Acceleration of resolution of cirrhosis by normalization of liver sinusoidal endothelial cell (LSEC) phenotype through the cGMP signaling pathway

Liver sinusoidal endothelial cells (LSEC) are endothelial cells lining the liver sinusoid. LSEC have a unique morphological phenotype among all mammalian endothelial cells, i.e. LSEC have open fenestrae grouped into sieve plates and also lack an organized basement membrane. In addition, LSEC possess both scavenger and antigen presentation functions, which are usually only observed in hematopoietic-derived cells. Given the many functions of LSEC, maintenance of normal LSEC phenotype has important implications.;Study 1: Sinusoidal obstruction syndrome (SOS) is initiated by damage to LSEC, which can be induced by either myeloablative chemo-irradiation or ingestion of pyrrolizidine alkaloids such as monocrotaline (Mct). To identify an additional marker for LSEC progenitor cells, this study identifies CD45 as a novel LSEC marker. Methods: Immunostaining was performed to confirm CD45 as an LSEC marker. Mct-treated female rats received infusions of male CD133 + bone marrow cells at the peak of sinusoidal injury. The y-chromosome was identified in isolated LSEC by fluorescent in situ hybridization. Results: LSEC in uninjured liver have both hematopoietic (CD45) and endothelial (CD31) markers. After Mct-induced SOS, infusion of bone marrow-derived CD133 + progenitor cells replace more than one-quarter of LSEC. Conclusion: LSEC have both hematopoietic and endothelial markers. Bone marrow-derived CD133+ progenitors replace LSEC after liver injury.;Study 2: The normal LSEC phenotype is maintained by VEGF-stimulated nitric oxide (NO). NO can work through the classical soluble guanylate cyclase (sGC)/cGMP/protein kinase G (PKG) pathway or through protein S-nitrosylation. Methods: LSEC were cultured with a variety of inhibitors and/or agonists and examined by scanning EM for fenestrae organized in sieve plates, the defining morphological marker for LSEC. Results: LSEC cultured with VEGF/ODQ (sGC inhibitor) or with VEGF/Rp-8-pCPT-PET-cGMPS (PKG inhibitor) defenestrated. LSEC grown with YC-1 (sGC activator) or 8-pCPT-cGMP (cGMP analog) defenestrated, but remained fully fenestrated when VEGF/L-NAME were added to YC-1 or VEGF/ODQ were added to 8-pCPT-cGMP. Conclusion: Both VEGF independent of NO plus VEGF-stimulated NO acting through the sGC/cGMP/PKG pathway are necessary and sufficient to maintain LSEC differentiation.;Study 3: Differentiated, but not capillarized, LSEC promote reversion of activated stellate cells (HSC) to quiescence. Thus restoring LSEC differentiation may promote resolution of fibrosis. This study examined whether in vivo activation of sGC accelerated reversal of capillarization and cirrhosis. Methods: Thioacetamide-induced cirrhosis was followed by BAY 60-2770 (kind gift from Bayer Schering), a sGC activator. Fibrosis was assessed by Sirius red staining and a-smooth muscle actin expression was measured by immunoblot. Results: After induction of cirrhosis with thioacetamide, one-week treatment with BAY 60-2770 restored normal fenestration, but HSC activation and cirrhosis were unchanged. One additional week without drug caused regression to bridging fibrosis vs persistent cirrhosis in controls. 3 weeks thioacetamide followed by 3 weeks thioacetamide plus BAY 60-2770 completely reversed capillarization and prevented progression to advanced cirrhosis. Conclusions: Activation of sGC reverses capillarization and the normalized LSEC alone then accelerates regression of cirrhosis. sGC activation also prevents progression of cirrhosis despite ongoing thioacetamide exposure.;In summary, our research provides novel insights into regulation of the liver sinusoidal endothelial cell phenotype, which suggests a possible anti-fibrotic treatment strategy. Furthermore, these findings may have broader implications with regards to cross talk between endothelial cells and pericytes in organs other than the liver.