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NO production by NOS-III. These include the female sexual steroid hormone, 17β-estradiol;36−38 lipid lowering agents, represented by HMG-CoA reductase inhibitors (“statins”);39 blood pressure lowering drugs, represented by ACE inhibitors;40 and antioxidants, represented by antioxidant vitamins (e.g. vitamin E and C).41 Some of these treatments lead to clinical efficacy in cardiovascular diseases and evidence have been accumulating for the role of NO as an important contributor to these therapeutic effects.

In a randomized, double-blind, placebo-controlled clinical trial (TREND = Trial on Reversing ENdothelial Dysfunction), the ACE inhibitor quinapril have been shown to improve endotheliumdependent relaxation after six months of treatment.42 In another study the effect of the HMG-CoA reductase inhibitor, lovastatin, was examined in patients with hyperlipidemia and coronary artery disease. Benefit on endothelium-dependent relaxation was shown after five and a half months of treatment.43

2. Nitric Oxide and Angiogenesis

2.1.VEGF causes endothelium-dependent vasodilation mediated by EDNO

The endothelial cell-specific vascular endothelial growth factor (VEGF) increases cytosolic free calcium in cultured endothelial cells.44 In isolated canine coronary artery segments, VEGF causes endotheliumdependent relaxation that can be attenuated by pretreatment with NG- monomethyl-L-arginine (L-NMMA).45 Isner’s group demonstrated the release of NO from arteries after treatment with VEGF46 and extended these studies to demonstrate that VEGF promoted recovery of endothelium-dependent relaxation in ischemic rabbit hindlimb. Subsequent work by the same group showed that VEGF infusion lead to EDNO-mediated hypotension in animals47 as well as in humans.48 The signaling link between VEGF and EDNO release include VEGF/KDR interaction-induced Akt phosphorylation (activation), which in turn phosphorylates NOS-III at Serine 1177, leading to increased NO production (Fig. 1).

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Fig. 1. Current concept of the role of the NOS-III/NO system in angiogenesis. The endothelial cell-specific angiogenic growth factor, VEGF, interacts with its receptor (KDR) on endothelial cells and via several mechanisms stimulates the proliferation, migration and survival of these cells, which ultimately leads to new vessel formation (angiogenesis). An intact NOS-III/NO system is required for these actions of VEGF (and of other growth factors, including FGF and HGF, not shown). NOS-III, localized to the caveolae in endothelial cell plasma membrane is activated by VEGF/KDR interaction via P13K-induced activation (phosphorylation, P) of Akt, which in turn phosphorylates serine 1177 in NOS-III enzyme (NOS-P), which augments EDNO production several-fold (for further details see text).

2.2. Tumor angiogenesis and NO

Indirect evidence that NO may be involved in the angiogenic process was provided by studies evaluating tumor angiogenesis. Increased levels of NO have been reported in human tumors,49,50 and it has been demonstrated that transfection of the inducible NOS (NOS-II) gene into an adenocarcinoma cell line gave rise to more vascularized tumors than the wild-type cells when injected into animals.51 Other experiments using inhibitors of NO synthase also strongly support the importance of NO in xenografted tumor neovascularization.52,53 Ziche and colleagues have demonstrated that vascularization of tumors that have

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been initiated with breast cancer cells overexpressing VEGF can be attenuated by treatment with NO synthase inhibitors.54

2.3. Evidence in cultured endothelial cells and in rabbit cornea

A more direct link between the production of NO induced by VEGF and angiogenesis have been demonstrated by Papapetropoulos et al.55,56 and Morbidelli et al.57 describing that many of the angiogenic responses to VEGF in vitro were associated with increases in cGMP in cultured endothelial cells. Inhibitors of NO synthesis attenuated VEGF-induced angiogenic responses, including cultured endothelial cell proliferation, migration and tube formation.

In vivo evidence for the link between NO and angiogenesis in adult animals was first described by Ziche and co-workers, who demonstrated that, in rabbits, corneal angiogenesis induced by VEGF can be inhibited by treatment of the animals with inhibitors of NO production.15,54

2.4. Role of NO in post-ischemic revascularization

In the rabbit ischemic hindlimb model, dietary L-arginine supplementation significantly decreased blood pressure, reduced arterial resistance, increased flow at rest, and increased flow reserve in the ischemic limb.58 In hypercholesterolemic mice, elevated level of plasma asymmetric dimethylarginine (ADMA), an endogenous inhibitor of NO synthesis,59 is associated with impaired angiogenic response to ischemia.60 This effect of ADMA can be reversed by administration of the NO precursor L-arginine or mimicked in normal animals by administration of an NO synthase inhibitor.61

Isner’s group was the first to investigate the effects of ischemia on angiogenesis in NOS-III-KO mice.58 The degree of angiogenesis (revascularization) was determined using both laser Doppler imaging in situ and by measuring capillary density. In the NOS-III-KO mice the spontaneous angiogenic response to ischemia was severely attenuated as compared to normal mice. The levels of VEGF were comparable in the ischemic limbs of both the control and NOS-III-KO mice, suggesting that the reason for the attenuated response to ischemia was not the lack of ischemia-induced upregulation of VEGF production. Although

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VEGF gene transfer has already been shown to stimulate the angiogenic response in Apo E-KO mice,62 treatment of NOS-III-KO mice with either a vector carrying the VEGF gene, or injection of recombinant VEGF protein, failed to improve post-ischemic revascularization.58

The ability to restore the effects of ischemia on angiogenesis in the NOS-III-KO mice with a systemically administered NO donor was not successful,58 so a direct proof for the therapeutic benefits of exogenously applied NO on angiogenesis still remained to be demonstrated.

2.5.Role of NO in exogenous VEGF and FGF-induced revascularization

Therapeutic angiogenesis in several animal models of myocardial and hindlimb ischemia have been demonstrated using VEGF and FGF protein or gene delivery. The essential role of NO in exogenous VEGFinduced post-ischemic revascularization have been demonstrated in NOS-III-KO mice.58 In a rat hindlimb ischemia model, treatment with both VEGF and FGF significantly augmented post-ischemic revascularization. Pretreatment with L-NAME prevented therapeutic arteriogenesis in this rat model by exogenous VEGF-121 and FGF-2 protein treatment63 or by exercise training,64 indicating that impaired EDNO production can reduce angiogenesis and arteriogenesis in response to both exogenously administered VEGF and FGF.

2.6. Molecular mechanisms

A number of studies indicate that NO is an endothelial cell survival factor, inhibiting apoptosis.65−67 Under certain experimental conditions NO enhances endothelial cell proliferation.57 Another prerequisite for the formation of new vessels is migration of endothelial cells. NO is known to enhance endothelial migration, by stimulating endothelial cell podokinesis,68 and/or by enhancing the expression of avβ3, an endothelial integrin involved in attachment and migration.69 Migration of endothelial cells also requires the dissolution of the surrounding extracellular matrix. NO may contribute to this process by increasing the production of urokinase-type plasminogen activator (uPA).70 Finally, the hemodynamic effects of this potent vasodilator may play a role in