Extracellular Matrix Turnover Research Articles

Protective Effects of Circulating TIMP3 on Coronary Artery Disease and Myocardial Infarction: A Mendelian Randomization Study.

Tissue inhibitor of metalloproteinase 3 (TIMP3) is a protease with high expression levels in the heart and plays an essential role in extracellular matrix turnover by maintaining equilibrium with matrix metalloproteinases. Considerable data in experimental models have demonstrated a protective role of TIMP3 in coronary artery disease (CAD) and myocardial infarction (MI). However, causality remains unexplored in population studies. Here, we sought to decipher the potential causality between TIMP3 and CAD/MI using the Mendelian randomization (MR) method. We extracted summary−level datasets for TIMP3 and CAD/MI from the genome−wide association studies performed in the KORA study and CARDIoGRAMplusC4D consortium, respectively. Seven independent SNPs were obtained as instrumental variables for TIMP3. The MR analyses were replicated using FinnGen datasets, and the main results were combined in meta−analyses. Elevated genetically predicted serum TIMP3 levels were causally associated with a lower risk of CAD [odds ratio (OR), 0.97; 95% confidence interval (CI), 0.95, 0.98; p = 5.29 × 10−5] and MI (OR, 0.96; 95% CI, 0.95, 0.98; p = 3.85 × 10−5). The association patterns persisted in the meta−analyses combining the different datasets (CAD: OR, 0.97; 95% CI, 0.96, 0.99; p = 4.37 × 10−5; MI: OR, 0.97; 95% CI, 0.96, 0.99; p = 9.96 × 10−5) and was broadly consistent across a set of complementary analyses. Evidence of heterogeneity and horizontal pleiotropy was limited for all associations considered. In conclusion, this MR study supports inverse causal associations between serum TIMP3 and the risk of CAD and MI. Strategies for raising TIMP3 levels may offer new avenues for the prevention strategies of atherosclerotic cardiovascular diseases.

Nintedanib modulates type III collagen turnover in viable precision-cut lung slices from bleomycin-treated rats and patients with pulmonary fibrosis

BackgroundAberrant extracellular matrix (ECM) deposition and remodelling is important in the disease pathogenesis of pulmonary fibrosis (PF). We characterised neoepitope biomarkers released by ECM turnover in lung tissue from bleomycin-treated rats and patients with PF and analysed the effects of two antifibrotic drugs: nintedanib and pirfenidone.MethodsPrecision-cut lung slices (PCLS) were prepared from bleomycin-treated rats or patients with PF. PCLS were incubated with nintedanib or pirfenidone for 48 h, and levels of neoepitope biomarkers of type I, III and VI collagen formation or degradation (PRO-C1, PRO-C3, PRO-C6 and C3M) as well as fibronectin (FBN-C) were assessed in the culture supernatants.ResultsIn rat PCLS, incubation with nintedanib led to a reduction in C3M, reflecting type III collagen degradation. In patient PCLS, incubation with nintedanib reduced the levels of PRO-C3 and C3M, thus showing effects on both formation and degradation of type III collagen. Incubation with pirfenidone had a marginal effect on PRO-C3. There were no other notable effects of either nintedanib or pirfenidone on the other neoepitope biomarkers studied.ConclusionsThis study demonstrated that nintedanib modulates neoepitope biomarkers of type III collagen turnover and indicated that C3M is a promising translational neoepitope biomarker of PF in terms of therapy assessment.

Solid fraction determines stiffness and viscosity in decellularized pancreatic tissues.

The role of extracellular matrix (ECM) composition and turnover in mechano-signaling and the metamorphic fate of cells seeded into decellularized tissue can be elucidated by recent developments in non-invasive imaging and biotechnological analysis methods. Because these methods allow accurate quantification of the composition and structural integrity of the ECM, they can be critical in establishing standardized decellularization protocols. This study proposes quantification of the solid fraction, the single-component fraction and the viscoelasticity of decellularized pancreatic tissues using compact multifrequency magnetic resonance elastography (MRE) to assess the efficiency and quality of decellularization protocols. MRE of native and decellularized pancreatic tissues showed that viscoelasticity parameters depend according to a power law on the solid fraction of the decellularized matrix. The parameters can thus be used as highly sensitive markers of the mechanical integrity of soft tissues. Compact MRE allows consistent and noninvasive quantification of the viscoelastic properties of decellularized tissue. Such a method is urgently needed for the standardized monitoring of decellularization processes, evaluation of mechanical ECM properties, and quantification of the integrity of solid structural elements remaining in the decellularized tissue matrix.

The role of Vitamin D3 in ocular fibrosis and its therapeutic potential for the glaucomatous trabecular meshwork.

Glaucoma is the leading cause of irreversible blindness globally. The most prevalent subtype, Primary Open Angle Glaucoma (POAG), is characterized by increased intraocular pressure (IOP), damage to the optic nerve head and irreversible visual loss. IOP increases aqueous humor (AqH) outflow is reduced through the trabecular meshwork (TM) and Schlemm's canal (SC). Increased outflow resistance is partly due to TM/SC dysregulation, including loss of normal trabecular meshwork cell (TMC) function, following increased levels of oxidative stress within TMC, dysregulated extracellular matrix (ECM) deposition and remodeling alongside alterations in TMC phenotype and apoptosis. Current widely available POAG treatments do not target the aberrant expression of ECM in the TM directly. As a result, most drug treatments can fail as the underlying pathological process continues unabated. Rho-kinase inhibitors have demonstrated the benefit of restoring TM/SC function, however there is a clear need to develop further treatment strategies that can target the underlying cellular processes which become dysregulated within the TMC during POAG pathogenesis. Vitamin D is suggested to be beneficial in alleviating the symptoms of fibrosis and inflammation in soft tissues. It has important functions in many major organ systems, including regulation of calcium, phosphate and parathyroid hormone. Evidence suggests that Vitamin D3 modulates ECM turnover through the conventional TGFβ-SMAD signaling, which is associated with the development of POAG. The link between Vitamin D3, inflammation and fibrosis within ocular tissues will be discussed and the potential roles of Vitamin D3 in the management of POAG patients will be explored within this review.

Serological Biomarkers of Extracellular Matrix Turnover and Neutrophil Activity Are Associated with Long-Term Use of Vedolizumab in Patients with Crohn's Disease.

Crohn’s disease (CD) is a relapsing-remitting inflammatory disease of the gastrointestinal (GI) tract characterized by increased extracellular matrix (ECM) remodeling. The introduction of the α4β7-integrin inhibitor vedolizumab (VEDO) has improved disease management, although there is a high rate of primary non-response in patients with CD. We studied whether ECM biomarkers of neutrophil activity and mucosal damage could predict long-term response to VEDO in patients with CD. Serum levels of human neutrophil elastase (HNE)-derived fragments of calprotectin (CPa9-HNE), and matrix metalloproteinase (MMP)-derived fragments of type I (C1M), III (C3M), IV (C4M), and VI (C6Ma3) collagen, type III collagen formation (PRO-C3), basement membrane turnover (PRO-C4) and T-cell activity (C4G), were measured using protein fingerprint assays in patients with CD (n = 32) before VEDO therapy. Long-term response was defined as VEDO treatment of at least 12 months. CPa9-HNE was significantly increased at baseline in non-responders compared with responders (p < 0.05). C1M, C3M, C4M, C6Ma3, and PRO-C4 were also significantly increased at baseline in non-responders compared with responders (all p < 0.05). All biomarkers were associated with response to VEDO (all p < 0.05). To conclude, baseline levels of serum biomarkers for neutrophil activity and mucosal damage are linked to the pathology of CD, and are associated with long-term use of VEDO in patients with CD. Therefore, these biomarkers warrant further validation and could aid in therapeutic decision-making concerning vedolizumab therapy.

Expression of CD44, Transforming Growth Factor-β, and Matrix Metalloproteinases in Women WithPelvic Organ Prolapse.

BackgroundDefects in the pelvic floor connective tissue may underlie the etiology of pelvic organ prolapse (POP). We hypothesized that the expression of proteins regulating extracellular matrix turnover is altered in the uterosacral ligament of women with POP. We compared the expression of CD44, transforming growth factor (TGF)-β, and matrix metalloproteinases (MMPs) 2/9 in women with and without POP.Methods and ResultsThis matched case-control study included 30 postmenopausal women, with POP stage 2 and higher according to the POP quantification system, and 30 postmenopausal women without POP. Immunohistochemical analyses of the uterosacral ligament specimens obtained after hysterectomy were performed to determine CD44, TGF-β, MMP-2, and MMP-9 expression. The expression was quantified using ImageJ software, and the association between prolapse occurrence and risk factors was evaluated using Spearman's correlation analysis. CD44 expressions were significantly lower (p < 0.05), whereas MMP-2 and MMP-9 expression was higher (p < 0.0001 and p < 0.05, respectively), in the POP group than in the control group. The expression of TGF-β was similar in both groups. The occurrence of uterine prolapse was positively correlated with age, postmenopausal age, and MMP-2 and MMP-9 expression (p < 0.01) and negatively correlated with CD44 expression (p < 0.05).ConclusionCD44, MMP-2, and MMP-9 may play critical roles in the pathogenesis of POP and may be candidate biomarkers of POP progression.

Serological Biomarkers of Intestinal Collagen Turnover Identify Early Response to Infliximab Therapy in Patients With Crohn's Disease.

BackgroundCrohn’s disease (CD) is characterized by excessive protease activity and extracellular matrix (ECM) remodeling. To date, 30–50% of patients experience non-response to anti-TNF-α treatment. This study aimed to assess whether serological biomarkers of ECM turnover could monitor or predict response to infliximab (IFX) induction therapy in patients with and without a surgical history.MethodsSerum biomarkers of type I (C1M), III (C3M), IV (C4M), and VI (C6Ma3) collagen degradation, type III (PRO-C3) and VI (PRO-C6) collagen formation, basement membrane turnover (PRO-C4), and T-cell activity (C4G), were measured at baseline and week 14, in 63 patients with CD undergoing IFX induction therapy. Patients were stratified according to surgical history.ResultsC4M was elevated at baseline in responders with a surgical history (n = 10) and associated with response at baseline (P < 0.05). Additionally, C6Ma3, PRO-C3, and PRO-C6 were elevated at week 14 in responders compared with non-responders (n = 8) and could differentiate between the two groups (P < 0.05). Two biomarker ratios (C4M/C4G and PRO-C4/C4G) were elevated at week 14 in non-responders (n = 5) without a surgical history compared with responders (n = 40) and could differentiate between the response groups (P < 0.05).ConclusionBaseline levels of a serological biomarker for type IV collagen degradation associated with response to IFX induction therapy, and biomarkers of type III and VI collagen formation may be used to monitor response at the end of induction therapy in patients with a surgical history. Biomarker ratios of type IV collagen turnover demonstrated promising results in monitoring treatment response in patients without a surgical history.

Aberrant TIMP-1 overexpression in tumor-associated fibroblasts drives tumor progression through CD63 in lung adenocarcinoma

Tissue inhibitor of metalloproteinase-1 (TIMP-1) is an important regulator of extracellular matrix turnover that has been traditionally regarded as a potential tumor suppressor owing to its inhibitory effects of matrix metalloproteinases. Intriguingly, this interpretation has been challenged by the consistent observation that increased expression of TIMP-1 is associated with poor prognosis in virtually all cancer types including lung cancer, supporting a tumor-promoting function. However, how TIMP-1 is dysregulated within the tumor microenvironment and how it drives tumor progression in lung cancer is poorly understood. We analyzed the expression of TIMP-1 and its cell surface receptor CD63 in two major lung cancer subtypes: lung adenocarcinoma (ADC) and squamous cell carcinoma (SCC), and defined the tumor-promoting effects of their interaction. We found that TIMP-1 is aberrantly overexpressed in tumor-associated fibroblasts (TAFs) in ADC compared to SCC. Mechanistically, TIMP-1 overexpression was mediated by the selective hyperactivity of the pro-fibrotic TGF-β1/SMAD3 pathway in ADC-TAFs. Likewise, CD63 was upregulated in ADC compared to SCC cells. Genetic analyses revealed that TIMP-1 secreted by TGF-β1-activated ADC-TAFs is both necessary and sufficient to enhance growth and invasion of ADC cancer cells in culture, and that tumor cell expression of CD63 was required for these effects. Consistently, in vivo analyses revealed that ADC cells co-injected with fibroblasts with reduced SMAD3 or TIMP-1 expression into immunocompromised mice attenuated tumor aggressiveness compared to tumors bearing parental fibroblasts. We also found that high TIMP1 and CD63 mRNA levels combined define a stronger prognostic biomarker than TIMP1 alone. Our results identify an excessive stromal TIMP-1 within the tumor microenvironment selectively in lung ADC, and implicate it in a novel tumor-promoting TAF-carcinoma crosstalk, thereby pointing to TIMP-1/CD63 interaction as a novel therapeutic target in lung cancer.

Molecular Mechanisms of Oral Submucous Fibrosis and Oral Cancer

Chewing betel quid (BQ) increases the risk of oral cancer and oral submucous fibrosis (OSMF), a potentially malignant oral premalignant condition (OPMD). BQ constituents including areca nut (AN), trauma by coarse AN fibre, catechin, copper, alkaloids, increased reactive oxygen species, inflammation, and cytotoxicity are hypothesised to be the causative factors. They may stimulate tissue inflammation, fibroblast proliferation and collagen deposition, myofibroblast differentiation and contraction, collagen cross-links, and impede collagenphagocytosis, ultimately leading to the formation of oral squamous cell carcinoma (OSMF) and oral cancer. Through modulation of transforming growth factor1, plasminogen activator inhibitor1, cystatin, lysyl oxidase, tissue inhibitors of metalloproteinases, and matrix metalloproteinases, BQ componentinduced alterations in extracellular matrix turnover facilitate these events. In addition, genetic predisposition plays a role in many disease processes. Understanding the molecular pathways underlying BQinduced OSMF and oral cancer can aid in the future prevention and treatment of disease. This paper provides a comprehensive review of the molecular processes involved in BQ-induced OSMF and oral cancer, as well as future preventative prospects.

Effect of pigmentation intensity of trabecular meshwork cells on mechanisms of micropulse laser trabeculoplasty

The intraocular pressure (IOP)-lowering mechanisms of micropulse laser trabeculoplasty (MLT) remain unclear. The present study was performed to investigate the mechanism of action of MLT, and to determine whether the pigmentation intensity of trabecular meshwork (TM) cells is associated with the treatment effects. Primary human TM cells were exposed to melanin granules to artificially introduce different levels of pigmentation. Micropulse (MP) laser irradiation was performed, and interleukin (IL)-1α/β, matrix metalloproteinases (MMPs), tissue inhibitor of metalloproteinases (TIMPs), and extracellular matrix (ECM) protein expression were evaluated by RT-qPCR and immunocytochemistry. IL-1α/β and MMP-1, -3, and -9 mRNA expression were significantly upregulated at 4 and 24 h after MP laser irradiation, respectively, but there were no significant changes in TIMP expression. The extent of these upregulation was greater in cells with strong pigmentation intensity. Protein expressions of fibronectin and collagen I were significantly decreased in cells with strong staining intensity. These results suggested that MP laser irradiation alter the MMP/TIMP ratio and enhance ECM turnover, resulting in increased outflow of aqueous humor. The pigmentation intensity of the TM tissues may affect the treatment efficacy of MLT, because TM cells with strong staining intensity showed a significantly enhanced response to MP laser irradiation.

Bioinformatic Prioritization and Functional Annotation of GWAS-Based Candidate Genes for Primary Open-Angle Glaucoma.

Background: Primary open-angle glaucoma (POAG) is the most prevalent glaucoma subtype, but its exact etiology is still unknown. In this study, we aimed to prioritize the most likely ‘causal’ genes and identify functional characteristics and underlying biological pathways of POAG candidate genes. Methods: We used the results of a large POAG genome-wide association analysis study from GERA and UK Biobank cohorts. First, we performed systematic gene-prioritization analyses based on: (i) nearest genes; (ii) nonsynonymous single-nucleotide polymorphisms; (iii) co-regulation analysis; (iv) transcriptome-wide association studies; and (v) epigenomic data. Next, we performed functional enrichment analyses to find overrepresented functional pathways and tissues. Results: We identified 142 prioritized genes, of which 64 were novel for POAG. BICC1, AFAP1, and ABCA1 were the most highly prioritized genes based on four or more lines of evidence. The most significant pathways were related to extracellular matrix turnover, transforming growth factor-β, blood vessel development, and retinoic acid receptor signaling. Ocular tissues such as sclera and trabecular meshwork showed enrichment in prioritized gene expression (>1.5 fold). We found pleiotropy of POAG with intraocular pressure and optic-disc parameters, as well as genetic correlation with hypertension and diabetes-related eye disease. Conclusions: Our findings contribute to a better understanding of the molecular mechanisms underlying glaucoma pathogenesis and have prioritized many novel candidate genes for functional follow-up studies.

MicroRNAs as potential indicators of the development and progression of uterine leiomyoma.

Recent studies demonstrated a significant role of several microRNAs (miRs) in the development of leiomyoma. Here, we investigated miR expression profiles using microarray and found a significantly higher expression of miRs in leiomyoma than in adjacent myometrium. We also confirmed the upregulation of five selected miRs including miR-181a-5p, 127-3p, 28-3p, 30b-5p and let-7c-5p in cellular proliferation, extracellular matrix turnover, and angiogenesis by RT-qPCR. Interestingly, the miRs showed a higher expression in cases of large leiomyoma or in patients with a history of transfusion due to anemia. We then analyzed the expression of the miR target molecules including Transforming Growth Factor Beta Receptor 2 (TGFBR2) and Insulin-like Growth Factor 2 mRNA Binding Protein 1 (IGF2BP1) via immunohistochemistry. TGFBR2 and IGF2BP1 were positively stained in 81% and 62.5% of leiomyoma tissues but not in adjacent myometrium. Both were more frequently positive in patients with ≥ 6 cm leiomyoma and mass effect. The mean expression levels of miR-181a-5p, 127-3p, 28-3p, 30b-5p and let-7c-5p were higher in cases with TGFBR2 and IGF2BP1 positive leiomyoma. We observed several miRs were overexpressed in leiomyoma tissues, and these results provide insight into the role of miRs in the development and progression of leiomyoma and underscore the need to validate their utility as diagnostic or therapeutic targets.

Quantitative Proteomics Reveals That ADAM15 Can Have Proteolytic-Independent Functions in the Steady State

A disintegrin and metalloproteinase 15 (ADAM15) is a member of the ADAM family of sheddases. Its genetic ablation in mice suggests that ADAM15 plays an important role in a wide variety of biological functions, including cartilage homeostasis. Nevertheless, while the substrate repertoire of other members of the ADAM family, including ADAM10 and ADAM17, is largely established, little is known about the substrates of ADAM15 and how it exerts its biological functions. Herein, we used unbiased proteomics to identify ADAM15 substrates and proteins regulated by the proteinase in chondrocyte-like HTB94 cells. ADAM15 silencing did not induce major changes in the secretome composition of HTB94 cells, as revealed by two different proteomic approaches. Conversely, overexpression of ADAM15 remodeled the secretome, with levels of several secreted proteins being altered compared to GFP-overexpressing controls. However, the analysis did not identify potential substrates of the sheddase, i.e., transmembrane proteins released by ADAM15 in the extracellular milieu. Intriguingly, secretome analysis and immunoblotting demonstrated that ADAM15 overexpression increased secreted levels of tissue inhibitor of metalloproteinases 3 (TIMP-3), a major regulator of extracellular matrix turnover. An inactive form of ADAM15 led to a similar increase in the inhibitor, indicating that ADAM15 regulates TIMP-3 secretion by an unknown mechanism independent of its catalytic activity. In conclusion, high-resolution quantitative proteomics of HTB94 cells manipulated to have increased or decreased ADAM15 expression did not identify canonical substrates of the proteinase in the steady state, but it revealed that ADAM15 can modulate the secretome in a catalytically-independent manner.

The Role of Mechanically-Activated Ion Channels Piezo1, Piezo2, and TRPV4 in Chondrocyte Mechanotransduction and Mechano-Therapeutics for Osteoarthritis.

Mechanical factors play critical roles in the pathogenesis of joint disorders like osteoarthritis (OA), a prevalent progressive degenerative joint disease that causes debilitating pain. Chondrocytes in the cartilage are responsible for extracellular matrix (ECM) turnover, and mechanical stimuli heavily influence cartilage maintenance, degeneration, and regeneration via mechanotransduction of chondrocytes. Thus, understanding the disease-associated mechanotransduction mechanisms can shed light on developing effective therapeutic strategies for OA through targeting mechanotransducers to halt progressive cartilage degeneration. Mechanosensitive Ca2+-permeating channels are robustly expressed in primary articular chondrocytes and trigger force-dependent cartilage remodeling and injury responses. This review discusses the current understanding of the roles of Piezo1, Piezo2, and TRPV4 mechanosensitive ion channels in cartilage health and disease with a highlight on the potential mechanotheraputic strategies to target these channels and prevent cartilage degeneration associated with OA.

MO063: A New Tool for Preclinical Research and Drug Discovery: Extracellular Matrix Remodeling Quantification in Human Precision-Cut Kidney Slices

Abstract BACKGROUND AND AIMS Accumulation of extracellular matrix (ECM) proteins is a hallmark of renal fibrosis, leading to altered tissue homeostasis, kidney failure and ultimately death. Current treatment strategies are ineffective in halting renal fibrogenesis, and drug discovery is hampered by the lack of clinically relevant biomarkers. Thus, there is an urgent need for early and pharmacodynamic biomarkers of renal fibrosis to support the development of new therapeutic treatments. In this study, we explored the use of non-invasive biomarkers of ECM turnover in human precision-cut kidney slices (PCKS): we observed the release of these markers over time in the slice supernatants and measured the changes following treatment with a prostaglandin E2 (PGE2) EP1 receptor antagonist. METHOD PCKS were prepared from healthy and fibrotic human kidney tissue obtained from patients treated at Aarhus University Hospital, Skejby. The slices were cultured in 48-well plates in medium (300 µl/well) and incubated for up to 72 hours to stimulate the development of renal fibrosis. The medium was collected at hours 0, 24, 48 and 72. Biomarkers of collagen type I (PRO-C1), III (PRO-C3), fibronectin (FN; FBN-C) and α-SMA formation and MMP-mediated degradation of collagen type I (C1M), III (C3M) and IV (Tumstatin; TUM) were measured in the medium by ELISA assays developed at Nordic Bioscience. mRNA expression levels of COL1A1, FN and α-SMA were measured by qPCR at all time points. Viability was assessed by measuring the ATP content of the slices at all time points. The anti-fibrotic effect of SC-19 220—a PGE2 EP1 receptor antagonist—was studied in fibrotic PCKS at hours 24 and 48 after treatment. RESULTS Fibrosis developed spontaneously in human kidney slices, as shown by increasing levels of PRO-C1 (P &amp;lt; 0.0001), PRO-C3 (P &amp;lt; 0.0001), FBN-C (P &amp;lt; 0.0001) and α-SMA (P &amp;lt; 0.0001) over time. Levels of C1M (not significant) and C3M (P &amp;lt; 0.01) did not increase to the same extent, and levels of TUM decreased over time (P &amp;lt; 0.001), indicating that this model reflects the process of fibrogenesis over fibrolysis. In addition, protein levels of PRO-C1 and FBN-C correlated significantly with mRNA levels of COL1A1 (r = 0.92, P = 0.0088) and FN (r = 0.86, P = 0.0277), respectively. Treatment with SC-19 220 reduced the gene expression of COL1A1, FN and α-SMA, and at a protein level it decreased levels of PRO-C1, PRO-C3 and FBN-C. PCKS retained constant ATP levels up to 72 hours. CONCLUSION In conclusion, we demonstrated that quantification of ECM remodeling in human PCKS via noninvasive biomarkers can be a valuable preclinical tool to study renal fibrogenesis and the effect of anti-fibrotic treatments. This study highlights that the EP1 receptor is a promising target for halting the production of ECM proteins. Since this is a human model, results may be translated to clinical care with higher confidence.

AKAP‐mediated regulation of extracellular matrix secretion in cardiac fibrosis

Cardiac fibroblasts represent the most abundant cell type in the myocardium and provide structural support through controlled proliferation and extracellular matrix (ECM) turnover. In response to chronic pressure overload, quiescent resident cardiac fibroblasts undergo transdifferentiation to myofibroblasts. These activated fibroblasts display increased ECM synthetic capacity and secrete large amounts of collagens. Excessive deposition of ECM results in massive interstitial fibrosis, which decreases myocardial compliance and affects electrical coupling between cardiomyocytes, causing diastolic dysfunctions and arrhythmias. In this context, deciphering the molecular mechanisms favouring increased ECM secretion and deposition in stressed hearts might be the key for the development of targeted approaches inhibiting excessive myocardial fibrosis and dysfunction.A‐kinase anchoring proteins (AKAPs) are molecular scaffolds that act as signal organizers. By recruiting and coordinating the activity of signalling enzymes in space and time they control physiological and pathological responses in cardiac cells. In this respect, our current findings suggest that AKAPs directly regulate the activity of the pathway that controls intracellular ECM protein transport and secretion in myofibroblasts. We are currently evaluating the use of molecular disruptors of selected AKAP complexes to reduce collagen secretion. These disruptors will be tested for their ability to prevent fibrosis and myocardial dysfunction in response to pressure overload.

Reciprocal Regulation between lncRNA ANRIL and p15 in Steroid-Induced Glaucoma.

Steroid-induced glaucoma (SIG) is the most common adverse steroid-related effect on the eyes. SIG patients can suffer from trabecular meshwork (TM) dysfunction, intraocular pressure (IOP) elevation, and irreversible vision loss. Previous studies have mainly focused on the role of extracellular matrix turnover in TM dysfunction; however, whether the cellular effects of TM cells are involved in the pathogenesis of SIG remains unclear. Here, we found that the induction of cellular senescence was associated with TM dysfunction, causing SIG in cultured cells and mouse models. Especially, we established the transcriptome landscape in the TM tissue of SIG mice via microarray screening and identified ANRIL as the most differentially expressed long non-coding RNA, with a 5.4-fold change. The expression level of ANRIL was closely related to ocular manifestations (IOP elevation, cup/disc ratio, and retinal nerve fiber layer thickness). Furthermore, p15, the molecular target of ANRIL, was significantly upregulated in SIG and was correlated with ocular manifestations in an opposite direction to ANRIL. The reciprocal regulation between ANRIL and p15 was validated using luciferase reporter assay. Through depletion in cultured cells and a mouse model, ANRIL/p15 signaling was confirmed in cellular senescence via cyclin-dependent kinase activity and, subsequently, by phosphorylation of the retinoblastoma protein. ANRIL depletion imitated the SIG phenotype, most importantly IOP elevation. ANRIL depletion-induced IOP elevation in mice can be effectively suppressed by p15 depletion. Analyses of the single-cell atlas and transcriptome dynamics of human TM tissue showed that ANRIL/p15 expression is spatially enriched in human TM cells and is correlated with TM dysfunction. Moreover, ANRIL is colocalized with a GWAS risk variant (rs944800) of glaucoma, suggesting its potential role underlying genetic susceptibility of glaucoma. Together, our findings suggested that steroid treatment promoted cellular senescence, which caused TM dysfunction, IOP elevation, and irreversible vision loss. Molecular therapy targeting the ANRIL/p15 signal exerted a protective effect against steroid treatment and shed new light on glaucoma management.

Effect of Basic Fibroblast Growth Factor on Expression of Let-7 MicroRNA in Proliferation of Human Dental Pulp Cells

Objective: Basic fibroblast growth factor (bFGF) plays a pivotal role in cell proliferation, differentiation and extracellular matrix turnover in various tissues. In human dental pulp cells (HDPCs), let-7 microRNA is involved in cell proliferation and differentiation. There is little information on the effect of bFGF-induced cell proliferation on let-7 microRNA in HDPCs. This study investigated the effect of bFGF on let-7g, let-7f, and let-7i microRNAs and some of the genes involved in cell proliferation including p53 and Ki67 in HDPCs.Material and Methods: HDPCs were cultured and treated with bFGF at 0, 1, and 5 ng/mL. Cell proliferation was examined using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay at 24 and 48 hours. Additionally, gene expressions of let-7g, let-7f, let-7i microRNAs, and p53 and Ki67 were examined by quantitative real-time polymerase chain reaction at 24 hours. All experiments were performed in triplicate.Results: The results showed that let-7g, let-7f, and let-7i microRNAs were expressed in HDPCs. MTT assays showed that bFGF induced greater cell proliferation than the controls at 24 and 48 hours (p-value&lt;0.050). HDPCs treated with bFGF showed a decrease in p53 expression (p-value&lt;0.001) while Ki67 expression increased (p-value&lt;0.001). The expression of let-7g microRNA decreased under the influence of bFGF (p-value&lt;0.050). However, bFGF had no effect on expression of let-7f and let-7i microRNAs (p-value&gt;0.050).Conclusion: Our preliminary study showed that exogenous bFGF could decrease let-7g microRNA expression suggesting that let-7g microRNA may be involved in bFGF-induced HDPCs proliferation.

Unravelling the distinct biological functions and potential therapeutic applications of TIMP2 in cancer.

Tissue inhibitors of metalloproteinases (TIMPs) are a conserved family of proteins that were originally identified as endogenous inhibitors of matrixin and adamalysin endopeptidase activity. The matrixins and adamalysins are the major mediators of extracellular matrix (ECM) turnover, thus making TIMPs important regulators of ECM structure and composition. Despite their high sequence identity and relative redundancy in inhibitory profiles, each TIMP possesses unique biological characteristics that are independent of their regulation of metalloproteinase activity. As our understanding of TIMP biology has evolved, distinct roles have been assigned to individual TIMPs in cancer progression. In this respect, data regarding TIMP2's role in cancer have borne conflicting reports of both tumor suppressor and, to a lesser extent, tumor promoter functions. TIMP2 is the most abundant TIMP family member, prevalent in normal and diseased mammalian tissues as a constitutively expressed protein. Despite its apparent stable expression, recent work highlights how TIMP2 is a cell stress-induced gene product and that its biological activity can be dictated by extracellular posttranslational modifications. Hence an understanding of TIMP2 molecular targets, and how its biological functions evolve in the progressing tumor microenvironment may reveal new therapeutic opportunities. In this review, we discuss the continually evolving functions of TIMP proteins, future perspectives in TIMP research, and the therapeutic utility of this family, with a particular focus on TIMP2.

Mechanical Response of Mouse Cervices Lacking Decorin and Biglycan During Pregnancy.

Cervical remodeling is critical for a healthy pregnancy. The proper regulation of extracellular matrix (ECM) turnover leads to remodeling throughout gestation, transforming the tissue from a stiff material to a compliant, extensible, viscoelastic tissue prepared for delivery. Small leucine-rich proteoglycans (SLRPs) regulate structural fiber assembly in the cervical ECM and overall tissue material properties. To quantify the SLRPs' mechanical role in the cervix, whole cervix specimens from nonpregnant and late pregnant knockout mice of SLRPs, decorin and biglycan, were subjected to cyclic load-unload, ramp-hold, and load-to-failure mechanical tests. Further, a fiber composite material model, accounting for collagen fiber bundle waviness, was developed to describe the cervix's three-dimensional large deformation equilibrium behavior. In nonpregnant tissue, SLRP knockout cervices have the same equilibrium material properties as wild-type tissue. In contrast, the load-to-failure and ramp-hold tests reveal SLRPs impact rupture and time-dependent relaxation behavior. Loss of decorin in nonpregnant (NP) cervices results in inferior rupture properties. After extensive remodeling, cervical strength is similar between all genotypes, but the SLRP-deficient tissue has a diminished ability to dissipate stress during a ramp-hold. In mice with a combined loss of decorin and biglycan, the pregnant cervix loses its extensibility, compliance, and viscoelasticity. These results suggest that decorin and biglycan are necessary for crucial extensibility and viscoelastic material properties of a healthy, remodeled pregnant cervix.