Discovery of 3,7-dimethoxyflavone that inhibits liver fibrosis based on dual mechanisms of antioxidant and inhibitor of activated hepatic stellate cell.

The important pathway toward liver fibrosis is the TGF-ß1-induced activation of hepatic stellate cells (HSCs). To discover chemicals to inhibit liver fibrosis, we screened 3000 chemicals using cell array system where human HSCs line LX2 cells are activated with TGF-ß1. We discovered 3,7-dimethoxyflavone (3,7-DMF) as a chemical to inhibit TGF-ß1-induced activation of HSCs. In the thioacetamide (TAA)-induced mouse liver fibrosis model, 3,7-DMF treatment via intraperitoneal or oral administration prevented liver fibrosis as well as reversed the established fibrosis in the separate experiments. It also reduced liver enzyme elevation, suggesting protective effect on hepatocytes because it has antioxidant effect. Treatment with 3,7-DMF induced antioxidant genes, quenches ROS away, and improved the hepatocyte condition that was impaired by H2O2 as reflected by restoration of HNF-4α and albumin. In the TAA-mouse liver injury model also, TAA significantly increased ROS in the liver which led to decrease of albumin and nuclear expression of HNF-4α, increase of TGF-ß1 and hepatocytes death, accumulation of lipid, and extra-nuclear localization of HMGB1. Treatment of 3,7-DMF normalized all these pathologic findings and prevented or resolved liver fibrosis. In conclusion, we discovered 3,7-DMF that inhibits liver fibrosis based on dual actions; antioxidant and inhibitor of TGF-ß1-induced activation of HSCs.

Discovery of chemerin as the new chemoattractant of human mesenchymal stem cells.

BACKGROUND: The homing capacity of human mesenchymal stem cells (hMSCs) to the injured sites enables systemic administration of hMSCs in clinical practice. In reality, only a small proportion of MSCs are detected in the target tissue, which is a major bottleneck for MSC-based therapies. We still don't know the mechanism how MSCs are chemo-attracted to certain target organ and engrafted through trans-endothelial migration. In this study, we aimed to determine the mechanism how the circulating hMSCs home to the injured liver. METHODS AND RESULTS: When we compare the cytokine array between normal and injured mouse liver at 1-day thioacetamide (TAA)-treatment, we found that chemerin, CXCL2, and CXCL10 were higher in the injured liver than normal one. Among three, only chemerin was the chemoattractant of hMSCs in 2D- and 3D-migration assay. Analysis of the signal transduction pathways in hMSCs showed that chemerin activated the phosphorylation of JNK1/2, ERK1/2 and p38, and finally upregulated CD44, ITGA4, and MMP-2 that are involved in the transendothelial migration and extravasation of MSCs. Upstream transcription regulators of CD44, ITGA4, and MMP-2 after chemerin treatment were MZF1, GATA3, STAT3, and STAT5A. To develop chemerin as a chemoattractant tool, we cloned gene encoding the active chemerin under the CMV promoter (CMV-aChemerin). We analyzed the migration of hMSCs in the 3D model for space of the Disse, which mimics transmigration of hMSCs in the liver. CMV-aChemerin-transfected hepatocytes were more effective to attract hMSC than control hepatocytes, leading to the enhanced transendothelial migration and homing of hMSCs to liver. The homing efficiency of the intravascularly-delivered hMSCs to liver was evaluated after systemic introduction of the CMV-aChemerin plasmid packed in liposome-vitamin A conjugates which target liver. CMV-aChemerin plasmid targeting liver significantly enhanced homing efficiency of hMSCs to liver compared with control plasmid vector. CONCLUSIONS: Chemerin is the newly found chemoattractant of hMSCs and may be a useful tool to manipulate the homing of the intravascularly-administered hMSC to the specific target organ.

Endothelin-1 enhances the regenerative capability of human bone marrow-derived mesenchymal stem cells in a sciatic nerve injury mouse model.

We expanded the application of endothelin-1 (EDN1) by treating human mesenchymal stem cell (hMSC) organotypic spinal cord slice cultures with EDN1. EDN1-treated hMSCs significantly enhanced neuronal outgrowth. The underlying mechanism of this effect was evaluated via whole-genome methylation. EDN1 increased whole-genome demethylation and euchromatin. To observe demethylation downstream of EDN1, deaminases and glycosylases were screened, and APOBEC1 was found to cause global demethylation and OCT4 gene activation. The sequence of methyl-CpG-binding domain showed similar patterns between EDN1- and APOBEC1-induced demethylation. SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin subfamily A member 4 (SMARC A4) and SMARC subfamily D, member 2 (SMARC D2) were screened via methyl-CpG-binding domain sequencing as a modulator in response to EDN1. Chromatin immunoprecipitation of the H3K9me3, H3K27me3, and H3K4me4 binding sequences on the APOBEC1 promoter was analyzed following treatment with or without siSMARC A4 or siSMARC D2. The results suggested that SMARC A4 and SMARC D2 induced a transition from H3K9me3 to H3K4me3 in the APOBEC1 promoter region following EDN1 treatment. Correlations between EDN1 pathways and therapeutic efficacy in hBM-MSCs were determined in a sciatic nerve injury mouse model. Thus, EDN1 may be a useful novel-concept bioactive peptide and biomaterial component for improving hMSC regenerative capability.