Influence of the NO - Synthesis - Pathway in Angiogenesis

Angiogenesis is one of the responses to tissue ischemia. Hypoxia is known to induce the expression of vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF). These growth factors cause endothelial cells from pre-existing vessels to proliferate and begin to migrate towards the angiogenic stimulus. Recent evidence suggests that the NO synthase pathway may participate in this phenomenon. NO arises from the five-electron oxidation of L-arginine to NO and L-citrulline by the enzyme nitric oxide synthase. The synthesis of NO may be competitively antagonized by methyl arginines such as asymmetric dimethylarginine (ADMA). Currently human umbilical vein endothelial cells were observed to grow on a fibrin matrix form capillary-like structures in response to bFGF. The effect of bFGF was blocked by antagonists of NO synthase, such as asymmetric dimethylarginine (ADMA), and was mimicked by NO donors. On the other hand administration of bFGF and VEGF are known to stimulate the release of NO from the endothelium, which may participate in their angiogenic effects. Most significantly, angiogenesis is dramatically retarded in mice lacking the gene for endothelial NO synthase. In disorders where NO synthesis or activity is known to be impaired (e.g., diabetes, aging and hypercholesterolemia), angiogenesis is also impaired. Accordingly it is speculated that perturbations of the NO synthase pathway may disrupt angiogenesis. ADMA, an endogenous competitive antagonist of NO synthase, appears to be elevated in many disorders associated with atherosclerosis. The elevation of ADMA in hypercholesterolemics or in those with atherosclerosis may explain the observation that administration of L-arginine can improve NO-mediated vasodilatation in patients. Of interest, preliminary studies indicate that L-arginine can enhance angiogenesis in the ischemic hind limb of rabbits . Accordingly, it is hypothesized that ADNIA is an endogenous anti-angiogenic agent. To test this hypothesis the following specific aims will be addressed: 1. Does ADMA inhibit angiogenesis in a murine model of hind limb ischemia of rabbits? Animals will be implanted with Alza pumps containing ADMA or vehicle in concentrations to achieve a two- or threefold increase in plasma levels (levels which are observed in patients with hypercholesterolemia or atherosclerosis). I will assess angiogenesis using treadmill exercise time, maximal VO 2 , skeletal muscle blood flow during rest and exercise, and capillary density using techniques already existing in Dr. Cooke`s lab . Animals will be studied at one-week intervals from 0-6 weeks after femoral artery ligation and implantation of the Alza pumps. To determine if the increase in ADMA is inhibiting endogenous NO production, I will measure urinary nitrate production as well as the elaboration of NO by aortic segments in vitro in some animals. In a separate series of studies it will be determined if supplemental L-arginine, administered in drinking water, can restore NO production in angiogenesis. Alternatively, I will administer an exogenous NO donor by Alza pump to determine if replacement of NO can restore angiogenesis. In a separate series of studies, I will determine if elevations in endogenous ADMA can inhibit angiogenesis. Animals will undergo the procedures above, except that they will be implanted with the antagonist of dimethyl-arginine/dimethyihydrolase (DDAH); (the enzyme that metabolizeses, ADMA to citrulline). I will document by HPLC and chemiluminescence the increase in ADMA and decrease in NO, respectively, in plasma and tissues from these animals, and will use the procedures above to assess angiogenesis. 2. Does hypercholesterolemia inhibit angiogenesis, and is this effect mediated by ADMA? I predict that the studies above will demonstrate that exogenous or endogenous ADMA impairs angiogenesis by reducing NO production. Recent observations suggest that angiogenesis may be impaired in hypercholesterolemia . 1 speculate that this impairment may be due to increased ADMA production and reduced NO elaboration. The above studies will probably consume the first year of my fellowship, a logical next step would be to employ the techniques discussed above to document : that angiogenesis is impaired in apo-E deficient mice compared to controls that this impairment of angiogenesis is associated with an increased ADMA and reduced NO production that the impairment is exacerbated by administration of the DDAH antagonist, and is attenuated by administration of L-arginine or NO donors.