α-SMA Protein Research Articles

OP21 MIF-CD74 signaling promotes intestinal LTi-like ILC3s activation and gut fibrosis via upregulating pSTAT3

Abstract Background Intestinal fibrosis is a common complication of inflammatory bowel disease, and its core pathogenesis is fibroblast activation and massive extracellular matrix (ECM) deposition. In recent years, innate lymphoid cells (ILCs) have been shown to be an important source of fibroblast activation signals in fibrotic diseases. This study aims to explore the heterogeneity of ILC3 (the main ILC subgroup in the human intestine) in the intestinal fibrosis and the mechanism of its interaction with stromal cells. Methods The intestinal fibrosis model was constructed with ILC3 deficiency mice (Rorc knock out) treated with chronic DSS/TNBS. Flow cytometry was performed to detect the proportion of ILC3 subsets and cytokine expression level. ILC3 were sorted out for transcriptome sequencing and co-culture experiment with primary intestinal fibroblasts. Results In chronic DSS/TNBS models, the colon CCR6+ILC3 significantly expanded (p<0.05), and the expression of cytokine IL-22 was upregulated (p<0.05). ILC3 deficiency mice showed lower degree of intestinal fibrosis, including decrease in collagen involvement area (p<0.05) and decrease in ECM-related genes expression(p<0.05). After adoptively transferred of CCR6+ILC3, the severity of intestinal fibrosis in mice increases again. Flow cytometry revealed that CCR6+ILC3 was the main source of IL-22 in the mouse colon. CCR6+ILC3 cells were then sorted by flow cytometry, and co-cultured with mouse intestinal fibroblasts in vitro. Results shown that CCR6+ILC3 can activate fibroblasts. The expression level of α-SMA protein was significantly upregulated (p<0.05). Cell migration ability was also improved in the scratch experiment (p<0.05). Blocking IL-22 could inhibit the effects above. Transcriptome sequencing analysis of sorted ILC3 revealed a significant upregulation in signaling pathways related to cell activation, proliferation and adhesion, with CD74 existing in all of them. Flow cytometry confirmed the expression of CD74 on ILC3 for the first time, which specifically present on CCR6+ILC3 within RORγt+ cells. Cell stimulation experiments in vitro showed that macrophage migration inhibitory factor (MIF)-CD74 signal upregulated IL-22 level of ILC3 cells and was associated with increased phosphorylation of STAT3 signaling pathway. And MIF, the CD74 ligand, significantly increased in both mouse intestinal fibrosis models and stenosis tissue samples from CD patients (p<0.05). Conclusion CCR6+ILC3 is enriched in the intestinal fibrotic microenvironment and can continuously promote disease progression by activating fibroblasts through IL-22. CCR6+ILC3 can sense the pathogenic inflammatory factor MIF in chronic colitis through CD74, and increase IL-22 expression by promote STAT3 phosphorylation.

Integrated metabolomics and mass spectrometry imaging analysis reveal the efficacy and mechanism of Huangkui capsule on type 2 diabetic nephropathy.

Huangkui capsule (HKC), a Chinese patent medicine, is clinically used for treating diabetic nephropathy. However, the core disease-specific biomarkers and targets of type 2 diabetic nephropathy (T2DN) and the therapeutic mechanism of HKC are not fully elucidated. This study aimed to investigate the therapeutic effects and underlying molecular mechanisms of HKC for T2DN. The db/db mouse model was used to evaluate the efficacy of HKC for T2DN, and the core pathways regulated by HKC were studied to determine its kidney protective mechanism. High-throughput UPLC-MS/MS and multivariate analysis were employed to analyze the serum and kidney metabolic profiles of db/db mice, identifying potential core biomarkers of T2DN. Atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry imaging was used to locate in situ spatial distribution of core biomarkers and drug active ingredients in kidney tissues. Biochemical indicators, histopathology, immunohistochemistry, immunofluorescence, molecular docking, and western blotting were combined to reveal therapeutic effects, pathways, and targets of HKC. HKC substantially improved pathological characteristics, kidney function, oxidative stress, inflammation, and lipid metabolism indicators of T2DN. Twelve core disease-specific biomarker that significantly influenced clustering were identified and its unique spatial distribution information in the kidneys was revealed. 3-dehydrosphinganine, retinyl ester, and 9-cis-retinoic acid (9cRA) could serve as novel disease-specific biomarkers for T2DN. Based on newly discovered biomarkers, quercetin, myricetin, and isorhamnetin were found to act on key enzymes SPT, ALDH1A1, AOX, LRAT, and DGAT1 in retinol and sphingolipid metabolism pathways. Western blotting showed that HKC ameliorated T2DN by targeting these enzymes, upregulating 9cRA and retinyl ester, downregulating 3-dehydrosphinganine, increasing TGF-β signal transduction, inhibiting the expression of the immune fibrosis proteins OX-8, Col-I and α-SMA, inhibiting Th17 cell development and ceramide synthesis, reducing IL-1β, TNF-α, MDA, TC, LDL-C, and TG levels, and increaseing SOD activity. HKC exerts significant therapeutic effects on T2DN. HKC corrects the metabolic disorder of sphingolipids and retinol, and improves T2DN by regulating the activities of SPT, ALDH1A1, AOX, LRAT, and DGAT1. This study provides valuable ideas and new mechanistic insights for the treatment of T2DN with HKC.

Cancer-associated fibroblasts induce almonertinib resistance in non-small cell lung cancer

BackgroundAlmonertinib is the initial third-generation EGFR-TKI in China, but its resistance mechanism is unknown. Cancer-associated fibroblasts (CAFs) are essential matrix components in the tumor microenvironment, but their impact on almonertinib resistance is unknown. This study aimed to explore the correlation between CAFs and almonertinib resistance in non-small cell lung cancer (NSCLC).MethodsThe anti-cancer effects of almonertinib on NSCLC cells, as well as the reversal of these effects mediated by CAFs, were validated through phenotypic experiments. Differential gene expression analysis, along with GO and KEGG enrichment analyses, was performed to predict the potential mechanisms underlying resistance to third-generation EGFR-TKIs. Finally, qPCR and Western blot analyses were used to explore the signaling pathways by which CAFs induce resistance to almonertinib in NSCLC cells.ResultsOur findings revealed that almonertinib significantly suppressed the invasion, migration, and proliferation of EGFR T790M-mutant NSCLC cells. TGF-β1 successfully induced the differentiation of CAFs and upregulated the expression of CAF markers, including α-SMA and fibroblast activation protein (FAP). Exposure of H1975 cells to almonertinib increased TGF-β1 secretion. Additionally, CAFs enhanced the survival of almonertinib-treated NSCLC cells, whereas normal fibroblasts (NFs) exerted the opposite effect. qPCR analysis demonstrated that the expression of the core molecules of the Hippo pathway, YAP and TAZ, was lower in A549 cells than in H1975 cells, and CAF intervention further reduced YAP/TAZ expression in H1975 cells. Western blot analysis confirmed a significant reduction in YAP/TAZ protein levels in cancer cells treated with CAF-conditioned medium (CAF-CM) compared to those treated with normal control-conditioned medium (NC-CM). Finally, we demonstrated that CAFs induced resistance to almonertinib in NSCLC cells, potentially through a mechanism involving YAP/TAZ.ConclusionThis study demonstrated that H1975 cells stimulated by almonertinib promoted the accumulation of CAFs in NSCLC cells, likely through increased secretion of TGF-β1. The accumulation of CAFs enhanced the survival of NSCLC cells undergoing almonertinib treatment and induced drug resistance. Additionally, the mechanism underlying CAF-induced drug resistance in NSCLC cells was potentially linked to the activation of the YAP/TAZ signaling pathway.

Expression of PAX6 and Keratocyte-Characteristic Markers in Human Limbal Stromal Cells of Congenital Aniridia and Healthy Subjects, In Vitro

Purpose Our aim was to examine the expression of PAX6 and keratocyte-specific markers in human limbal stromal cells (LSCs) in congenital aniridia (AN) and in healthy corneas, in vitro. Methods Primary human LSCs were extracted from individuals with aniridia (AN-LSCs) (n = 8) and from healthy corneas (LSCs) (n = 8). The cells were cultured in either normal-glucose serum-containing cell culture medium (NGSC-medium) or low-glucose serum-free cell culture medium (LGSF-medium). Analysis of PAX6 and keratocyte-specific markers was conducted using qPCR and Western blotting. The keratocyte-specific markers included Collagen I (COL1A1), Collagen III (COL3A1), Collagen V (COL5A1), α-smooth muscle actin (ACTA2), Aldehyde Dehydrogenase 3 Family, Member A1 (ALDH3A1), Keratocan (KER), Lumican (LUM), and CD34. Results PAX6 mRNA expression exhibited a significant decrease in AN-LSCs compared to LSCs in both NGSC- and LGSF-medium (p = 0.04; p = 0.014). There was a marked reduction in COL5A1 mRNA expression (p = 0.011), accompanied by notably higher ALDH3A1 and KER mRNA levels (p = 0.007; p = 0.013) in AN-LSCs compared to LSCs when using NGSC-medium. In LGSF-medium, AN-LSCs showed a significant increase in COL1A1 and COL5A1 mRNA expression compared to LSCs (p = 0.048; p = 0.002). Moreover, COL1A1 and α-SMA protein expression were significantly elevated in AN-LSCs compared to LSCs in LGSF-medium (p = 0.048, p = 0.008). Conclusions Our investigation affirms the altered expression of PAX6 and keratocyte-specific markers in AN-LSCs relative to healthy controls. Both NGSC- and LGSF-medium exerted distinct effects on both LSCs and AN-LSCs. The observed variations in PAX6 and keratocyte-specific marker expression in AN-LSCs may play a pivotal role in the development and progression of aniridia-associated keratopathy.

Design, synthesis, and biological evaluation of novel 3-naphthylthiophene derivatives as potent SIRT2 inhibitors for the treatment of myocardial fibrosis.

SIRT2 (sirtuin2) is a NAD+-dependent deacetylase implicated in fibrosis and inflammation of the liver, kidney, and heart. In this study, we designed and synthesized a series of 3-naphthylthiophene derivatives and evaluated their inhibitory activity against the SIRT2 enzyme. Among them, Z18 demonstrated outstanding SIRT2 inhibitory activity and selectivity. It significantly inhibited both the proliferation of cardiac fibroblasts (CFs) and the activity and expression of SIRT2 in CFs. Moreover, compound Z18 effectively suppressed TGF-β1-induced increases in α-SMA and CoL-1A1 protein expression, as well as hydroxyproline levels. Pharmacological mechanism tests demonstrated that Z18 inhibited SIRT2, thereby suppressing the TGF-β1-induced autocrine production of TGF-β1 and the phosphorylation of Smad2/3 in CFs. In MTT assays, Z18 exhibited a significant inhibitory effect on the proliferation of CFs induced by TGF-β1. In vivo, Z18 effectively ameliorated TAC- and ISO-induced declines in cardiac function, histopathological morphological changes, and collagen deposition. It also inhibited SIRT2 activity and reduced the expression of α-SMA and p-Smad2/3. In hepatorenal toxicity assays, Z18 exhibited an excellent safety profile. These results support the further development of the selective SIRT2 inhibitor Z18 as a potential lead compound for the treatment of myocardial fibrosis.

Receptor activity-modifying protein 1 regulates the differentiation of mouse skin fibroblasts by downregulating α-SMA expression via suppression of high mobility group AT-hook 1 to promote skin wound repair.

Skin innervation is very important for normal wound healing, and receptor activity-modifying protein 1 (RAMP1) has been reported to modulate calcitonin gene-related peptide (CGRP) receptor function and thus be a potential treatment target. This study aimed to elucidate the intricate regulatory effect of RAMP1 on skin fibroblast function, thereby addressing the existing knowledge gap in this area. Immunohistochemical staining and immunofluorescence (IF) staining were used to measure the dynamic changes in the expression of RAMP1 and α-smooth muscle actin (α-SMA) in skin wound tissue in mice. Mouse skin fibroblasts (MSFs) stably transfected with Tet-on-Flag-RAMP1 overexpression (OE) and Tet-on-Flag control (Ctrl) lentiviruses were constructed for in vitro experiments. High mobility group AT-hook 1 (HMGA1) plasmids and α-SMA plasmids were used to overexpress HMGA1 and α-SMA, respectively. An α-SMA siRNA was used to silence α-SMA. Quantitative real-time polymerase chain reaction (qPCR), western blot and IF staining analyses were used to determine the mRNA and protein levels in the cells in different groups. A scratch wound healing assay was used to evaluate the cell migration ability of different groups. Cleavage under targets and release using nuclease (CUT & RUN) assays and dual-luciferase reporter assays were used to predict and verify the interaction between HMGA1 and the α-SMA promoter. RAMP1 and α-SMA protein expression levels in the dermis changed dynamically and were negatively correlated during dorsal skin wound healing in mice. RAMP1 OE in vitro inhibited the differentiation and promoted the migration of MSFs by decreasing α-SMA expression via the suppression of HMGA1, which was shown for the first time to bind to the α-SMA promoter and increase α-SMA transcription. RAMP1 OE also modulated extracellular matrix (ECM) synthesis and remodeling by promoting collagen III and MMP9 expression and decreasing collagen I, MMP2, and tissue inhibitor of metalloproteinases 1 expression. Our findings suggest that RAMP1 OE decreases differentiation and promotes migration in MSFs by downregulating α-SMA expression via the suppression of HMGA1 and modulates ECM synthesis and remodeling, revealing a novel mechanism regulating α-SMA transcription, providing new insights into the RAMP1-mediated regulation of fibroblast function, and identifying effective nerve-related targets for skin wound repair.

The interplay between lung galectins and pro-fibrotic markers in post-COVID-19 fibrogenesis: A pilot study.

COVID-19, caused by the SARS-CoV-2 virus, can lead to serious lung conditions, notably interstitial pulmonary fibrosis. Our study tracked the progression of fibrosis markers in serial bronchoalveolar lavage (BAL) measurements collected from 16 COVID-19 patients at 1, 3, and 6 months post-infection. Additionally, BAL samples from 10 healthy control subjects were included. Using RT-PCR, ELISA, and immunofluorescence, we monitored molecular markers of fibrosis and investigated the interplay between galectins-1 and -3 and key pro-fibrotic mediators. We found increased α-smooth muscle actin (αSMA)-positive macrophages and heightened levels of αSMA, TGFβ, and CTGF mRNA and proteins at six months compared to controls. Furthermore, galectin-1 and galectin-3 concentrations showed a time-dependent increase and correlated significantly with pro-fibrotic markers. These findings suggest that galectins contribute to fibrotic progression following COVID-19 and highlight their potential as therapeutic targets.

Curcumin Alleviates Arecoline-induced Oral Submucous Fibrosis via the FOSL1/MAPK8 Axis.

Oral submucous fibrosis (OSF) is a precancerous lesion of the oral cavity. Areca nut consumption can cause OSF through sustained activation of buccal mucosal fibroblasts (BMFs). This study explored the effect of curcumin on arecoline-induced BMF activation and its mechanism of action. BMFs were isolated and identified by immunofluorescence detection of fibroblast surface markers vimentin and S100A4. After transfection with FOSL1- or MAPK8-related vectors, BMFs were activated by arecoline and treated with curcumin. Scratch and transwell assays were performed to detect cell migration. ChIP and luciferase reporter assays were conducted to detect the binding of FOSL1 to the MAPK8 promoter. RT-qPCR was used to detect FOSL1 and MAPK8 mRNA expression. Western blotting was used to detect FOSL1, MAPK8, COL1A1, α-SMA, Smad2, and p-Smad2 proteins. Curcumin treatment inhibited arecoline-induced fibroblast migration, reduced the expression of myofibroblast markers COL1A1, α-SMA, and p-Smad2, and downregulated the expression of FOSL1 and MAPK8. FOSL1 or MAPK8 overexpression enhanced migration and increased COL1A1, α-SMA, and p-Smad2 expression in curcumin-treated cells. FOSL1 bound to the MAPK8 promoter and promoted MAPK8 expression. Simultaneous FOSL1 overexpression and MAPK8 knockdown, compared to FOSL1 overexpression, reduced cell migration and inhibited COL1A1, α-SMA, and p-Smad2 expression. In conclusion, curcumin targets FOSL1 to reduce MAPK8 expression, thereby suppressing arecoline-induced fibroblast activation.

Exposure of Primary Human Skin Fibroblasts to Carbon Dioxide-Containing Solution Significantly Reduces TGF-β-Induced Myofibroblast Differentiation In Vitro.

Wound healing as a result of a skin injury involves a series of dynamic physiological processes, leading to wound closure, re-epithelialization, and the remodeling of the extracellular matrix (ECM). The primary scar formed by the new ECM never fully regains the original tissue's strength or flexibility. Moreover, in some cases, due to dysregulated fibroblast activity, proliferation, and differentiation, the normal scarring can be replaced by pathological fibrotic tissue, leading to hypertrophic scars or keloids. These disorders can cause significant physical impairment and psychological stress and represent significant challenges in medical management in the wound-healing process. The present study aimed to investigate the therapeutic effects of exogenously applied carbon dioxide (CO2) on fibroblast behavior, focusing on viability, proliferation, migration, and differentiation to myofibroblasts. We found that CO2 exposure for up to 60 min did not significantly affect fibroblast viability, apoptosis rate, or proliferation and migration capacities. However, a notable finding was the significant reduction in α-smooth muscle actin (α-SMA) protein expression, indicative of myofibroblast differentiation inhibition, following CO2 exposure. This effect was specific to CO2 and concentration as well as time-dependent, with longer exposure durations leading to greater reductions in α-SMA expression. Furthermore, the inhibition of myofibroblast differentiation correlated with a statistically significantly reduced glycolytic and mitochondrial energy metabolism, and as a result, with a reduced ATP synthesis rate. This very noticeable decrease in cellular energy levels seemed to be specific to CO2 exposure and could not be observed in the control cultures using nitrogen (N2)-saturated solutions, indicating a unique and hypoxia-independent effect of CO2 on fibroblast metabolism. These findings suggest that exogenously applied CO2 may possess fibroblast differentiation-reducing properties by modulating fibroblast's energy metabolism and could offer new therapeutic options in the prevention of scar and keloid development.

Design and synthesis of glycyrrhetinic acid glycosides against acute lung injury and pulmonary fibrosis.

HMGB1 mediated signalling pathway plays an important role in acute injury and fibrosis in lung tissues. Glycyrrhizic acid (GL) is a HMGB1 inhibitor, and its aglycone (glycyrrhetinic acid, GA) is the major pharmacophore and plays the main role during binding to HMGB1. To improve selectivity for these lung diseases, a series of novel glycyrrhetinic acid glycosides targeting mannose acceptors in the respiratory tract and lung tissues were synthesised, and their biological activities were evaluated in vitro and in vivo. For normal lung cell lines WI-38 and Beas-2B, all the compounds but c6 showed reduced cytotoxicity vs the positive controls (GA and GL), IC50 values were > 800µM. For three cancer cells, c1 exhibited high selectivity for lung cancer cells A549. In the inflammation assays, compound c1 displayed the strongest activity of NO inhibition, and c4 was next; both them not only down-regulated the expression levels of IL-1β and TNF-α in RAW264.7 cells, but also decreased the levels of TNF-α, IL-1β, HMGB1, RAGE and ROS in A549 cells in a dose-dependent manner. Noteworthy, compound c1 of 50μM reduced the levels of HMGB1 and RAGE to 38.4 and 37.0% of the LPS group, and it showed much higher binding affinity with HMGB1 than GL, which confirmed by molecular docking; in addition, c1 also inhibited the deposition of α-SMA and Col-1 proteins in TGF-β1-activated A549 cells. In the bleomycin-induced lung fibrosis mouse model, c1 decreased fibrous protein production and deposition in the lung tissues; at a 30mg/kg dose, it reduced the levels of α-SMA and Col-1 to 48.12 and 56.37% of the BLM group, respectively. The pharmacokinetics tests showed c1 relative distribution rate in lung tissue (at 1h, 18.86%; at 2h, 12.80%) is much higher than that of GA (at 1h, 2.8%; at 2h, 1.9%). These results show compound c1 is likely to be a candidate for acute lung injury and pulmonary fibrosis.

Metformin Attenuates Hepatic Fibrosis in Mice by Altering the Gut Microbiota

Background: Metformin is a first-line drug widely used in the treatment of type 2 diabetes. It has been shown to improve liver fibrosis; however, the underlying mechanism remains unclear. Objectives: This study investigates the relationship between metformin and gut microbiota in the treatment of liver fibrosis. Methods: Carbon tetrachloride (CCl₄) was injected intraperitoneally to induce liver fibrosis in mice, followed by metformin treatment for 4 weeks. A CCl₄-induced hepatic fibrosis mouse model was established. The mice were divided into two groups: Metformin treatment and control groups. Hematoxylin-eosin (HE) staining was used to evaluate the overall liver condition, Sirius red staining to assess the degree of hepatic fibrosis, α-smooth muscle actin (α-SMA) immunohistochemical staining to determine the activation of hepatic stellate cells (HSCs), oil red O staining to observe intracellular lipid droplets in hepatocytes, and 16sRNA sequencing to analyze fecal microbiota. Results: In the untreated CCl₄-induced hepatic fibrosis model, hepatic tissue exhibited edema, degeneration, intracellular lipid deposition in hepatocytes, and inflammatory cell infiltration between lobules. Metformin treatment attenuated hepatic fibrosis (area reduced from 1.383% to 0.669%, P < 0.001), decreased inflammatory cell infiltration, reduced intracellular lipid accumulation in hepatocytes (area reduced from 0.457% to 0.086%, P < 0.001), and decreased α-SMA protein expression (area reduced from 1.509% to 0.598%, P < 0.05). Additionally, metformin reduced harmful bacteria and increased the abundance of beneficial bacteria in the intestinal tract of the mice. Conclusions: These findings indicate that metformin reduces liver inflammation, alleviates liver fibrosis, and alters the intestinal microbiota. The modification of gut flora may be a significant mechanism by which metformin exerts its therapeutic effects on liver fibrosis.

Study on anti-adhesion effect and mechanism of dynamic and static stress stimulation during early healing process of rat Achilles tendon injury

To investigate the anti-adhesive effect and underlying mechanism of dynamic and static stress stimulation on the early healing process of rat Achilles tendon injury. Achilles tendon tissues of 15 male Sprague Dawley (SD) rats aged 4-6 weeks were isolated and cultured by enzyme digestion method. Rat Achilles tendon cells were treated with tumor necrosis factor α to construct the Achilles tendon injury cell model, and dynamic stress stimulation (dynamic group) and static stress stimulation (static group) were applied respectively, while the control group was not treated. Live/dead cell double staining was used to detect cell activity, ELISA assay was used to detect the expression of α smooth muscle actin (α-SMA), and real-time fluorescence quantitative PCR was used to detect the mRNA expression of collagen type Ⅰ (COL1A1), collagen type Ⅲ (COL3A1), and Scleraxis (SCX). Thirty male SD rats aged 4-6 weeks underwent Achilles tendon suture and were randomly divided into dynamic group (treated by dynamic stress stimulation), static group (treated by static stress stimulation), and control group (untreated), with 10 rats in each group. HE staining and scoring were performed to evaluate the healing of Achilles tendon at 8 days after operation. COL1A1 and COL3A1 protein expressions were detected by immunohistochemical staining, α-SMA and SCX protein expressions were detected by Western blot, and maximum tendon breaking force and tendon stiffness were detected by biomechanical stretching test. In vitro cell experiment, when compared to the static group, the number of living cells in the dynamic group was higher, the expression of α-SMA protein was decreased, the relative expression of COL3A1 mRNA was decreased, and the relative expression of SCX mRNA was increased, and the differences were all significant ( P<0.05). In the in vivo animal experiment, when compared to the static group, the tendon healing in the dynamic group was better, the HE staining score was lower, the expression of COL1A1 protein was increased, the expression of COL3A1 protein was decreased, the relative expression of SCX protein was increased, the relative expression of α-SMA protein was decreased, and the tendon stiffness was increased, the differences were all significant ( P<0.05). Compared with static stress stimulation, the dynamic stress stimulation improves the fibrosis of the scar tissue of the rat Achilles tendon, promote the recovery of the biomechanical property of the Achilles tendon, and has obvious anti-adhesion effect.

Alanyl-Glutamine Inhibits the Epithelial-Mesenchymal Transition of Airway Epithelial Cells in Asthmatic Mice via DPP4-SIRT1 Pathway

Introduction: Alanyl-glutamine (Ala-Gln) is a compound known for its protective effects in various tissue injuries. However, its role in asthma-related lung injuries remains underexplored. This study investigates the mechanisms by which Ala-Gln modulates sDPP4-induced airway epithelial-mesenchymal transition and ovalbumin (OVA)-induced asthma in a mouse model. Methods: An asthma model was established in female C57BL/6 J mice by using OVA. CD4+ T cells and bronchial epithelial cells (BECs) were isolated from the spleen and bronchi of the mice, respectively. Interventions included recombinant sCD26/sDPP4 protein, Ala-Gln, and EX527 (a SIRT1 inhibitor). Flow cytometry was used to assess Th17 and Treg cell populations. Mice were treated with Ala-Gln, EX527, and budesonide (BUD). Histopathological changes in lung tissues were evaluated using hematoxylin-eosin and Masson staining. White blood cell counts were measured with a hematology analyzer. The expression levels of DPP4, IL-17, SIRT1, SMAD2/3, N-cadherin, E-cadherin, MMP9, and α-SMA proteins were analyzed. Results: Treatment with recombinant sCD26/sDPP4 resulted in decreased E-cadherin expression in BECs and increased levels of α-SMA, MMP9, and N-cadherin, effects that were mitigated by Ala-Gln. Ala-Gln also prevented the reduction in SIRT1 expression in BECs and the increase in Th17 cell differentiation induced by recombinant sCD26/sDPP4. EX527 administration alongside Ala-Gln reversed these changes and enhanced the phosphorylation of SMAD2/3 through SIRT1 signaling. BUD alone reduced inflammation and fibrosis in bronchial tissue and lowered the Th17/Treg ratio in peribronchial lymph nodes. The therapeutic effect of BUD was further improved with concurrent Ala-Gln treatment. Conclusion: Ala-Gln can inhibit BEC fibrosis and Th17 cell differentiation mediated by recombinant sCD26/sDPP4 through the SIRT1 pathway. Combined with BUD, Ala-Gln enhanced therapeutic efficacy in OVA-induced asthma in mice, which could offer improved outcomes for asthmatic patients with elevated DPP4 levels.

High α-SMA expression in the tumor stroma is associated with adverse clinical parameters in mismatch repair-proficient colorectal cancers only.

As mismatch repair status confers differential prognosis in colorectal cancers, this study aimed to determine associations of α-smooth muscle actin (α-SMA) protein expression in mismatch repair-proficient (pMMR) and mismatch repair-deficient (dMMR) colorectal tumors with clinicopathologic and prognostic features. Tissue microarrays from patients with colorectal cancer, immunostained with α-SMA, were assessed through digital image analysis. Total (n=962), pMMR (n=782), and dMMR (n=156) stromal H-scores were assessed for associations with clinicopathologic and survival data. Higher α-SMA expression was correlated with pMMR status (P=5.2223×10-8). In the pMMR subgroup, higher α-SMA stromal expression at the tumor periphery was correlated with higher T stage (P=.002), perineural invasion (P=.038), infiltrative tumor edge (P=.01), involved nodal status (P=.036), metastases (P=.013), synchronous metastases (P=.007), recurrence (P=.004), and both 3-year and 5-year survival (P=.018). dMMR tumors showed no significant correlations with α-SMA staining. The findings highlight that immunostaining with α-SMA in pMMR colorectal tumors, especially at the tumor periphery, has the potential to identify patients with adverse prognostic features. Digital assessment of α-SMA may offer improved objectivity, accuracy, economy of time, and risk stratification for management.

The hematopoietic activity of EPO is unfavorable to the treatment of bleomycin-induced pulmonary fibrosis in mice

The main function of erythropoietin (EPO) is to promote hematopoiesis and improve anemia. In addition, EPO also has many non-hematopoietic effects such as anti-inflammation, anti-apoptosis and anti-oxidation. To achieve the protective effects, large doses of EPO are required, so the probability of side effects increases. Previous studies have revealed that EPO can improve pulmonary fibrosis in mice, but it has not been clarified whether the hematopoiesis of EPO contributes to amelioration of pulmonary fibrosis and whether EPO improves overall mortality. Our results show that EPO decreases hydroxyproline content, α-sma and col-1 protein levels in mice with bleomycin-induced pulmonary fibrosis. However, compared with the control group, the weight loss and mortality rate of the EPO group were not improved, while the number of red blood cells (RBCs), hemoglobin (Hb), red cell width distribution-coefficient of variation (RDW-CV) and hematocrit (HCT) were significantly higher. Furthermore, we observed massive thrombosis in the lung of EPO treated lung fibrosis mice but not in control mice. Therefore, our results show that in the condition of lung fibrosis, the hematopoietic activity of exogenous EPO is not conducive to its tissue protective effect.

Salirasib Inhibits the Expression of Genes Involved in Fibrosis in Fibroblasts of Systemic Sclerosis Patients.

Fibrosis is a principal sign of systemic sclerosis (SSc) which can affect several organs including the lung, heart, and dermis. Dermal fibroblasts of SSc patients are characterized by persistent and activated Ras and ERK1/2 signaling which stimulates extreme collagen and extracellular matrix synthesis. Salirasib is a Ras inhibitor that competitively prevents the adherence of GTP-bound Ras to the plasma membrane, that inhibits Ras signaling. This study intended to clarify whether salirasib can influence fibrotic mediators in SSc fibroblasts. Dermal fibroblasts from 10 SSc patients were treated with salirasib in the presence of TGF-β1, and mRNA levels of H-Ras and genes related to fibrosis, such as COL1A1, COL1A2, CTGF, TGF-β1, fibronectin, ACTA2, and MMP1 was measured by real-time PCR. The α-SMA protein expression was analyzed by immunofluorescence staining. In dermal fibroblasts of SSc patients, salirasib treatment, markedly downregulated the H-Ras gene expression. In addition, the protein expression of α-SMA and gene expression of ACTA2 were inhibited upon salirasib treatment. Salirasib also significantly reduced the expression of COL1A1, and COL1A2 genes and augmented the gene expression of MMP1. The mRNA levels of other genes related to fibrosis such as FN1, CTGF, and TGF-β1 were significantly decreased upon salirasib treatment. Considering salirasib significantly reduced the expression of genes related to the fibrosis process and α-SMA gene and protein expression, and given significant upregulation of MMP1 by salirasib, it can be considered as a new curative strategy for fibrotic diseases like SSc.

Periostin Induces Epithelial-Mesenchymal Transition via p38-MAPK Pathway in Human Renal Tubular Cells by High Glucose.

Periostin mediates inflammation and fibrosis by regulating extracellular matrix adhesion, migration, and differentiation in multiple organ diseases. Studies have shown periostin mainly located in the dilated mesangium, tubulointerstitial and fibrotic regions of the diabetic kidney disease, which was negatively correlated with renal function. However, the underlying mechanism remains poorly explored. The expression of periostin in HK-2 cells was investigated under high glucose and high concentration of TGF-β1. The signaling pathway of periostin involved in epithelial-mesenchymal transdifferentiation of HK-2 cells was also validated. The expression of periostin were investigated by RT-PCR, western blot analysis and immunofluorescence assays with different concentrations of glucose and TGF-β1. The expression of E-Cad, α-SMA and p38 proteins were also detected. The effects of periostin, E-Cad, and α-SMA in high glucose were investigated by p38 inhibitors. To demonstrate the interaction among periostin, p38 and EMT markers, periostin under high glucose and high TGF-β1 was knocked down, resulting p38 and phosphorylated p38 was evaluated. The combined of high glucose (HG, 22 mmol/L) and high TGF-β1 (10 ng/mL) upregulated the expression of periostin obviously, stimulating the expression of α-SMA and p38 while inhibiting the expression of E-Cad. p38 inhibitors reduced the expression of periostin and α-SMA while promoted E-Cad protein expression in HK-2 cells under HG conditions. Additionally, p38-MAPK signal pathway was involved in epithelial-mesenchymal transition of human renal tubules in high glucose environment. Significant, knockdown periostin expression effectively inhibited the expression of p38 and phosphorylated p38 under the combination of HG and high TGF-β1, verifying the interaction of periostin with the p38-MAPK signaling pathway. Periostin, a downstream factor of TGF-β1, is positively regulated by TGF-β1 under HG condition, affecting the epithelial-interstitial differentiation of HK-2 cells via p38-MAPK signaling pathway. Therefore, periostin may serve as a biomarker of renal fibrosis in diabetic kidney disease.

Epigenetic BET inhibitor apabetalone counters inflammatory and fibrotic processes in activated cardiac fibroblasts providing insight into reduced hospitalizations for heart failure in BETonMACE trial

Abstract Background After myocardial infarction (MI), sustained crosstalk between monocytes and cardiac fibroblasts (CFs) promotes chronic inflammation and interstitial fibrosis that can contribute to heart failure (HF). Resident and myocardium-infiltrating immune cells produce proinflammatory cytokines IL-1β and TNFα that stimulate CFs to produce monocyte chemoattractants. Monocytes secrete TGF-β1 that transforms CFs into contractile myofibroblasts overproducing the extracellular matrix (ECM) responsible for cardiac fibrosis. Epigenetic BET inhibitors (BETi) reduce CF transdifferentiation to prevent cardiac fibrosis and HF in animal models. In patients with cardiovascular disease, type 2 diabetes and recent MI, administration of the BETi apabetalone (APA) resulted in less hospitalization due to HF vs. placebo (BETonMACE phase 3 trial; hazard ratio 0.59, p=0.03). Purpose To examine APA’s in vitro effects on inflammatory and profibrotic processes in CFs associated with cardiac fibrosis and HF. Methods Immortalized and primary human CFs were stimulated with cytokines (10ng/mL IL-1β, TNFα, TGF-β1) or with conditioned media from 10ng/mL IFNγ and 100ng/mL lipopolysaccharide-treated THP-1 macrophages ± 5μM APA on plastic or in 3D collagen gels. Gene expression was analyzed by PCR, protein levels by FACS or ELISA, collagen deposition with picrosirius red, THP-1 monocyte migration in Boyden chambers, and CF contraction with 3D collagen gels. Results IL-1β or TNFα stimulation of CFs upregulated monocyte chemoattractant 1 (CCL2) and vascular cell adhesion protein 1 (VCAM1) expression, promoting THP-1 cell migration and adhesion to CFs. APA treatment reduced cytokine-induced CCL2 (&amp;gt;20%) and VCAM1 (&amp;gt;70%) gene expression as well as THP-1 cell migration and adhesion to stimulated CFs (&amp;gt;60%). TGF-β1 treatment induced CF transdifferentiation into myofibroblasts as shown by increased expression of α smooth muscle actin (α-SMA) protein, and secretion of ECM proteins fibronectin and periostin. APA treatment reduced myofibroblast mRNA and protein expression of α-SMA (~30%), fibronectin (~30%) and periostin (97%). Myofibroblast-mediated collagen deposition was also reduced by APA by ~20%. α-SMA-dependent CF contraction can be visualized in attached 3D collagen gels. TGF-β1, IL-1β, TNFα or macrophage-conditioned media promoted CF-mediated gel contraction by 2.5, 1.4, 2.1, or 1.8-fold, respectively. APA treatment countered gel contraction by 30-60%, thereby reducing the myofibroblast-like behaviour in organotypic cultures. Conclusions In vitro, APA treatment reduced proinflammatory crosstalk between monocytes and CFs, resulting in less monocyte migration, monocyte - CF adhesion and CF contraction. APA also reduced signaling by TGF-β1, thus reducing profibrotic and contractile behaviour of CFs responsible for cardiac fibrosis. Since fibrosis contributes to HF, this data provides insight into the observed reduction in hospitalization due to HF in the BETonMACE trial.

Investigating the role of an orphan nuclear receptor NR4A1 in atrial fibrillation

Abstract Introduction Atrial fibrosis is a major remodelling process in atrial fibrillation (AF). The lack of clinically effective drug targets, due to insufficient understanding of the mechanisms of cardiac fibrosis, hampers the treatment of AF. Orphan nuclear receptor subfamily 4 group A member 1 (NR4A1) is linked to fibrotic disease in multiple organs, however, its role in cardiac fibrosis and AF is yet to be demonstrated. Recent transcriptomic profiling revealed that NR4A1 expression is downregulated in human atrial cardiac fibroblasts (ACFs) in the presence of persistent AF. However, its mechanistic role in atrial fibrogenesis and AF is unknown. Purpose To investigate the expression profile and function of NR4A1 in human ACFs and provide insights into the role of NR4A1 in the context of persistent AF. Methods Human ACFs were isolated (enzymatic digestion) from right and left atrial appendages of 24 patients with persistent AF and controls in sinus rhythm (SR), who had elective heart surgery. NR4A1 knockdown in ACFs was achieved using NR4A1-siRNA. Gene and protein expression were determined by qPCR and western blot respectively, while BrdU assay and scratch assay were used to evaluate cell proliferation and migration. Atrial fibroblasts subpopulations were identified by single-nucleus RNA-sequencing. Subcellular localisation of NR4A1 was assessed by immunostaining, analysed by ImageJ. Results Validation of RNA-sequencing data confirmed a reduction of NR4A1 gene expression in human ACFs in persistent AF by 25% (p=0.029, Fig. 1A) and the NR4A1 predominant abundance in one of three identified subclusters of human ACFs (NR4A1+NAMPT+). The siRNA-mediated knockdown of NR4A1 in ACFs suppressed gene and protein expression of important component of extracellular matrix collagen-1 (Fig. 1B and Fig. 1C) and fibronectin protein (but not mRNA). The NR4A1-deficient cells also had decreased alpha-smooth muscle actin (αSMA) protein and gene expression (Fig. 1D and Fig. 1E), reduced periostin gene expression in the absence of changes in its protein. Furthermore, the NR4A1-deficiency reduced proliferation (by 18%, p=0.005, Fig. 1F) and accelerated migration (p=0.019, Fig. 1G) of human ACFs. These effects were independent of TGF-β1 stimulation, unlike reported in rat neonatal cardiac and dermal fibroblasts. Despite decreased NR4A1 gene expression, patients with AF had doubled NR4A1 protein levels compared to SR-ACFs (p=0.049, Fig. 2A). This was associated with abnormal 1.8-fold increase in the receptor subcellular localisation (p=0.032, Fig. 2B and Fig. 2C), as opposed to the physiological nuclear localisation observed in ACFs in the absence of AF. Conclusion While under physiological conditions, NR4A1 potently activates profibrotic processes in human ACFs, persistent AF patients have increased but abnormally distributed NR4A1 protein in atrial fibroblasts, whose consequences are yet to be determined in order to develop new therapies for AF.Figure 1Figure 2

Mitochondrial Oxidative Stress and Cardiac Dysfunction in TERT Knockout Mice

Abstract Background Heart failure (HF) is a major cause of hospitalization in the elderly, with its incidence increasing due to aging and associated risk factors like hypertension, diabetes, and myocardial fibrosis. Aging-related myocardial fibrosis, characterized by alterations in the extracellular matrix and myocardial structure, impairs cardiac function. Mitochondrial oxidative stress plays a pivotal role in cardiac electrical and structural remodeling, necessitating a deeper understanding of its involvement in aging-related heart failure. Purpose The purpose of this study is to investigate the mechanisms of aging-related ventricular electrical and structural remodeling by constructing an aging mouse model. This research aims to provide potential therapeutic targets for heart failure associated with aging. Methods We developed a third-generation homozygous telomerase reverse transcriptase (TERT) knockout mouse model. Comparing third-generation homozygous TERT knockout mice (F3 group) with age-matched wild-type males (WT group). The study encompassed blood pressure measurement, electrocardiographic analysis, echocardiography, ELISA for cardiac biomarkers, ventricular tissue electrophysiology, fibrosis assessment through staining, transcriptomic profiling via RNA-seq, and electron microscopy of ventricular mitochondria. Results Compared to the WT group, the F3 group demonstrated increased P53 and P16 protein expression. Decreased LVEF and FS, along with increased interventricular septum thickness, left ventricular diameter, and left ventricular volume. Elevated serum NT-pro-BNP and troponin I (cTnI) levels, with prolonged QRS duration. Decreased left and right ventricular conduction velocity, accompanied by increased conduction dispersion. Notable elevation in COLI, TGFβ, and α-SMA gene transcription and protein expression in ventricular tissue. Significant differences in mitochondrial oxidative stress signal pathways via RNA-seq analysis. Elevated serum MDA levels and decreased SOD levels, with up-regulated Mn-SOD gene transcription and protein expression, and down-regulated MFN2 and Drp1 gene transcription and protein expression in ventricular tissue. Electron microscopy revealed mitochondrial abnormalities such as loose arrangement, decreased number, swelling, vacuolation, and myofilament disintegration or breakage in the F3 group. Conclusion Third-generation TERT-/- senescent mice exhibit notable cardiac impairments, including electrical remodeling, structural changes, and mitochondrial dysfunction. These findings suggest a potential link between mitochondrial oxidative stress and aging-related heart failure, indicating novel therapeutic opportunities targeting mitochondrial dysfunction for managing such conditions.