ABSTRACT
Endoglin, a transforming growth factor ß (TGF-ß) receptor type III, is co-expressed with endothelial nitric oxide synthase (eNOS) in aortic endothelium in atherosclerotic plaques of mice. Interestingly, atorvastatin (ATV) is able to increase both endoglin and eNOS expression and reduce plaque size beyond its lipid lowering effects but by unknown mechanisms. We hypothesized whether inflammation modulates ATV-dependent induction of endoglin and eNOS expression in vitro in endothelial cells and whether ATV-induced eNOS expression is regulated via endoglin. After treatment of human umbilical vein endothelial cells (HUVECs) with TNF-α, endoglin and eNOS protein expression was reduced, concomitantly with increased levels of cell surface VCAM-1 and soluble endoglin, as determined by flow cytometry, Western blot and ELISA analyses. By contrast, ATV treatment increased endoglin and eNOS protein expression, while preventing TNF-α-mediated downregulation of endoglin and eNOS protein levels. Moreover, suppression of endoglin using small interfering RNA (siRNA), but not inhibition of TGF-ß signaling with SB431542, abrogated ATV-induced eNOS expression. These results suggest that ATV treatment prevents inflammation-reduced endoglin and eNOS expression in endothelial cells and that ATV-induced eNOS expression strongly depends on the proper expression of endoglin in HUVECs. Possible implications of these findings might be reflected in pathological conditions characterized by reduced expression of endoglin and eNOS as for example in hereditary hemorrhagic telangiectasia or in other endothelial dysfunctions.
Subject(s)
Antigens, CD/metabolism , Atorvastatin/pharmacology , Human Umbilical Vein Endothelial Cells/drug effects , Nitric Oxide Synthase Type III/metabolism , Receptors, Cell Surface/metabolism , Antigens, CD/genetics , Cells, Cultured , Endoglin , Gene Expression , Human Umbilical Vein Endothelial Cells/metabolism , Humans , RNA, Small Interfering/genetics , Reactive Oxygen Species/metabolism , Receptors, Cell Surface/genetics , Tumor Necrosis Factor-alpha/pharmacologyABSTRACT
Platinum-based chemotherapeutic agents induce the formation of crosslinks in DNA, which are accepted as being responsible for the cytotoxicity of these agents. In this study, we used a modification of the alkaline comet assay for detection of the presence of DNA crosslinks in vitro caused by cisplatin, and in peripheral lymphocytes of patients with non-small cell lung carcinoma undergoing chemotherapy with platinum derivatives. The comet technique modified for the detection of DNA crosslinks was calibrated in vitro by treating HeLa cells and human lymphocytes from healthy donors with different concentrations of cisplatin. A cisplatin dose-dependent formation of DNA crosslinks was observed in in vitro measurements using 10-200 µM concentrations of cisplatin. Lymphocytes from cancer patients were also assayed for the formation and repair of DNA crosslinks. Evidence of crosslink formation and repair was observed in peripheral blood lymphocytes of all cancer patients in this study, although some inter-individual differences were observed in the response to chemotherapy and in repair of DNA crosslinks. We propose that monitoring the number of DNA crosslinks in peripheral blood lymphocytes might be a quick and sensitive method for monitoring a patient's sensitivity to this agent. Modification of the method by incubation of analysed cells with styrene oxide before crosslink analysis by comet assay extends the use of the method also to laboratories which have no facilities to use ionizing irradiation for introducing DNA breaks into the cells.
Subject(s)
Antineoplastic Agents/pharmacology , Carcinoma, Non-Small-Cell Lung/drug therapy , Cisplatin/pharmacology , Comet Assay/methods , DNA Damage , Lung Neoplasms/drug therapy , Lymphocytes/drug effects , Aged , Carcinoma, Non-Small-Cell Lung/genetics , Female , HeLa Cells , Humans , Lung Neoplasms/genetics , Lymphocytes/metabolism , Male , Middle AgedABSTRACT
Various reactive oxygen species (ROS) may be produced from normal biochemical, essential metabolic processes or from external sources as exposure to a variety of agents presented in the environment. Lipids, proteins, carbohydrates and DNA are all capable of reacting with ROS and can be implicated in etiology of various human disorders (rheumatoid arthritis, reperfusion injury, atherosclerosis, lung diseases etc.). In the organism damage by ROS is counteracted with natural antioxidants (glutathione peroxidases, superoxide dismutases, catalase, glutathione, ubiquinol, uric acid, and essential minerals) and nutritional antioxidants from diet (i.e. vitamins E, C, carotenoids). Possible mechanisms of nutritional depletion and side effects of high intake are in the article described.
