Extracellular Matrix-related Genes Research Articles

Dimethyl sulfoxide (DMSO) enhances direct cardiac reprogramming by inhibiting the bromodomain of coactivators CBP/p300

AimsDirect cardiac reprogramming represents an attractive way to reversing heart damage caused by myocardial infarction because it removes fibroblasts, while also generating new functional cardiomyocytes. Yet, the main hurdle for bringing this technique to the clinic is the lack of efficacy with current reprogramming protocols. Here, we describe our unexpected discovery that DMSO is capable of significantly augmenting direct cardiac reprogramming in vitro. Methods and resultsUpon induction with cardiac transcription factors- Gata4, Hand2, Mef2c and Tbx5 (GHMT), the treatment of mouse embryonic fibroblasts (MEFs) with 1% DMSO induced ~5 fold increase in Myh6-mCherry+ cells, and significantly upregulated global expression of cardiac genes, including Myh6, Ttn, Nppa, Myh7 and Ryr2. RNA-seq confirmed upregulation of cardiac gene programmes and downregulation of extracellular matrix-related genes. Treatment of TGF-β1, DMSO, or SB431542, and the combination thereof, revealed that DMSO most likely targets a separate but parallel pathway other than TGF-β signalling. Subsequent experiments using small molecule screening revealed that DMSO enhances direct cardiac reprogramming through inhibition of the CBP/p300 bromodomain, and not its acetyltransferase property. ConclusionIn conclusion, our work points to a direct molecular target of DMSO, which can be used for augmenting GHMT-induced direct cardiac reprogramming and possibly other cell fate conversion processes.

A model combing an immune-related genes signature and an extracellular matrix-related genes signature in predicting prognosis of left- and right-sided colon cancer.

3533 Background: Primary tumor sidedness has been found to be prognostic in colorectal cancer (CRC), with right-sided colon cancer (RCC) having a worse survival than left-sided tumors (LCC), even after controlling for known negative prognostic factors. Our previous proteomic study identified differences in protein profiles between LCC and RCC. Immune-related proteins were found to be up-regulated in LCC while the differentially expressed proteins in RCC were mainly enriched in extracellular matrix-related proteins. Herein we aim to construct a prognostic prediction model for LCC and RCC patients by using immune-related genes (IRGs) and extracellular matrix-related genes (ECMGs). Methods: A total of 1,868 CRC patients with complete follow-up data from 1 training cohort (n = 562) and 3 independent validation cohorts (n = 622, n = 403, n = 281, respectively) were enrolled in our study. Tumors located in the splenic flexure, descending colon, sigmoid colon, and rectum are defined as LCC. In contrast, tumors located in the region from the hepatic flexure to the cecum are defined as RCC. The Least Absolute Shrinkage and Selection Operator (LASSO) algorithm was used to construct the multi-gene signatures. Univariate and multivariate analyses were used to test the prognostic value of these models. Results: Our biomarker discovery effort identified a 9-gene IRGs signature that significantly associated with poor DFS for LCC (HR = 3.46, 95%CI = 2.38-5.01, P < 0.001) and a 21-gene ECMGs signature associated with prognosis for RCC (HR = 4.53, 95%CI = 2.84-7.22, P < 0.001). For LCC, the IRGs signature was significantly correlated with worse prognosis in three independent validation cohort (Validation-1 cohort: HR = 2.08, 95%CI = 1.41-3.09, P < 0.001; Validation-2 cohort: HR = 2.19, 95%CI = 1.26-3.81, P = 0.004; Validation-3 cohort: HR = 2.94, 95%CI = 1.53-5.63, P < 0.001). Similarly, the ECMGs signature also robustly predicted survival for RCC in three independent validation (Validation-1 cohort: HR = 1.86, 95%CI = 1.22-2.83, P = 0.003; Validation-2 cohort: HR = 1.96, 95%CI = 1.18-3.26, P = 0.008; Validation-3 cohort: HR = 2.8, 95%CI = 1.27-6.17, P = 0.007). When compared with Oncotype DX, we found IRGs achieved an improved survival correlation in LCC (C-index, validation-3 cohort: 0.75 vs 0.64) and ECMGs got a better survival correlation in RCC (C-index, validation-3 cohort: 0.74 vs 0.58). Conclusions: Combing a 9-gene IRGs signature for LCC and a 21-gene ECMGs signature for RCC, we established a prognostic model that can robustly stratify CRC patients into high- and low- risk groups of tumor recurrence and predict prognosis.

Identification and validation of a risk signature based on extracellular matrix-related genes in gliomas.

Gliomas have the highest incidence among primary brain tumors, and the extracellular matrix (ECM) plays a vital role in tumor progression. We constructed a risk signature using ECM-related genes to predict the prognosis of patients with gliomas.mRNA and clinical data from glioma patients were downloaded from The Cancer Genome Atlas (TCGA), Genotype-Tissue Expression (GTEx) and Chinese Glioma Genome Atlas (CGGA) databases. Differentially expressed ECM-related genes were screened, and a risk signature was built using least absolute shrinkage and selection operator (LASSO) Cox regression. Cell type identification by estimating relative subsets of RNA transcripts (CIBERSORT) was used to assess immune infiltration in different risk groups. Gene set enrichment analysis (GSEA) was performed to explore the molecular mechanisms of the genes employed in the risk score.Differentially expressed ECM-related genes were identified, and their associated regulatory mechanisms were predicted via analysis of protein–protein interaction (PPI), transcription factor (TF) regulatory and TF coexpression networks. The established risk signature considered 17 ECM-related genes. The prognosis of the high-risk group was significantly worse than that of the low-risk group. We used the CGGA database to validate the signature. CIBERSORT indicated that the levels of naive B cells, activated memory CD4 T cells, regulatory T cells, gamma delta T cells, activated NK cells, monocytes, activated dendritic cells and activated mast cells were higher in the high-risk group. The levels of plasma cells, CD8 T cells, naive CD4 T cells, resting memory CD4 T cells, M0 macrophages, M1 macrophages, resting mast cells, and neutrophils were lower in the high-risk group. Ultimately, GSEA showed that the terms intestinal immune network for IgA production, primary immunodeficiency, and ECM receptor interaction were the top 3 terms enriched in the high-risk group. The terms Wnt signaling pathway, ErbB signaling pathway, mTOR signaling pathway, and calcium signaling pathway were enriched in the low-risk group.We built a risk signature to predict glioma prognosis using ECM-related genes. By evaluating immune infiltration and biofunctions, we gained a further understanding of this risk signature. This risk signature could be an effective tool for predicting glioma prognosis.This study did not require ethical approval. We will disseminate our findings by publishing results in a peer-reviewed journal.

701 Caloric restriction during aging alters expression of dermal extracellular matrix-related genes in mice

701 Caloric restriction during aging alters expression of dermal extracellular matrix-related genes in mice

Temporarily Epigenetic Repression in Bergmann Glia Regulates the Migration of Granule Cells.

Forming tight interaction with both Purkinje and granule cells (GCs), Bergmann glia (BG) are essential for cerebellar morphogenesis and neuronal homeostasis. However, how BG act in this process is unclear without comprehensive transcriptome landscape of BG. Here, high temporal‐resolution investigation of transcriptomes with FACS‐sorted BG revealed the dynamic expression of genes within given functions and pathways enabled BG to assist neural migration and construct neuron‐glia network. It is found that the peak time of GCs migration (P7‐10) strikingly coincides with the downregulation of extracellular matrix (ECM) related genes, and the disruption of which by Setdb1 ablation at P7‐10 in BG leads to significant migration defect of GCs emphasizing the criticality of Nfix‐Setdb1 mediated H3K9me3 repressive complex for the precise regulation of GCs migration in vivo. Thus, BG's transcriptomic landscapes offer an insight into the mechanism by which BG are in depth integrated in cerebellar neural network.

Pathogenic alleles in microtubule, secretory granule and extracellular matrix-related genes in familial keratoconus.

Keratoconus is a common corneal defect with a complex genetic basis. By whole exome sequencing of affected members from 11 multiplex families of European ancestry, we identified 23 rare, heterozygous, potentially pathogenic variants in 8 genes. These include nonsynonymous single amino acid substitutions in HSPG2, EML6 and CENPF in two families each, and in NBEAL2, LRP1B, PIK3CG and MRGPRD in three families each; ITGAX had nonsynonymous single amino acid substitutions in two families and an indel with a base substitution producing a nonsense allele in the third family. Only HSPG2, EML6 and CENPF have been associated with ocular phenotypes previously. With the exception of MRGPRD and ITGAX, we detected the transcript and encoded protein of the remaining genes in the cornea and corneal cell cultures. Cultured stromal cells showed cytoplasmic punctate staining of NBEAL2, staining of the fibrillar cytoskeletal network by EML6, while CENPF localized to the basal body of primary cilia. We inhibited the expression of HSPG2, EML6, NBEAL2 and CENPF in stromal cell cultures and assayed for the expression of COL1A1 as a readout of corneal matrix production. An upregulation in COL1A1 after siRNA inhibition indicated their functional link to stromal cell biology. For ITGAX, encoding a leukocyte integrin, we assayed its level in the sera of 3 affected families compared with 10 unrelated controls to detect an increase in all affecteds. Our study identified genes that regulate the cytoskeleton, protein trafficking and secretion, barrier tissue function and response to injury and inflammation, as being relevant to keratoconus.

Alendronate promotes the gene expression of extracellular matrix mediated by SP-1/SOX-9.

Osteoarthritis (OA) is a disease with significant degenerative changes of articular cartilage, which is reported to be closely related to the integrity of chondrocytes extracellular matrix (ECM). Alendronate belongs to the family of bisphosphonates with promising cartilage repair function. In the present study, the effects of Alendronate on the gene expression of chondrocytes ECM and the potential mechanism will be investigated to explore the potential therapeutic property of Alendronate on OA. Human SW1353 chondrocytes were stimulated with 1 and 2 μM Alendronate for 12 h. The gene expression of Col2α1, COL9α2, and Acan in the treated chondrocytes was determined by qRT-PCR. QRT-PCR and western blot analysis were used to evaluate the expression level of SOX-9 in the treated chondrocytes. The expression level of SP-1 was checked by qRT-PCR and immunostaining. SiRNA against SP-1 was transfected into chondrocytes to knockdown the expression of SP-1. The levels of p-ERK1/2 and total ERK1/2 were examined using western blot analysis. TNF-α was used to induce an OA-like in vitro model in the chondrocytes for therapeutic evaluations. Treatment with Alendronate increased the levels of ECM related genes (Col2α1, COL9α2, and Acan) in a dose-dependent manner through increasing the expression of SOX-9, a central regulator of ECM genes. Additionally, our findings demonstrate that the effects of Alendronate in the expression of SOX-9 are mediated by SP-1 as silencing of SP-1 abolished these effects. Notably, Alendronate increased the phosphorylation of ERK1/2 and inhibition of ERK1/2 using its specific inhibitor U0126 blocked the expression of SP-1. Finally, we found that treatment with Alendronate could rescue TNF-α-induced reduction of Col2α1, COL9α2, Acan and SOX-9. Our data indicated that Alendronate might promote the gene expression of extracellular matrix through SOX-9 mediated by the ERK1/2/SP1 signaling pathway.

Effect of Metformin on Cardiac Metabolism and Longevity in Aged Female Mice.

The antidiabetic drug metformin exerts pleiotropic effects on multiple organs, including the cardiovascular system. Evidence has shown that metformin improves healthspan and lifespan in male mice, yet its lifespan lengthening effect in females remains elusive. We herein demonstrated that metformin fails to extend the lifespan in female mice. Compared to 2-month-old young controls, 20-month-old female mice showed a spectrum of degenerative cardiac phenotypes alongside significant alterations in the extracellular matrix composition. Despite lowered reactive oxygen species production, long-term metformin treatment did not improve cardiac function in the aged female mice. In contrast, RNA sequencing analyses demonstrated that metformin treatment elevated the extracellular matrix-related gene while lowering oxidative phosphorylation-related gene expression in the heart. In addition, metformin treatment induced metabolic reprogramming that suppressed mitochondrial respiration but activated glycolysis (i.e., Warburg effect) in cultured primary cardiomyocytes and macrophages, thereby sustaining an inflammatory status and lowering ATP production. These findings suggest the unexpected detrimental effects of metformin on the regulation of cardiac homeostasis and longevity in female mice, reinforcing the significance of comprehensive testing prior to the translation of metformin-based novel therapies.

Integrin \u03b12\u03b21 plays an important role in the interaction between human articular cartilage-derived chondrocytes and atelocollagen gel

Although atelocollagen gel is used as a scaffold for culturing human articular cartilage-derived chondrocytes, little is known about cell–gel interactions. In this study, we investigated the mechanism via which atelocollagen gel affects human articular cartilage-derived chondrocytes. Two types of three-dimensional cultures of human articular cartilage-derived chondrocytes (i.e., with and without atelocollagen gel) were compared. While the amount of atelocollagen gel in culture gradually decreased with time, it promoted the expression of matrix metalloproteinases (MMPs) during the early stages of culture. Genome-wide differential gene expression analysis revealed that cell membrane- and extracellular matrix-related genes were highly ranked among up- and down-regulated groups in cells cultured in the presence of atelocollagen gel. Among the integrin family of genes, the expression of integrin subunit alpha 2 and integrin subunit alpha 10 was significantly increased in the presence of atelocollagen gel. Blocking α2β1 integrin with the specific inhibitor BTT 3033 had a significant effect on cell proliferation, MMP expression, and cell shape, as well as on the response to mechanical stimulation. Taken together, our findings indicate that the α2β1 integrin pathway plays an important role in the interaction of atelocollagen gel with human articular cartilage-derived chondrocytes and may be a potential therapeutic target for articular cartilage disorders.

Glabridin inhibits osteoarthritis development by protecting chondrocytes against oxidative stress, apoptosis and promoting mTOR mediated autophagy

Osteoarthritis (OA) is a common chronic degenerative disease that affects the elderly. Thus far, no pharmacological therapy approved by regulators has shown a convincing effect on OA. Glabridin, a small molecule, is a well-known and powerful natural antioxidant, which has a strong scavenging effect on free radicals. This study attempted to explore the role and underlying mechanisms of Glabridin on OA both in vitro and in vivo. In the in vitro study, Glabridin was found to increase the expression levels of extracellular matrix (ECM) related genes, Collagen II, Aggrecan (ACAN), SRY-box 9 (SOX9) and proteoglycan 4 (PRG4). Moreover, Glabridin was observed to significantly reduce the level of oxidative stress in OA chondrocytes while effectively reducing the apoptosis of chondrocytes. Glabridin was also found to significantly increase the autophagy of human OA chondrocytes. During the in vivo study, intraarticular injection of Glabridin was observed to alleviate OA progression and protect chondrocytes against apoptosis following anterior cruciate ligament transection (ACLT) in rats. Furthermore, the mammalian target of rapamycin (mTOR) mediated autophagy was identified as one of the potential mediators of Glabridin activity. Overall, Glabridin protects articular cartilage from damage in rats with OA by protecting chondrocytes against oxidative stress, apoptosis and promoting mTOR mediated autophagy.

Arteriovenous fistula-induced cardiac remodeling shows cardioprotective features in mice

ObjectiveArteriovenous fistulae (AVF) placed for hemodialysis have high flow rates that can stimulate left ventricular (LV) hypertrophy. LV hypertrophy generally portends poor cardiac outcomes, yet clinical studies point to superior cardiac-specific outcomes for patients with AVF when compared with other dialysis modalities. We hypothesize that AVF induce physiologic cardiac hypertrophy with cardioprotective features. MethodsWe treated 9- to 11-week-old C57Bl/6 male and female mice with sham laparotomy or an aortocaval fistula via a 25G needle. Cardiac chamber size and function were assessed with serial echocardiography, and cardiac computed tomography angiography. Hearts were harvested at 5 weeks postoperatively, and the collagen content was assessed with Masson's trichrome. Bulk messenger RNA sequencing was performed from LV of sham and AVF mice at 10 days. Differentially expressed genes were analyzed using Ingenuity Pathway Analysis (Qiagen) to identify affected pathways and predict downstream biological effects. ResultsMice with AVF had similar body weight and wet lung mass, but increased cardiac mass compared with sham-operated mice. AVF increased cardiac output while preserving LV systolic and diastolic function, as well as indices of right heart function; all four cardiac chambers were enlarged, with a slight decrement in the relative LV wall thickness. Histology showed preserved collagen density within each of the four chambers without areas of fibrosis. RNA sequencing captured 19 384 genes, of which 857 were significantly differentially expressed, including transcripts from extracellular matrix-related genes, ion channels, metabolism, and cardiac fetal genes. The top upstream regulatory molecules predicted include activation of angiogenic (Vegf, Akt1), procardiomyocyte survival (Hgf, Foxm1, Erbb2, Lin9, Areg), and inflammation-related (CSF2, Tgfb1, TNF, Ifng, Ccr2, IL6) genes, as well as the inactivation of cardiomyocyte antiproliferative factors (Cdkn1a, FoxO3, α-catenin). The predicted downstream effects include a decrease in heart damage, and increased arrhythmia, angiogenesis, and cardiogenesis. There were no significant sex-dependent differences in the AVF-stimulated cardiac adaptation. ConclusionsAVF stimulate adaptive cardiac hypertrophy in wild-type mice without heart failure or pathologic fibrosis. Transcriptional correlates suggest AVF-induced cardiac remodeling has some cardioprotective, although also arrhythmogenic features. (JVS–Vascular Science 2021;2:110-28.) Clinical RelevanceArteriovenous fistulae (AVF) are commonly used as access for hemodialysis in patients with end-stage renal disease. AVF induce a high-output state that is associated with long-term structural cardiac remodeling, including left ventricle hypertrophy, but this element has uncertain clinical significance. Although left ventricle hypertrophy has traditionally been associated with an increased risk of cardiovascular disease, clinical studies have suggested that cardiac-specific outcomes of patients with end-stage renal disease were better with AVF compared with other dialysis modalities. This study uses a mouse model of AVF to study the structural, functional, and molecular correlates of AVF-induced cardiac remodeling. It finds that AVF causes an adaptive cardiac hypertrophy without functional decline or fibrosis. Transcriptional correlates suggest an electrical remodeling and the upregulation of proangiogenic, procardiogenic, and prosurvival factors, implying that AVF-induced cardiac hypertrophy is potentially cardioprotective, but also arrhythmogenic.

MXD1 is a Potential Prognostic Biomarker and Correlated With Specific Molecular Change and Tumor Microenvironment Feature in Esophageal Squamous Cell Carcinoma.

Background: Identification of novel biomarkers is crucial for the diagnosis and treatment of esophageal squamous cell carcinoma (ESCC). This study aimed to reveal the clinical significance and molecular characteristics of MYC-associated factor X dimerization protein 1 (MXD1) in ESCC. Patients and methods: We collected 3 ESCC cohorts to investigate the effect of MXD1 on clinical outcomes. In addition, we compared and analyzed the possible transcription changes between MXD1-low and MXD1-high ESCC patients using bioinformatics. Moreover, immunohistochemical analysis was conducted to confirm the potential impact of MXD1 on the prognosis and tumor immune microenvironment (TIME). Results: MXD1 messenger RNA (mRNA) expression was significantly lower in tumors than in normal tissues. Low expression of MXD1 in ESCC was associated with a more aggressive tumor stage and worse prognosis at both the mRNA and protein levels. Moreover, MXD1-low ESCC showed upregulation of epithelial–mesenchymal transition and extracellular matrix-related gene sets, and significantly higher NFE2L2 and KIAA1324L mutation frequencies. In contrast, MXD1-high ESCC showed upregulation of tumor differentiation and immune-related gene sets. Furthermore, the CIBERSORT approach showed that high expression of MXD1 was associated with a higher proportion of neutrophils but a lower proportion of M2 macrophages. At the protein level, MXD1 expression was positively correlated with programmed cell death 1 ligand 1 (PDL1) and CD8 expression. In silico analysis predicted that MXD1-high ESCC was more likely to benefit from immunotherapy. Conclusion: This study suggests that MXD1 is a crucial prognostic factor in ESCC patients and is closely associated with specific transcriptional changes and TIME features.

Dysregulation of epigenetic related genes in Diabetic Trigger finger Patients; preliminary analysis of Patient-Derived Samples.

Trigger finger (TF), a painful condition involving a finger flexor tendon, is a common problem with a prevalence of ~2-3% in the general population. However, the TF prevalence is higher among diabetic patients-ranges from 6.7% to 10%. We have analyzed the expression of the extracellular matrix, inflammation, and epigenetic related genes in diabetic and non-diabetes TF. We hypothesized that Diabetes condition induces alter the expression of epigenetic modification genes in diabetic patients and one of the underlying determinants for more prevalence of TF in diabetic patients. Tissues from the fingers of patients with symptomatic trigger fingers were collected. We had three groups: carpal tunnel syndrome (as a control), trigger finger, and diabetic trigger finger. A quantitative real-time polymerase chain reaction was performed. The gene expression of Extracellular matrix (ECM) components [COL-I, COL-II, COL-X, Aggrecan], DNA methyltransferases enzymes (DNMT1, DNMT3), growth factors (TGF-b, IGF), and Histone deacetylase enzymes (HDAC1, HDAC2) were evaluated in all groups. The mRNA expression of COL-I, COL-II, Aggrecan was significantly higher in the pully A1 of diabetic patients (p= 0.0164, p=0.0351, p=0.0399, respectively) as compared to non-diabetic TF patients. Diabetes was associated with a significant increase in the DNMT3 expression compared to non-diabetic TF patients (p=0.0485). HDAC1 and HDAC2 gene expression were up-regulated in diabetic TF than non-diabetic TF. The chronic state of hyperglycemia induces epigenetic modification of gene expressions in trigger fingers. This seems to have a significant impact on the development, recurrence, and progression of trigger finger in diabetic patients.

Mechanisms Underlying the Antifibrotic Potential of Estradiol for Vocal Fold Fibrosis.

Vocal fold fibrosis remains a significant clinical challenge. Estrogens, steroid hormones predominantly responsible for secondary sexual characteristics in women, have been shown to alter wound healing and limit fibrosis, but the effects on vocal fold fibrosis are unknown. We sought to elucidate the expression of estrogen receptors and the effects of estrogens on TGF-β1 signaling in rat vocal fold fibroblasts (VFFs). In vitro. VFFs were isolated from 10-week-old, male Sprague-Dawley rats, and estrogen receptor alpha (ERα) and G protein-coupled receptor 30 (GPR30) were examined via immunostaining and quantitative polymerase chain reaction (qPCR). VFFs were treated with estradiol (E2, 10-7 , 10-8 or 10-9 M) ± transforming growth factor beta 1 (TGF-β1, 10 ng/mL). ICI 182,780 (ICI, 10-7 M) or G36 (10-7 M) were employed as antagonists of ERα or GPR30, respectively. qPCR was employed to determine estrogen receptor-mediated effects of E2 on genes related to fibrosis. ERα and GPR30 were expressed in VFFs at both the protein and the mRNA levels. E2 (10-7 M) did not alter Smad3, Smad7, Acta2 mRNA, or extracellular matrix related genes. However, the combination of E2 (10-8 M) and TGF-β1 significantly increased Smad7 (P=.03) and decreased Col1a1 (P=.04) compared to TGF-β1 alone; this response was negated by the combination of ICI and G36 (P=.009). E2 regulated TGF-β1/Smad signaling via estrogen receptors in VFFs. These findings provide insight into potential mechanisms of estrogens on vocal fold injury with the goal of enhanced therapeutics for vocal fold fibrosis. NA Laryngoscope, 131:2285-2291, 2021.

Expression of Extracellular Matrix-Related Genes and Their Regulatory microRNAs in Problematic Colorectal Polyps.

Simple SummaryDuring bowel cancer screening programs, many diagnostically problematic polyps are removed. The greatest challenge is to distinguish between adenomas with epithelial misplacement and adenomas with early carcinoma, considering the diagnosis affects prognosis and treatment. Our aim was to analyze the expression of extracellular matrix related genes and proteins DCN, EPHA4, FN1, SPARC, SPON2, and SPP1, in 44 biopsies. Differences were observed in most of the analyzed genes and proteins in adenoma with epithelial misplacement in comparison to adenoma with early carcinoma, reflecting inflammatory stromal reaction to traumatisation and misplacement of dysplastic glands in the submucosa in the former, and desmoplastic stromal reaction to true invasion of dysplastic glands in the submucosa in the latter. The observed expression patterns are too complex to be used in diagnostic work, but might contribute to better understanding extracellular matrix changes in colorectal cancerogenesis and help to find new diagnostic markers in the future.Colorectal carcinoma usually evolves gradually, forming a spectrum of lesions, due to accumulation of genetic mutations and epigenetic alterations. Many early lesions are detected since the introduction of screening programs. The greatest challenge is to distinguish between adenomas with epithelial misplacement (AEM) and adenomas with early carcinoma (AEC), considering the diagnosis affects prognosis and treatment. We analyzed the expression of selected extracellular matrix (ECM)-related genes and proteins, and their regulatory microRNAs using RT-qPCR and immunohistochemistry in biopsies from 44 patients. Differences were observed in AEM in comparison to AEC for DCN, EPHA4, FN1, SPON2, and SPP1, reflecting inflammatory stromal reaction to traumatisation and misplacement of dysplastic glands in the submucosa in the former, and desmoplastic stromal reaction to true invasion of dysplastic glands in the submucosa in the latter. Expression of regulatory microRNAs hsa-miR-200c and hsa-miR-146a significantly negatively correlated with the expression of their regulated genes, while significant difference between AEM and AEC was observed only for hsa-miR-29c. The described expression patterns are too complex to be used in diagnostic work, but might contribute to better understanding ECM changes in colorectal carcinoma development, helping to find new markers in the future.

MiR-21 is upregulated, promoting fibrosis and blocking G2/M in irradiated rat cardiac fibroblasts.

BackgroundRadiation exposure of the thorax is associated with a greatly increased risk of cardiac morbidity and mortality even after several decades of advancement in the field. Although many studies have demonstrated the damaging influence of ionizing radiation on cardiac fibroblast (CF) structure and function, myocardial fibrosis, the molecular mechanism behind this damage is not well understood. miR-21, a small microRNA, promotes the activation of CFs, leading to cardiac fibrosis. miR-21 is overexpressed after irradiation; however, the relationship between increased miR-21 and myocardial fibrosis after irradiation is unclear. This study was conducted to investigate gene expression after radiation-induced CF damage and the role of miR-21 in this process in rats.MethodsWe sequenced irradiated rat CFs and performed weighted correlation network analysis (WGCNA) combined with differentially expressed gene (DEG) analysis to observe the effect on the expression profile of CF genes after radiation.ResultsDEG analysis showed that the degree of gene changes increased with the radiation dose. WGCNA revealed three module eigengenes (MEs) associated with 8.5-Gy-radiation—the Yellow, Brown, Blue modules. The three module eigengenes were related to apoptosis, G2/M phase, and cell death and S phase, respectively. By blocking with the cardiac fibrosis miRNA miR-21, we found that miR-21 was associated with G2/M blockade in the cell cycle and was mainly involved in regulating extracellular matrix-related genes, including Grem1, Clu, Gdf15, Ccl7, and Cxcl1. Stem-loop quantitative real-time PCR was performed to verify the expression of these genes. Five genes showed higher expression after 8.5 Gy-radiation in CFs. The target genes of miR-21 predicted online were Gdf15 and Rsad2, which showed much higher expression after treatment with antagomir-miR-21 in 8.5-Gy-irradiated CFs. Thus, miR-21 may play the role of fibrosis and G2/M blockade in regulating Grem1, Clu, Gdf15, Ccl7, Cxcl1, and Rsad2 post-irradiation.

Transcriptional heterogeneity between primary adult grey and white matter astrocytes underlie differences in modulation of in vitro myelination

BackgroundMultiple sclerosis (MS) is an inflammation-mediated demyelinating disease of the central nervous system that eventually results in secondary axonal degeneration due to remyelination failure. Successful remyelination is orchestrated by astrocytes (ASTRs) and requires sequential activation, recruitment, and maturation of oligodendrocyte progenitor cells (OPCs). In both MS and experimental models, remyelination is more robust in grey matter (GM) than white matter (WM), which is likely related to local differences between GM and WM lesions. Here, we investigated whether adult gmASTRs and wmASTRs per se and in response to MS relevant Toll-like receptor (TLR) activation differently modulate myelination.MethodsDifferences in modulation of myelination between adult gmASTRs and wmASTRs were examined using an in vitro myelinating system that relies on a feeding layer of ASTRs. Transcriptional profiling and weighted gene co-expression network analysis were used to analyze differentially expressed genes and gene networks. Potential differential modulation of OPC proliferation and maturation by untreated adult gmASTRs and wmASTRs and in response to TLR3 and TLR4 agonists were assessed.ResultsOur data reveal that adult wmASTRs are less supportive to in vitro myelination than gmASTRs. WmASTRs more abundantly express reactive ASTR genes and genes of a neurotoxic subtype of ASTRs, while gmASTRs have more neuro-reparative transcripts. We identified a gene network module containing cholesterol biosynthesis enzyme genes that positively correlated with gmASTRs, and a network module containing extracellular matrix-related genes that positively correlated with wmASTRs. Adult wmASTRs and gmASTRs responding to TLR3 agonist Poly(I:C) distinctly modulate OPC behavior, while exposure to TLR4 agonist LPS of both gmASTRs and wmASTRs results in a prominent decrease in myelin membrane formation.ConclusionsPrimary adult gmASTRs and wmASTRs are heterogeneous at the transcriptional level, differed in their support of in vitro myelination, and their pre-existing phenotype determined TLR3 agonist responses. These findings point to a role of ASTR heterogeneity in regional differences in remyelination efficiency between GM and WM lesions.

Single-Cell RNA Sequencing Unravels Heterogeneity of the Stromal Niche in Cutaneous Melanoma Heterogeneous Spheroids

Simple SummaryCutaneous malignant melanoma is one of the most dangerous forms of skin cancer affecting humans. Frequently, it is linked to DNA damage due to ultraviolet radiation. Photoageing along with chronological ageing are therefore critically important factors in melanoma biology. The tissue microenvironment is also heavily affected by induced senescence. There is growing evidence that senescent dermal fibroblasts can consequently promote tumour progression. We focused on the analysis of microenvironmental factors represented in melanoma heterogeneous spheroids by either photodamaged or normal dermal fibroblasts. Our effort was primarily focused on the determination of the functional diversity of fibroblasts in heterogeneous spheroids. Therefore, we analysed these 3D models by single-cell RNA sequencing and advanced bioinformatic analysis. We aimed to map the fibroblast diversity resulting from previously acquired damage caused by exposure to extrinsic and intrinsic stimuli. Using this robust methodology, we highlighted molecules that could become important for the control of melanoma cell–cancer-associated fibroblast interaction as an essential part of the tumour microenvironment.Heterogeneous spheroids have recently acquired a prominent position in melanoma research because they incorporate microenvironmental cues relevant for melanoma. In this study, we focused on the analysis of microenvironmental factors introduced in melanoma heterogeneous spheroids by different dermal fibroblasts. We aimed to map the fibroblast diversity resulting from previously acquired damage caused by exposure to extrinsic and intrinsic stimuli. To construct heterogeneous melanoma spheroids, we used normal dermal fibroblasts from the sun-protected skin of a juvenile donor. We compared them to the fibroblasts from the sun-exposed photodamaged skin of an adult donor. Further, we analysed the spheroids by single-cell RNA sequencing. To validate transcriptional data, we also compared the immunohistochemical analysis of heterogeneous spheroids to melanoma biopsies. We have distinguished three functional clusters in primary human fibroblasts from melanoma spheroids. These clusters differed in the expression of (a) extracellular matrix-related genes, (b) pro-inflammatory factors, and (c) TGFβ signalling superfamily. We observed a broader deregulation of gene transcription in previously photodamaged cells. We have confirmed that pro-inflammatory cytokine IL-6 significantly enhances melanoma invasion to the extracellular matrix in our model. This supports the opinion that the aspects of ageing are essential for reliable melanoma 3D modelling in vitro.

Inhibition of eNOS by L-NAME resulting in rat hind limb developmental defects through PFKFB3 mediated angiogenetic pathway

l-arginine/NOS/NO signaling pathway plays a critical role in controlling variety of vascular diseases. However, whether NOS inhibition by L-NAME suppresses late embryonic development is undefined. The aim of this study is to determine whether NOS inhibition by L-NAME is critical for late embryonic rat hind limb development. The pregnant rat at E13.5 administrated L-NAME by consecutive intraperitoneal injection. The embryos been harvested from E16.5 to E 20.5. Hematoxylin and Eosin Staining, Immunofluorescence and Immunohistochemistry performed to determine hind limb Vasculogenesis, HUVEC culture, Adenoviral PFKFB3 infection, Real time PCR and western blot were performed to determine whether l-arginine/NOS/NO pathway controlling late embryonic hind limb development through PFKFB3 mediated angiogenetic pathway. NOS inhibition by L-NAME resulting in late embryonic hind limb developmental defects characterized by severe hemorrhage. The in vivo studies showed that NOS inhibition strongly suppressed hind limb angiogenetic remodeling by impairing differentiation of endothelial cells and smooth muscle cells, and extracellular matrix synthesis. For underlie mechanism, our studies indicated that L-NAME treatment dramatically suppresses PFKFB3 expression in hematopoietic progenitor cells, tubulogenetic endothelial cells and smooth muscle cells. Knockdown of PFKFB3 dramatically inhibits the expression of angiogenetic genes, as well as tubulogenesis and extracellular matrix related genes. Taken together, our data in this study demonstrated that l-arginine-eNOS-NO pathway is important for rat hind limb development during late embryonic stage. This could be both a useful animal model and a promising therapeutic treatment for defects of late embryonic developmental hind limbs.

Smad‑binding decoy reduces extracellular matrix expression in human hypertrophic scar fibroblasts.

The exact mechanisms underlying hypertrophic scarring is yet to be fully understood. However, excessive collagen deposition by fibroblasts has been demonstrated to result in hypertrophic scar formation, and collagen synthesis in dermal fibroblasts is regulated by the transforming growth factor-β1/Smad signaling pathway. In view of this, a Smad-binding decoy was designed and its effects on hypertrophic scar-derived human skin fibroblasts was evaluated. The results of the present study revealed that the Smad decoy attenuates the total amount of collagen, collagen I and Smad2/3 expression in scar fibroblasts. Data from RNA sequencing indicated that the Smad decoy induced more than 4-fold change in 178 genes, primarily associated with to the extracellular matrix, compared with the untreated control. In addition, results from quantitative real-time polymerase chain reaction further confirmed that the Smad decoy significantly attenuated the expression of extracellular matrix-related genes, including COL1A1, COL1A2 and COL3A1. Furthermore, the Smad decoy reduced transforming growth factor-β1-induced collagen deposition in scar fibroblasts. Data generated from the present study provide evidence supporting the use of the Smad decoy as a potential hypertrophic scar treatment.