Regression of atherosclerosis - Role of nitric oxide and apoptosis

Background-We have recently found that administration of L-arginine to hype rcholesterolemic rabbits induces regression of preexisting lesions. Others have previously shown that activation of the L-arginine/nitric oxide (NO) s ynthase pathway can induce apoptosis of vascular cells in vitro. Accordingl y, the current study was designed to determine if dietary supplementation o f L-arginine induces apoptosis of intimal lesions and if this effect is med iated through the NO synthase pathway. Methods and Results-Male New Zealand White rabbits were fed a 0.5% choleste rol diet for 10 weeks and subsequently placed on 2.5% L-arginine HCl in the drinking water, and the cholesterol diet was continued for 2 weeks, at whi ch time the aortas were harvested for histological studies. L-Arginine trea tment increased the number of apoptotic cells (largely macrophages) in the intimal lesions by 3-fold (11.9+/-3.9 vs 3.9+/-1.4 apoptotic cells/mm(2), P <0.01). In subsequent studies, aortas were harvested for ex vivo studies. A ortic segments were incubated in cell culture medium for 4 to 24 hours with modulators of the NO synthase pathway. The tissues were then collected for histological studies and the conditioned medium collected for measurement of nitrogen oxides by chemiluminescence. Addition of sodium nitroprusside ( 10(-5) mol/L) to the medium caused a time-dependent increase in apoptosis o f vascular cells (largely macrophages) in the intimal lesion. L-Arginine (1 0(-3) mol/L) had an identical effect on apoptosis, which was associated wit h an increase in nitrogen oxides released into the medium. These effects we re not mimicked by D-arginine, and they were antagonized by the NO synthase inhibitor L-nitro-arginine (10(-4) mol/L), The effect of L-arginine was no t influenced by an antagonist of cGMP-dependent protein kinase, nor was the effect mimicked by the agonist of protein kinase G or 8-BR cGMP, Conclusions-These results indicate that supplemental L-arginine induces apo ptosis of macrophages in intimal lesions by its metabolism to NO, which act s through a cGMP-independent pathway. These studies are consistent with our previous observation that supplementation of dietary arginine induces regr ession of atheroma in this animal model. These studies provide a rationale for further investigation of the therapeutic potential of manipulating the NO synthase pathway in atherosclerosis.