Inhibition of mesangial cell proliferation by antisense oligodeoxynucleotide targeting preproendothelin-1 mRNA in vitro

To investigate the effects of endothelin-1 (ET-1) on mesangial cell proliferation in vitro. Antisense oligodeoxynucleotide (As-ODN) and its control sequences, sense (Se-ODN) and mismatch (Mis-ODN) oligodeoxynucleotides, targeting preproendothelin-1 (ppET-1) mRNA were delivered into cultured human mesangial cells (HMC) with lipofectin-mediated gene transfer method. The cytotoxicity of lipofectin was determined with lactate dehydrogenase (LDH) release assay. The efficiency of ODNs transfer into HMC was examined with biotinylated As-ODN staining. The effect of As-ODN on expression of ppET-1 mRNA was analyzed with semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). The action of As-ODN on HMC ET-1 secretion was tested by radioimmunoassay (RIA). The influence of As-ODN on HMC proliferation was evaluated with MTT method. As-ODN was transferred into HMC with lipofectin without any impairment of cell viability. The ppET-1 mRNA expression, the ET-1 secretion and the cell proliferation were inhibited by As-ODN transferred into HMC, while Se-ODN and Mis-ODN transferred into HMC did not show any effects on all of these. Decrease of ET-1 secretion by HMC, caused by the down-regulation of ppET-1 mRNA expression after As-ODN transfer, led to inhibition of HMC proliferation. These results suggest ET-1 is one of the autocrine growth factors of HMC, and may play an important role in the pathogenesis of proliferative glomerulonephritis.