Background-NO is a major regulator of cardiovascular physiology that reduces vascular and cardiac contractility.Accumulating evidence indicates that endogenous inhibitors may regulate NOS. The NOS inhibitors asymmetric dimethylarginine (ADMA) and N-monomethylarginine are metabolized by the enzyme dimethylarginine dimethylaminohydrolase (DDAH). This study was designed to determine if increased expression of DDAH could reduce tissue and plasma levels of the NOS inhibitors and thereby increase NO synthesis. Methods and Results-We used gene transfer and transgenic approaches to overexpress human DDAH I in vitro and in vivo. The overexpression of DDAH in cultured endothelial cells in vitro induced a 2-fold increase in NOS activity and NO production. In the hDDAH-1 transgenic mice, we observed Ϸ2-fold increases in tissue NOS activity and urinary nitrogen oxides, associated with a 2-fold reduction in plasma ADMA. The systolic blood pressure of transgenic mice was 13 mm Hg lower than that of wild-type controls (PϽ0.05). The systemic vascular resistance and cardiac contractility were decreased in response to the increase in NO production. Conclusions-DDAH

Recently, we have discovered an endogenous cholinergic pathway for angiogenesis mediated by endothelial nicotinic acetylcholine receptors (nAChRs). Since angiogenesis plays a major role in wound repair, we hypothesized that activation of nAChRs with nicotine would accelerate wound healing in a murine excisional wound model. In genetically diabetic and control mice full-thickness skin wounds (0.8 cm) were created on the dorsum and topically treated over 7 days with either vehicle (phosphate-buffered saline, PBS) or nicotine (10 ؊8 mol/L, 10 ؊9 mol/L; each, n ‫؍‬ 5). Wound size was measured over 14 days followed by resection, histological analysis, and quantitation of vascularity. In diabetic animals an agonist (epibatidine, 10 ؊10 mol/L) or antagonist (hexamethonium, 10 ؊4 mol/L) of nAChRs as well as the positive control basic fibroblast growth factor (bFGF, 25 g/kg) were also tested. To further study the role of endothelial nAChRs in angiogenesis, we used an ex vivo vascular explant model. In diabetic mice wound healing was markedly impaired. Nicotine significantly accelerated wound healing as assessed by closure rate and histological score. The effects of nicotine were equal to bFGF and were mimicked by epibatidine and blocked by hexamethonium. Histomorphometry revealed increased neovascularization in animals treated with nicotine. Furthermore, capillary-like sprouting from vascular explants was significantly enhanced by nicotine. In conclusion, agonist-induced stimulation of nAChRs accelerates wound healing in diabetic mice by promoting angiogenesis. We have discovered a cholinergic pathway for angiogenesis that is involved in wound healing, and which is a potential target for therapeutic angiogenesis.

Background-This study was designed to determine whether overexpression of the enzyme dimethylarginine dimethylaminohydrolase (DDAH) could enhance angiogenesis by reducing levels of the endogenous nitric oxide synthase (NOS) inhibitor asymmetric dimethylarginine (ADMA). Methods and Results-In DDAH1 transgenic (TG) and wild-type mice (each nϭ42), the role of DDAH overexpression on angiogenesis was studied by use of the disk angiogenesis system and a murine model of hindlimb ischemia (each nϭ21). After surgery, animals were treated with either PBS or the NOS inhibitors ADMA or N -nitro-L-arginine methyl ester (L-NAME; each 250 mol · kg Ϫ1 · d Ϫ1 ) by use of osmotic minipumps (each nϭ7). L-NAME was chosen to study an inhibitor that is not degraded by DDAH. Neovascularization in the disk angiogenesis system was impaired by both NOS inhibitors; however, TG animals were resistant to the effects of ADMA on neovascularization. Similarly, TG mice were more resistant to the inhibitory effect of ADMA on angioadaptation (angiogenesis and arteriogenesis) after hindlimb ischemia, as assessed by fluorescent microsphere studies and postmortem microangiograms. Enhanced neovascularization and limb perfusion in TG mice were associated with reduced plasma and tissue ADMA levels and enhanced tissue NOS enzyme activity. Conclusions-We describe a novel mechanism by which DDAH regulates postnatal neovascularization. Therapeutic manipulation of DDAH expression or activity may represent a novel approach to restore tissue perfusion. (Circulation.