Impaired Angiogenic Signaling in Models of Regional Ischemia

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The long-term existence of a diabetic milieu leads to the impairment of several cellular and molecular processes in angiogenesis and arteriogenesis. These aberrations were extensively studied in several animal models of DM. Animal models of angiogenesis showed an altered expression of several angiogenic growth factors and their receptors in the presence of DM, which was associated with an impairment of ischemia-driven angiogenesis and arteriogenesis [68, 80, 81]. The adverse contribution of DM-induced chemical modifications of growth factors and ECM proteins could be established [82, 83]. Moreover, there were attempts to "rescue" the impaired angiogenesis/arteriogenesis by adding abundant amounts of angiogenic growth factors or cells (discussed in 3.1.1). Hindlimb ischemia created by ligation of the femoral artery showed a reduced restoration of perfusion secondary to decreased capillary formation in several animal models of DM. In specific, this was shown in non-obese diabetic (NOD) mice [84], in streptozotocin (STZ)-induced mice [80] and rats [81].

A plethora of evidence suggests that the decreased expression of pro-angiogenic factors and/or their corresponding receptors is critical for the impaired angiogenic response in the diabetic situation. VEGF-A is a key proangiogenic factor exerting its function via specific receptors on several cell types involved in the process of angiogenesis and arteriogenesis [1]. Importantly, new vessel formation partly correlates with hypoxia-induced increases in VEGF-A levels in the myocardium in the non-diabetic state [85]. This process is apparently blunted in DM as both mRNA and protein for VEGF are decreased in mouse ischemic diabetic hindlimbs [84]. This correlates with the decreased expression of VEGF-A and both VEGF receptors (VEGFR-1 and VEGFR-2) in the ventricular myocardium of diabetic rats as well as diabetic human beings [41]. One potential mechanism for decreased VEGF-A expression in the myocardium in diabetic and late insulin-resistant states could be the loss of insulin-induced VEGF expression. Studies have shown that insulin can increase VEGF mRNA expression by activating the PI3-kinase/Akt pathway [86], which is reduced in the vasculature from insulin-resistant rats [87]. Similarly, the expression of the angiogenic growth factors, VEGF-A and PlGF, and their co-receptors, neuropilin-1 (NP-1) and neuropilin-2 (NP-2), is decreased in ischemic limbs of two diabetic mouse models [88].

A different situation was observed in coronary vessels from OLETF rats, a model of human NIDDM, with regard to the mRNA and protein levels of VEGF-A

and VEGFR-2 [89]. Their expression was increased and, following treatment with the angiotensin II receptor antagonist candesartan was dramatically reduced. Thus, upregulation of VEGF and its receptor VEGFR-2 implies that VEGF may participate in promoting new capillary growth from existing coronary vessels in the NIDDM rat heart [89]. Alternatively, it could be possible that an increased expression of both VEGF and VEGFR-2 may reflect an activated status of coronary EC and may rather bear a pro-atherosclerotic potential of coronary vessels in DM. This apparent discrepancy in VEGF(R) expression [41, 84, 88, 89] may be attributed to differences of the diabetic animal models or may be explained by the different age of the analyzed animals.

While most of the experimental DM studies claimed a decreased expression of many proangiogenic factors, the local renin-angiotensin system (RAS) including the synthesis of angiotensin II (ANG II) were upregulated in streptozotocin-induced diabetic rats [90]. In parallel, it was shown that ANG II expression increased about 2-3-fold in coronary vessels of diabetic hearts [89]. It appears as both IDDM and NIDDM activate the local RAS and enhance generation of ANG II in vascular tissues. It was also found that ATX receptors, but not AT2 receptors, were more abundantly expressed in coronary vessels of NIDDM than in non-diabetic rats [89]. The relevance of this observation together with the up-regulation of VEGF-A and VEGFR-2 remain unclear, as the observed up-regulation appears to be limited to the large coronary arteries that do not directly participate in arteriogenesis and angiogenesis, but rather represent a substrate for the atherosclerotic process.

bFGF has been recognized as a multifunctional protein that stimulates angio-genesis and the proliferation of EC and SMC in vitro and in vivo [1, 59]. In a rat model of STZ-induced diabetes, it was able to promote angiogenesis (capillary density) in ischemic hindlimbs in a dose-dependent fashion [91]. There is no direct or indirect evidence that bFGF expression was affected by DM in the presence of ischemic conditions.

Following hyperglycemia, glycation of bFGF with intracellular sugars reduces its high-affinity heparin-binding capacity and its mitogenic activity [82]. Similarly, glycation of bFGF lowered its chemotactic potential towards EC, and when injected into normoglycemic mice, bFGF displayed a weaker angiogenic effect compared to non-glycated bFGF [92]. These results suggest that reduced new vessel growth observed in DM may be a consequence of growth factor glycation. In addition to glycation of growth factors, glycation of ECM proteins has been shown to reduce their proteolysis and, therefore, affect angiogenic processes [80].

In a mouse hind-limb ischemia model, diabetic animals showed a more reduced angiographic perfusion in the ischemic leg, which was paralleled by a tremendous increase in AGE blood levels [80]. Treatment with aminoguanidine, an inhibitor of AGE formation, decreased the elevated AGE blood and normalized the impaired ischemia-induced angiogenesis in diabetic mice. This effect is probably mediated by restoration of matrix degradation that was disturbed secondary to AGE accumulation [80] (see chapter 4).

The neurotrophin nerve growth factor (NGF), initially recognized to be involved in nerve regeneration, can elicit a pro-angiogenic activity and promotes EC survival [93] and proliferation [94]. Studies have demonstrated that diabetes reduces the levels of NGF in several peripheral tissues including skeletal muscles [95, 96]. Moreover, DM impairs the up-regulation of NGF and expression of its receptor p75 in response to hindlimb ischemia [97]. Interestingly, the NGF-induced angio-genic response appears to be mediated by VEGF-A, as a neutralizing antibody for VEGF-A inhibits the effect of NGF in ischemic limb muscles [98]. In parallel with an impaired expression of NGF and its receptor, there was an impaired formation of new capillaries and arterioles in the presence of DM [97]. This, at least in part, may be due to the increased rate of apoptosis of EC and myocytes due to the lack of functional NGF signals.

Hepatocyte growth factor (HGF) has been reported to stimulate therapeutic angio-genesis in experimental animal models [99, 100]. In turn, tissue HGF levels are decreased in the myocardium of diabetic mice [101], and a dramatic down-regulation of HGF expression was detected in the skeletal muscle of ischemic hindlimbs [81]. The impaired pattern of HGF coincided with a reduction of MMP-1 and Ets-1 expression. Ets-1 is an important transcription factor for MMP-1, stromelysin 1 and urokinase plasminogen activator (uPA) [102, 103]. Downregulation of Ets-1 might therefore play a pivotal role in the impairment of angiogenesis in DM. Indeed, the decreased expression of tissue HGF in ischemic tissues of diabetic animals was associated with a significantly lower number of newly formed capillaries [81].

Another molecular system of potential relevance for diabetic angiopathy is the kallikrein-kinin system (KKS). KKS exhibits pleiotropic effects during angiogenesis. Human tissue kallikrein (hTK) can induce angiogenesis in a hindlimb ischemic model [104]; kinins generated by tissue kallikrein (TK) from kininogen stimulate EC proliferation and survival [105] through the release of the vasodilator autacoids NO and prostacyclin [106, 107]. Experimental and clinical evidence suggests involvement of the KKS in the pathogenesis of diabetic complications, as the B2-knockout phenotype mimics characteristics of syndrome X as well as impaired insulin-dependent glucose transport [108, 109]. TK is downregulated in cardiac tissue in DM type 1 [110]. Among patients with PVD, the diabetics show lower circulating TK levels compared to nondiabetic individuals [111].

3.1.1. Experimental therapeutic angiogenesis in DM

Several attempts have been made to rescue the impaired "diabetic" phenotype of angiogenesis. External supplementation with proangiogenic proteins, the transfer of angiogenic genes as well as cell transplantation led to a significant improvement of the angiogenic response in several animal models (summarized in Table 2). Exogenous adenovirus-mediated supplementation with VEGF-A could restore the impaired blood flow in DM [84]. This supports the importance of VEGF-A in promoting peripheral angiogenesis and the detrimental consequences of DM. Local supplementation of NGF [97] and HGF by gene transfer [81] to the ischemic tissue normalized angiogenesis and arteriogenesis in diabetic animals. Furthermore, NGF

was able to prevent the increased apoptosis of EC in the ischemic tissue, which may in part contribute to the improved angiogenic response. TK gene transfer led to a pleiotropic improvement in ischemic hindlimb perfusion initially abrogated by DM [105, 112]. TK improved the formation of new capillaries and arterioles, indicating the simulation of both angiogenesis and arteriogenesis. TK exerts angiogenic action via enzymatic cleavage of kininogen leading to the release of kinin peptides; the latter targeting the kinin B(1) receptor to exert proliferative and anti-apoptotic effects on EC [105, 112]. In addition, kinin peptides stimulate the production of prostacyclin [105]. TK prevented DM-associated apoptosis of EC and restored impaired eNOS expression in ischemic diabetic muscles [112].

Recent studies demonstrated the possibility to use bone marrow cells or circulating cells to promote vessel growth [113, 114]. It was also shown that DM is associated with an impaired function of bone marrow cells and circulating cells [68, 71]. Bone marrow mononuclear cells (BM-MNC) isolated from diabetic individuals revealed a reduced proangiogenic potential. They appeared to be less potent in their ability to improve blood flow recovery and new capillary growth as compared to non-diabetic BM-MNC, when administered in non-diabetic animals [68]. The reduced proangiogenic potential was accompanied by a reduced capacity of diabetic BM-MNC to differentiate into EPCs and by an impaired angiogenic potential in vitro. Placenta growth factor PlGF, a member of VEGF family, was capable of improving the impaired function of diabetic BM-MNC and to promote angiogenesis in ischemic diabetic muscles [68].

Certain CD34+ populations of MNC carry an angiogenic potential [66]. Different types of DM were shown to negatively affect CD34+ cell physiology [71]. CD34+ cells isolated from type 2 diabetic individuals were able to produce more endothelial-like cells (ELC) than from type 1 diabetic counterparts. This was probably due to preconditioning effect of insulin, which stimulated ELC production in vitro (a "burst" effect). This might be attributed to the ability of insulin to improve VEGF synthesis as shown in neonatal cardiomyocytes [41]. Overall, human CD34+ cells had a favorable effect on perfusion recovery in the ischemic hindlimb muscles of diabetic mice [71]. Interestingly, CD34+ cells were able to stimulate flow restoration to a larger extent in diabetic animals as compared to non-diabetic animals. This is in contrary to the impaired proangiogenic capacity of non-diabetic BM-MNC when applied in diabetic animals [68]. This implies that endogenous angiogenic potential of BM-MNC is affected when brought into a diabetic environment [68]. Differently, the autologous BM-MNC improved blood flow restoration in ischemic muscles to a higher degree in rats with STZ-induced DM [115]. A decreased intrinsic potential for blood flow recovery in the settings of DM was linked to a decreased production of NO, as the plasma NO levels declined blood levels with progression of DM [115].

Epidermal progenitor cells (EpPC) have previously been considered to give rise only to keratinocytes [116]. When injected into ischemic muscles, however, they apparently were able to adopt an endothelial phenotype and improved blood flow

Table 2. Animal studies on angiogenesis/arteriogenesis focusing on experimental diabetes mellitus

Author, date [ref]

Animal model used

Primary endpoint with

Effect on angio

Mode of

Effect of the

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