Effects of a High-Fat Diet and a Plant Extract Composition on Extracellular Matrix Remodeling in Rat Liver.

Recent data have emphasized the role of inflammation and intestinal immunoglobulin A (IgA) responses in the pathogenesis of alcoholic liver disease (ALD). In order to further explore such associations, we compared IgA titers against antigens targeted to ethanol metabolites and tissue transglutaminase with pro- and anti-inflammatory mediators of inflammation, markers of liver status, transferrin protein desialylation and extracellular matrix metabolism in alcohol-dependent patients with or without liver disease and in healthy controls. Serum IgAs against protein adducts with acetaldehyde (HbAch-IgA), the first metabolite of ethanol, and tissue transglutaminase (tTG-IgA), desialylated transferrin (CDT), pro- and anti-inflammatory cytokines, markers of liver status (GT, ALP) and extracellular matrix metabolism (PIIINP, PINP, hyaluronic acid, ICTP and CTx) were measured in alcohol-dependent patients with (n = 83) or without (n = 105) liver disease and 88 healthy controls representing either moderate drinkers or abstainers. In ALD patients, both tTG-IgA and HbAch-IgA titers were significantly higher than those in the alcoholics without liver disease (p < 0.0005 for tTG-IgA, p = 0.006 for Hb-Ach-IgA) or in healthy controls (p < 0.0005 for both comparisons). The HbAch-IgA levels in the alcoholics without liver disease also exceeded those found in healthy controls (p = 0.0008). In ROC analyses, anti-tTG-antibodies showed an excellent discriminative value in differentiating between ALD patients and healthy controls (AUC = 0.95, p < 0.0005). Significant correlations emerged between tTG-IgAs and HbAch-IgAs (rs = 0.462, p < 0.0005), CDT (rs = 0.413, p < 0.0001), GT (rs = 0.487, p < 0.0001), alkaline phosphatase (rs = 0.466, p < 0.0001), serum markers of fibrogenesis: PIIINP (rs = 0.634, p < 0.0001), hyaluronic acid (rs = 0.575, p < 0.0001), ICTP (rs = 0.482, p < 0.0001), pro-inflammatory cytokines IL-6 (rs = 0.581, p < 0.0001), IL-8 (rs = 0.535, p < 0.0001) and TNF-α (rs = 0.591, p < 0.0001), whereas significant inverse correlations were observed with serum TGF-β (rs = -0.366, p < 0.0001) and CTx, a marker of collagen degradation (rs = -0.495, p < 0.0001). The data indicate that the induction of IgA immune responses toward ethanol metabolites and tissue transglutaminaseis a characteristic feature of patients with AUD and coincides with the activation of inflammation, extracellular matrix remodeling and the generation of aberrantly glycosylated proteins. These processes appear to work in concert in the sequence of events leading from heavy drinking to ALD.

Extracellular matrix (ECM) remodeling in skeletal muscle is a potential mechanism linking obesity with metabolic dysfunction. It is also a constructive feature of skeletal muscle adaptation to exercise training. PURPOSE: To test the hypothesis that skeletal muscle ECM remodeling associated with insulin resistance can be minimized by exercise training. METHODS: Six-week-old male C57/BL6J mice (n=48) were divided into two groups, high-fat (60% calories from fat) diet (HF, n=36) and normal chow-fed control (C, n=12) group. After 12 weeks of feeding, HF mice developed insulin resistance, as confirmed by insulin and glucose tolerance tests (ITT and GTT). HF mice were then randomly assigned to three groups: high-fat diet only group (HFS, n=12), high-fat diet + aerobic exercise group (HF+AE, n=12), high-fat diet + resistance training group (HF+RT, n=12). The HF+AE and HF+RT groups were subject to aerobic (treadmill running) and resistive (vertical ladder climbing) training, for 12 weeks. After training, gastrocnemius muscle was harvested and analyzed for ECM factors using immunohistochemistry, ECM PCR array, and western blotting. ANOVA was performed to test the significance of group differences at p<0.05. RESULTS: High-fat feeding induced higher deposition of collagens (COLI, III and IV) in the skeletal muscle of HFS group, and increased gene and protein expression of MMP3, CDH1, ITGAL and SELL and decreased the expression of TIMP3 in HFS group, as compared to group C. These changes were minimized even reversed by either aerobic or resistance exercise training (mRNA fold change relative to C in HF+AE and HF+RT vs. HFS: MMP3, 1.1 and 1.6 vs. 2.6; CDH1, 1.7 and 1.1 vs. 2.0; ITGAL, 1.9 and -1.0 vs. 2.0; SELL, 1.4 and 1.0 vs. 2.1; TIMP3, 1.2 and -1.0 vs. -1.2; p<0.05). These effects were accompanied by a significant improvement in insulin sensitivity (GTT AUC glucose in mmol/l x 120 min: C, 27.2±3.0; HFS, 39.7 ± 7.0; HF+AE, 32.4± 7.5; HF+RT, 30.3± 6.1; p<0.05). CONCLUSION: Both aerobic and resistive exercise training can minimize changes in skeletal muscle ECM associated with insulin resistance. Skeletal muscle ECM remodeling may play a significant role in mediating the metabolic benefits of exercise training. This study was supported by National Nature Science Foundation of China (31470060).

Significance: The vascular extracellular matrix (ECM) not only provides mechanical stability but also manipulates vascular cell behaviors, which are crucial for vascular function and homeostasis. ECM remodeling, which alters vascular wall mechanical properties and exposes vascular cells to bioactive molecules, is involved in the development and progression of hypertension. Recent Advances: This brief review summarized the dynamic changes in ECM components and their modification and degradation during hypertension and after antihypertensive treatment. We also discussed how alterations in the ECM amount, assembly, mechanical properties, and degradation fragment generation provide input into the pathological process of hypertension. Critical Issues: Although the relevance between ECM remodeling and hypertension has been recognized, the underlying mechanism by which ECM remodeling initiates the development of hypertension remains unclear. Therefore, the modulation of ECM remodeling on arterial stiffness and hypertension in genetically modified rodent models is summarized in this review. The circulating biomarkers based on ECM metabolism and therapeutic strategies targeting ECM disorders in hypertension are also introduced. Future Directions: Further research will provide more comprehensive understanding of ECM remodeling in hypertension by the application of matridomic and degradomic approaches. The better understanding of mechanisms underlying vascular ECM remodeling may provide novel potential therapeutic strategies for preventing and treating hypertension. Antioxid. Redox Signal. 34, 765-783.

Building a pathway to recovery: Targeting ECM remodeling in CNS injuries

The role of extracellular matrix (ECM) remodeling in fibrosis progression in nonalcoholic fatty liver disease (NAFLD) is complex and dynamic, involving the synthesis and degradation of different ECM components, including tenascin C (TNC). The aim was to analyze the influence of inducible nitric oxide synthase (iNOS) deletion on inflammation and ECM remodeling in the liver of ob/ob mice, since a functional relationship between leptin and iNOS has been described. The expression of molecules involved in inflammation and ECM remodeling was analyzed in the liver of double knockout (DBKO) mice simultaneously lacking the ob and the iNOS genes. Moreover, the effect of leptin was studied in the livers of ob/ob mice and compared to wild-type rodents. Liver inflammation and fibrosis were increased in leptin-deficient mice. As expected, leptin treatment reverted the obesity phenotype. iNOS deletion in ob/ob mice improved insulin sensitivity, inflammation, and fibrogenesis, as evidenced by lower macrophage infiltration and collagen deposition as well as downregulation of the proinflammatory and profibrogenic genes including Tnc. Circulating TNC levels were also decreased. Furthermore, leptin upregulated TNC expression and release via NO-dependent mechanisms in AML12 hepatic cells. iNOS deficiency in ob/ob mice improved liver inflammation and ECM remodeling-related genes, decreasing fibrosis, and metabolic dysfunction. The activation of iNOS by leptin is necessary for the synthesis and secretion of TNC in hepatocytes, suggesting an important role of this alarmin in the development of NAFLD.

Aberrant extracellular matrix (ECM) remodelling in muscle, liver and adipose tissue is a key characteristic of obesity and insulin resistance. Despite its emerging importance, the effective ECM targets remain largely undefined due to limitations of current approaches. Here, we developed a novel ECM-specific mass spectrometry-based proteomics technique to characterise the global view of the ECM changes in the skeletal muscle and liver of mice after high fat (HF) diet feeding. We identified distinct signatures of HF-induced protein changes between skeletal muscle and liver where the ECM remodelling was more prominent in the muscle than liver. In particular, most muscle collagen isoforms were increased by HF diet feeding whereas the liver collagens were differentially but moderately affected highlighting a different role of the ECM remodelling in different tissues of obesity. Moreover, we identified a novel association between collagen 24α1 and insulin resistance in the skeletal muscle. Using quantitative gene expression analysis, we extended this association to the white adipose tissue. Importantly, collagen 24α1 mRNA was increased in the visceral adipose tissue, but not the subcutaneous adipose tissue of obese diabetic subjects compared to lean controls, implying a potential pathogenic role of collagen 24α1 in obesity and type 2 diabetes.

We report intrauterine growth restriction (IUGR) increases vascular stiffening in both male and female rats through increased collagen content and altered elastin structure more than a high-fat diet (HFD) alone. Our study shows the importance of stiffness supporting the hypothesis that there are physiologic differences and potential windows for early intervention targeting vascular remodeling mechanisms.

Distinct Roles for Matrix Metalloproteinases 2 and 9 in Embryonic Hematopoietic Stem Cell Emergence, Migration, and Niche Colonization.

In this study, the effect of gender and physiological ageing on circulating concentrations of plasma sulfated glycosaminoglycans (sGAG) as well as molecules involved in pro- (tumor necrosis factor-α; TNF-α) and anti-inflammatory responses (soluble tumor necrosis factor receptor-1, sTNF-RI) were assessed. The relationships between sGAG and molecules involved in age-dependent extracellular matrix (ECM) remodeling during physiological ageing were also investigated. Circulating TNF-α and sTNF-RI were measured in 91 healthy volunteers using enzyme-linked immunosorbent assays. sGAG were quantified using an Alcian blue-binding assay. A linear age-related decline in plasma sGAG was found during the first five decades of life (r=-0.61, p<0.05), followed by an increase occurring only in females (r=0.46, p<0.05). Circulating TNF-α concentrations were inversely correlated with age (r=-0.24, p<0.05) over the lifetime. For TNF-α, the observed changes were gender specific. Serum sTNF-RI concentrations were not affected by age in either men or women. A significant positive correlation was found between the concentrations of TNF-α and both sGAG (r=0.22, p<0.05) and sTNF-RI (r=0.21, p<0.05). Our data demonstrate that physiological ageing is associated with ECM remodeling, reflected by plasma sGAGs concentrations. Changes in the ECM metabolism during the ageing process were influenced by circulating TNF-α. Furthermore, serum concentrations of biomolecules involved in pro- and anti-inflammatory responses are not increased in healthy elderly subjects.

Background: Increased extracellular matrix (ECM) metabolism is associated with poor outcomes in numerous cardiovascular diseases. However, its relationship to outcomes in patients with single right ventricle physiology (SRV) is unknown. The objective of this study was to investigate the association of measures of ECM metabolism to outcomes in SRV patients under consideration for stage 3 palliation (S3P). Methods: SRV patients undergoing routine pre-S3P catheterization were prospectively enrolled. Serum matrix metalloproteinase 2 (MMP-2), cardiac magnetic resonance T1 mapping derived extracellular volume (ECV), and pressure-volume loop derived ventricular stiffness (β) by micro-conductance catheter were collected. Controls were age-matched children undergoing closure of clinically insignificant patent ductus arteriosus. SRV were divided into those who had an optimal outcome or suboptimal outcome (hemodynamic concern causing delay in S3P, length of stay ≥ 14 days, heart transplant or death in first year after S3P). Results: Of 30 patients, 15 had SRV and 15 were controls; 7 SRV patients had suboptimal outcome. Mean age was 4.2 ± 0.7 years. SRV patients with suboptimal outcome had lower β (p = 0.05) and ECV (p = 0.04) than patients with optimal outcome (Figure A and B, respectively). MMP-2 was lower in SRV patients with suboptimal outcome compared to SRV patients with optimal outcome and controls (Figure C) (p = 0.05). In SRV patients, MMP-2 correlated with β (r = 0.56, p = 0.03). Conclusion: In biventricular heart disease characterized by chronically increased afterload, MMP-2, ECV, and ventricular stiffness are elevated. In contrast, this study shows that SRV patients with lower MMP-2, ECV, and ventricular stiffness have worse outcomes at the time of S3P; this suggests immature ECM metabolism leads to poor outcomes in SRV patients. Future studies exploring the mechanism by which immature ECM and dysregulated ECM metabolism influence SRV outcomes are warranted.

Understanding the role of the extracellular matrix in cardiovascular development and disease: Where do we go from here?

Background: The extracellular matrix (ECM) is a highly organized non-cellular network consisting of structural proteins, growth factors, cytokines and other secreted molecules. The ECM is highly dynamic and is a critical player in the regulation of local invasion and metastasis. The ECM network is ever-changing and an important immunosuppressive component of the tumor microenvironment (TME). However, the reciprocal effects of the immune system on the tumor-associated ECM are elusive and largely unexplored. Regulatory T (Treg) cells function to enforce peripheral tolerance and are potent suppressors of tumor immunity. We have previously shown that Treg cells promote tumor growth in murine breast cancer models by favoring alternative activation of tumor-associated macrophages (TAMs) via suppression of IFN-g. Aims: In this work, we sought to assess whether Treg cell-mediated immune suppression is associated with changes in the ECM, and whether those ECM changes functionally impact metastatic dissemination. Further, we assessed the requirement of TAMs and IFN-g in the ECM remodeling. Design Methods: In this study, we used spontaneous and transplantable models of breast cancer susceptible to genetic ablation of Treg cells. We used a combination of histopathological analysis, tumor decellularization, bioengineering 3D-chip modeling, in vivo manipulations and bioinformatic analysis, to evaluate remodeling of the ECM, cancer cell invasive behavior and metastatic dissemination. Results Summary: Treg cell ablation resulted in significant ECM remodeling, with reduced amounts and organization of collagen fibers, and increased amounts of fibronectin and laminin. Tumor cells seeding on Treg cell ablated tumor- derived decellularized ECM matrices results in reduced epithelial-mesenchymal transition (EMT) transcription factors, and profound impairment of collective migration. In vivo, Treg cell ablation in a neo-adjuvant setting followed by primary tumor resection resulted in significant reduction of circulating tumor cells (CTC) and impairment of lung metastatic disease. Importantly, we demonstrated that Treg cell ablation-driven changes in the matrisome correlate with long-term improved survival in a cohort of breast cancer patient samples. Through genetic knock-out models and in-vivo neutralization, we observed that Treg cell-dependent changes in ECM-related phenotypes are dependent on IFN-g-signaling. Single-cell RNA-sequencing (scRNA-seq) of murine breast tumors revealed that the matrisome signature is highly upregulated among TAMs, compared to other TME cell types. Lastly, using conditional genetic knock-out models we show these IFN-g-dependent changes are mostly driven by TAMs. Conclusions: Altogether, we identified a novel metastasis-promoting effect of Treg cells in the breast cancer microenvironment through regulation of ECM dynamics beyond their described effects on primary tumor growth. Citation Format: Ailen D. Garcia-Santillan, Jessanne Y. Lichtenberg, He Shen, Jasmine M Rodriguez, Jonathan Barra, Nicholas M Clark, Wei Du, Leandro M Martinez, Ashley D Hadjis, Taylor Calicchia, Mikhail G Dozmorov, Jose J Bravo-Cordero, Amy L Olex, Priscilla Y Hwang, Paula D Bos. Regulatory T (Treg) cell contributes to tumor cell dissemination via extracellular matrix (ECM) remodeling, which is driven by TAMs in an IFNg-dependent manner [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr B023.

BackgroundBladder cancer (BCA) shows significant prognostic differences between non-muscle-invasive (NMIBC) and muscle-invasive (MIBC) forms. While NMIBC frequently recurs and can progress to invasive disease, reliable biomarkers to monitor this transition are lacking. Extracellular matrix (ECM) remodeling is a critical factor influencing tumor aggressiveness, yet the key regulators of ECM changes across BCA stages remain unclear. In this study, we investigate the role of COL1A2 in ECM-related tumor biology and its potential as a prognostic biomarker for BCA progression.MethodsWe utilized a multi-step bioinformatics pipeline, analyzing RNA-seq data from TCGA and GEO datasets to identify molecular differences between NMIBC and MIBC. Prognostic markers were prioritized via differential expression analysis, Cox regression, and Kaplan–Meier survival analysis. The regulatory network was explored using protein-protein interaction analysis, and ECM-related activity was quantified through ssGSEA. Cell-type-specific insights were gained through single-cell RNA-seq analysis, and intercellular communication was deciphered using CellChat. Functional validation was performed through in vitro knockdown experiments in BCA cell lines.ResultsCOL1A2 emerged as a key prognostic ECM-related gene associated with MIBC. Single-cell RNA-seq analysis revealed that COL1A2 and ECM components were predominantly enriched in matrix cancer-associated fibroblasts (CAFs), with PTK2 (FAK, focal adhesion kinase) upregulated in epithelial cells undergoing epithelial-mesenchymal transition (EMT). CellChat analysis uncovered a dominant COL1A2-mediated signaling axis from matrix CAFs to EMT epithelial cells via COL1A1/2–SDC1/4 ligand-receptor interactions. Functional assays confirmed that COL1A2 knockdown significantly impaired MIBC cell invasion and migration by suppressing ECM remodeling and EMT.ConclusionOur results suggest that the COL1A2–ECM–FAK signaling axis plays a critical role in MIBC progression, and COL1A2 could serve as a potential biomarker and therapeutic target for muscle-invasive bladder cancer.

Bladder dysfunction has one of the highest prevalences as a comorbidity of obesity in industrialized countries. The aetiopathogenesis of obesity-associated bladder dysfunction is still obscure, but there is growing evidence that general metabolic changes in obese patients may be in part responsible. As demonstrated recently, high fat diet (HFD) significantly alters the protein expression in the urinary bladder, activates multiple signalling pathways associated with cell survival and inflammation and ultimately provokes bladder fibrosis in an obese rat model. The study aimed to elucidate the role of matrix metalloproteases (MMPs) and their specific tissue inhibitors of metalloproteases (TIMPs) in obesity-related bladder extracellular matrix (ECM) remodelling and the effect of weight loss surgery via sleeve gastrectomy (SG) on phenotype and molecular parameters. Twenty-four male Sprague-Dawley rats were used for (i) characterization of the HFD phenotype and (ii) evaluation of alterations following SG. Metabolic status, the degree of bladder fibrosis and tissue expression and activity of MMP2, MMP9, MMP14, TIMP1 and TIMP2 were analysed by immunohistochemistry, enzyme-linked immunosorbent assay and activity assays. Statistical differences were calculated by analysis of variance or independent Student's t-test. A P-value <0.05 was considered statistically significant. In HFD rats, we found significant alterations in lipid metabolism, fat mass, free fatty acid profile, insulin resistance and inflammatory markers. Voided volume was significantly decreased, and bladder showed marked fibrosis. MMPs and TIMPs were differentially regulated depending on animal status (controls, chow diet, HFD, and SG- and sham-operated animals) in both urothelium and detrusor smooth muscle. Although animal weight and most metabolic parameters were positively affected by SG, bladder fibrosis persisted. The limitations of this study were 1 month follow-up and lack of direct measurement of bladder function. Early diagnosis of the bladder dysfunction associated with obesity is essential to allow targeted early intervention, that is, before manifestation of potentially irreversible ECM fibrotic alterations.

Liver donor age affects hepatocyte function through age-dependent changes in decellularized liver matrix