Effects of Antihypertensive Drugs on Alcohol-Induced Functional Responses of Cultured Human Endothelial Cells

345 Hypertens Res Vol.31 (2008) No.2 p.345-351 Original Article Effects of Antihypertensive Drugs on Alcohol-Induced Functional Responses of Cultured Human Endothelial Cells Giorgio SOARDO1), Debora DONNINI1), Massimo MORETTI2), Carla MILOCCO1), Cristiana CATENA1), and Leonardo A. SECHI1) Alcohol-induced endothelial changes might contribute to an increase in blood pressure in regular alcohol consumers. Some antihypertensive drugs affect oxidative stress and endothelial function and might counteract the effects of alcohol at the cellular level. The aim of this study was to investigate in vitro the effects of three different types of antihypertensive agents on alcohol-induced endothelial responses and oxidative stress. Cultured human endothelial cells were exposed to increasing concentrations (1, 10, 60 µmol/L) of zofenoprilat, carvedilol, and lacidipine in the absence and in the presence of ethanol (140 mmol/L). Concentrations of endothelin (ET) and nitric oxide (NO) were measured in the culture media as markers of endothelial function, and malondialdehyde (MDA) and intracellular glutathione (GSHi) were measured as markers of oxidative stress. Exposure to alcohol increased the levels of ET, NO, and MDA, and decreased GSHi. Carvedilol and zofenoprilat were more effective than lacidipine in counteracting the effects of alcohol on ET production. Alcohol-induced NO production was enhanced by carvedilol, whereas zofenoprilat and lacidipine did not have a significant effect. The alcohol-induced increase in MDA concentrations was blunted by all three drugs, but only carvedilol restored a normal response. All three drugs increased GSHi levels, with the effect being greater for carvedilol and lacidipine than zofenoprilat. Carvedilol is more effective than zofenoprilat and lacidipine in counteracting alcohol-induced endothelial responses in vitro and in decreasing oxidative stress. These effects might be particularly beneficial in patients with alcohol-related hypertension. (Hypertens Res 2008; 31: 345–351) Key Words: carvedilol, endothelium, lacidipine, oxidative stress, zofenoprilat Introduction Many cross-sectional and prospective epidemiological studies have reported that regular intake of alcoholic beverages is closely associated with increased prevalence of hypertension (1). This association occurs in both sexes at different ages (2) and has been observed across various types of alcoholic beverages (3) in patients of different ethnicity (4–6). Intervention studies performed in moderate to heavy drinkers with either normal (7, 8) or high (9, 10) blood pressure have generally demonstrated that withdrawal of alcohol consumption exerts a significant hypotensive effect. It is known that the vascular endothelium generates many biologically active substances that contribute to the regulation of vascular tone (11), and data obtained in recent years have clearly indicated that alcohol affects endothelial function (12). Experiments performed in animal models and cultured endothelial cells indicate that alcohol modulates both endothelin (13, 14) and nitric oxide (14, 15) production, an effect From the 1)Division of Internal Medicine-Liver Unit and 2)Institute of General Pathology, Department of Experimental and Clinical Pathology and Medicine, University of Udine School of Medicine, Udine, Italy. Address for Reprints: Giorgio Soardo, M.D., Clinica Medica, Department of Experimental and Clinical Pathology and Medicine, University of Udine, P. le S. Maria della Misericordia, 1 33100, Udine, Italy. E-mail: Received May 10, 2007; Accepted in revised form September 2, 2007. 346 Hypertens Res Vol. 31, No. 2 (2008) that might be related to the demonstration of an impaired endothelium-dependent vasodilatation in chronic alcohol abusers (16). In both heavy alcohol consumers and cultured endothelial cells, we have recently demonstrated that alcoholinduced endothelial changes are associated with activation of oxidative stress (14), as ascertained by detection of decreased intracellular glutathione and increased malondialdehyde concentrations. Beneficial effects on oxidative stress and endothelial function have been reported for some drugs belonging to different classes of antihypertensive agents such as zofenoprilat (17), carvedilol (18), and lacidipine (19). The effects of these drugs might contribute to a reduction of blood pressure and organ protection and might be particularly beneficial in patients with alcohol-related hypertension. The present study was designed to compare in vitro the effects of zofenopril, carvedilol, and lacidipine on alcohol-induced endothelial responses and oxidative stress. Methods Endothelial Cell Culture Experiments on cultured human endothelial cells were carried out with a procedure that has been described in a previous publication, to which readers are referred for further details (14). Briefly, aortic endothelial cells were isolated from a segment of the human ascending aorta, characterized as described previously (20), and maintained as a stable cell line. Cells were originally isolated after incubation of the minced aortic tissue for 2 h in a sterile tube, in the presence of a solution containing collagenase–trypsin–chicken serum (CTC), serum containing 30 U/L collagenase (CLS-2, 287 U/mg; Worthington Biochemical Corp., Freehold, USA), 0.75 g/L trypsin (trypsin 1-300, porcine pancreas, 683 U/mg; ICN Biochemicals, Cleveland, USA), and 20 mL/L heat inactivated and dialyzed chicken serum. After incubation, fragments were centrifuged at 2,000 × g for 5 min and resuspended in HECM (human endothelial cell medium) containing 60% of 199 medium (Sigma Chemical Co., St. Louis, USA), 40% Coon’s modified HAM’S F12 (Life Technologies, Paisley, UK), 0.36 g/L glucose, 0.53 g/L CaCl2, 0.049 g/L MgCl2, 8% fetal calf serum (Intergen Company, Purchase, USA), 1 μg/ mL insulin (Elanco, Indianapolis, USA), 10 − 8 nmol/L hydrocortisone, 5 μg/mL transferrin, 10 ng/mL glycin-L-histidyl-Llysine acetate, 10 μg/mL somatostatin, 50 mg/mL pituitary extract (Pel Freez Biologicals, Rogers, USA), and 75 mg/mL hypothalamus extract (Pel Freez Biologicals) (21). The obtained cell cultures were amplified in vitro and characterized by staining for Factor VIII–related antigen (A082) and for CD31 (Dako Corp., Carpinteria, USA). Five × 10 5 endothelial cells were cultured in 6 mL of HECM medium and experiments were performed with cells in a stationary state. Two days after plating, the media were supplemented with three different concentrations (1, 10, and 60 μmol/L) of zofenoprilat (the active form of zofenopril) (Menarini Ricerche S.p.A., Florence, Italy), carvedilol (Roche Pharma, Mannheim, Germany), or lacidipine (GlaxoSmithKline, Durham, UK) in the absence or the presence of 140 mmol/L ethanol. The concentrations of zofenoprilat (22), carvedilol (23), and lacidipine (24) that were used in these experiments were on the same order of magnitude as the concentrations reached by adminis (...truncated)