Abstract
Background: Atherosclerosis is a chronic inflammatory disease characterised by monocyte/macrophage and T cell infiltration of the vascular wall in response to a variety of stimuli including oxidised low density lipoprotein (oxLDL). Studies on human blood vessels and on mouse models of the disease, as well as population studies, have led to the hypothesis that connexins (Cx) and gap junction channels play an important role in the pathogenesis of atherosclerosis. Three major Cxs (Cx37, Cx40 and Cx43) have been shown to be differentially expressed within atherosclerotic plaques at different stages of its development. Objectives: We sought to characterise the expression of Cxs in macrophages and T lymphocytes induced by oxLDL in the presence or absence of statins, we aimed to assess the molecular pathways linking leukocyte stimulation and junctional channel formation and function under the above mentioned conditions. Finally, we aimed to correlate these effects with T cell functionality in vitro. Methods: The expression of Cx37, Cx40 and Cx43 in human monocytes derived from peripheral blood mononuclear cells was determined after pre-treatment with oxLDL (100μg/ml), for 48 hours, with/without Simvastatin (100μM) and Pravastatin (100μM) for 24 hours using RT-PCR, western blotting and confocal fluorescence microscopy. T cell proliferation was assessed by the CyQuant assay (that measures the content of DNA of the culture) after co-culturing human naive CD4+ T lymphocytes (also derived from peripheral blood mononuclear cells) with macrophages. Macrophages were first pre-treated with Tetanus Toxin (TTox; 4μg/ml) to induce a rapid, adaptive immune response. Then CD4+ T lymphocyte were added for 48 hours with or without 100μg/ml of oxLDL. The influence of the gap junction inhibitors (peptides 37,43GAP27 and 40GAP27, 300μM), 18-α-glycyrrhetinic acid (100μM) and/or statins (Simvastatin, 100μM; Pravastatin, 100μM) on T cell proliferation was examined. Macrophage-T cell junctional coupling was assessed by calcein AM dye transfer with the same treatments as for T cell proliferation using flow cytometry. The effects of oxLDL and statins on T lymphocytes signal transduction pathways was assessed by immunoprecipitation and western blots to measure the synthesis and phosphorylation of PKCθ, IkBα, IKKα, IKKβ and NFkBp50 intracellular proteins. Results: Our results show the expression of Cx37, Cx40 and Cx43 mRNA and protein in macrophages. Cx43 was predominantly expressed by macrophages when treated with oxLDL. Conversely, Simvastatin had the opposite effect on Cx40 and Cx43 mRNA and Cx37, Cx40 and Cx43 protein expression whereas Pravastatin reduced the expression of both mRNA and protein of the three Cxs. Both statins also significantly inhibited T cell proliferation. Simvastatin reduced the expression of IKKβ while Pravastatin had a similar effect but on IKKα expression. Both statins were found to specifically halt the expression of NFkBp50 and PKCθ in oxLDL activated CD4+ T lymphocytes. A differential effect of oxLDL and statins on dye transfer coupling was also observed mirroring the change in Cxs mRNA and protein expression levels. Conclusions: Our results show for the first time that statins regulate the levels of Cxs expression in macrophages and CD4+ T lymphocytes and the levels of junctional communication between these two cell types after exposure to atherogenic insult. The athero-protective role attributed to statins may be mediated by gap junction expression and function and is likely linked to key specific protein kinase phosphorylation pathways associated to lymphocyte immune function. Abrogation of the oxLDL effect on Cxs expression by statins further supports these conclusions.