Broad-spectrum Matrix Metalloproteinases Inhibitor Research Articles

Matrix metalloproteinases: a dual role in breast cancer?

353 ISSN 1758-1923 10.2217/BMT.13.36 © 2013 Future Medicine Ltd Breast Cancer Manage. (2013) 2(5), 353–356 Matrix metalloproteinases as cancer targets Breast cancer 5-year survival rates in the UK have improved up to 85% over the last decade due to earlier detection, better diagnosis and an increasing range of treatment options. Today, the majority of breast cancer-related deaths are due to the spread of tumor cells in the body. Therefore, the search for markers of aggressive disease and tumor cell dissemination is ongoing, along with ways to prevent or treat invasive spread. Matrix metalloproteinases (MMPs) have long been regarded as potential targets for anticancer therapy. In humans, the MMPs are a family of 24 secreted endopeptidases that can collectively degrade all protein components of the extracellular matrix. Although they play important roles in normal physiological processes, including wound healing and embryonic development, they are also often found to be expressed at higher levels in cancer tissues compared with their normal counterparts. Numerous studies have demonstrated their involvement in tumor invasion and metastasis in various cancer types [1]. The tumorspecific upregulation of MMPs, together with their prominent roles in cancer dissemination and a high protein sequence homology at their catalytic sites, led to the development in the 1980s and 1990s of smallmolecule active site-directed inhibitors for cancer therapy [2]. Initial preclinical studies had generated promising findings and these broadspectrum inhibitors went into clinical trials accompanied by great optimism. Unfortunately, these trials proved disappointing, with some of the inhibitors in fact promoting tumor progression and decreasing survival rates in some types of cancer [3,4]. Subsequent in-depth analysis of in vitro and in vivo studies revealed a number of factors that may in part explain the limited beneficial effects of treatment with broad-spectrum MMP inhibitors. The discrepancy in preclinical and clinical efficacy of these drugs is most probably related to the different disease stages used. While in vivo animal studies were most

Fibulin-3, -4, and -5 Are Highly Susceptible to Proteolysis, Interact with Cells and Heparin, and Form Multimers

Extracellular short fibulins, fibulin-3, -4, and -5, are components of the elastic fiber/microfibril system and are implicated in the formation and homeostasis of elastic tissues. In this study, we report new structural and functional properties of the short fibulins. Full-length human short fibulins were recombinantly expressed in human embryonic kidney cells and purified by immobilized metal ion affinity chromatography. All three fibulins showed various levels of degradation after the purification procedure. N-terminal sequencing revealed that all three fibulins are highly susceptible to proteolysis within the N-terminal linker region of the first calcium-binding epidermal growth factor domain. Proteolytic susceptibility of the linker correlated with its length. Exposure of these fibulins to matrix metalloproteinase (MMP)-1, -2, -3, -7, -9, and -12 resulted in similar proteolytic fragments with MMP-7 and -12 being the most potent proteases. Fibulin-3 proteolysis was almost completely inhibited in cell culture by the addition of 25 μm doxycycline (a broad spectrum MMP inhibitor). Reducible fibulin-4 dimerization and multimerization were consistently observed by SDS-PAGE, Western blotting, and mass spectrometry. Atomic force microscopy identified monomers, dimers, and multimers in purified fibulin-4 preparations with sizes of ∼10-15, ∼20-25, and ∼30-50 nm, respectively. All short fibulins strongly adhered to human fibroblasts and smooth muscle cells. Although only fibulin-5 has an RGD integrin binding site, all short fibulins adhere at a similar level to the respective cells. Solid phase binding assays detected strong calcium-dependent binding of the short fibulins to immobilized heparin, suggesting that these fibulins may bind cell surface-located heparan sulfate.

TNFα cleavage beyond TACE/ADAM17: matrix metalloproteinase 13 is a potential therapeutic target in sepsis and colitis

For decades, matrix metalloproteinases (MMPs) have been described as rather unspecific matrix‐ and collagen‐degrading enzymes. Accordingly, this group of proteases was mainly associated with tissue remodeling and pathologic conditions such as tumour development and metastasis. However, broad spectrum MMP inhibitors completely failed in cancer therapy. Recent studies with gene deficient mice revealed more specific functions of MMPs, e.g . the cleavage of chemokines, thereby stimulating inflammatory responses (Dufour & Overall, 2013). Additionally, mass spectrometry‐based proteomic techniques allowed for the identification of hundreds of new substrates of MMPs, giving rise to unexpected biological activity of these proteases in health and disease. MMP13 is known to be one of few collagenases capable of cleaving rigid collagen fibrils. This ability is based on a complex unfolding mechanism of the triple‐helical collagen structure. Therefore, MMP13 biology is well studied in bone and cartilage turnover but little is known about other functions. In this issue, Libert and coworkers report on a pro‐inflammatory activity of MMP13 not by ‘simply’ degrading extracellular matrix but through the release of biologically active soluble TNFα from its membrane bound precursor form. Ectodomain shedding of TNFα is a paracrine signaling event in inflammation performed by the metalloprotease tumour necrosis factor α converting enzyme (TACE, also known as ADAM17) (Scheller et al, 2011). Although TACE/ADAM17 is believed to be the major sheddase of TNFα, Vandenbroucke et al demonstrated that after LPS‐induced sepsis and DSS‐induced colitis, MMP13 is up‐regulated and cleaves TNFα. This leads to ER‐stress and mucus depletion resulting in an increased interaction of bacteria with the intestinal epithelium. Moreover, elevated levels of bioactive TNFα affect the intestinal permeability through endocytosis of tight junction proteins, thereby increasing systemic inflammation. All these events were abolished in MMP13 deficient mice, resulting in increased survival of MMP13 knockout animals, when treated with LPS or DSS. …

Expression and inhibition of matrix metalloproteinase (MMP)-8, MMP-9 and MMP-12 in early colonic anastomotic repair

Submucosal collagen is paramount for colonic anastomotic integrity. Matrix metalloproteinases (MMPs) mediate collagen degradation that increases the risk of wound dehiscence. Although broad-spectrum MMP inhibitors are beneficial for anastomotic strength, they can cause adverse reactions. Knowledge of specific MMPs responsible for the weakening of anastomoses can be used to optimise MMP inhibition therapy. We aimed to quantify transcript and protein levels of multiple MMPs in colonic anastomoses and evaluate the effect of inhibiting the MMPs that displayed the highest expression levels on anastomotic repair. Left-sided colonic anastomoses were made in male Sprague-Dawley rats. After 3 days when biomechanical strength is lowest, MMP mRNA and protein levels were measured by quantitative real-time polymerase chain reaction, enzyme-linked immunosorbent assays and gelatin zymography. The effects of the MMP-8, MMP-9 and MMP-12 synthetic inhibitor AZD3342 was also studied. MMP-8, MMP-9 and MMP-12 gene and protein expression increased profoundly (p < 0.01), and MMP-13 mRNA and MMP-2 mRNA and protein modestly (p < 0.001) in the anastomoses. MMP-3 mRNA levels were not up-regulated significantly compared with adjacent uninjured colon. Increased anastomotic MMP-12 levels paralleled macrophage infiltration by immunohistochemical analyses. AZD3342 (50 mg/kg) treatment increased the anastomotic breaking strength by 29% (p = 0.015) day 3 compared with vehicle. Improved anastomotic strength was not accompanied with alterations of type I or type III procollagen mRNA but was possibly due to inhibition of the concerted digestive action on the existent submucosal collagens by the targeted MMPs. The present findings justify the concept of selective MMP inhibition to enhance anastomotic strength in colon.

Abstract 1397: BMP4 inhibits the proliferation of breast cancer cells in 3D and induces a migratory phenotype in MDA-MB-231 cells.

Abstract Bone morphogenetic protein 4 (BMP4) is an extracellular signaling molecule that belongs to the bone morphogenetic protein (BMP) family, which in turn is part of the transforming growth factor β (TGF-β) -superfamily. BMPs regulate cell proliferation, differentiation and motility, and have also been shown to be involved in cancer pathogenesis. We have previously shown that BMP4 is able to consistently reduce breast cancer cell proliferation through G1 cell cycle arrest and simultaneously to induce the migration in a subset of these cell lines. In order to study the effects of BMP4 in a more physiological setting, we used two different 3D culture systems: Matrigel, a basement membrane extract from mouse sarcoma cells, and a synthetic polyethylene glycol (PEG) gel with matrix metalloproteinase (MMP) degradable cross-links and RGD peptides. MCF-10A normal breast epithelial cells formed round acini with a hollow lumen and correct apicobasal localization of α-6 integrin in Matrigel whereas irregular structures without lumen formation or apicobasal integrin localization were seen in PEG gel. Thus the two matrices do not provide identical 3D environments. In Matrigel, BMP4 treatment reduced the proliferation of MCF-10A cells and T-47D breast cancer cells. In PEG gel, reduced growth was also seen in two other breast cancer cell lines, MDA-MB-231 and MDA-MB-361. To clarify the mechanisms behind the growth suppressive effects of BMP4, we first examined the expression levels of 11 known cell cycle regulators in T-47D and MDA-MB-361cells grown in 2D culture using Western blot. Based on its consistent upregulation in both cell lines, p21 was selected for detailed analyses. In 2D culture, BMP4 induced the expression of p21 in all three breast cancer cell lines but not in MCF-10A cells. Unexpectedly, there was no correlation between p21 expression and the BMP4 induced growth suppression in 3D. These data seem to indicate that different mechanisms are involved in BMP4 mediated cell growth regulation in 2D and 3D environments. Finally, in response to BMP4 treatment MDA-MB-231 cells formed large branching, stellate structures in Matrigel, suggestive of increased cell migration. This effect was not seen in PEG gel. Gremlin, a BMP antagonist, reversed the stellate phenotype, confirming that the effect was indeed dependent on BMP4. Similarly, a broad-spectrum MMP inhibitor, Batimastat, was also able to reverse the stellate appearance. These results imply that the actions of MMPs are required for this migratory phenotype. Taken together, our results show that BMP4 reduced the growth of breast epithelial cells and breast cancer cells in 3D culture. In addition, the highly metastatic breast cancer cell line MDA-MB-231 formed stellate, branching structures upon BMP4 treatment in Matrigel. Further research is needed to elucidate the molecular mechanisms behind these BMP4 induced phenotypes and their clinical relevance. Citation Format: Minna E. Ampuja, Riikka Jokimäki, Emma-Leena Alarmo, Kati Juuti-Uusitalo, Anne Kallioniemi. BMP4 inhibits the proliferation of breast cancer cells in 3D and induces a migratory phenotype in MDA-MB-231 cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1397. doi:10.1158/1538-7445.AM2013-1397

Abstract 2134: Bone-targeted MMP inhibitors for the treatment of multiple myeloma.

Abstract Multiple Myeloma (MM) is a plasma cell malignancy that is hallmarked by areas of extensive bone destruction. Our group, and others have recently shown that individual host or tumor derived MMP-2 and MMP-9 play an important role in the progression of many cancers in the bone microenvironment, including MM. These data support the design of selective inhibitors of individual MMPs for the treatment of multiple myeloma. Previous clinical trials with broad-spectrum matrix metalloproteinase (MMP) inhibitors failed, arguably because the precise roles of individual MMPs had not been fully appreciated. Therefore, in the current study, we have developed novel bone seeking MMP inhibitors (BMMPIs) that are highly selective to MMP-2 and structurally are based on the bone targeting bisphosphonate, Tiludronate. In vitro, BMMPIs showed greater enzyme inhibition, and specificity for MMP-2 than previously synthesized bisphosphonates (e.g. Zoledronate, Etidronate and Tiludronate) with Ki values in the nanomolar range. Further treatment of the 5TGM1 mouse MM cell line with low micromolar concentrations of the BMMPI inhibitors significantly reduced cell viability through inhibition of cell growth (p&amp;lt;0.05). In vivo, 5TGMI tumor-bearing mice receiving BMMP inhibitors three times a week, showed significant increase in overall survival compared to vehicle and tiludronate treated mice over 80 days (p&amp;lt;0.05). This was associated with a reduction in tumor burden and protection from tumor-associated bone loss compared to vehicle treated mice as measured by x-ray and histomorphometry. These data suggest that given the roles for MMPs in tumor progression in bone, that our novel BMMPIs may be effective in the treatment of MM. We predict that MMP-2 specific BMMPIs may be more effective than current clinically used bisphosphonates for the treatment of MM, and may eliminate undesirable side effects of broad-spectrum MMP inhibitors due to their high specificity and bone seeking nature. We are currently continuing to characterize these BMMPI both as single agents, and in combination with existing MM treatments (eg. Bortezomib) for the treatment and eradication of MM. Citation Format: Gemma Shay, Marilena Tauro, Antonio Laghezza, Paolo Tortorella, Lori A. Hazlehurst, Conor C. Lynch. Bone-targeted MMP inhibitors for the treatment of multiple myeloma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2134. doi:10.1158/1538-7445.AM2013-2134

Early VEGFR2 activation in response to flow is VEGF-dependent and mediated by MMP activity

Although several potential mechanosensors/mechanotransducers have been proposed, the precise mechanisms by which ECs sense and respond to mechanical forces and translate them into biochemical signals remains unclear. Here, we report that two major ligand-dependent tyrosine autophosphorylation sites of VEGFR2, Y1175 and Y1214, are rapidly activated by shear stress in human coronary artery endothelial cells (HCAECs). Neutralizing antibody against VEGFR2 not only abrogates flow-induced phosphorylation of these tyrosine residues, but also has a marked inhibitory effect on downstream eNOS activation. In situ proximity ligation assay revealed that VEGF and VEGFR2 are closely associated in HCAECs, and more importantly, this association is increased with flow. Finally, we show that flow-induced VEGFR2 activation is attenuated in the presence of the broad spectrum matrix metalloproteinase (MMP) inhibitor, GM6001. Taken together, our results suggest that a ligand-dependent mechanism involving the activity of MMPs plays a key role in the early, shear stress-induced activation of VEGFR2.

Matrix Metalloproteinase-9 Reduces Islet Amyloid Formation by Degrading Islet Amyloid Polypeptide

Deposition of islet amyloid polypeptide (IAPP) as amyloid is a pathological hallmark of the islet in type 2 diabetes, which is toxic to β-cells. We previously showed that the enzyme neprilysin reduces islet amyloid deposition and thereby reduces β-cell apoptosis, by inhibiting fibril formation. Two other enzymes, matrix metalloproteinase (MMP)-2 and MMP-9, are extracellular gelatinases capable of degrading another amyloidogenic peptide, Aβ, the constituent of amyloid deposits in Alzheimer disease. We therefore investigated whether MMP-2 and MMP-9 play a role in reducing islet amyloid deposition. MMP-2 and MMP-9 mRNA were present in mouse islets but only MMP-9 activity was detectable. In an islet culture model where human IAPP (hIAPP) transgenic mouse islets develop amyloid but nontransgenic islets do not, a broad spectrum MMP inhibitor (GM6001) and an MMP-2/9 inhibitor increased amyloid formation and the resultant β-cell apoptosis. In contrast, a specific MMP-2 inhibitor had no effect on either amyloid deposition or β-cell apoptosis. Mass spectrometry demonstrated that MMP-9 degraded amyloidogenic hIAPP but not nonamyloidogenic mouse IAPP. Thus, MMP-9 constitutes an endogenous islet protease that limits islet amyloid deposition and its toxic effects via degradation of hIAPP. Because islet MMP-9 mRNA levels are decreased in type 2 diabetic subjects, islet MMP-9 activity may also be decreased in human type 2 diabetes, thereby contributing to increased islet amyloid deposition and β-cell loss. Approaches to increase islet MMP-9 activity could reduce or prevent amyloid deposition and its toxic effects in type 2 diabetes.

Abstract WP298: Matrix Metalloproteinases Are Involved In The Regulation Of Angiogenesis After Intracerebral Hemorrhage

Background and purpose: Intracerebral hemorrhage (ICH) is one of the most devastating subtypes of stroke. And our previous work has demonstrated that ICH induces angiogenesis, accompanied by up-regulation of pro-angiogenic factors. Matrix metalloproteinases (MMPs) can cause blood-brain barrier dysfunction by degrading the extracellular cellular matrix (ECM) around the vessels after ICH, but opening a way for the prolonging newborn vessels is a key step for their functional structure, therefore, the purpose of the study is to investigate whether MMPs are involved in the process of angiogenesis after ICH. Methods: Thirty Kunming mice were randomly divided into sham group, ICH group and doxycycline (DOX)-treated group. And then 5 mice were randomly selected for Western Blot to detect the expression of MMP9, and the other five for the immunohistochemistry to detect vWF. ICH model was induced by injection collagenase type VII into right globus pallidus stereotaxically, and DOX, a broad-spectrum MMP inhibitor, was injected by intraperitoneally at 7 days after ICH induction. Neurological severity score (NSS), corner turn test and foot-fault test were used to investigate the neurological function. And vWF-positive vessels were counted around the hematoma. Results: At 7 days, there is no difference between the two ICH-induced groups in NSS, corner turn test, foot-fault test; while at 14 days, the NSS in ICH group is significantly lower than that of DOX-treated group ( P &lt;0.05), and the times for right-turn and foot-fault in ICH group are notably fewer than those of DOX-treated group ( P &lt;0.05); At 14d, the number of vWF-postive microvessel in ICH group was significantly larger than that of DOX-treated group ( P &lt;0.01), and Western Blot revealed that DOX decreased MMP9 expression remarkably( P &lt;0.01). Conclusion: Matrix metalloproteinases were involved in the regulation of angiogenesis after ICH.

Abstract TP256: Targets of Vascular Protection in Acute Ischemic Stroke Differ in Type 2 Diabetes

Background: Hemorrhagic transformation (HT) is an important complication of acute ischemic stroke particularly in diabetic patients receiving thrombolytic treatment with tissue plasminogen activator (tPA), the only approved drug for the treatment of AIS. The objective of the current study was to determine the effects of acute inhibition of the mediators of HT (i.e., NFkB, peroxynitrite, and matrix metalloproteinases) on vascular injury and functional outcome in a diabetic model of cerebral ischemia. Methods: Ischemia was induced by middle cerebral artery occlusion in control and type 2 diabetic Goto-Kakizaki rats. Treatment groups received a single dose of peroxynitrite decomposition catalyst FeTPPs, non-specific NFkB inhibitor curcumin, or broad-spectrum matrix metalloproteinase (MMP) inhibitor minocycline at reperfusion. Post-stroke infarct volume, edema, hemorrhage, neurological deficits, and MMP-9 activity were evaluated. Results: All therapies reduced MMP-9 and HT in diabetic groups. In addition, acute curcumin and minocycline therapy reduced edema in these animals. Improved neurological function was observed in varying degrees with treatment as indicated by beam-walk performance, modified Bederson scores and grip strength; however, infarct size was similar to untreated diabetic animals. In control animals, all treatments reduced MMP-9 activity yet bleeding was not improved. Neuroprotection was only conferred by curcumin and minocycline. Conclusion: Uncovering underlying mechanisms contributing to the success of acute therapy in diabetes will advance tailored stroke therapies. Table 1 . Summary of experimental stroke data. SE are shown in parentheses. a p&lt;0.05 vs. untreated control, b p&lt;0.05 vs. untreated, c p&lt;0.05 vs. untreated diabetes, d p&lt;0.001 vs. untreated control, e p&lt;0.01 vs. untreated control, * All values in this data set were below detection.

Doxycycline-Mediated Inhibition of Corneal Angiogenesis: An MMP-Independent Mechanism

We explored the underlying mechanisms of doxycycline inhibition of corneal angiogenesis. Human umbilical vein endothelial cells (HUVECs) were cultured in vitro in the presence or absence of VEGF, doxycycline, or broad-spectrum matrix metalloproteinase (MMP) inhibitors (1,10-phenanthroline [1PT] and batimastat). HUVEC proliferation was determined using Cell Counting Kit-8. Rats with VEGF-induced corneal neovascularization (CNV) were treated with saline solution, 0.1% doxycycline, 0.1% 1PT, or 50 μM batimastat (n = 7/group). The length and area of CNV were measured on day 7. The activity of MMP-2 and MMP-9 was determined by a fluorogenic peptide substrate. Western blotting and ELISA were used to analyze the expression of phosphorylated eNOS and Akt, and PI3K activity. Our results showed that doxycycline inhibited HUVEC proliferation induced by VEGF in a dose-dependent manner in vitro, and decreased CNV induced by VEGF in vivo in terms of vessel length and area. 1PT and batimastat showed similar MMP inhibitory functions with doxycycline in vitro and in vivo, but they had no effects on HUVEC proliferation, and only partially mimicked the inhibitory properties of doxycycline (∼45%) on angiogenesis induced by VEGF. In addition, although doxycycline is capable of modulating the PI3K/Akt-eNOS pathway in vitro and in vivo in an MMP-independent manner, 1PT and batimastat were not. The mechanism of doxycycline-mediated inhibition of angiogenesis occurs through MMP inhibitory activity and modulation of the PI3K/Akt-eNOS pathway, an MMP-independent mechanism.

Identification of a Novel Metalloproteinase and Its Role in Juvenile Development of the Tobacco Hornworm, Manduca sexta (Linnaeus)

Matrix metalloproteinases (MMPs) are a class of zinc-dependent endopeptidases that are highly conserved across numerous taxa, from bacteria to humans. Recently, MMPs have been identified in several insect species and are hypothesized to function in immunity and development. In this study, we identify a putative MMP and correlate its proteolytic activity and gene and protein expression in the tracheae with developmental stage. Ms-MMP gene expression increases 10-fold during molting, which is accompanied by an increase in both protein expression and gelatinolytic activity. To directly test the hypothesis that Ms-MMP plays a critical role in juvenile development of Manduca sexta, we injected a broad-spectrum MMP inhibitor and recorded its effects on growth and development. Inhibition of MMPs caused a delay in juvenile development and decreased growth rates. Understanding the function of MMPs will help us better understand molting and control of body size in insects. Furthermore, elucidating functions for MMPs in lower taxa may yield critical information about the evolution of the numerous MMPs found in vertebrates. J. Exp. Zool. (Mol. Dev. Evol.) 320B:105–117, 2013. © 2013 Wiley Periodicals, Inc.

Targets of vascular protection in acute ischemic stroke differ in type 2 diabetes

Hemorrhagic transformation is an important complication of acute ischemic stroke, particularly in diabetic patients receiving thrombolytic treatment with tissue plasminogen activator, the only approved drug for the treatment of acute ischemic stroke. The objective of the present study was to determine the effects of acute manipulation of potential targets for vascular protection [i.e., NF-κB, peroxynitrite, and matrix metalloproteinases (MMPs)] on vascular injury and functional outcome in a diabetic model of cerebral ischemia. Ischemia was induced by middle cerebral artery occlusion in control and type 2 diabetic Goto-Kakizaki rats. Treatment groups received a single dose of the peroxynitrite decomposition catalyst 5,10,15,20-tetrakis(4-sulfonatophenyl)prophyrinato iron (III), the nonspecific NF-κB inhibitor curcumin, or the broad-spectrum MMP inhibitor minocycline at reperfusion. Poststroke infarct volume, edema, hemorrhage, neurological deficits, and MMP-9 activity were evaluated. All acute treatments reduced MMP-9 and hemorrhagic transformation in diabetic groups. In addition, acute curcumin and minocycline therapy reduced edema in these animals. Improved neurological function was observed in varying degrees with treatment, as indicated by beam-walk performance, modified Bederson scores, and grip strength; however, infarct size was similar to untreated diabetic animals. In control animals, all treatments reduced MMP-9 activity, yet bleeding was not improved. Neuroprotection was only conferred by curcumin and minocycline. Uncovering the underlying mechanisms contributing to the success of acute therapy in diabetes will advance tailored stroke therapies.

A role for matrix metalloproteinases in nicotine-induced conditioned place preference and relapse in adolescent female rats.

Reconfiguration of extracellular matrix proteins appears to be necessary for the synaptic plasticity that underlies memory consolidation. The primary candidates involved in controlling this process are a family of endopeptidases called matrix metalloproteinases (MMPs); however, the potential role of MMPs in nicotine addiction-related memories has not been adequately tested. Present results indicate transient changes in hippocampal MMP-2, -3, and -9 expression following context dependent learning of nicotine-induced conditioned place preference (CPP). Members of a CPP procedural control group also indicated similar MMP changes, suggesting that memory activation occurred in these animals as well. However, hippocampal MMP-9 expression was differentially elevated in members of the nicotine-induced CPP group on days 4 and 5 of training. Inhibition of MMPs using a broad spectrum MMP inhibitor (FN439) during nicotine-induced CPP training blocked the acquisition of CPP. Elevations in hippocampal and prefrontal cortex MMP-3 expression—but not MMP-2 and -9—accompanied reactivation of a previously learned drug related memory. Decreases in the actin regulatory cytoskeletal protein cortactin were measured in the HIP and PFC during the initial two days of acquisition of CPP; however, no changes were seen following re-exposure to the drug related environment. These results suggest that MMP-9 may be involved in facilitating the intracellular and extracellular events required for the synaptic plasticity underlying the acquisition of nicotine-induced CPP. Furthermore, MMP-3 appears to be important during re-exposure to the drug associated environment. However, rats introduced into the CPP apparatus and given injections of vehicle rather than nicotine during training also revealed a pattern of MMP expression similar to nicotine-induced CPP animals.

Matrix metalloproteinase-9 in the initial injury after hepatectomy in mice

To investigate the role of matrix metalloproteinase (MMP)-9 in the pathogenesis of postoperative liver failure (PLF) after extended hepatectomy (EH). An insufficient volume of the remnant liver (RL) results in higher morbidity and mortality, and a murine model with 80%-hepatectomy was used. All investigations were performed 6 h after EH. Mice were first divided into two groups based on the postoperative course (i.e., the PLF caused or did not), and MMP-9 expression was measured by Western blotting. The source of MMP-9 was then determined by immunohistological stainings. Tissue inhibitor of metalloproteinase (TIMP)-1 is the endogenous inhibitor of MMP-9, and MMP-9 behavior was assessed by the experiments in wild-type, MMP-9(-/-) and TIMP-1(-/-) mice by Western blotting and gelatin zymography. The behavior of neutrophils was also assessed by immunohistological stainings. An anti-MMP-9 monoclonal antibody and a broad-spectrum MMP inhibitor were used to examine the role of MMP-9. Symptomatic mice showed more severe PLF (histopathological assessments: 2.97 ± 0.92 vs 0.11 ± 0.08, P < 0.05) and a higher expression of MMP-9 (71085 ± 18274 vs 192856 ± 22263, P < 0.01). Nonnative leukocytes appeared to be the main source of MMP-9, because MMP-9 expression corresponding with CD11b positive-cell was observed in the findings of immunohistological stainings. In the histopathological findings, the PLF was improved in MMP-9(-/-) mice (1.65% ± 0.23% vs 0.65% ± 0.19%, P < 0.01) and it was worse in TIMP-1(-/-) mice (1.65% ± 0.23% vs 1.78% ± 0.31%, P < 0.01). Moreover, neutrophil migration was disturbed in MMP-9(-/-) mice in the immunohistological stainings. Two methods of MMP-9 inhibition revealed reduced PLF, and neutrophil migration was strongly disturbed in MMP-9-blocked mice in the histopathological assessments (9.6 ± 1.9 vs 4.2 ± 1.2, P < 0.05, and 9.9 ± 1.5 vs 5.7 ± 1.1, P < 0.05). MMP-9 is important for the process of PLF. The initial injury is associated with MMP-9 derived from neutrophils, and MMP-9 blockade reduces PLF. MMP-9 may be a potential target to prevent PLF after EH and to overcome an insufficient RL.

Axonal growth towards Xenopus skin in vitro is mediated by matrix metalloproteinase activity

We have previously demonstrated that the growth of peripheral nervous system axons is strongly attracted towards limb buds and skin explants in vitro. Here, we show that directed axonal growth towards skin explants of Xenopus laevis in matrigel is associated with expression of matrix metalloproteinase (MMP)-18 and also other MMPs, and that this long-range neurotropic activity is inhibited by the broad-spectrum MMP inhibitors BB-94 and GM6001. We also show that forced expression of MMP-18 in COS-7 cell aggregates enhances axonal growth from Xenopus dorsal root ganglia explants. Nidogen is the target of MMPs released by cultured skin in matrigel, whereas other components remain intact. Our results suggest a novel link between MMP activity and extracellular matrix breakdown in the control of axonal growth.

Loss of Timp3 Gene Leads to Abdominal Aortic Aneurysm Formation in Response to Angiotensin II

Aortic aneurysm is dilation of the aorta primarily due to degradation of the aortic wall extracellular matrix (ECM). Tissue inhibitors of metalloproteinases (TIMPs) inhibit matrix metalloproteinases (MMPs), the proteases that degrade the ECM. Timp3 is the only ECM-bound Timp, and its levels are altered in the aorta from patients with abdominal aortic aneurysm (AAA). We investigated the causal role of Timp3 in AAA formation. Infusion of angiotensin II (Ang II) using micro-osmotic (Alzet) pumps in Timp3(-/-) male mice, but not in wild type control mice, led to adverse remodeling of the abdominal aorta, reduced collagen and elastin proteins but not mRNA, and elevated proteolytic activities, suggesting excess protein degradation within 2 weeks that led to formation of AAA by 4 weeks. Intriguingly, despite early up-regulation of MMP2 in Timp3(-/-)Ang II aortas, additional deletion of Mmp2 in these mice (Timp3(-/-)/Mmp2(-/-)) resulted in exacerbated AAA, compromised survival due to aortic rupture, and inflammation in the abdominal aorta. Reconstitution of WT bone marrow in Timp3(-/-)/Mmp2(-/-) mice reduced inflammation and prevented AAA in these animals following Ang II infusion. Treatment with a broad spectrum MMP inhibitor (PD166793) prevented the Ang II-induced AAA in Timp3(-/-) and Timp3(-/-)/Mmp2(-/-) mice. Our study demonstrates that the regulatory function of TIMP3 is critical in preventing adverse vascular remodeling and AAA. Hence, replenishing TIMP3, a physiological inhibitor of a number of metalloproteinases, could serve as a therapeutic approach in limiting AAA development or expansion.

A Pan Photoaffinity Probe for Detecting Active Forms of Matrix Metalloproteinases

A photoaffinity probe based on the scaffold of a potent broad-spectrum phosphinic peptide inhibitor of matrix metalloproteinases (MMPs) has been developed. A photolabile diazirine group for covalent modification of MMP active forms was incorporated at the P(1) ' position, and a tritium radioactive label for the sensitive detection of MMP covalent adducts by radioimaging was attached. The probe was characterized on seven catalytic domains of human MMPs (MMP-2, -3, -8, -9, -12, -13 and -14) and was found to display nanomolar affinities towards this set of MMPs, covalently modifying them with crosslinking yields varying from 12 to 58 %, thus leading to highly sensitive detection of these MMPs. In a complex proteome complemented with four recombinant MMPs (MMP-2, -9, -12 and -13), this probe enabled their simultaneous detection with a threshold of few femtomoles and low background labelling. Those properties should make this new pan-activity-based MMP probe a valuable tool for the detection of MMP active forms from biological fluids or tissue extracts.

Grafting of functional motifs onto protein scaffolds identified by PDB screening - an efficient route to design optimizable protein binders

Artificial miniproteins that are able to target catalytic sites of matrix metalloproteinases (MMPs) were designed using a functional motif-grafting approach. The motif corresponded to the four N-terminal residues of TIMP-2, a broad-spectrum protein inhibitor of MMPs. Scaffolds that are able to reproduce the functional topology of this motif were obtained by exhaustive screening of the Protein Data Bank (PDB) using STAMPS software (search for three-dimensional atom motifs in protein structures). Ten artificial protein binders were produced. The designed proteins bind catalytic sites of MMPs with affinities ranging from 450 nm to 450 μm prior to optimization. The crystal structure of one artificial binder in complex with the catalytic domain of MMP-12 showed that the inter-molecular interactions established by the functional motif in the artificial binder corresponded to those found in the MMP-14-TIMP-2 complex, albeit with some differences in geometry. Molecular dynamics simulations of the ten binders in complex with MMP-14 suggested that these scaffolds may allow partial reproduction of native inter-molecular interactions, but differences in geometry and stability may contribute to the lower affinity of the artificial protein binders compared to the natural protein binder. Nevertheless, these results show that the in silico design method used provides sets of protein binders that target a specific binding site with a good rate of success. This approach may constitute the first step of an efficient hybrid computational/experimental approach to protein binder design.

Metalloproteinases and Their Associated Genes Contribute to the Functional Integrity and Noise-Induced Damage in the Cochlear Sensory Epithelium

Matrix metalloproteinases (MMPs) and their related gene products regulate essential cellular functions. An imbalance in MMPs has been implicated in various neurological disorders, including traumatic injuries. Here, we report a role for MMPs and their related gene products in the modulation of cochlear responses to acoustic trauma in rats. The normal cochlea was shown to be enriched in MMP enzymatic activity, and this activity was reduced in a time-dependent manner after traumatic noise injury. The analysis of gene expression by RNA sequencing and qRT-PCR revealed the differential expression of MMPs and their related genes between functionally specialized regions of the sensory epithelium. The expression of these genes was dynamically regulated between the acute and chronic phases of noise-induced hearing loss. Moreover, noise-induced expression changes in two endogenous MMP inhibitors, Timp1 and Timp2, in sensory cells were dependent on the stage of nuclear condensation, suggesting a specific role for MMP activity in sensory cell apoptosis. A short-term application of doxycycline, a broad-spectrum inhibitor of MMPs, before noise exposure reduced noise-induced hearing loss and sensory cell death. In contrast, a 7 d treatment compromised hearing sensitivity and potentiated noise-induced hearing loss. This detrimental effect of the long-term inhibition of MMPs on noise-induced hearing loss was further confirmed using targeted Mmp7 knock-out mice. Together, these observations suggest that MMPs and their related genes participate in the regulation of cochlear responses to acoustic overstimulation and that the modulation of MMP activity can serve as a novel therapeutic target for the reduction of noise-induced cochlear damage.