Potential effect of uremic medium on cell proliferation and apoptosis and the activation of nuclear factor-kappa B pathway of aortic endothelial cells in rabbit culture

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To investigate the potential effect of uremic medium on cell proliferation and apoptosis of aortic endothelial cell (AEC), two key processes in the development of atherosclerosis, in rabbit culture. And to understand the effects of uremic medium on the activation of nuclear factor-kappa B (NF-κB) pathway and cytokines expression of AEC. Rabbit AEC were cultured with growth media supplemented with pooled sera from normal rabbits or those with chronic renal failure. The 80% confluent AEC were incubated for 24 h with media supplemented with pools of control or uremic sera. Cell proliferation was assessed by a MTT assay and cell cycle detected by flow cytometry. Hoechst33342 assay and flow cytometry were used to investigate the apoptotic effect of uremic medium in AEC. The expression of mRNA and protein levels for NF-κB, IκBα were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. NF-κB P65 nuclear translocation was analyzed by immunofluorescence. The activity of NF-κB was measured by electrophoretic mobility shift assay (EMSA). Concentrations of TNF-α and IL-6 in culture supernatants were evaluated by ELISA, and the expression of protein for TNF-α in cell lysates by Western blot. Uremic medium induced proliferation in the lower concentration range of 3%-10% while promoted apoptosis in the higher concentrations (> 10%). Uremic serum increased NF-κB mRNA (0.35 ± 0.05 vs 0.26 ± 0.02, P < 0.01) and protein (1.67 ± 0.15 vs 0.41 ± 0.05, P < 0.01) expression, decreased IκBα mRNA (0.13 ± 0.03 vs 0.24 ± 0.04, P < 0.01) and protein (0.29 ± 0.06 vs 0.65 ± 0.08, P < 0.01) expression. Uremic serum enabled NF-κB p65 nuclear translocation and increased NF-κB DNA binding activity. An increased secretion of cytokines IL-6 and TNF-α. in AEC was observed after a treatment of 10% uremic sera in a time dependent manner. The expression of TNF-α in AEC exposed to 10% uremic sera also increased significantly (0.37 ± 0.04 vs 0.14 ± 0.03, P < 0.01). Uremic medium induces the activation of AEC. A lower level of uremic medium accelerates the proliferation of AEC while a higher level induces the apoptosis of AEC. The increased proliferation may be related to a higher NF-κB activity and the expression of inflammation cytokines. Although the enhanced atherosclerosis can not be explained on the basis of an apoptotic process, the proliferative status can contribute to intimal proliferation, an earlier step in the development of atherosclerosis.