Silencing IQGAP1 alleviates hepatic fibrogenesis via blocking bone marrow mesenchymal stromal cell recruitment to fibrotic liver.
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ABSTRACT:
SUBMITTER: Ma Y
PROVIDER: S-EPMC8728523 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Recently, circular RNAs (circRNAs) have been frequently reported to be involved in hepatocellular carcinoma (HCC) development and progression. However, the role of circRNAs in hepatic fibrosis (HF) is still unclear. Our previous high-throughput screen revealed changes in many circRNAs in mice with carbon tetrachloride (CCl4)-induced HF. For instance, the expression of circPSD3, a circRNA derived from the Pleckstrin and Sec7 domain-containing 3 (PSD3) gene, was considerably downregulated in primary hepatic stellate cells (HSCs) and liver tissues of mice with CCl4-induced HF compared to those in the vehicle group. In vivo overexpression of circPSD3 using AAV8-circPSD3 arrested the deterioration of CCl4-induced HF as indicated by reduced serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) content, liver hydroxyproline level, collagen deposition, and pro-fibrogenic gene and pro-inflammatory cytokine levels. Moreover, in vitro loss-of-function and gain-of-function analyses suggested that circPSD3 inhibited the activation and proliferation of HSCs. Mechanistically, circPSD3 served as a sponge for miR-92b-3p, subsequently promoting the expression of Smad7. In conclusion, our present findings reveal a novel mechanism by which circPSD3 alleviates hepatic fibrogenesis by targeting the miR-92b-3p/Smad7 axis, and they also indicate that circPSD3 may serve as a potential biomarker for HF.
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Project description:Activated hepatic stellate cells are reported to play a significant role in liver fibrogenesis. Beside the phenotype reversion and apoptosis of activated hepatic stellate cells, the senescence of activated hepatic stellate cells limits liver fibrosis. Our previous researches have demonstrated that interleukin-10 could promote hepatic stellate cells senescence via p53 signaling pathway in vitro. However, the relationship between expression of p53 and senescence of activated hepatic stellate cells induced by interleukin-10 in fibrotic liver is unclear. The purpose of present study was to explore whether p53 plays a crucial role in the senescence of activated hepatic stellate cells and degradation of collagen mediated by interleukin-10. Hepatic fibrosis animal model was induced by carbon tetrachloride through intraperitoneal injection and transfection of interleukin-10 gene to liver was performed by hydrodynamic-based transfer system. Depletions of p53 in vivo and in vitro were carried out by adenovirus-based short hairpin RNA against p53. Regression of fibrosis was assessed by liver biopsy and collagen staining. Cellular senescence in the liver was observed by senescence-associated beta-galactosidase (SA-β-Gal) staining. Immunohistochemistry, immunofluorescence double staining, and Western blot analysis were used to evaluate the senescent cell and senescence-related protein expression. Our data showed that interleukin-10 gene treatment could lighten hepatic fibrosis induced by carbon tetrachloride and induce the aging of activated hepatic stellate cells accompanied by up-regulating the expression of aging-related proteins. We further demonstrated that depletion of p53 could abrogate up-regulation of interleukin-10 on the expression of senescence-related protein in vivo and vitro. Moreover, p53 knockout in fibrotic mice could block not only the senescence of activated hepatic stellate cells, but also the degradation of fibrosis induced by interleukin-10 gene intervention. Taken together, our results suggested that interleukin-10 gene treatment could attenuate carbon tetrachloride-induced hepatic fibrosis by inducing senescence of activated hepatic stellate cells in vivo, and this induction was closely related to p53 signaling pathway.
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Project description:The therapeutic potential of bone marrow stromal cells (BMSCs) has been demonstrated in different models of stroke. Although it is well established that BMSCs selectively migrate to the site of brain injury, the mechanisms underlying this process are poorly understood. This study addresses the hypothesis that selectins mediate the recruitment of BMSCs into the postischemic cerebral microvasculature.Focal ischemic stroke was induced by middle cerebral artery occlusion and reperfusion. Cell recruitment was monitored using either fluorescent- or radiolabeled BMSCs detected by intravital microscopy or tissue radioactivity. Mice were treated with either a blocking antibody against P- or E-selectin or with the nonselective selectin antagonist, fucoidin. The role of CD44 in cell recruitment was evaluated using BMSCs from CD44 knockout mice.Middle cerebral artery occlusion and reperfusion was associated with a significantly increased adhesion of BMSCs in cerebral venules compared with sham mice. Immunoneutralization of either E- or P-selectin blocked the middle cerebral artery occlusion and reperfusion-induced recruitment of adherent BMSCs. An attenuated recruitment response in the postischemic hemisphere was also noted after fucoidin treatment or administration of CD44-deficient BMSCs.Cerebral vascular endothelium assume a proadhesive phenotype after ischemic stroke that favors the recruitment of BMSCs, which use both P- and E-selectin to home into the infarct site. CD44 may serve as the critical ligand for selectin-mediated BMSC recruitment.
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Project description:Hepatic fibrosis is a common pathological consequence of a sustained wound healing response to continuous liver injury, characterized by increased production and accumulation of extracellular matrix. If unresolved, the fibrotic process results in organ failure, and eventually death after the development of cirrhosis. It has been suggested that macrophages play central role in the progression of hepatic fibrosis, which is related to inflammation and pyroptosis, a novel programmed and proinflammatory cell death. However, it remains far less clear if, or how, lncRNAs regulates the activation and pyroptosis of macrophage in hepatic fibrosis. In the present study, we demonstrated that the liver-enriched lncRNA Lfar1, which has been reported to promote hepatic fibrosis through inducing hepatic stellate cells activation and hepatocytes apoptosis, was dysregulated during proinflammatory M1 activation and pyroptosis of macrophage. Our study revealed that silencing lnc-Lfar1 by a lentivirus-shRNA alleviated CCl4- and BDL-induced proinflammatory M1 macrophage activation and NLRP3 inflammasome-mediated pyroptosis. Furthermore, the in vitro experiments demonstrated that lnc-Lfar1 knockdown significantly suppressed LPS- and IFN-γ-induced proinflammatory activation of macrophages, and inhibited LPS/ATP- and LPS/Nigericin-induced NLRP3 inflammasome-mediated pyroptosis. Mechanistically, lnc-Lfar1 regulated LPS- and IFN-γ-induced proinflammatory activation of macrophages through the NF-ĸB pathway. All these data supported our conclusion that lnc-Lfar1 plays a vital role in controlling the activation and pyroptosis of macrophage, thus providing a possible therapeutic target against inflammation-related disorders including hepatic fibrosis.
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Project description:This study is performed to explore the role of P2X4 in intracerebral hemorrhage (ICH) and the association between P2X4 and the NLRP1/Caspase-1 pathway. The mouse ICH model was established via collagenase injection into the right basal ganglia. P2X4 expression in brain tissues was knocked down via intracerebroventricular injection with adeno-associated virus (AAV) harboring shRNA against shP2X4. The gene expression of P2X4 and protein levels related to NLRP1 inflammasome were detected using qRT-PCR and Western blot analysis, respectively. Muramyl dipeptide (an activator of NLRP1) was used to activate NLRP1 in brain tissues. ICH induced high expression of P2X4 in mouse brain tissues. The knockdown of P2X4 alleviated short- and long-term neurological deficits of ICH mice, as well as inhibited the tissue expression and serum levels of pro-inflammatory cytokines, including TNF-α, interleukin (IL)-6, and IL-1β. Additionally, the expressions of NLRP1, ASC, and pro-Caspase-1 were down-regulated upon P2X4 silencing. Moreover, neurological impairment and the expression and secretion of cytokines after P2X4 silencing were aggravated by the additional administration of MDP. P2X4 knockdown represses neuroinflammation in brain tissues after ICH. Mechanistically, P2X4 inhibition exerts a neuroprotective effect in ICH by blocking the NLRP1/Caspase-1 pathway.
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Project description:Myosin 1c (Myo1c) is an unconventional myosin that modulates signaling pathways involved in tissue injury and repair. In this study, we observed that Myo1c expression is significantly upregulated in human chronic liver disease such as nonalcoholic steatohepatitis (NASH) and in animal models of liver fibrosis. High throughput data from the GEO-database identified similar Myo1c upregulation in mice and human liver fibrosis. Notably, transforming growth factor-β1 (TGF-β1) stimulation to hepatic stellate cells (HSCs), the liver pericyte and key cell type responsible for the deposition of extracellular matrix, upregulates Myo1c expression, whereas genetic depletion or pharmacological inhibition of Myo1c blunted TGF-β-induced fibrogenic responses, resulting in repression of α-smooth muscle actin (α-SMA) and collagen type I α 1 chain (Col1α1) mRNA. Myo1c deletion also decreased fibrogenic processes such as cell proliferation, wound healing response, and contractility when compared with vehicle-treated HSCs. Importantly, phosphorylation of mothers against decapentaplegic homolog 2 (SMAD2) and mothers against decapentaplegic homolog 3 (SMAD3) were significantly blunted upon Myo1c inhibition in GRX cells as well as Myo1c knockout (Myo1c-KO) mouse embryonic fibroblasts (MEFs) upon TGF-β stimulation. Using the genetic Myo1c-KO mice, we confirmed that Myo1c is critical for fibrogenesis, as Myo1c-KO mice were resistant to carbon tetrachloride (CCl4)-induced liver fibrosis. Histological and immunostaining analysis of liver sections showed that deposition of collagen fibers and α-SMA expression were significantly reduced in Myo1c-KO mice upon liver injury. Collectively, these results demonstrate that Myo1c mediates hepatic fibrogenesis by modulating TGF-β signaling and suggest that inhibiting this process may have clinical application in treating liver fibrosis.NEW & NOTEWORTHY The incidences of liver fibrosis are growing at a rapid pace and have become one of the leading causes of end-stage liver disease. Although TGF-β1 is known to play a prominent role in transforming cells to produce excessive extracellular matrix that lead to hepatic fibrosis, the therapies targeting TGF-β1 have achieved very limited clinical impact. This study highlights motor protein myosin-1c-mediated mechanisms that serve as novel regulators of TGF-β1 signaling and fibrosis.
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Project description:BackgroundBiologics containing growth factors are frequently used to enhance healing after musculoskeletal injuries. One mechanism of action is thought to be though the ability of biologics to induce homing and migration of endogenous mesenchymal stromal cells (MSCs) to a target tissue. However, the ability of biologics to stimulate chemotaxis (directed migration of cells) and chemokinesis (increase rate of cell migration) of MSCs is unknown.Hypothesis/purposeThe aim of this study was to directly compare the ability of biologics including platelet rich plasma (PRP) and bone marrow concentrate (BMC) to induce MSC migration. The hypothesis was that leukocyte-low platelet rich plasma (Llo PRP) would induce migration to a greater extent than leukocyte-high platelet rich plasma (Lhi PRP) or BMC.MethodsBone marrow-derived MSCs were isolated from 8 horses. Migration of MSCs toward a biologic (BMC, Llo PRP, and Lhi PRP) or the positive control platelet derived growth factor (PDGF) was continuously traced and measured for 24hrs using time-lapse microscopy and a microfluidics device. Cell migration, chemotaxis and chemokinesis were determined by measurements of displacement, number of cells migrated, and cell flux.ResultsAll biologics resulted in a significantly greater percentage of MSCs migrated compared to the positive control (PDGF). MSCs migrated further toward BMC compared to Llo PRP. Cell migration, measured as cell flux, was greater toward BMC and Lhi PRP than Llo PRP.ConclusionThe biologics BMC and Lhi PRP elicit greater chemotaxis and chemokinesis of MSCs than Llo PRP. However, all biologics recruited the same number of MSCs suggesting that differences in other regenerative effects, such as growth factor concentration, between biologics should be strongly considered when choosing a biologic for treatment of musculoskeletal injuries. The results of this study have the potential to reduce the need, risks, and costs associated with MSC culture and delivery.
| S-EPMC5864018 | biostudies-literature
Project description:IntroductionThe number of patients with end stage kidney disease (ESKD) are increasing world-side. While interstitial fibrosis (IF) is a common step for the progression to ESKD, therapeutic options for IF is still limited in clinical settings. We have reported that bone marrow-derived fibrotic cell, fibrocyte, is involved in the pathogenesis of kidney fibrosis. Also recent studies revealed that erythropoietin has protective effect on kidney diseases. However, it is unknown whether erythropoietin (EPO) inhibits fibrosis in progressive kidney injury. Therefore, we explored the impacts of EPO on kidney fibrosis with focusing on fibrocyte.MethodFibrocyte was differentiated from peripheral mononuclear cells of healthy donor. Fibrocyte was stimulated with transforming growth factor beta (TGF)-β with/without EPO treatment. Moreover, the therapeutic effect of EPO was evaluated in murine unilateral ureteral obstruction (UUO) model.ResultTGF-β stimulation increased the expression of COL1 mRNA in fibrocyte. EPO signal reduced the expression of COL1 mRNA in dose dependent manner. EPO reduced mitochondrial oxidative stress and ameliorated mitochondrial membrane depolarization induced by TGF-β stimulation. Moreover, EPO reduced the mRNA expression of mitochondria related molecules, TRAF6, in fibrocyte. In addition, the count of CD45+/αSMA + double-positive fibrocyte was decreased in the EPO-administered UUO kidneys.ConclusionEPO signals function to prevent kidney fibrosis, particularly in fibrocyte. Regulating the renal accumulation of fibrocyte is a part of the anti-fibrotic functions of EPO.
| S-EPMC8167964 | biostudies-literature
Project description:For bone marrow stromal cells (BMSC) to be useful in cartilage repair their propensity for hypertrophic differentiation must be overcome. A single day of TGF-β1 stimulation activates intrinsic signaling cascades in BMSCs which subsequently drives both chondrogenic and hypertrophic differentiation. TGF-β1 stimulation upregulates SP7, a transcription factor known to contribute to hypertrophic differentiation, and SP7 remains upregulated even if TGF-β1 is subsequently withdrawn from the chondrogenic induction medium. Herein, we stably transduced BMSCs to express an shRNA designed to silence SP7, and assess the capacity of SP7 silencing to mitigate hypertrophy. SP7 silencing dampened both hypertrophic and chondrogenic differentiation processes, resulting in diminished microtissue size, impaired glycosaminoglycan production and reduced chondrogenic and hypertrophic gene expression. Thus, while hypertrophic features were dampened by SP7 silencing, chondrogenic differentation was also compromised. We further investigated the role of SP7 in monolayer osteogenic and adipogenic cultures, finding that SP7 silencing dampened characteristic mineralization and lipid vacuole formation, respectively. Overall, SP7 silencing affects the trilineage differentiation of BMSCs, but is insufficient to decouple BMSC hypertrophy from chondrogenesis. These data highlight the challenge of promoting BMSC chondrogenesis whilst simultaneously reducing hypertrophy in cartilage tissue engineering strategies.
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Project description:IQGAP1 is a scaffolding protein previously implicated in adherens junction formation. However, its role in the establishment or maintenance of tight junctions (TJs) has not been explored. We hypothesized that IQGAP1 could regulate TJ formation by modulating the expression and/or localization of junctional proteins, and we systematically tested this hypothesis in the model Madin-Darby canine kidney (MDCK) cell line. We find that IQGAP1 silencing enhances a transient increase in transepithelial electrical resistance (TER) observed during the early stages of TJ formation (Cereijido et al., 1978). Quantitative microscopy and biochemical experiments suggest that this effect of IQGAP1 on TJ assembly is accounted for by reduced expression and TJ recruitment of claudin 2, and increased TJ recruitment of claudin 4. Furthermore, we show that IQGAP1 also regulates TJ formation through its interactor CDC42, because IQGAP1 knockdown increases the activity of the CDC42 effector JNK and dominant-negative CDC42 prevents the increase in TER caused by IQGAP1 silencing. Hence, we provide evidence that IQGAP1 modulates TJ formation by a twofold mechanism: (1) controlling the expression and recruitment of claudin 2 and recruitment of claudin 4 to the TJ, and (2) transient inhibition of the CDC42-JNK pathway.
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