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Non-alcoholic steatohepatitis (NASH) is the most severe stage of non-alcoholic fatty liver disease (NAFLD) with extensive fibrosis, inflammation and steatosis. To date, there are no approved therapies for NAFLD/NASH. To identify novel therapeutic targets against hepatic fibrosis, we investigated the role of thrombospondin-I (TSP-1), a protein that regulates transforming growth factor beta 1 (TGF-β1) signaling and subsequent effects on fibrosis, angiogenesis, and inflammation. We first determined that shRNA-mediated TSP-1 ablation caused attenuated expression of fibrotic markers in activated primary human hepatic stellate cells (HSCs), which are the major myofibroblast precursor cells in the liver. To investigate whether TSP-1 has a role in NASH in vivo, we examined the effect of TSP-1 genetic deficiency in a mouse model that utilizes a choline-deficient L-amino acid defined high fat diet (CDAHFD). We demonstrated that CDAHFD-fed TSP-1-deficient mice were less prone to hepatic fibrosis, as indicated by histology and reduced expression of inflammation and fibrosis mRNA markers. In addition, we observed that serum lipid levels were lower in CDAHFD-fed TSP-1-deficient mice. Transcriptomic analysis of hepatic mRNA from TSP-1 deficient mice fed control and CDAHFD diets revealed upregulation of the peroxisome proliferator-activator receptor alpha (PPARα) pathway, which is involved in fatty acid metabolism and inflammation in the liver. Published studies indicate that PPARα activation alleviates NAFLD/NASH symptoms in mouse models and patients. Our results reveal a previously unknown relationship between TSP-1 and PPARα and suggest that TSP-1 could be a candidate target for NASH treatment. In a second set of studies, we investigated the molecular pathways involved in activation of primary human HSCs. We performed transcriptomic analysis of TGF-β1activated human HSCs and also characterized the anti-fibrotic mechanisms of the bromodomain inhibitor JQ1, which is known to attenuate fibrosis in experimental models. Our transcriptomic analysis identified an activated signal transducer and activator of transcription 3 (STAT3) signature in TGF-β1-treated HSCs. We determined that Cpd188-mediated STAT3 inhibition promotes HSC apoptosis, enabling resolution of fibrosis. STAT3 overexpression attenuated Cpd188-mediated apoptosis. Our findings highlight an important role for STAT3 in HSC activation. Therefore, STAT3 inhibition constitutes a promising direction for hepatic fibrosis treatment.

CD36 is a multi-ligand scavenger receptor present in many cells including microvascular endothelial cells (MVEC) in which it induces anti-angiogenic signaling via its cognate protein ligand thrombospondin-1 (TSP-1). Cell-derived microparticles (MP), less than or equal to 1µm vesicles shed from many cell types during activation or apoptosis bind CD36 via phosphatidylserine (PS) exposed on their surface. We hypothesized that CD36-MP interactions on MVEC inhibits angiogenesis. We found that MP generated in vitro from different cellular sources (THP1, human umbilical vein endothelial cells (HUVEC), MVEC and RBCs) and endogenous circulating human MP inhibit angiogenic activities of MVEC using in vitro and in vivo migration, and in vitro tube-formation assays. Experiments on different passages and batches of MVEC with different levels of CD36 expression, murine CD36 transfection in HUVEC that do not express CD36 and cd36-/- mice showed a CD36 dependent inhibition of MVEC migration by MP. Studies with annexin-V showed a PS dependent inhibitory effect. Mechanistically, MP-mediated inhibition was via induction of reactive oxygen species (ROS) in a NADPH oxidase and Fyn kinase dependent manner. CD36 forms a complex with vascular endothelial growth factor receptor-2 (VEGFR2) in MVEC and inhibits VEGF signaling. Src homology 2 domain containing protein tyrosine phosphatase (SHP)-2 is constitutively associated with the VEGFR2-CD36 signaling complex and TSP-1-CD36 interaction induces SHP-1 association with the complex. Mechanism(s) of TSP-1-mediated inhibition of VEGFR2 signaling was not known. We hypothesized that CD36-TSP-1 interaction recruits SHP-1 to VEGFR2-CD36 complex, to dephosphorylate VEGFR2 and attenuates VEGF signaling. Immunohistochemistry of MVEC exposed to a recombinant protein containing the CD36 binding domain of thrombospondin-1 (known as the TSR domain) induced association of SHP-1 with CD36. Silencing CD36 expression in MVEC by siRNA showed that TSR-induced SHP-1/VEGFR2 complex formation required CD36. Silencing SHP-1 expression in MVEC by siRNA abrogated TSR-mediated inhibition of VEGFR2 phosphorylation as well as TSR-mediated inhibition of VEGF-induced endothelial cell migration. These studies reveal MP as an anti-angiogenic ligand for CD36 and identify different anti-angiogenic signaling pathways of CD36 ligands via ROS generation and SHP-1 mediated inhibition of VEGFR2 signaling providing novel targets to modulate angiogenesis therapeutically.