1 CGMP Signaling Regulates Liver Sinusoidal Endothelial Cell (SEC) Phenotype and Accelerates Reversal of Cirrhosis

Differentiated, but not capillarized, SEC promote reversion of stellate cells (HSC) to quiescence.Capillarization precedes fibrosis In Vivo.Thus restoring SEC differentiation may promote resolution of fibrosis.This study examines whether NO maintains SEC phenotype In Vitro through the soluble guanylate cyclase (sGC)/cGMP/protein kinase G (PKG) pathway and whether In Vivo activation of sGC accelerates reversal of capillarization and cirrhosis.Methods Rat SEC were cultured for 2 days with inhibitors or agonists and examined by scanning EM for fenestrae in sieve plates, defining features of SEC differentiation.Controls: SEC cultured alone (complete defenestration) or with 40 ng/ml VEGF (fully fenestrated).Thioacetamide-induced (200mg/kg i.p. biw for 3 weeks) cirrhosis was followed by 0.3 mg/ kg p.o. BAY 60-2770 (kind gift from Bayer Schering Pharma), an NO-independent activator of sGC.Sirius red staining for fibrosis was assessed blindly (semi-quantitative score 0-6).In Vitro studies 1.SEC cultured with VEGF plus L-NAME (nitric oxide synthase inhibitor), with VEGF/ODQ (sGC inhibitor) or with VEGF/Rp-8-pCPT-PET-cGMPS (PKG inhibitor) defenestrated completely.Thus the NO/cGMP pathway is necessary to maintain fenestrated SEC.2.SEC grown with DETA-NONOate (6μM) produced similar levels of NO and cGMP to SEC cultured with VEGF, but SEC defenestrated.SEC grown with YC-1 (sGC activator) or 8-pCPT-cGMP (cGMP analog) defenestrated.Thus the NO/cGMP pathway alone is insufficient to maintain SEC phenotype.3.SEC grown with VEGF, L-NAME and DETA-NONOate, with VEGF, L-NAME and YC-1, or with VEGF, ODQ and 8-pCPT-cGMP remained fully fenestrated.Thus maintenance of SEC phenotype requires both VEGF and the NO/cGMP pathway.4.BAY60-2770 alone was not sufficient to reverse HSC activation, but reversal required co-cultured SEC and was NO-independent.In Vivo studies with BAY 60-2770 1-week treatment normalized cGMP level in SEC [0.82± 0.02 fmol/μg protein in normal vs 0.83± 0.05 fmol/μg protein in treated vs 0.47±0.06fmol/μg protein in solvent controls (p= 0.011)] and completely reversed capillarization [normal porosity 6.80 ± 0.31%; 6.24±0.37% in treated vs 3.14 ± 0.22% in solvent controls (p<0.01)],but cirrhosis persisted.2-week treatment caused reversal to bridging fibrosis vs persistent cirrhosis in solvent controls.Conclusions Both VEGF independent of NO plus VEGF-stimulated NO acting through the sGC/cGMP/PKG pathway are necessary and sufficient to maintain SEC differentiation.Reversal of HSC activation by sGC activation requires the presence of SEC.Stimulation of sGC reversed capillarization, which promoted reversal of cirrhosis.