RAR‐Related Orphan Receptor Gamma (ROR‐γ) Mediates Epithelial‐Mesenchymal Transition Of Hepatocytes During Hepatic Fibrosis

The epithelial‐mesenchymal transition (EMT) is involved in many different types of cellular behavior, including liver fibrosis. In this report, we studied a novel function of RAR‐related orphan receptor gamma (ROR‐γ) in hepatocyte EMT during liver fibrosis. To induce EMT in vitro, primary hepatocytes and FL83B cells were treated with TGF‐β1. Expression of ROR‐γ was analyzed by Western blot in the fibrotic mouse livers and human livers with cirrhosis. To verify the role of ROR‐γ in hepatocyte EMT, we silenced ROR‐γ in FL83B cells using a lentiviral short hairpin RNA (shRNA) vector. The therapeutic effect of ROR‐γ silencing was investigated in a mouse model of TAA‐induced fibrosis by hydrodynamic injection of plasmids. ROR‐γ expression was elevated in hepatocyte cells treated with TGF‐β1, and ROR‐γ protein levels were elevated in the fibrotic mouse livers and human livers with cirrhosis. Knockdown of ROR‐γ resulted in the attenuation of TGF‐β1‐induced EMT in hepatocytes. Strikingly, ROR‐γ bound to ROR‐specific DNA response elements (ROREs) in the promoter region of TGF‐β type I receptor (Tgfbr1) and Smad2, resulting in the downregulation of Tgfbr1 and Smad2 after silencing of ROR‐γ. Therapeutic delivery of shRNA against ROR‐γ attenuated hepatocyte EMT and ameliorated liver fibrosis in a mouse model of TAA‐induced liver fibrosis. Overall, our results suggest that ROR‐γ regulates TGF‐β‐induced EMT in hepatocytes during liver fibrosis. We suggest that ROR‐γ may become a potential therapeutic target in treating liver fibrosis. J. Cell. Biochem. 118: 2026–2036, 2017. © 2016 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals Inc.     

Periostin down‐regulation attenuates the pro‐fibrogenic response of hepatic stellate cells induced by TGF‐β1

Liver fibrosis is characterized by an exacerbated accumulation of deposition of the extracellular matrix (ECM), and the activation of hepatic stellate cells (HSC) plays a pivotal role in the development of liver fibrosis. Periostin has been shown to regulate cell adhesion, proliferation, migration and apoptosis; however, the involvement of periostin and its role in transforming growth factor (TGF)‐β1‐induced HSC activation remains unclear. We used RT‐PCR and Western blot to evaluate the expression level of periostin in hepatic fibrosis tissues and HSCs, respectively. Cell proliferation was determined using the Cell Proliferation ELISA BrdU kit, cell cycle was analysed by flow cytometry. The expression of α‐smooth muscle actin (α‐SMA), collagen I, TGF‐β1, p‐Smad2 and p‐Smad3 were determined by western blot. Our study found that periostin was up‐regulated in liver fibrotic tissues and activated HSCs. In addition, siRNA‐periostin suppressed TGF‐β1‐induced HSC proliferation. The HSC transfected with siRNA‐periostin significantly inhibited TGF‐β1‐induced expression levels of α‐SMA and collagen I. Furthermore, TGF‐β1 stimulated the expression of periostin, and siRNA‐periostin attenuated TGF‐β1‐induced Smad2/3 activation in HSCs. These results suggest that periostin may function as a novel regulator to modulate HSC activation, potentially by promoting the TGF‐β1/Smad signalling pathway, and propose a strategy to target periostin for the treatment of liver fibrosis.     

EZH2‐mediated repression of Dkk1 promotes hepatic stellate cell activation and hepatic fibrosis

EZH2, a histone H3 lysine‐27‐specific methyltransferase, is involved in diverse physiological and pathological processes including cell proliferation and differentiation. However, the role of EZH2 in liver fibrosis is largely unknown. In this study, it was identified that EZH2 promoted Wnt pathway‐stimulated fibroblasts in vitro and in vivo by repressing Dkk‐1, which is a Wnt pathway antagonist. The expression of EZH2 was increased in CCl₄‐induced rat liver and primary HSCs as well as TGF‐β1‐treated HSC‐T6, whereas the expression of Dkk1 was reduced. Silencing of EZH2 prevented TGF‐β1‐induced proliferation of HSC‐T6 cells and the expression of α‐SMA. In addition, knockdown of Dkk1 promoted TGF‐β1‐induced activation of HSCs. Moreover, silencing of EZH2 could restore the repression of Dkk‐1 through trimethylation of H3K27me3 in TGF‐β1‐treated HSC‐T6 cells. Interestingly, inhibition of EZH2 had almost no effect on the activation of HSC when Dkk1 was silenced. Collectively, EZH2‐mediated repression of Dkk1 promotes the activation of Wnt/β‐catenin pathway, which is an essential event for HSC activation.     

Angiotensin II increases mRNA levels of all TGF‐β isoforms in quiescent and activated rat hepatic stellate cells

AII (angiotensin II) is a vasoactive peptide that plays an important role in the development of liver fibrosis mainly by regulating profibrotic cytokine expression such as TGF‐β (transforming growth factor‐β). Activated HSCs (hepatic stellate cells) are the major cell type responsible for ECM (extracellular matrix) deposition during liver fibrosis and are also a target for AII and TGF‐β actions. Here, we studied the effect of AII on the mRNA levels of TGF‐β isoforms in primary cultures of rat HSCs. Both quiescent and activated HSCs were stimulated with AII for different time periods, and mRNA levels of TGF‐β1, TGF‐β2 and TGF‐β3 isoforms were evaluated using RNaseI protection assay. The mRNA levels of all TGF‐β isoforms, particularly TGF‐β2 and TGF‐β3, were increased after AII treatment in activated HSCs. In addition, activated HSCs were able to produce active TGF‐β protein after AII treatment. The mRNA expression of TGF‐β isoforms induced by AII required both ERK1/2 and Nox (NADPH oxidase) activation but not PKC (protein kinase C) participation. ERK1/2 activation induced by AII occurs via AT1 receptors, but independently of either PKC and Nox activation or EGFR (epidermal growth factor receptor) transactivation. Interestingly, AII has a similar effect on TGF‐β expression in quiescent HSCs, although it has a smaller but significant effect on ERK1/2 activation in these cells.     

Placental growth factor silencing ameliorates liver fibrosis and angiogenesis and inhibits activation of hepatic stellate cells in a murine model of chronic liver disease

Placental growth factor (PlGF) is a member of the vascular endothelial growth factor (VEGF) family and is involved in pathological angiogenesis associated with chronic liver diseases. However, the precise mechanisms underlying PlGF signalling contributing to liver fibrosis and angiogenesis remain largely unexplored. This study aimed to assess the effect of reducing PlGF expression using small interfering RNA (siRNA) on experimental liver fibrosis and angiogenesis, and to elucidate the underlying molecular mechanisms. Fibrosis was induced in mice by carbon tetrachloride (CCl₄) for 8 weeks, and mice were treated with PlGF siRNA or non‐targeting control siRNA starting two weeks after initiating CCl₄ injections. The results showed that PlGF was highly expressed in cirrhotic human and mice livers; which mainly distributed in activated hepatic stellate cells (HSCs). PlGF silencing robustly reduced liver inflammation, fibrosis, intrahepatic macrophage recruitment, and inhibited the activation of HSCs in vivo. Moreover, PlGF siRNA‐treated fibrotic mice showed diminished hepatic microvessel density and angiogenic factors, such as hypoxia‐inducible factor‐1α (HIF‐1α), VEGF and VEGF receptor‐1. Moreover, down‐regulation of PlGF with siRNA in HSCs inhibited the activation and proliferation of HSCs. Mechanistically, overexpression of PlGF in activated HSCs was induced by hypoxia dependent on HIF‐1α, and PlGF induces HSC activation and proliferation via activation the phosphatidylinositol 3‐kinase (PI3K)/Akt signalling pathways. These findings indicate that PlGF plays an important role in liver fibrosis‐associated angiogenesis and that blockage of PlGF could be an effective strategy for chronic liver disease.     

GATA binding protein 3 is correlated with leptin regulation of PPARγ1 in hepatic stellate cells

Accumulating evidence reveals that hormone leptin, mainly produced by adipocyte, plays a unique role in promotion of liver fibrosis. Hepatic stellate cell (HSC) activation is a key step in liver fibrosis and peroxisome‐proliferator activated receptor γ (PPARγ) exerts a crucial role in inhibition of HSC activation. Our previous researches demonstrated that leptin reduced PPARγ1 (a major subtype of PPARγ in HSCs) expression through GATA binding protein 2 (GATA2) binding to a site around ?2323 in PPARγ1 promoter. The present researches aimed to examine the effect of GATA3 on leptin‐induced inhibition of PPARγ1 and elucidate the relationship between GATA3 and GATA2. Gene expressions were analysed by real‐time PCR, western blot, luciferase assay and immunostaining. C57BL/6J ob/ob mouse model of thioacetamide‐induced liver injury was used in vivo. Results demonstrate that leptin significantly induces GATA3 expression in HSCs by multiple signalling pathways including NADPH oxidase pathway. There exist crosstalks between NADPH oxidase pathway and the other pathways. GATA3 can bind to GATA2‐binding site in PPARγ1 promoter and interacts with GATA2, contributing to leptin inhibition of PPARγ1 expression in HSCs. These data demonstrated novel molecular events for leptin inhibition of PPARγ1 expression in HSCs and thus might have potential implications for clarifying the detailed mechanisms underlying liver fibrosis in diseases in which circulating leptin levels are elevated such as non‐alcoholic steatohepatitis in obese patients.     

miR‐193a/b‐3p relieves hepatic fibrosis and restrains proliferation and activation of hepatic stellate cells

MicroRNAs (miRNAs) have been confirmed to participate in liver fibrosis progression and activation of hepatic stellate cells (HSCs). In this study, the role of miR‐193a/b‐3p in concanavalin A (ConA)‐induced liver fibrosis in mice was evaluated. According to the results, the expression of miR‐193a/b‐3p was down‐regulated in liver tissues after exposure to ConA. Lentivirus‐mediated overexpression of miR‐193a/b‐3p reduced ConA‐induced liver injury as demonstrated by decreasing ALT and AST levels. Moreover, ConA‐induced liver fibrosis was restrained by the up‐regulation of miR‐193a/b‐3 through inhibiting collagen deposition, decreasing desmin and proliferating cell nuclear antigen (PCNA) expression and lessening the content of hydroxyproline, transforming growth factor‐β1 (TGF‐β1) and activin A in liver tissues. Furthermore, miR‐193a/b‐3p mimics suppressed the proliferation of human HSCs LX‐2 via inducing the apoptosis of LX‐2 cells and lowering the levels of cell cycle‐related proteins Cyclin D1, Cyclin E1, p‐Rb and CAPRIN1. Finally, TGF‐β1 and activin A‐mediated activation of LX‐2 cells was reversed by miR‐193a/b‐3p mimics via repressing COL1A1 and α‐SMA expression, and restraining the activation of TGF‐β/Smad2/3 signalling pathway. CAPRIN1 and TGF‐β2 were demonstrated to be the direct target genes of miR‐193a/b‐3p. We conclude that miR‐193a/b‐3p overexpression attenuates liver fibrosis through suppressing the proliferation and activation of HSCs. Our data suggest that miR‐193a‐3p and miR‐193b‐3p may be new therapeutic targets for liver fibrosis.     

PI3‐kinase/Akt pathway‐regulated membrane transportation of acid‐sensing ion channel 1a/Calcium ion influx/endoplasmic reticulum stress activation on PDGF‐induced HSC Activation

Acid‐sensing ion channel 1a (ASIC1a) allows Na⁺ and Ca²⁺ flow into cells. It is expressed during inflammation, in tumour and ischaemic tissue, in the central nervous system and non‐neuronal injury environments. Endoplasmic reticulum stress (ERS) is caused by the accumulation of misfolded proteins that interferes with intracellular calcium homoeostasis. Our recent reports showed ASIC1a and ERS are involved in liver fibrosis progression, particularly in hepatic stellate cell (HSC) activation. In this study, we investigated the roles of ASIC1a and ERS in activated HSC. We found that ASIC1a and ERS‐related proteins were up‐regulated in carbon tetrachloride (CCl₄)‐induced fibrotic mouse liver tissues, and in patient liver tissues with hepatocellular carcinoma with severe liver fibrosis. The results show silencing ASIC1a reduced the expression of ERS‐related biomarkers GRP78, Caspase12 and IREI‐XBP1. And, ERS inhibition by 4‐PBA down‐regulated the high expression of ASIC1a induced by PDGF, suggesting an interactive relationship. In PDGF‐induced HSCs, ASIC1a was activated and migrated to the cell membrane, leading to extracellular calcium influx and ERS, which was mediated by PI3K/AKT pathway. Our work shows PDGF‐activated ASIC1a via the PI3K/AKT pathway, induced ERS and promoted liver fibrosis progression.     

PLK1 regulates hepatic stellate cell activation and liver fibrosis through Wnt/β‐catenin signalling pathway

As an outcome of chronic liver disease, liver fibrosis involves the activation of hepatic stellate cells (HSCs) caused by a variety of chronic liver injuries. It is important to explore approaches to inhibit the activation and proliferation of HSCs for the treatment of liver fibrosis. PLK1 is overexpressed in many human tumour cells and has become a popular drug target in tumour therapy. Therefore, further study of the function of PLK1 in the cell cycle is valid. In the present study, we found that PLK1 expression was elevated in primary HSCs isolated from CCl₄‐induced liver fibrosis mice and LX‐2 cells stimulated with TGF‐β1. Knockdown of PLK1 inhibited α‐SMA and Col1α1 expression and reduced the activation of HSCs in CCl₄‐induced liver fibrosis mice and LX‐2 cells stimulated with TGF‐β1. We further showed that inhibiting the expression of PLK1 reduced the proliferation of HSCs and promoted HSCs apoptosis in vivo and in vitro. Furthermore, we found that the Wnt/β‐catenin signalling pathway may be essential for PLK1‐mediated HSCs activation. Together, blocking PLK1 effectively suppressed liver fibrosis by inhibiting HSC activation, which may provide a new treatment strategy for liver fibrosis.     

Depletion of β‐arrestin2 in hepatic stellate cells reduces cell proliferation via ERK pathway

β‐Arrestins are multifunctional adaptor proteins. Recently, some new roles of β‐arrestins in regulating intracellular signaling networks have been discovered, which regulate cell growth, proliferation, and apoptosis. Though, the role of β‐arrestins expression in the pathology of hepatic fibrosis remains unclear. In this study, the possible relationship between the expression of β‐arrestins with the experimental hepatic fibrosis and the proliferation of hepatic stellate cells (HSCs) were investigated. Porcine serum induced liver fibrosis was established in this study. At five time points, the dynamic expression of β‐arrestin1, β‐arrestin2, and α‐smooth muscle actin (α‐SMA) in rat liver tissues, was measured by immunohistochemical staining, double immunofluorescent staining, and Western blotting. This study showed that aggravation of hepatic fibrosis with gradually increasing expression of β‐arrestin2 in the hepatic tissues, but not β‐arrestin1. Further, as hepatic fibrosis worsens, β‐arrestin2‐expressing activated HSCs accounts for an increasingly larger percentage of all activated HSCs. And the expression of β‐arrestin2 had a significant positive correlation with the expression of α‐SMA, an activated HSCs marker. In vitro studies, the dynamic expression of β‐arrestin1 and β‐arrestin2 in platelet derived growth factor‐BB (PDGF‐BB) stimulated HSCs was assessed by Western blotting. The expression of β‐arrestin2 was remarkably increased in PDGF‐BB stimulated HSCs. Furthermore, the small interfering RNA (siRNA) technique was used to explore the effect of β‐arrestins on the proliferation of HSCs and the activation of ERK1/2. Transfection of siRNA targeting β‐arrestin2 mRNA (siβ‐arrestin2) into HSCs led to a 68% and 70% reduction of β‐arrestin2 mRNA and protein expression, respectively. siβ‐arrestin2 abolished the effect of PDGF‐BB on the proliferation of HSCs. In addition, siβ‐arrestin2 exerted the inhibition of the activation of ERK1/2 in HSCs. The present study provided strong evidence for the participation of the β‐arrestin2 in the pathogenesis of hepatic fibrosis. The β‐arrestin2 depletion diminishes HSCs ERK1/2 signaling and proliferation stimulated by PDGF‐BB. Selective targeting of β‐arrestin2 inhibitors to HSCs might present as a novel strategy for the treatment of hepatic fibrosis. J. Cell. Biochem. 114: 1153–1162, 2013. © 2012 Wiley Periodicals, Inc.     

MicroRNA‐708 modulates Hepatic Stellate Cells activation and enhances extracellular matrix accumulation via direct targeting TMEM88

Transmembrane protein 88 (TMEM88) is a potential 2‐transmembrane‐type protein that interacts with the PDZ domain of Dishevelled‐1 (DVL‐1), a crucial component of Wnt signalling pathway through its C‐terminal Val‐Trp‐Val (VWV) motif in Xenopus embryo cells. Since the significant function of β‐catenin in liver fibrosis, it is urgent to study the TMEM88 mechanism in liver fibrosis. The current research was for evaluating the function of TMEM88 in the process of the liver fibrosis and clarifying the inherent mechanism. The study found that TMEM88 is decreased in human fibrotic liver tissues. Functionally, TMEM88 significantly reduced the expression levels of α‐smooth muscle actin (α‐SMA) and collagen type I (Col.I) and repressed extracellular matrix (ECM) accumulation by restoring the balance between matrix metalloproteinases (MMPs) and TIMPs (tissue inhibitor of metalloproteinases). TMEM88 inhibited HSCs proliferation and evaluated the apoptosis of activated LX‐2 cells by regulating Wnt3a, Wnt2b and β‐catenin of Wnt/β‐catenin signalling pathway. Moreover, we demonstrated that miR‐708 particularly targeted TMEM88 3′‐UTR regions and down‐regulated the expression level of TMEM88 in TGF‐β1‐stimulated LX‐2 cells. MiR‐708 promoted the generation of ECM and cell activation in activated LX‐2 cells. These results determined that miR‐708 could promote HSCs activation and enhance ECM accumulation via direct targeting TMEM88 by Wnt/β‐catenin signalling pathway. This will provide a potential target for future research in the process of liver fibrosis.     

MeCP2‐421‐mediated RPE epithelial‐mesenchymal transition and its relevance to the pathogenesis of proliferative vitreoretinopathy

Proliferative vitreoretinopathy (PVR) is a blinding eye disease. Epithelial‐mesenchymal transition (EMT) of RPE cells plays an important role in the pathogenesis of PVR. In the current study, we sought to investigate the role of the methyl‐CpG‐binding protein 2 (MeCP2), especially P‐MeCP2‐421 in the pathogenesis of PVR. The expressions of P‐MeCP2‐421, P‐MeCP2‐80, PPAR‐γ and the double labelling of P‐MeCP2‐421 with α‐SMA, cytokeratin, TGF‐β and PPAR‐γ in human PVR membranes were analysed by immunohistochemistry. The effect of knocking down MeCP2 using siRNA on the expressions of α‐SMA, phospho‐Smad2/3, collagen I, fibronectin and PPAR‐γ; the expression of α‐SMA stimulated by recombinant MeCP2 in ARPE‐19; and the effect of TGF‐β and 5‐AZA treatment on PPAR‐γ expression were analysed by Western blot. Chromatin immunoprecipitation was used to determine the binding of MeCP2 to TGF‐β. Our results showed that P‐MeCP2‐421 was highly expressed in PVR membranes and was double labelled with α‐SMA, cytokeratin and TGF‐β, knocking down MeCP2 inhibited the activation of Smad2/3 and the expression of collagen I and fibronectin induced by TGF‐β. TGF‐β inhibited the expression of PPAR‐γ, silence of MeCP2 by siRNA or using MeCP2 inhibitor (5‐AZA) increased the expression of PPAR‐γ. α‐SMA was up‐regulated by the treatment of recombinant MeCP2. Importantly, we found that MeCP2 bound to TGF‐β as demonstrated by Chip assay. The results suggest that MeCP2 especially P‐MeCP2‐421 may play a significant role in the pathogenesis of PVR and targeting MeCP2 may be a potential therapeutic approach for the treatment of PVR.     

Effect of human amniotic epithelial cells on pro‐fibrogenic resident hepatic cells in a rat model of liver fibrosis

Myofibroblasts are key fibrogenic cells responsible for excessive extracellular matrix synthesis characterizing the fibrotic lesion. In liver fibrosis, myofibroblasts derive either from activation of hepatic stellate cells (HSC) and portal fibroblasts (PF), or from the activation of fibroblasts that originate from ductular epithelial cells undergoing epithelial–mesenchymal transition. Ductular cells can also indirectly promote myofibroblast generation by activating TGF‐β, the main fibrogenic growth factor, through αvβ6 integrin. In addition, after liver injury, liver sinusoidal cells can lose their ability to maintain HSC quiescence, thus favouring HSC differentiation towards myofibroblasts. The amniotic membrane and epithelial cells (hAEC) derived thereof have been shown to decrease hepatic myofibroblast levels in rodents with liver fibrosis. In this study, in a rat model of liver fibrosis, we investigated the effects of hAEC on resident hepatic cells contributing to myofibroblast generation. Our data show that hAEC reduce myofibroblast numbers with a consequent reduction in fibronectin and collagen deposition. Interestingly, we show that hAEC strongly act on specific myofibroblast precursors. Specifically, hAEC reduce the activation of PF rather than HSC. In addition, hAEC target reactive ductular cells by inhibiting their proliferation and αvβ6 integrin expression, with a consequent decrease in TGF‐β activation. Moreover, hAEC counteract the transition of ductular cells towards fibroblasts, while it does not affect injury‐induced and fibrosis‐promoting sinusoidal alterations. In conclusion, among the emerging therapeutic applications of hAEC in liver diseases, their specific action on PF and ductular cells strongly suggests their application in liver injuries involving the expansion and activation of the portal compartment.     

Transforming growth factor‐β1 requires NADPH oxidase 4 for angiogenesis in vitro and in vivo

Angiogenesis, the formation of new blood vessels, is a key physiological event in organ development and tissue responses to hypoxia but is also involved in pathophysiologies such as tumour growth and retinopathies. Understanding the molecular mechanisms involved is important to design strategies for therapeutic intervention. One important regulator of angiogenesis is transforming growth factor‐β1 (TGF‐β1). In addition, reactive oxygen species (ROS) and the ROS‐forming NADPH oxidase type 4 (Nox4) have been implicated as additional regulators such as during hypoxia. Here, we show that both processes are indeed mechanistically linked. TGF‐β1‐stimulated Nox4 expression and ROS formation in endothelial cells. In cells from Nox4‐deficient mice, TGF‐β1‐induced cell proliferation, migration and tube formation were abolished. In vivo, TGF‐β1 stimulated growth of blood vessels into sponges implanted subcutaneously, and this angiogenesis was markedly reduced in Nox4 knockout mice. Thus, endothelial cells are regulated by a TGF‐β1 signalling pathway involving Nox4‐derived ROS to promote angiogenesis. In order to abrogate pathological angiogenesis triggered by a multitude of factors, such as TGF‐β1 and hypoxia, Nox4 may thus be an ideal therapeutic target.     

Thyroid hormone induces sprouting angiogenesis in adult heart of hypothyroid mice through the PDGF‐Akt pathway

Study of physiological angiogenesis and associated signalling mechanisms in adult heart has been limited by the lack of a robust animal model. We investigated thyroid hormone‐induced sprouting angiogenesis and the underlying mechanism. Hypothyroidism was induced in C57BL/6J mice by feeding with propylthiouracil (PTU). One year of PTU treatment induced heart failure. Both 12 weeks‐ (young) and 1 year‐PTU (middle age) treatment caused a remarkable capillary rarefaction observed in capillary density. Three‐day Triiodothyronine (T3) treatment significantly induced cardiac capillary growth in hypothyroid mice. In cultured left ventricle (LV) tissues from PTU‐treated mice, T3 also induced robust sprouting angiogenesis where pericyte‐wrapped endothelial cells formed tubes. The in vitro T3 angiogenic response was similar in mice pre‐treated with PTU for periods ranging from 1.5 to 12 months. Besides bFGF and VEGF₁₆₄, PDGF‐BB was the most robust angiogenic growth factor, which stimulated notable sprouting angiogenesis in cultured hypothyroid LV tissues with increasing potency, but had little effect on tissues from euthyroid mice. T3 treatment significantly increased PDGF receptor beta (PDGFR‐β) protein levels in hypothyroid heart. PDGFR inhibitors blocked the action of T3 both on sprouting angiogenesis in cultured LV tissue and on capillary growth in vivo. In addition, activation of Akt signalling mediated in T3‐induced angiogenesis was blocked by PDGFR inhibitor and neutralizing antibody. Our results suggest that hypothyroidism leads to cardiac microvascular impairment and rarefaction with increased sensitivity to angiogenic growth factors. T3‐induced cardiac sprouting angiogenesis in adult hypothyroid mice was associated with PDGF‐BB, PDGFR‐β and downstream activation of Akt.     

MicroRNA‐30a ameliorates hepatic fibrosis by inhibiting Beclin1‐mediated autophagy

We explored the role of microRNA‐30a (miR‐30a) and the mechanism involved in hepatic fibrosis. MiR‐30a overexpression was achieved by miR‐30a mimics transfection in hepatic stellate cells (HSCs) (HSC‐T6, LX‐2), and miR‐30a agomir (ago‐miR‐30a) treatment in mice. MiR‐30a levels were measured using TaqMan miRNA assay system, and the localization of miR‐30a was detected by fluorescence in situ hybridization (FISH). The interaction of miR‐30a and Beclin1 was confirmed by dual‐luciferase reporter assay. Autophagic flux was analysed using tandem mRFP‐GFP‐LC3 fluorescence microscopy, electron microscopy and Western blot of LC3‐II/I ratio. MiR‐30a was notably down‐regulated in activated HSCs and LX‐2‐exosomes induced by TGF‐β1; overexpression of miR‐30a down‐regulated extracellular matrix (ECM), such as α‐SMA, TIMP‐1, and Collagen I expression, and suppressed cell viability in HSCs. MiR‐30a was significantly down‐regulated in hepatic fibrosis mice and overexpression of miR‐30a prevented BDL‐induced fibrogenesis, concomitant with the down‐regulation of ECM. MiR‐30a inhibited HSCs autophagy and increased lipid accumulation in HSCs and in mice fibrotic hepatic tissues. MiR‐30a inhibited its downstream effector of Beclin1 by direct targeting its 3′‐UTR region. Moreover, Knock‐down of Beclin1 by small interfering RNA (siRNA) inhibited HSC autophagy and activation in LX‐2 cells. In conclusion, miR‐30a is down‐regulated in hepatic fibrosis models and its overexpression prevents liver fibrogenesis by directly suppressing Beclin1‐mediated autophagy; therefore, miR‐30a may be a new potential therapeutic target for controlling hepatic fibrosis.     

A protective role of ciglitazone in ox‐LDL‐induced rat microvascular endothelial cells via modulating PPARγ‐dependent AMPK/eNOS pathway

Thiazolidinediones, the antidiabetic agents such as ciglitazone, has been proved to be effective in limiting atherosclerotic events. However, the underlying mechanism remains elucidative. Ox‐LDL receptor‐1 (LOX‐1) plays a central role in ox‐LDL‐mediated atherosclerosis via endothelial nitric oxide synthase (eNOS) uncoupling and nitric oxide reduction. Therefore, we tested the hypothesis that ciglitazone, the PPARγ agonist, protected endothelial cells against ox‐LDL through regulating eNOS activity and LOX‐1 signalling. In the present study, rat microvascular endothelial cells (RMVECs) were stimulated by ox‐LDL. The impact of ciglitazone on cell apoptosis and angiogenesis, eNOS expression and phosphorylation, nitric oxide synthesis and related AMPK, Akt and VEGF signalling pathway were observed. Our data showed that both eNOS and Akt phosphorylation, VEGF expression and nitric oxide production were significantly decreased, RMVECs ageing and apoptosis increased after ox‐LDL induction for 24 hrs, all of which were effectively reversed by ciglitazone pre‐treatment. Meanwhile, phosphorylation of AMP‐activated protein kinase (AMPK) was suppressed by ox‐LDL, which was also prevented by ciglitazone. Of interest, AMPK inhibition abolished ciglitazone‐mediated eNOS function, nitric oxide synthesis and angiogenesis, and increased RMVECs ageing and apoptosis. Further experiments showed that inhibition of PPARγ significantly suppressed AMPK phosphorylation, eNOS expression and nitric oxide production. Ciglitazone‐mediated angiogenesis and reduced cell ageing and apoptosis were reversed. Furthermore, LOX‐1 protein expression in RMVECs was suppressed by ciglitazone, but re‐enhanced by blocking PPARγ or AMPK. Ox‐LDL‐induced suppression of eNOS and nitric oxide synthesis were largely prevented by silencing LOX‐1. Collectively, these data demonstrate that ciglitazone‐mediated PPARγ activation suppresses LOX‐1 and moderates AMPK/eNOS pathway, which contributes to endothelial cell survival and function preservation.