Synergism between melatonin and atorvastatin against endothelial cell damage induced by lipopolysaccharide Introduction Atherosclerosis is the result of a chronic inflammation perpetuated by a dysfunctional endothelium. Disrupted production and release of endothelium-derived nitric oxide (NOÆ), which is produced by endothelial nitric oxide synthase (eNOS), is a major cause of endothelial dysfunction [1]. There is a strong association between elevated plasma cholesterol levels and atherosclerosis [2]. Consequently, inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase such as the statins are often utilized in the prevention of coronary heart disease because of their efficacy for lowering lipid levels. Additional benefits of statins have been identified. These actions are independent of their cholesterol-lowering effect and in- clude, among others, the improvement of endothelial function by promoting the production of eNOS-dependent NOÆ [1]. There is a general agreement that reactive oxygen species (ROS) play a vital role in the pathogenesis of coronary atherosclerosis and its complications [3, 4]. The importance of ROS in the development and maintenance of hyperten- sion has been documented in a number of studies [5, 6], and there is increasing evidence that oxidative injury induces endothelial cell damage [7]. Because oxidative stress contributes to endothelial dysfunction and development of atherosclerosis, the use of antioxidant supplementation to improve endothelial function has been reported [8]. Several clinical trials, however, did not show beneficial actions of antioxidant vitamin supplementation [9]. A number of drugs used clinically to reduce hypertension inflict collateral damage, and it has been suggested that these damaging effects are mediated by free radicals and related reactants [10, 11]. Melatonin and several of its metabolites are known to protect against oxidative stress in all cells, tissues, and organs [12–16]. Antioxidative actions of melatonin are observed at multiple levels including attenuation of radical formation, which is referred to as radical avoidance [17]. Melatonin exhibits immunomodu- latory properties by inhibiting COX-2 and iNOS in inflammatory process [18, 19]. Previously, the protective effects of melatonin against ROS-induced endothelial cell death have been documented [20]. There is also evidence of the existence for the presence of melatonin receptors in bovine cerebral arteries, suggesting a potential role for melatonin in regulating blood flow [20]. Moreover, Abstract: The beneficial effects of atorvastatin are based on both cholesterol-dependent and independent mechanisms. The latter probably include the ability of the estatin to enhance the expression of endothelial nitric oxide synthase (eNOS) and to cause a vasodilatation. In turn, the antioxidant and anti-inflammatory actions of melatonin are related to its vascular protection. In the present study, we investigated the efficacy of the combination of melatonin plus atorvastatin against endothelial cell damage induced by inflammation and oxidative stress injury. Human umbilical vein endothelial cells (HUVEC) were cultured with bacterial lipopolysaccharide (LPS) in the presence or absence of melatonin and/or atorvastatin. LPS inhibited eNOS mRNA and protein expression, which was reversed by atorvastatin and, to a lesser extent, by melatonin. Together, melatonin + atorvastatin induced higher eNOS protein expression than either compound alone. Melatonin, but not atorvastatin, reduced free radical generation, lipid peroxidation, and interleukin-6 levels induced by LPS. In the presence of atorvastatin, the effects of melatonin were maintained or even improved. These data suggest that melatonin improves the beneficial effects of atorvastatin and reduces its side effects in endothelial cells during inflammation and under conditions of oxidative stress. Jose ´ C. Dayoub 1,2 , Francisco Ortiz 1,2 , Luis C. Lo ´ pez 1,2 , Carmen Venegas 1,2 , Alberto del Pino- Zumaquero 1,2 , Olga Roda 3 , Indalecio Sa ´ nchez-Montesinos 3 , Darı ´o Acun ˜ a-Castroviejo 1,2 and Germaine Escames 1,2 1 Centro de Investigacio ´n Biome ´ dica, Parque Tecnolo ´gico de Ciencias de la Salud, Universidad de Granada and RETICEF, Granada, Spain; 2 Departamento de Fisiologı ´a, Facultad de Medicina, Universidad de Granada, Granada, Spain; 3 Departamento de Anatomı ´a; Facultad de Medicina, Universidad de Granada, Granada, Spain Key words: atorvastatin, endothelial nitric oxide synthase, human umbilical vein endothelial cells, melatonin, oxidative stress Address reprint requests to Germaine Esca- mes, Centro de Investigacio ´n Biome ´ dica, Parque Tecnolo ´ gico de Ciencias de la Salud, Avenida del Conocimiento s/n, 18100 Armilla, Granada, Spain. E-mail: gescames@ugr.es Received January 20, 2011; Accepted April 8, 2011. J. Pineal Res. 2011; 51:324–330 Doi:10.1111/j.1600-079X.2011.00892.x Ó 2011 John Wiley & Sons A/S Journal of Pineal Research 324 Molecular, Biological, Physiological and Clinical Aspects of Melatonin