Fibroblast activation and extracellular matrix (ECM) deposition play an important role in the tracheal abnormal repair process and fibrosis. As a transcription factor, SOX9 is involved in fibroblast activation and ECM deposition. However, the mechanism of how SOX9 regulates fibrosis after tracheal injury remains unclear. We investigated the role of SOX9 in TGF-β1-induced fibroblast activation and ECM deposition in rat tracheal fibroblast (RTF) cells. SOX9 overexpression adenovirus (Ad-SOX9) and siRNA were transfected into RTF cells. We found that SOX9 expression was up-regulated in RTF cells treated with TGF-β1. SOX9 overexpression activated fibroblasts and promoted ECM deposition. Silencing SOX9 inhibited cell proliferation, migration, and ECM deposition, induced G2 arrest, and increased apoptosis in RTF cells. RNA-seq and chromatin immunoprecipitation sequencing (ChIP-seq) assays identified MMP10, a matrix metalloproteinase involved in ECM deposition, as a direct target of SOX9, which promotes ECM degradation by increasing MMP10 expression through the Wnt/β-catenin signaling pathway. Furthermore, in vivo, SOX9 knockdown ameliorated granulation proliferation and tracheal fibrosis, as manifested by reduced tracheal stenosis. In conclusion, our findings indicate that SOX9 can drive fibroblast activation, cell proliferation, and apoptosis resistance in tracheal fibrosis via the Wnt/β-catenin signaling pathway. The SOX9-MMP10-ECM biosynthesis axis plays an important role in tracheal injury and repair. Targeting SOX9 and its downstream target MMP10 may represent a promising therapeutic approach for tracheal fibrosis.

BACKGROUND: While Alzheimer\'s disease (AD) is defined by amyloid-β plaques and tau tangles in the brain, it is evident that many other pathophysiological processes such as inflammation, neurovascular dysfunction, oxidative stress, and metabolic derangements also contribute to the disease process and that varying contributions of these pathways may reflect the heterogeneity of AD. Here, we used a previously validated panel of cerebrospinal fluid (CSF) biomarkers to explore the degree to which different pathophysiological domains are dysregulated in AD and how they relate to each other.
METHODS: Twenty-five CSF biomarkers were analyzed in individuals with a clinical diagnosis of AD verified by positive CSF AD biomarkers (AD, n = 54) and cognitively unimpaired controls negative for CSF AD biomarkers (CU-N, n = 26) using commercial single- and multi-plex immunoassays.
RESULTS: We noted that while AD was associated with increased levels of only three biomarkers (MMP-10, FABP3, and 8OHdG) on a group level, half of all AD participants had increased levels of biomarkers belonging to at least two pathophysiological domains reflecting the diversity in AD. LASSO modeling showed that a panel of FABP3, 24OHC, MMP-10, MMP-2, and 8OHdG constituted the most relevant and minimally correlated set of variables differentiating AD from CU-N. Interestingly, factor analysis showed that two markers of metabolism and oxidative stress (24OHC and 8OHdG) contributed independent information separate from MMP-10 and FABP3 suggestive of two independent pathophysiological pathways in AD, one reflecting neurodegeneration and vascular pathology, and the other associated with metabolism and oxidative stress.
CONCLUSIONS: Better understanding of the heterogeneity among individuals with AD and the different contributions of pathophysiological processes besides amyloid-β and tau will be crucial for optimizing personalized treatment strategies.
UNASSIGNED: A panel of 25 highly validated biomarker assays were measured in CSF.MMP10, FABP3, and 8OHdG were increased in AD in univariate analysis.Many individuals with AD had increased levels of more than one biomarker.Markers of metabolism and oxidative stress contributed to an AD multianalyte profile.Assessing multiple biomarker domains is important to understand disease heterogeneity.

BACKGROUND: Non-alcoholic steatohepatitis (NASH), the most severe form of non-alcoholic fatty liver disease (NAFLD), is currently untreatable with a clinically validated treatment. Matrix Metallopeptidase 10 (MMP10) is a common host-response-gene involved in the immune response. However, it remains unknown whether and how MMP10 influences NASH development by modulating macrophage function.
METHODS: In vitro, MMP10 overexpression (MMP10-OE), MMP10 knockout (MMP10-KO), proliferator-activated receptor γ (PPARγ)-OE, and control plasmids were transfected into primary Kupffer cells, which were then cultured with or without Interleukin (IL)-4 stimulation. MMP10-OE mice and MMP10-KO mice were fed a normal chow diet (NCD) or a high-fat diet (HFD) for 30 weeks to study the role of MMP10 in NASH model. Hepa1-6 cells were cultured with or without free fatty acid (FFA) treatment for 24 h.
RESULTS: MMP10 is downregulated in NASH, and M1/M2 indicators are significantly imbalanced. MMP10 is triggered in response to M2 macrophages polarization. MMP10 overexpression diminishes hepatic steatosis and inflammation in HFD-induced NASH. Mechanistically, PPARγ can bind to the MMP10 promoter and then up-regulates MMP10 expression, which is engaged when IL-4 stimulates M2 macrophage polarization. The downstream STAT3 signaling pathway is further activated to induce M2 polarization, which results in a decreased expression of the pro-inflammatory IL-1β and tumor necrosis factor (TNF)-a and an increased expression of the anti-inflammatory IL-10, ultimately alleviating NASH progression.
CONCLUSIONS: We demonstrate that IL-4 effectively promotes MMP10 expression via PPARγ, and MMP10 overexpression modulates macrophage polarization, hepatic steatosis, and fibrosis, offering prospective targets for NASH treatment.

Identification of molecular regulators of hepatocellular carcinoma (HCC) initiation and progression is not well understood. We chemically induced HCC in male Wistar rats by administration of diethyl nitrosamine (DEN) and 2-acetylaminofluorene (2-AFF). Using 2D-electrophoresis and MALDI-TOF-MS/MS analyses, we characterized differentially expressed proteins in liver tissues at early stage of HCC progression. Using RT-PCR analysis, we quantified the mRNA expression of the characterized proteins and validated the transcript expression with tumor tissues of clinically confirmed HCC patients. Using bioinformatic tools, we analyzed a network among the introduced proteins that identified their interacting partners and analyzed the molecular mechanisms associated with signaling pathways during HCC progression. We characterized a protein, namely, pre-mRNA splicing factor 1 homolog (ISY1), which is upregulated at both transcriptome and proteome levels at HCC initiation, progression, and tumor stages. We analyzed the interacting partners of ISY1, namely, APOA-1, SYNE1, MMP10, and MTG1. Real-time PCR analysis confirmed elevated expression of APOA-1 mRNA at HCC initiation, progression, and tumor stages in animals undergoing tumorigenesis. The mRNA expression of the interacting partners was validated with tumor tissues of clinically confirmed liver cancer patients; the analysis revealed significant elevation in expression of transcripts. The transcriptome and proteome analyses complement each other and dysregulation in mRNA and protein expression of these regulators may play critical role in HCC initiation and progression.

The transition from gonocytes into spermatogonia takes place during the homing process. A subpopulation of undifferentiated spermatogonia in niche then shifts to spermatogonial stem cells (SSCs), accompanied by the self-renewal ability to maintain life-long fertility in males. Enormous changes in cell morphology, gene expression, and epigenetic features have been reported during spermatogenesis. However, little is known about the difference of these features in SSCs during aging. Here, we examined the dynamics of SET domain bifurcated 1 (SETDB1) expression in porcine testes. SETDB1 was expressed in postnatal undifferentiated spermatogonia, while gradually disappeared after being packed within the basal compartment of seminiferous tubules. In addition, the cell-adhesion ability, proliferative activity, and trimethylation of the histone H3 lysine 9 (H3K9me3) level were significantly altered in SETDB1-deficient porcine SSCs. Moreover, the matrix metalloproteinases 3/10 (MMP3/10) was upregulated at both mRNA and protein levels. These results illustrate the significance of SETDB1 in modulating early male germ cell development.

Taxifolin (TXL), also known as dihydroquercetin, is one of the most important flavonoids prevalent across the plant kingdom. Increasing evidence has demonstrated its critical role in respiratory diseases. The present study aims to reveal the detailed mechanism in TNF-α-stimulated BEAS-2B cells by which TXL might exert effects on the development of asthma. Cell viability detection of BEAS-2B treated with TXL before and after TNF-α induction employed MMT. The expressions of inflammatory cytokines, MUC5AC and ICAM-1 were determined by quantitative reverse transcription PCR (RT-qPCR), enzyme-linked immunosorbent assay (ELISA) and Western blot after TXL was exposed to an in vitro asthma model. Then, light transmittance and apoptosis were then measured employing fluorescein transmittance, TUNEL and Western blot. After overexpressing MMP10, the abovementioned assays were performed again. Finally, the association between Wnt/β-catenin pathway and MMP10 was confirmed by detecting the proteins in this pathway. TXL increases the cell viability of TNF-induced BEAS-2B cells. TXL suppressed the inflammation, mucus formation, and apoptosis in TNF-α-induced BEAS-2B cells. Furthermore, after the prediction of binding sites between TXL and MMP10, it was found that overexpression of MMP10 reversed the effects of TXL on suppressing the progression of TNF-α-induced BEAS-2B cells. Finally, TXL blocked Wnt/β-catenin pathway by inhibiting MMP10 expression.TXL can be a promising drug for the treatment of asthma due to its inhibition of MMP10 expression by blocking Wnt/β-catenin pathway. Future experimental in vivo studies of asthma on this commonly used bioactive flavonoid could open new avenues for the therapies of asthma.

Energy metabolism and extracellular matrix (ECM) function together orchestrate and maintain tissue organization, but crosstalk between these processes is poorly understood. Here, we used single-cell RNA-Seq (scRNA-Seq) analysis to uncover the importance of the mitochondrial respiratory chain for ECM homeostasis in mature cartilage. This tissue produces large amounts of a specialized ECM to promote skeletal growth during development and maintain mobility throughout life. A combined approach of high-resolution scRNA-Seq, mass spectrometry/matrisome analysis, and atomic force microscopy was applied to mutant mice with cartilage-specific inactivation of respiratory chain function. This genetic inhibition in cartilage results in the expansion of a central area of 1-month-old mouse femur head cartilage, showing disorganized chondrocytes and increased deposition of ECM material. scRNA-Seq analysis identified a cell cluster-specific decrease in mitochondrial DNA-encoded respiratory chain genes and a unique regulation of ECM-related genes in nonarticular chondrocytes. These changes were associated with alterations in ECM composition, a shift in collagen/noncollagen protein content, and an increase of collagen crosslinking and ECM stiffness. These results demonstrate that mitochondrial respiratory chain dysfunction is a key factor that can promote ECM integrity and mechanostability in cartilage and presumably also in many other tissues.

OBJECTIVE: Preclinical ulcerative colitis is poorly defined. We aimed to characterize the preclinical systemic inflammation in ulcerative colitis, using a comprehensive set of proteins.
METHODS: We obtained plasma samples biobanked from individuals who developed ulcerative colitis later in life (n = 72) and matched healthy controls (n = 140) within a population-based screening cohort. We measured 92 proteins related to inflammation using a proximity extension assay. The biologic relevance of these findings was validated in an inception cohort of patients with ulcerative colitis (n = 101) and healthy controls (n = 50). To examine the influence of genetic and environmental factors on these markers, a cohort of healthy twin siblings of patients with ulcerative colitis (n = 41) and matched healthy controls (n = 37) were explored.
RESULTS: Six proteins (MMP10, CXCL9, CCL11, SLAMF1, CXCL11 and MCP-1) were up-regulated (P < .05) in preclinical ulcerative colitis compared with controls based on both univariate and multivariable models. Ingenuity Pathway Analyses identified several potential key regulators, including interleukin-1β, tumor necrosis factor, interferon-gamma, oncostatin M, nuclear factor-κB, interleukin-6, and interleukin-4. For validation, we built a multivariable model to predict disease in the inception cohort. The model discriminated treatment-naïve patients with ulcerative colitis from controls with leave-one-out cross-validation (area under the curve = 0.92). Consistently, MMP10, CXCL9, CXCL11, and MCP-1, but not CCL11 and SLAMF1, were significantly up-regulated among the healthy twin siblings, even though their relative abundances seemed higher in incident ulcerative colitis.
CONCLUSIONS: A set of inflammatory proteins are up-regulated several years before a diagnosis of ulcerative colitis. These proteins were highly predictive of an ulcerative colitis diagnosis, and some seemed to be up-regulated already at exposure to genetic and environmental risk factors.

Early osteoarthritis (OA)-like symptoms are difficult to study owing to the lack of disease samples and animal models. In this study, we generated induced pluripotent stem cell (iPSC) lines from a patient with a radiographic early-onset finger osteoarthritis (efOA)-like condition in the distal interphalangeal joint and her healthy sibling. We differentiated those cells with similar genetic backgrounds into chondrogenic pellets (CPs) to confirm efOA. CPs generated from efOA-hiPSCs (efOA-CPs) showed lower levels of COL2A1, which is a key marker of hyaline cartilage after complete differentiation, for 21 days. Increase in pellet size and vacuole-like morphologies within the pellets were observed in the efOA-CPs. To analyze the changes occurred during the development of vacuole-like morphology and the increase in pellet size in efOA-CPs, we analyzed the expression of OA-related markers on day 7 of differentiation and showed an increase in the levels of COL1A1, RUNX2, VEGFA, and AQP1 in efOA-CPs. IL-6, MMP1, and MMP10 levels were also increased in the efOA-CPs. Taken together, we present proof-of-concept regarding disease modeling of a unique patient who showed OA-like symptoms.

Rheumatoid arthritis (RA) is a common chronic autoimmune disease featured by synovial inflammation. miR-496 is closely involved in various pathologic conditions. However, its role in RA has not yet been elucidated. Expression of miR-496 and MMP10 was determined based on the clinical samples with RA retrieved from the Gene Expression Omnibus (GEO) datasets. In vitro model of RA was constructed in MH7A cells stimulated by IL-1β (10 ng/mL). Cell counting kit 8 (CCK-8) and flow cytometry experiments were implemented to investigate the cell viability and apoptosis rate of MH7A cells. TargetScan was applied to identify the targets of miR-496, and the regulation of miR-496 on MMP10 expression was validated by a dual-luciferase reporter gene assay. qRT-PCR and western blot analyses were conducted to examine the expression. miR-496 expression was decreased in RA tissues and MH7A cells after IL-1β treatment. Overexpression of miR-496 significantly inhibited IL-1β-treated MH7A cell viability. MMP10 was identified as a target of miR-496 and its expression was negatively regulated by miR-496. The effects of miR-496 on MH7A cell proliferation and apoptosis were reversed by MMP10. The activity of NF-κB pathway was associated with the miR-496/MMP10 axis in IL-1β-stimulated MH7A cells. To summarize, this study demonstrated that miR-496 can impair the proliferative ability and facilitate the apoptosis of IL-1β-treated MH7A through regulating MMP10 expression and NF-κB signaling pathway.