Hyaluronan synthase 2, a target of miR-200c, promotes carbon tetrachloride-induced acute and chronic liver inflammation via regulation of CCL3 and CCL4.

Liver fibrosis occurs during wound healing after repeated liver injury and is characterized by extensive extracellular matrix deposition. We previously identified hyaluronan synthase 2 (HAS2) as a driver of liver fibrosis and hepatic stellate cell (HSC) activation. Developing strategies to suppress HSC activation is key to alleviating liver fibrosis, and HAS2 is an attractive candidate for intervention. To gain insight into the molecular function of HAS2, we investigated its posttranscriptional regulation. We found that miR-200c directly targets the 3' untranslated regions of HAS2. Moreover, miR-200c and HAS2 were inversely expressed in fibrotic human and mouse livers. After establishing the direct interaction between miR-200c and HAS2, we investigated the functional outcome of regulating HAS2 expression in three murine models: CCl4-induced acute liver injury, CCl4-induced chronic liver fibrosis, and bile duct ligation-induced liver fibrosis. Hepatic Has2 expression was induced by acute and chronic CCl4 treatment. In contrast, miR-200c expression was decreased after CCl4 treatment. HSC-specific Has2 deletion reduced the expression of inflammatory markers and infiltration of macrophages in the models. Importantly, hyaluronidase-2 (HYAL2) but not HYAL1 was overexpressed in fibrotic human and murine livers. HYAL2 is an enzyme that can cleave the extracellular matrix component hyaluronan. We found that low-molecular-weight hyaluronan stimulated the expression of inflammatory genes. Treatment with the HA synthesis inhibitor 4-methylumbelliferone alleviated bile duct ligation-induced expression of these inflammatory markers. Collectively, our results suggest that HAS2 is negatively regulated by miR-200c and contributes to the development of acute liver injury and chronic liver inflammation via hyaluronan-mediated immune signaling

The matricellular protein CCN5 inhibits fibrotic deformation of retinal pigment epithelium

Retinal pigment epithelium (RPE) plays an essential role in maintaining retinal function, and its defect is thought to be critically implicated in various ocular disorders. This study demonstrated that the matricellular protein CCN5 was down-regulated in ARPE-19 cells treated with the pro-fibrotic agent transforming growth factor (TGF)-beta. A recombinant adenovirus expressing CCN5 (AdCCN5) was used to restore the level of CCN5 in these cells. AdCCN5 prevented TGF-beta-induced fibrotic changes, including disruption of tight junctions, up-regulation of mesenchymal marker proteins, and down-regulation of epithelial marker proteins. In addition, AdCCN5 prevented TGF-beta-induced functional defects, including increased migratory activity and reduced phagocytic activity. Notably, AdCCN5 reversed morphological and functional defects pre-established by TGF-beta prior to viral infection. The CCN5 level was down-regulated in RPE of 18-month-old Ccl2(-/)(-) mice, which exhibited retinal defects. Restoration of the CCN5 level via intravitreal injection of a recombinant adeno-associated virus expressing CCN5 (AAV9-CCN5) normalized the altered expression of mesenchymal, epithelial, and functional marker proteins, as assessed by western blotting and immunohistochemistry. Taken together, these data suggest that down-regulation of CCN5 is associated with fibrotic deformation of RPE under pathological conditions and that restoration of the CCN5 level effectively promotes recovery of deformed RPE.Y

A Food Effect Study of an Oral Thrombin Inhibitor and Prodrug Approach To Mitigate It

LB30870, a new direct thrombin inhibitor, showed 80% reduction in oral bioavailability in fed state. The present study aims to propose trypsin binding as a mechanism for such negative food effect and demonstrate a prodrug approach to mitigate food effect. Effect of food composition on fed state oral bioavailability of LB30870 was studied in dogs. Various prodrugs were synthesized, and their solubility, permeability, and trypsin binding affinity were measured. LB30870 and prodrugs were subject to cocrystallization with trypsin, and the X-ray structures of cocrystals were determined. Food effect was studied in dogs for selected prodrugs. Protein or lipid meal appeared to affect oral bioavailability of LB30870 in dogs more than carbohydrate meal. Blocking both carboxyl and amidine groups of LB30870 resulted in trypsin <i>K</i>_i values orders of magnitude higher than that of LB30870. Prodrugs belonged to either Biopharmaceutical Classification System I, II, or III. X-ray crystallography revealed that prodrugs did not bind to trypsin, but instead their hydrolysis product at the amidine blocking group formed cocrystal with trypsin. A prodrug with significantly less food effect than LB30870 was identified. Binding of prodrugs to food components such as dietary fiber appeared to counteract the positive effect brought with the prodrug approach. Further formulation research is warranted to enhance the oral bioavailability of prodrugs. In conclusion, this study is the first to demonstrate that the negative food effect of LB30870 can be attributed to trypsin binding. Trypsin binding study is proposed as a screening tool during lead optimization to minimize food effect