alpha-SMA Levels Research Articles

#2660 WWP2-induced pericytes to myofibroblast transition contributes to the progression of fibrosis in CKD in diabetes

Abstract Background and Aims CKD in diabetes can be characterized by different clinical and histological phenotypes, associated to different molecular pathways specifically involved in the progression of kidney disease. CKD in diabetes includes different phenotypes, commonly classified according to Mazzucco et al (PMID:11920336) as diabetic Nephropathy (DN), non-diabetic renal disease (NDRD) and mixed forms (DN+NDRD). Among these, DN represents the primary cause of end stage renal disease (ESRD) and our group demonstrated that renal damage in DN is characterized by a specific molecular feature, represented by the activation of lysine63 (K63)-ubiquitination (Ub) pathway. In particular, we demonstrated that accumulation of K63-Ub proteins is involved in the progression of renal fibrosis (PMID: 27881486). WWP2 is an E3 ubiquitin-protein ligase that modulates myofibroblast pro-fibrotic activation in cardiac fibrosis (PMID: 31399586), however its specific role in kidney fibrosis in diabetes and its subsequent involvement in K63-Ub pathway has not been described so far. Thus aim of this study was to evaluate the involvement of WWP2 in the progression of kidney fibrosis in diabetes and its involvement in K63-Ub pathway. Method WWP2, K-63Ub, alpha-sma and PDGFbeta-receptor expression were evaluated in: i) 22 kidney biopsies from patients with a biopsy proven diagnosis of DN (n = 11), NDRD (n = 6) and CKD without diabetes (n = 5), by immunohistochemistry and immunofluorescence; ii) streptozotocin (STZ)-treated DBA/2J mice, a model of human DN, in the presence or absence of a specific inhibitor of K63Ub (NSC697923) by immunohistochemistry (n = 3 for each group); iii) in HK2 tubular cells, EAHY926 endothelial cells and in human-derived pericytes under hyperglycemic conditions by western blotting and qPCR. Results Immunohistochemistry showed that WWP2 was expressed both in CKD and in DN patients’ biopsies when compared to patients with NDRD (p < 0.05 and p < 0.01 respectively), both at tubular, glomerular and in the vascular compartment. Immunofluorescence confirmed that WWP2 increased expression in DN patients compared to NDRD, was associated to accumulation of K63Ub proteins in the tubular compartment as well as in the vascular compartment in particular in pericytes, cells that envelop the surface of vessels and have been described as the major source of scar-forming myofibroblasts in CKD (PMID: 19008372). The involvement of WWP2 in the K63-Ub pathway was confirmed in vivo in DBA2J diabetic mice in which the specific inhibition by NSC697923 of the K63Ub pathway, significantly reduced WWP2 expression in kidney tissues from diabetic mice (p < 0.05). These results were confirmed in vitro where specific inhibition of K63Ub pathway by NSC697923 significantly reduced hyperglycaemia-induced WWP2 expression in HK2 cells by western blotting and qPCR (p < 0.01). Hyperglycaemic conditions did not influence WWP2 expression in EAHY926 endothelial cells (p = n.s.). Conversely, using qPCR, in pericytes we observed an hyperglycemia-induced expression of both WWP2 levels (RR>2) and alpha-sma levels (RR>3) at different time points. Moreover, western blotting showed that hyperglycemia also induced a decrease of PDGF-beta-receptor marker of pericytes, thus suggesting their transition to myofibroblasts. Conclusion These findings demonstrate the influence of WWP2 on the k63-Ub pathway thus driving fibrogenesis in DN patients, in particular through pericyte to myofibroblast transition. WWP2 could represent a potential novel target for therapeutic intervention in the treatment of chronic kidney disease in diabetes.

The Effect of Activated Growth Factor (AGF) from Platelets on Alpha-SMA Levels in Osteoarthritis: Invivo Study

Background: The most prevalent joint condition and leading contributor to disability is osteoarthritis (OA), resulting in a significant socio-economic impact. It occurs due to an imbalance of pro-inflammatory and anti-inflammatory mediators, influencing anabolic and catabolic activities. This study aims to determine the effect of activated growth factor (AGF) on alpha-SMA levels.
 Methods: This research is an in vivo post-test-only control group experimental study conducted at the Eureka Research Laboratory. Thirty male Wistar rats were divided into five treatment groups: normal control group, negative control group, AGF I group (TGF-β 100 pg/mL), AGF II group (TGF-β 1000 pg/mL), and AGF III group (TGF-β 10,000 pg/mL). AGF was obtained from rat blood intravenously and centrifuged at a predetermined speed. Growth factor activation was performed by adding 10% CaCl2, and then TGF-β levels were measured. All groups of rats were acclimatized for 7 days. After that, osteoarthritis was induced with intra-articular injection of monoiodoacetate (MIA) 4.8 mg/60μL in all groups of rats except the normal control group. Next, rats were given treatment according to the group for 21 days. On the 21st day, rats were euthanized, and alpha-SMA levels were measured using the sandwich method ELISA kit.
 Results: Sequentially, the mean Alpha-SMA levels for each group in pg/mL were: 91.495 ± 2.36; 10.682 ± 1.09; 30.502 ± 2.00; 52.892 ± 1.29; and 76.180 ± 2.65. In the AGF group, there was an increase in alpha-SMA levels directly proportional to the dose of TGF-β injected.
 Conclusion: AGF has an effect on increasing alpha-SMA levels in the joints of rats with a model of osteoarthritis.

THE EXPRESSION OF ALPHA-SMOOTH MUSCLE ACTIN IN CONTRACTED SOCKET

Abstract
 Introduction & Objectives : A contracted socket is considered a common complication in anophthalmic patients, with the resulting poor fitting of an ocular prosthesis. In severe cases where patients have had multiple previous reconstructive attempts, further socket contraction may result from fibrosis, leading to eventual surgical failure. Alpha-smooth muscle actin (alpha-SMA) is often used to identify pathologic fibroblasts. It may serve as a reliable marker for myofibroblast cells. The aim of the study was to find alpha-SMA protein expression in myofibroblasts derived from orbital socket contracture.
 Methods : In this study, we observed five patients with contracted sockets who underwent fornicoplasty and buccal graft surgery from October 2022 to February 2023. Tissue samples were taken from the conjunctival fornix area during the surgery and then examined for alpha-SMA expression.
 Results : In this study, there were 2 out of 5 (40%) positive alpha-SMA expressions with history of recurrent contraction, 2 (40%) negative expressions with no history of recurrent contraction, and 1 result that could not be assessed due to the limited number of samples containing fibroblasts in patient with history of recurrent contraction.
 Conclusion : Alpha-SMA levels can be detected in patients with contracted sockets. Patients with a history of recurrently contracted sockets have a tendency to express positive alpha-SMA. This may allow for the need for the administration of antimetabolites in patients with recurrent contracted sockets to avoid recurrence of this contracted socket.

P0555HYPOTHERMIA ATTENUATES CHRONIC RENAL INJURY AFTER ISCHEMIA-REPERFUSION IN KIDNEY

Abstract Background and Aims: Hypothermia attenuates the renal injury induced by ischemia-reperfusion(IR). However, the detailed molecular pathway remains unknown. Although there has been reported that hypothermia protects acute renal damage, there is little known that hypothermia reduce renal fibrosis in IR kidney injury. We evaluated hypothermic protection against renal fibrosis in renal IR injury. Method: C57Bl/6 mice were divided into the following groups: sham-operated (cold, 32C) vs normal temperature (37C); IR mice (32C vs 37C). Kidneys were harvested 10 and 27 minutes after induction of renal ischemia and 24, 72, 168 hours after iIR injury. Functional and molecular markers of kidney injury were evaluated. Results: The blood urea nitrogen and serum creatinine levels and the histologic renal injury scores were significantly lower in 32C IR than 37C IR kidneys (all P values < .05).In kidney harvested 10 and 27 min, the extent of renal AMPK,ERK, and HIF1 phosphorylation was significantly increased in the kidneys of 32C compared to 37C IR mice. In kidney harvested 27 min, 24hr, 72hr, and 168hr, the expression levels of SOX9, TGF beta, alpha SMA, collagen IV, and fibrinogen decreased in 32C IR compared to 37C IR mice. In addition, in kidney harvested 72hr, and 168hr, the stained area of TGF beta, alpha SMA, and masson trichrome, were significantly decreased in 32C IR compared to 37C IR mice. Conclusion: Hypothermia attenuates the renal fibrosis induced by Ischemia reperfusion.

Obesity Enhances the Conversion of Adipose-Derived Stromal/Stem Cells into Carcinoma-Associated Fibroblast Leading to Cancer Cell Proliferation and Progression to an Invasive Phenotype.

Obesity is associated with enhanced tumor growth and progression. Within the adipose tissue are adipose-derived stromal/stem cells (ASCs) that have been shown to convert into carcinoma-associated fibroblast (CAFs) in the presence of tumor-derived factors. However, the impact of obesity on the ASCs and on the conversion of ASCs into CAFs has not been demonstrated. In the current study, ASCs isolated from lean donors (BMI < 25; lnASCs) were compared with ASCs isolated from obese donors (BMI > 30, obASCs). The contribution of tumor-derived factors on the conversion of ASCs to CAFs was investigated. Following exposure to cancer cells, obASCs expressed higher levels of CAF markers, including NG2, alpha-SMA, VEGF, FAP, and FSP, compared to lnASCs. To investigate the crosstalk between ASCs and breast cancer cells, MCF7 cells were serially cocultured with lnASCs or obASCs. After coculture with lnASCs and obASCs, MCF7 cells demonstrated enhanced proliferation and expressed an invasive phenotype morphologically, with more pronounced effects following exposure to obASCs. Long-term exposure to obASCs also enhanced the expression of protumorgenic factors. Together, these results suggest that obesity alters ASCs to favor their rapid conversion into CAFs, which in turn enhances the proliferative rate, the phenotype, and gene expression profile of breast cancer cells.

Rapamycin inhibits transforming growth factor beta 1 induced myofibroblast differentiation via the phosphorylated-phosphatidylinositol 3-kinase mammalian target of rapamycin signal pathways in nasal polyp-derived fibroblasts.

Rapamycin has antiproliferative and antifibrogenic effects in vitro and in vivo. The purpose of this study was to evaluate the effects of rapamycin on transforming growth factor (TGF) beta 1 induced myofibroblast differentiation (alpha smooth-muscle actin [SMA]), extracellular matrix production, and collagen contraction in nasal polyp-derived fibroblasts (NPDF). The underlying molecular mechanisms of rapamycin were also determined in NPDFs. NPDFs were grown in culture and transformed into myofibroblasts by using TGF beta 1 (5 ng/mL). For cytotoxicity evaluation, a 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide assay was used. Expression levels of alpha SMA, phosphorylated phosphatidylinositol 3-kinase (PI3K), and phosphorylated mammalian target of rapamycin (mTOR) were determined by using Western blot, reverse transcription-polymerase chain reaction, and immunofluorescence staining. The total amount of collagen was analyzed by using the Sircol collagen assay, and contractile activity was measured with a collagen gel contraction assay. Silencing mTOR with mTOR-specific small interference RNA was determined by using reverse transcription-polymerase chain reaction. Whereas rapamycin (range, 0-400 nM) had no significant cytotoxic effects on TGF beta 1 induced NPDFs, it significantly reduced the expression levels of alpha-SMA in TGF beta 1 induced NPDFs in a dose-dependent manner. TGF beta 1 induced collagen production and collagen contraction were significantly inhibited by rapamycin treatment. Rapamycin also attenuated the TGF beta 1 induced activation of PI3K and mTOR, and its inhibitory effects were similar to those of mTOR silencing and a specific PI3K inhibitor. Rapamycin inhibited TGF beta 1 induced myofibroblast differentiation, extracellular matrix production, and collagen contraction through the PI3K/mTOR signal pathways in NPDFs.

Increased expression of the Glucose-transporter GLUT1 during hepatic stellate cell activation promotes hepatic fibrosis

Normal liver reveals only low expression of the facilitative glucose transporter (GLUT) 1. Recently, we have shown that GLUT1 expression is increased in hepatocellular carcinoma (HCC), where GLUT1 acts as a tumor promotor. Hyperglycemia is one of the factors known to induce and promote hepatic fibrogenesis, and the activation of hepatic stellate cells (HSCs) is the key event of hepatic fibrosis. Furthermore, these cells form and transduce the HCC stroma. The aim of this study was to assess the expression and functional role of GLUT1 in hepatic fibrosis. Methods and Results: Hepatic GLUT1 expression was significantly increased in different murine models of hepatic fibrosis, namely chronic TAA or CCl4-application, bile-duct-ligation and dietary models of non-alcoholic steatohepatitis (NASH). Also in cirrhotic human livers and human NASH GLUT1 expression was increased. Interestingly, hepatic GLUT1 expression revealed a significant correlation with collagen I and alpha-sma levels indicating activated HSCs as cellular source of GLUT1, which was confirmed by immunohistochemical analysis. In line with this, we found that GLUT1 expression increased during in vitro activation of primary murine and human HSCs. To gain insight in the functional role of GLUT1 in activated HSCs we inhibited GLUT1 expression with siRNA or the specific GLUT1 inhibitor WZB117 and found that GLUT1 suppression impaired glucose uptake and lactate secretion of HSCs indicative for reduced anaerobic glycolysis. Functional analysis demonstrated that reduced GLUT1 expression led to lower apoptosis resistance of HSCs. Transgenic GLUT1 knockdown mice subjected to TAA induced liver fibrosis model revealed less pronounced liver damage than WT animals confirmed by hepatic sirius red staining and alpha-sma protein expression. In line with this, treatment with GLUT1 inhibitor WZB117 in combination with the NASH inducing MCD diet resulted in less hepatic alpha-sma and collagen I expression than animals treated with vehicle. Conclusions: Increased GLUT1 expression during HSC activation functionally affects HSCs, and herewith, likely promotes the development and progression of hepatic fibrosis. Therefore and based on its known role as tumor-promotor, GLUT1 appears as attractive novel target to inhibit the progression of chronic liver disease. Corresponding author: Czech, Barbara E-Mail:barbara.czech@ukr.de

Fibroblast phenotypes in different lung diseases.

BackgroundThe “seed and soil” hypothesis emphasizes the importance of interactions between tumor cells and their microenvironment. CAFs (Cancer associated fibroblasts) are important components of the tumor microenvironment. They were widely involved in cancer cells growth and metastasis. Fibroblasts may also play a role in inflammatory disease. The phenotype conversion of fibroblasts in lung diseases has not been investigated previously. We hypothesized that fibroblasts phenotypes may vary among different types of lung disease.MethodsThe study included six types of lung tissues, ranging from normal lung to lung adenocarcinoma with lymphatic metastasis. Para-carcinoma tissues which were 2-cm-away from the tumor focus were also included in the analysis. The expression of target proteins including alpha-SMA (smooth muscle actin), FAP (fibroblast activation protein), vimentin, E-cadherin, and CK-19 (cytokeratin-19) were examined by immunohistochemistry. TGF-beta(transforming growth factor) and Twist were detected simultaneously in all samples.ResultsA progressive increase in the levels of alpha-SMA, vimentin and CK-19 was observed in correlation to the degree of malignancy from normal lung tissue to lung adenocarcinoma with lymphatic metastasis, whereas E-cadherin expression showed the opposite trend. TGF-beta and Twist were detected in cancer tissues and inflammatory pseudotumors. None of the proteins were detected in para-carcinoma tissues.ConclusionsFibroblast phenotypes varied according to the type and degree of lung malignancy and fibroblasts phenotypic conversion occurs as a gradual process with specific spatiotemporal characteristics. Similar fibroblast phenotypes in inflammatory diseases and cancer tissues suggested a correlation between inflammation and cancer and implied a common mechanism underlying the formation of fibroblasts in inflammatory diseases and lung cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s13019-014-0147-z) contains supplementary material, which is available to authorized users.

Defective Wound-healing in Aging Gingival Tissue

Aging may negatively affect gingival wound-healing. However, little is known about the mechanisms underlying this phenomenon. The present study examined the cellular responses associated with gingival wound-healing in aging. Primary cultures of human gingival fibroblasts were obtained from healthy young and aged donors for the analysis of cell proliferation, cell invasion, myofibroblastic differentiation, and collagen gel remodeling. Serum from young and old rats was used to stimulate cell migration. Gingival repair was evaluated in Sprague-Dawley rats of different ages. Data were analyzed by the Mann-Whitney and Kruskal-Wallis tests, with a p value of .05. Fibroblasts from aged donors showed a significant decrease in cell proliferation, migration, Rac activation, and collagen remodeling when compared with young fibroblasts. Serum from young rats induced higher cell migration when compared with serum from old rats. After TGF-beta1 stimulation, both young and old fibroblasts demonstrated increased levels of alpha-SMA. However, alpha-SMA was incorporated into actin stress fibers in young but not in old fibroblasts. After 7 days of repair, a significant delay in gingival wound-healing was observed in old rats. The present study suggests that cell migration, myofibroblastic differentiation, collagen gel remodeling, and proliferation are decreased in aged fibroblasts. In addition, altered cell migration in wound-healing may be attributable not only to cellular defects but also to changes in serum factors associated with the senescence process.

Down‐regulation of Smurf1 and Smurf2 is involved in the Initiation and Progression of Hepatic Fibrotic rats

The detail mechanisms undergoing degradation of TGFbeta/Smad molecules are poorly understood. This study was designed to determine if novel Smurfs‐dependent degradation machinery is involved in the hepatic fibrosis. Rats with hepatic fibrosis were induced by 40% carbon tetrachloride. Immunohistochemistry staining and western blot were performed to quantify the expression levels of Smurf1 and Smurf 2 in normal and fibrotic liver tissues. In parallel, the hepatic stellate cells (HSCs) marker Desmin and alpha‐SMA were dynamically measured as well. Furthermore, in vitro study was performed to elucidate the relationship among Smad3, Smad7, alpha (2) I collagen and Smurf1&amp;2 levels in cultured HSCs by using specific siRNA to block the Smurf1&amp;2 mRNA. It was found that either Smurf1 or Smurf2 was enriched in normal hepatic Disse space. However, the expression levels of alpha‐SMA but not Desmin, Smurf1 or Smurf2 have significantly increased in fibrotic rats (P&lt;0.01). In those rats with hepatic fibrosis, both Smurf1 and Smurf2, particular Smurf2, were found to markedly decrease (P&lt;0.01). Both decrement of Smurf1 and Smurf2 are associated to the increasing levels in phosphorylation of Smad3 as well as alpha (2) I collagen mRNA in these cells. Our data indicate that down‐regulation of Smurf1&amp;2 in liver may importantly initialize and participate in the process of hepatic fibrosis. (Supported by NSFC 30960145 and GHF 200958, zhangfacan@126.com)

Bcl‐2 overexpression in hepatic stellate cell line CFSC‐2G, induces a pro‐fibrotic state

Development of hepatic fibrosis is a complex process that involves oxidative stress (OS) and an altered balance between pro- and anti-apoptotic molecules. Since Bcl-2 overexpression preserves viability against OS, our objective was to address the effect of Bcl-2 overexpression in the hepatic stellate cells (HSC) cell-line CFSC-2G under acetaldehyde and H(2)O(2) challenge, and explore if it protects these cells against OS, induces replicative senescence and/or modify extracellular matrix (ECM) remodeling potential. To induce Bcl-2 overexpression, HSC cell line CFSC-2G was transfected by lipofection technique. Green fluorescent protein-only CFSC-2G cells were used as a control. Cell survival after H(2)O(2) treatment and total protein oxidation were assessed. To determine cell cycle arrest, proliferation-rate, DNA synthesis and senescence were assessed. Matrix metalloproteinases (MMP), tissue-inhibitor of MMP (TIMP), transglutaminases (TG) and smooth muscle a-actin (alpha-SMA) were evaluated by western blot in response to acetaldehyde treatment as markers of ECM remodeling capacity in addition to transforming growth factor-beta (TGF-beta) mRNA. Cells overexpressing Bcl-2 survived approximately 20% more than control cells when exposed to H(2)O(2) and approximately 35% proteins were protected from oxidation, but Bcl-2 did not slow proliferation or induced senescence. Bcl-2 overexpression did not change alpha-SMA levels, but it increased TIMP-1 (55%), tissue transglutaminases (tTG) (25%) and TGF-beta mRNA (49%), when exposed to acetaldehyde, while MMP-13 content decreased (47%). Bcl-2 overexpression protected HSC against oxidative stress but it did not induce replicative senescence. It increased TIMP-1, tTG and TGF-beta mRNA levels and decreased MMP-13 content, suggesting that Bcl-2 overexpression may play a key role in the progression of fibrosis in chronic liver diseases.

Scaffold-Free In Vitro Arterial Mimetics: The Importance of Smooth Muscle–Endothelium Contact

We have developed an in vitro endothelial cell (EC)-smooth muscle cell (SMC) coculture platform that can mimic either the healthy or diseased state of blood vessels. Transforming growth factor-beta1 (TGF-beta1) and heparin were introduced to the SMC cultures to upregulate the SMC differentiation markers, alpha-smooth muscle actin (alpha-SMA) and calponin (homotypic model). Interestingly, seeding of near-confluent concentrations of ECs on the SMCs (heterotypic model) induced higher levels of alpha-SMA and calponin expression in the SMC cultures than did the addition of heparin and TGF-beta1 alone. The expression levels increased further on pretreating the SMCs with TGF-beta1 and heparin before adding a near-confluent monolayer of ECs. In contrast, seeding of sparse concentrations of ECs forced the SMCs into a more hyperplastic state as determined by alpha-SMA and calponin expression. This study highlights the importance of both soluble factors and EC seeding densities when considering culture conditions; in vivo SMCs are in close proximity with and interact with a monolayer of ECs. Our study suggests that this architecture is important for healthy vascular tissue function. In addition, it shows that disruption of this architecture can be used to mimic diseased states. As the EC-SMC coculture model can mimic either a diseased or a healthy blood vessel it may be useful as a test bed for evaluating cardiovascular therapeutics.

Sustained expression of NADPH oxidase 4 by p38 MAPK-Akt signaling potentiates radiation-induced differentiation of lung fibroblasts

Radiation-induced fibrosis (RIF) is a long-term adverse effect of curative radiotherapy; however, the distinct molecular mechanisms of RIF in neighboring normal tissue are not fully understood. We investigated the mechanisms underlying radiation-induced fibroblast differentiation into myofibroblasts. Lung fibroblasts produced reactive oxygen species (ROS) immediately after irradiation, the level of which remained increased for 24 h. The NADPH oxidase inhibitor, diphenyleneiodonium (DPI), suppressed ROS production and significantly decreased the radiation-induced expression of alpha-smooth muscle actin (alpha-SMA) and fibronectin (FN). The mRNA and protein expression of Nox4 was increased by radiation, and siRNA knockdown of Nox4 reduced alpha-SMA and FN levels. Increased phosphorylation of p38MAPK, Erk, and PI3k/Akt was observed after irradiation. Inhibitors of p38 MAPK and Akt, but not of Erk, reduced radiation-induced fibroblast differentiation and Nox4 expression. Notably, DPI partially decreased phosphorylation of p38MAPK and Akt, suggesting that p38MAPK, Akt, and Nox4 may cooperate in a positive feedback loop. Nox4 expression was also increased during bleomycin-induced fibroblast differentiation, and downregulation of Nox4 reduced alpha-SMA levels and extracellular matrix (ECM) accumulation. These results demonstrate that interfering Nox4 activation can be a potential strategy to disrupt fibrotic process.

Effects of salvianolic acids on oxidative stress and hepatic fibrosis in rats

Enhanced oxidative stress is associated with hepatic fibrosis. Salvianolic acids A (Sal A) and B (Sal B) have been reported to be strong polyphenolic antioxidants and free radical scavengers. The present study is to investigate if Sal A and B could attenuate oxidative stress and liver fibrosis in rats. A cell line of rat hepatic stellate cells (HSCs) was stimulated with platelet-derived growth factor (PDGF, 10 ng/ml). The inhibitory effects of Sal A and B on intracellular hydrogen peroxide levels were measured with dichlorofluorescein diacetate (DCF-DA) dye assay. alpha-Smooth muscle actin (alpha-SMA), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits were measured by Western blotting. Liver fibrosis was induced by intraperitoneal injections of thioacetamide (TAA, 200 mg/kg) twice per week for 6 weeks. Sal A (10 mg/kg), Sal B (50 mg/kg) or S-adenosylmethionine (SAMe, 10 mg/kg), was given by gavage twice per day consecutively for 4 weeks starting 2 weeks after TAA injection. In vitro, PDGF increased the accumulation of hydrogen peroxide in HSCs, which was attenuated by Sal A (10 muM) and Sal B (200 muM). Sal A and B attenuated the PDGF-stimulated expressions of alpha-SMA and NADPH oxidase subunits gp91(phox) and p47(phox) in membrane fractions. In vivo studies showed that the hepatic levels of collagen, malondialdehyde, TNF-alpha, IL-6, and IL-1beta, fibrosis scores and protein expressions of alpha-SMA, heme-oxygenase-1, iNOS, and gp91(phox), and serum levels of ALT, AST, IL-6, and IL-1beta were increased in TAA-intoxicated rats, all of which were attenuated by 4-week treatment of Sal A or Sal B. Our results showed that Sal A and B attenuated PDGF-induced ROS formation in HSCs, possibly through inhibition of NADPH oxidase. Sal A and B treatments were also effective against hepatic fibrosis in TAA-intoxicated rats.

BMP6 reverses TGF-β1-induced changes in HK-2 cells: implications for the treatment of renal fibrosis

The aim of the study was to investigate the potential role of BMP6 in TGF-beta1-mediated changes in HK-2 cells. BMP6 was purified via heparin affinity and reverse phase liquid chromatography. The purity, specificity, and bioactivity of BMP6 were determined by SDS-PAGE, Western blot assays, and the induction of alkaline phosphatase (ALP) activity, respectively. Cell proliferation, morphology, and expression levels of alpha-SMA and E-cadherin were assessed by cell viability, microscopy, and Western blot assays, respectively. In addition, cell adhesion abilities were determined by counting the number of attached cells. The expression of fibronectin, collagen IV, matrix metalloproteinases 2 (MMP-2), and tissue inhibitors of matrix metalloproteinases 2 (TIMP-2) were analyzed using RT-PCR. MMP-2 activity was analyzed by zymography, whereas the activation of the MAPKs and Smad signaling were analyzed using Western blot assays and a reporter gene assay, respectively. Our results indicated that recombinant BMP6 induced ALP activity in a dose-dependent and time-course-dependent manner. Treatment with TGF-beta1 reduced both the cell proliferation and the expression of E-cadherin, induced a morphological transformation, decreased the expression and activity of MMP-2, and increased the expression levels of alpha-SMA, fibronectin, and TIMP-2 in HK-2 cells. All of these effects were inhibited when cells were treated with TGF-beta1 in combination with rhBMP6, whereas rhBMP6 alone demonstrated no such effect. Treatment with TGF-beta1, rhBMP6, or a combination of both had no effect on the expression of collagen IV. In addition, the administration of rhBMP6 prevented the enhanced adhesion behavior triggered by TGF-beta1. Furthermore, the addition of rhBMP6 abrogated the JNK and Smad2/3 signaling that was activated by TGF-beta1. BMP6 ameliorated the TGF-beta1-induced changes in HK-2 cells. The suppression of TGF-beta1-mediated JNK and Smad2/3 signaling activation were implicated in these effects.Acta Pharmacologica Sinica (2009) 30: 994-1000; doi: 10.1038/aps.2009.56; published online 22 June 2009.

Transforming growth factor-β1 induces intestinal myofibroblast differentiation and modulates their migration

To investigate the effects of transforming growth factor beta 1 (TGF-beta 1) on the differentiation of colonic lamina propria fibroblasts (CLPF) into myofibroblasts in vitro. Primary CLPF cultures were incubated with TGF-beta 1 and analyzed for production of alpha-smooth muscle actin (alpha-SMA), fibronectin (FN) and FN isoforms. Migration assays were performed in a modified 48-well Boyden chamber. Levels of total and phosphorylated focal adhesion kinase (FAK) in CLPF were analyzed after induction of migration. Incubation of CLPF with TGF-beta 1 for 2 d did not change alpha-SMA levels, while TGF-beta 1 treatment for 6 d significantly increased alpha-SMA production. Short term incubation (6 h) with TGF-beta 1 enhanced CLPF migration, while long term treatment (6 d) of CLPF with TGF-beta 1 reduced migration to 15%-37% compared to untreated cells. FN and FN isoform mRNA expression were increased after short term incubation with TGF-beta 1 (2 d) in contrast to long term incubation with TGF-beta 1 for 6 d. After induction of migration, TGF-beta 1-preincubated CLPF showed higher amounts of FN and its isoforms and lower levels of total and phosphorylated FAK than untreated cells. Long term incubation of CLPF with TGF-beta 1 induced differentiation into myofibroblasts with enhanced alpha-SMA, reduced migratory potential and FAK phosphorylation, and increased FN production. In contrast, short term contact (6 h) of fibroblasts with TGF-beta 1 induced a dose-dependent increase of cell migration and FAK phosphorylation without induction of alpha-SMA production.

Regulation of Scleral Cell Contraction by Transforming Growth Factor-β and Stress: COMPETING ROLES IN MYOPIC EYE GROWTH

Reduced extracellular matrix accumulation in the sclera of myopic eyes leads to increased ocular extensibility and is related to reduced levels of scleral transforming growth factor-beta (TGF-beta). The current study investigated the impact of this extracellular environment on scleral cell phenotype and cellular biomechanical characteristics. Scleral cell phenotype was investigated in vivo in a mammalian model of myopia using the myofibroblast marker, alpha-smooth muscle actin (alpha-SMA). In eyes developing myopia alpha-SMA levels were increased, suggesting increased numbers of contractile myofibroblasts, and decreased in eyes recovering from myopia. To understand the factors regulating this change in scleral phenotype, the competing roles of TGF-beta and mechanical stress were investigated in scleral cells cultured in three-dimensional collagen gels. All three mammalian isoforms of TGF-beta altered scleral cell phenotype to produce highly contractile, alpha-SMA-expressing myofibroblasts (TGF-beta3>TGF-beta2>TGF-beta1). Exposure of cells to the reduced levels of TGF-beta found in the sclera in myopia produced decreased cell-mediated contraction and reduced alpha-SMA expression. These findings are contrary to the in vivo gene expression data. However, when cells were exposed to both the increased stress and the reduced levels of TGF-beta found in myopia, increased alpha-SMA expression was observed, replicating in vivo findings. These results show that although reduced scleral TGF-beta is a major contributor to the extracellular matrix remodeling in the myopic eye, it is the resulting increase in scleral stress that dominates the competing TGF-beta effect, inducing increased alpha-SMA expression and, hence, producing a larger population of contractile cells in the myopic eye.

Increased Expression of Myo-inositol Oxygenase is Involved in the Tubulointerstitial Injury of Diabetic Nephropathy

Myo-inositol oxygenase (MIOX) catalyzes the oxidative cleavage of myo-inositol (MI) to give D-glucuronic acid, a committed step in MI catabolism. Previous studies have shown that increased mRNA and protein levels of MIOX in the cortex of the kidney in diabetic mice. The implication of MIOX expression in diabetic nephropathy, however, has not been revealed. In the present study, we demonstrate for the first time that the expression of MIOX was increased at both the mRNA and protein levels in the kidney of rats with diabetic nephropathy. In addition, alpha-smooth muscle actin (alpha-SMA) and fibronectin levels were increased and E-cadherin levels decreased in the same diabetic kidneys. In vitro, studies have shown that high concentrations of glucose significantly increased MIOX secretion in rat renal tubular epithelial cells NRK-52E in a dose-dependent manner, suggesting that hyperglycemia is a direct cause of the MIOX increase in the kidney. With respect to the function of MIOX, we have shown that overexpression of MIOX induces greater levels of alpha-SMA, increased fibronectin expression, and lower levels of E-cadherin expression relative to normal NRK-52E cells. The blockade of MIOX by antisense oligonucleotide (ODN) inhibits high glucose-induced production of alpha-SMA and fibronectin in normal NRK-52E cells. This suggests that increased expression of MIOX in diabetic kidneys may contribute to tubulointerstitial injury and the development of diabetic nephropathy.

GSK-3β in mouse fibroblasts controls wound healing and fibrosis through an endothelin-1–dependent mechanism

Glycogen synthase kinase-3 (GSK-3) is a widely expressed and highly conserved serine/threonine protein kinase encoded by 2 genes, GSK3A and GSK3B. GSK-3 is thought to be involved in tissue repair and fibrogenesis, but its role in these processes is currently unknown. To investigate the function of GSK-3beta in fibroblasts, we generated mice harboring a fibroblast-specific deletion of Gsk3b and evaluated their wound-healing and fibrogenic responses. We have shown that Gsk3b-conditional-KO mice (Gsk3b-CKO mice) exhibited accelerated wound closure, increased fibrogenesis, and excessive scarring compared with control mice. In addition, Gsk3b-CKO mice showed elevated collagen production, decreased cell apoptosis, elevated levels of profibrotic alpha-SMA, and increased myofibroblast formation during wound healing. In cultured Gsk3b-CKO fibroblasts, adhesion, spreading, migration, and contraction were enhanced. Both Gsk3b-CKO mice and fibroblasts showed elevated expression and production of endothelin-1 (ET-1) compared with control mice and cells. Antagonizing ET-1 reversed the phenotype of Gsk3b-CKO fibroblasts and mice. Thus, GSK-3beta appears to control the progression of wound healing and fibrosis by modulating ET-1 levels. These results suggest that targeting the GSK-3beta pathway or ET-1 may be of benefit in controlling tissue repair and fibrogenic responses in vivo.

Deficient α-smooth muscle actin as a cause of functional intestinal obstruction in childhood

Most functional intestinal obstruction in childhood is due to defects in the enteric innervation. Functional intestinal obstruction due to smooth muscle disorders is rare. The aim of this study was to describe four patients with functional intestinal obstruction where the only histopathological abnormality was deficient, alpha-smooth muscle actin (alpha-SMA) in the circular muscle of their small bowel. Resected intestinal segments from four children with functional intestinal obstruction were either snap frozen or embedded in paraffin wax, and transversely sectioned. Sections were examined by routine H&E staining, AChE and NADPH-diaphorase histochemistry, and immunohistochemistry using PGP9.5 and alpha-SMA antibodies. Normal age-matched tissue was used as controls. All four patients demonstrated normal innervation in the small and large bowel. We observed striking differences in the expression of alpha-SMA within the intestinal muscle layers in all four patients. There was a markedly decreased level of alpha-SMA in the smooth muscle compared to the longitudinal muscle in the small bowel in each patient. All other regions of the intestine examined showed normal levels of alpha-SMA, with similar levels expressed in both the circular and longitudinal muscles. The decreased level of alpha-SMA in the smooth muscle of these patients causes a serious mechanical defect, thus interfering with normal intestine motility. These findings suggest that patients with functional intestinal obstruction should have a comprehensive investigation of the intestinal smooth muscle in addition to the assessment of the enteric nervous system.