Inhibition Of Matrix Metalloproteinase-13 Research Articles

Astaxanthin: A Marine Drug That Ameliorates Cerebrovascular-Damage-Associated Alzheimer's Disease in a Zebrafish Model via the Inhibition of Matrix Metalloprotease-13.

Alzheimer's disease (AD) is a major type of dementia disorder. Common cognitive changes occur as a result of cerebrovascular damage (CVD) via the disruption of matrix metalloproteinase-13 (MMP-13). In diabetic cases, the progress of vascular dementia is faster and the AD rate is higher. Patients with type 2 diabetes are known to have a higher risk of the factor for AD progression. Hence, this study is designed to investigate the role of astaxanthin (AST) in CVD-associated AD in zebrafish via the inhibition of MMP-13 activity. CVD was developed through the intraperitoneal and intracerebral injection of streptozotocin (STZ). The AST (10 and 20 mg/L), donepezil (1 mg/L), and MMP-13 inhibitor (i.e., CL-82198; 10 μM) were exposed for 21 consecutive days in CVD animals. The cognitive changes in zebrafish were evaluated through light and dark chamber tests, a color recognition test, and a T-maze test. The biomarkers of AD pathology were assessed via the estimation of the cerebral extravasation of Evans blue, tissue nitrite, amyloid beta-peptide aggregation, MMP-13 activity, and acetylcholinesterase activity. The results revealed that exposure to AST leads to ameliorative behavioral and biochemical changes. Hence, AST can be used for the management of AD due to its multi-targeted actions, including MMP-13 inhibition.

MMP-13 Regulates Matrix Binding and Internalization of NG2/CSGP4 in Mandibular Fibrochondrocytes

Objective: Post-traumatic osteoarthritis (OA) is associated with cartilage degradation resulting in changes in cell-matrix mediated signaling after injury. Matrix metalloproteinase-13 (MMP13) is a critical enzyme in this process, degrading the extracellular matrix and modifying receptors. One of the receptors altered by MMP13 is a transmembrane proteoglycan, Neuron Glial antigen 2 (NG2/CSPG4). In the temporomandibular joint (TMJ), NG2/CSPG4 is membrane bound and binds with type VI collagen. During OA, the NG2/CSPG4 intracellular domain is internalized into the cytosol. While NG2/CSPG4 is a known substrate for MMP13, there are important gaps in knowledge related to how MMP-13 modifies NG2/CSPG4 after injury. The hypothesis of this study is that MMP13 regulates NG2/CSPG4 interactions with the extracellular matrix, the internalization of the intracellular domain, and key cartilage signaling pathways during mechanical loading and OA progression. Methods: To characterize MMP13 and NG2/CSPG4 levels during the progression of TMJ OA, we used a surgical destabilization preclinical murine model (approved under UIC ACC #20-068) and compared them with clinical samples from human patients receiving total joint replacement for advanced stage TMJ OA (approved under UIC IRB #2017-0033). To mechanistically interrogate MMP-13-NG2/CSPG4, we cultured primary mandibular fibrochondrocytes and treated them with an MMP13 inhibitor (CAS 544678-85-5) at 15 nM for 24 hours or vehicle control. To evaluate matrix binding, we performed immunocytochemistry using an antibody against the NG2/CSPG4 ectodomain on decellularized coverslips. To mechanically load samples, cell-agarose-collagen scaffolds received a constrained static compression treatment for 2 hours at 2.5 N with and without the MMP13. Gene expression changes were quantified using RT-qPCR and protein changes were quantified with western blot. Data were evaluated using a one-way ANOVA. Results: Our results illustrate that MMP13 is present in preclinical and clinical samples a spatiotemporally consistent pattern with NG2/CSPG4 internalization. In vitro, the inhibition of MMP13 impairs the ability of the NG2/CSPG4 ectodomain to be retained in the extracellular matrix. Mechanical loading of the cells activates MMP13, leading to clearance of membrane associated NG2/CSPG4. Inhibition of MMP13 during mechanical loading promotes the accumulation of membrane associated NG2/CSPG4 and a decrease in internalized NG2/CSPG4 (n = 4; p < 0.05). MMP13 did not cause the complete proteolytic degradation of the ectodomain. This mechanically sensitive MMP-13-NG2/CSPG4 axis regulates the expression of key mineralization and OA genes including BMP2 and PTHrP (n = 4; p < 0.05). Conclusions: Together, these findings indicate that MMP13 mediated cleavage of NG2/CSPG4 potentiates the internalization of the intracellular domain and regulates genes implicated in the mineralization signaling axis during growth and degenerative diseases such as OA. Funding support from NIH/NIDCR 1R01DE029835-01. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Amelioration of post-traumatic osteoarthritis via nanoparticle depots delivering small interfering RNA to damaged cartilage.

The progression of osteoarthritis (OA) is associated with inflammation triggered by the enzymatic degradation of extracellular matrix in injured cartilage. Here, we show that a locally injected depot of nanoparticles functionalized with an antibody targeting collagen type-II and carrying small interfering RNA for matrix metalloproteinase 13 (MMP13), which breaks down collagen type-II, substantially reduced the expression of MMP13 and protected cartilage integrity and overall joint structure in acute and severe mouse models of post-traumatic OA. MMP13 inhibition suppressed clusters of genes associated with tissue restructuring, angiogenesis, innate immune responses and proteolysis. We also show that intra-articular injections of the nanoparticles led to higher reductions in disease progression than either a single injection or weekly injections of the steroid methylprednisolone. Sustained drug retention by targeting collagen in the damaged extracellular matrix of osteoarthritic cartilage may also be an effective strategy for the treatment of OA with other disease-modifying drugs.

Investigation on Chemical Constituents of Foeniculum vulgare Essential Oil and the Molecular Docking Studies of its Components for Possible Matrix Metalloproteinase-13 Inhibition

Background: Foeniculum vulgare (Fennel) has a wide range of applications. Previous studies revealed the presence of different compounds in the essential oil (EO) of fennel fruit (FF). Matrix metalloproteinase-13 (MMP-13) participates in several human biological processes including the degradation of extracellular matrix proteins, activation or degradation of some significant regulatory proteins, and tumor cell invasion. Furthermore, the up-regulation of MMP-13 is associated with many disorders such as tooth caries and periodontitis, as well as the degradation of enamel and tissues around the implant and Alzheimer’s disease. Therefore, the aims of the present study were to investigate the compounds of the EO of FF (EOFF) from the Hamedan district, along with performing molecular docking analysis to assess the binding affinity of four compounds originated from F. vulgare with the MMP-13. Finally, the study focused on evaluating the pharmacokinetic and toxicity characteristics of the compounds. Methods: Hydrodistillation method was used for obtaining the EO from FF. Then, gas chromatography-mass spectrometry was applied to identify the components of the EO. Molecular docking analysis was carried out using AutoDock software. Eventually, the pharmacokinetic and toxicity features of compounds were evaluated using bioinformatics webservers. Results: The results revealed the presence of fourteen compounds, among which e-anethole (86.86%), fenchone (743%), estragole (165%), and thymol (1.21%) were the main components. Based on the results, thymol, fenchone, e-anethole, and estragole could potentially bind to the MMP-13 active site, respectively. Conclusion: Regardless of several studies on the chemical constituents of EOFF, the subject has its own pharmacognostical importance. According to computational studies, EOFF has the potential for study on several human disorders such as cancer, tooth decay, and Alzheimer’s disease.

Molecular docking study of flavonoid compounds for possible matrix metalloproteinase-13 inhibition

Matrix metalloproteinase-13 (MMP-13) has been reported to be involved in different biological processes such as degradation of extracellular matrix proteins, activating or degrading some significant regulatory proteins, wound healing, tissue remodeling, cartilage degradation, bone development, bone mineralization, ossification, cell migration, and tumor cell invasion. Further, MMP-13 participates in many oral diseases such as tooth decay, gingivitis, and degradation of enamel and tissue around the implant. In addition, inhibition of MMP-13 has shown therapeutic properties for Alzheimer's disease (AD). We performed molecular docking to assess the binding affinity of 29 flavonoid compounds with the MMP-13. Additionally, pharmacokinetic and toxicity characteristics of the top-ranked flavonoids were studied. The current study also intended to identify the most important amino acids involved in the inhibition of MMP-13 based on topological feature (degree) in the ligand-amino acid network for MMP-13. Molecular docking and network analysis werestudied using AutoDock and Cytoscape software, respectively. Pharmacokinetic and toxicity characteristics of compounds were predicted using bioinformatics webtools. The results revealed that nine of the studied flavonoids had considerable estimated free energy of binding and inhibition constant: Rutin, nicotiflorin, orientin, vitexin, apigenin-7-glucoside, quercitrin, isoquercitrin, quercitrin-3-rhamnoside, and vicenin-2. Proline-242 was found to be the most important amino acid inhibiting the enzyme. The results of the current study may be helpful in the prevention and therapeutic procedures of many disorders such as cancer, tooth caries, and AD. Nevertheless, validation tests are required in the future.

Candesartan Treatment Associates With Reduced Expression of Collagen and Matrix Metalloproteinase Genes in Severely Stenotic Human Aortic Valves: A Prospective, Placebo-controlled, Randomized, Double-blinded Clinical Trial

Introduction: Calcific aortic valve stenosis (AS) is a progressive disease resulting from long-lasting inflammatory, fibrotic, and calcifying processes in the aortic valve leaflets. The renin-angiotensin-aldosterone system (RAAS) has been demonstrated to be active in human stenotic aortic valves. In experimental animals, administration of an angiotensin II type 1 receptor blocker has exerted both anti-inflammatory and antifibrotic effects on stenotic aortic leaflets. In human aortic smooth muscle cells, angiotensin II increases the expression of matrix metalloproteinase. The migration of vascular smooth muscle cells (VSMCs) is an early event of atherosclerosis. Indeed, angiotensin II increases matrix metalloproteinase 13 (MMP13)'s expression in aortic smooth muscle cells (SMCs) and the silencing or inhibition of MMP13 results in reduced SMCs' migration. Here were examined whether candesartan would beneficially affect the major pathogenic valvular mechanisms active in severe AS. Methods: In this placebo-controlled, randomized, double-blinded, prospective study a total of 43 AS patients were randomized to receive candesartan (target dose: 16 mg/d) or placebo for an average of about 5 months (164 ± 67 days) before undergoing aortic valve replacement surgery. In the explanted valves, the mRNA levels of markers for fibrosis were assessed using an mRNA assay panel and quantitative polymerase chain reaction (qPCR). Moreover, markers of fibrosis, inflammation, and neovascularization were analyzed immunohistologically. Results: In the candesartan group, the mRNA expression levels of Collagen Type X Alpha 1 Chain (Col10a1) (p = 0.003), Col11a1 (p = 0.002), Col12a1 (p = 0.016), and of MMP8 (p = 0.024) and MMP13 (p = 0.007) were lower than in the placebo group. During this short intervention period, immunohistological analysis did not show differences in fibrosis, calcification, neovascularization, or inflammation of the valves. Conclusion: In the present study, we found the potential mechanism for candesartan to interact with the progression of aortic valve stenosis by modeling the mRNA expressions of collagens and matrix metalloproteinases. Provided these potentially beneficial antifibrotic effects candesartan could retard the progression of AS. However, additional longer interventional studies are needed to prove possible beneficial clinical effects of candesartan in AS. Disclosure: Nothing to disclose

A systematic computational analysis of human matrix metalloproteinase 13 (MMP-13) crystal structures and structure-based identification of prospective drug candidates as MMP-13 inhibitors repurposable for osteoarthritis

Osteoarthritis (OA) is the most common form of arthritis with no available disease-modifying treatments, and is a major cause of disability. Matrix metalloproteinase 13 (MMP-13) is vital for OA progression and thus, inhibition of MMP-13 is an effective strategy to treat OA. Since the past few decades, drug repurposing has gained substantial popularity worldwide as a time- and cost-effective approach to find new indications for the existing drugs. Therefore, more than 40 X-ray co-crystal structures of the human MMP-13 with bound inhibitors are investigated to gain the structural insights such as conserved direct interactions with binding site residues, namely Ala-238, Thr-245 and Thr-247. Afterwards, enrichment study using active and decoy set of ligands revealed three MMP-13 structures (PDB-IDs: 1XUC, 3WV1 and 5BPA) with optimal enrichment performance. Docking-based screening of existing drugs against the three crystal structures followed by binding free-energy calculation suggested drugs namely eltrombopag, cilostazol and domperidone as potential MMP-13 inhibitors that need further experimental validation. These insights may serve as a potential starting point of further experimental validation and structure-based drug design/repurposing of MMP-13 inhibitors for the treatment of OA. Abbreviations 2D two-dimensional 3D three-dimensional FDA Food and Drug Administration MM-GBSA Molecular Mechanics Generalized Born Surface Area MMPs matrix metalloproteinases MMP-13 matrix metalloproteinase 13 NMR nuclear magnetic resonance OA osteoarthritis PDB Protein Data Bank PDB-ID Protein Data Bank ID PLIP protein–ligand interaction profiler ROC receiver operating characteristic, RMSD root mean square deviation Communicated by Ramaswamy H. Sarma

FOXO3a (Forkhead Transcription Factor O Subfamily Member 3a) Links Vascular Smooth Muscle Cell Apoptosis, Matrix Breakdown, Atherosclerosis, and Vascular Remodeling Through a Novel Pathway Involving MMP13 (Matrix Metalloproteinase 13).

Vascular smooth muscle cell (VSMC) apoptosis accelerates atherosclerosis and promotes breakdown of the extracellular matrix, but the mechanistic links between these 2 processes are unknown. The forkhead protein FOXO3a (forkhead transcription factor O subfamily member 3a) is activated in human atherosclerosis and induces a range of proapoptotic and other transcriptional targets. We, therefore, determined the mechanisms and consequences of FOXO3a activation in atherosclerosis and arterial remodeling after injury. Expression of a conditional FOXO3a allele (FOXO3aA3ER) potently induced VSMC apoptosis, expression and activation of MMP13 (matrix metalloproteinase 13), and downregulation of endogenous TIMPs (tissue inhibitors of MMPs). mmp13 and mmp2 were direct FOXO3a transcriptional targets in VSMCs. Activation of endogenous FOXO3a also induced MMP13, extracellular matrix degradation, and apoptosis, and MMP13-specific inhibitors and fibronectin reduced FOXO3a-mediated apoptosis. FOXO3a activation in mice with VSMC-restricted FOXO3aA3ER induced MMP13 expression and activity and medial VSMC apoptosis. FOXO3a activation in FOXO3aA3ER/ApoE-/- (apolipoprotein E deficient) mice increased atherosclerosis, increased necrotic core and reduced fibrous cap areas, and induced features of medial degeneration. After carotid artery ligation, FOXO3a activation increased VSMC apoptosis, VSMC proliferation, and neointima formation, all of which were reduced by MMP13 inhibition. FOXO3a activation induces VSMC apoptosis and extracellular matrix breakdown, in part, because of transcriptional activation of MMP13. FOXO3a activation promotes atherosclerosis and medial degeneration and increases neointima after injury that is partly dependent on MMP13. FOXO3a-induced MMP activation represents a direct mechanistic link between VSMC apoptosis and matrix breakdown in vascular disease.

Functional characterization of selective exosite-binding inhibitors of matrix metalloproteinase-13 (MMP-13) - experimental validation in human breast and colon cancer.

Considering the pathological significance of MMP-13 in breast and colon cancers, exosite-based inhibition of the C-terminal hemopexin (Hpx) domain could serve as an alternative strategy to develop selective inhibitors for MMP-13.Two of six lead compounds, compound 5 (2,3-dihydro-1,4-benzodioxine-5-carboxylic acid) and compound 6 (1-acetyl-4-hydroxypyrrolidine-2-carboxylic acid) exhibited considerable inhibitory activity against MMP-13. Complementing to this study, we have also shown the gene expression levels of MMP-13 within the subtypes of colon and breast cancers classified from patients' tissue samples to provide a better understanding on which subtype of breast cancer patients would get benefited by MMP-13 inhibitors.Our current results show that compounds 5 and 6 could effectively inhibit MMP-13 and provide specific therapeutic possibilities in the treatment of inflammatory disorders and cancers. The characterization of these lead compounds would provide a better mechanistic understanding of exosite-based inhibition of MMP-13, which could overcome the challenges in the identification of other MMP catalytic domain-specific inhibitors.

Abstract 409: Matrix Metalloproteinase-13 Inhibition is Protective in a Pressure Overload Model of Heart Failure

Heart failure (HF), the leading cause of morbidity and mortality in the United States, is characterized by pathologic remodeling, fibrosis and deteriorating cardiac function. Cardiac fibrosis occurs due to imbalanced production and degradation of extracellular matrix (ECM) proteins. Cardiac fibroblasts (CF) are largely responsible for the secretion of ECM proteins in the heart, and upon injury, transition to a migratory and proliferative myofibroblast (MF) phenotype, leading to excess ECM deposition. Elevated expression of matrix metalloproteinases (MMPs), proteolytic enzymes responsible for degradation of the ECM, is common in HF. Specifically, MMP13 is known to be upregulated in human HF patients. Therefore, we hypothesized that MMP13 plays an important role in pathologic cardiac remodeling, and that inhibition of MMP13 would prevent the development of HF in a pressure overload model, transverse aortic constriction (TAC). Mice were subjected to TAC and treated with the MMP13 inhibitor, WAY170523 (WAY), or vehicle 4 weeks post-TAC until 12 weeks post-TAC. Mice treated with WAY display decreased cardiac hypertrophy and preserved cardiac function compared to vehicle treated mice. WAY treatment may also attenuate interstitial and perivascular fibrosis as well as expression of pro-fibrotic genes. To determine the effect of MMP13 inhibition in cardiac cells, CF and MF were isolated from healthy mice or mice 5 days post-ischemia/reperfusion injury, respectively, and treated with WAY. MMP13 inhibition led to decreased CF invasion but did not affect migration, proliferation or adhesion. Interestingly, inhibition of MMP13 in MF attenuated migration, proliferation and invasion. Moreover, WAY treatment reduced collagen and fibronectin deposition in the ECM of MF. MMP13 inhibition also appeared to decrease Angiotensin II-induced hypertrophy in ventricular cardiomyocytes (CM). These data suggest a role for MMP13 in pressure overload-induced HF, CM hypertrophy and CF behavior. MMP13 inhibition after injury may attenuate cardiac hypertrophy as well as the CF to MF transition, leading to decreased cardiac fibrosis and improved cardiac function. Further understanding of the role of MMP13 could lead to a novel therapeutic target in the treatment of HF.

High molecular weight hyaluronic acid regulates MMP13 expression in chondrocytes via DUSP10/MKP5.

To determine the effect of high molecular weight hyaluronic acid (HA) on matrix metalloproteinase 13 (MMP13) expression induced by tumor necrosis factor α (TNF-α) in chondrocytes. Human chondrocytic C28/I2 cells were incubated with TNF-α and HA. In some experiments, the cells were pre-incubated with a CD44 function-blocking monoclonal antibody (CD44 mAb) prior to addition of TNF-α and HA. The expression of MMP13 was determined by real-time reverse-transcription polymerase chain reaction (RT-PCR) and an enzyme linked immunosorbent assay, while the phosphorylation of signaling molecules was measured by western blot analysis. The transcriptional activity of activator protein 1 (AP-1) was analyzed by a reporter assay. To further clarify the molecular mechanisms of HA in MMP13 regulation, the expression level of dual-specificity protein phosphatase 10 (DUSP10)/mitogen-activated protein kinases phosphatase 5 (MKP5) in HA-treated chondrocytes was assessed by real-time RT-PCR, western blotting, and immunofluorescence microscopy. HA decreased MMP13 mRNA and protein expression induced by TNF-α. Blockage of HA-CD44 binding by CD44 mAb suppressed HA-mediated inhibition of MMP13. HA inhibited transient phosphorylation of p38 mitogen-activated protein kinase (MAPK) and c-jun NH2 -terminal kinase (JNK) induced by TNF-α. Reporter assay findings also revealed that pre-treatment with HA inhibited the transcriptional activity of AP-1 mediated by TNF-α. Moreover, HA induced the expression of DUSP10/MKP5, a negative regulator of p38 MAPK and JNK pathways. These results indicate that HA-CD44 interactions downregulate TNF-α-induced MMP13 expression via regulation of DUSP10/MKP5, suggesting that HA plays an important role as a regulatory factor in cartilage degradation. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:331-339, 2017.

A novel role of MMP-13 for murine DC function: its inhibition dampens T-cell activation

Dendritic cells (DCs) have been shown to express matrix metalloproteinase 13 (MMP-13), but little is known about its specific function in DCs and its role in inflammatory conditions. In the present study, we describe a novel role of MMP-13 in regulating the immunostimulatory function of murine DCs through moderating MHC-I surface presentation, endocytosis and cytokine/chemokine secretion. MMP-13 expression was confirmed in bone marrow-derived DCs at both the mRNA and the protein level and, furthermore, at the activity level. Remarkably, LPS treatment strongly enhanced MMP-13 mRNA expression as well as MMP-13 activity, indicating an important role of MMP-13 in inflammatory processes. Functionally, MMP-13 inhibition did not influence the DC migratory capacity, while endocytosis of ovalbumin was significantly decreased. Inhibition of MMP-13 lowered the capability of murine DCs to activate CD8+ T cells, apparently through reducing MHC-I surface presentation. Decreased surface expression of CD11c on DCs, as well as changes in the DC cytokine/chemokine profile after MMP-13 inhibition, emphasizes the influence of MMP-13 on DC function. Moreover, T-cell-targeting cytokines such as IL-12, IL-23 and IL-6 were significantly reduced. Collectively, our data reveal a novel involvement of MMP-13 in regulating DC immunobiology through moderating MHC-I surface presentation, endocytosis and cytokine/chemokine secretion. Furthermore, the reduced MHC-I surface presentation by DCs resulted in a poor CD8+ T-cell response in vitro This novel finding indicates that MMP-13 might be a promising target for therapeutic intervention in inflammatory diseases.

Inhibition of matrix metalloproteinase-13 expression in IL-1β-treated articular chondrocytes by a steroidal saponin, spicatoside A, and its cellular mechanisms of action

Matrix metalloproteinase-13 (MMP-13) plays a critical role in degrading major collagens in human cartilage under some pathological conditions such as osteoarthritis. To establish the therapeutic potential against cartilage degradation, the effects of 12 naturally-occurring triterpenoids and steroids on MMP-13 induction were examined in the human chondrocyte cell line, SW1353. They included coreanoside F1, suavissimoside R1, spicatoside A, 25(S)-ruscogenin, methyl protogracillin, hederagenin, loniceroside A, loniceroside B, loniceroside C, smilaxin A, smilaxin C, and ursolic acid. Among these, only spicatoside A and 25(S)-ruscogenin were found to inhibit MMP-13 expression in IL-1β-treated SW1353 cells at a pharmacologically-relevant concentration of 10 μM. These effects were also supported by the finding that spicatoside A (20 μM) reduced glycosaminoglycan release from IL-1α-treated rabbit joint cartilage culture to some degree. When the cellular mechanisms of action of spicatoside A in MMP-13 inhibition were investigated, the blocking point was not found among the MMP-13 signaling molecules examined such as mitogen-activated protein kinases, activator protein-1, and nuclear transcription factor-κB. Instead, spicatoside A was found to reduce MMP-13 mRNA stability. All of these findings suggest that spicatoside A and 25(S)-ruscogenin have a therapeutic potential for protecting against cartilage breakdown in arthritic disorders.

Cancer progression by breast tumors with Pit-1-overexpression is blocked by inhibition of metalloproteinase (MMP)-13.

IntroductionThe POU class 1 homeobox 1 transcription factor (POU1F1, also known as Pit-1) is expressed in the mammary gland and its overexpression induces profound phenotypic changes in proteins involved in cell proliferation, apoptosis, and invasion. Patients with breast cancer and elevated expression of Pit-1 show a positive correlation with the occurrence of distant metastasis. In this study we evaluate the relationship between Pit-1 and two collagenases: matrix metalloproteinase-1 (MMP-1) and matrix metalloproteinase-13 (MMP-13), which have been related to metastasis in breast cancer.MethodsWe began by transfecting the MCF-7 and MDA-MB-231 human breast adenocarcinoma cell lines with the Pit-1 overexpression vector (pRSV-hPit-1). Afterward, the mRNA, protein, and transcriptional regulation of both MMP-1 and MMP-13 were evaluated by real-time PCR, Western blot, chromatin immunoprecipitation (ChIP), and luciferase reporter assays. We also evaluated Pit-1 overexpression with MMP-1 and MMP-13 knockdown in a severe combined immunodeficiency (SCID) mouse tumor xenograft model. Finally, by immunohistochemistry we correlated Pit-1 with MMP-1 and MMP-13 protein expression in 110 human breast tumors samples.ResultsOur data show that Pit-1 increases mRNA and protein of both MMP-1 and MMP-13 through direct transcriptional regulation. In SCID mice, knockdown of MMP-13 completely blocked lung metastasis in Pit-1-overexpressing MCF-7 cells injected into the mammary fat pad. In breast cancer patients, expression of Pit-1 was found to be positively correlated with the presence of both MMP-1 and MMP-13.ConclusionsOur data indicates that Pit-1 regulates MMP-1 and MMP-13, and that inhibition of MMP-13 blocked invasiveness to lung in Pit-1-overexpressed breast cancer cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-014-0505-8) contains supplementary material, which is available to authorized users.

Abstract 311: Inhibition Of Matrix Metalloproteinase-13 Dependent Protease-activated Receptor-1 Activation Attenuates Fibrotic Signaling

Heart failure (HF) is the leading cause of morbidity and mortality in the United States and is characterized by progressive myocardial fibrosis, pathologic remodeling and deteriorating cardiac function. Cardiac fibroblasts (CF) are largely responsible for the secretion of ECM proteins as well as cytokines and growth factors in the heart. Upon injury or pathologic stimulation, CF transition to a myofibroblast phenotype, leading to excess production of ECM proteins and pro-inflammatory cytokines. Previous studies in our lab have indicated a role for protease-activated receptor-1 (PAR-1), the most highly expressed GPCR on CF, in pathologic cardiac remodeling. In particular, we reported the novel cleavage of PAR-1 via matrix metalloproteinase-13 (MMP-13) and cardioprotective effects of MMP-13 inhibition in an acute model of HF. Therefore, we hypothesize that MMP-13 plays an important role in cardiac remodeling through activation of PAR-1, particularly in the pathologic transition of CF to myofibroblasts. To investigate this hypothesis, RNA was collected from hearts of mice infused with isoproterenol (ISO) for 7 days and concurrently treated with a specific MMP-13 inhibitor, WAY170523, or vehicle. To evaluate the effect of WAY170523, we used qRT-PCR to measure changes in the expression of fibrotic markers, COL1a1, COL3a1, and TGF-β. Inhibition of MMP-13 with WAY170523 attenuated expression of these genes compared to vehicle treated animals. We previously reported that stimulation of CF and cardiomyocytes with MMP-13 induces phosphorylation of ERK1/2, a member of the MAPK family known to play a role in cardiac hypertrophy, and treatment with a direct PAR-1 antagonist decreased the activation of ERK1/2. We have found that ERK1/2 phosphorylation is directly attenuated following inhibition of MMP-13 with WAY170523. Overall, these data suggest a role for MMP-13 dependent PAR-1 activation in pathologic myofibroblast transition and a potential therapeutic role for MMP-13 inhibition, possibly through its inhibition of ERK1/2 phosphorylation. Treatment with WAY170523 also attenuates markers of fibrosis in vivo, indicating a potential salutary role for MMP-13 inhibition in the treatment of HF.

Predicting and Virtually Screening the Selective Inhibitors of MMP-13 over MMP-1 by Molecular Descriptors and Machine Learning Methods

基质金属蛋白酶-13（MMP-13）为预防和治疗骨关节炎（OA）提供了充满希望的靶标．通过抑制剂来阻断MMP-13的活性将会对治疗OA疾病产生潜在的作用．然而，宽谱抑制剂同样抑制MMP家族的其它成员，特别是MMP-1，这将会导致肌与骨的综合症．因此，设计和发现潜在的MMP-13相对于MMP-1的高效选择性抑制剂，在对治疗OA新型药物的研发中具有相当重要的现实意义．本研究通过两种机器学习方法（ML）：支持向量机（SVM）和随机森林（RF）来建立分类模型，用于预测不同结构的MMP-13对MMP-1的选择性抑制剂．所建这些模型的预测效果都已经达到了令人满意的精度．在这两种ML模型中，RF对于MMP-13选择性抑制剂和非抑制剂的精度分别达到97-58％和100％．同时，与MMP-13对MMP-1的选择性抑制最相关的分子描述符也基于不同的特征选择方法被两种模型挑选出来．最后，用预测效果最好的RF模型虚拟筛选了ZINC数据库的“fragment—like”子集，从而得到了一系列潜在的候选药物．研究表明，机器学习方法，特别是RF方法，对于发现潜在的MMP-13选择性抑制剂十分有效．同时还得到了一些与MMP-13的选择性抑制相关的分子描述符．

MiR-126-5p regulates osteoclast differentiation and bone resorption in giant cell tumor through inhibition of MMP-13

Giant cell tumor (GCT) of bone is an aggressive skeletal tumor characterized by localized bone resorption. Matrix metalloproteinase-13 (MMP-13) is the principal proteinase expressed by the stromal cells of GCT (GCTSCs) and also considered to play a crucial role in formation of the osteolytic lesion in GCT. However, the exact mechanism of the regulation of MMP-13 expression in GCTSCs was unknown. In this study, we identified miR-126-5p was significantly downregulated in GCTSCs and affect osteoclast (OC) differentiation and bone resorption by repressing MMP-13 expression at the post-transcriptional level. Thus, our studies show that miR-126-5p plays an important physiological role in multinucleated giant cell formation and osteolytic lesion in GCT.

QSAR Rationale of Matrix Metalloproteinase Inhibition Activity in a Class of Carboxylic Acid Based Compounds

The matrix metalloproteinase-13 (MMP-13) inhibitory activities of carboxylic acid based compounds, in presence and absence of bovine serum albumin (BSA), have been analyzed quantitatively in terms of chemometric descriptors. The statistically validated quantitative structure-activity relationship (QSAR) models obtained through combinatorial protocol in multiple linear regression (CP-MLR) analysis and the participated descriptors in these models provided rationales to explain the inhibitory activities of these congeners. For MMP-13 inhibition activity, the identified descriptors (BEHm1, BELm1 and BEHm8) have highlighted the role of the atomic mass in terms of the highest and lowest eigenvalues derived from Burden matrix. The positive correlation with activity suggested that their higher values are desirable in improving the activity of a compound. Additionally, the descriptor C-027 representing R-CH-X type fragment in a molecular structure advocates the absence of such type of fragment for the improved activity. On the other hand presence of RCO-N< or >N-X=X type fragment (descriptor N-072) would be beneficiary to the MMP-13 inhibitory activity. The structural features, rationalized by the descriptors MSD (Balaban’s mean square distance index), nCrHR (number of ring tertiary C (sp3), H-047 (H attached to C1(sp3)/C0(sp2)) and H-050 (H attached to heteroatom) have imparted positive impact on the MMP-13 w/BSA inhibition activity. The atomic properties such as atomic polarizability and atomic Sanderson’s electronegativity have shown their positive impact on the activity via descriptors BELp4 and GATS3e in respective eigenvalues or lag. The other descriptors, MATS1m and MATS3e, have revealed the negative influence of atomic mass and electronegativity on the of MMP-13 w/BSA inhibition activity. The results obtained from CP-MLR analysis have been supported further through partial least-squares (PLS) study.

Nebulized hypertonic saline attenuates acute lung injury following trauma and hemorrhagic shock via inhibition of matrix metalloproteinase-13*

We hypothesized that aerosolized inhaled hypertonic saline given at the onset of resuscitation will decrease acute lung injury following hemorrhagic shock, by inhibiting the release of epithelial derived proinflammatory mediators. Animal study. Animal-care facility procedure room in a medical center. Adult male Sprague-Dawley rats. Rats underwent hemorrhagic shock followed by 2 hrs of resuscitation and 1 hr of observation. In the study group, nebulized hypertonic saline was delivered at the end of the shock period and after 1 hr and 2 hrs of resuscitation. Shock provoked acute lung injury, which was attenuated with inhaled hypertonic saline (1.56 ± 0.2 mg protein/mL vs. 0.95 ± 0.3 mg protein/mL bronchoalveolar lavage fluid, shock vs. shock + hypertonic saline, p < .01). Nebulized hypertonic saline reduced inflammation (cytokine-induced neutrophil chemoattractant-1 accumulation in bronchoalveolar lavage fluid 5999 ± 1267 pg/mL vs. 3342 ± 859 pg/mL, shock vs. shock + hypertonic saline, p = .006). Additionally, nebulized hypertonic saline inhibited matrix -metalloproteinase-13 accumulation in the bronchoalveolar lavage fluid (1513 ± 337 pg/mL bronchoalveolar lavage fluid vs. 230 ± 19 pg/mL, shock vs. shock + hypertonic saline, p = .009) and pretreatment with a matrix metalloproteinase-13 inhibitor was sufficient to attenuate postshock acute lung injury (1.42 ± 0.09 mg/mL vs. 0.77 ± 0.23 mg/mL bronchoalveolar lavage protein, shock vs. shock + matrix metalloproteinase-13 inhibitor CL-82198, p = .002). Inhaled hypertonic saline attenuates postshock acute lung injury by exerting an anti-inflammatory effect on the pulmonary epithelium, suggesting a new clinical strategy to treat acute lung injury/acute respiratory distress syndrome.

Selective Inhibition of Matrix Metalloproteinase-13 Increases Collagen Content of Established Mouse Atherosclerosis

Evidence has linked collagen loss with the onset of acute coronary events. This study tested the hypothesis that selective matrix metalloproteinase-13 (MMP-13) collagenase inhibition increases collagen content in already established and nascent mouse atheromas. In vitro and in situ experiments documented the selectivity and efficacy of an orally available MMP-13 inhibitor (MMP13i-A). In vivo observations monitored macrophage accumulation and MMP-13 activity using molecular imaging. After 10 weeks of MMP13i-A treatment, apolipoprotein E-deficient mice with evolving or established lesions exhibited reduced MMP-13 activity without affecting macrophage content, measured either by intravital microscopy or fluorescence reflectance imaging. Histological analysis indicated that MMP13-iA did not affect plaque size or macrophage or smooth muscle cell accumulation. Administration of MMP13i-A to mice with evolving or established atheromas substantially increased plaque interstitial collagen content in the intima and locally in the fibrous cap, compared with vehicle-treated controls. Analysis of collagen revealed thicker collagen fibers within the plaques of treated groups. Pharmacological MMP-13 inhibition yields collagen accumulation in plaques (a feature associated in humans with resistance to rupture), even in established plaques. This study, of considerable clinical relevance, furnishes new mechanistic insight into regulation of the plaque's extracellular matrix and validates molecular imaging for studying plaque biology.