ABSTRACT
It is well documented that there are gender differences in the incidence and patterns of cardiovascular diseases but the reasons are unclear. Sex steroids may modulate the behaviour of vascular endothelial cells, which in turn act by paracrine processes to alter adjacent vascular smooth muscle activity. We hypothesised that the sex steroids alter the percentage of vascular endothelial cells that secrete the vasodilator peptide, adrenomedullin and modify the adrenomedullin-stimulating action of angiotensin-II. The percentage of adrenomedullin-secreting human aortic endothelial cells was determined using the cell immunoblot method. Cells were incubated with selected concentrations of angiotensin-II, oestradiol and testosterone alone and sex steroids in combination with angiotensin-II. Adrenomedullin mRNA expression in endothelial cells was quantified by real-time PCR. It was observed that at 4 h, angiotensin-II increased the percentage of adrenomedullin-secreting cells in a concentration-dependent manner. Testosterone at physiological concentrations was observed to increase the number of adrenomedullin-secreting cells whilst oestradiol had no effect. Addition of testosterone to angiotensin-II resulted in less than additive increases in the number of cells secreting adrenomedullin. Oestradiol reduced the angiotensin-II-induced increase in adrenomedullin-secreting cells. Adrenomedullin mRNA expression was significantly increased by testosterone and there was also a trend for an increase in adrenomedullin mRNA expression, which occurred when cells were incubated with angiotensin-II. Our results point to a complex interplay between the sex steroids and angiotensin-II in regulating adrenomedullin production by human endothelial cells, which may contribute to gender-related differences in vascular disease in humans.
Subject(s)
Adrenomedullin/metabolism , Angiotensin II/pharmacology , Endothelial Cells/metabolism , Endothelium, Vascular/metabolism , Gonadal Steroid Hormones/pharmacology , Adrenomedullin/genetics , Aorta , Cardiovascular Diseases/metabolism , Dose-Response Relationship, Drug , Endothelial Cells/drug effects , Endothelium, Vascular/drug effects , Estradiol/pharmacology , Female , Humans , Immunoblotting/methods , Male , RNA, Messenger/analysis , Radioimmunoassay/methods , Reverse Transcriptase Polymerase Chain Reaction , Stimulation, Chemical , Testosterone/pharmacologyABSTRACT
Monocyte cells are exposed to a range of reactive oxygen species (ROS) when they are recruited to a site of inflammation. In this study, we have examined the damage caused to the monocyte-like cell line U937 by peroxyl radicals and characterised the protective effect of the macrophage synthesised compound 7,8-dihydroneopterin. Exposure of U937 cells to peroxyl radicals, generated by the thermolytic breakdown of 2,2'-azobis(amidinopropane) dihydrochloride (AAPH), resulted in the loss of cell viability as measured by thiazolyl blue (MTT) reduction, and lactate dehydrogenase (LDH) leakage. The major form of cellular damage observed was cellular thiol loss and the formation of reactive protein hydroperoxides. Peroxyl radical oxidation of the cells only caused a small increase in cellular lipid oxidation measured. Supplementation of the media with increasing concentrations of 7,8-dihydroneopterin significantly reduced the cellular thiol loss and inhibited the formation of the protein hydroperoxides. High performance liquid chromatography (HPLC) analysis showed 7,8-dihydroneopterin was oxidised by both peroxyl radicals and preformed protein hydroperoxides to predominately 7,8-dihydroxanthopterin. The possibility that 7,8-dihydroneopterin is a cellular antioxidant protecting macrophage proteins during inflammation is discussed.
