Alpha-smooth Muscle Actin mRNA Research Articles

Curcumin Interferes with TGF- β 1-Induced Fibrosis in NRK-49F Cells by Reversing ADAMTS18 Gene Methylation.

To explore the molecular mechanism by which curcumin affects renal interstitial fibrosis (RIF) progression by regulating ADAM metallopeptidase with thrombospondin type 1 motif 18 (ADAMTS18) methylation. NRK-49F cells RIF model were induced with transforming growth factor β 1 (TGF- β 1). Effects of different concentrations of curcumin (0, 10, 20, and 30 μmol/L) on cell proliferation, cell cycle, cell apoptosis as well as cyclin D1 expression were analyzed by cell counting kit-8, flow cytometry and Western blot, respectively. ADAMTS18 methylation levels were determined by methylation-specific polymerase chain reaction. ADAMTS18, fibronectin (FN), type I collagen (Col- I) and alpha-smooth muscle actin (α -SMA) mRNA and protein expressions were analyzed by real-time PCR (RT-PCR) and Western blot, respectively. Meanwhile, cells were treated with 50 mmol/L 5-aza-2'-deoxycytidine (5-aza-dC, demethylation agent) for 72 h. Effect of curcumin on extracellular matrix (ECM) deposition was evaluated by immunochemical staining and Western blot. NRK-49F cells were transfected with ADAMTS18 small interfering RNA and grouped into a normal control, ADAMTS18-knock-out (KO), and ADAMTS18-KO+ 30 μmol/L curcumin groups, and whether curcumin can reverse the effect of ADAMTS18 knockdown on RIF was evaluated. Compared with the control group, TGF-β 1 significantly inhibited the proliferation of NRK-49F cells, blocked the G1/G0 phase, promoted cell apoptosis and inhibited cyclin D1 expression (P<0.01). Among the different concentrations of curcumin, 30 μmol/L curcumin significantly reversed these processes (P<0.01). Immunochemical staining and Western blot results showed that curcumin significantly inhibited the deposition of FN, Col- I and α-SMA (P<0.01). Curcumin and 5-zaz-dC had synergistic effects, promoting ADAMTS18 expression, removing ADAMTS18 methylation, and reducing ECM deposition. ADAMTS18 knockdown promoted ECM accumulation, and curcumin reversed this process (P<0.01). TGF-β 1-induced fibrosis in NRK-49F cells. Curcumin promoted ADAMTS18 expression, reduced ECM accumulation, and alleviated RIF progression by inhibiting ADAMTS18 methylation.

Andrographolide Inhibits Corneal Fibroblast to Myofibroblast Differentiation In Vitro.

Corneal opacification due to fibrosis is a leading cause of blindness worldwide. Fibrosis occurs from many causes including trauma, photorefractive surgery, microbial keratitis (infection of the cornea), and chemical burns, yet there is a paucity of therapeutics to prevent or treat corneal fibrosis. This study aimed to determine if andrographolide, a labdane diterpenoid found in Andrographis paniculate, has anti-fibrotic properties. Furthermore, we evaluated if andrographolide could prevent the differentiation of fibroblasts to myofibroblasts in vitro, given that the transforming growth factor beta-1(TGF-β1) stimulated persistence of myofibroblasts in the cornea is a primary component of fibrosis. We demonstrated that andrographolide inhibited the upregulation of alpha smooth muscle actin (αSMA) mRNA and protein in rabbit corneal fibroblasts (RCFs), thus, demonstrating a reduction in the transdifferentiation of myofibroblasts. Immunofluorescent staining of TGF-β1-stimulated RCFs confirmed a dose-dependent decrease in αSMA expression when treated with andrographolide. Additionally, andrographolide was well tolerated in vivo and had no impact on corneal epithelialization in a rat debridement model. These data support future studies investigating the use of andrographolide as an anti-fibrotic in corneal wound healing.

Renoprotective Effect of KLF2 on Glomerular Endothelial Dysfunction in Hypertensive Nephropathy.

Kruppel-like factor 2 (KLF2) regulates endothelial cell metabolism; endothelial dysfunction is associated with hypertension and is a predictor of atherosclerosis development and cardiovascular events. Here, we investigated the role of KLF2 in hypertensive nephropathy by regulating KLF2 expression in human primary glomerular endothelial cells (hPGECs) and evaluating this expression in the kidney tissues of a 5/6 nephrectomy mouse model as well as patients with hypertension. Hypertension-mimicking devices and KLF2 siRNA were used to downregulate KLF2 expression, while the expression of KLF2 was upregulated by administering simvastatin. After 4 mmHg of pressure was applied on hPGECs for 48 h, KLF2 mRNA expression decreased, while alpha-smooth muscle actin (αSMA) mRNA expression increased. Apoptosis and fibrosis rates were increased under pressure, and these phenomena were aggravated following KLF2 knockdown, but were alleviated after simvastatin treatment; additionally, these changes were observed in angiotensin II, angiotensin type-1 receptor (AT1R) mRNA, and interleukin-18 (IL-18), but not in angiotensin type-2 receptor mRNA. Reduced expression of KLF2 in glomerular endothelial cells due to hypertension was found in both 5/6 nephrectomy mice and patients with hypertensive nephropathy. Thus, our study demonstrates that the pressure-induced apoptosis and fibrosis of glomerular endothelial cells result from angiotensin II, AT1R activation, and KLF2 inhibition, and are associated with IL-18.

Evaluation of a novel combination of TRAM-34 and ascorbic acid for the treatment of corneal fibrosis in vivo.

Corneal injury and aberrant wound healing commonly result in corneal fibrosis and subsequent vision loss. Intermediate-conductance calmodulin/calcium-activated K+ channels (KCa3.1) have been shown to promote fibrosis in non-ocular and ocular tissues via upregulation of transforming growth factor beta (TGFβ). TRAM-34 is a selective inhibitor of KCa3.1 and reduces fibrosis by downregulation of TGFβ-induced transdifferentiation of stromal fibroblasts to myofibroblasts. Ascorbic acid has been demonstrated to be effective in promoting corneal re-epithelialization and reduction of neovascularization via anti-VEGF and anti-MMP mechanisms. This study evaluates tolerability and efficacy of a novel combination of TRAM-34 (25μM) and ascorbic acid (10%) topical treatment for corneal fibrosis using an established in vivo rabbit model and conducting clinical eye examinations. Markers of corneal fibrosis were evaluated in all corneas at study endpoint via histopathology, immunofluorescence, and quantitative real-time PCR. The eyedrop treated eyes showed significantly improved clinical outcomes based on modified McDonald Shadduck scores, reduction of clinical haze on Fantes scores, and reduction of central corneal thickness (CCT). At cellular and molecular levels, eyedrop treatment also significantly reduced expression of alpha smooth muscle actin (α-SMA) mRNA and protein, collagen III mRNA, and fibronectin mRNA compared to non-treated eyes. Our study suggests that a tested new bimodal eyedrop is well tolerated and effectively reduces corneal fibrosis/haze in rabbits in vivo.

Tanshinone IIA attenuates high glucose-induced epithelial-to-mesenchymal transition in HK-2 cells through VDR/Wnt/β-catenin signaling pathway.

The progression of diabetic kidney disease (DKD) is closely related to renal tubular epithelial- to-mesenchymal transition (EMT) and tubulointerstitial fibrosis. Tanshinone IIA (TSIIA), extracted from a traditional Chinese medicine named Salvia miltiorrhiza, has been proved to have anti-fibrosis effects. The aim of this study was to investigate the effect of TSIIA on high glucose-induced EMT in human proximal tubular cells (HK-2 cells) and its possible mechanism. The proliferation of cells exposed to different concentrations of glucose was measured by light microscopy and CCK-8 test. The cells were stimulated with 30 mM glucose and different concentrations of TSIIA (5 μM or 10 μM) for 48 h. Vitamin D receptor (VDR)-siRNA was used to transfect cells, and high glucose and TSIIA treatment were further used to treat cells. The expression of alpha smooth muscle actin (a-SMA) mRNA was detected by qPCR to ensure successful induction of EMT, and the expression of VDR mRNA was detected by qPCR to ensure successful transfection of VDR-siRNA. Protein expression of a-SMA, E-cadherin, VDR, b-catenin and glycogen synthase kinase 3b (GSK-3b) was detected by Western blot analysis. The results showed that high glucose concentration inhibited cell proliferation and promoted EMT in HK-2 cells. TSIIA could reverse high glucose-induced EMT by increasing the level of VDR protein and inhibiting the levels of b-catenin and GSK-3b proteins suggestive of a negative correlation between VDR and the Wnt/b-catenin pathway. After VDR-siRNA transfection and incubation of cells at high glucose concentration, the inhibitory effect of VDR on the expression of b-catenin and GSK-3b of Wnt pathway was suppressed and the b-catenin pathway was activated. When VDR level was restored by TSIIA, the inhibitory effect of VDR on the pathway was also restored and the activation of the pathway was suppressed. TSIIA was able to attenuate high glucose-induced EMT in HK-2 cells by up-regulating VDR levels, which might be related to the inhibitory effect of VDR on the Wnt pathway.

Dendrobium Mixture Ameliorates Diabetic Nephropathy in db/db Mice by Regulating the TGF-β1/Smads Signaling Pathway.

Dendrobium mixture (DMix) is an effective treatment for diabetic nephropathy (DN), but the molecular mechanism underlying its action remains unclear. In this study, we investigated whether DMix regulates the transforming growth factor-β1 (TGF-β1)/Smads signal transduction pathway. Twenty-four db/db mice were randomly divided into three groups: the model, DMix, and gliquidone groups, while eight db/m mice were selected as the normal control group. The drug was administered by continuous gavage for 8 weeks. Body weight (BW), kidney weight (KW), kidney index, fasting blood glucose (FBG), blood lipid, 24-hour urinary albumin excretion rate, blood urea nitrogen, and serum creatinine levels were measured. Pathological changes in the renal tissue were observed under a light microscope. Real-time quantitative PCR and immunohistochemical staining were used to detect the mRNA and protein expression levels of TGF-β1 and alpha-smooth muscle actin (α-SMA), respectively, in renal tissues. TGF-β1, Smad2, p-Smad2, Smad3, p-Smad3, and α-SMA expression levels were measured using western blotting. The results showed that DMix significantly reduced the FBG level, BW, KW, and blood lipid level and improved renal function in db/db mice. Histopathology showed that DMix alleviated glomerular mesangial cell proliferation and renal interstitial fibrosis in db/db mice. Additionally, DMix reduced the protein and mRNA expression levels of TGF-β1 and α-SMA and inhibited Smad2 and Smad3 phosphorylation. We conclude that DMix may inhibit renal fibrosis and delay the progression of DN by regulating the TGF-β1/Smads signaling pathway.

The effect of inhibiting exosomes derived from adipose-derived stem cells via the TGF-β1/Smad pathway on the fibrosis of keloid fibroblasts.

The main mechanism of keloid formation is that keloid fibroblasts (KFs) apoptosis is inhibited, leading to excessive proliferation. Transforming growth factor-β1 (TGF-β1) is a key signal molecule in the process of regulating cell fibrosis. This paper discusses the effect of adipose-derived stem cell exosomes (ADSCs-EXO) on the proliferation and apoptosis of KFS and its possible mechanism, in order to provide reference for the clinical intervention of hypertrophic scar. ADSCs were isolated and cultured from human adipose tissue, the supernatant was collected, and the exosomes secreted by ADSCs-EXO were extracted by ultracentrifugation. At the same time, KFs were cultured from human keloid tissue to P3 generation, and then divided into four groups: control group, experimental group A, experimental group B and experimental group C. KFs were then cultured with four concentrations of ADSCs-EXO (0, 1, 10, and 100 µg/mL, respectively). After 24 hours, cells in each group were taken to detect the following: proliferation of cells in each group using the cell counting Kit 8 (CCK-8) method, cell migration ability via the Transwell test, cell apoptosis by flow cytometry, collagen synthesis using the hydroxyproline method, messenger ribonucleic acid (mRNA) expression of fibrosis-related genes in each group by real-time fluorescent polymerase chain amplification, and the expression of fibrosis-related proteins in the cells of each group by western blotting. Compared with the control group, the proliferation rate, migration rate, and collagen synthesis levels in the three experimental groups decreased with the increase of ADSCs-EXO concentration, while the apoptosis rate in the three experimental groups increased with the increase of ADSCs-EXO concentration, and the differences were statistically significant (P<0.05). Also, compared with the control group, the relative mRNA and protein expression of alpha-smooth muscle actin (α-SMA), TGF-β1, and Smad3 in the three groups decreased significantly, while the expression of three kinds of mRNA and protein decreased with the increase of ADSCs-EXO concentration, and the differences were statistically significant (P<0.05). ADSCs-EXO may inhibit the proliferation and migration, and promote the apoptosis of KFs by inhibiting the expression of the TGF-β1/Smad pathway.

Thymocytes induce renal tubular epithelial cells to undergo the epithelial-to-mesenchymal transition.

Renal tubulointerstitial fibrosis is known to occur as a result of epithelial cell transformation into myofibroblasts via the epithelial-to-mesenchymal transition (EMT) process. It has been reported that macrophages, regulatory T (Treg) cells, and gamma delta T (γδ T) cells can promote fibrosis via EMT in vivo. Our study intended to detect whether thymocytes can induce renal tubular cells to undergo the EMT. Rat thymocytes were activated by phytohemagglutinin and concanavalin A. The rat renal tubular epithelial cells (NRK-52E) were incubated in a conditioned medium harvested from activated thymocytes or co-cultured with freshly isolated thymocytes for 48 hours. Real-time reverse transcription-polymerase chain reaction, immunofluorescence, and western blotting analysis were used to test the expression of the epithelial and mesenchymal markers in NRK-52E cells. Scratch assay was designed to test the cell migration abilities of NRK-52E cells. Student's t test and oneway analysis of variance test were used for statistical analysis. The combined stimulation with phytohemagglutinin and concanavalin A activated the primary isolated rat thymocytes. After treatment with conditioned medium or freshly isolated thymocytes, the expression levels of cytokeratin 19 and E-cadherin were downregulated in NRK-52E cells, while the mRNA and protein expression levels of alpha-smooth muscle actin, desmin, and vimentin were upregulated (P < 0.05). We found that the cell migration abilities of the induced NRK-52E cells were significantly improved. Both activated rat thymocytes (more percentage of CD8+ T cells) and freshly isolated thymocytes have promoting effects on the EMT of NRK-52E cells.

Botulinum toxin type A attenuates hypertrophic scar formation via the inhibition of TGF-β1/Smad and ERK pathways.

Hypertrophic scar is a common complication in would healing process, and how to effectively prevent and treat it has been a hot and difficult research issue. Previous studies have showed that botulinum toxin type A (BTA) has effects on the prevention and treatment of hypertrophic scar, but little is known about the specific mechanisms. This study aimed to explore the potential mechanisms of BTA on the inhibition of hypertrophic scar formation. Hypertrophic scar-derived human fibroblasts were cultured and then treated with transforming growth factor-β1 (TGF-β1) and various concentrations of BTA. Cell proliferation and viability were measured by CellTiter 96® AQueous One Solution Cell Proliferation Assay and trypan blue staining, respectively. The total amount of collagen was examined using Sirius red staining. Collagen I and Collagen III in the culture supernatant were evaluated by enzyme-linked immunosorbent assay. Reverse transcription-quantitative polymerase chain reaction and Western blot analysis were performed to detect the transcription and translation levels. Our results revealed that BTA decreased the proliferation of hypertrophic scar-derived human fibroblasts. The mRNA and protein expression levels of alpha-smooth muscle actin, collagen I, and collagen III induced by TGF-β1 were inhibited by BTA in a dose-dependent manner. BTA also inhibited the phosphorylation of Smad2/3 and ERK. BTA decreased the proliferation of fibroblasts and prevented overdeposition of ECM through the inhibition of the TGF-β1/Smad and ERK pathways. The findings of this study provide new scientific reference for the prevention and treatment of hypertrophic scar.

Hawthorn Herbal Preparation from Crataegus oxyacantha Attenuates In Vivo Carbon Tetrachloride -Induced Hepatic Fibrosis via Modulating Oxidative Stress and Inflammation.

Hawthorn (HAW) is a herbal preparation extracted from Crataegus oxyacantha. HAW has cardioprotective, antioxidants, anti-inflammatory, and anti-hypotensive effects. HAW’s effect on hepatic fibrosis remains, however, unknown. This study evaluated the impact of HAW on carbon tetrachloride (CCl4)-induced hepatic fibrosis in rats and elucidated its mechanisms. HAW reduced liver index and the serum liver enzyme markers and reduced liver damage, and fibrosis as confirmed by histopathological scoring of hematoxylin-eosin staining. Collagen deposition was reduced in HAW group compared to CCl4 group as confirmed by Masson staining, hydroxyproline content, and both mRNA and protein levels of alpha-smooth muscle actin, collagen 1 and 3. HAW also down regulated the gene expressions of inflammatory markers including interleukin-IL-1β, tumor necrosis factor-α, transforming growth factor-β 1, nuclear factor kappa-B, and cyclooxygenase-2 and decreased the myeloperoxidase activity. The effects of HAW was also associated with decreased levels of hepatic oxidative stress markers (malondialdehyde and P.Carbonyl) and with increased activity of superoxide dismutase. Those effects are possibly mediated by blocking the pro-oxidant machinery and down regulating the inflammatory and profibrotic responses. Finally, chlorogenic acid, epicatechin, rutin, vitexin quercetin, and iso quercetin were identified as the major species of polyphenols of the HAW herbal preparation used here. Therefore, HAW’s potent protecting effects against liver fibrosis predicts a significant beneficial application.

Collagen release by human hepatic stellate cells requires vitamin C and is efficiently blocked by hydroxylase inhibition.

Liver fibrosis is characterized by the accumulation of extracellular matrix proteins, mainly composed of collagen. Hepatic stellate cells (HSCs) mediate liver fibrosis by secreting collagen. Vitamin C (ascorbic acid) is a cofactor of prolyl-hydroxylases that modify newly synthesized collagen on the route for secretion. Unlike most animals, humans cannot synthesize ascorbic acid and its role in liver fibrosis remains unclear. Here, we determined the effect of ascorbic acid and prolyl-hydroxylase inhibition on collagen production and secretion by human HSCs. Primary human HSCs (p-hHSCs) and the human HSCscell line LX-2 were treated with ascorbic acid, transforming growth factor-beta (TGFβ) and/or the pan-hydroxylase inhibitor dimethyloxalylglycine (DMOG). Expression of collagen-I was analyzed by RT-qPCR (COL1A1), Western blotting, and immunofluorescence microscopy. Collagen secretion was determined in the medium by Western blotting for collagen-I and by HPLC for hydroxyproline concentrations. Expression of solute carrier family 23 members 1 and 2 (SLC23A1/SLC23A2), encoding sodium-dependent vitamin C transporters 1 and 2 (SVCT1/SVCT2) was quantified in healthy and cirrhotic human tissue. In the absence of ascorbic acid, collagen-I accumulated intracellularly in p-hHSCs and LX-2 cells, which was potentiated by TGFβ. Ascorbic acid co-treatment strongly promoted collagen-I excretion and enhanced extracellular hydroxyproline concentrations, without affecting collagen-I (COL1A1) mRNA levels. DMOG inhibited collagen-I release even in the presence of ascorbic acid and suppressed COL1A1 and alpha-smooth muscle actin (αSMA/ACTA2) mRNA levels, also under hypoxic conditions. Hepatocytes express both ascorbic acid transporters, while p-hHSCs and LX-2 express the only SVCT2, which is selectively enhanced in cirrhotic livers. Human HSCs rely on ascorbic acid for the efficient secretion of collagen-I, which can be effectively blocked by hydroxylase antagonists, revealing new therapeutic targets to treat liver fibrosis.

Galangin Reverses Hepatic Fibrosis by Inducing HSCs Apoptosis via the PI3K/Akt, Bax/Bcl-2, and Wnt/β-Catenin Pathway in LX-2 Cells.

Hepatic fibrosis (HF) is a common disease, with currently no available treatment. Galangin, a natural flavonoid extracted from Alpinia officinaruim Hance, has multiple effects demonstrated in previous studies. The aim of the present study was to explore the anti-fibrogenic effect of galangin in vitro, and research its potential molecular mechanisms. LX-2 cells were chosen as an in vitro HF model, and were treated with galangin in different concentrations. Cell viability was analyzed using Cell Counting Kit-8 (CCK-8) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, cell apoptosis was measured using flow cytometry, and the anti-fibrogenic effect of galangin was determined using RT-quantitative (q)PCR, immunofluorescence, and Western blotting. The results show that the proliferation of LX-2 cells was efficiently inhibited by galangin, and apoptosis was induced in a dose-dependent manner. Both the mRNA and protein expression of alpha-smooth muscle actin (α-SMA) and collagen I were markedly downregulated. Galangin also inhibited the phosphatidylinositol 3-kinase (PI3K)/Akt and Wnt/β-catenin signaling pathways and increased the Bax/Bcl-2 ratio. The results of this study suggest that galangin has an anti-fibrogenic effect and may represent a promising agent in the treatment of hepatic fibrosis.

Tropomyosin 3.1 Association With Actin Stress Fibers is Required for Lens Epithelial to Mesenchymal Transition.

PurposeEpithelial to mesenchymal transition (EMT) is a cause of anterior and posterior subcapsular cataracts. Central to EMT is the formation of actin stress fibers. Selective targeting of actin stress fiber-associated tropomyosin (Tpm) in epithelial cells may be a means to prevent stress fiber formation and repress lens EMT.MethodsWe identified Tpm isoforms in mouse immortalized lens epithelial cells and epithelial and fiber cells from whole lenses by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) followed Sanger sequencing. We focused on the role of one particular tropomyosin isoform, Tpm3.1, in EMT. To induce EMT, we treated cells or native lenses with TGFβ2. To test the function of Tpm3.1, we exposed cells or whole lenses to a Tpm3.1-specific chemical inhibitor, TR100, as well as investigated lenses from Tpm3.1 knockout mice. We examined stress fiber formation by confocal microscopy and assessed EMT progression by analysis of alpha-smooth muscle actin (αSMA) mRNA (real-time RT-PCR), and protein (Western immunoassay [WES]).ResultsLens epithelial cells express eight Tpm isoforms. Cell culture studies showed that TGFβ2 treatment results in the upregulation of Tpm3.1, which associates with actin in stress fibers. TR100 prevents stress fiber formation and reduces αSMA in TGFβ2-treated cells. Using an ex vivo lens culture model, TGFβ2 treatment results in stress fiber formation at the basal regions of the epithelial cells. Genetic knockout of Tpm3.1 or treatment of lenses with TR100 prevents basal stress fiber formation and reduces epithelial αSMA levels.ConclusionsTargeting specific stress fiber associated tropomyosin isoform, Tpm3.1, is a means to repress lens EMT.

Paclitaxel alleviates liver fibrosis induced by bile duct ligation in rats: Role of TGF-β1, IL-10 and c-Myc

Liver fibrosis is a global health issue that causes morbidity and mortality with no currently available treatment. It has been shown that low dose paclitaxel (PTX) can stabilize microtubules and inhibit the profibrotic transforming growth factor-beta 1 (TGF-β1) signaling pathway. In this study the effect of treatment with low dose PTX was examined using a model of cholestatic liver fibrosis. Bile-duct ligation (BDL) was induced in rats for 2 weeks then PTX (0.3 mg/kg/ip) was administered three times a week for 2 weeks. Administration of PTX ameliorated BDL-induced elevation in biomarkers of hepatocellular damage (alanine transaminase; ALT and aspartate transaminase; AST) and obstructive cholestatic injury (total bilirubin and gamma glutamyl transferase; γ-GT). PTX was able to correct the increase in liver weight to body weight ratio and the bile duct proliferation induced by BDL. Additionally, PTX treatment corrected the BDL-induced fibrosis of portal tracts, elevation of hydroxyproline content and increased alpha smooth muscle actin (α-SMA) mRNA and protein expression. This antifibrotic effect of PTX was further examined through its inhibitory effect on TGF-β1 mRNA and protein expression in addition to c-Myc mRNA expression. Furthermore, PTX rectified the BDL-induced decrease in interleukin-10 (IL-10) mRNA and protein expression. In conclusion, this study suggests that PTX at low dose has the potential to treat BDL-induced liver fibrosis in rats possibly through suppression of TGF-β1 and c-Myc and activation of IL-10 pathways.

Human adipose-derived stem cells pre-treated with platelet-rich plasma and hepatocyte growth factor alleviate liver fibrosis in mice

Background: Transplantation of adipose-derived stem cells (ADSCs) is a potential therapy for a variety of liver diseases. Previous studies have shown that ADSC-based therapy is promising for liver fibrosis treatment. Recently, many publications have suggested that pretreating ADSCs with growth factors before transplantation can elevate the effectiveness of the therapy. Therefore, we hypothesize that human ADSCs (hADSCs) pretreated with platelet-rich plasma (PRP) and hepatocyte growth factor (HGF), compared to ADSCs alone, would accelerate the treatment effects on liver fibrosis in mice.&#x0D; Methods: The hADSCs were cultured solely with conventional media, or with HGF (human recombinant; 20 ng/ml), or with HGF and PRP (from healthy human blood, 10%), concomitantly added to the medium for 7 days before transplantation. Eight-week-old male mice were treated with CCl4 (1 ml/kg) for 11 weeks to induce liver fibrosis. The mice were then subsequently divided into: 1) Placebo group (PBS injection); 2) ADSCs/HGF+PRP (5x105HGF and PRP pre-treated cells/mice); 3) ADSCs/HGF (5x105HGF pre-treated cells/mice) and 4) ADSCs (5x105non-pretreated cells/mice).&#x0D; Results: Seven days post-transplantation, the alanine transaminase (ALT) level in the placebo was notably elevated (143.10±14.96 IU/L), compared to ALT levels of ADSCs-, ADSCs/HGF+PR-, and ADSCs/HGF-transplanted mice, which showed an improvement (67.94±18.57 IU/L, 49.44±7.56 IU/L, and 57.93±5.75 IU/L, respectively). The procollagen-α1 and alpha-smooth muscle actin (α-SMA) mRNA levels were significantly down-regulated in the ADSCs-transplanted group compared to those of the placebo. Importantly, these levels were lower in ADSCs/HGF+PR (procollagen-α1: 75.64±45.89; α-SMA: 36.17±36.09) than those of ADSCs/HGF (procollagen-α1: 212.8±84.35; α-SMA: 52.41±7.93) and ADSCs only (procollagen-α1: 310.50 ± 55.36; α-SMA: 184.70±14.06). Stem cell transplantation also improved histological index, reducing inflammation and collagen/necrotic structure accumulation. However, there were no statistical differences between three ADSCs treatment groups after 14 days after transplantation.&#x0D; Conclusion: Pre-treatment with PRP and HGF for 7 days enhanced the efficacy of ADSCs in alleviating liver fibrosis in vivo.

Type I collagen induces mesenchymal cell differentiation into myofibroblasts through YAP-induced TGF-β1 activation

In organ fibrosis, mechanical stress and transforming growth factor beta-1 (TGF-β1) promote differentiation into myofibroblast from mesenchymal cells, leading to extracellular matrix (ECM) remodeling or active synthesis, deposition or degradation of ECM components. A major component of ECM, type I collagen (col I) triple helical molecules assemble into fibrils or are denatured to gelatin without triple-helicity in remodeling. However, whether changes of ECM components in remodeling have influence on mesenchymal cell differentiation remains elusive. This study adopted three states of collagen I existing in ECM remodeling: molecular collagen, fibrillar collagen and gelatin to see what are characteristics in the effects on two cell lines of mesenchymal origin, murine 3T3-L1 embryonic fibroblast and murine C2C12 myoblasts. The results showed that all three forms of collagen I were capable of inducing these two cells to differentiate into myofibroblasts characterized by increased expression of alpha-smooth muscle actin (α-SMA) mRNA. The expression of α-SMA is positively regulated by TGF-β1. Nuclear translocation of Yes-associated protein (YAP) is involved in this process. Focal adhesion kinase (FAK) is activated in the cells cultured on molecular collagen-coated plates, contributing to YAP activation. On the other hand, in the cells cultured on fibrillar collagen gel or gelatin-coated plates, oxidative stress but not FAK induce YAP activation. In conclusion, the three physicochemically distinct forms of col I induce the differentiation of mesenchymal cells into myofibroblasts through different pathways.

Exposure of neonatal mice to bromine impairs their alveolar development and lung function.

The halogen bromine (Br2) is used extensively in industry and stored and transported in large quantities. Its accidental or malicious release into the atmosphere has resulted in significant casualties. The pathophysiology of Br2-induced lung injury has been studied in adult animals, but the consequences of Br2 exposure to the developing lung are completely unknown. We exposed neonatal mouse littermates on postnatal day 3 (P3) to either Br2 at 400 ppm for 30 min (400/30), to Br2 at 600 ppm for 30 min (600/30), or to room air, then returned them to their dams and observed until P14. Mice exposed to Br2 had decreased survival (S) and had decreased weight (W) at P14 in the 400/30 group (S = 63.5%, W = 6.67 ± 0.08) and in the 600/30 group (S = 36.1%, W = 5.13 ± 0.67) as compared with air breathing mice (S = 100%, W = 7.96 ± 0.30). Alveolar development was impaired, as evidenced by increased mean linear intercept at P14. At P14, Br2 exposed mice also exhibited a decrease of arterial partial pressure of oxygen, decreased quasi-static lung compliance, as well as increased alpha smooth muscle actin mRNA and protein and increased mRNA for IL-1β, IL-6, CXCL1, and TNFα. Global gene expression, evaluated by RNA sequencing and Ingenuity Pathway Analysis, revealed persistent abnormalities in gene expression profiles at P14 involving pathways of "formation of lung" and "pulmonary development." The data indicate that Br2 inhalation injury early in life results in severe lung developmental consequences, wherein persistent inflammation and global altered developmental gene expression are likely mechanistic contributors.

Active immunization against transforming growth factor beta1 prevents hepatic fibrosis in a rat model of liver disease.

Transforming growth factor beta1 (TGF-β1) plays an important role in hepatic fibrogenesis. In this study, we documented the effects of active immunization against TGF-β1 on hepatic fibrosis in an animal model of chronic liver disease. BALB/c mice were immunized against 3 different peptides of TGF-β1 ligated into hepatitis B virus core protein (HBVc). Titers of TGF-β1 antibodies were documented by enzyme linked immunoassays and antibody activity by cell membrane receptor binding and proliferation assays. The most immunogenic recombinant HBVc + TGF-β1 peptide (HBVc + C) then served as a vaccine in Sprague-Dawley rats with dimethylnitrosamine-induced chronic liver disease. Hepatic fibrosis was documented by serum hyaluronic acid levels, liver histology, and reverse transcriptase polymerase chain reaction for hepatic collagen I (α1) and smooth muscle alpha actin mRNA expression. Relative to control rats vaccinated with HBVc alone, recombinant HBVc + C vaccinated animals had significantly lower serum hyaluronic acid levels, less histologic evidence of hepatic fibrosis, and reduced expression of collagen I (α1) and smooth muscle alpha actin mRNA in the liver. The results of this proof-of-concept study suggest that active immunization against TGF-β1 is a worthwhile strategy to pursue in efforts to prevent hepatic fibrosis associated with chronic liver disease.

Dissecting the Development of Dupuytren Disease Through Human Genetics

Objective: Dupuytren disease (DD) is a polygenic fibroproliferative disorder of the palmar fascia of the hand. Building on our previous work, we aimed to further characterize the genetic predisposition to DD by performing a genome-wide association study (GWAS) on a total of 7934 DD cases and 12°861 controls from the United Kingdom, Holland, and Germany. We then aimed to dissect the functional effects of the statistically associated variant within DD-derived myofibroblasts. Materials and Methods: We extracted DNA from saliva or blood of DD patients who had undergone fasciectomy or dermofasciectomy. The genotypes of each sample were investigated using Illumina HumanExomeCore microarray chips in the discovery phase, and other platforms for replication phase. For our functional analysis, we used single-gene TaqMan assays to measure messenger RNA (mRNA) expression of myofibroblast cells that were disaggregated from DD nodules. Primary myofibroblasts were stimulated with a combination of recombinant WNT3A, SFRP4, and DKK1 proteins for 48 hours, and mRNA expression was measured. Results: Our GWAS meta-analysis revealed a total of 24 DD-associated regions, 15 of which were novel ( P &lt; 5 × 10−8). We observed a 4-fold difference in SFRP4 mRNA expression between the 2 homozygous genotypes of our most associated single-nucleotide polymorphism rs1687975. Interestingly, an inverse correlation was observed in extracellular SFRP4 protein secretion. Our WNT signaling pathway stimulation experiments demonstrated that SFRP4 attenuates WNT3A-induced connective tissue growth factor (CTGF) expression, showing a 20% decrease ( P &lt; .05). SFRP4 alone has no effect on alpha-smooth muscle actin, COL1A1, and COL3A1 mRNA expression. Conclusions: We have performed a large 3-stage GWAS and have discovered 15 novel Dupuytren-associated regions. We have identified that our most significantly associated variant, rs16879765, has an effect on the mRNA expression of a WNT antagonist gene SFRP4. Further functional analysis on SFRP4 has revealed that it affects the expression of CTGF. This implies its anti-fibrotic effect is mediated by the non-canonical WNT signaling pathway, potentially via the YAP/TAZ pathway that is known to interact with transforming growth factor β (TGF BETA) pathway, and this plays a critical role in the development of DD. Our GWAS has also revealed multiple potential new therapeutic targets for DD. Genes within the associated regions include MMP14 and WNT4 and have effects on diverse functions such as inflammation, extracellular matrix remodeling, cellular differentiation, and proliferation, which are likely to be important processes in the development of DD and fibrotic diseases in general.

Alpha-Smooth Muscle Actin mRNA and Protein Are Increased in Isolated Brain Vessel Extracts of Alzheimer Mice

Alzheimer's disease (AD) is a severe neurodegenerative disorder of the brain, characterized by extracellular beta-amyloid (Aβ) plaques, intracellular tau pathology, neurodegeneration and inflammation. There is clear evidence that the blood-brain barrier is damaged in AD and that vessel function is impaired. Alpha-smooth muscle actin (αSMA) is a prominent protein expressed on brain vessels, especially in cells located closer to the arteriole end of the capillaries, which possibly influences the blood vessel contraction. The aim of the present study was to observe αSMA protein and mRNA expression in isolated brain vessel extracts and cortex in an Alzheimer mouse model with strong Aβ plaque deposition. Our data revealed a prominent expression of αSMA protein in isolated brain vessel extracts of AD mice by Western blot analysis. Immunostaining showed that these vessels were associated with Aβ plaques. Quantitative real-time PCR analysis confirmed this increase at the mRNA expression level and showed a significant increase of transforming growth factor beta-1 mRNA expression in AD mice. In situ hybridization demonstrated a strong expression pattern of αSMA mRNA in the whole cortex and hippocampus. In conclusion, our data provide evidence that αSMA protein and mRNA are enhanced in vessels in an AD mouse model, possibly counteracting vessel malfunction in AD.