MON-212 COMPLETE ABROGATION OF ALPHA SMOOTH MUSCLE ACTIN ON DUAL INHIBITION OF PHOSPHODIESTERASE 5 AND 5-HT2B INHIBITORS IN HUMAN PERITONEAL FIBROBLASTS ISOLATED FROM CAPD PATIENTS

Abstract

Peritoneal fibrosis is a major cause of ultrafiltration failure in continuous ambulatory peritoneal dialysis (CAPD) patients. Transforming growth factor beta 1 (TGF-β1) activates resident fibroblasts which trans-differentiate into myofibroblasts (MFBs), characterized by increased alpha-smooth muscle actin (α-SMA) expression and extracellular matrix (ECM) proteins production. Our objective was to evaluate anti-fibrotic efficacy on dual inhibition of Phosphodiesterase 5 (PDE5) inhibitors, Sildenafil & Zaprinast, plus 5-HT2B inhibitor, SB204741, respectively in human peritoneal fibroblasts (HPFB). Biopsy from parietal peritoneum (PB) of control patients (n=8) and CAPD patients (n=6) excised during laparotomy was incubated overnight in dispase (2.4 U/mL)/37°C. In disease mimicking strategy, HPFB were incubated with TGF-β1 (10 ng/ml)/1 hr, later with TGF-β1 (10 ng/ml)/[Sildenafil (10 µM) plus SB204741 (1 µM) or Zaprinast (10 µM) plus SB204741 (1 µM)]/24 hr. In pre-treatment strategy, HPFB were pretreated with [Sildenafil (10 µM) plus SB204741 (1 µM) or Zaprinast (10 µM) plus SB20474 (1 µM)]/1 hr and later with only TGF-β1 (10 ng/ml)/24 hr. Similar strategies were followed for individual treatments of inhibitors. Real-time qPCR for pro-fibrotic (COL1A1, COL1A2, ACTA2, CTGF and FN1) and anti-fibrotic genes (MMP2/TIMP1) was performed. Immunoblotting was performed for expression of α-SMA. In TGF-β1 stimulated HPFB, upregulated expression of pro-fibrotic genes was observed (p<0.05). Expression of pro-fibrotic genes in HPFB cells stimulated with TGF-β1 was significantly (p<0.05) reduced by dual inhibition strategy with complete amelioration of ACTA2 in comparison to their respective individual treatments (figure 1). Ratio of anti-fibrotic genes (MMP2/TIMP1) was restored significantly (p<0.05). In TGF-β1 stimulated HPFB, dual inhibition strategy decreased expression of α-SMA protein significantly (p<0.05). Complete inhibition of α-SMA on dual inhibition strategy of PDE5 and 5-HT2B receptor antagonists suggest inhibition of conversion of resident fibroblasts to MFBs.