FoxO3 restricts liver regeneration by suppressing the proliferation of hepatocytes.

Upon injury, the liver is capable of substantial regeneration from the original tissue until an appropriate functional size. The underlying mechanisms controlling the liver regeneration processes are not well elucidated. Previous studies have proposed that the transcription factor FoxO3 is involved in various liver diseases, but its exact role in the regulation of liver regeneration remains largely unclear. To directly test the detailed role of FoxO3 in liver regeneration, both a constitutive Albumin-Cre driver line and adeno-associated virus serotype 8 (AAV8)-Tbg-Cre (AAV-Cre)-injected adult FoxO3fl/fl mice were subjected to 70% partial hepatectomy (PH). Our data demonstrate that FoxO3 deletion accelerates liver regeneration primarily by limiting polyploidization and promoting the proliferation of hepatocytes during liver regeneration. RNA-seq analysis indicates that FoxO3 deficiency greatly alters the expression of gene sets associated with cell proliferation and apoptosis during liver regeneration. Chromatin immunoprecipitation-PCR (ChIP-PCR) and luciferase reporter assays reveal that FoxO3 promotes the expression of Nox4 but suppresses the expression of Nr4a1 in hepatocytes. AAV8 virus-mediated overexpression of Nox4 and knockdown of Nr4a1 significantly suppressed hepatocyte proliferation and liver regeneration in FoxO3-deficient mice. We demonstrate that FoxO3 negatively controls hepatocyte proliferation through Nox4 upregulation and Nr4a1 downregulation, thereby ensuring appropriate functional regeneration of the liver. Our findings provide novel mechanistic insight into the therapeutic mechanisms of FoxO3 in liver damage and repair.

CpG oligodeoxynucleotide preconditioning improves cardiac function after myocardial infarction via modulation of energy metabolism and angiogenesis.

Unmethylated CpG oligodeoxynucleotide (CpG-ODN), a Toll-like receptor 9 (TLR9) ligand, has been shown to protect against myocardial ischemia/reperfusion injury. However, the potential effects of CpG-ODN on myocardial infarction (MI) induced by persistent ischemia remains unclear. Here, we investigated whether and how CpG-ODN preconditioning protects against MI in mice. C57BL/6 mice were treated with CpG-ODN by i.p. injection 2 hr prior to MI induction, and cardiac function, and histology were analyzed 2 weeks after MI. Both 1826-CpG and KSK-CpG preconditioning significantly improved the left ventricular (LV) ejection fraction (LVEF) and LV fractional shortening (LVFS) when compared with non-CpG controls. Histological analysis further confirmed the cardioprotection of CpG-ODN preconditioning. In vitro studies further demonstrated that CpG-ODN preconditioning increases cardiomyocyte survival under hypoxic/ischemic conditions by enhancing stress tolerance through TLR9-mediated inhibition of the SERCA2/ATP and activation of AMPK pathways. Moreover, CpG-ODN preconditioning significantly increased angiogenesis in the infarcted myocardium compared with non-CpG. However, persistent TLR9 activation mediated by lentiviral infection failed to improve cardiac function after MI. Although CpG-ODN preconditioning increased angiogenesis in vitro, both the persistent stimulation of CpG-ODN and stable overexpression of TLR9 suppressed the tube formation of cardiac microvascular endothelial cells. CpG-ODN preconditioning significantly protects cardiac function against MI by suppressing the energy metabolism of cardiomyocytes and promoting angiogenesis. Our data also indicate that CpG-ODN preconditioning may be useful in MI therapy.

Gene delivery of hypoxia-inducible VEGF targeting collagen effectively improves cardiac function after myocardial infarction.

Vascular endothelial growth factor (VEGF) plays important roles in improvement of cardiac function following myocardial infarction (MI). However, the lack of a steerable delivery system of VEGF targeting the infarcted myocardium reduces the therapeutic efficacy and safety. Here, we constructed a series of lentiviral vector systems which could express a fusion protein consisted of a collagen-binding domain (CBD) and hVEGF (CBDhVEGF), under the control of 5HRE-hCMVmp (5HRE), the hypoxia-inducible promoter consists of five copies of the hypoxia-responsive element (HRE) and a human cytomegalovirus minimal promoter (hCMVmp). We demonstrated that 5HRE has the comparable ability to strongly drive CBDhVEGF under hypoxic condition as the ubiquitous CMV promoter, but it can hardly drive target gene under normoxic condition. 5HRE-drived CBDhVEGF specifically bound to type I collagen and significantly promoted the viability of HUVEC cells. Moreover, after injection of lentivirus into heart of mouse with MI, CBDhVEGF was mainly retained in infarcted myocardium where containing rich collagen and significantly improved angiogenesis and cardiac function when compared with hVEGF. Moreover, CBDhVEGF mediated by lentivirus has little leakage from infarcted zone into blood than hVEGF. Taken together, our results indicate that 5HRE-CBDhVEGF lentiviral vector system could improve cardiac function in the collagen-targeting and hypoxia-inducible manners.

Expression profile of rrbp1 genes during embryonic development and in adult tissues of Xenopus laevis.

Recent studies suggest that ribosome-binding protein 1 (RRBP1) is involved in multiple diseases such as tumorigenesis and cardiomyopathies. However, its function during embryonic development remains largely unknown. We searched Xenopus laevis database with human RRBP1 protein sequence and identified two cDNA sequences encoding Xenopus orthologs of RRBP1 including rrbp1a (NM_001089623) and rrbp1b (NM_001092468). Both genes were firstly detected at blastula stage 8 with weak signals in animal hemisphere by whole mount in situ hybridization. Evident expression of rrbp1 was mainly detected in cement gland and notochord at neurula and tailbud stages. Heart expression of rrbp1 was detected at stage 36. RT-PCR results indicated that very weak expression of rrbp1a was firstly detected in oocytes, followed by increasing expression until stage 39. Differently, very weak expression of rrbp1b was firstly observed at stage 2, and then maintained at a lower level to stage 17 followed by an intense expression from stages 19-39. Moreover, both expression profiles were also different in adult tissues. This study reports Xenopus rrbp1 expression during early embryonic development and in adult tissues. Our study will facilitate the functional analysis of Rrbp1 family during embryonic development.

The CXCL10/CXCR3 axis promotes cardiac microvascular endothelial cell migration via the p38/FAK pathway in a proliferation-independent manner.

CXCL10 is a chemokine with potent chemotactic activity for immune and non-immune cells expressing its receptor CXCR3. Previous studies have demonstrated that CXCL10 is involved in myocardial infarction. However, the role of CXCL10 in cardiac microvascular endothelial cell (CMEC) regulation and related mechanisms remains unclear. In this study, we investigated the effects of CXCL10 on the CMEC migration and explored its potential molecular mechanism by wound healing, cell proliferation and viability analysis. Furthermore, migration-related signaling pathways, including FAK, Erk, p38 and Smad, were examined by Western blotting. We found that CXCL10 significantly promotes CMEC migration under normal conditions and during hypoxia/ischemia. However, no significant differences in CMEC proliferation and viability were observed with or without CXCL10 treatment. CXCL10-mediated CMEC migration was greatly blocked by treatment with an anti-CXCR3 antibody. Although CXCL10 treatment promoted phosphorylation and activation of the FAK, Erk, and p38 pathways during hypoxia/ischemia, CXCL10-mediated CMEC migration was significantly blocked by p38 and FAK inhibitors, but not by an Erk inhibitor. Furthermore, CXCL10-mediated FAK activation was suppressed by the p38 inhibitor. These findings indicated that the CXCL10/CXCR3 pathway promotes the migration of CMECs under normal conditions and during hypoxia/ischemia in a proliferation-independent manner, at least in part, through regulation of the p38/FAK pathways.

Hypoxia/ischemia promotes CXCL10 expression in cardiac microvascular endothelial cells by NFkB activation.

CXCL10, the chemokine with potent chemotactic activity on immune cells and other non-immune cells expressing its receptor CXCR3, has been demonstrated to involve in myocardial infarction, which was resulted from hypoxia/ischemia. The cardiac microvascular endothelial cells (CMECs) are the first cell type which is implicated by hypoxia/ischemia. However, the potential molecular mechanism by which hypoxia/ischemia regulates the expression of CXCL10 in CMECs remains unclear. In the present study, the expression of CXCL10 was firstly examined by real-time PCR and ELISA analysis. Several potential binding sites (BS) for transcription factors including NF-kappaB (NFkB), HIF1 alpha (HIF1α) and FoxO3a were identified in the promoter region of CXCL10 gene from -2000 bp to -1 bp using bioinformatics software. Luciferase reporter gene vectors for CXCL10 promoter and for activation of above transcription factors were constructed. The activation of NFkB, hypoxia-inducible transcription factor-1 alpha (HIF-1α) and FoxO3a was also analyzed by Western blotting. It was shown that the production of CXCL10 in CMECs was significantly increased by hypoxia/ischemia treatment, in parallel with the activation of CXCL10 promoter examined by reporter gene vector system. Furthermore, transcription factors including NFkB, HIF1α and FoxO3a were activated by hypoxia/ischemia in CMECs. However, over-expression of NFkB, but not that of HIF1α or FoxO3a, significantly promoted the activation of CXCL10 promoter reporter gene. These findings indicated that CXCL10 production in CMECs was significantly increased by hypoxia/ischemia, at least in part, through activation of NFkB pathway and subsequently binding to CXCL10 promoter, finally promoted the transcription of CXCL10 gene.