COL1A1 mRNA Levels Research Articles

Screening and expression validation of key proteins for secondary hair follicle growth in cashmere goats based on iTRAQ quantitative proteomics technology.

The growth of secondary hair follicles (SHFs) in cashmere goats has periodic changes, including telogen, anagen, and catagen, during which proteins play important roles as the executor of life activities. In this study, the skin tissues of cashmere goats at three different growth stages of SHFs were collected for proteome sequencing and validation experiments. Through protein differential expression analysis and time series analysis, FKBP prolyl isomerase 10 (FKBP10) and fibrillin 2 (FBN2) were screened as the key proteins for SHF cycle growth of cashmere goats, and albumin (ALB), collagen type I alpha 1 chain (COL1A1) and elastin (ELN) were predicted to be their interacting proteins. The results of quantitative real-time PCR (qRT-PCR), western blot, and immunohistochemistry experiments showed that the mRNA and protein expression levels of FKBP10, FBN2, COL1A1, ELN and ALB were higher in anagen and lower in telogen. They were all highly expressed in the outer root sheath of SHFs in anagen. FKBP10, FBN2, COL1A1, ELN, and ALB can promote the growth of SHFs in cashmere goats. This study lays the foundation for analyzing the growth cycle regulatory mechanism of SHFs in cashmere goats, and provides new ideas for further improving cashmere yield and quality.

PIEZO1 mediates matrix stiffness-induced tumor progression in kidney renal clear cell carcinoma by activating the Ca2+/Calpain/YAP pathway

ObjectiveThe significance of physical factors in the onset and progression of tumors has been increasingly substantiated by a multitude of studies. The extracellular matrix, a pivotal component of the tumor microenvironment, has been the subject of extensive investigation in connection with the advancement of KIRC (Kidney Renal Clear Cell Carcinoma) in recent years. PIEZO1, a mechanosensitive ion channel, has been recognized as a modulator of diverse physiological processes. Nonetheless, the precise function of PIEZO1 as a transducer of mechanical stimuli in KIRC remains poorly elucidated. MethodsA bioinformatics analysis was conducted using data from The Cancer Genome Atlas (TCGA) and the Clinical Proteomic Tumor Analysis Consortium (CPTAC) to explore the correlation between matrix stiffness indicators, such as COL1A1 and LOX mRNA levels, and KIRC prognosis. Expression patterns of mechanosensitive ion channels, particularly PIEZO1, were examined. Collagen-coated polyacrylamide hydrogel models were utilized to simulate varying stiffness environments and study their effects on KIRC cell behavior in vitro. Functional experiments, including PIEZO1 knockdown and overexpression, were performed to investigate the molecular mechanisms underlying matrix stiffness-induced cellular changes. Interventions in the Ca2+/Calpain/YAP Pathway were conducted to evaluate their effects on cell growth, EMT, and stemness characteristics. ResultsOur findings indicate a significant correlation between matrix stiffness and the prognosis of KIRC patients. It is observed that higher mechanical stiffness can facilitate the growth and metastasis of KIRC cells. Notably, we have also observed that the deficiency of PIEZO1 hinders the proliferation, EMT, and stemness characteristics of KIRC cells induced by a stiff matrix. Our study suggests that PIEZO1 plays a crucial role in mediating KIRC growth and metastasis through the activation of the Ca2+/Calpain/YAP Pathway. ConclusionThis study elucidates a novel mechanism through which the activation of PIEZO1 leads to calcium influx, subsequent calpain activation, and YAP nuclear translocation, thereby contributing to the progression of KIRC driven by matrix stiffness.

Pregnane X receptor inhibits the transdifferentiation of hepatic stellate cells by down-regulating periostin expression.

Pregnane X receptor (PXR) is a xenobiotic-sensing nuclear receptor that plays a key role in drug metabolism. Recently, PXR was found to attenuate the development of liver cancer by suppressing epithelial-mesenchymal transition (EMT) in liver cancer cells in a mouse model of two-stage chemical carcinogenesis. To elucidate the role of PXR in the EMT of liver cancer cells, we focused on its role in hepatic stellate cells (HSCs), which are components of the tumor microenvironment in hepatocellular carcinoma (HCC). Human HSC-derived LX-2 cells stably expressed destabilization domain (DD)-fused human PXR (hPXR-LX2 cells). Human HCC-derived HepG2 cells were transfected with the EMT marker VIM promoter-regulated reporter plasmid and co-cultured with hPXR-LX2 cells or treated with hPXR-LX2-derived conditioned medium (CM). Co-culture or CM treatment increased reporter activity in HepG2 cells. This induction was attenuated upon PXR activation in hPXR-LX2 cells by treatment with the DD-stabilizing chemical Shield-1 and the human PXR ligand rifampicin. PXR activation in hPXR-LX2 cells exhibited inhibition of TGF-β1-induced transdifferentiation, supported by observations of morphological changes and protein or mRNA levels of the transdifferentiation markers COL1A1 and FN1. PXR activation in hPXR-LX2 cells also attenuated the mRNA levels of the key transdifferentiation factor, POSTN. Treatment of hPXR-LX2 cells with recombinant POSTN restored the PXR-mediated suppression of transdifferentiation. Reporter assays with the POSTN promoter showed that PXR inhibited the NF-κB-mediated transcription of POSTN. Consequently, PXR activation in HSCs is expected to inhibit transdifferentiation by down-regulating POSTN expression, thereby suppressing EMT of liver cancer cells.

PPARβ/δ attenuates hepatic fibrosis by reducing SMAD3 phosphorylation and p300 levels via AMPK in hepatic stellate cells

The role of peroxisome proliferator-activated receptor (PPAR)β/δ in hepatic fibrosis remains a subject of debate. Here, we examined the effects of a PPARβ/δ agonist on the pathogenesis of liver fibrosis and the activation of hepatic stellate cells (HSCs), the main effector cells in liver fibrosis, in response to the pro-fibrotic stimulus transforming growth factor-β (TGF-β). The PPARβ/δ agonist GW501516 completely prevented glucose intolerance and peripheral insulin resistance, blocked the accumulation of collagen in the liver, and attenuated the expression of inflammatory and fibrogenic genes in mice fed a choline-deficient high-fat diet (CD-HFD). The antifibrogenic effect of GW501516 observed in the livers CD-HFD-fed mice could occur through an action on HSCs since primary HSCs isolated from Ppard-/- mice showed increased mRNA levels of the profibrotic gene Col1a1. Moreover, PPARβ/δ activation abrogated TGF-β1-mediated cell migration (an indicator of cell activation) in LX-2 cells (immortalized activated human HSCs). Likewise, GW501516 attenuated the phosphorylation of the main downstream intracellular protein target of TGF-β1, suppressor of mothers against decapentaplegic (SMAD)3, as well as the levels of the SMAD3 co-activator p300 via the activation of AMP-activated protein kinase (AMPK) and the subsequent inhibition of extracellular signal-regulated kinase-1/2 (ERK1/2) in LX-2 cells. Overall, these findings uncover a new mechanism by which the activation of AMPK by a PPARβ/δ agonist reduces TGF-β1-mediated activation of HSCs and fibrosis via the reduction of both SMAD3 phosphorylation and p300 levels.

L-Carnitine relieves cachexia-related skeletal muscle fibrosis by inducing deltex E3 ubiquitin ligase 3L to negatively regulate the Runx2/COL1A1 axis.

Cancer cachexia-induced skeletal muscle fibrosis (SMF) impairs muscle regeneration, alters the muscle structure and function, reduces the efficacy of anticancer drugs, diminishes the patient's quality of life and shortens overall survival. RUNX family transcription factor 2 (Runx2), a transcription factor, and collagen type I alpha 1 chain (COL1A1), the principal constituent of SMF, have been linked previously, with Runx2 shown to directly modulate COL1A1 mRNA levels. l-Carnitine, a marker of cancer cachexia, can alleviate fibrosis in liver and kidney models; however, its role in cancer cachexia-associated fibrosis and the involvement of Runx2 in the process remain unexplored. Female C57 mice (48weeks old) were inoculated subcutaneously with MC38 cells to establish a cancer cachexia model. A 5mg/kg dose of l-carnitine or an equivalent volume of water was administered for 14days via oral gavage, followed by assessments of muscle function (grip strength) and fibrosis. To elucidate the interplay between the deltex E3 ubiquitin ligase 3L(DTX3L)/Runx2/COL1A1 axis and fibrosis in transforming growth factor beta 1-stimulated NIH/3T3 cells, a suite of molecular techniques, including quantitative real-time PCR, western blot analysis, co-immunoprecipitation, molecular docking, immunofluorescence and Duolink assays, were used. The relevance of the DTX3L/Runx2/COL1A1 axis in the gastrocnemius was also explored in the in vivo model. l-Carnitine supplementation reduced cancer cachexia-induced declines in grip strength (>88.2%, P<0.05) and the collagen fibre area within the gastrocnemius (>57.9%, P<0.05). At the 5mg/kg dose, l-carnitine also suppressed COL1A1 and alpha-smooth muscle actin (α-SMA) protein expression, which are markers of SMF and myofibroblasts. Analyses of the TRRUST database indicated that Runx2 regulates both COL1A1 and COL1A2. In vitro, l-carnitine diminished Runx2 protein levels and promoted its ubiquitination. Overexpression of Runx2 abolished the effects of l-carnitine on COL1A1 and α-SMA. Co-immunoprecipitation, molecular docking, immunofluorescence and Duolink assays confirmed an interaction between DTX3L and Runx2, with l-carnitine enhancing this interaction to promote Runx2 ubiquitination. l-Carnitine supplementation restored DTX3L levels to those observed under non-cachectic conditions, both in vitro and in vivo. Knockdown of DTX3L abolished the effects of l-carnitine on Runx2, COL1A1 and α-SMA in vitro. The expression of DTX3L was negatively correlated with the levels of Runx2 and COL1A1 in untreated NIH/3T3 cells. This study revealed a previously unrecognized link between Runx2 and DTX3L in SMF and demonstrated that l-carnitine exerted a significant therapeutic impact on cancer cachexia-associated SMF, potentially through the upregulation of DTX3L.

Insulin-like growth factor-1 infusion in preterm piglets does not affect growth parameters of skeletal muscle or tendon tissue.

Prematurity has physical consequences, such as lower birth weight, decreased muscle mass and increased risk of adult-onset metabolic disease. Insulin-like growth factor 1 (IGF-1) has therapeutic potential to improve the growth and quality of muscle and tendon in premature births, and thus attenuate some of these sequalae. We investigated the effect of IGF-1 on extensor carpi radialis muscle and biceps brachii tendon of preterm piglets. The preterm group consisted of 19-day-old preterm (10 days early) piglets, treated with either IGF-1 or vehicle. Term controls consisted of groups of 9-day-old piglets (D9) and 19-day-old piglets (D19). Muscle samples were analysed by immunofluorescence to determine the cross-sectional area (CSA) of muscle fibres, fibre type composition, satellite cell content and central nuclei-containing fibres in the muscle. Tendon samples were analysed for CSA, collagen content and maturation, and vascularization. Gene expression of the tendon was measured by RT-qPCR. Across all endpoints, we found no significant effect of IGF-1 treatment on preterm piglets. Preterm piglets had smaller muscle fibre CSA compared to D9 and D19 control group. Satellite cell content was similar across all groups. For tendon, we found an effect of age on tendon CSA, and mRNA levels of COL1A1, tenomodulin and scleraxis. Immunoreactivity for elastin and CD31, and several markers of tendon maturation, were increased in D9 compared to the preterm piglets. Collagen content was similar across groups. IGF-1 treatment of preterm-born piglets does not influence the growth and maturation of skeletal muscle and tendon.

Semaglutide May Ameliorate Fibrosis and Inhibit Epithelial-Mesenchymal Transition in Intrauterine Adhesion Models.

The purpose of this study was to explore the effect of Semaglutide on intrauterine adhesions and discover new drugs for such adhesions. In this study, the cell model was simulated by TGF-β1-induced human endometrial epithelial cells, and the animal model was established through mechanical curettage and inflammatory stimulation. After co-culturing with TGF-β1 with or without different concentrations of Semaglutide for 48 h, cells were collected for RT-qPCR and Western blotting analyses. Three doses were subcutaneously injected into experimental mice once a day for two weeks, while the control group received sterile ddH2O. The serum and uterine tissues of the mice were collected. HE and Masson staining were used for the uterine histomorphological and pathological analyses. RT-qPCR and Western blotting were used for mRNA and protein expression analyses. Serum indicators were detected using ELISA kits. The results showed that Semaglutide significantly reduced the mRNA levels of fibrosis indicators ACTA2, COL1A1, and FN and inflammatory indicators TNF-α, IL-6, and NF-κB in the two models. Semaglutide improved endometrium morphology, increased the number of endometrial glands, and reduced collagen deposition in IUA mice. The results also showed that Semaglutide could inhibit vimentin, E-Cadherin, and N-Cadherin in the two models. In summary, Semaglutide can ameliorate fibrosis and inflammation of intrauterine adhesions as well as inhibit epithelial-mesenchymal transition in IUA models.

Heat shock protein 72 supports extracellular matrix production in metastatic mammary tumors.

This study identified tumorigenic processes most dependent on murine heat shock protein 72 (HSP72) in the mouse mammary tumor virus-PyMT mammary tumor model, which give rise to spontaneous mammary tumors that exhibit HSP72-dependent metastasis to the lung. RNA-seq expression profiling of Hspa1a/Hspa1b (Hsp72) WT and Hsp72-/- primary mammary tumors discovered significantly lower expression of genes encoding components of the extracellular matrix (ECM) in Hsp72 knockout mammary tumors compared to WT controls. In vitro studies found that genetic or chemical inhibition of HSP72 activity in cultured collagen-expressing human or murine cells also reduces mRNA and protein levels of COL1A1 and several other ECM-encoding genes. In search of a possible mechanistic basis for this relationship, we found HSP72 to support the activation of the tumor growth factor-β-suppressor of mothers against decapentaplegic-3 signaling pathway and evidence of suppressor of mothers against decapentaplegic-3 and HSP72 coprecipitation, suggesting potential complex formation. Human COL1A1 mRNA expression was found to have prognostic value for HER2+breast tumors over other breast cancer subtypes, suggesting a possible human disease context where targeting HSP72 may have a therapeutic rationale. Analysis of human HER2+breast tumor gene expression data using a gene set comprising ECM-related gene and protein folding-related geneas an input to the statistical learning algorithm, Galgo, found a subset of these genes that can collectively stratify patients by relapse-free survival, further suggesting a potential interplay between the ECM and protein-folding genes may contribute to tumor progression.

Antisense oligonucleotide-mediated terminal intron retention of endoglin: A potential strategy to inhibit renal interstitial fibrosis

TGF-β is considered an important cytokine in the development of interstitial fibrosis in chronic kidney disease. The TGF-β co-receptor endoglin (ENG) tends to be upregulated in kidney fibrosis. ENG has two membrane bound isoforms generated via alternative splicing. Long-ENG was shown to enhance the extent of renal fibrosis in an unilateral ureteral obstruction mouse model, while short-ENG inhibited renal fibrosis. Here we aimed to achieve terminal intron retention of endoglin using antisense-oligo nucleotides (ASOs), thereby shifting the ratio towards short-ENG to inhibit the TGF-β1-mediated pro-fibrotic response. We isolated mRNA from kidney biopsies of patients with chronic allograft disease (CAD) (n = 12) and measured total ENG and short-ENG mRNA levels. ENG mRNA was upregulated 2.3 fold (p < 0.05) in kidneys of CAD patients compared to controls, while the percentage short-ENG of the total ENG mRNA was significantly lower (1.8 fold; p < 0.05). Transfection of ASOs that target splicing regulatory sites of ENG into TK173 fibroblasts led to higher levels of short-ENG (2 fold; p < 0.05). In addition, we stimulated these cells with TGF-β1 and measured a decrease in upregulation of ACTA2, COL1A1 and FN1 mRNA levels, and protein expression of αSMA, collagen type I, and fibronectin. These results show a potential for ENG ASOs as a therapy to reduce interstitial fibrosis in CKD.

A novel network pharmacology strategy to decode mechanism of Wuling Powder in treating liver cirrhosis

BackgroundLiver cirrhosis is a chronic liver disease with hepatocyte necrosis and lesion. As one of the TCM formulas Wuling Powder (WLP) is widely used in the treatment of liver cirrhosis. However, it’s key functional components and action mechanism still remain unclear. We attempted to explore the Key Group of Effective Components (KGEC) of WLP in the treatment of Liver cirrhosis through integrative pharmacology combined with experiments.MethodsThe components and potential target genes of WLP were extracted from published databases. A novel node importance calculation model considering both node control force and node bridging force is designed to construct the Function Response Space (FRS) and obtain key effector proteins. The genetic knapsack algorithm was employed to select KGEC. The effectiveness and reliability of KGEC were evaluated at the functional level by using gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Finally, the effectiveness and potential mechanism of KGEC were confirmed by CCK-8, qPCR and Western blot.Results940 effective proteins were obtained in FRS. KEGG pathways and GO terms enrichments analysis suggested that effective proteins well reflect liver cirrhosis characteristics at the functional level. 29 components of WLP were defined as KGEC, which covered 100% of the targets of the effective proteins. Additionally, the pathways enriched for the KGEC targets accounted for 83.33% of the shared genes between the targets and the pathogenic genes enrichment pathways. Three components scopoletin, caryophyllene oxide, and hydroxyzinamic acid from KGEC were selected for in vivo verification. The qPCR results demonstrated that all three components significantly reduced the mRNA levels of COL1A1 in TGF-β1-induced liver cirrhosis model. Furthermore, the Western blot assay indicated that these components acted synergistically to target the NF-κB, AMPK/p38, cAMP, and PI3K/AKT pathways, thus inhibiting the progression of liver cirrhosis.ConclusionIn summary, we have developed a new model that reveals the key components and potential mechanisms of WLP for the treatment of liver cirrhosis. This model provides a reference for the secondary development of WLP and offers a methodological strategy for studying TCM formulas.

The XPO1 inhibitor selinexor ameliorates bleomycin-induced pulmonary fibrosis in mice via GBP5/NLRP3 inflammasome signaling

Pulmonary fibrosis is an irreversible and progressive lung disease with limited treatments available. Selinexor (Sel), an orally available, small-molecule, selective inhibitor of XPO1, exhibits notable antitumor, anti-inflammatory and antiviral activities. However, its potential role in treating pulmonary fibrosis is unknown. C57BL/6J mice were used to establish a pulmonary fibrosis model by intratracheal administration of bleomycin (BLM). Subsequently, Sel was administered intraperitoneally. Our data demonstrated that Sel administration ameliorated BLM-induced pulmonary fibrosis by increasing mouse body weights; reducing H&E staining, Masson staining scores, and shadows in mouse lung computed tomography (CT) images, decreasing the total cell and neutrophil counts in the lung and bronchoalveolar lavage fluid (BALF); and decreasing the levels of TGF-β1. We next confirmed that Sel reduced the deposition of extracellular matrix (ECM) components in the lungs of BLM-induced pulmonary fibrosis mice. We showed that collagen I, alpha-smooth muscle actin (α-SMA), and hydroxyproline levels and the mRNA levels of Col1a1, Eln, Fn1, Ctgf, and Fgf2 were reduced. Mechanistically, tandem mass tags (TMT)- based quantitative proteomics analysis revealed a significant increase in GBP5 in the lungs of BLM mice but a decrease in that of BLM + Sel mice; this phenomenon was confirmed by western blotting and RT-qPCR. NLRP3 inflammasome signaling was significantly enriched in both the BLM group and BLM + Sel group based on GO and KEGG analyses of differentially expressed proteins between the groups. Furthermore, Sel reduced the expression of NLRP3, cleaved caspase 1, and ASC in vivo and in vitro, and decreased the levels of IL-1β, IL-18, and IFN-r in lung tissue and BALF. SiRNA-GBP5 inhibited NLRP3 signaling in vitro, and overexpression of GBP5 inhibited the protective effect of Sel against BLM-induced cellular injury. Taken together, our findings indicate that Sel ameliorates BLM-induced pulmonary fibrosis by targeting GBP5 via NLRP3 inflammasome signaling. Thus, the XPO1 inhibitor – Sel might be a potential therapeutic agent for pulmonary fibrosis.

Stromal Type I Collagen in Breast Cancer: Correlation to Prognostic Biomarkers and Prediction of Chemotherapy Response

IntroductionFibrillar collagens accumulate in the breast cancer stroma and appear as poorly defined spiculated masses in mammography imaging. The prognostic value of tissue type I collagen remains elusive in treatment-naïve and chemotherapy-treated breast cancer patients. Here, type I collagen mRNA and protein expression were analysed in 2 large independent breast cancer cohorts. Levels were related to clinicopathological parameters, prognostic biomarkers, and outcome. MethodCOL1A1 mRNA expression was analysed in 2509 patients with breast cancer obtained from the cBioPortal database. Type I collagen protein expression was studied by immunohistochemistry in 1395 women diagnosed with early invasive breast cancer. ResultsLow COL1A1 mRNA and protein levels correlated with poor prognosis features, such as hormone receptor negativity, high histological grade, triple-negative subtype, node positivity, and tumour size. In unadjusted analysis, high stromal type I collagen protein expression was associated with improved overall survival (OS) (HR = 0.78, 95% CI = 0.61-0.99, p = .043) and trended towards improved breast cancer–specific survival (BCSS) (HR = 0.65, 95% CI = 0.42-1.01, P = 0.053), although these findings were lost after adjustment for other clinical variables. In unadjusted analysis, high expression of type I collagen was associated with better OS (HR = 0.70, 95% CI = 0.55-0.90, P = .006) and BCSS (HR = 0.55, 95% CI = 0.34-0.88, P = .014) among patients not receiving chemotherapy. Strikingly, the opposite was observed among patients receiving chemotherapy. There, high expression of type I collagen was instead associated with worse OS (HR = 1.83, 95% CI = 0.65-5.14, P = .25) and BCSS (HR = 1.72, 95% CI = 0.54-5.50, P = .357). ConclusionLow stromal type I collagen mRNA and protein expression are associated with unfavourable tumour characteristics in breast cancer. Stromal type I collagen might predict chemotherapy response.

Pharmacological Stimulation of Soluble Guanylate Cyclase Counteracts the Profibrotic Activation of Human Conjunctival Fibroblasts.

Conjunctival fibrosis is a serious clinical concern implicated in a wide spectrum of eye diseases, including outcomes of surgery for pterygium and glaucoma. It is mainly driven by chronic inflammation that stimulates conjunctival fibroblasts to differentiate into myofibroblasts over time, leading to abnormal wound healing and scar formation. Soluble guanylate cyclase (sGC) stimulation was found to suppress transforming growth factor β (TGFβ)-induced myofibroblastic differentiation in various stromal cells such as skin and pulmonary fibroblasts, as well as corneal keratocytes. Here, we evaluated the in vitro effects of stimulation of the sGC enzyme with the cell-permeable pyrazolopyridinylpyrimidine compound BAY 41-2272 in modulating the TGFβ1-mediated profibrotic activation of human conjunctival fibroblasts. Cells were pretreated with the sGC stimulator before challenging with recombinant human TGFβ1, and subsequently assayed for viability, proliferation, migration, invasiveness, myofibroblast marker expression, and contractile properties. Stimulation of sGC significantly counteracted TGFβ1-induced cell proliferation, migration, invasiveness, and acquisition of a myofibroblast-like phenotype, as shown by a significant downregulation of FAP, ACTA2, COL1A1, COL1A2, FN1, MMP2, TIMP1, and TIMP2 mRNA levels, as well as by a significant reduction in α-smooth muscle actin, N-cadherin, COL1A1, and FN-EDA protein expression. In addition, pretreatment with the sGC stimulator was capable of significantly dampening TGFβ1-induced acquisition of a contractile phenotype by conjunctival fibroblasts, as well as phosphorylation of Smad3 and release of the proinflammatory cytokines IL-1β and IL-6. Taken together, our findings are the first to demonstrate the effectiveness of pharmacological sGC stimulation in counteracting conjunctival fibroblast-to-myofibroblast transition, thus providing a promising scientific background to further explore the feasibility of sGC stimulators as potential new adjuvant therapeutic compounds to treat conjunctival fibrotic conditions.

High fat diet has a protective sex-dependent effect on aortic aneurysm severity in a Marfan syndrome mouse model.

Marfan syndrome (MFS) is a genetic disorder caused by mutations in fibrillin-1 and is characterized by thoracic aortic aneurysms and other complications. Previous studies revealed sexual dimorphisms in aortic aneurysm formation in patients with MFS. The present study aimed to investigate the combined role of a high-fat diet (HFD) and biological sex in aortic disease using the mgR/mgR MFS mouse model. Male and female mgR/mgR mice as well as wild-type (WT) littermate mice were fed a control diet (CD, 10% fat) or HFD (60% fat) from 4 to 12 weeks of age. Key aortic disease parameters analyzed included the diameter of the aortic wall; elastic fiber fragmentation; proteoglycan content; mRNA levels of Mmp12, Col1a1, Col3a1, and Fbn1; and fibrillin-1 deposition in the aortic wall. HFD-fed female mgR/mgR mice had significantly reduced aortic diameters (35%), elastic fiber fragmentation (56%), pathologically enhanced proteoglycans (45%), and expression of Mmp12 (64%), Col1a1 (41%), and Col3a1 (43%) compared to male mgR/mgR mice on HFD. Fibrillin-1 deposition and Fbn1 mRNA levels were unaffected. The data reveal a protective effect of HFD in female mice. In contrast, CD did not exert any protective effects. This study demonstrates a specific sexual dimorphism in MFS mice, with HFD exerting an explicit protective effect on aortic disease severity in female mice. These preclinical data may be useful for developing nutritional recommendations for individuals with MFS in the longer term.

Adipose tissue depot specific expression and regulation of fibrosis-related genes and proteins in experimental obesity.

Transforming growth factor beta (Tgfb) is a well-studied pro-fibrotic cytokine, which upregulates cellular communication network factor 2 (Ccn2), collagen, and actin alpha 2, smooth muscle (Acta2) expression. Obesity induces adipose tissue fibrosis, which contributes to metabolic diseases. This work aimed to analyze the expression of Tgfb, Ccn2, collagen1a1 (Col1a1), Acta2 and BMP and activin membrane-bound inhibitor (Bambi), which is a negative regulator of Tgfb signaling, in different adipose tissue depots of mice fed a standard chow, mice fed a high fat diet (HFD) and ob/ob mice. Principally, these genes were low expressed in brown adipose tissues and this difference was less evident for the ob/ob mice. Ccn2 and Bambi protein as well as mRNA expression, and collagen1a1 mRNA were not induced in the adipose tissues upon HFD feeding whereas Tgfb and Acta2 mRNA increased in the white fat depots. Immunoblot analysis showed that Acta2 protein was higher in subcutaneous and perirenal fat of these mice. In the ob/ob mice, Ccn2 mRNA and Ccn2 protein were upregulated in the fat depots. Here, Tgfb, Acta2 and Col1a1 mRNA levels and serum Tgfb protein were increased. Acta2 protein was, however, not higher in subcutaneous and perirenal fat of these mice. Col6a1 mRNA was shown before to be higher in obese fat tissues. Current analysis proved the Col6a1 protein was induced in subcutaneous fat of HFD fed mice. Notably, Col6a1 was reduced in perirenal fat of ob/ob mice in comparison to the respective controls. 3T3-L1 cells express Ccn2 and Bambi protein, whose levels were not changed by fatty acids, leptin, lipopolysaccharide, tumor necrosis factor and interleukin-6. All of these factors led to higher Tgfb in 3T3-L1 adipocyte media but did not increase its mRNA levels. Free fatty acids induced necrosis whereas apoptosis did not occur in any of the in vitro incubations excluding cell death as a main reason for higher Tgfb in cell media. In summary, Tgfb mRNA is consistently induced in white fat tissues in obesity but this is not paralleled by a clear increase of its target genes. Moreover, discrepancies between mRNA and protein expression of Acta2 were observed. Adipocytes seemingly do not contribute to higher Tgfb mRNA levels in obesity. These cells release more Tgfb proteinwhen challenged with obesity-related metabolites connecting metabolic dysfunction and fibrosis.

Dangui Huoxue Preparation (DHP) Ameliorates Skin Fibrosis, Inflammation, and Vasculopathy in the Bleomycin-Induced Murine Model of Systemic Sclerosis.

Systemic sclerosis (SSc) is an immune-mediated rheumatic disease that is characterized by fibrosis of the skin and internal organs and vasculopathy with poor prognosis. Dangui Huoxue Preparation (DHP) is a clinically effective traditional Chinese herbal formula for the treatment of SSc in the hospital. This study aims to investigate the therapeutic effects and underlying molecular mechanisms of DHP in the treatment of SSc. SSc mice models are induced by bleomycin (BLM). Tissues of DHP group, normal control group, and positive control drug Sanqi Tongshu Capsule (STC) group are collected for inflammation, fibrosis, and vasculopathy. Also, the human dermal fibroblasts (HDF) stimulated with TGF-β1 are analyzed for in vitro study. The expression levels of MCP-1, IFN-γ, IL-1β, IL-10, Fizz1, iNOS, and IL12p40, and the mRNA levels of Col1a1, Col1a2, Col3a1, and Col5a1 are significantly decreased in all DHP groups and STC group compare with those in the BLM group. The main drug of DHP inhibits the proliferation and migration of HDF, reduces Ctgf, Itgb3, Itgb5 expression, and also inhibits the Smad3 pathway. In conclusion, DHP can ameliorate SSc skin inflammation, fibrosis, and vasculopathy, possibly suppressing the TGF-β1/Smad3 signaling pathway through extracellular and intracellular mechanisms.

Effects of Brimonidine, Omidenepag Isopropyl, and Ripasudil Ophthalmic Solutions to Protect against H2O2-Induced Oxidative Stress in Human Trabecular Meshwork Cells

Purpose We investigated whether hydrogen peroxide (H2O2)-induced oxidative stress causes human trabecular meshwork (HTM) cell dysfunction observed in open angle glaucoma (OAG) in vitro, and the effects of topical glaucoma medications on oxidative stress in HTM cells. Methods We used commercially available ophthalmic solutions of brimonidine, omidenepag isopropyl, and ripasudil in the study. HTM cells were exposed to H2O2 for 1 h, with or without glaucoma medications. We assessed cell viability and senescence via WST-1 and senescence-associated-β-galactosidase (SA-β-Gal) activity assays. After exposure to H2O2 and glaucoma medications, we evaluated changes in markers of fibrosis and stress by using real-time quantitative polymerase chain reaction (qPCR) to measure the mRNA levels of collagen type I alpha 1 chain (COL1A1), fibronectin, alpha-smooth muscle actin (α-SMA), matrix metalloproteinase-2 (MMP-2), endoplasmic reticulum stress markers of C/EBP homologous protein (CHOP), 78-kDa glucose-regulated protein (GRP78), and splicing X-box binding protein-1 (sXBP-1). Results HTM cell viability decreased and SA-β-Gal activity increased significantly after exposure to H2O2. Treatment with three ophthalmic solutions attenuated these changes. Real-time qPCR revealed that H2O2 upregulated the mRNA levels of COL1A1, fibronectin, α-SMA, CHOP, GRP78, and sXBP-1, whereas it downregulated MMP-2 mRNA expression significantly. Brimonidine suppressed the upregulation of stress markers CHOP and GRP78. Additionally, omidenepag isopropyl and ripasudil decreased the upregulation of COL1A1 and sXBP-1. Furthermore, ripasudil significantly suppressed fibrotic markers fibronectin and α-SMA, compared with the other two medications. Conclusion In vitro, H2O2 treatment of HTM cells induced characteristic changes of OAG, such as fibrosis changes and the upregulation of stress markers. These glaucomatous changes were attenuated by additional treatments with brimonidine, omidenepag isopropyl, and ripasudil ophthalmic solutions.

Epigenetic regulation of collagen in the mare's placenta: putative relationship with maternal age

Placental plasticity influences fetal development, pregnancy success, and foal growth. Older fertile mares deliver heavier foals and placentas, with increased collagen deposition. In humans, epigenetics regulates placental development and function. Epigenetics, through DNA methylation, also modulates endometrial fibrosis. As such, we hypothesized that as mares get older, collagen deposition in the placenta might be epigenetically modulated. This study assessed if transcription of collagen genes in the equine placenta was epigenetically modulated through DNA methyltransferases (DNMTs), and affected by aging. Immediately after foaling, samples from different chorioallantoic areas (gravid horn, non-gravid horn, and body) were collected from young (n=10; 4-6 years) and older mares (n=10; 12-18 years). Gene transcription of collagen type 1 (COL1A1), collagen type 3 (COL3A1), and DNA methyltransferases (DNMT1, DNMT3A and DNMT3B) was determined (qRT-PCR). Unpaired T-test and Mann-Whitney test were used with statistical significance set a p <0.05. In older mares, COL1A1 mRNA levels were higher in the gravid than in the non-gravid horn (p<0.05), and also upregulated in comparison to young mares’ placentas (p<0.05). In young mares, these transcripts were increased in the placenta body in comparison to the gravid (p<0.05) and non-gravid horns (p<0.01). In older mares, COL3A1 transcriptionwas higher in gravid and non-gravid horns, with respect to the same placenta regions in young mares (p<0.05). In older mares, DNMT1 mRNA expression in the body was lower than in younger mares (p<0.05). For all placentas, DNMT1 transcripts in the non-gravid horn were lowest, compared to the gravid horn (p<0.01) and body (p<0.001). In young mares, DNMT1 mRNA expression in the body was also higher than in the gravid horn (p<0.05). With mares aging, there was a downregulation of DNMT3A transcripts in gravid and non-gravid placenta horns, compared to young mares’ placentas (p< 0.05). In older mares, its levels were lowest in the gravid horn, compared to the non-gravid horn (p<0.05) and body (p<0.01). Similarly, in older mares’ placentas, DNMT3B transcripts were downregulated in the gravid horn (p<0.05) and placenta body (p<0.001), compared to young mares. DNMT3B mRNA levels were lowest in the non-gravid horn, both in young mares, in comparison to placenta body (p<0.01) and in older mares, with respect to the gravid horn and body (p<0.01). Considering mares aging, hypomethylation indicated by decreased DNMTs transcripts, in parallel with upregulation of collagen gene expression in gravid and non-gravid horns, may suggest epigenetic involvement on placental plasticity. However, further research is needed to unravel the intricacies of epigenetic modulation of collagen in equine placentas.

Decitabine effect on COL1, COL3, MMP2 and MMP9 in TGF-β1 treated mare endometrial fibroblasts

Endometrial chronic degenerative fibrosis in mares, known as endometrosis is detrimental to fertility. Endometrium type I (COL1) and III (COL3) collagen accumulation and periglandular fibrosis characterize endometrosis. Transforming growth factor β1 (TGFβ1), is a main pro-fibrotic signal for myofibroblast differentiation, promoting collagen synthesis by epigenetic mechanisms in humans. Collagen turnover is controlled by metalloproteinase-2 (MMP-2), MMP-9 and their inhibitors. Because epigenetic alterations may change fibroblast phenotype and control fibroproliferative diseases, we hypothesized that endometrial fibroblasts might be under epigenetic regulation and modulate endometrosis. Epigenetic changes can be reversed and are promising for therapy. Nevertheless, this has not been demonstrated in mares. DNA methylation, a stable epigenetic marker, can be assessed through DNA methyltransferases (DNMTs) action. This study aimed to evaluate if transcription of some genes involved in endometrosis (COL1A1, COL3A1, MMP2 and MMP9) was altered with endometrial fibrosis, and if they were epigenetically modulated, through DNA methylation. Therefore, in vitro epigenetic modulation of equine endometrial fibroblasts treated with TGF-β1 was assessed, and the epigenetic inhibitor, 5-aza-2′-deoxycytidine (5-aza-dc or decitabine), was used to impair fibrogenesis. Fibroblasts from mare endometria (n=5) were treated with TGF-β1 (10 ng/mL) for 48h, followed by decitabine (1 µM) for 48h. As controls, fibroblasts were incubated in culture medium, or with decitabine or TGF-β1, for 96h. TGF-β1 and decitabine effects on DNA methyltransferases(DNMT1, DNMT3A and DNMT3B), COL1A1, COL3A1, MMP2 and MMP9 mRNA levels (qPCR) were evaluated in fibroblasts, and collagen (EIA) and metalloproteinases MMP-2 and -9 (zymography) in conditioned medium. TGF-β1 increased DNMT3A mRNA levels (P<0.05), COL1 and COL3 mRNA (P<0.01 and P<0.001, respectively) and protein (P<0.05). It also decreased MMP2 mRNA levels and enzymatic activity (P<0.0001 and P<0.05, respectively). Decitabine diminished DNMT3A mRNA levels (P<0.001), COL1 (P<0.001) and COL3 (P<0.01) mRNA and protein (P<0.01 and P<0.05, respectively). It increased MMP2 (P<0.01) and MMP9 mRNA (P<0.001), and enzymatic activity (P<0.05 and P<0.01, respectively), in TGF-β1 challenged fibroblasts. Data show an epigenetic involvement in the pathogenesis of endometrosis, as depicted by downregulation of TGF-β1 profibrotic effect in endometrial fibroblasts treated with decitabine. Decitabine influences MMPs reflected by increasing MMP2 and 9 mRNA and enzymatic activity in TGF-β1 treated endometrial fibroblasts. Furthermore, the hypermethylation (indicated by increased DNMT3A mRNA levels) that occurred with TGF-β1 treatment, seems to inhibit the expression and activity of MMP2. Data are in agreement with previous results and suggest that endometrosis is epigenetically modulated by anti-fibrotic genes (MMP2 and MMP9) inhibition, rather than fibrotic genes activation. Further studies will allow to better understand endometrosis epigenetics modulation. UIDB/00276/2020; LA/P/0059/2020; 2022.09161.PTDC

Antiphotoaging Effect of AGEs Blocker™ in UVB-Irradiated Cells and Skh:HR-1 Hairless Mice.

Chronic exposure to ultraviolet (UV) radiation is a major cause of photoaging. It involves extrinsic aging, wrinkle formation, and skin dehydration, and leads to excessive production of active oxygen that adversely affects the skin. Here, we investigated the antiphotoaging effect of AGEs BlockerTM (AB), which comprises Korean mint aerial part and fig and goji berry fruits. Compared to its individual components, AB was more potent at increasing the expression of collagen and hyaluronic acid and decreasing MMP-1 expression in UVB-irradiated Hs68 fibroblasts and HaCaT keratinocytes. In Skh:HR-1 hairless mice exposed to 60 mJ/cm2 UVB for 12 weeks, oral administration of 20 or 200 mg/kg/day AB restored skin moisture by improving UVB-induced erythema, skin moisture, and transepidermal water loss, and alleviated photoaging by improving UVB-induced elasticity and wrinkles. Moreover, AB upregulated the mRNA levels of hyaluronic acid synthase and collagen-related Col1a1, Col3a1, and Col4a1 genes, increasing hyaluronic acid and collagen expression, respectively. AB inhibited UVB-induced MAPK and AP-1 (c-fos) activation, resulting in significantly downregulated expression of MMP-1 and -9, which are responsible for collagen degradation. AB also stimulated the expression and activity of antioxidative enzymes and reduced lipid peroxidation. Thus, AB is a potential preventive and therapeutic agent for photoaging.