Elevated Matrix Metalloproteinase Activity Research Articles

LOX-1 and MMP-9 Inhibition Attenuates the Detrimental Effects of Delayed rt-PA Therapy and Improves Outcomes After Acute Ischemic Stroke.

Acute ischemic stroke triggers endothelial activation that disrupts vascular integrity and increases hemorrhagic transformation leading to worsened stroke outcomes. rt-PA (recombinant tissue-type plasminogen activator) is an effective treatment; however, its use is limited due to a restricted time window and hemorrhagic transformation risk, which in part may involve activation of MMPs (matrix metalloproteinases) mediated through LOX-1 (lectin-like oxLDL [oxidized low-density lipoprotein] receptor 1). This study's overall aim was to evaluate the therapeutic potential of novel MMP-9 (matrix metalloproteinase 9) ± LOX-1 inhibitors in combination with rt-PA to improve stroke outcomes. A rat thromboembolic stroke model was utilized to investigate the impact of rt-PA delivered 4 hours poststroke onset as well as selective MMP-9 (JNJ0966) ±LOX-1 (BI-0115) inhibitors given before rt-PA administration. Infarct size, perfusion, and hemorrhagic transformation were evaluated by 9.4-T magnetic resonance imaging, vascular and parenchymal MMP-9 activity via zymography, and neurological function was assessed using sensorimotor function testing. Human brain microvascular endothelial cells were exposed to hypoxia plus glucose deprivation/reperfusion (hypoxia plus glucose deprivation 3 hours/R 24 hours) and treated with ±tPA and ±MMP-9 ±LOX-1 inhibitors. Barrier function was assessed via transendothelial electrical resistance, MMP-9 activity was determined with zymography, and LOX-1 and barrier gene expression/levels were measured using qRT-PCR (quantitative reverse transcription PCR) and Western blot. Stroke and subsequent rt-PA treatment increased edema, hemorrhage, MMP-9 activity, LOX-1 expression, and worsened neurological outcomes. LOX-1 inhibition improved neurological function, reduced edema, and improved endothelial barrier integrity. Elevated MMP-9 activity correlated with increased edema, infarct volume, and decreased neurological function. MMP-9 inhibition reduced MMP-9 activity and LOX-1 expression. In human brain microvascular endothelial cells, LOX-1/MMP-9 inhibition differentially attenuated MMP-9 levels, inflammation, and activation following hypoxia plus glucose deprivation/R. Our findings indicate that LOX-1 inhibition and ± MMP-9 inhibition attenuate negative aspects of ischemic stroke with rt-PA therapy, thus resulting in improved neurological function. While no synergistic effect was observed with simultaneous LOX-1 and MMP-9 inhibition, a distinct interaction is evident.

Plasminogen application improves plastic closure of wound defects in patients with chronic diabetic wounds

Background. Chronic diabetic foot ulcers and wounds are significant complications associated with diabetes, comprising approximately 85% of purulent-necrotic lesions affecting the lower extremities. The development of these wounds is influenced by pathogenetic factors such as hyperglycemia, neuropathy, and existing infections, which contribute to metabolic disturbances, including tissue hypoxia and the activation of proteolytic enzymes known as matrix metalloproteinases (MMPs). Aim. To explore the therapeutic potential of autologous plasminogen in facilitating the healing process of diabetic wounds through the modulation of MMP activity. Materials and Methods. The study enrolled 45 patients diagnosed with chronic diabetic wounds, who were assigned to two distinct groups. The control group (n=25) received conventional treatment approaches, while the intervention group consisted of 20 patients treated with autologous plasminogen applications. Results. After 18 days of treatment, a substantial reduction of 3.5-fold in MMP-2 and MMP-9 activity was observed within the intervention group, accompanied by complete wound closure in 16 patients. Additionally, four patients underwent autodermoplasty, successfully achieving wound defect closure through effective graft integration. In contrast, the control group exhibited consistently elevated MMP activity levels throughout the entire observation period. Conclusions. The activity of matrix metalloproteinases (MMPs) in chronic diabetic wounds reaches dramatic levels, making spontaneous wound healing impossible. The application of autologous Pg allows modulation of this activity and creates favorable conditions for wound healing by reducing excessive MMP activity, improving blood supply, and resolving inflammatory processes. Keywords: chronic wounds, diabetes mellitus, matrix metalloproteinases, plasminogen, autodermoplasty.

Multilevel Assessment of Stent-Induced Inflammation in the Adjacent Vascular Tissue.

A recent U.S. Food and Drug Administration report presented the currently available scientific information related to biological response to metal implants. In this work, a multilevel approach was employed to assess the implant-induced and biocorrosion-related inflammation in the adjacent vascular tissue using a mouse stent implantation model. The implications of biocorrosion on peri-implant tissue were assessed at the macroscopic level via in vivo imaging and histomorphology. Elevated matrix metalloproteinase activity, colocalized with the site of implantation, and histological staining indicated that stent surface condition and implantation time affect the inflammatory response and subsequent formation and extent of neointima. Hematological measurements also demonstrated that accumulated metal particle contamination in blood samples from corroded-stetted mice causes a stronger immune response. At the cellular level, the stent-induced alterations in the nanostructure, cytoskeleton, and mechanical properties of circulating lymphocytes were investigated. It was found that cells from corroded-stented samples exhibited higher stiffness, in terms of Young's modulus values, compared to noncorroded and sham-stented samples. Nanomechanical modifications were also accompanied by cellular remodeling, through alterations in cell morphology and stress (F-actin) fiber characteristics. Our analysis indicates that surface wear and elevated metal particle contamination, prompted by corroded stents, may contribute to the inflammatory response and the multifactorial process of in-stent restenosis. The results also suggest that circulating lymphocytes could be a novel nanomechanical biomarker for peri-implant tissue inflammation and possibly the early stage of in-stent restenosis. Large-scale studies are warranted to further investigate these findings.

Role of matrix metalloproteinase-9 in neurodevelopmental deficits and experience-dependent plasticity in Xenopus laevis.

Matrix metalloproteinase-9 (MMP-9) is a secreted endopeptidase targeting extracellular matrix proteins, creating permissive environments for neuronal development and plasticity. Developmental dysregulation of MMP-9 may also lead to neurodevelopmental disorders (ND). Here, we test the hypothesis that chronically elevated MMP-9 activity during early neurodevelopment is responsible for neural circuit hyperconnectivity observed in Xenopus tadpoles after early exposure to valproic acid (VPA), a known teratogen associated with ND in humans. In Xenopus tadpoles, VPA exposure results in excess local synaptic connectivity, disrupted social behavior and increased seizure susceptibility. We found that overexpressing MMP-9 in the brain copies effects of VPA on synaptic connectivity, and blocking MMP-9 activity pharmacologically or genetically reverses effects of VPA on physiology and behavior. We further show that during normal neurodevelopment MMP-9 levels are tightly regulated by neuronal activity and required for structural plasticity. These studies show a critical role for MMP-9 in both normal and abnormal development.

Development of Brain Targeting Peptide Based MMP-9 Inhibiting Nanoparticles for the Treatment of Brain Diseases with Elevated MMP-9 Activity

Latent and active levels of cerebral matrix metalloproteinase 9 (MMP-9) are elevated in neurological diseases and brain injuries, contributing to neurological damage and poor clinical outcomes. This study aimed developing peptide-based nanoparticles with ability to cross the blood-brain-barrier (BBB) and inhibit MMP-9.Three amphiphilic peptides were synthesised containing brain-targeting ligands (HAIYPRH or CKAPETALC) conjugated with MMP-9 inhibiting peptide (CTTHWGFTLC) linked by glycine (spacer) at the N-terminus, and the peptide sequences were conjugated at the N- terminus to cholesterol. 19F NMR assay was developed to measure MMP-9 inhibition. Cell toxicity was evaluated by the LDH assay, and dialysis studies were conducted with/without fetal bovine serum. An in vitro model was employed to evaluate the ability of nanoparticles crossing the BBB.The amphiphilic peptide (Cholesterol-GGGCTTHWGFTLCHAIYPRH) formed nanoparticles (average size of 202.8 nm) with ability to cross the BBB model. MMP-9 inhibiting nanoparticles were non-toxic to cells, and reduced MMP-9 activity from kobs of 4.5 × 10−6s−1 to complete inhibition. Dialysis studies showed that nanoparticles did not disassemble by extreme dilution (40 folds), but gradually hydrolysed by serum enzymes.In conclusion, the MMP-9 inhibiting nanoparticles reduced the activity of MMP-9, with acceptable serum stability, minimal cell toxicity and ability to cross the in vitro BBB model.

Targeting of an antecedent proteinase by an activatable probe with deep tissue penetration facilitates early visualization and dynamic malignancy evaluation of orthotopic pancreatic ductal adenocarcinoma (PDAC).

Pancreatic ductal adenocarcinoma (PDAC) is highly lethal and most commonly diagnosed at an advanced stage; therefore, early detection with an effective approach is vital. However, due to the anatomical stealthiness and hypovascular features of PDAC, clinically available imaging techniques lack specificity and sensitivity for early detection. As important components of the tumour microenvironment, elevated matrix metalloproteinase (MMP) levels during the early stages of tumour formation lead to tumour invasion and metastases by degrading the extracellular matrix. Thus, in the current study, we hypothesized that MMPs might be promising markers for early visualization and prognosis evaluation of PDAC. An MMP activatable probe, I780BP-PEG12, was synthesized utilizing a long wavelength near-infrared (NIR) fluorophore that could map the dynamic development of orthotopic PDAC in vivo with deep tissue penetration. Elevated MMP activity in tumours was detected as early as 4 days after tumour transplantation. At that time, the tumour diameter was approximately 3 mm, which is much smaller than the size visualized by clinical approaches. Furthermore, much higher levels of MMP activity were detected in PDAC under diabetic conditions, which promote the malignant actions of tumours. By noninvasively monitoring MMP alteration, tumour growth, and prognostic evaluation, we found that malignant actions under diabetic conditions were reversed by inhibition of MMPs. Generally, in addition to earlier visualization of PDAC, a probe targeting MMPs can facilitate dynamic monitoring of tumour progression and inhibitory treatment in vivo, which is beneficial for personal therapeutic strategy planning and optimization of PDAC management, especially for diabetic individuals.

Downregulation of IDH2 exacerbates the malignant progression of osteosarcoma cells via increased NF-κB and MMP-9 activation.

Isocitrate dehydrogenase 2 (IDH2) is a mitochondrial NADP-dependent isocitrate dehydrogenase. It is considered to be a novel tumor suppressor in several types of tumors. However, the role and related mechanism of IDH2 in osteosarcoma remain unknown. The expression and significance of IDH2 were investigated by immunohistochemistry in formalin-fixed paraffin sections from 44 osteosarcoma patients. IDH2 was downregulated via lentiviral vector‑mediated RNA interference (RNAi) in the Saos-2 and MG-63 human osteosarcoma cell lines. The effect of IDH2 downregulation on human osteosarcoma was studied in vitro by MTT, flow cytometry and invasion assays. Nuclear factor-κB (NF-κB) and matrix metalloproteinase-9 (MMP-9) assays were also used to study the likely molecular mechanism of IDH2 downregulation on the malignant progression of osteosarcoma cells. The results revealed that the expression of IDH2 was inversely correlated with pathological grade and metastasis in osteosarcoma. IDH2 downregulation promoted a pro-proliferative effect on the Saos-2 and MG-63 osteosarcoma cell lines. IDH2 downregulation accelerated cell cycle progression from S to G2/M phase. The pro-proliferative effect induced by IDH2 downregulation may be ascribed to increased NF-κB activity via IκBα phosphorylation. The invasive activity of osteosarcoma cells was also significantly promoted by IDH2 downregulation and may result from elevated MMP-9 activity. In conclusion, IDH2 downregulation may exacerbate malignant progression via increased NF-κB and MMP-9 activity and may implicate the potential biological importance of IDH2 targeting in osteosarcoma cells. Downregulation of IDH2 exacerbates the malignant progression of osteosarcoma cells via increased NF-κB and MMP-9 activation.

Abstract 720: Dual MMP-7-proximity-activated and folate-targeted nanoparticles for siRNA delivery

Abstract We have developed a dual, folic acid (FA) and matrix metalloproteinase (MMP), proximity-activated targeting (PAT) smart polymeric nanoparticle (SPN) for tumor-specific siRNA delivery. The nanocarrier was designed to expose concealed FA ligands specifically in regions of elevated MMP activity, resulting in targeted uptake by folate receptor-expressing cells. Site-selective chemistry and reversible addition-fragmentation chain transfer (RAFT) polymerization were used to prepare diblock copolymers {p(DMAEMA)-b-p(DMAEMA-PAA-BMA) (SPN)} terminated with either MMP-7 peptide coupled to a Y-shaped 20 kDa PEG (PAT-SPN) or a 2 kDa linear PEG coupled to FA (FA-SPN). These amphiphilic polymers were co-assembled in aqueous media, forming mixed micelles consisting of 0, 25, 50, 75, 100 mol% FA and presenting zeta potentials of -0.21, -0.20, +1.75, +6.71 and +12.2 mV, respectively. Cleavage by MMP-7 was confirmed by decreased particle diameter (e.g., 25% FA micelle diameter decreased from 54 to 48 nm) and simultaneous increase in zeta potential (-0.20 to +8.11 mV). These data suggest that MMP-7 removal of the PEG corona from the nanocarriers revealed the underlying polycationic charge of the pDMAEMA core. Intracellular uptake of PAT/FA-SPNs was investigated in FA receptor-expressing breast cancer cells by confocal microscopy and flow cytometry. Pre-treatment with exogenous MMP-7 to mimic the proteolytically active microenvironment of human breast cancers, elevated cellular uptake 2.3-fold for single targeting PAT-SPN and 4.6-fold for dual-targeting PAT/FA-SPN compared to intact PAT-SPN. Cell uptake of uncleaved PAT/FA-SPN was statistically equivalent to untreated cells. These data suggest that the 20 kDa Y-shaped PEG effectively shields both the underlying cationic charge and the FA, and also blocks nonspecific cellular interactions of the SPN. 75-100% FA-SPN polymer reduced the dependency of cellular uptake on MMP-7 activity. Excess FA reduced the uptake of FA-SPN-containing micelles, consistent with FA receptor-dependent uptake following MMP activation. Functional protein knockdown delivered by PAT/FA-SPN was validated using luciferase-expressing MDA-MB-231 cells. 50% PAT/FA-SPN, pre-activated with MMP-7and loaded with luciferase siRNA, resulted in significant suppression of luciferase expression relative to controls (no MMP activation or competitive inhibition with free FA or scrambled siRNA), confirming the efficient dual PAT/FA targeting and endosomal escape of this designed nanoparticle. Unlike other similar technologies, this micelle design is optimized for use in vivo. Animal studies confirm an extended blood half-life, appropriate organ distribution, minimal toxicity and significant siRNA activity in mammary tumors. 1. Li, H. et al. Adv. Func. Mater. 2013. 10.1002/adfm.201202215 Support DoD CDMRP (W81XWH-10-1-0445/0446); Vanderbilt Institute of Nanoscale Science & Engineering Citation Format: Todd D. Giorgio, Hongmei Li, Martina Miteva, Kellye C. Kirkbride, Ming Cheng, Chris E. Nelson, Craig L. Duvall. Dual MMP-7-proximity-activated and folate-targeted nanoparticles for siRNA delivery. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 720. doi:10.1158/1538-7445.AM2014-720

Melatonin reduces airway inflammation in ovalbumin-induced asthma

Asthma is a common chronic inflammatory airway disease that is recognized as a major public health problem. In this study, we evaluated the effects of melatonin on allergic asthma using a murine model of ovalbumin (OVA)-induced allergic asthma and BEAS-2B cells. To induce allergic asthma, the mice were sensitized and airway-challenged with OVA. Melatonin was administered by intraperitoneal injection once per day at doses of 10 and 15mg/kg from days 21 to 23 after the initial OVA sensitization. We investigated the effects of melatonin on proinflammatory cytokines and matrix metalloproteinase-9 (MMP-9) activity and expression in tumor necrosis factor (TNF)-α-stimulated BEAS-2B cells. The administration of melatonin significantly decreased the number of inflammatory cells, airway hyperresponsiveness, and immunoglobulin (Ig) E with reductions in interleukin (IL)-4, IL-5, and IL-13. Melatonin attenuated the airway inflammation and the mucus production in lung tissue and significantly suppressed elevated MMP-9 expression and activity induced by an OVA challenge. In TNF-α-stimulated BEAS-2B cells, treatment with melatonin significantly reduced the levels of proinflammatory cytokines and lowered the expression and activity of MMP-9. These results indicate that melatonin effectively suppressed allergic asthma induced by an OVA challenge. The results suggest a potential role for melatonin in treating asthma.

Analysis of the MMP-dependent and independent functions of tissue inhibitor of metalloproteinase-2 on the invasiveness of breast cancer cells

Matrix metalloproteinases (MMPs) are secreted endopeptidases that play an essential role in remodeling the extracellular matrix (ECM). MMPs are primarily active during development, when the majority of ECM remodeling events occurs. In adults, elevated MMP activity has been observed in many pathological conditions such as cancer and osteoarthritis. The proteolytic activity of MMPs is controlled by their natural inhibitors - the tissue inhibitor of metalloproteinases (TIMPs). In addition to blocking MMP-mediated proteolysis, TIMPs have a number of MMP-independent functions including binding to cell surface proteins thereby stimulating signaling cascades. TIMP-2, the most studied member of the family, can both inhibit and activate MMPs directly, as well as inhibit MMP activity indirectly by upregulating expression of RECK, a membrane anchored MMP regulator. While TIMP-2 has been shown to play important roles in breast cancer, we describe how the MMP-independent effects of TIMP-2 can modulate the invasiveness of MCF-7, T47D and MDA-MB-231 breast cancer cells. Using an ALA + TIMP-2 mutant which is devoid of MMP inhibition, but still capable of initiating specific cell signaling cascades, we show that TIMP-2 can differentially affect MMP activity and cellular invasiveness in both an MMP dependent and independent manner. More specifically, MMP activity and invasiveness is increased with the addition of exogenous TIMP-2 in poorly invasive cell lines whereas it is decreased in highly invasive cells lines (MDA-MB-231). Conversely, the addition of ALA + TIMP-2 resulted in decreased invasiveness regardless of cell line.

Multispectral optoacoustic tomography of matrix metalloproteinase activity in vulnerable human carotid plaques.

AimsElevated expression of cathepsins, integrins and matrix metalloproteinases (MMPs) is typically associated with atherosclerotic plaque instability. While fluorescent tagging of such molecules has been amply demonstrated, no imaging method was so far shown capable of resolving these inflammation-associated tags with high fidelity and resolution beyond microscopic depths. This study is aimed at demonstrating a new method with high potential for noninvasive clinical cardiovascular diagnostics of vulnerable plaques using high-resolution deep-tissue multispectral optoacoustic tomography (MSOT) technology.Methods and resultsMMP-sensitive activatable fluorescent probe (MMPSense™ 680) was applied to human carotid plaques from symptomatic patients. Atherosclerotic activity was detected by tuning MSOT wavelengths to activation-dependent absorption changes of the molecules, structurally modified in the presence of enzymes. MSOT analysis simultaneously provided morphology along with heterogeneous MMP activity with better than 200 micron resolution throughout the intact plaque tissue. The results corresponded well with epi-fluorescence images made from thin cryosections. Elevated MMP activity was further confirmed by in situ zymography, accompanied by increased macrophage influx.ConclusionsWe demonstrated, for the first time to our knowledge, the ability of MSOT to provide volumetric images of activatable molecular probe distribution deep within optically diffuse tissues. High-resolution mapping of MMP activity was achieved deep in the vulnerable plaque of intact human carotid specimens. This performance directly relates to pre-clinical screening applications in animal models and to clinical decision potential as it might eventually allow for highly specific visualization and staging of plaque vulnerability thus impacting therapeutic clinical decision making.

The effect of a hydroxamic acid-containing polymer on active matrix metalloproteinases

Matrix metalloproteinase (MMP) sequestering polymer microspheres were prepared by a post-polymerization hydroxamic acid derivatization of poly(methyl methacrylate- co-methacrylic acid). The microspheres were designed to selectively bind MMPs from solutions on contact through a direct interaction between the polymer-bound hydroxamic acid groups and the characteristic catalytic site zinc atom common to all MMPs. MMP activity assays showed that the hydroxamic acid microspheres reduce MMP activity on contact in a time and concentration-dependent fashion. This effect was observed for several MMP subclasses (MMP-2, -3, -8 and -13) suggesting that the microspheres possess a broad-spectrum MMP binding capacity. However, inactive pro-forms of MMPs showed little binding affinity for the microspheres indicating that the interaction was dependent on MMP activation. The preferential binding of active MMPs was confirmed by MMP-3 and MMP-8 activation studies, which demonstrated significant increases in microsphere binding on activation. The MMP sequestering effect of the microspheres was also demonstrated in a physiologically relevant solution (human chronic wound fluid extract) indicating that the binding interaction was effective in a multi-component, competitive adsorption environment. Thus, the hydroxamic acid-containing microspheres may find use as localized, broad-spectrum MMP inhibitors for the treatment of a number of disease conditions characterized by elevated MMP activity.

Imatinib mesylate attenuates fibrosis in coxsackievirus b3-induced chronic myocarditis

Coxsackievirus B3 (CVB3)-induced chronic myocarditis in mice is accompanied by severe fibrosis and by sustained elevation of platelet-derived growth factor (PDGF)-A, -B, and -C levels in the cardiac tissue. To test if PDGF stimulation of resident fibroblasts causally contributes to fibrosis, we employed inhibition of PDGF receptor signalling with the orally available kinase inhibitor Imatinib. Chronic myocarditis was induced by CVB3 infection of major histocompatibility complex (MHC) class II knockout (B6Aa(0)/Aa(0)) mice. The mice were treated with 100 mg/kg Imatinib or vehicle, respectively, twice daily for 34 days. Expression of PDGF-C and of inflammatory cytokines were analysed by semi-quantitative RT-PCR. PDGFalpha receptor phosphorylation was detected by immunoblotting of cardiac tissue extracts and in situ by immunohistochemistry. Fibrosis formation was analysed by Sirius-Red staining and hydroxyproline (HP) determination. Fibronectin, and tenascin expression was analysed by RT-PCR and immunohistochemistry. Matrix metalloproteinase (MMP) activity was assessed with collagen, synthetic peptides, and gelatine as substrates. Imatinib significantly inhibited the myocarditis-related PDGFalpha receptor activation in the heart tissue. The virus titres in the hearts, inflammatory infiltrations, and elevated PDGF levels were unaffected by the Imatinib treatment. A significant attenuation of fibrosis occurred in Imatinib-treated animals. The Sirius Red-stained fibrotic area was reduced from 5.30 +/- 0.50 to 3.21 +/- 0.35%, and the HP content was reduced from 362 +/- 43 to 238 +/- 32 microMol/10 mg dry weight vs. 190 +/- 27 in uninfected controls. The expression of fibronectin, EIIIA+ fibronectin, and tenascin C were likewise reduced. The diminished matrix protein deposition was not caused by elevated MMP activity, since MMP activity was not changed or even reduced under Imatinib. The data suggest a causal role for elevated PDGF expression and PDGF receptor activity in the pathogenesis of cardiac fibrosis.

Changes in MMP‐2 and ‐9 Activity and MMP‐8 Reactivity After Amphotericin B Induced Synovitis and Treatment with Bufexamac

The objective here was to evaluate the acute effects of induced arthritis on synovial fluid (SF) levels of matrix metalloproteinases (MMP) -2, -8 and -9 in horses. To evaluate MMP-2 and -9 activities and the effect of non-steroidal anti-inflammatory drug (NSAID) bufexamac during remission from acute arthritis. Aseptic arthritis was induced in 24 Standardbred horses using 20 mg of amphotericin B as a single intra-articular (IA) injection in the right intercarpal joint. After 1 week and 2 weeks, horses were treated intra-articularly with 10, 20, or 40 mg of bufexamac suspension or with sterile saline solution as control. SF was sampled prior to induction and at weekly intervals for 5 weeks. Fluids were evaluated for MMP-2 and MMP-9 activity by gelatin zymography or for MMP-8 immunoreactivity by Western Blotting. IA injection of amphotericin B consistently resulted in significant increase in the immunoreactivity of MMP-8 and activity of both the latent and the active forms of MMP-2 and -9, among which the active form of MMP-2 increased the most. MMP-9 levels declined to pre-induction levels within 2 weeks, whereas levels of MMP-2 remained still high after 5 weeks. Treatment with bufexamac did not significantly affect levels of gelatinolytic MMP. Results suggest that after acute arthritis of horses, elevated MMP activity is present in the joint, for several weeks, to a degree that could promote cartilage degradation, and treatment with the NSAID bufexamac is not likely to affect that. Furthermore, analysing levels of MMP-9 activity and especially levels of active forms of MMP-2 activity may be valuable to predict the time of occurrence of arthritis in horses.

Lesional Overexpression of Matrix Metalloproteinase-9 Promotes Intraplaque Hemorrhage in Advanced Lesions But Not at Earlier Stages of Atherogenesis

Matrix metalloproteinase-9 (MMP-9) is involved in atherosclerosis and elevated MMP-9 activity has been found in unstable plaques, suggesting a crucial role in plaque rupture. This study aims to assess the effect of MMP-9 on plaque stability in apolipoprotein E-deficient mice at different stages of plaque progression. Atherosclerotic lesions were elicited in carotid arteries by perivascular collar placement. MMP-9 overexpression in intermediate or advanced plaques was effected by intraluminal incubation with an adenovirus (Ad.MMP-9). A subset was coincubated with Ad.TIMP-1. Mock virus served as a control. Plaques were analyzed histologically. In intermediate lesions, MMP-9 overexpression induced outward remodeling, as shown by a 30% increase in media size (p=0.03). In both intermediate and advanced lesions, prevalence of vulnerable plaque morphology tended to be increased. Half of MMP-9-treated lesions displayed intraplaque hemorrhage, whereas in controls and the Ad.MMP-9/Ad.TIMP-1 group this was 8% and 16%, respectively (p=0.007). Colocalization with neovessels may point to neo-angiogenesis as a source for intraplaque hemorrhage. These data show a differential effect of MMP-9 at various stages of plaque progression and suggest that lesion-targeted MMP-9 inhibition might be a valuable therapeutic modality in stabilizing advanced plaques, but not at earlier stages of lesion progression.

Disruption of the TIMP-1 gene product is associated with accelerated endometrial gland formation during early postnatal uterine development.

Postnatal uterine development is marked by periods of tissue remodeling. The objective of the present study was to examine the role of tissue inhibitor of metalloproteinase-1 (TIMP-1), a regulator of tissue remodeling events, during postnatal uterine development and to assess the phenotypic consequences of disruption of the TIMP-1 gene product during this time period. To accomplish this goal, wild-type and TIMP-1 null mice were sacrificed at Postnatal Days (PNDs) 5, 10, 15, 20, and 25 and uterine morphology, TIMP expression and matrix metalloproteinase (MMP) activity were assessed. In wild-type mice, TIMP-1 mRNA steady-state levels were highest at PND 5, after which expression decreased. TIMP-2 and TIMP-3 expression in wild-type mice showed no significant changes from PND 5 to 25. In TIMP-1 null mice, TIMP-2 and TIMP-3 expression patterns were similar to those in wild-type counterparts with the exception that, at PND 10, TIMP-2 and TIMP-3 expression was significantly lower in the null mice. Endometrial gland number and uterine histology were similar between genotypes at PNDs 5 and 10, but at PNDs 15 and 20, endometrial glands were more abundant in TIMP-1 null mice. Associated with the increased gland density in the null mice was an increase in total MMP activity above the levels expressed in wild-type mice. In summary, disruption of the TIMP-1 gene product is associated with reduced TIMP-2 and TIMP-3 steady-state mRNA levels, elevated MMP activity, and accelerated endometrial gland formation. We conclude that, during early postnatal uterine development, TIMP-1 may be critical for proper endometrial gland development.

The dynamic interaction between matrix metalloproteinase activity and adverse myocardial remodeling.

The process of cardiac remodeling in response to cardiac injury and/or persistent elevations in wall stress generally relates to the progressive changes that occur in ventricular chamber dimensions and the various components of the myocardium, in particular the cardiomyocytes and the extracellular matrix. Volume overload, pressure overload or myocardial injury produces a sustained abnormal elevation in myocardial wall stress which initiates cardiac remodeling that frequently results in ventricular decompensation and heart failure. Regardless of the inciting cause, there appear to be three distinct phases to this process. In the initial phase, fibrillar collagen is partially degraded secondary to increased matrix metalloproteinase (MMP) activity. Following this, there is a chronic compensatory phase during which MMP activity and collagen concentration return to normal while cardiomyocyte size continues to progressively increase. The final phase is attained once the compensatory hypertrophic mechanisms are exhausted and is characterized by elevated MMP activity, marked ventricular dilatation and prominent fibrosis. Details of this progressive, dynamic remodeling process and its effect on ventricular function during chronic volume overload, chronic pressure overload and following myocardial infarction will be the focus of this article.

Lipodermatosclerosis and the significance of proteolytic remodeling in the pathogenesis of venous ulceration (Review).

The preceding stage of venous ulceration represents a scleroderma-like hardening of the skin called lipodermatosclerosis. Clinical stages such as lipodermatosclerosis and venous ulceration, which succeed one another are highly associated to chronic venous insufficiency. Lipodermatosclerosis is characterized by fibrous scar tissue of the reticular dermis built up of collagen bundles and loss of cellular components, whereas venous ulceration is characterized by total loss of epidermis and partially of matrix structures in the upper dermis. There is a growing recognition that an excessive proteolytic activity by proteases, in particular that of matrix metalloproteinases and fibrinolytic factors of the plasminogen activation system may be a key feature in the pathophysiological understanding of venous leg ulcer formation. Lipodermatosclerosis displays an intense ongoing proteolytic process by elevated matrix metalloproteinase activity, as recently shown on different molecular and biological levels. Elevated expression on mRNA and protein level of matrix metalloproteinases and fibrinolytic factors of the plasminogen activation system have been detected in liposclerotic skin lesions. In addition, matrix metalloproteinases were proteolytically activated confirmed by zymography experiments and collagen degradation assays. Therefore it is well conceivable, that proteolytic enzymes of matrix metalloproteinases could initiate an elevated turnover of the extracellular matrix with subsequent breakdown of the matrix scaffold finally resulting in venous ulceration.

Increased gelatinase A (MMP-2) and gelatinase B (MMP-9) activities in human brain after focal ischemia

Matrix metalloproteinases (MMPs) are involved in remodelling extracellular matrix. Gelatinase B (MMP-9) is an inducible 92 kDa MMP expressed by neutrophils, microglia, and endothelial cells. Gelatinase A (MMP-2) is a 72 kDa MMP, constitutively expressed in brain. Elevated MMP activity has been linked to various pathologic conditions, and the therapeutic benefit of MMP inhibitors is under study in a few experimental models. Using gelatin zymography, we have compared activities of these MMPs in infarcted and matched non-infarcted cerebral tissue from eight subjects dying at intervals of less than 2 h to several years after a stroke. Gelatinase B activity was markedly elevated in the infarcted tissue at two days post-infarction, and remained elevated in cases dying months after the event. Increases in gelatinase A activity were subtle at 2–5 days; they were marked and significant in cases dying at 4 months and later. The findings indicate distinct temporal profiles of post-ischemic gelatinase activity in human brain, with earlier but equally persistent elevation in gelatinase B when compared to gelatinase A.

Cardiac collagen remodeling in the cardiomyopathic Syrian hamster and the effect of losartan.

Although increased deposition of collagen proteins has been described in cardiomyopathy, little is known of the temporal relationship between events in collagen gene transcription and the occurrence of cardiac fibrosis, the removal of collagen by matrix metalloproteinases (MMPs), or of the regulation of these events by angiotensin AT1 receptors in this disease. We sought to study steady-state collagen mRNA abundance and the deposition of specific collagen subtypes in right and left ventricular muscle of Syrian cardiomyopathic (CMP) hamsters at different stages of cardiomyopathy. Using zymography, we also investigated the gelatinolytic activities of different MMPs to gain some information about collagen removal in experimental hearts. Finally, we investigated the effect of AT1 receptor blockade (losartan) on collagen remodeling. We observed that the mRNA levels of types I and III collagens were significantly increased in all four experimental groups (35, 65, 120, and 200 day) in left ventricular tissue when compared to control (F1-beta strain) values. The mRNA levels of these collagen species in experimental right ventricular tissue samples were only elevated significantly in the 35 and 200 day experimental groups when compared to controls. Fibrillar collagen deposition was elevated in left and right ventricular CMP samples after a lag period from the occurrence of corresponding increases in mRNA abundance. Although 2-week losartan treatment of 65, 120 and 200 day experimental groups had no significant effect on left ventricular fibrillar collagen concentration or collagen mRNA abundance when compared to vehicle-infused CMP hamsters, AT1 receptor blockade was associated with complete regression of cardiac hypertrophy. Both MMP-1 (54 kDa band) and MMP-2 (58 and 62 kDa bands) activities were increased in left ventricular CMP tissues at 65, 120 and 200 days when compared to F1-beta controls. Losartan treatment was associated with significant attenuation of MMP activities in cardiomyopathic samples at 65 and 120 days. Thus, elevation of mRNA abundance of fibrillar collagen genes occurs at very early stages in this model of cardiomyopathy, and corresponding collagen proteins were subsequently deposited in the cardiac interstitium at later stages. As collagen concentration was significantly increased in later stages of cardiomyopathy studied herein (120 and 200 day groups), our data support the hypothesis that collagen synthesis exceeds the capacity of collagen removal during the progression of cardiomyopathy. Nevertheless, cardiac collagen remodeling may be facilitated by elevated MMP activity in cardiomyopathic stages in this experimental model, and we suggested that attenuation of MMP activity in the presence of losartan may be a cardioprotective mechanism of this agent.