Human Matrix Metalloproteinase Research Articles

Murine matrix metalloproteinase 13 and human matrix metalloproteinase 1 are involved in remodelling processes after myocardial infarction

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): German Centre for Cardiovascular Research (DZHK) Background Cardiovascular diseases such as myocardial infarction (MI) remain a leading cause of death in the world. Matrix metalloproteinases (MMPs) are not only essential for the cleavage of collagen, but are also modifying inflammatory cytokines. Thus, MMPs are playing a substantial role in the context of fibrotic as well as inflammatory processes in tissue remodelling after MI. Purpose Mmp13 is the highest expressed collagenase in the murine left ventricle (LV). After ligation of the left anterior descending artery (LAD) in mice, Mmp13 is highly upregulated in the LV. Therefore, the aim of this study was to characterize the role of MMP13 in MI. In the human LV, the highest expressed collagenase is MMP1. Single nucleotide polymorphisms (SNPs) in the promotor region of MMP1 can lead to alterations in gene expression level. Therefore, we analysed the genotype of 3 SNPs in about 2000 patients admitted to the emergency department with suspected MI to reveal associations with the development of MI and outcome after MI. Methods A MMP13-knockout (KO) mouse model was examined after induction of MI and gene expression analysis, histological staining, in-situ hybridisation and hemodynamic measurements were conducted. Mmp13 expression in different cardiac cell types was investigated at quiescent stage and under ischaemic conditions, to determine the cellular origin of Mmp13 expression. Out of the human cohort, 2 patient groups (non-MI and MI) were restricted. Hazard ratios adjusted to age and male sex were assessed, to evaluate risk for MI and risk for death after MI in dependency of the SNPs. Results After activation with ischaemic secretome of cardiomyocytes, the Mmp13 expression of macrophages (6.6-fold; p=0.0286) and fibroblasts (4.9-fold; p=0.0079) was significantly increased. Under stimulation with ischaemic secretome of fibroblasts, Mmp13 expression of macrophages (4.3-fold; p=0.0286) and leukocytes (2.3-fold; p=0.0260) was significantly elevated. Additionally, in situ hybridisation revealed Mmp13 expression in immune cells in the border zone of the infarct region. While the survival of WT mice was about 50%, only very few KO mice died after MI (p=0.0107). Moreover, KO mice showed benefits in cardiac function compared to WT littermates 28 days post-MI. In the human cohort, risk for death was significantly altered between the examined genotypes in 2 of 3 investigated SNPs, indicating a role of MMP1 in the remodelling process after MI. Conclusion High levels of Mmp13 after MI originate mainly from activated macrophages and from activated fibroblasts in the infarct border zone. MMP13-deficiency is beneficial for mice after MI, leading to an improved cardiac function 28 days post-MI compared to WT controls. Analysis of SNPs of the human functional homologue of Mmp13 – MMP1 – revealed an association of MMP1 with remodelling processes after MI.

Spectroscopic and computational studies on the binding interaction of biologically active thioridazine and perphenazine with human Matrix metalloproteinases 9

Matrix metaloproteinase (MMP)-9, as a neuroinflammation-facilitating element, plays a significant role in neurodegenerative disorders and could be considered an inflammation control target. This study is intended to survey the effects of thioridazine (TRZ) and phenothiazines (PRZ) as neuroleptic drugs on the performance of recombinant human MMP-9 (rhMMP-9) by using experimental (ultraviolet visible (UV–vis), fluorescence, synchronous fluorescence, fluorescence resonance energy transfer (FRET) spectroscopy, circular dichroism (CD), surface plasmon resonance (SPR), and theoretical techniques (molecular docking). For this purpose, rhMMP-9 was expressed and purified by utilizing recombinant E. coli bacteria. The kinetic analysis illustrated that the different concentrations of TRZ and PRZ could considerably decrease the catalytic function of rhMMP-9 through competitive and mixed inhibition mechanisms, respectively. The results illustrated the higher affinity of TRZ-MMP-9 with an inhibitory IC50 value of 10.7 µM in comparison with PRZ (12.1 µM) following the conformational alteration of the enzyme. In addition, hydrophobic binding and hydrogen bonds/van der Waals forces were suggested for TRZ-MMP-9 and PRZ-MMP-9, respectively. Finally, the molecular docking study demonstrated that TRZ and PRZ bind to the cavity in the catalytic and fibronectin domains, respectively, which supports the experimental data. This study provides some experimental and structural insights into the possible anti-inflammatory mechanisms of TRZ and PRZ drugs that suggest both drugs could be used as anti-MMP-9 drugs in neuroinflammatory conditions.

Multiple integrated computational approach to analyse wound healing potential of Symplocos racemosa bark as Matrix metalloproteinase inhibitors

The healing of wounds is the flagging concern in chronic wound cases especially when accompanied by pathogenic, diabetic comorbidities. Matrix metalloproteinases are associated with widespread pathological ailments, and the selective inhibitors for metalloproteinases can be of great interest in wound healing strategies. In the present research study, six constituents of Symplocos racemosa Roxb were evaluated for the docking aptitudes on human matrix metalloproteinase MMP 2 (PDB ID: 1QIB) and MMP 9 (PDB ID: 4H1Q) utilising Autodock Vina followed by the visualisation using Discovery studio (DS). The Pymol was used to generate the poses and the best binding pose was chosen for the docking aptitudes. 2D interactions and the 3D poses of the docked complex were accomplished using DS and LigPlot + software respectively. Working on SWISS ADME and OSIRIS software accomplished the physicochemical characteristics, absorption, distribution, metabolism, excretion, molecular properties, bioactivity score, and toxicity predictions. The molecule’s physiochemical investigations discovered that all of the ligands comply with Lipinski’s rule of five except compound 6, which deviated with two violations. Docking studies against 4H1Q revealed that compounds 1, 3, 5 and 6 exhibited maximum interactions with the target protein, with the free binding energies of −8.3 kJ Mol−1, −9.3 kJ Mol−1, −7.2 kJ Mol−1 and −11.0 kJ Mol−1 respectively. In case of the 1QIB target, compounds 1, 3 and 6 displayed remarkable binding energies of −8.7 kJ mol−1, −9.0 kJ mol−1 and −8.8 kJ mol−1. Bioactivity prediction study revealed that all of the selected Phytoconstituents displayed incredible Bioactivity scores. None of the selected chemical compounds was found to be irritant to the skin as discovered by toxicity studies. The contacts of the ligand-protein complex during the simulation studies revealed that the H-bond interactions of the ligands with LEU188, ALA189, GLN402, ARG420, MET422, PRO421, and ARG424 of 4H1Q were stable for more than 30% of the simulation time. It was thus concluded that the tested compounds predominantly compounds 1, 5 and 6 might rank among the vital supplementary lead drugs in chronic wounds and healing complexities. It is also worth noting the potential aptitude of the compound 3, however, its toxicity concern must be considered.

Discovery of 1-(5-bromopyrazin-2-yl)-1-[3-(trifluoromethyl)benzyl]urea as a promising anticancer drug via synthesis, characterization, biological screening, and computational studies

Cancer and different types of tumors are still the most resistant diseases to available therapeutic agents. Finding a highly effective anticancer drug is the first target and concern of thousands of drug designers. In our attempts to address this concern, a new pyrazine derivative, 1-(5-bromopyrazin-2-yl)-1-[3-(trifluoromethyl)benzyl]urea (BPU), was designed via structural optimization and synthesized to investigate its anticancer/antitumor potential. The in-vitro anticancer properties of BPU were evaluated by MTT assay using selected cell lines, including the Jurkat, HeLa, and MCF-7 cells. The Jurkat cells were chosen to study the effect of BPU on cell cycle analysis using flow cytometry technique. BPU exhibited an effective cytotoxic ability in all the three cell lines assessed. It was found to be more prominent with the Jurkat cell line (IC50 = 4.64 ± 0.08 µM). When it was subjected to cell cycle analysis, this compound effectively arrested cell cycle progression in the sub-G1 phase. Upon evaluating the antiangiogenic potential of BPU via the in-vivo/ex-vivo shell-less chick chorioallantoic membrane (CAM) assays, the compound demonstrated very significant findings, revealing a complementary supportive action for the compound to act as a potent anticancer agent through inhibiting blood vessel formation in tumor tissues. Moreover, the docking energy of BPU computationally scored − 9.0 kcal/mol with the human matrix metalloproteinase 2 (MMP-2) and − 7.8 kcal/mol with the human matrix metalloproteinase 9 (MMP-9), denoting promising binding results as compared to the existing drugs for cancer therapy. The molecular dynamics (MD) simulation outcomes showed that BPU could effectively bind to the previously-proposed catalytic sites of both MMP-2 and MMP-9 enzymes with relatively stable statuses and good inhibitory binding abilities and parameters. Our findings suggest that the compound BPU could be a promising anticancer agent since it effectively inhibited cell proliferation and can be selected for further in-vitro and in-vivo investigations. In addition, the current results can be extensively validated by conducting wet-lab analysis so as to develop novel and better derivatives of BPU for cancer therapy with much less side effects and higher activities.

Loss of MTA2-mediated downregulation of PTK7 inhibits hepatocellular carcinoma metastasis progression by modulating the FAK-MMP7 axis.

The expression of metastasis tumor-associated protein 2 (MTA2) and protein tyrosine kinase 7 (PTK7) is associated with hepatocellular carcinoma (HCC) progression. However, the functional effect and mechanism through which MTA2 regulates PTK7-mediated HCC progression remains unclear. Here, we found that MTA2 knockdown significantly down-regulated PTK7 expression in HCC cells (SK-Hep-1 and PLC/PRF/5). Data from the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases show that the PTK7 expression level was higher in HCC tissues than in normal liver tissues. In HCC patients, the PTK7 expression level clearly correlated with tumor stage and grade, lower overall survival (OS) correlated positively with MTA2 level, and PTK7 expression acted as a downstream factor for MTA2 expression. In addition, matrix metalloproteinase 7 (MMP7) expression was closely regulated by PTK7, and the mRNA and protein expression levels of MTA2 and PTK7 correlated positively with lower OS. MMP7 downregulation by PTK7 knockdown clearly decreased the migration and invasion abilities of HCC cells. In HCC cells, recombinant human MMP7 reversed the PTK7 knockdown-induced suppression of migration and invasion. Furthermore, deactivation of FAK using siFAK or FAK inhibitor (PF-573228, PF) synergistically contributed to PTK7 knockdown-inhibited FAK activity, MMP7 expression, and the migration and invasion abilities of HCC cells. Collectively, our findings show that PTK7 mediates HCC progression by regulating the MTA2-FAK-MMP7 axis and may be a diagnostic value for HCC patients.

Generation of Protease Inhibitory Antibodies by Functional In Vivo Selection.

Targeting dysregulated protease expression and/or abnormal substrate proteolysis, highly selective inhibition of pathogenic proteases by monoclonal antibodies (mAbs) presents an attractive therapeutic approach for the treatment of diseases including cancer. Herein, we report a functional selection methodfor protease inhibitory mAbs by periplasmic co-expression of three recombinant proteins-a protease of interest, an antibody Fab library, and a modified β-lactamase TEM-1. We validate this approach by isolation of highly selective and potent mAbs inhibiting human matrix metalloproteinase 9 (MMP9).

In vitro screening of ninety-five plant extracts for inhibition of Clostridium histolyticum collagenase and Bacillus polymyxa metalloprotease- analogous human MMPs

Introduction and Aim: Matrix metalloproteinases (MMPs) are involved in many human diseases. Medicinal plants and their different constituents are utilized as therapeutic agents. In the present work we report the total phenolics content and screening of ninety five plant extracts for inhibitory activities against Clostridium histolyticum collagenase (ChC) and Bacillus polymyxa metalloprotease (BpM). Materials and Methods: Total phenolics content from plant methanolic extracts were assessed using Folin-Ciocalteau assay. Screening of plant extracts for ChC and BpM inhibitory activities was performed using dot blot assay on X-ray film. Results: G. superba fruit was observed to have the highest phenolics (257.58 ± 0.75 mg GAE /g tissue) content while C. nurvala stem was observed to have the lowest phenolics (0.468 ± 0.003 mg GAE /g tissue) content. Thirty two plants showed ChC inhibitory activities while twenty seven plants showed BpM inhibitory activities. From those plants twenty five plants have common ChC and BpM inhibitory activities. Most of plant barks were found to have a high amount of total phenolics as compared to other plant tissues while the most of plant leaves were found to have low amounts of total phenolics. Most of barks were found to exhibit inhibitory activities as compared to other tissue. Only 6 different leaves were found to exhibit inhibitory activity out of 42 different leaves. None of the root extracts exhibited inhibitory activities. Conclusion: The inhibitory activity exhibiting plant extracts could be used as a significant source for the determination of novel drug candidates to treat MMPs related diseases.

Upregulated TIMP1 facilitates and coordinates myometrial contraction by decreasing collagens and cell adhesive capacity during human labor.

Myometrial contraction is one of the key events involved in parturition. Increasing evidence suggests the importance of the extracellular matrix (ECM) in this process, in addition to the functional role of myometrial smooth muscle cells, and our previous study identified an upregulated tissue inhibitor of metalloproteinase 1 (TIMP1) in human laboring myometrium compared to nonlabor samples. This study aimed to further explore the potential role of TIMP1 in myometrial contraction. First, we confirmed increased myometrial TIMP1 levels in labor and during labor with cervical dilation using transcriptomic and proteomic analyses, followed by real-time PCR, western blotting, and immunohistochemistry. Then, a cell contraction assay was performed to verify the decreased contractility after TIMP1 knockdown in vitro. To further understand the underlying mechanism, we used RNA-sequencing analysis to reveal the upregulated genes after TIMP1 knockdown; these genes were enriched in collagen fibril organization, cell adhesion, and ECM organization. Subsequently, a human matrix metalloproteinase (MMP) array and collagen staining were performed to determine the TIMPs, MMPs and collagens in laboring and nonlabor myometrium. A real-time cell adhesion assay was used to detect cell adhesive capacity. The results showed upregulated MMP8 and MMP9, downregulated collagens, and attenuated cell adhesive capacity in laboring myometrium, while lower MMP levels and higher collagen levels and cell adhesive capacity were observed in nonlabor. Moreover, TIMP1 knockdown led to restoration of cell adhesive capacity. Together, these results indicate that upregulated TIMP1 during labor facilitates and coordinates myometrial contraction by decreasing collagen and cell adhesive capacity, which may provide effective strategies for the regulation of myometrial contraction.

Role of some structural features in EPS from microalgae stimulating collagen production by human dermal fibroblasts

ABSTRACTExopolysaccharides (EPS) from the microalgae Porphyridium cruentum, Chrysotila dentata, Pavlova sp., Diacronema sp., Glossomastix sp., Phaeodactylum tricornutum, and Synechococcus sp. were isolated and depolymerized. First, EPS were submitted to a high pressure pre-treatment step, followed by a solid acid-catalyzed hydrolysis step carried out in a batch or recycle fixed-bed reactor, using a strong acidic cation-exchange resin. Twenty-eight different EPS forms were thus obtained. After characterization of their main structural features (weight- and number-averaged molecular weight, polydispersity index, sulfate and uronic acid contents), we investigated the structure–function relationship of their pro-collagen activity. We found that native microalgae EPS were able to inhibit until 27% of human matrix metalloproteinase-1 (MMP-1) activity while the depolymerized forms were able to enhance collagen production by two different human fibroblast lines, used as cell models due to their major role in dermal collagen biosynthesis. The most active EPS forms, obtained by depolymerization in the recycle fixed-bed reactor of D. ennorea and Glossomastix sp. EPS, led to 390% increase in collagen production. Finally, principal component (PCA) and Pearson analyses indicated that MMP-1 inhibition was strongly correlated to the sulfate group content of EPS whereas collagen production by fibroblasts was mostly related to their proportion of low molecular weight polysaccharides (<10 kDa). Uronic acid content of EPS was also shown essential but only if the size of EPS was reduced in the first place. Altogether, these results gave new insights of the dermo-cosmetic potential of microalgae EPS as well as the key parameters of their activity.

Green Synthesis of Genistein-Fortified Zinc Ferrite Nanoparticles as a Potent Hepatic Cancer Inhibitor: Validation through Experimental and Computational Studies.

In hepatic cancer, precancerous nodules account for damage and inflammation in liver cells. Studies have proved that phyto-compounds based on biosynthetic metallic nanoparticles display superior action against hepatic tumors. This study targeted the synthesis of genistein-fortified zinc ferrite nanoparticles (GENP) trailed by anticancer activity assessment against diethylnitrosamine and N-acetyl-2-aminofluorene induced hepatic cancer. The process of nucleation was confirmed by UV/VIS spectrophotometry, X-ray beam diffraction, field-emission scanning electron microscopy, and FT-IR. An in vitro antioxidant assay illustrated that the leaves of Pterocarpus mildbraedii have strong tendency as a reductant and, in the nanoformulation synthesis, as a natural capping agent. A MTT assay confirmed that GENP have a strong selective cytotoxic potential against HepG2 cancer cells. In silico studies of genistein exemplified the binding tendency towards human matrix metalloproteinase comparative to the standard drug marimastat. An in vivo anticancer evaluation showed that GENP effectively inhibit the growth of hepatic cancer by interfering with hepatic and non-hepatic biochemical markers.

Effect of Sodium Hypochlorite 0.05% on MMP-9 Extracellular Release in Chronic Wounds.

In chronic wounds, high concentrations of matrix metalloproteinases (MMPs) can cause excessive proteolysis and slow wound healing. Consequently, restoring a proper MMP balance can help reduce the risk of a chronic wound. An antiseptic solution containing 0.05% sodium hypochlorite (Amukine Med 0.05%, Angelini S.p.A.; hereafter termed NaClO solution) is available on the market. The NaClO solution was proven effective and safe in managing infected skin wounds. To further characterize its activity, this study evaluated the in vitro activity of the NaClO solution on the monocyte release of MMPs. Human monocytic THP-1 (ATCC® TIB-202™) cell lines were differentiated into macrophages and treated with different concentrations of NaClO (from 0.05% to 5 × 10-7%). In addition, the THP-1 cell line was stimulated with wound fluid (WF) from patients with active venous leg ulcers in the inflammatory phase. The effect of NaClO (0.025-0.0062%) was also evaluated on healthy human peripheral blood serum samples. The effects of treatments on the gelatinolytic activity of MMP-9 were evaluated by gelatin zymography. The effects on MMPs release were evaluated through the Pro™ Human MMP 9-plex Assay. An exploratory scratch wound healing assay was also performed. The NaClO solution reduced the gelatinolytic activity of MMP-9 and its activated form. The downregulation of MMP-9 gelatinolytic activity was also observed in peripheral blood serum. The MMPs profile showed a reduction in MMP-1 release (p < 0.05) and a slight reduction of the release of MMP-9 and MMP-12 after the treatment with LPS and the NaClO solution. A slight improvement in wound healing was observed after macrophage activation and treatment with the NaClO solution. The results obtained suggest a possible ability of the NaClO solution to modulate the proteolytic pathways in the wound microenvironment, further characterizing its activity and use in clinical practice during wound care.

Identification of small molecule inhibitors against MMP-14 via High-Throughput screening

Matrix metalloproteinases (MMPs) are involved in various cellular events in physiology and pathophysiology through endopeptidases activity. The expression levels and activities of most MMPs remain minimal in the normal conditions, whereas some MMPs are significantly activated in pathological conditions such as cancer and neovascularization. Hence, MMPs are considered as both diagnostic markers and potential targets for therapeutic agents. Twenty-three known human MMPs share a similar active site structure with a zinc-binding motif, resulting in lack of specificity. Therefore, the enhancement of target specificity is a primary goal for the development of specific MMP inhibitors. MMP-14 regulates VEGFA/VEGFR2-system through cleavage of the non-functional VEGFR1 in vascular angiogenesis. In this study, we developed a fluorescence-based enzymatic assay using a specific MMP-14 substrate generated from VEGFR1 cleavage site. This well optimized assay was used as a primary screen method to identify MMP-14 specific inhibitors from 1,200 Prestwick FDA-approved drug library. Of ten initial hits, two compounds showed IC50 values below 30 µM, which were further validated by direct binding analysis using surface plasmon resonance (SPR). Clioquinol and chloroxine, both of which contain a quinoline structure, were identified as MMP-14 inhibitors. Five analogs were tested, four of which were found to be completely devoid of inhibitory activity. Clioquinol exhibited selectivity towards MMP-14, as it showed no inhibitory activity towards four other MMPs.

Amelioration of Pulmonary Fibrosis by Matrix Metalloproteinase-2 Overexpression

Idiopathic pulmonary fibrosis is a progressive and fatal disease with a poor prognosis. Matrix metalloproteinase-2 is involved in the pathogenesis of organ fibrosis. The role of matrix metalloproteinase-2 in lung fibrosis is unclear. This study evaluated whether overexpression of matrix metalloproteinase-2 affects the development of pulmonary fibrosis. Lung fibrosis was induced by bleomycin in wild-type mice and transgenic mice overexpressing human matrix metalloproteinase-2. Mice expressing human matrix metalloproteinase-2 showed significantly decreased infiltration of inflammatory cells and inflammatory and fibrotic cytokines in the lungs compared to wild-type mice after induction of lung injury and fibrosis with bleomycin. The computed tomography score, Ashcroft score of fibrosis, and lung collagen deposition were significantly reduced in human matrix metalloproteinase transgenic mice compared to wild-type mice. The expression of anti-apoptotic genes was significantly increased, while caspase-3 activity was significantly reduced in the lungs of matrix metalloproteinase-2 transgenic mice compared to wild-type mice. Active matrix metalloproteinase-2 significantly decreased bleomycin-induced apoptosis in alveolar epithelial cells. Matrix metalloproteinase-2 appears to protect against pulmonary fibrosis by inhibiting apoptosis of lung epithelial cells.

Matrix Metalloproteinase-1 Expression in Fibroblasts Accelerates Dermal Aging and Promotes Papilloma Development in Mouse Skin

Fragmentation, disorganization, and depletion of the collagen-rich dermal extracellular matrix are hallmarks of aged human skin. These deleterious alterations are thought to critically mediate many of the prominent clinical attributes of aged skin, including thinning, fragility, impaired wound healing, and a propensity for carcinoma. Matrix metalloproteinase-1 (MMP1) initiates the cleavage of collagen fibrils and is significantly increased in dermal fibroblasts in aged human skin. To investigate the role of elevated MMP1 in skin aging, we generated a conditional bitransgenic mouse (type I collagen alpha chain 2; human MMP1 [Col1a2;hMMP1]) that expresses full-length, catalytically active hMMP1 in dermal fibroblasts. hMMP1 expression is activated by a tamoxifen-inducible Cre recombinase that is driven by the Col1a2 promoter and upstream enhancer. Tamoxifen induced hMMP1 expression and activity throughout the dermis Col1a2:hMMP1 mice. At 6 months of age, Col1a2;hMMP1 mice displayed loss and fragmentation of dermal collagen fibrils, which was accompanied by many of the features of aged human skin, such as contracted fibroblast morphology, reduced collagen production, increased expression of multiple endogenous MMPs, and proinflammatory mediators. Interestingly, Col1a2;hMMP1 mice displayed substantially increased susceptibility to skin papilloma development. These data demonstrate that fibroblast expression of hMMP1 is a critical mediator of dermal aging and creates a dermal microenvironment that promotes keratinocyte tumor development.

An MMP-9 exclusive neutralizing antibody attenuates blood-brain barrier breakdown in mice with stroke and reduces stroke patient-derived MMP-9 activity

Rapid upregulation of matrix metalloproteinase 9 (MMP-9) leads to blood-brain barrier (BBB) breakdown following stroke, but no MMP-9 inhibitors have been approved in clinic largely due to their low specificities and side effects. Here, we explored the therapeutic potential of a human IgG monoclonal antibody (mAb), L13, which was recently developed with exclusive neutralizing specificity to MMP-9, nanomolar potency, and biological function, using mouse stroke models and stroke patient samples. We found that L13 treatment at the onset of reperfusion following cerebral ischemia or after intracranial hemorrhage (ICH) significantly reduced brain tissue injury and improved the neurological outcomes of mice. Compared to control IgG, L13 substantially attenuated BBB breakdown in both types of stroke model by inhibiting MMP-9 activity-mediated degradations of basement membrane and endothelial tight junction proteins. Importantly, these BBB-protective and neuroprotective effects of L13 in wild-type mice were comparable to Mmp9 genetic deletion and fully abolished in Mmp9 knockout mice, highlighting the in vivo target specificity of L13. Meanwhile, ex vivo co-incubation with L13 significantly neutralized the enzymatic activities of human MMP-9 in the sera of ischemic and hemorrhagic stroke patients, or in the peri-hematoma brain tissues from hemorrhagic stroke patients. Overall, we demonstrated that MMP-9 exclusive neutralizing mAbs constitute a potential feasible therapeutic approach for both ischemic and hemorrhagic stroke.

Inhibition of matrix metalloproteinases by HIV-1 integrase strand transfer inhibitors.

More than fifteen million women with the human immunodeficiency virus type-1 (HIV-1) infection are of childbearing age world-wide. Due to improved and affordable access to antiretroviral therapy (ART), the number of in utero antiretroviral drug (ARV)-exposed children has exceeded a million and continues to grow. While most recommended ART taken during pregnancy suppresses mother to child viral transmission, the knowledge of drug safety linked to fetal neurodevelopment remains an area of active investigation. For example, few studies have suggested that ARV use can be associated with neural tube defects (NTDs) and most notably with the integrase strand transfer inhibitor (INSTI) dolutegravir (DTG). After risk benefit assessments, the World Health Organization (WHO) made recommendations for DTG usage as a first and second-line preferred treatment for infected populations including pregnant women and those of childbearing age. Nonetheless, long-term safety concerns remain for fetal health. This has led to a number of recent studies underscoring the need for biomarkers to elucidate potential mechanisms underlying long-term neurodevelopmental adverse events. With this goal in mind, we now report the inhibition of matrix metalloproteinases (MMPs) activities by INSTIs as an ARV class effect. Balanced MMPs activities play a crucial role in fetal neurodevelopment. Inhibition of MMPs activities by INSTIs during neurodevelopment could be a potential mechanism for adverse events. Thus, comprehensive molecular docking testing of the INSTIs, DTG, bictegravir (BIC), and cabotegravir (CAB), against twenty-three human MMPs showed broad-spectrum inhibition. With a metal chelating chemical property, each of the INSTI were shown to bind Zn++ at the MMP's catalytic domain leading to MMP inhibition but to variable binding energies. These results were validated in myeloid cell culture experiments demonstrating MMP-2 and 9 inhibitions by DTG, BIC and CAB and even at higher degree than doxycycline (DOX). Altogether, these data provide a potential mechanism for how INSTIs could affect fetal neurodevelopment.

Plant-derived Bioactive Compounds Targeting MMP-9 for Treatment of Endometrial Carcinoma: An In silico Study

Endometrial cancer, also called uterine cancer, is the second most common and fourth leading cause of death among gynecological problems in the world. Matrix metalloproteinase-9 (MMP-9) has been found to play an important role in the formation and metastases of endometrial cancer. Thus, this study investigates the naturally anti-inflammatory compounds available from plant sources that can target the MMP-9 by various in silico approaches. The target 1L6J (Crystal structure of human matrix metalloproteinase-9) structure was retrieved from the PDB database. Five plant compounds were selected based on Lipinski’s rule of five using the PubChem database. The pharmacokinetics, ADMET properties, and biological activity of these compounds were predicted computationally using databases such as SWISS-ADME, pkCSM, and PASS. Comparative analysis of the bioactive compounds with the target was performed by AutoDock 4.2.6. The target residues interacting with the compounds were visualized in a 2D manner using LigPlot v.2.2. Based on the docking results, the compounds namely lupeol, ß amyrin, ursolic acid, kaempferol, and quercetin exhibited binding energy value of -10.68 kcal/mol, -9.4 kcal/mol, -9.02 kcal/mol, -8.72 kcal/mol and -8.45 kcal/mol respectively; whereas the control drug, Megestrol acetate showed -8.84 kcal/mol kcal thus, indicating that few of the selected compounds were seen to have better binding energy comparative to the control drug. We conclude that lupeol, ß amyrin, and ursolic acid can be potent inhibitors of target MMP-9 with the least side effects. Therefore, by in vitro and in vivo studies, these compounds can be effectively explored to evaluate the level of toxicity and efficacy to develop more effective treatments for endometrial carcinoma.

Sodium carboxymethyl cellulose-based extrafibrillar demineralization to optimize dentin bonding durability.

To investigate the effects of a sodium carboxymethyl cellulose (SCMC)-based extrafibrillar demineralization conditioner on dentin bonding durability and explore the possible mechanisms. The SCMC-based extrafibrillar demineralization conditioner was facilely developed by dissolving SCMC into deionized water at an appropriate concentration. A single layer collagen mineralization/demineralization model was designed to visualize extrafibrillar demineralization in detail. Dentin surfaces of human third molars were conditioned with 3 % SCMC or 37 % phosphoric acid (PA). The morphology, composition, and mechanical properties of conditioned dentin from each group were characterized. To evaluate dentin bonding performance, SCMC- and PA- conditioned dentin were applied with adhesive restoration using the dry-bonding technique. The microtensile bond strength (MTBS), interface nanoleakage, and in situ zymography were measured after 24h of water storage, 10,000 thermocycles, or one month of collagenase aging. The inhibitory effect of SCMC on recombinant human matrix metalloproteinase-2 (rhMMP-2) and cell toxicity were also investigated. After SCMC conditioning, both demineralization of extrafibrillar minerals and retention of intrafibrillar minerals were observed in the single layer collagen model and the dentin ultrastructure. The mechanical properties of SCMC-conditioned dentin were largely preserved. Compared with PA, SCMC conditioning produced greater MTBS values and less nanoleakage expression after aging. Endogenous gelatinolytic activity was suppressed in SCMC-conditioned dentin. In addition to being nontoxic, the inhibition of rhMMP-2 by SCMC was confirmed to be dose-dependent. From the perspective of minimal intervention, the SCMC-based extrafibrillar demineralization conditioner could improve dentin bonding durability, suggesting a promising strategy to extend the service life of adhesive restorations.

Activation and Inhibition of Human Matrix Metalloproteinase-9 (MMP9) by HOCl, Myeloperoxidase and Chloramines.

Matrix metalloproteinase-9 (MMP9, gelatinase B) plays a key role in the degradation of extracellular-matrix (ECM) proteins in both normal physiology and multiple pathologies, including those linked with inflammation. MMP9 is excreted as an inactive proform (proMMP9) by multiple cells, and particularly neutrophils. The proenzyme undergoes subsequent processing to active forms, either enzymatically (e.g., via plasmin and stromelysin-1/MMP3), or via the oxidation of a cysteine residue in the prodomain (the “cysteine-switch”). Activated leukocytes, including neutrophils, generate O2− and H2O2 and release myeloperoxidase (MPO), which catalyzes hypochlorous acid (HOCl) formation. Here, we examine the reactivity of HOCl and a range of low-molecular-mass and protein chloramines with the pro- and activated forms of MMP9. HOCl and an enzymatic MPO/H2O2/Cl− system were able to generate active MMP9, as determined by fluorescence-activity assays and gel zymography. The inactivation of active MMP9 also occurred at high HOCl concentrations. Low (nM—low μM) concentrations of chloramines formed by the reaction of HOCl with amino acids (taurine, lysine, histidine), serum albumin, ECM proteins (laminin and fibronectin) and basement membrane extracts (but not HEPES chloramines) also activate proMMP9. This activation is diminished by the competitive HOCl-reactive species, methionine. These data indicate that HOCl-mediated oxidation and MMP-mediated ECM degradation are synergistic and interdependent. As previous studies have shown that modified ECM proteins can also stimulate the cellular expression of MMP proteins, these processes may contribute to a vicious cycle of increasing ECM degradation during disease development.

New insights into the role of matrix metalloproteinase 3 (MMP3) in bone.

The Matrix Metalloproteinases are important regulators of bone metabolism and can influence bone mass and bone remodeling. We investigate the role of Matrix Metalloproteinase 3 (MMP3) on bone in mice, by using Mmp3 knockout (Mmp3 KO) in the context of estrogen deficiency, and in human, by analyzing the association of promoter polymorphism with bone mineral density in postmenopausal women and with MMP3 expression. We presented evidence in this paper that Mmp3 KO significantly increases trabecular bone mass and trabecular number and does not affect cortical bone thickness. We also found that Mmp3 KO protects from the deleterious effects of ovariectomy on bone mineral density in mice by preventing deterioration of bone microarchitecture. The effect of Mmp3 KO does not involve bone formation parameters but instead acts by inhibition of bone resorption, leading to a reduced bone loss associated to ovariectomy. By studying a human cohort, we found that a polymorphism located in the promoter of the human MMP3 gene is associated with bone mineral density in postmenopausal women and found that MMP3 rs632478 promoter variants are associated with change in promoter activity in transfection experiments. In conclusion MMP3, although weakly expressed in bone cells, could be one of the important regulators of sex hormone action in bone and whose activity could be targeted for therapeutic applications such as in Osteoporosis.