L-arginine reverses the anti-angiogenic effects of asymmetric dimethylarginine

Background: Endothelium derived nitric oxide (NO) plays a significant role in native angiogenesis and basic fibroblast growth factor (bFGF) induced angiogenesis. Recently, asymmetric dimethylarginine (ADMA), an endogenous competitive antagonist of NO synthase, has been found to be elevated in many disorders associated with impaired NO activity, including hypercholesterolemia. We hypothesized that by inhibiting NO synthesis, ADMA may act an endogenous anti-angiogenic agent. Methods: To study systemic and local effects of ADMA on angiogenesis, we used a disc angiogenesis system (DAS). The DAS, a synthetic polyvinyl alcohol disc, was implanted subcutaneously in female C57BL/6J mice. To study systemic effects of ADMA, the DAS was implanted into four groups of mice: (a) wildtype [E⁺], (b) apoE-deficient hypercholesterolemic [E], (c) E⁺+L-nitro-arginine(LNA, 6mg/100ml drinking water), and (d) E^-+L-arginine (LA, 6g/100ml drinking water). To study local effects of ADMA on angiogenesis, we used E⁺ mice and placed inside the center of the discs pellets which contained: (a) control(PBS), (b) ADMA(40μg), (c) ADMA(400μg) ± LA(6g/100ml drinking water), (d) bFGF(2μg), (e) bFGF(20μg) + ADMA(400μg) ± LA (6g/100ml drinking water). The pellets were coated with a copolymer to allow a controlled release of the agent. After 14 days, discs were extracted, sectioned, and quantitated for vascular growth occurring centripetally into the disc. Arterial blood was collected and plasma ADMA was measured by high-performance liquid chromatography (HPLC). Results: Plasma ADMA was increased 20-fold in the E^- as compared to E⁺ mice (0.9 ± 0.4 vs 0.04 ± 0.08 μM, p<0.05). Concurrently, E^- mice showed a decrease in fibrovascular growth as compared to E⁺ mice (5.9 ± 2.1 vs 10.8 ± 2.2 mm², p<0.05). Impaired angiogenesis in E⁺ mice was reversed with supplementation of the NO precursor, LA (11.0 ± 2.4 vs 5.9 ± 2.1 mm ², p<0.05), while E⁺ treated with LNA mimicked the ADMA anti-angiogenic effect (6.0 ± 0.4 vs 10.8 ± 2.2 mm², p<0.05). Local administration of ADMA (40μg, 400μg) showed a dose dependent anti-angiogenic effect as compared to control (7.2 ± 1.6, 4.3 ± 1.2 vs 12.2 ± 1.0 mm², p<0.05, p<0.007, respectively). Local ADMA effect was reversed with dietary LA (19.8 ± 2.6 vs 4.3 ± 1.2 mm², p<0.005). bFGF induced angiogenesis was also inhibited by 400μg ADMA (23.3 ± 4.0 vs 12.3 ± 4.0 mm², p<0.006). However, dietary LA reversed the inhibitory effects of local ADMA on bFGF induced discs (30.5 ± 3.6 vs 12.3 ± 4.0 mm², p<0.0001). Conclusion: Endogenous ADMA is markedly elevated in hypercholesterolemic mice. By competitively inhibiting NO synthase, systemic and local ADMA significantly blocks angiogenesis, but can be reversed by dietary supplementation of the NO precursor, L-arginine.