Background: Interleukin-6 (IL-6)/soluble IL-6 receptor (sIL-6R) promotes peritoneal angiogenesis by stimulating SP4-mediated vascular endothelial growth factor (VEGF) production in peritoneal dialysis (PD). Moreover, histone methyltransferase enhancer of zeste homologue 2 (EZH2) is involved in IL-6/sIL-6R signalling via the acceleration of vascular endothelial growth factor (VEGF)-induced angiogenesis. However, the molecular mechanism underlying how EZH2 epigenetically activates VFGF expression in IL-6/sIL-6R signalling during PD is still unclear. Methods and Results: In this study, we measured the expression of EZH2, DNMT3B and SP4 in human peritoneal mesothelial cells (HPMCs) treated with IL-6/sIL-6R stimulation and/or EZH2 overexpression, silencing or inhibition. Methylation of the CpG site in the SP4 promoter region and VEGF production were measured under these treatments in HPMCs. Moreover, tube formation in human umbilical vein endothelial cells (HUVECs) was detected following treatment with conditioned media from these stimulated HPMCs. The 5/6 nephrectomy (5/6Nx) rat model was established, and the rats were injected with peritoneal dialysate. EZH2, DNMT3B and SP4 expression and microvessels were analysed in 5/6Nx + PD rats treated with IL-6/sIL-6R and EZH2 overexpression. The results showed that IL-6/sIL-6R and EZH2 overexpression enhanced the expression of EZH2, DNMT3B and SP4, but EZH2 silencing/inhibition reduced these expression levels. The results for VEGF production and tube formation in vitro followed the same trend. IL-6/sIL-6R and EZH2 overexpression increased the methylation percentage of the -170 bp CpG site in the SP4 promoter region in HPMCs. Moreover, IL-6/sIL-6R and EZH2 overexpression stimulated EZH2, DNMT3B and SP4 expression and promoted angiogenesis in 5/6Nx + PD rats. Conclusions: Thus, this study indicated that EZH2 is involved in IL-6/sIL-6R signalling and epigenetically regulates SP4 expression, thereby stimulating VEGF production and angiogenesis in PD. Targeting EZH2 is expected to be a novel therapeutic approach for end-stage renal disease (ESRD) patients with PD treatment.
Background: To investigate the effects and mechanism of high concentration glucose (HG), exogenous hydrogen peroxide (H 2 O 2 ), and antioxidants on the cell growth (cell proliferation) of human peritoneal mesothelial cells (HPMCs).Methods: All tests were conducted on cultured HPMCs (HMrSV5) in vitro. Various concentrations of glucose (0.1%, 1.35%, and 3.86%), H 2 O 2 (0.5 and 0.1 mmol/L), and antioxidants (pyruvate and catalase) were used in cell culture. Moreover, in order to study the interaction between these factors, HG and H 2 O 2 , HG and antioxidants, HG, H 2 O 2 , and antioxidants, were used respectively. After 12-24 h, phase-contrast microscopy was used to examine the morphological changes of HPMCs. DNA synthesis was detected by 3 H-thymidine incorporation to measure cell proliferation, and flow cytometry was used to evaluate the proportion of cells in G 1 phase. Furthermore, semiquantitative reverse-transcription polymerase chain reaction (RT-PCR) was utilized to determine the mRNA expression of p21 Waf1 and p27 Kip1 [cyclin-dependent kinase inhibitors (CKIs)], while immunocytochemistry (ICC) and Western blotting were employed to measure the protein expression of p21 Waf1 and p27 Kip1 .Results: HG or low-dose exogenous H 2 O 2 resulted in hypertrophy and senescence of HPMCs, resulting in similar morphological changes. Both HG and exogenous H 2 O 2 (0.5 mmol/L) inhibited the proliferation of HPMCs and led to G1 phase arrest of HPMCs. The proportion of cells in G 1 phase increased. Moreover, HG enhanced the toxic effects of exogenous H 2 O 2 . Both HG and exogenous H 2 O 2 increased the expression of p21 Waf1 and p27 Kip1 . The addition of an antioxidant in HG medium arrested cells in the G 1 phase and improved the inhibited cell proliferation.Conclusions: Both HG and exogenous H 2 O 2 treatments can induce growth inhibition of HPMCs by arresting cell cycle progression, which is partially due to the increased expression of p21 Waf1 and p27 Kip1 . Thus, the effects of HG might be associated with endogenous reactive oxygen species (ROS), and it might be beneficial to use antioxidants in peritoneal dialysis (PD).
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