COL1A1 Expression Research Articles

Effect of COL11A1 on oral squamous cell carcinoma

BackgroundOral squamous cell carcinoma (OSCC) is the most common malignant tumor of the oral cavity, which is mainly a series of atypical hyperplasia of oral epithelial cells, and the overall prognosis remains poor. MethodsGSE37991 and GSE38517 were downloaded from gene expression omnibus (GEO) to identify differentially expressed genes (DEGs). Functional enrichment analysis of DEGs was performed, weighted gene co-expression network analysis (WGCNA) was used. Protein-protein interaction (PPI) network was constructed. Core gene expression was visualized using a heatmap. Comparative toxicogenomics database (CTD) and miRNA analyses identified related diseases and regulatory miRNAs. Western blot (WB) was conducted to examine expression of COL11A1 and TGF-SMAD signaling components in OSCC samples. Results5163 DEGs were identified. DEGs were enriched in metabolic processes and signaling pathways, including TGF-β/SMAD and PI3K-Akt. WGCNA identified 11 core modules. PPI network analysis revealed five core genes: COL11A1, AURKA, MELK, CCNA2, and BUB1. Heatmap analysis showed that COL11A1 is highly expressed in OSCC. CTD analysis indicated that COL11A1 is associated with OSCC. miRNA prediction identified potential regulatory factors. Western blot analysis demonstrated that COL11A1 is overexpressed in OSCC and is associated with TGF-SMAD signaling, inflammation, and cell cycle progression. ConclusionCOL11A1 is highly expressed in OSCC and may serve as a target gene interacting with the TGF-SMAD signaling pathway.

COL8A1 overexpression promotes glioma cell growth by activating focal adhesion kinase signaling cascade

We explored expression and biological roles of collagen type VIII alpha-1 chain (COL8A1) in glioma. Bioinformatics analyses unveiled COL8A1 overexpression within glioma tissues correlates with adverse clinical outcomes of patients. COL8A1 overexpression was also detected in local glioma tissues and various glioma cells. In primary and immortalized glioma cells, COL8A1 shRNA or knockout (KO) reduced cell viability, proliferation and mobility, disrupted cell cycle, and prompted apoptosis. While COL8A1 overexpression augmented the malignant behaviors in glioma cells. COL8A1 shRNA or KO in primary glioma cells decreased phosphorylation of FAK and downstream targets Akt and Erk1/2. Conversely, elevating COL8A1 expression increased their phosphorylations. In vivo experiments confirmed growth inhibition of patient-derived glioma xenografts within the mouse brain following COL8A1 KO. Hindered proliferation, lowered phosphorylation levels of FAK, Akt, and Erk1/2, as well as increased apoptosis were observed within the COL8A1 KO intracranial glioma xenografts. Thus, COL8A1 overexpression promotes glioma cell growth.

Discovery of Sporachelins by Genome Mining of a Micromonospora Strain.

Myxochelins are a group of catecholate siderophores encoded by mxc biosynthetic gene clusters (BGCs). They are mainly produced by myxobacteria and display a wide variety of bioactivities. Herein, we report a group of new myxochelins produced not by a myxobacterial strain but by an actinobacteria strain, Micromonospora sp. TMD166. They consisted of six new compounds, designated as sporachelins A (1), A1 (2), B (3), C (4), D (5), and E (6), and the known compound myxochelin A (7). The planar structures were determined by comprehensive analyses of 1D and 2D NMR spectroscopic data, and the absolute configurations were confirmed by Marfey's analysis and chemical synthesis. The six sporachelins are the first examples of acylated derivatives at the primary alcohol of myxochelin A. These molecules were found to inhibit human 5-lipoxygenase. In addition, 1-7 exhibited antifibrotic activity in the TGFβ1-induced human hepatic cell line LX-2 by suppressing fibrosis-related genes COL1A1, ACTA2, and TGFB1 expression. This is the first report of antifibrotic activity by myxochelins.

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.

Aberrant expression of collagen type X in solid tumor stroma is associated with EMT, immunosuppressive and pro-metastatic pathways, bone marrow stromal cell signatures, and poor survival prognosis.

Collagen type X (ColXα1, encoded by COL10A1 ) is expressed specifically in the cartilage-to-bone transition, in bone marrow cells, and in osteoarthritic (OA) cartilage. We have previously shown that ColXα1 is expressed in breast tumor stroma, correlates with tumor-infiltrating lymphocytes, and predicts poor adjuvant therapy outcomes in ER + /HER2 + breast cancer. However, the underlying molecular mechanisms for these effects are unknown. In this study, we performed bioinformatic analysis of COL10A1 -associated gene modules in breast and pancreatic cancer as well as in cells from bone marrow and OA cartilage. These findings provide important insights into the mechanisms of transcriptional and extracellular matrix changes which impact the local stromal microenvironment and tumor progression. Immunohistochemistry was performed to examine collagen type X expression in solid tumors. WGCNA was used to generate COL10A1 -associated gene networks in breast and pancreatic tumor cohorts using RNA-Seq data from The Cancer Genome Atlas. Computational analysis was employed to assess the impact of these gene networks on development and progression of cancer and OA. Data processing and statistical analysis was performed using R and various publicly-available computational tools. Expression of COL10A1 and its associated gene networks highlights inflammatory and immunosuppressive microenvironments, which identify aggressive breast and pancreatic tumors and contribute to metastatic potential in a sex-dependent manner. Both cancer types are enriched in stroma, and COL10A1 implicates bone marrow-derived fibroblasts as drivers of the epithelial-to-mesenchymal transition (EMT) in these tumors. Heightened expression of COL10A1 and its associated gene networks is correlated with poorer patient outcomes in both breast and pancreatic cancer. Common transcriptional changes and chondrogenic activity are shared between cancer and OA cartilage, suggesting that similar microenvironmental alterations may underlie both diseases. COL10A1 -associated gene networks may hold substantial value as regulators and biomarkers of aggressive tumor phenotypes with implications for therapy development and clinical outcomes. Identification of tumors which exhibit high expression of COL10A1 and its associated genes may reveal the presence of bone marrow-derived stromal microenvironments with heightened EMT capacity and metastatic potential. Our analysis may enable more effective risk assessment and more precise treatment of patients with breast and pancreatic cancer. ColX highlights features of EMT in breast and pancreatic cancerColX gene modules are immunosuppressive and pro-metastaticColX-associated gene networks contribute to sex differences in pancreatic cancerColX-positive fibroblasts define more aggressive tumors with poorer survivalColX is emerging as a biomarker for bone marrow-derived cells in cancer.

Effect of nHA/CS/PLGA delivering adipose stem cell-derived exosomes and bone marrow stem cells on bone healing—in vitro and in vivo studies

Adipose stem cell-derived exosomes (ADSC-EXO) have been demonstrated to promote osteogenic differentiation of bone marrow stem cells (BMSCs) and facilitate bone regeneration. The present study aims to investigate the effect of ADSC-EXO-loaded nano-hydroxyapatite/chitosan/poly-lactide-co-glycolide (nHA/CS/PLGA) scaffolds on maxillofacial bone regeneration using tissue engineering. ADSC-EXO was isolated and co-cultured with BMSCs, and the osteogenic differentiation of BMSCs was assessed through the detection of mineralized nodule formation, alkaline phosphatase (ALP) activity, and mRNA expression of COL1A1 and runt-related transcription factor 2 (RUNX2). The nHA/CS/PLGA scaffolds were fabricated and loaded with ADSC-EXO and BMSCs, and these tissue engineering complexes were applied to the maxillofacial bone defect region of rabbits to elucidate their bone regeneration effect. The osteogenic differentiation of BMSCs was markedly enhanced when they were co-cultured with ADSC-EXO. This was evidenced by an increase in the formation of mineralized nodule formation, ALP activity, and mRNA expression of COL1A1 and runt-related transcription factor 2 (RUNX2). In vivo experiments demonstrated that the application of ADSC-EXO and BMSCs loaded nHA/CS/PLGA scaffolds effectively repaired maxillofacial bone defects in rabbits. ADSC-EXO has been demonstrated to promote the osteogenic differentiation of BMSCs. The ADSC-EXO and BMSCs loaded nHA/CS/PLGA scaffolds have been shown to facilitate the regeneration of maxillofacial bone defects. This may serve as a potential therapeutic strategy for large-scale bone defects.

Extracellular vesicles derived from bone marrow mesenchymal stem cells ameliorate liver fibrosis via micro-7045-5p.

Liver fibrosis is a crucial pathological factor in the persistence and progression of chronic liver disease. Increasing evidence has demonstrated the significant potential of extracellular vesicles (EVs) secreted by bone marrow mesenchymal stem cells (BMSCs) in the clinical treatment of liver fibrosis. This study aimed to mechanistically investigate the impact of BMSC-derived EVs (BMSC-EVs) containing miR-7045-5p on the autophagy of activated hepatic stellate cells (HSCs) during liver fibrosis. BMSCs were isolated from the bilateral femurs and tibiae of mice. Their identity was confirmed via immunofluorescence staining for the BMSC marker CD44. EVs were harvested from BMSC culture medium at passages 3-5 and then DiR-labeled. Labeled BMSC-EVs were co-cultured with the HSC-T6 cell line to determine their uptake and sub-cellular localization in HSCs. Various methods, such as western blotting, qRT-PCR, and ELISA, were employed to assess the effects of BMSC-EVs on the fibrotic activation (marked by COL1-A1 and α-SMA expression) and autophagy (p62, Atg16L1, Beclin-1, and LC3 expression) of HSC-T6 cells. Additionally, the BMSC-EV-induced changes in autophagy-related signaling pathways (PI3K, AKT, and mTOR pathways) in these cells were evaluated. Finally, the gene-chip detection technology was utilized to predict the involvement of BMSC-EV-derived miRNAs (BMSC-EV-miRs) in the observed effects, with a focus on miR-7045-5p, and our findings were validated in HSCs transfected with a miR-7045-5p mimic. The gene-chip detection results indicated that miR-7045-5p was enriched in BMSC-EVs compared with BMSCs and targeted Akt. In the CCl4-induced mouse model of liver fibrosis, BMSC-EV-miR-7045-5p ameliorated the fibrosis and enhanced liver function by suppressing the PI3K/Akt/mTOR signaling pathway. Additionally, miR-7045-5p inhibited TGF-β1-induced fibrotic activation of HSC-T6 cells. BMSC-EVs promote autophagy in HSC-T6 cells and alleviate liver fibrosis by inhibiting the PI3K/Akt/mTOR signaling pathway at least in part by delivering anti-fibrotic miRNAs, such as miR-7045-5p.

Structural Variants in COL1A1 and COL1A2 in Osteogenesis Imperfecta.

Osteogenesis Imperfecta (OI) is a heterogeneous skeletal dysplasia characterized by bone fragility, skeletal deformities, and short stature. Most commonly, it is caused by autosomal dominant variants in the type I collagen genes, COL1A1 or COL1A2. Type I collagen is the main protein of the extracellular matrix in the skeleton and changes in its structure or quantity may lead to OI. 85%-90% of OI cases occur due to sequence variants in type I collagen genes, while OI caused by structural abnormalities in type I collagen genes is less common. In most cases, haploinsufficiency of type I collagen is associated with a milder OI phenotype. Large genomic deletions often involve several genes within the same chromosomal region, leading to microdeletion syndromes with OI features. Here, we report eight Swedish patients from five unrelated families with OI due to structural variants in the COL1A1 and COL1A2 genes. One patient with OI type III had a complex rearrangement with a deletion and duplication event in COL1A2, leading to reduced COL1A2 expression. Three other patients from two different families with OI type I had whole gene deletions involving COL1A1. In one family, three affected individuals with OI type I had a small intragenic deletion of exons 11-12 in COL1A2. One patient had a 2.1 Mb de novo deletion encompassing COL1A1 and DLX3 genes and features of OI and tricho-dento-osseous syndrome. Overall, this study highlights the importance of investigating gene dosage abnormalities in patients with OI and further delineates clinical and genetic variability of OI caused by structural variants in type I collagen genes.

Plasma COL10A1 Level, a Potential Diagnostic and Prognostic Biomarker for Pancreatic Ductal Adenocarcinoma.

COL10A1 expression was up-regulated and could promote tumor development in pancreatic cancer. As a secreted protein, plasma COL10A1 level was proven to have certain diagnostic efficacy in gastric cancer, breast cancer, and colorectal cancer. It is still unknown whether it has a biomarker role for pancreatic cancer. To explore and analyze the diagnostic and prognostic value of plasma COL10A1 level in pancreatic ductal adenocarcinoma (PDAC). The RNA-seq dataset of PDAC from The Cancer Genome Atlas (TCGA) and six expression profiling microarray datasets from Gene Expression Omnibus (GEO) were downloaded to analyze the expression of COL10A1 in tissues. Thirty-six patients with PDAC and eighteen healthy volunteers were enrolled to measure COL10A1 levels in tissues and plasmas, and the relationship between clinical characteristics and the COL10A1 levels was analyzed. The diagnostic and prognostic efficacy of plasma COL10A1 levels were calculated. Aspects of COL10A1 expression level in tissues, COL10A1 expression was significantly higher in PDAC tissue than adjacent normal tissue. The expression of COL10A1 was correlated with T, M, and AJCC stages. Patients with high COL10A1 expression had worse recurrence-free survival (RFS) and overall survival (OS) than those with low expression. Aspects of COL10A1 expression levels in plasma, its diagnostic area under the curve (AUC) for PDAC was 0.926 (95% CI 0.853-0.999), diagnostic sensitivity was 81% (95% CI 64-92%), and specificity was 100% (95% CI 81-100%). The time-dependent AUCs at 1-year and 3-year were 0.71 (95% CI 0.51-0.90) and 0.74 (95% CI 0.48-1.00), respectively. In PDAC, plasma COL10A1 levels showed certain diagnostic and prognostic efficacy. COL10A1 may be a diagnostic and prognostic biomarker for PDAC and play a role in liquid biopsy of this disease.

A new paradigm of bone active risedronate-functionalized polycaprolactone/gelatin scaffold synergistically promotes osteogenic differentiation in rat bone marrow-derived mesenchymal stem cells for bone tissue engineering

Modern developments in bone tissue engineering focus on designing biomimetic materials that exhibit suitable mechanical and physiochemical properties. In this study, we present the development of a new biomaterial scaffold using a newly constructed polymer that contains bisphosphonate in combination with gelatin (GAT). To achieve scaffolding, a chemical grafting technique was used to develop risedronate (RS)-functionalized polycaprolactone (PCL-RS). The freeze-casting process was used to construct the porous PCL-RS/GAT scaffold after incorporating gelatin into the PCL-RS polymer solution. A scaffold with pores that are in perfect position and a porosity of 81.84% was successfully acquired. The biodegradability assessment, 48% of its early weight was lost after five weeks. The PCL-RS/GAT scaffold exhibited an elastic modulus of 32.01 MPa and a tensile strength of 3.9 MPa. The MTT analysis showed the PCL-RS/GAT favorable cytocompatibility with rat bone marrow-derived mesenchymal stem cells (BM-MSC). In addition, the cells cultured in the PCL-RS/GAT scaffold exhibited the greatest ALP activity and mineralization. The RT-PCR test results indicated that the PCL-RS/GAT scaffold had a high RUNX2, COL 1A1, and OCN gene expression, suggesting a strong osteoinductive capacity. The results indicate that the PCL-RS/GAT scaffold is a suitable biomimetic platform for tissue engineering in bone.

Evaluating the antifibrotic potential of naringenin, asiatic acid, and icariin using murine and human precision-cut liver slices.

Liver fibrosis is an exaggerated wound healing response defined by the excessive accumulation of extracellular matrix. This study investigated the antifibrotic potential of naringenin (NRG), asiatic acid (AA), and icariin (ICA) using murine and human precision-cut liver slices (PCLS). These natural products have shown promise in animal models, but human data are lacking. In this study, PCLS prepared from male mouse liver tissue (mPCLS), healthy human liver tissue (hhPCLS), and cirrhotic human liver tissue (chPCLS) were cultured for 48 h with varying concentrations of the three compounds. Our findings indicate that NRG reduced collagen type 1 (COL1A1) expression in a concentration-dependent manner in both mPCLS and chPCLS, decreased fibrosis-related gene expression, and significantly lowered pro-collagen type 1 (PCOL1A1) levels in the culture medium by 54 ± 21% (mPCLS) and 78 ± 35% (chPCLS). Furthermore, NRG effectively inhibited IL-1β and TNF-α in mPCLS and IL-1β in chPCLS on both gene and protein levels. AA specifically reduced COL1A1 and PCOL1A1 in chPCLS, while ICA selectively downregulated Col1a1 and Acta2 gene expression in mPCLS. This study suggests NRG's potential as an effective antifibrotic agent, warranting further investigation into its mechanisms and therapeutic applications in liver fibrosis.

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.

Interleukin-22 inhibits right ventricular remodelling by a unique mechanism in a pulmonary artery constriction model

Abstract Background Fibrosis and remodeling of right ventricle (RV) are associated with prognosis in patients with RV failure. Interleukin-22 (IL-22) is a member of the IL-10 cytokine family, play roles in tissue protection and wound repair. We have previously shown that IL-22 plays an important role in cardiac remodeling after acute myocardial infarction; however, the role of IL-22 in RV fibrosis and remodeling remains elusive. Purpose We investigated the role of IL-22 in the pathogenesis of RV remodeling during pressure overload. Methods Pulmonary artery banding (PAB) is performed by placing a 6-0 suture around the pulmonary artery over a 24 G needle in wild type mice (C57BL/6J) and IL-22 knockout mice (IL-22 KO). Only mice with moderate pulmonary artery stenosis (peak pressure gradient across the pulmonary band: 25–40 mmHg at 1 week after surgery by echo Doppler) were included in the study protocol. Four weeks after PAB, RV function was measured by echocardiography and real-time PCR analysis was performed to measure Col1a1, Col3a1, BNP, and transcriptome analysis was performed. Survival confirmation of mice with moderate pulmonary artery stenosis was also performed up to 56 days. Results IL-22 KO mice had significantly higher 56-day mortality rate compared with WT mice after PAC (p&amp;lt;0.01). Four weeks after PAB, IL-22 KO mice showed markedly increased RV weight (IL-22 KO mice vs. wild-type mice; RV/LV+IVS: 0.48±0.05 vs 0.40±0.02, P=0.002). IL-22 KO mice exhibited significant RV enlargement and RV dysfunction 4 weeks after PAB. (IL-22 KO mice vs wild type mice; RVEDV: 10±2.3mm2 vs 12.9±3.1mm2, P=0.005, TAPSE: 10.8±1.8mm vs 9.5±1.7mm, P=0.04, FAC: 35.2±7.8% vs 23±6.0%, P&amp;lt;0.001). The expressions of Col1a1 and Col3a1 were no difference between in IL22-KO mice and Wild mice. However, BNP, a cardioprotective marker, tended to be lower in IL-22KO mice. The transcriptome analysis also showed that the group of fibrosis-related genes that are elevated in PAC in WT mice were less elevated in IL-22 KO mice. Conclusion These results suggest that IL-22 is involved in RV remodelling during RV pressure loading without a general mechanism.

Signature of diabetic cardiomyopathy progression using high-fat diet and streptozotocin mouse model: from subclinical to clinical with three-hit mouse model

Abstract Background Diabetic cardiomyopathy (DbCM) is a pre-heart failure (pre-HF) condition that usually exhibits early diastolic dysfunction and evolving systolic dysfunction, along with left ventricular (LV) remodelling. Although DbCM shows distinct pathophysiology, the transition from the subclinical to the symptomatic stage is not straightforward and usually involves multifaceted processes which are not fully understood. Purpose To define the signature of DbCM progression in a novel mouse model using longitudinal echocardiography and multi-omics approaches. Methods Male mice (C57BL/6) were fed with high-fat diet (HFD) or control diet (CD) for 24 weeks, with single dose of streptozotocin (STZ) or vehicle injection at 8 weeks (n=10 each). Serial functional and metabolic parameters were measured prior to endpoint single nuclei RNA sequencing (snRNA-seq) and mass spectrometry-based proteomics analysis with comparison to cardiac remodelling to define detailed mechanisms underlying DbCM progression. Results HFD/STZ mice developed type 2 diabetes mellitus (T2DM) based on higher fasting blood glucose and HbA1C levels, insulin resistance and decreased beta-cell function (HOMA-IR and HOMA-beta, respectively) compared to controls. Progressive diastolic dysfunction was evident in HFD/STZ mice compared to CD mice based on enlarged LVPW;d from 12 weeks (P&amp;lt;0.05), reduced MV E/A ratio (P&amp;lt;0.01) and IVRT (P&amp;lt;0.05) from 16 weeks, and increased cardiac filling pressure (Left atrial (LA) area and LA volume) at 24 weeks, with preserved systolic function. This was paralleled by elevated collagen deposition (Picrosirius red), myocyte cross-sectional area and Col1a1 expression in HFD/STZ versus CD mice (all P&amp;lt;0.05). snRNA-seq analysis indicated preferential monocyte migration into diabetic hearts (P&amp;lt;0.01) whilst inflammatory pathways were activated across all cardiac cell types. Proteomics analysis revealed elevated expression of the established inflammation marker, plasma C-reactive protein (CRP), in HFD/STZ versus CD mice (P&amp;lt;0.05), which was correlated with worsening diastolic function (MV E/A ratio: r=-0.839, P&amp;lt;0.01) and LV hypertrophy (LVPW;d: r=0.861, P&amp;lt;0.01). Conclusion Our HFD/STZ mouse model offers a ‘three-hit’ approach which effectively captures key stages of clinical DbCM progression: 1) T2DM with insulin resistance, 2) progressive diastolic dysfunction with elevated chronic filling pressure and cardiac remodelling, and 3) systemic inflammation. This improved HFD/STZ mouse model is therefore appropriate to support a detailed study of mechanisms underlying DbCM progression and subsequent translation.

Anti-fibrotic effects of the sirt6-activator mdl-800 in cardiac stromal cells

Abstract Background Epigenetic changes are among the molecular substrates of cellular stress responses and tissue remodeling in heart failure progression. The sirtuin family of NAD+-dependent histone and non-histone protein deacetylases exert a protective anti-fibrotic role by negatively modulating the inflammatory response and by positive regulation of autophagy-related genes. Sirtuin6 (SIRT6) stands out as a key cardiac protector that can mitigate aging-induced cardiomyocyte senescence and cardiac hypertrophy. Cardiac stromal cells (CSCs) play a pivotal role in fibrosis and remodeling, regulating the composition and stiffness of the extracellular matrix (ECM). Autophagy enhancement has anti-fibrotic effects in CSCs, but the specific role of SIRT6 modulation in CSCs remains unexplored. Purpose To investigate the biological effects of MDL-800, a synthetic allosteric SIRT6 activator, on the stress response and fibrotic activation of CSCs. Methods CSCs were isolated from 4-week-old C57Bl6J mice by explant culture and stimulated with 10ng/ml of TGF-beta1 (TGFb1) up to 72h to induce fibrotic activation. CSCs were treated with MDL-800 up to 10µM and up to 72h, analyzed for cell viability, gene and protein expression levels. Results TGFb1 treatment led to a 0.64-fold reduction in SIRT6 gene expression after 24h. The MTS assay was used to assess a viability dose-response to MDL-800 up to 72h of treatment. The non-toxic concentrations of 2.5µM and 5µM were selected for further experiments (2.5µM vs CTR 0.97-fold and 5µM vs CTR 0.92-fold, normalized OD vs t0). Co-treatment of 5µM MDL-800 with TGFb1 for 72h resulted in a dose-dependent decrease in the expression of TGFb1-induced fibroblast activation markers, as confirmed by immunofluorescence staining for aSMA (TGFb1 vs CTR 2.21-fold, p&amp;lt;0.05; 5µM vs TGFb1 0.46-fold, p&amp;lt;0.05, N=4; mean fluorescence intensity). The anti-fibrotic effect mediated by MDL-800 was further validated by real-time PCR indicating a reduction in the mRNA expression of multiple fibrotic markers, including ACTA2 (TGFb1 vs CTR 1.67-fold, 5µM vs TGFB 0.29-fold; N=2), COL1a1 (TGFb1 vs CTR 1.32-fold, 5µM vs TGFb1 0.87-fold; N=2) COL3a1 (TGFb1 vs CTR 1.06-fold, 5µM vs TGFb1 0.43-fold; N=2) and POSTN (TGFb1 vs CTR 6.37-fold, 5µM vs TGFb1 0.23-fold; N=2). Treatment with 5µM MDL-800 demonstrated an increase in SIRT6 gene expression, suggesting a transcriptional modulation (TGFb1 vs CTR 0.95-fold, 5µM vs TGFb1 1.68-fold; N=2). Western Blot analyses showed an increase in protein levels of the autophagosome marker LC3-II following treatment with 5µM MDL-800 (TGFb1 vs CTR 0.95-fold; 5µM vs TGFb1 3.01-fold), suggesting that autophagy enhancement may be a potential mechanism for the observed anti-fibrotic effects. Conclusion MDL-800 treatment in CSCs is associated with a dose-dependent reduction of TGFb1-induced fibrotic markers and to increased levels of the autophagy marker, suggesting its potential use as an anti-fibrotic molecule for the heart.

The development of small-molecule ERBB4 agonists as a treatment for heart failure

Abstract The neuregulin-1 (NRG1)/ERBB4 signaling pathway has emerged as a cardioprotective pathway and is a promising target for the treatment of chronic heart failure. Activation of ERBB4 signaling is known to decrease cardiomyocyte cell death and hypertrophy, and fibroblast collagen synthesis. Recombinant NRG1 (rNRG1) is currently tested in phase III clinical trials for heart failure, but its need for intravenous administration is a disadvantage. In an attempt to circumvent this, we hypothesized that small-molecule-induced activation of ERBB4 is feasible and would recapitulate the effects of its natural ligand on myocytes and fibroblasts. To this end, we screened 10,240 compounds for their ability to induce homodimerization of ERBB4. We identified a series of 8 similar compounds (named EF-1 – EF-8) that concentration-dependently induced ERBB4 dimerization, with EF-1 being the most potent and effective compound (n = 4-5 independent repeats in each group; Emax = 27.9 ± 4.8% relative to NRG1, EC50 = 10.5 ± 4.5 x 10-6). EF-1 showed neither cytotoxicity nor increased cell proliferation of tumor cell lines. In vitro, EF-1 significantly decreased in a concentration-dependent manner hydrogen peroxide–induced cardiomyocyte cell death (n = 4 independent repeats in each group; P&amp;lt;0.0001 compared to siERBB4, Fig. 1A), angiotensin-II (AngII)-induced cardiomyocyte hypertrophy (n = 20 individual cardiomyocytes in each group; P&amp;lt;0.0001 compared to AngII/vehicle, Fig. 1B), and collagen expression in cultured human fibroblasts (n = 3 independent repeats in each group; P=0.03 compared to siERBB4, Fig. 1C). The observed effects in cultured cardiomyocytes and fibroblasts could be abrogated by siRNA targeting ERBB4, indicating that they were mediated by ERBB4. Moreover, when used in vivo, EF-1 (2 mg/kg/day) significantly decreased AngII-induced left ventricular Col1a1 and Col3a1 mRNA expression (n = 4-5 mice in each group; P=0.02 and P=0.004 compared to AngII/vehicle, respectively) and inhibited AngII-induced myocardial total and interstitial fibrosis in wild-type mice (n = 4-5 mice in each group, Fig. 2), but not in Erbb4-null mice. Moreover, EF-1 decreased acute cardiotoxicity (assessed by troponin release) in wild-type mice treated with doxorubicin (DOX; n = 8-9 mice in each group; P&amp;lt;0.0001 compared to DOX/vehicle), but not in Erbb4-null mice. In conclusion, we show that small-molecule-induced ERBB4 dimerization and activation is feasible, and recapitulates anti-fibrotic and cardiomyocyte protective effects in the heart in an ERBB4-dependent manner, both in vitro and in vivo. This could be the start for the further development of small-molecule ERBB4 agonists as a novel class of drugs to treat heart failure. Cardiomyocyte-protective effect in vitroAnti-fibrotic effect in vivo

Genistein inhibited endocytosis and fibrogenesis in keloid via CTGF signaling pathways

BackgroundThis study aimed to evaluate soy isoflavones' effect and potential use—specifically genistein—in treating human keloid fibroblast cells (KFs) and in a keloid tissue culture model.MethodsTo investigate the effects of genistein on keloid, a wound-healing assay was performed to detect cell migration. Flow cytometry was used to measure apoptosis. Western blotting and immunofluorescence staining were performed to detect the expression of target proteins. KF tissues were isolated, cultured, and divided into the control, silenced connective tissue growth factor (CTGF) proteins, and shNC (negative control) groups.ResultsGenistein suppressed cell proliferation and migration, triggering the cell cycle at the G2/M phase and increasing the expression of p53 dose-dependent in keloids. Genistein inhibited the expression of COL1A1, FN, and CTGF mRNA and protein. Knockdown CTGF reduced the migrated ability in KFs. Genistein also abated TGF-β1-induced keloid fibrosis through the endocytosis model. Separated and cultured the keloid patient’s tissues decreased the cell migration ability by genistein treatment and was time-dose dependent.ConclusionsThis study indicated that genistein-induced p53 undergoes cell cycle arrest via the CTGF pathway-inhibited keloid cultured cells, and genistein suppressed the primary keloid cell migration, suggesting that our research provides a new strategy for developing drugs for treating keloids.

The role of IL15 in angiotensin II-mediated cardiac remodelling

Abstract Hypertension (HTN) mediated target organ damage, such as cardiac remodelling, is a major cause of death and disability. Cytokines are important regulators of cardiac function, inflammation, and remodelling in cardiovascular disease. Increased levels of interleukin 15 (IL15) have been noted in HTN, but its role in HTN heart disease remains unknown. This study investigates the role of the IL15 axis in the pathogenesis of cardiac remodelling in HTN. Male 12-week-old IL15 receptor α knock-out (IL15RAKO), IL15 knock-out (IL15KO) and matching wild type (WT) mice were administered vehicle or angiotensin II (AngII) (490 ng/min/kg) for 2 weeks by osmotic minipump. Blood pressure was measured by tail cuff (n=5-8). Cardiac function was analysed by echocardiography (n=3-4). Cardiac infiltrating immune cells were analysed by flow cytometry (n=2-3 for IL15RAKO, n=6-10 for IL15KO). Cardiac remodelling was assessed by picrosirius red, WGA staining (n=5-8) and expression of markers of remodelling using RT-qPCR and western blots (n=5-8). Data are presented as mean±SEM and analysed using two-way ANOVA. AngII infusion caused a significant increase in systolic blood pressure in WT and IL15KO animals. In contrast, in IL15RAKO, the response to AngII was blunted. This was in line with a decrease in ejection fraction, however only WT mice had an increase in global longitudinal strain (-20.3±2.0 vs -10.9±1.2, p=0.0039 in WT and 19.9±0.8 vs -16.0±2.1, p=0.1878 in IL15RAKO). HTN was associated with increased cardiac IL15 protein levels in mice. The heart-to-body ratio, along with cardiomyocyte cross-sectional, were significantly increased in WT, but not in IL15RAKO HTN mice. Furthermore, histological analysis showed significantly higher cardiac collagen accumulation in WT but not IL15RAKO upon AngII infusion (5.8±1.0% in WTvvs. 2.1±0.5% in IL15RAKO, p=0.0021). This was in line with a higher expression of col1a1 in WT but not IL15RAKO HTN hearts (20.0±6.0 vs 4.5±1.7, p=0.0173). Similarly, the FN1 protein level was significantly increased in WT but not IL15RAKO AngII-treated mice. These changes were associated with a higher number of Cd11b+ macrophages accumulated in WT hypertensive hearts only. In contrast to IL15RAKO, mice lacking IL15 showed similar increases in global longitudinal strain, heart hypertrophy, and cardiomyocyte cross-sectional area as WT mice upon AngII infusion. Similarly, IL15KO hypertensive hearts showed a significant increase of col1a1 and FN1 in comparison to vehicle-treated mice. A similar level of Cd11b+ macrophages was found in hypertensive IL15KO and WT mice's hearts. In conclusion, IL15RA, but not IL15, plays a critical role in cardiac remodelling mediated by AngII infusion. This suggests that the IL15-IL15RA axis may play an important yet complex role in hypertensive heart disease.

Impact of paracrine effects of different clonal hematopoiesis-driver mutations in human macrophages on cardiac cells

Abstract Background Age-acquired, non-malignant somatic mutations in hematopoietic stem cells can lead to expansion of blood cells, known as clonal hematopoiesis of indeterminate potential (CHIP). CHIP-driving mutations in a spectrum of genes are correlated with an increased incidence of and poor prognosis in patients with cardiovascular disease. Recent reports suggest increased inflammation in carriers of frequent mutations in DNMT3A and TET2. Less frequent mutations in splicing factors, signaling molecules and epigenetic regulators were found to be associated with greater myeloid malignancy and cardiovascular mortality risks, but paracrine effects of these mutations on cardiac tissue is not known. Purpose We aim to assess the impact of less frequent CHIP-driver mutations on paracrine activities of macrophages on cardiac cells. Methods &amp; Results In order to identify interesting CHIP-driver mutations, we analysed frequency and all-cause mortality of 56 potential CHIP driver genes in two cohorts of patients with heart failure and aortic stenosis (1168 patients). Besides the well-studied mutations in TET2 and DNMT3A, we identified nine mutations with a relative higher frequency and impact on prognosis, among them splicing factors SF3B1, SRSF2, ZRSR2, TP53-related genes PPM1D and PHF6, signaling mediators CALR and GNAS, and epigenetic regulators EZH2, KDM6A. To mimic CHIP-driving conditions, we assessed the effect of siRNA-mediated silencing of the so far understudied genes on the paracrine activity of primary human macrophages, which are differentiated from primary CD14+ sorted monocytes. We achieved &amp;gt;40% reductions in mRNA expression for each of the targeted genes (p&amp;lt;0.05). To gain first insights into a potential paracrine activity of individual mutations, we incubated cardiomyocytes with the conditioned medium collected from macrophages. Notably, supernatants from ZRSR2-, PPM1D- or CALR-silenced macrophages significantly reduced cardiomyocyte beating frequencies (53.1%, 64.9% and 63.7% of control, respectively; all p&amp;lt;0.05). Interestingly, only supernatants of ZRSR2-, CALR-silenced macrophages induced hypertrophy (all p&amp;lt;0.05). Next, we assessed the effects on primary human cardiac fibroblasts. Treatment with conditioned media did not affect collagen 1A1 expression and cell counts, but conditioned media of EZH2-, and CALR-silenced macrophages induced fibroblast senescence evaluated by senescence-associated β-galactosidase staining (158.3% and 154.1% of control, respectively; both p&amp;lt;0.05). The mechanisms driving the different effects are currently under investigation. Conclusion Loss of genes from a broad spectrum of clonal hematopoiesis drivers differentially affects the paracrine activity of human monocyte-derived macrophages. Silencing of ZRSR2 or CALR in macrophages had most profound effects on cardiomyocytes. Surprisingly, EZH2 and CALR-silenced macrophages augmented senescence of cardiac fibroblasts, a finding that further needs to be explored.

The Functional Role of the Long Non-Coding RNA LINCMD1 in Leiomyoma Pathogenesis.

Existing evidence indicates that LINCMD1 regulates muscle differentiation-related gene expression in skeletal muscle by acting as a miRNA sponge, though its role in leiomyoma development is still unknown. This study investigated LINCMD1's involvement in leiomyoma by analyzing paired myometrium and leiomyoma tissue samples (n = 34) from patients who had not received hormonal treatments for at least three months prior to surgery. Myometrium smooth muscle cells (MSMCs) were isolated, and gene expression of LINCMD1 and miR-135b was assessed via qRT-PCR, while luciferase assays determined the interaction between LINCMD1 and miR-135b. To examine the effects of LINCMD1 knockdown, siRNA transfection was applied to a 3D MSMC spheroid culture, followed by qRT-PCR and Western blot analyses of miR-135b, APC, β-Catenin and COL1A1 expression. The results showed that leiomyoma tissues had significantly reduced LINCMD1 mRNA levels, regardless of patient race or MED12 mutation status, while miR-135b levels were elevated compared to matched myometrium samples. Luciferase assays confirmed LINCMD1's role as a sponge for miR-135b. LINCMD1 knockdown in MSMC spheroids increased miR-135b levels, reduced APC expression, and led to β-Catenin accumulation and higher COL1A1 expression. These findings highlight LINCMD1 as a potential therapeutic target to modulate aberrant Wnt/β-Catenin signaling in leiomyoma.