Connective tissue growth factor knockdown attenuated matrix protein production and vascular endothelial growth factor expression induced by transforming growth factor-beta1 in cultured human peritoneal mesothelial cells.
Connective tissue growth factor (CTGF), a downstream mediator of transforming growth factor-beta1 (TGF-beta1) inducing fibrosis, has recently been implicated in peritoneal fibrosis. Extracellular matrix (ECM) accumulation and angiogenesis are characteristic changes in peritoneal fibrosis. In this study we investigated the effect of CTGF knockdown via interference RNA (RNAi) on ECM production and vascular endothelial growth factor (VEGF) expression induced by TGF-beta1 in human peritoneal mesothelial cells (HPMCs). Four CTGF short hairpin RNA (shRNA) expression constructs were generated using the pRetroSuper vector, and infectious retroviral particles were prepared to infect HPMCs. Expression levels of CTGF, fibronectin(FN), collagen 1 (col 1), laminin, and VEGF mRNA and protein were measured by semi-quantitative reverse transcription polymerase chain reaction and western blot assay. CTGF expression was increased after stimulation with TGF-beta1, but inhibited using each of the four independent CTGF shRNA constructs (P < 0.01). Moreover, expression of ECM proteins (FN, col 1, and laminin) and VEGF were upregulated after incubation with TGF-beta1, but elevated levels of ECM and VEGF induced by TGF-beta1 were significantly inhibited by RNAi (P < 0.01), but not by the empty retroviral vector (P > 0.05). From these results, we concluded that retrovirus-mediated CTGF shRNA can effectively inhibit ECM production and VEGF expression induced by TGF-beta1 in HPMCs. This study suggests that downregulation of CTGF may represent a potential therapeutic approach for peritoneal fibrosis through decreasing ECM accumulation and angiogenesis.
Xiao
Li
L
Department of Nephrology, the 2nd Xiang-Ya Hospital, Nephrology Institute of Central South University, Changsha, 410011, Hunan, China.
Sun
Lin
L
Liu
Fu-You
FY
Peng
You-Ming
YM
Duan
Shao-Bin
SB
D015153:Blotting, Western / Q000379:methods; D002478:Cells, Cultured; D055513:Connective Tissue Growth Factor / Q000235:genetics*; D015536:Down-Regulation / Q000235:genetics; D004848:Epithelium; D016326:Extracellular Matrix Proteins / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression / Q000235:genetics; D055785:Gene Knockdown Techniques / Q000379:methods*; D006801:Humans; D010537:Peritoneum / Q000187:drug effects / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology*; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism*
D016326:Extracellular Matrix Proteins; D053773:Transforming Growth Factor beta1; D042461:Vascular Endothelial Growth Factor A; D055513:Connective Tissue Growth Factor