ABSTRACT
Title
Rosuvastatin improves endothelial dysfunction and wall remodeling of resistance arteries in rats with angiotensin II-induced hypertension
Authors
I. Rugani1, A. Virdis1, R. Colucci1,L. Antonioli1, M. Fornai1, E. Duranti1, N. Bernardini2, C. Blandizzi1, S. Taddei1
1Dept. of Internal Medicine, University of Pisa
2Dept. of Human Morphology and Applied Biology, University of Pisa
1Dept. of Internal Medicine, University of Pisa
2Dept. of Human Morphology and Applied Biology, University of Pisa
Abstract
Introduction. Endothelial dysfunction is an early step in the development of atherosclerosis. Angiotensin II plays a primary role in the induction of both endothelial dysfunction and pathological wall remodeling in resistance arterial vessels, an effect which involves COX-1 activity. Statins can exert beneficial vascular effects through mechanisms independent of their lipid lowering action. The present study examines the effects of rosuvastatin on endothelial dysfunction and wall remodeling in mesenteric resistance arteries from rats with angiotensin II-induced hypertension.
Methods. Male Sprague Dawley rats, subjected to angiotensin-II infusion (120 ng/kg/min by subcutaneous osmotic minipumps for 2 weeks), were employed. Rosuvastatin was administered by intragastric route for 2 weeks, starting the first day after minipump implantation (1-20 mg/kg/day). At the end of treatment, third-order mesenteric arteries were isolated and mounted on pressurized myographs. Endothelial-dependent relaxations were elicited by acetylcholine (ACh, 0.001-100 μM). Nitric oxide synthase (NOS) activity and reactive oxygen species (ROS) production were evaluated by testing ACh under incubation with NG-nitro-L-arginine methylester (L-NAME, 100 μM, NOS blocker) and ascorbic acid (VitC, 10 mM), respectively. ACh was assayed also in the presence of the cyclooxygenase inhibitors SC-560 (1 μM, COX-1 blocker) or DFU (1 μM, COX-2 blocker). Media thickness and lumen diameter of vessels were measured to estimate media/lumen ratio (M/L) and media cross-sectional area (CSA) as indexes of vascular remodeling. Fibronectin expression and density were examined by immunohistochemistry as indexes of vascular fibrosis. ROS production was evaluated by measurement of malondialdehyde (MDA) and dihydroethidium levels. COX-1 expression was assessed by RT-PCR.
Results. In vessels from normotensive rats, relaxation to ACh was blunted by L-NAME, while SC-560, DFU or VitC were without effects. In vessels from angiotensin II-treated rats, ACh-evoked relaxations were significantly impaired and not sensitive to L-NAME. Moreover, SC-560, but not DFU, improved endothelium-dependent relaxations. In this setting, VitC ameliorated ACh-induced relaxations and restored the inhibitory effects of L-NAME. Rosuvastatin normalized the response to ACh in vessels from Ang II-treated rats and restored the inhibitory effects of L-NAME, while SC-560, DFU, and VitC were without effects. RT-PCR revealed a COX-1 induction in Ang II-treated rats arteries, which was downregulated by rosuvastatin. Ang II infusion was associated with a significant increase in vascular fibronectin density and M/L, but not CSA, indicating an eutrophic remodeling. The M/L increment was partly prevented by rosuvastatin, while CSA was not affected. The Ang II-mediated enhanced vascular fibronectin deposition was prevented by rosuvastatin. Rosuvastatin reversed the increment of MDA and superoxide generation induced by angiotensin-II.
Conclusions. Ang II-induced endothelial dysfunction in resistance arteries results from an increase in ROS production and subsequent reduction of NO availability. COX-1 activity, leading to arterial constriction, contributes to the impairment of endothelial function in this model. Rosuvastatin exerts beneficial effects on endothelial dysfunction, through a restoration of NO availability and a reduction of COX-1-dependent contracting activity. Such favourable effects depend, at least in part, on the ability of rosuvastatin to interfere with intravascular ROS production. The inhibitory effects of rosuvastatin on COX-1 activity also appear to account for its ability to counteract the concomitant eutrophic remodeling occurring in resistance arteries.
Methods. Male Sprague Dawley rats, subjected to angiotensin-II infusion (120 ng/kg/min by subcutaneous osmotic minipumps for 2 weeks), were employed. Rosuvastatin was administered by intragastric route for 2 weeks, starting the first day after minipump implantation (1-20 mg/kg/day). At the end of treatment, third-order mesenteric arteries were isolated and mounted on pressurized myographs. Endothelial-dependent relaxations were elicited by acetylcholine (ACh, 0.001-100 μM). Nitric oxide synthase (NOS) activity and reactive oxygen species (ROS) production were evaluated by testing ACh under incubation with NG-nitro-L-arginine methylester (L-NAME, 100 μM, NOS blocker) and ascorbic acid (VitC, 10 mM), respectively. ACh was assayed also in the presence of the cyclooxygenase inhibitors SC-560 (1 μM, COX-1 blocker) or DFU (1 μM, COX-2 blocker). Media thickness and lumen diameter of vessels were measured to estimate media/lumen ratio (M/L) and media cross-sectional area (CSA) as indexes of vascular remodeling. Fibronectin expression and density were examined by immunohistochemistry as indexes of vascular fibrosis. ROS production was evaluated by measurement of malondialdehyde (MDA) and dihydroethidium levels. COX-1 expression was assessed by RT-PCR.
Results. In vessels from normotensive rats, relaxation to ACh was blunted by L-NAME, while SC-560, DFU or VitC were without effects. In vessels from angiotensin II-treated rats, ACh-evoked relaxations were significantly impaired and not sensitive to L-NAME. Moreover, SC-560, but not DFU, improved endothelium-dependent relaxations. In this setting, VitC ameliorated ACh-induced relaxations and restored the inhibitory effects of L-NAME. Rosuvastatin normalized the response to ACh in vessels from Ang II-treated rats and restored the inhibitory effects of L-NAME, while SC-560, DFU, and VitC were without effects. RT-PCR revealed a COX-1 induction in Ang II-treated rats arteries, which was downregulated by rosuvastatin. Ang II infusion was associated with a significant increase in vascular fibronectin density and M/L, but not CSA, indicating an eutrophic remodeling. The M/L increment was partly prevented by rosuvastatin, while CSA was not affected. The Ang II-mediated enhanced vascular fibronectin deposition was prevented by rosuvastatin. Rosuvastatin reversed the increment of MDA and superoxide generation induced by angiotensin-II.
Conclusions. Ang II-induced endothelial dysfunction in resistance arteries results from an increase in ROS production and subsequent reduction of NO availability. COX-1 activity, leading to arterial constriction, contributes to the impairment of endothelial function in this model. Rosuvastatin exerts beneficial effects on endothelial dysfunction, through a restoration of NO availability and a reduction of COX-1-dependent contracting activity. Such favourable effects depend, at least in part, on the ability of rosuvastatin to interfere with intravascular ROS production. The inhibitory effects of rosuvastatin on COX-1 activity also appear to account for its ability to counteract the concomitant eutrophic remodeling occurring in resistance arteries.