ABSTRACT FINAL ID: 1667
TITLE: Role of Incomplete Stem Cell Maturation in Hepatic Fibrosis
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Background: Capillarization, lack of liver sinusoidal endothelial cell (LSEC) fenestration with formation of an organized basement membrane, precedes and is permissive for hepatic stellate cell (HSC) activation. Capillarization is thought to be a de-differentiation of injured LSECs. We have shown that injured LSECs are repaired by bone marrow (BM)-derived Sinusoidal endothelial cell PROgenitor Cells (sprocs). Aims: This study examines the nature of capillarization and why capillarized LSECs do not maintain HSC quiescence. Methods: Rats transplanted with transgenic EGFP+ BM were treated for 3 weeks with thioacetamide. LSECs from cirrhotic liver were sorted into GFP+ (BM-derived) or GFP- (resident) LSECs. LSECs and BM sprocs healthy rats served as controls. Results: In early cirrhosis, 66% of LSECs were BM-derived and 32% were resident LSECs. BM sprocs and BM-derived LSECs were similar, with absence of fenestration (=capillarized), low eNOS activity, and low presence of LYVE-1 (6.9 and 33%, respectively) and CD32b (2.8 and 6.4%, resp). Both resident LSECs and control LSECs had fenestrae organized in sieve plates, high eNOS activity, and high positivity for LYVE-1 (72.5% and 75.8%, resp) and CD32b (61.5% and 68.6%, resp). Conditioned media (CM) from control LSECs maintained HSC quiescence, but CM from cirrhotic LSECs did not. Proteomic analysis revealed that CM from LSECs, but not from cirrhotic LSECs, contained HB-EGF, a protein that maintains HSC quiescence (Huang et al, Lab Invest 2012). Neutralizing antibody to HB-EGF prevented maintenance of HSC quiescence by control LSECs. Pro-HB-EGF gene expression was high in control LSECs, intermediate in resident LSECs and low in BM-derived LSECs (p<0.001). Pro-HB-EGF was detected in the cytoplasmic membrane of 27% of BM-derived LSECs but in < 1% of resident LSECs. Expression of HB-EGF sheddases (ADAM10, ADAM17, ADAM19, ADAMTS1 and MMP14) was downregulated in BM-derived LSECs compared to control LSECs (p<0.05). Conclusions: 1. “Capillarized” LSECs are BM sprocs recruited to injured liver that fail to fully mature, i.e. immature LSECs. 2. HB-EGF is the signal from LSECs that maintains HSC quiescence. 3. Pro-HB-EGF gene expression and shedding of HB-EGF are decreased in immature LSECs. In fibrotic injury, replacement of mature LSECs by immature LSECs decreases release of HB-EGF and this is permissive for HSC activation.