L-arginine, the precursor of endogenous nitric oxide (NO), has been shown to enhance endothelial function and to reduce the progression of atherosclerosis in cholesterol-fed rabbits. In the present study, we investigated whether myointimal cell proliferation is enhanced in hypercholesterolaemic rabbit aorta and whether chronic treatment of the rabbits with L-arginine or with the NO synthase inhibitor L-NAME influences this proliferative response and vascular monocyte accumulation. Rabbits were fed 1% cholesterol or normal rabbit chow for 12 weeks. Subgroups of cholesterol-fed rabbits were treated with oral L-arginine (2.25%) or L-NAME (3 mg/dl) in drinking water. Myointimal cell proliferation was quantified in aortic segments by immunohistochemical detection of bromodeoxyuridine (BrdU) incorporation into nuclear DNA; vascular monocyte accumulation was assessed by immunohistochemistry using a monoclonal anti-macrophage/monocyte antibody (RAM-11). Plasma levels of L-arginine and the endogenous NO synthase inhibitor, ADMA, were quantified by high-performance liquid chromatography (HPLC). Cholesterol feeding increased the aortic intima/media (I/M) ratio, which was not measurable in the control group, to 1.9 +/- 0.3. This was paralleled by enhanced cell proliferation (cholesterol, 2.4 +/- 0.2%; P < 0.05; control, 0.02 +/- 0.001% BrdU-positive cells per 72 h) and vascular monocyte accumulation. Double immunostaining for BrdU and alpha-actin showed that about two thirds of the proliferating cells were smooth muscle cells. ADMA levels increased from 0.8 +/- 0.1 micromol/l to 2.2 +/- 0.2 micromol/l in cholesterol-fed rabbits, but were unchanged by L-arginine or L-NAME treatment. Myointimal proliferation and intima/media ratios were correlated with ADMA plasma levels. Dietary L-arginine reduced monocyte accumulation by 85 +/- 2% (P < 0.05 vs cholesterol), myointimal cell proliferation (1.8 +/- 0.3% per 72 h; P < 0.05) and intimal thickening (I/M ratio: 0.7 +/- 0.2), whereas the inhibitor of NO synthase, L-NAME, further increased cell proliferation to 3.1 +/- 0.4% per 72 h (P < 0.05). No significant difference was observed in vascular monocyte infiltration between the cholesterol and L-NAME groups. We conclude that cell proliferation and vascular monocyte accumulation are enhanced in hypercholesterolaemic rabbit aorta. These atherogenic effects can be attenuated by dietary L-arginine. Decreased NO formation might underlie the enhanced monocyte accumulation and cell proliferation in hypercholesterolaemic rabbit aorta. The observed inhibition of cell proliferation adds to our understanding of the antiatherosclerotic effects of L-arginine in vivo.
Objectives: L-arginine exerts anti-atherosclerotic effects in hypercholesterolaemic rabbits via modulating endogenous NO production. We investigated whether L-arginine inhibits thromboxane formation in vivo and platelet aggregation ex vivo in this animal model. Ž . Ž Methods: The urinary excretion rates of 2,3-dinor-6-keto-PGF major urinary metabolite of PGI and 2,3-dinor-TXB major urinary . N s 4 for 12 weeks. Urine samples were collected in weekly intervals. At the end of the study period platelet aggregation ex vivo and endothelium-dependent and -independent vascular function of isolated aortic rings in vitro was assessed. Results: Urinary 2,3-dinor-TXB 2 Ž . Ž excretion significantly increased in the cholesterol group p -0.05 , and endogenous NO formation measured as urinary nitrate . Ž . Ž . excretion decreased p -0.05 . Both parameters were significantly correlated with each other R s 0.48, p -0.01 . L-arginine partly restored urinary nitrate excretion and significantly reduced TXA production to values even below those in the control group 2 Ž . p -0.001 . Urinary 2,3-dinor-6-keto-PGF excretion increased in early hypercholesterolaemia and returned to control values in the 1a second half of the study period. The early increase in urinary 2,3-dinor-6-keto-PGF excretion was attenuated by L-arginine. Platelet 1a aggregation was significantly enhanced in cholesterol-fed rabbits and attenuated by dietary L-arginine. L-arginine also improved the impaired endothelium-dependent relaxations to ADP, and normalized the vasoconstrictor effects of 5-HT in isolated aortic rings. Conclusions: Cholesterol-feeding enhances platelet aggregation and TXA formation, and stimulates platelet-endothelial cell interaction 2 in rabbits. These effects are probably due to impaired NO elaboration, as indicated by decreased urinary nitrate excretion. Chronic dietary supplementation with L-arginine elevates systemic NO elaboration and significantly increases the PGI rTXA ratio. It thus beneficially 2 2 influences the homeostasis between vasodilator and vasoconstrictor prostanoids in vivo. q 1998 Elsevier Science B.V.
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