plays a vital part in the pathogenesis and treatment of CVDs and has become one of the hotspots that are being discussed in the past decades. Therapeutic angiogenesis provides a valuable tool for treating cardiovascular diseases by stimulating the growth of new blood vessels from pre-existing vessels.

This avenue needs to be explored with caution however, as the role of angiogenesis in atherosclerosis remains a very contentious topic, and currently, there is no consensus as to whether angiogenesis is a way to treat coronary heart disease or in fact is a key causative factor in the pathogenesis of atherosclerotic plaque formation. The controversy surrounding the role of angiogenesis in ischemic heart disease reflects, in part, the complexity of the underlying disease process. There are lot of studies supporting the therapeutic role of angiogenesis in atherosclerosis since a key therapeutic objective has been to use the angiogenic cytokines such as VEGF or FGF to stimulate collateral blood vessel formation in the ischemic heart and limb [52]. But, on the other hand, the pathogenic role of angiogenesis has been suggested as VEGF, and other angiogenic growth factors can promote atherosclerosis in certain animal models and potentially destabilise coronary plaques by promoting intralesion angiogenesis [53].

Apolipoprotein E-deficient (ApoE−/−) mice, created by homologous recombination in ES cells, was first described in 1992 [54, 55]. Since then, this model becomes the most commonly used mouse model of atherosclerosis that is able to develop severe hypercholesterolemia and lesions of atherosclerosis highly similar to those observed in humans. Endogenous SIRT1 has been shown to decrease macrophage foam cell formation and atherogenesis in ApoE−/−mice [56], while endothelial-specific overexpression of human SIRT1 reduces atherogenesis in ApoE−/− mice and improves vascular function [57]. So, in the vasculature, *SIRT1* gain-of-function using *SIRT1* overexpression has been shown to improve endothelial function in mice. Subsequently, it was described that SIRT1 does not directly influence endothelium-dependent vascular function in ApoE−/− mice, but it improves vascular function by preventing superoxide production in ECs and reduces the expression of inflammatory adhesion molecules by suppressing NF-κB signalling [58].

HDACi TSA has been shown to exert contradictory role in the formation of atherosclerotic lesion. TSA successfully prevents neointima formation after injury [59, 60]. In contrast, however, several reports have elucidated the proatherogenic effects of TSA [61]. Another example of the discrepancies in TSA roles is the reduction of angiogenesis through the decrease of NO level (a key second messenger in angiogenesis signalling) through downregulation of eNOS [62]. These contrasting findings reinforce the theory of the contesting role angiogenesis plays in atherosclerosis. In addition, it was reported that TSA can reduce the cholesterol biosynthesis by repressing the genes involved in the cholesterol, fatty acids, and glycolysis pathways [63]. These evidences suggest that TSA could be used as a potential therapeutic agent for the control of cholesterol levels as high cholesterol level is one of the main triggers of atherosclerosis.

## **6.2. Myocardial infarction**

there is a recent progress on therapeutic angiogenesis by utilising polymeric biomaterials, combined with stem cell and gene therapy as well as stimulation of endogenous stem cell

Owing to the disadvantage of invasiveness, limited drug diffusion, and lack of selectivity towards targeted tissues, treatments with traditional drugs and surgery are becoming less commonly used [48]. An emerging technique, ultrasound-targeted microbubble destruction (UTMD), has been proposed as a non-invasive and specific targeting approach in angiogenic therapy of CVDs. UTMD might create a series of biological effects, including improving recovery of local tissue damages, improving transient membrane permeability, and extravasation to facilitate the entering of targeted genes or drugs into the tissues or cells of interest. There are several approaches indicating that inhibition of HDAC protects the heart against injury in different cardiovascular-related diseases, including myocardial infarction, myocardial hypertrophy, and diabetic cardiomyopathy. In addition, HDACi also seem to play a therapeutic role in other CVDs with vascular remodelling as one of their main manifestations. Here, we review the role of HDACs in these diseases one by one in order to better understand

the context-dependent effects of HDACs in angiogenesis regulation in these diseases.

results in acute stenosis of the arteries, leading to clinical manifestations [49–51].

Because many patients are not candidates for the standard treatments such as angioplasty or bypass surgery, a great enthusiasm has emerged for the utilisation of angiogenesis as a

Atherosclerosis of the arteries is a main causative pathogenesis of various CVDs including coronary artery disease (CAD), peripheral vascular disease (PVD), and stroke. It is a chronic pathological condition of the arteries that is characterised by the accumulation of lipids, chronic inflammation, generation of a fibrous cap, proliferation of SMCs, calcification in vascular smooth muscle layer, with the resultant loss of elasticity of arteries. In addition, disturbed shear stress (the tangential force of the flowing blood on the endothelial surface of the blood vessel) contributes to several elements of atherosclerotic disease. As a result of the growth of atheroma, the lumen of the artery is gradually narrowed, which changes the local environment. Activated ECs within the injury lesions produce adhesion molecules [intercellular adhesion molecule (ICAM) and vascular cell adhesion molecule-1 (VCAM-1)], chemotactic proteins [monocyte chemotactic protein-1 (MCP-1)], E- and P-selectin, and growth factors [macrophage colony-stimulating factor (M-CSF)] that create a pro-inflammatory environment. The inflammatory molecules then recruit monocytes to the vessel wall and promote their transmigration across the endothelial monolayer into the intima, where they proliferate, differentiate into macrophages, and foam cells by taking up the lipoproteins, leading to neointimal formation. With time, the foam cells and macrophages die and release lipid-filled contents and tissue factors, contributing to the formation of the lipid-rich necrotic core, which is a key component of unstable plaques. Meanwhile, SMCs migrate from the medial layer and accumulate within the intima, where they synthesise and secrete interstitial collagen and elastin and form the fibrous cap over the lesion. Ultimately, the thin fibrous caps rupture, then expose, and release procoagulant materials into the blood, triggering the thrombosis that impedes blood flow and

homing (see Ref. [47] for a more comprehensive review).

158 Physiologic and Pathologic Angiogenesis - Signaling Mechanisms and Targeted Therapy

**6.1. Atherosclerosis**

Myocardial infarction (MI) occurs when blood flow stops to part of the heart causing damage to the cardiomyocytes. In physiological conditions, oxygen and nutrients are supplied to the ventricular myocytes by the coronary arteries. Under pathological condition, the coronary artery is often occluded by various pathological condition such as the growth of atheroma in the coronary artery, rupture of vulnerable plaque, thrombi from proximal lesions, emboli secondary to atrial fibrillation, or vegetation after endocarditis.

Several gene or protein therapies to deliver angiogenic factors such as VEGF, FGF2, or FGF4, as well as cell therapy using endothelial progenitor cells (EPCs), mesenchymal stem cells (MSCs), or induced pluripotent stem cells (iPSCs), have been developed as potential proangiogenic therapeutics for ischemic heart disease and peripheral vascular disease [64, 65]. HDAC4 inhibition has been demonstrated to promote cardiac stem cells mediated cardiac regeneration and improve the restoration of cardiac function in mice [4]. Granger et al. observed that ischemia induces HDAC activity in the heart resulting in increased deacetylation of histones H3/4 in vitro and in vivo that leads to injured cardiomyocytes [66]. Furthermore, HDACi exert direct antifibrotic activities that alter the response to ischemic cardiac injury and reduce infarct size, which are accompanied by improvement in cardiac functions in the mouse infarcted heart. However, it is unclear whether these therapeutic effects have any links with angiogenesis in these earlier studies.

TSA have exerted an increased angiogenic response in vivo in the mouse infarcted hearts. This indicates that TSA preserves cardiac performance and mitigates myocardial remodelling through stimulating cardiac endogenous regeneration that could be dependent on enhanced angiogenesis within the infarcted heart tissues [67]. The repression of ischemia-induced gene expression such as HIF-1α and VEGF has been suggested as possible mechanisms mediated by HDACi to stabilise vascular permeability [66]. Recruitment of stem cells has also been suggested as another main mechanism that TSA mediates through. After 8 weeks of TSA treatment in MI mice with or without c-kit deficiency, significantly improved neovascularisation and cardiac repair accompanied by cardiac functions and reduced cardiac remodelling can be observed in the wildtype infarcted heart compared to the c-kit-deficient mice [68]. It is also important to distinguish between the timing of the HDACi effects. Many reports show that long-term (8 weeks) administration of HDACi induces neovascularisation [67], while acute treatments (12 h) with HDACi inhibit angiogenesis [66].

#### **6.3. Cardiac hypertrophy**

Cardiac hypertrophy is a form of remodelling and is an adaptive response to the request for high workload from peripheral tissue or from intrinsic underlying disease conditions such as valvular dysfunction, hypertension, and MI. The heart responds to stresses by undergoing a remodelling process that is associated with myocyte hypertrophy, myocyte death, inflammation, and fibrosis, which often result in impaired cardiac function and heart failure. These are accompanied by activation of the myocyte enhancer factor-2 (MEF2) transcription factor and reprogramming of cardiac gene expression. Recent studies have revealed key roles for HDACs as both positive and negative regulators of pathological cardiac remodelling (**Figure 2**).

Members of MEF2 transcription factors family are some of the key regulators of myocardial hypertrophy. The first connection between HDACs and the regulation of pathological cardiac remodelling was provided by the discovery that class IIa HDACs interact with members of Angiogenesis and Cardiovascular Diseases: The Emerging Role of HDACs http://dx.doi.org/10.5772/66409 161

ventricular myocytes by the coronary arteries. Under pathological condition, the coronary artery is often occluded by various pathological condition such as the growth of atheroma in the coronary artery, rupture of vulnerable plaque, thrombi from proximal lesions, emboli

Several gene or protein therapies to deliver angiogenic factors such as VEGF, FGF2, or FGF4, as well as cell therapy using endothelial progenitor cells (EPCs), mesenchymal stem cells (MSCs), or induced pluripotent stem cells (iPSCs), have been developed as potential proangiogenic therapeutics for ischemic heart disease and peripheral vascular disease [64, 65]. HDAC4 inhibition has been demonstrated to promote cardiac stem cells mediated cardiac regeneration and improve the restoration of cardiac function in mice [4]. Granger et al. observed that ischemia induces HDAC activity in the heart resulting in increased deacetylation of histones H3/4 in vitro and in vivo that leads to injured cardiomyocytes [66]. Furthermore, HDACi exert direct antifibrotic activities that alter the response to ischemic cardiac injury and reduce infarct size, which are accompanied by improvement in cardiac functions in the mouse infarcted heart. However, it is unclear whether these therapeutic effects have any links with

TSA have exerted an increased angiogenic response in vivo in the mouse infarcted hearts. This indicates that TSA preserves cardiac performance and mitigates myocardial remodelling through stimulating cardiac endogenous regeneration that could be dependent on enhanced angiogenesis within the infarcted heart tissues [67]. The repression of ischemia-induced gene expression such as HIF-1α and VEGF has been suggested as possible mechanisms mediated by HDACi to stabilise vascular permeability [66]. Recruitment of stem cells has also been suggested as another main mechanism that TSA mediates through. After 8 weeks of TSA treatment in MI mice with or without c-kit deficiency, significantly improved neovascularisation and cardiac repair accompanied by cardiac functions and reduced cardiac remodelling can be observed in the wildtype infarcted heart compared to the c-kit-deficient mice [68]. It is also important to distinguish between the timing of the HDACi effects. Many reports show that long-term (8 weeks) administration of HDACi induces neovascularisation [67], while acute

Cardiac hypertrophy is a form of remodelling and is an adaptive response to the request for high workload from peripheral tissue or from intrinsic underlying disease conditions such as valvular dysfunction, hypertension, and MI. The heart responds to stresses by undergoing a remodelling process that is associated with myocyte hypertrophy, myocyte death, inflammation, and fibrosis, which often result in impaired cardiac function and heart failure. These are accompanied by activation of the myocyte enhancer factor-2 (MEF2) transcription factor and reprogramming of cardiac gene expression. Recent studies have revealed key roles for HDACs as both positive and negative regulators of pathological cardiac remodelling (**Figure 2**).

Members of MEF2 transcription factors family are some of the key regulators of myocardial hypertrophy. The first connection between HDACs and the regulation of pathological cardiac remodelling was provided by the discovery that class IIa HDACs interact with members of

secondary to atrial fibrillation, or vegetation after endocarditis.

160 Physiologic and Pathologic Angiogenesis - Signaling Mechanisms and Targeted Therapy

angiogenesis in these earlier studies.

**6.3. Cardiac hypertrophy**

treatments (12 h) with HDACi inhibit angiogenesis [66].

**Figure 2.** HDACs in cardiovascular disease. Different risk factors lead to the appearance of atheromatous plaques in the coronary arteries, and from these, other cardiovascular diseases such as myocardial infarction occur. The discovery that different HDAC enzymes are involved in processes such as angiogenesis has led to the development of inhibitors to modulate its effect as a therapeutic treatment (means induction; ⟞means inhibition).

MEF2 transcription factor family [69]. The transcriptional activity of MEF2 factors is upregulated in response to pathological stress in the heart, and ectopic overexpression of constitutively active forms of MEF2 in mouse heart causes dilated cardiomyopathy. It was reported that class II HDACs are substrates for a stress-responsive kinase specific for conserved serines that regulate MEF2-HDAC interactions. Those kinases phosphorylate the signal-responsive sites in class II HDACs, and mutant proteins lacking these phosphorylation sites can act as signal-resistant repressors of cardiomyocyte hypertrophy and fetal cardiac gene expression in vitro [70]. These studies support a role for class IIa HDACs as endogenous repressors of cardiac hypertrophy. Conversely, the function of class IIb HDACs in the heart remains largely unknown in heart hypertrophy.

Nevertheless, administration of HDACi TSA 2 weeks after the induction of pressure overload can reverse cardiac hypertrophy in mice [71]. The class I selective HDACi MPT0E014 also improves cardiac contractibility and attenuates structural remodelling in isoproterenolinduced dilated cardiomyopathy [72]. As there is an intrinsic relationship between decreased capillary density and the transition of cardiac hypertrophy to cardiac failure [73], it remains to be investigated whether the cardioprotective effects exerted by HDACi are related to increased angiogenesis within the hypertrophic heart.

## **6.4. Peripheral artery disease**

Peripheral artery disease (PAD) can be defined as the narrowing of the peripheral arteries that are not directly linked to the supply to the heart or the brain. PAD development is a multifactorial process with many different forms [74].

Different action mechanisms have been proposed for different HDACi in terms of regulating angiogenesis in the case of vascular diseases. It was reported in a mouse model of hindlimb ischaemia that the inhibition of class IIa HDACs is pro-angiogenic while class I HDAC inhibition is anti-angiogenic in mouse models of hindlimb ischemia [75].

#### **6.5. Stroke**

Stroke is a devastating illness and the second cause of death and disability worldwide after cardiac ischemia. A stroke occurs when a blood vessel that carries oxygen and nutrients to the brain is either blocked by a clot or bursts. As a consequence, part of the brain can die. Post-mortem studies have revealed that angiogenesis can be observed several days after cerebral ischemic stroke; it is noteworthy that higher microvessel density correlates with longer patient survival [76]. Enhanced angiogenesis facilities neurovascular remodelling processes and promotes brain functional recovery after stroke.

There are several studies testing the effects of HDACi in neurovascular remodelling processes and in brain functional recovery after stroke. Sun et al. showed that VPA treatment enhanced post-ischemic angiogenesis by increasing microvessel density, facilitating EC proliferation, and up-regulating rate of cerebral blood flow in the ipsilateral cortex. These events may be associated with up-regulation of HIF-1α and its downstream proangiogenic target VEGF as well as extracellular MMP2/9 [77]. Similar results were obtained by treating rats with VPA during permanent middle cerebral artery occlusion (pMCAO). They exhibit reduced infarct volume, promote functional recovery, enhance angiogenesis by upregulating VEGF [78], and reduce monocytes infiltration [79]. SIRT1 is proangiogenic and increases EC tube formation, especially in post-natal angiogenesis [46]. So loss of SIRT1 reduces angiogenesis and increases brain infarction, while SIRT1 was also demonstrated to play an important role in neuroprotection against brain ischemia by deacetylation and subsequent inhibition of p53-induced and nuclear factor κB-induced inflammatory and apoptotic pathways [80].

After pMCAO, sodium butyrate and TSA induce neurogenesis via HDACi in multiple ischemic brain regions in rats. Sodium butyrate also strongly upregulated VEGF, increasing angiogenesis and functional recovery after stroke. It was also described that sodium butyrate exhibits neuroprotective/neurogenic effects in rat model of neonatal hypoxia-ischemia [81]. All these results highlight that the inhibition of HDAC in brain after stroke enhances angiogenesis, and this may contribute to the long-term functional recovery after stroke.

#### **6.6. HDACs role in angiogenesis in diabetes**

Diabetes mellitus is a chronic disease where the lack of insulin leads to anomalies in the substrate metabolism, causing a range of acute and long-term complications. One of the main complication is the loss of small blood vessels. Another related secondary disease is diabetic glomerulomegaly or kidney disease. One of the predominant feature of diabetic glomerulomegaly is an increase in glomerular capillary volume [82], which can be controlled by anti-angiogenic therapies. As there is evidence of genetic association between diabetes and HDACs, treatment with HDACi exerts a reduction in glomerular endothelial markers expression, which demonstrates the anti-angiogenic benefit [83]. This effect seems to be opposite when it applies to the diabetic heart failure model, as another HDACi sodium butyrate exerts improved cardiac functions and increased microvessel density within the diabetic myocardium [84]. Moreover, HDACi also modulates cardiac peroxisome proliferator-activated receptors (PPARs) and fatty acid metabolism in diabetic cardiomyopathy [85].

## **7. Pathogenic role of angiogenesis**

#### **7.1. Cancer**

capillary density and the transition of cardiac hypertrophy to cardiac failure [73], it remains to be investigated whether the cardioprotective effects exerted by HDACi are related to

Peripheral artery disease (PAD) can be defined as the narrowing of the peripheral arteries that are not directly linked to the supply to the heart or the brain. PAD development is a multifac-

Different action mechanisms have been proposed for different HDACi in terms of regulating angiogenesis in the case of vascular diseases. It was reported in a mouse model of hindlimb ischaemia that the inhibition of class IIa HDACs is pro-angiogenic while class I HDAC inhibi-

Stroke is a devastating illness and the second cause of death and disability worldwide after cardiac ischemia. A stroke occurs when a blood vessel that carries oxygen and nutrients to the brain is either blocked by a clot or bursts. As a consequence, part of the brain can die. Post-mortem studies have revealed that angiogenesis can be observed several days after cerebral ischemic stroke; it is noteworthy that higher microvessel density correlates with longer patient survival [76]. Enhanced angiogenesis facilities neurovascular remodelling processes

There are several studies testing the effects of HDACi in neurovascular remodelling processes and in brain functional recovery after stroke. Sun et al. showed that VPA treatment enhanced post-ischemic angiogenesis by increasing microvessel density, facilitating EC proliferation, and up-regulating rate of cerebral blood flow in the ipsilateral cortex. These events may be associated with up-regulation of HIF-1α and its downstream proangiogenic target VEGF as well as extracellular MMP2/9 [77]. Similar results were obtained by treating rats with VPA during permanent middle cerebral artery occlusion (pMCAO). They exhibit reduced infarct volume, promote functional recovery, enhance angiogenesis by upregulating VEGF [78], and reduce monocytes infiltration [79]. SIRT1 is proangiogenic and increases EC tube formation, especially in post-natal angiogenesis [46]. So loss of SIRT1 reduces angiogenesis and increases brain infarction, while SIRT1 was also demonstrated to play an important role in neuroprotection against brain ischemia by deacetylation and subsequent inhibition of p53-induced and nuclear factor κB-induced inflammatory and apoptotic path-

After pMCAO, sodium butyrate and TSA induce neurogenesis via HDACi in multiple ischemic brain regions in rats. Sodium butyrate also strongly upregulated VEGF, increasing angiogenesis and functional recovery after stroke. It was also described that sodium butyrate exhibits neuroprotective/neurogenic effects in rat model of neonatal hypoxia-ischemia [81]. All these results highlight that the inhibition of HDAC in brain after stroke enhances angio-

genesis, and this may contribute to the long-term functional recovery after stroke.

increased angiogenesis within the hypertrophic heart.

162 Physiologic and Pathologic Angiogenesis - Signaling Mechanisms and Targeted Therapy

tion is anti-angiogenic in mouse models of hindlimb ischemia [75].

torial process with many different forms [74].

and promotes brain functional recovery after stroke.

**6.4. Peripheral artery disease**

**6.5. Stroke**

ways [80].

There are more than 200 different kinds of cancers, and each type behaves and responds to treatments in different ways. Epigenetic enzymes are dysregulated in tumours through mutation or altered expression. More importantly, tumourigenesis is largely due to overexpression of oncogenes or the loss of function of tumour suppressor genes. The identification of these proteins has driven the rapid development of small-molecule inhibitors.

As we mention above, the function of HDACs is not solely on modifying histones, but they can also target many different cellular substrates and proteins, including those that are involved in tumour progression. Currently, many HDACi are in clinical trials for cancer therapeutics as HDACi result in hyperacetylation (and therefore repression) of genes related to tumour cell apoptosis, growth arrest, senescence, differentiation, cell invasion, and metastasis [86].

An exemplary role of HDACi play in modulating the tumour cells directly is its action on vasculogenic mimicry (VM). VM refers to the process by which highly aggressive tumour cells mimic ECs to form vessel-like structures that aid in supplying enough nutrients to rapidly growing tumours [87]. HDAC3 has demonstrated an important facilitative role on VM in gliomas, as HDAC3 expression is directly correlated with the number of VM in tumours with worsen tumour grade [88]. HDACi such as SAHA exert significant anti-VM effect in the progressive pancreatic cancer cells through its inhibition of AKT and ERK signalling pathways [89].

The role of HDACs play in tumour angiogenesis has also been studied. It is widely known that hypoxia induces tumour angiogenesis and cell survival through the up-regulation of VEGF expression in tumour cells [90]. Different studies have reported that inhibition of HDAC activity by TSA blocks hypoxia-induced tumour angiogenesis [91]. Other HDACi also exert similar effects, as exemplified by MPT0G157, a potent inhibitor of HDAC1, 2, 3, and 6, which was found to promote HIF-1α degradation followed by the downregulation of VEGF expression [92]. There are also reports of the anti-tumoural effects of other HDACi (TSA, sodium butyrate, and VPA) that are also partly mediated by the reduction of VEGFR-2 expression that might be related to repressing tumour angiogenesis [93].

SIRT1, a class III HDAC, also plays an important role in tumour initiation, progression, and development of drug resistance by hindering senescence, stress-induced apoptosis [94, 95], and activating cell growth and angiogenesis. MiR-34a, whose expression level was found to be reduced in various tumour cell lines [96, 97], was reported to exert its tumour suppression effect via direct binding onto SIRT1 mRNA and regulate cell apoptosis via SIRT1-p53 pathway [98]. miR-34a also exerts its anti-tumoural effect through inhibiting SIRT1 to induce the senescence of EPCs to suppress EPC-mediated tumour angiogenesis [99].

There are emerging HDACi for cancer therapy. HDACi-targeting class I, II, and IV HDACs to be used as anticancer agents are currently under development. One of them, vorinostat, has been approved by FDA for treating cutaneous T-cell lymphoma for patients with persistent or recurrent disease or following two systemic therapies. Other inhibitors, for example, FK228, PXD101, PCI-24781, ITF2357, MGCD0103, MS-275, valproic acid, and LBH589 have also demonstrated therapeutic potential as monotherapy or combination with other anti-tumour drugs [86, 100].

## **7.2. Age-related macular degeneration**

Age-related macular degeneration (AMD) is the leading cause of blindness worldwide. AMD is characterised by the deposition of drusen aggregates under the retinal epithelium. Clusterin is one of the major proteins in drusens [101], and during aging, the expression of clusterin increases [102]. The impact of epigenetic modifications on the pathogenesis of AMD has been reported. It is known that aging affects histone acetylation status, so it is reasonable to presume that the epigenetic regulation might have a role in clusterin expression. It was reported that the treatment with HDACi induces prominent increases in the expression levels of clusterin mRNA and the secretion of clusterin protein. This result indicates that epigenetic factors regulate clusterin expression which could be affecting the pathogenesis of AMD via the inhibition of angiogenesis and inflammation [103].

## **7.3. Pulmonary arterial hypertension**

Pulmonary arterial hypertension (PAH) is a condition characterised by increased pulmonary vascular resistance and pulmonary artery pressure leading to right heart failure and premature death [104]. During the process, there is a vascular remodelling caused by dysregulated cell proliferation, migration, and survival. The cause of PAH is complex, but the excessive proliferation of SMCs and ECs within the pulmonary artery is thought to play an essential role in its pathogenesis.

Elevated levels of HDAC1 and HDAC5 have been observed in the PAH lungs, and treatments with HDACi such as SAHA and VPA reduce disease worsening in rat models of pulmonary hypertension [105]. In addition, MEF2 might have a protective role in PAH progression as the expression of MEF2 and its transcriptional targets are significantly decreased in pulmonary artery ECs from patients with PAH. The impaired MEF2 activity in ECs from PAH was associated with increased nuclear accumulation of HDAC4 and HDAC5. So, increasing MEF2 activity by the selective inhibition of class IIa HDACs by MC1568 seems to suppress excessive EC migration and proliferation by PAH-ECs and can rescue experimental PAH model [106]. Although the increased migration and proliferation of pulmonary artery ECs in PAH are also hallmarks of angiogenesis, it is still contentious to link excessive angiogenesis with the pathogenesis of PAH [107], and any potential anti-angiogenic therapy for PAH should be proceeded with caution.
