MMP inhibition as a novel strategy for extracellular matrix preservation during whole liver decellularization.

As the only reliable treatment option for end-stage liver diseases, conventional liver transplantation confronts major supply limitations. Accordingly, the decellularization of discarded livers to produce bioscaffolds that support recellularization with progenitor/stem cells has emerged as a promising translational medicine approach. The success of this approach will substantially be determined by the extent of extracellular matrix (ECM) preservation during the decellularization process. Here, we assumed that the matrix metalloproteinase (MMP) inhibition could reduce the ECM damage during the whole liver decellularization of an animal model using a perfusion-based system. We demonstrated that the application of doxycycline as an MMP inhibitor led to significantly higher preservation of collagen, glycosaminoglycans, and hepatic growth factor (HGF) contents, as well as mechanical and structural features, including tensile strength, fiber integrity, and porosity. Notably, produced bioscaffolds were biocompatible and efficiently supported cell viability and proliferation in vitro. We also indicated that produced bioscaffolds efficiently supported HepG2 cell function upon seeding onto liver ECM discs using albumin and urea assay. Additionally, MMP inhibitor pretreated decellularized livers were more durable in contact with collagenase digestion compared to control bioscaffolds in vitro. Using zymography, we confirmed the underlying mechanism that results in these promising effects is through the inhibition of MMP2 and MMP9. Overall, we demonstrated a novel method based on MMP inhibition to ameliorate the ECM structure and composition preservation during liver decellularization as a critical step in fabricating transplantable bioengineered livers.

Matrix Metalloproteinase-9 inhibitors as therapeutic drugs for traumatic brain injury.

Traumatic brain injury (TBI) is one of the leading causes of morbidity and mortality among young adults and the elderly. In the United States, TBI is responsible for around 30 percent of all injuries brought on by injuries in general. Vasogenic cerebral edema due to blood-brain barrier (BBB) dysfunction and the associated elevation of intracranial pressure (ICP) are some of the major causes of secondary injuries following traumatic brain injury. Matrix metalloproteinase-9 (MMP-9) is a therapeutic target for being an enzyme that degrades the proteins that make up a part of the microvascular basal lamina as well as inter-endothelial tight junctions of the blood-brain barrier. MMP-9-mediated BBB dysfunctions and the compromise of the BBB is a major pathway that leads the development of vasogenic cerebral edema, elevation of ICP, poor cerebral perfusion and brain herniation following traumatic brain injury. That makes MMP-9 an effective therapeutic target and endogenous or exogenous MMP-9 inhibitors as therapeutic drugs for preventing secondary brain damage after traumatic brain injury. Although our understanding of the mechanisms that underlie the primary and secondary stages of damage following a TBI has significantly improved in recent years, such information has not yet resulted in the successful development of novel pharmacological treatment options for traumatic brain injury. Recent pre-clinical and/or clinical studies have demonstrated that there are several compounds with specific or non-specific MMP-9 inhibitory properties either directly binding and inhibiting MMP-9 or by indirectly inhibiting MMP-9, with potential as therapeutic agents for traumatic brain injury. This article reviews the efficacy of several such medications and potential agents that include endogenous and exogeneous compounds that are at various levels of research and development. MMP-9-based therapeutic drug development has enormous potential in the pharmacological treatment of cerebral edema and/or neuronal injury resulting from traumatic brain injury.

Engineering Selective TIMPs Using a Counter-Selective Screening Strategy.

The yeast surface display platform provides a powerful approach for screening protein diversity libraries to identify binders with an enhanced affinity toward a binding partner. Here, we describe an adaptation of the approach to identify binders with enhanced specificity toward one among multiple closely related binding partners. Specifically, we describe methods for engineering selective matrix metalloproteinase (MMP) inhibitors via yeast surface display of a tissue inhibitor of metalloproteinase (TIMP) diversity library coupled with a counter-selective screening strategy. This protocol may also be employed for developing selective protein binders or inhibitors toward other targets.

Acute hypoxia promotes the liver angiogenesis of largemouth bass (Micropterus salmoides) by HIF - Dependent pathway.

A 24-h hypoxia exposure experiment was conducted to determine how hypoxia exposure induce liver angiogenesis in largemouth bass. Nitrogen (N2) was pumped into water to exclude dissolved oxygen into 1.2 ± 0.2 mg/L, and liver tissues were sampled during hypoxia exposure of 0 h, 4 h, 8 h, 12 h, 24 h and re-oxygenation for 12 h. Firstly, the results showed that hypoxia exposure promoted the angiogenesis occurrence by immunohistochemical analysis of vascular endothelial growth factor receptor 2 (VEGFR2). Secondly, the concentration of vasodilation factor increased and it's activity was elevated during 8 h exposure, such as nitric oxide (NO) and nitric oxide synthase (NOS) (p < 0.05). Thirdly, hypoxia exposure promoted angiogenesis through up-regulation the expression of matrix metalloproteinase 2 (MMP-2), jagged, protein kinase B (AKT), phosphoinositide-3-kinase (PI3K), mitogen-activated protein kinase (MAPK) at 4 h; contrarily, the expression of inhibiting angiogenesis genes presented up-regulated at 8 h (p < 0.05), such as matrix metalloproteinase inhibitor-2 (TIMP-2), matrix metalloproteinase inhibitor-3 (TIMP-3). Finally, the genes and proteins that regulate angiogenesis presented obvious chronological order. Parts of them promoted the budding and extension of blood vessels were up-regulated during 4 h-8 h (p < 0.05), such as vascular endothelial growth factor a (VEGFA), VEGFR2, monocarboxylic acid transporter 1 (MCT1), CD147, prolyl hydroxylase (PHD), nuclear factor kappa-B (NF-κB); other part of them promoted blood vessel maturation were highly expressed during 12 h-24 h (p < 0.05), such as angiogenin-1 (Ang-1) and angiogenin-2 (Ang-2). In short, acute hypoxia can promote the liver angiogenesis of largemouth bass by HIF - dependent pathway.

Verapamil inhibited the development of ureteral stricture by blocking CaMK II-mediated STAT3 and Smad3/JunD pathways.

BACKGROUND: Ureteral stricture (US) is a fibrotic process that leads to urinary tract obstruction and even kidney damage, with the characteristic of reduced extracellular matrix (ECM) degradation and increased collagen synthesis. Verapamil, as a calcium channel blocker, was reported to prevent scar formation. Our work aimed to investigate the biological effects and mechanism of verapamil in US. METHODS: Fibroblasts were subjected to transforming growth factor-beta 1 (TGF-ß1) to stimulate collagen synthesis, and the messenger ribonucleic acid (mRNA) and protein expressions in fibroblasts were assessed using quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. The location of phosphorylation-signal transducer and activator of transcription 3 (p-STAT3) and Jund proto-oncogene subunit (JunD) in fibroblasts were determined by immunofluorescence (IF). The binding relationship between signal transducer and activator of transcription 3 (STAT3) and collagen type I alpha1 (COL1A1)/collagen type III alpha 1 chain (COL3A1) and the binding relationship between JunD and tissue inhibitor of metalloproteinases-1 (TIMP-1) were verified by dual luciferase reporter gene and chromatin Immunoprecipitation (ChIP) assays. RESULTS: Herein, we found that verapamil could inhibit TGF-ß1/Ca2 + /calmodulin-dependent protein kinase II (CaMK II)-mediated STAT3 activation in fibroblasts, and STAT3 inhibition repressed collagen production. In addition, verapamil could inhibit TGF-ß1/CaMK II-mediated Mothers against DPP homolog 3 (Smad3)/JunD pathway activation in fibroblasts, and JunD silencing inhibited TIMP1 (a matrix metalloproteinase inhibitor) expression. Our subsequent experiments revealed that STAT3 bound with COL1A1 promoter and COL3A1 promoter and activated their transcription, and JunD bound with TIMP1 promoter and activated its transcription. Moreover, as expected, STAT3 activation could eliminate the inhibitory effect of verapamil treatment on TGF-ß1-induced collagen production in fibroblasts, and JunD overexpression reversed the inhibitory effect of verapamil treatment on TGF-ß1-induced TIMP1 expression in fibroblasts. CONCLUSION: Verapamil inhibited collagen production and TIMP-1 expression in US by blocking CaMK II-mediated STAT3 and Smad3/JunD pathways.

Matrix metaloproteinases in vascular pathology.

Vascular diseases are the main cause of morbidity and mortality. The vascular extracellular matrix (ECM) is essential in mechanical support, also regulating the cellular behavior fundamental to vascular function and homeostasis. Vascular remodeling is an adaptive response to various physiological and pathological changes and is associated with aging and vascular diseases. The aim of this review is provide a general overview of the involvement of MMPs in the pathogenesis of vascular diseases, namely, arterial hypertension, atherosclerosis, aortic aneurysms and myocardial infarction. The change in the composition of the ECM by matrix metalloproteinases (MMPs) generates a pro-inflammatory microenvironment that modifies the phenotypes of endothelial cells and vascular smooth muscle cells. They play a central role in morphogenesis, tissue repair and remodeling in response to injury, e.g., after myocardial infarction, and in progression of diseases such as atherosclerosis. Alterations in specific MMPs could influence arterial remodeling and lead to various pathological disorders such as hypertension and aneurysm formation. MMPs are regulated by endogenous tissue inhibitors of metalloproteinases (TIMPs), and the MMP/TIMP ratio generally determines the extent of ECM protein degradation and tissue remodeling. Studies are currently focused on improving the diagnostic and prognostic value of MMPs involved in the pathogenic process, increasing their therapeutic potential, and monitoring the disease. New selective MMP inhibitors may improve the specificity of these inhibitors, target specific MMPs in relevant pathological conditions and mitigate some of the side effects.

Matrix Metalloproteinases: From Molecular Mechanisms to Physiology, Pathophysiology, and Pharmacology.

The first matrix metalloproteinase (MMP) was discovered in 1962 from the tail of a tadpole by its ability to degrade collagen. As their name suggests, matrix metalloproteinases are proteases capable of remodeling the extracellular matrix. More recently, MMPs have been demonstrated to play numerous additional biologic roles in cell signaling, immune regulation, and transcriptional control, all of which are unrelated to the degradation of the extracellular matrix. In this review, we will present milestones and major discoveries of MMP research, including various clinical trials for the use of MMP inhibitors. We will discuss the reasons behind the failures of most MMP inhibitors for the treatment of cancer and inflammatory diseases. There are still misconceptions about the pathophysiological roles of MMPs and the best strategies to inhibit their detrimental functions. This review aims to discuss MMPs in preclinical models and human pathologies. We will discuss new biochemical tools to track their proteolytic activity in vivo and ex vivo, in addition to future pharmacological alternatives to inhibit their detrimental functions in diseases. SIGNIFICANCE STATEMENT: Matrix metalloproteinases (MMPs) have been implicated in most inflammatory, autoimmune, cancers, and pathogen-mediated diseases. Initially overlooked, MMP contributions can be both beneficial and detrimental in disease progression and resolution. Thousands of MMP substrates have been suggested, and a few hundred have been validated. After more than 60 years of MMP research, there remain intriguing enigmas to solve regarding their biological functions in diseases.

A novel glyceroglycolipid from brown algae Ishige okamurae improve photoaging and counteract inflammation in UVB-induced HaCaT cells.

BACKGROUND: Excessive exposure to Ultraviolet (UV) rays can cause premature skin aging. Ishigoside (IGS) is a new glyceroglycolipid compound isolated from brown algal Ishige okamurae, However, whether it can protect the skin from (Ultraviolet-B) UVB damage has not been illuminated. METHODS: The in vitro anti-photoaging effect of IGS was conducted in UVB-induced HaCaT. The HaCaT cells were divided into the following five groups: (1) cells didn't suffer from UVB irradiation or IGS treatment. (2-5) Cells were treated with various concentrations of IGS (0, 10, 50, and 100 µM) and irradiated by 40 mJ/cm2 UVB. The Matrix metalloproteinase (MMP) of photoaging process was determined by ELISA kits and the latent interaction between IGS and MMP was further performed by molecular docking. The crucial signaling pathway proteins involved in the collagen synthesis and degradation were subsequently evaluated by Western blotting, immunofluorescence and EMSA. RESULTS: IGS effectively suppresses the high expressions and secretions of matrix metalloproteinases (MMPs) and photo-inflammation by blocking MAPKs, AP-1 and NF-κB. Meanwhile, increasing antioxidant enzyme expression. Molecular docking results suggest that inhibition of IGS on MMPs may be attributed to its hydrogen supply and hydrophobic capacity. In addition, IGS enhanced procollagen production by upregulating the TGF-ß/Smad pathways. CONCLUSIONS: IGS exhibited anti-photoaging activity in UVB-damage HaCaT. These effects might be a contribution by its suppression of MMPs expression via MAPKs, AP-1 and NF-κB pathway and have anti-oxidative and anti-inflammatory effects. Therefore, IGS has the great potential to become skin-care products or functional foods for preventing skin photoaging.

Structure-based molecular insights into matrix metalloproteinase inhibitors in cancer treatments.

Protease inhibitors are of considerable interest as anticancer agents. Matrix metalloproteinases (MMPs) were the earliest type of proteases considered as anticancer targets. The developments of MMP inhibitors (MMPIs) by pharmaceutical companies can be dated from the early 1980s. Thus far, none of the over 50 MMPIs entering clinical trials have been approved. This work summarizes the reported studies on the structure of MMPs and complexes with ligands and inhibitors, based on which, the authors analyzed the clinical failures of MMPIs in a structural biological manner. Furthermore, MMPs were systematically compared with urokinase, a protease-generating plasmin, which plays similar pathological roles in cancer development; the reasons for the clinical successes of urokinase inhibitors and the clinical failures of MMPIs are discussed.

Extended Cheese Whey Fermentation Produces a Novel Casein-Derived Antibacterial Polypeptide That Also Inhibits Gelatinases MMP-2 and MMP-9.

Our previous works produced a whey fermentation methodology that yielded antibacterial activity and potential inhibition of matrix metalloproteases (MMP)-2 and -9. Here, we evaluated if these activities were due to fermentation-produced peptides. Prolonged fermentation was carried out in the presence of our specific lactic acid bacteria (LAB) consortium. LAB fermentation yielded a total of 11 polypeptides, which were predominantly produced after 6 days of fermentation. One which was derived from beat casein presented a particularly high antibacterial activity against food pathogenic bacteria and was more effective than standard food disinfectants. This polypeptide was further studied and was also found to be active against several strains of pathogenic bacteria, including methicillin-resistant Staphylococcus aureus (MRSA), in a dose-dependent manner. It also inhibited MMP-2 and MMP-9 whilst reducing HT29 cancer cell migration in vitro. Overall, this novel whey-derived polypeptide presents dual antibacterial and anti-inflammatory activity, revealing a strong potential to be used in functional foods or as a nutraceutical. Its identification and further characterization can open novel perspectives in the field of preventive/curative diets related to gut microbiota, gut inflammation, and cancer prevention, particularly if used in in vivo studies.

MMPs and TIMPs levels are correlated with anthropometric parameters, blood pressure, and endothelial function in obesity.

The association between matrix metalloproteinases (MMPs), tissue inhibitor of metalloproteinases (TIMPs) and obesity as well as obesity-related disease including metabolic syndrome is not fully explored. Our aims are that: (i) to evaluate the plasma levels of MMP-1, MMP-2, MMP-3, MMP-9, TIMP-1, TIMP-2 and their ratios in non-obese people, overweight and obese people with or without metabolic syndrome, (ii) to investigate correlations between MMPs or TIMPs levels and several anthropometric parameters, blood pressure, endothelial function. Anthropometric and biochemical parameters were determined in 479 randomly selected participants, subdividing according to body mass index (BMI) and metabolic syndrome status. Plasma MMPs and TIMPs levels were measured. The assessment of endothelial function was characterized in people with obesity, overweight and non-obese, using laser Doppler Flowmetry. Obese people have elevated MMP-1, MMP-2, TIMP-1, TIMP-2 levels and decreased MMP-3/TIMP-1 and MMP-9/TIMP-1 ratios compared with non-obese people. MMP-1 levels and MMP-1/TIMP-1 ratio were positively correlated with BMI and waist circumference (WC) while MMP-2 levels were negatively correlated with BMI and WC values in obese people. MMP-3 levels and MMP-3/TIMP-1 ratio were positively correlated with systolic blood pressure (SBP) or diastolic blood pressure (DBP) in obese and metabolic syndrome people. Additionally, MMP-9 levels and MMP-9/TIMP-1 ratio were negatively correlated with endothelium-dependent response in obese and metabolic syndrome people. MMP-1, MMP-2, TIMP-1, TIMP-2 levels were increased in obese subjects. Significant correlations between anthropometric parameters and MMP-1 as well as MMP-1/TIMP-1 ratio supported these results. MMP-3 and -9 levels as well as their ratios with TIMP-1 were associated with blood pressure and endothelial-dependent response, respectively. In conclusion, our results demonstrated that MMP-1, MMP-3 and MMP-9 levels were correlated with several obesity-related parameters including BMI, WC, blood pressure and endothelial-dependent response. Our findings will hopefully provide new aspects for the use of MMPs and TIMPs as clinical biomarkers in obesity-related cardiovascular diseases such as metabolic syndrome and hypertension. The lack of measure of MMPs activity in plasma and relevant organs/tissues in obesity and metabolic syndrome is considered as a limitation in this report.

Cytokine Signaling and Matrix Remodeling Pathways Associated with Cardiac Sarcoidosis Disease Activity Defined Using FDG PET Imaging.

While cardiac imaging has improved the diagnosis and risk assessment for cardiac sarcoidosis (CS), treatment regimens have consisted of generalized heart failure therapies and non-specific anti-inflammatory regimens. The overall goal of this study was to perform high-sensitivity plasma profiling of specific inflammatory pathways in patients with sarcoidosis and with CS.Specific inflammatory/proteolytic cascades were upregulated in sarcoidosis patients, and certain profiles emerged for CS patients.Plasma samples were collected from patients with biopsy-confirmed sarcoidosis undergoing F-18 fluorodeoxyglucose positron emission tomography (n = 47) and compared to those of referent control subjects (n = 6). Using a high-sensitivity, automated multiplex array, cytokines, soluble cytokine receptor profiles (an index of cytokine activation), as well as matrix metalloproteinase (MMP), and endogenous MMP inhibitors (TIMPs) were examined.The plasma tumor necrosis factor (TNF) and soluble TNF receptors sCD30 and sTNFRI were increased using sarcoidosis, and sTNFRII increased in CS patients (n = 18). The soluble interleukin sIL-2R and vascular endothelial growth factor receptors (sVEGFR2 and sVEGFR3) increased to the greatest degree in CS patients. When computed as a function of referent control values, the majority of soluble cytokine receptors increased in both sarcoidosis and CS groups. Plasma MMP-9 levels increased in sarcoidosis but not in the CS subset. Plasma TIMP levels declined in both groups.The findings from this study were the identification of increased activation of a cluster of soluble cytokine receptors, which augment not only inflammatory cell maturation but also transmigration in patients with sarcoidosis and patients with cardiac involvement.

Insight into the structural requirement of aryl sulphonamide based gelatinases (MMP-2 and MMP-9) inhibitors - Part I: 2D-QSAR, 3D-QSAR topomer CoMFA and Naïve Bayes studies - First report of 3D-QSAR Topomer CoMFA analysis for MMP-9 inhibitors and jointly inhibitors of gelatinases together.

Gelatinases [gelatinase A - matrix metalloproteinase-2 (MMP-2), gelatinase B - matrix metalloproteinase-9 (MMP-9)] play key roles in many disease conditions including cancer. Despite some research work on gelatinases inhibitors both jointly and individually had been reported, challenges still exist in achieving potency as well as selectivity. Here in part I of a series of work, we have reported the structural requirement of some arylsulfonamides. In particular, regression-based 2D-QSARs, topomer CoMFA (comparative molecular field analysis) and Bayesian classification models were constructed to refine structural features for attaining better gelatinase inhibitory activity. The 2D-QSAR models exhibited good statistical significance. The descriptors nsssN, SHBint6, SHBint7, PubchemFP629 were directly correlated with the MMP-2 binding affinities whereas nsssN, SHBint10 and AATS2i were directly proportional to MMP-9 binding affinities. The topomer CoMFA results indicated that the steric and electrostatic fields play key roles in gelatinase inhibition. The established Naïve Bayes prediction models were evaluated by fivefold cross validation and an external test set. Furthermore, important molecular descriptors related to MMP-2 and MMP-9 binding affinities and some active/inactive fragments were identified. Thus, these observations may be helpful for further work of aryl sulphonamide based gelatinase inhibitors in future.

Sodium hyaluronate-g-2-((N-(6-aminohexyl)-4-methoxyphenyl)sulfonamido)-N-hydroxyacetamide with enhanced affinity towards MMP12 catalytic domain to be used as visco-supplement with increased degradation resistance.

The present paper describes the functionalization of sodium hyaluronate (NaHA) with a small molecule (2-((N-(6-aminohexyl)-4-methoxyphenyl)sulfonamido)-N-hydroxyacetamide) (MMPI) having proven inhibitory activity against membrane metalloproteins involved in inflammatory processes (i.e. MMP12). The obtained derivative (HA-MMPI) demonstrated an increased resistance to the in-vitro degradation by hyaluronidase, viscoelastic properties close to those of healthy human synovial fluid, cytocompatibility towards human chondrocytes and nanomolar affinity towards MMP 12. Thus, HA-MMPI can be considered a good candidate as viscosupplement in the treatment of knee osteoarticular disease.

Matrix metalloproteinase contribution in management of cancer proliferation, metastasis and drug targeting.

Matrix metalloproteinases (MMPs) or matrixins, are members of a zinc-dependent endopeptidase family. They cause remodeling of the extracellular matrix (ECM) leading to numerous diseases. MMPs subfamilies possess: collagenases, gelatinases, stromelysins and membrane-type MMPs (MT-MMP). They consist of several domains; pro-peptide, catalytic, linker peptide and the hemopexin (Hpx) domains. MMPs are involved in initiation, proliferation and metastasis of cancer through the breakdown of ECM physical barriers. Overexpression of MMPs is associated with poor prognosis of cancer. This review will discuss both types of MMPs and current inhibitors, which target them in different aspects, including, biosynthesis, activation, secretion and catalytic activity. Several synthetic and natural inhibitors of MMPs (MMPIs) that can bind the catalytic domain of MMPs have been designed including; peptidomimetic, non-peptidomimetic, tetracycline derivatives, off-target MMPI, natural products, microRNAs and monoclonal antibodies.

Biphenyl substituted lysine derivatives as recognition elements for the matrix metalloproteinases MMP-2 and MMP-9.

Matrix metalloproteinases (MMPs) are an important factor in cancer progression and metastasis, especially gelatinases MMP-2 and MMP-9. A simple methodology for their detection and monitoring is highly desirable. Molecular probes have been very widely and successfully applied to study the activity of MMPs in cellular processes in vitro. We thus synthesized a small compound library of MMP-2 and MMP-9 binding probes based on drug molecules and endowed with free amine groups for the functionalization of transducer surfaces. In this study, we combined experimental results obtained by a kinetic fluorogenic peptide substrate cleavage assay with molecular modeling studies in order to assess the ability of the probe to bind to their target enzymes. The synthesized biphenyl substituted lysine derivatives showed IC50-values in the low nanomolar concentration range against MMP-2 (ligands 3a-d: 3 nM to 8 µM, ligands 4a-d: 45 nM to 350 µM) and low micromolar range against MMP-9 (ligands 3a-d: 350 nM to 60 µM, ligands 4a-d: 5 µM to 600 µM), with a selectivity up to more than 160-fold for MMP-2. The experimental results correlated well with molecular modelling with FleXAID and X-score functions. We showed that in our compound series, the side chain remained far away from the S1' cavity and the ligand for all the docked minima. Ligands 4a-d with their free amine group on the side chain may thus be bound to transducer surfaces for the fabrication of sensors, while retaining their activity against their target enzymes.

Downregulation of microRNA-21-5p from macrophages-derived exosomes represses ventricular remodeling after myocardial infarction via inhibiting tissue inhibitors of metalloproteinase 3.

OBJECTIVE: Exosomes are known to transfer microRNAs (miRNAs) to affect the progression of human diseases. We aim to explore the role of M1 macrophages-derived exosomes (M1 exosomes) conveying miR-21-5p in ventricular remodeling in mice with myocardial infarction (MI) by regulating tissue inhibitors of metalloproteinase 3 (TIMP3). METHODS: Macrophages were isolated and co-cultured with miR-21-5p antagomir to extract the exosomes. The modeled mice were injected with relative exosomes to investigate their roles in the cardiac function, pathology of myocardial tissue, myocardial fibrosis, cardiomyocyte apoptosis and ventricular remodeling in MI mice. The expression of miR-21-5p and TIMP3 was detected and their targeting relationship was analyzed. RESULTS: MiR-21-5p was upregulated while TIMP3 was downregulated in MI mouse myocardial tissues. M1 exosomes impaired cardiac function, aggravated pathology of myocardial tissue, myocardial fibrosis and ventricular remodeling, and promoted cardiomyocyte apoptosis in MI mice. M1 exosomes containing miR-21-5p antagomir alleviated the above alterations, while the role of exosomes containing miR-21-5p antagomir was reversed by silencing TIMP3. TIMP3 was targeted by miR-21-5p. CONCLUSION: Downregulation of miR-21-5p from macrophages-derived exosomes suppresses ventricular remodeling after MI via inhibiting TIMP3.

IFN-λ Modulates the Migratory Capacity of Canine Mammary Tumor Cells via Regulation of the Expression of Matrix Metalloproteinases and Their Inhibitors.

Interactions between neoplastic and immune cells taking place in tumors drive cancer regulatory mechanisms both in humans and animals. IFN-λ, a potent antiviral factor, is also secreted in the tumor; however, its role in tumor development is still unclear. In our study, we investigate the influence of IFN-λ on the canine mammary tumor (CMT) cell survival and their metastatic potential in vitro. First, we examined, by Western blot, the expression of the IFN-λ receptor complex in three CMT cell lines (P114, CMT-U27 and CMT-U309). We showed that only two cell lines (P114 and CMT-U27) express both (IL-28RA and IL-10Rb) receptor subunits and respond to IFN-λ treatment by STAT phosphorylation and the expression of interferon-stimulated genes. Using MTT, crystal violet and annexin-V assays, we showed a minimal role of IFN-λ in CMT viability. However, IFN-λ administration had a contradictory effect on cell migration in the scratch test, namely, it increased P114 and decreased CMT-U27 motility. Moreover, we demonstrated that this process is related to the expression of extracellular matrix metalloproteinases and their inhibitors; furthermore, it is independent of Akt and ERK signaling pathways. To conclude, we showed that IFN-λ activity is reliant on the expression of two receptor subunits and tumor type, but further investigations are needed.

Activation of the Interleukin-33/ST2 Pathway Exerts Deleterious Effects in Myxomatous Mitral Valve Disease.

Mitral valve disease (MVD) is a frequent cause of heart failure and death worldwide, but its etiopathogenesis is not fully understood. Interleukin (IL)-33 regulates inflammation and thrombosis in the vascular endothelium and may play a role in the atherosclerotic process, but its role in mitral valve has not been investigated. We aim to explore IL-33 as a possible inductor of myxomatous degeneration in human mitral valves. We enrolled 103 patients suffering from severe mitral regurgitation due to myxomatous degeneration undergoing mitral valve replacement. Immunohistochemistry of the resected leaflets showed IL-33 and ST2 expression in both valve interstitial cells (VICs) and valve endothelial cells (VECs). Positive correlations were found between the levels of IL-33 and molecules implicated in the development of myxomatous MVD, such as proteoglycans, extracellular matrix remodeling enzymes (matrix metalloproteinases and their tissue inhibitors), inflammatory and fibrotic markers. Stimulation of single cell cultures of VICs and VECs with recombinant human IL-33 induced the expression of activated VIC markers, endothelial-mesenchymal transition of VECs, proteoglycan synthesis, inflammatory molecules and extracellular matrix turnover. Our findings suggest that the IL-33/ST2 system may be involved in the development of myxomatous MVD by enhancing extracellular matrix remodeling.

Matrix metalloproteinases and their inhibitors in Fuchs endothelial corneal dystrophy.

Fuchs endothelial corneal dystrophy (FECD) is characterized by a progressive loss of corneal endothelial cells (CECs) and an abnormal accumulation of extracellular matrix in Descemet's membrane leading to increased thickness and formation of excrescences called guttae. Extracellular matrix homeostasis is modulated by an equilibrium between matrix metalloproteinases (MMPs) and their endogenous tissue inhibitors (TIMPs). This study aimed to investigate MMPs and TIMPs profile in FECD, taking into account cell morphology. Populations of FECD and healthy CECs were cultured and their conditioned media collected for analysis. The presence of proteases in the conditioned media was studied using a semi-quantitative proteome profiler array, and MMPs levels were assessed using quantitative assays (ELISA and quantitative antibody array). MMP activity was determined by zymography and fluorometry. The expression pattern of the membrane type 1-MMP (MT1-MMP, also known as MMP-14) was examined by immunofluorescence on ex vivo FECD and healthy explants of CECs attached to Descemet's membrane. Finally, MMPs and TIMPs protein expression was compared to gene expression obtained from previously collected data. FECD and healthy CEC populations generated cultures of endothelial, intermediate, and fibroblastic-like morphology. Various MMPs (MMP-1, -2, -3, -7, -8, -9, -10, and -12) and TIMPs (TIMP-1 to -4) were detected in both FECD and healthy CECs culture supernatants. Quantitative assays revealed a decrease in MMP-2 and MMP-10 among FECD samples. Both these MMPs can degrade the main extracellular matrix components forming guttae (fibronectin, laminin, collagen IV). Moreover, MMPs/TIMPs ratio was also decreased among FECD cell populations. Activity assays showed greater MMPs/Pro-MMPs proportions for MMP-2 and MMP-10 in FECD cell populations, although overall activities were similar. Moreover, the analysis according to cell morphology revealed among healthy CECs, both increased (MMP-3 and -13) and decreased (MMP-1, -9, -10, and -12) MMPs proteins along with increased MMPs activity (MMP-2, -3, -9, and -10) in the fibroblastic-like subgroup when compared to the endothelial subgroup. However, FECD CECs did not show similar behaviors between the different morphology subgroups. Immunostaining of MT1-MMP on ex vivo FECD and healthy explants revealed a redistribution of MT1-MMP around guttae in FECD explants. At the transcriptional level, no statistically significant differences were detected, but cultured FECD cells had a 12.2-fold increase in MMP1 and a 4.7-fold increase in TIMP3. These results collectively indicate different, and perhaps pathological, MMPs and TIMPs profile in FECD CECs compared to healthy CECs. This is an important finding suggesting the implication of MMPs and TIMPs in FECD pathophysiology.