Guangcun Huang

Over-expressed microRNA-27a and 27b influence fat accumulation and cell proliferation during rat hepatic stellate cell activation

Hepatic stellate cells (HSCs) activation is an initial event in liver fibrosis. MicroRNAs (miRNAs) have been found to play essential roles in cell differentiation, proliferation, and fat metabolism. In this study, we showed that down‐regulation of two over‐expressed miRNAs, miR‐27a and 27b allowed culture‐activated rat HSCs to switch to a more quiescent HSC phenotype, with restored cytoplasmic lipid droplets and decreased cell proliferation. Mechanistically, retinoid X receptor α was confirmed to be the target of miR‐27a and 27b. These results indicated a new role and mechanism of miR‐27a and 27b in regulating fat metabolism and cell proliferation during HSCs activation.

Regulation of hepatic stellate cells by connective tissue growth factor

Connective tissue growth factor (CTGF/CCN2) regulates cell proliferation, differentiation, adhesion, chemotaxis, migration, apoptosis and extracellular matrix production. Through these diverse actions, CTGF/CCN2 plays a major role in important physiological and pathophysiological processes such as embryogenesis, implantation, angiogenesis, chondrogenesis, tumorigenesis, differentiation, wound healing and fibrosis. Whereas hepatic levels of CTGF/CCN2 are usually low, elevated levels of hepatic CTGF/CCN2 occur in patients with liver fibrosis and in experimental animal models of liver fibrosis. In fibrotic liver, CTGF/CCN2 is produced by multiple cell types but its sustained expression by and action on hepatic stellate cells is particularly important because these cells assume an activated phenotype during fibrosing injury and are principally responsible for the excessive production of fibrillar collagens, a process that is driven by CTGF/CCN2. Through its direct actions and interactions with other molecules such as fibronectin or transforming growth factor beta-1, CTGF/CCN2 promotes proliferation, survival, migration, adhesion, and extracellular matrix production in activated hepatic stellate cells, thereby promoting hepatic fibrogenic pathways. This review focuses on the regulation of hepatic stellate cell function by CTGF/CCN2.

Over-expression of C/EBP-{alpha} induces apoptosis in cultured rat hepatic stellate cells depending on p53 and peroxisome proliferator-activated receptor-{gamma}

Hepatic stellate cells (HSCs) play a key role in the pathogenesis of hepatic fibrosis. In our previous studies, CCAAT enhancer binding protein-alpha (C/EBP-alpha) has been shown to be involved in the activation of HSCs and to have a repression effect on hepatic fibrosis in vivo. However, the mechanisms are largely unknown. In this study, we show that the infection of adenovirus vector expressing C/EBP-alpha gene (Ad-C/EBP-alpha) could induce HSCs apoptosis in a dose- and time-dependent manner by Annexin V/PI staining, caspase-3 activation assay, and flow cytometry. Also, over-expression of C/EBP-alpha resulted in the up-regulation of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) and P53, while P53 expression was regulated by PPAR-gamma. In addition, Fas, FasL, DR4, DR5, and TRAIL were studied. The results indicated that the death receptor pathway was mainly involved and regulated by PPAR-gamma and p53 in the process of apoptosis triggered by C/EBP-alpha in HSCs.

Integrin expression and function in the response of primary culture hepatic stellate cells to connective tissue growth factor (CCN2)

Production of connective tissue growth factor (CCN2, also known as CTGF) is a hallmark of hepatic fibrosis. This study examined early primary cultures of hepatic stellate cells (HSC) for (i) CCN2 regulation of its cognate receptor integrin subunits; and (ii) interactions between CCN2 and integrin α5β1, heparan sulphate proteoglycans (HSPG) or fibronectin (FN) in supporting cell adhesion. HSC were isolated from healthy male Balb/c mice. mRNA levels of CCN2 or α5, β1, αv or β3 integrin subunits were measured in days 1–7 primary culture HSC, and day 3 or day 7 cells treated with recombinant CCN2 or CCN2 small interfering RNA. Interactions between CCN2 and integrin α5β1, HSPG or FN were investigated using an in vitro cell adhesion assay. Co‐incident with autonomous activation over the first 7 days, primary culture HSC increasingly expressed mRNA for CCN2 or integrin subunits. Addition of exogenous CCN2 or knockdown of endogenous CCN2 differentially regulated integrin gene expression in day 3 versus day 7 cells. Either full length CCN2 (‘CCN21–4’) or residues 247–349 containing module 4 alone (‘CCN24’) supported day 3 cell adhesion in an integrin α5β1‐ and HSPG‐dependent fashion. Adhesion of day 3 cells to FN was promoted in an integrin α5β1‐dependent manner by CCN21–4 or CCN24, whereas FN promoted HSPG‐dependent HSC adhesion to CCN21–4 or CCN24. These findings suggest CCN2 regulates integrin expression in primary culture HSC and supports HSC adhesion via its binding of cell surface integrin α5β1, a novel CCN2 receptor in primary culture HSC which interacts co‐operatively with HSPG or FN.

PERK Activation Preserves the Viability and Function of Remyelinating Oligodendrocytes in Immune-Mediated Demyelinating Diseases

Remyelination occurs in multiple sclerosis (MS) lesions but is generally considered to be insufficient. One of the major challenges in MS research is to understand the causes of remyelination failure and to identify therapeutic targets that promote remyelination. Activation of pancreatic endoplasmic reticulum kinase (PERK) signaling in response to endoplasmic reticulum stress modulates cell viability and function under stressful conditions. There is evidence that PERK is activated in remyelinating oligodendrocytes in demyelinated lesions in both MS and its animal model, experimental autoimmune encephalomyelitis (EAE). In this study, we sought to determine the role of PERK signaling in remyelinating oligodendrocytes in MS and EAE using transgenic mice that allow temporally controlled activation of PERK signaling specifically in oligodendrocytes. We demonstrated that persistent PERK activation was not deleterious to myelinating oligodendrocytes in young, developing mice or to remyelinating oligodendrocytes in cuprizone-induced demyelinated lesions. We found that enhancing PERK activation, specifically in (re)myelinating oligodendrocytes, protected the cells and myelin against the detrimental effects of interferon-γ, a key proinflammatory cytokine in MS and EAE. More important, we showed that enhancing PERK activation in remyelinating oligodendrocytes at the recovery stage of EAE promoted cell survival and remyelination in EAE demyelinated lesions. Thus, our data provide direct evidence that PERK activation cell-autonomously enhances the survival and preserves function of remyelinating oligodendrocytes in immune-mediated demyelinating diseases.

Heparin-binding epidermal growth factor-like growth factor suppresses experimental liver fibrosis in mice

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a cytoprotective agent in several organ systems but its roles in liver fibrosis are unclear. We studied the roles of HB-EGF in experimental liver fibrosis in mice and during hepatic stellate cell (HSC) activation. Thioacetamide (TAA; 100 mg/kg) was administered by intraperitoneal injection three times a week for 4 weeks to wild-type HB-EGF+/+ or HB-EGF-null (HB-EGF−/−) male mice. Livers were examined for histology and expression of key fibrotic markers. Primary cultured HSCs isolated from untreated HB-EGF+/+ or HB-EGF−/− mice were examined for fibrotic markers and/or cell migration either during culture-induced activation or after exogenous HB-EGF (100 ng/ml) treatment. TAA induced liver fibrosis in both HB-EGF+/+ and HB-EGF−/− mice. Hepatic HB-EGF expression was decreased in TAA-treated HB-EGF+/+ mice by 37.6% (P<0.05) as compared with animals receiving saline alone. HB-EGF−/− mice treated with TAA showed increased hepatic α-smooth muscle actin-positive cells and collagen deposition, and, as compared with HB-EGF+/+ mice, TAA-stimulated hepatic mRNA levels in HB-EGF−/− mice were, respectively, 2.1-, 1.7-, 1.8-, 2.2-, 1.2- or 3.3-fold greater for α-smooth muscle actin, α1 chain of collagen I or III (COL1A1 or COL3A1), transforming growth factor-β1, connective tissue growth factor or tissue inhibitor of metalloproteinase-1 (P<0.05). HB-EGF expression was detectable in primary cultured HSCs from HB-EGF+/+ mice. Both endogenous and exogenous HB-EGF inhibited HSC activation in primary culture, and HB-EGF enhanced HSC migration. These findings suggest that HB-EGF gene knockout in mice increases susceptibility to chronic TAA-induced hepatic fibrosis and that HB-EGF expression or action is associated with suppression of fibrogenic pathways in HSCs.

Impaired Eukaryotic Translation Initiation Factor 2B Activity Specifically in Oligodendrocytes Reproduces the Pathology of Vanishing White Matter Disease in Mice

Vanishing white matter disease (VWMD) is an inherited autosomal-recessive hypomyelinating disease caused by mutations in eukaryotic translation initiation factor 2B (eIF2B). eIF2B mutations predominantly affect the brain white matter, and the characteristic features of VWMD pathology include myelin loss and foamy oligodendrocytes. Activation of pancreatic endoplasmic reticulum kinase (PERK) has been observed in oligodendrocytes in VWMD. PERK activation in response to endoplasmic reticulum stress attenuates eIF2B activity by phosphorylating eIF2α, suggesting that impaired eIF2B activity in oligodendrocytes induced by VWMD mutations or PERK activation exploit similar mechanisms to promote selective white matter pathology in VWMD. Using transgenic mice that allow for temporally controlled activation of PERK specifically in oligodendrocytes, we discovered that strong PERK activation in oligodendrocytes during development suppressed eIF2B activity and reproduced the characteristic features of VWMD in mice, including hypomyelinating phenotype, foamy oligodendrocytes, and myelin loss. Notably, impaired eIF2B activity induced by PERK activation in oligodendrocytes of fully myelinated adult mice had minimal effects on morphology or function. Our observations point to a cell-autonomous role of impaired eIF2B activity in myelinating oligodendrocytes in the pathogenesis of VWMD.

HB-EGF Protects the Lungs after Intestinal Ischemia/Reperfusion Injury

BACKGROUND Acute respiratory distress syndrome continues to be a major source of morbidity and mortality in critically-ill patients. Heparin binding EGF-like growth factor (HB-EGF) is a biologically active protein that acts as an intestinal cytoprotective agent. We have previously demonstrated that HB-EGF protects the intestines from injury in several different animal models of intestinal injury. In the current study, we investigated the ability of HB-EGF to protect the lungs from remote organ injury after intestinal ischemia/reperfusion (I/R). METHODS Mice were randomly assigned to one of the following groups: (1) sham-operated; (2) sham+HB-EGF (1200 microg/kg in 0.6 mL administered by intra-luminal injection at the jejuno-ileal junction immediately after identification of the superior mesenteric artery); (3) superior mesenteric artery occlusion for 45 min followed by reperfusion for 6 h (I/R); or (4) I/R+HB-EGF (1200 microg/kg in 0.6 mL) administered 15 min after vascular occlusion. The severity of acute lung injury was determined by histology, morphometric analysis and invasive pulmonary function testing. Animal survival was evaluated using Kaplan-Meier analysis. RESULTS Mice subjected to intestinal I/R injury showed histologic and functional evidence of acute lung injury and decreased survival compared with sham-operated animals. Compared with mice treated with HB-EGF (I/R+HB-EGF), the I/R group had more severe acute lung injury, and decreased survival. CONCLUSION Our results demonstrate that HB-EGF reduces the severity of acute lung injury after intestinal I/R in mice. These data demonstrate that HB-EGF may be a potential novel systemic anti-inflammatory agent for the prevention of the systemic inflammatory response syndrome (SIRS) after intestinal injury.

Ethanol-mediated expression of connective tissue growth factor (CCN2) in mouse pancreatic stellate cells

Activated pancreatic stellate cells (PSC) play a central role in the pathogenesis of pancreatic fibrosis, a common feature of chronic pancreatitis which is often caused by excessive alcohol consumption. In view of the central role of connective tissue growth factor (CCN2) in fibrosis, we investigated the mechanisms by which CCN2 is regulated in PSC following their exposure to ethanol or acetaldehyde. Primary cultures of PSC from Balb/c mice were treated with 0–50 mM ethanol or 0–200 μM acetaldehyde in the presence or absence of 4-methylpyrazole (4MP; an inhibitor of alcohol dehydrogenase), diallyl sulfide (DAS; an inhibitor of cytochrome P4502E1) or anti-oxidant catalase or vitamin D. CCN2 production, assessed by reverse-transcriptase polymerase chain reaction to measure CCN2 mRNA levels or by fluorescence activated cell sorting to assess CCN2 protein, was enhanced in a dose-dependent manner by ethanol or acetaldehyde. In the presence of 4MP, DAS, or the anti-oxidants vitamin D or catalase, there was a substantial decrease in the ability of ethanol to stimulate CCN2 mRNA expression and a concomitant decrease in CCN2-positive PSC. Accumulation of reactive oxygen species in PSC after exposure to ethanol was verified by loading the cells with dichlorofluorescin diacetate and showing that there was a stimulation of its oxidized fluorescent product, the latter of which was diminished in the presence of catalase or vitamin D. These results show the production of acetaldehyde and oxidant stress in mouse PSC are the cause of increased CCN2 mRNA and protein production after exposure of the cells to ethanol. The potential therapeutic effects of inhibitors of ethanol metabolism or anti-oxidants in alcoholic pancreatitis may arise in part through their ability to attenuate CCN2 production by PSC.

Adrenomedullin regulates expressions of transforming growth factor‐β1 and β1‐induced matrix metalloproteinase‐2 in hepatic stellate cells

Adrenomedullin (AM), a peptide isolated from human pheochromocytoma, can be produced and secreted by various types of cells including hepatic stellate cells (HSCs), and its possible role in HSCs is not clear now. In the present study, the interactive regulation between transforming growth factor (TGF)‐β1 and AM and the effect of AM on TGF‐β1‐induced matrix metalloproteinase (MMP)‐2 expression in HSCs were investigated. TGF‐β1 and AM inhibited gene transcript level mutually (real‐time reverse transcription‐polymerase chain reaction). AM suppressed the protein expression level of TGF‐β1 (Western blot), but TGF‐β1 might have no effect on AM secretion level. MMP‐2 protein expression in HSCs was increased in response to TGF‐β1, and upregulation of MMP‐2 expression stimulated with TGF‐β1 was suppressed by AM in dose‐dependent manner (Western blot). AM decreased the phosphorylation level of extracellular signal‐regulated kinase (ERK) in HSCs treated with TGF‐β1, and TGF‐β1‐induced MMP‐2 expression was suppressed by adding Mitogen‐activated protein Kinase/ERK (MEK) inhibitor U0126 (Western blot). Our results suggest that AM may intervene the activation of HSCs by inhibiting TGF‐β1 production and TGF‐β1‐induced MMP‐2 expression; AM may suppress the upregulation of MMP‐2 expression induced by TGF‐β1 partially through ERK pathway.