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Concept: Tissue inhibitor of metalloproteinases

172

Infection with Wuchereria bancrofti can cause severe disease characterized by subcutaneous fibrosis and extracellular matrix remodeling. Matrix metalloproteinases (MMPs) are a family of enzymes governing extracellular remodeling by regulating cellular homeostasis, inflammation, and tissue reorganization, while tissue-inhibitors of metalloproteinases (TIMPs) are endogenous regulators of MMPs. Homeostatic as well as inflammation-induced balance between MMPs and TIMPs is considered critical in mediating tissue pathology.

Concepts: Inflammation, Extracellular matrix, Infection, Matrix metalloproteinase, Diethylcarbamazine, Filariasis, Wuchereria bancrofti, Tissue inhibitor of metalloproteinases

167

Tissue inhibitors of metalloproteinases (TIMPs) while originally characterized as inhibitors of matrix metalloproteinases (MMPs) have recently been shown to have a wide range of functions that are independent of their MMP inhibitory properties. Tissue inhibitor of metalloproteinases-3 (TIMP-3) is a potent inhibitor of VEGF-mediated angiogenesis and neovascularization through its ability to block the binding of VEGF to its receptor VEGFR-2. To identify and characterize the anti-angiogenic domain of TIMP-3, structure function analyses and synthetic peptide studies were performed using VEGF-mediated receptor binding, signaling, migration and proliferation. In addition, the ability of TIMP-3 peptides to inhibit CNV in a mouse model was evaluated. We demonstrate that the anti-angiogenic property resides in the COOH-terminal domain of TIMP-3 protein which can block the binding of VEGF specifically to its receptor VEGFR-2, but not to VEGFR-1 similar to the full-length wild-type protein. Synthetic peptides corresponding to putative loop 6 and tail region of TIMP-3 have anti-angiogenic properties as determined by inhibition of VEGF binding to VEGFR-2, VEGF-induced phosphorylation of VEGFR-2 and downstream signaling pathways as well as endothelial cell proliferation and migration in response to VEGF. In addition, we show that intravitreal administration of TIMP-3 peptide could inhibit the size of laser-induced choroidal neovascularization lesions in mice. Thus, we have identified TIMP-3 peptides to be efficient inhibitors of angiogenesis and have a potential to be used therapeutically in diseases with increased neovascularization.

Concepts: Protein, Signal transduction, Angiogenesis, Peptide, Enzyme inhibitor, Inhibitor, Matrix metalloproteinase, Tissue inhibitor of metalloproteinases

167

Synthetic inhibitors of matrix metalloproteinases (MMPs) designed previously as well as tissue inhibitors of metalloproteinases (TIMPs) lack enzyme selectivity, which has been a major obstacle for developing the inhibitors into safe and effective MMP-targeted drugs. Here we designed a fusion protein named APP-IP-TIMP-2, in which the ten amino acid residues sequence of APP-derived MMP-2-selective inhibitory peptide (APP-IP) is added to the N terminus of TIMP-2. The APP-IP and TIMP-2 regions of the fusion protein are designed to interact with the active site and the hemopexin-like domain of MMP-2, respectively. The reactive site of the TIMP-2 region, which has broad specificity against MMPs, is blocked by the APP-IP adduct. The recombinant APP-IP-TIMP-2 showed strong inhibitory activity toward MMP-2 (Ki(app) = 0.68 pM), whereas its inhibitory activity toward MMP-1, MMP-3, MMP-7, MMP-8, MMP-9 or MT1-MMP was six orders of magnitude or more weaker (IC(50) > 1 μM). The fusion protein inhibited the activation of pro-MMP-2 in the concanavalin A-stimulated HT1080 cells, degradation of type IV collagen by the cells, and the migration of the stimulated cells. As compared with the decapeptide APP-IP (t1/2 = 30 min), APP-IP-TIMP-2 (t1/2 > 96 h) showed much longer half-life in cultured tumor cells. Therefore, the fusion protein may be a useful tool to evaluate contributions of proteolytic activity of MMP-2 in various pathophysiological processes. It may also be developed as an effective anti-tumor drug that has restricted side effects.

Concepts: Protein, Amino acid, Acid, Collagen, Peptide, Matrix metalloproteinase, Glycine, Tissue inhibitor of metalloproteinases

147

Subjects susceptible to chronic periodontitis (CP) show a high risk for the development of peiimplantitis (PI). Both diseases are multifactorial, presenting similarities in their pathophysiology and polygenic profile. MMP-13 (matrix metalloproteinases 13/ collagenase 3) is a collagenolytic enzyme, which expression is induced by TGF beta 3 (transforming growth factor type 3) in human gingival fibroblasts and inhibited by TIMP-2 (tissue inhibitor of metalloproteinase type 2). The aim of this study was to investigate the occurrence of peiimplantitis (PI) in subjects with history of chronic periodontitis (CP) and polymorphisms frequency in MMP13, TIMP2 and TGFB3 genes. One hundred and sixty-three volunteers received dental implant placement were submitted to oral and radiographic examination in order to identify past history of CP or presence of PI. Volunteers were divided into 4 groups: Control (without PI and CP, n=72), CP (with CP and without PI, n=28), PI (with PI and without CP, n=28) and diseased (with CP and PI, n=35). The chi-square test correlated genotypes in specific regions of MMP13 (rs2252070), TIMP2 (rs7501477) and TGFB3 (rs2268626) genes, considering the interaction between CP and PI. The results showed that volunteers with CP had 3.2 times more susceptibility to develop PI (p=0.0004) compared to those without CP. No significant association was observed in MMP13, TIMP2 and TGFB3 genes with CP or PI. CP is a risk factor to develop PI, however, there is no association of both diseases with polymorphisms in the MMP13, TIMP2 and TGFB3 genes.

Concepts: Gene, Genetics, Dental implant, Dentistry, Tissue inhibitor of metalloproteinases, Transforming growth factor, TGF beta, TGF beta 3

28

UVB exposure is well known to induce skin photodamage and photoaging that correlates with qualitative and quantitative deterioration of the dermal extracellular matrix (ECM) because of the upregulation of matrix metalloproteinases (MMPs). Although inhibitory effects of tissue inhibitor of metalloproteinases (TIMPs) on most MMPs have been reported, the protective role of TIMP-1 against photodamage is poorly understood. To address this, TIMP-1 function was augmented or abolished in a human skin xenograft photodamage model after the confirmation of significantly diminished TIMP-1 expression both in photoaged and intrinsically aged skins. During a chronic UVB exposure regimen, pre-treatment with a lentiviral vector overexpressing TIMP-1 or concomitant administration of an anti-TIMP-1-neutralizing antibody (NAB) led to photoprotection or more severe photodamage, respectively. Overexpression of TIMP-1 resulted in significant inhibition of UVB-induced ECM degradation, as well as suppression of decreased skin elasticity and roughness, whereas the NAB-mediated inhibition of TIMP-1 had opposite effects. Furthermore, UVB-induced production of the pro-inflammatory cytokine, tumor necrosis factor α, was inhibited by TIMP-1 treatment of human keratinocytes. Taken together, these data shed light on the important role of TIMP-1 in protection and recovery from cutaneous photodamage because of its suppression of ECM degradation and inflammation.

Concepts: Extracellular matrix, Skin, Qualitative research, Tissue, Matrix metalloproteinase, Tumor necrosis factor-alpha, Epidermis, Tissue inhibitor of metalloproteinases

27

Background: Matrix metalloproteinases (MMPs) are key players in proteolytic blood-brain barrier (BBB) disruption during ischemic stroke, leading to vascular edema, hemorrhagic transformation and infiltration by leukocytes. Their effect is dampened by the endogenous tissue inhibitors of metalloproteinases (TIMPs). The respective cellular source of specific MMPs and TIMPs during BBB breakdown is still under investigation. Methods: We analyzed the MMP and TIMP release of human brain microvascular endothelial cells (BMECs) under oxygen glucose deprivation (OGD). Cultured human BMECs (the hCMEC/D3 cell line) were subjected to OGD (6, 12, 18 and 24 h). Gene expression of MMP-2, MMP-9, TIMP-1 and TIMP-2 were serially measured by quantitative real time-PCR and compared to ELISA-detected cell culture medium levels. Results: OGD induced a significant and long-lasting increase in MMP-2 gene expression, reaching a plateau after 12 h. Medium protein levels of MMP-2 were correspondingly elevated at 12 h of OGD. The MMP-9 synthesis rate was detectable at very low levels and remained unaffected by OGD. TIMP-1 gene expression and secretion declined under OGD, whereas both expression and secretion of TIMP-2 remained stable. Contrary to the respective gene expression rate, medium levels of MMP-2, TIMP-1 and TIMP-2 started a simultaneous decline after 12 h of OGD. This is most likely due to an impaired synthesis and enhanced consumption rate under OGD. Conclusions: The objective of our study was to determine the contribution of human BMECs to the MMP metabolism under in vitro OGD conditions simulating ischemic stroke. Our results suggest that human BMECs switch to a proinflammatory state by means of an enhanced production of MMP-2, attenuated release of TIMP-1, and unaffected production of TIMP-2. Thus, human BMECs might participate in the MMP-mediated BBB breakdown during ischemic stroke. However, our data does not support human BMECs to be a source of MMP-9.

Concepts: Inflammation, Cell, Bacteria, Blood vessel, Stroke, Cell culture, Matrix metalloproteinase, Tissue inhibitor of metalloproteinases

27

Matrix metalloproteinases (MMPs) and their natural tissue inhibitors (TIMPs) are involved in cell signaling processes and the release of extracellular matrix (ECM) and non-ECM molecules. Nonregulated MMP activity and an imbalance between metalloproteinases and their inhibitors might contribute to various disorders, including neurodegenerative diseases such as Alzheimer’s disease (AD), which is the most common cause of dementia. There is a complex relationship between MMPs and TIMPs with AD. It has been shown that MMPs and TIMPs are localized in neuritic senile plaques and neurofibrillary tangles in the postmortem brains of patients with AD. Some MMPs have also been shown to induce tau aggregation and the formation of neurofibrillary tangles in vitro. Moreover, MMPs contribute to AD pathogenesis via the disruption of the blood-brain barrier and promotion of neurodegeneration. However, MMPs can degrade both soluble and fibrillar forms of amyloid-β (Aβ). It has also been shown that Aβ enhances the expression of MMPs in neuroglial cultures and induces the release of TIMP-1 by brain cells. Inhibition of Aβ-induced MMP activity resulted in an improvement of performance tests in mice. Moreover, simultaneous examination of MMP-9, MMP-2, and TIMP-1 in the CSF contributed to the ability to differentiate between AD and other types of dementia. Thus, the aim of this literature study was to describe the role of MMPs and TIMPs in neurodegeneration, as well as their potential usefulness as CSF or plasma biomarkers in the diagnosis of AD as well as other neurodegenerative disorders and vascular dementia.

Concepts: Alzheimer's disease, Extracellular matrix, Neurology, Neurodegeneration, Neurodegenerative disorders, Matrix metalloproteinase, Tissue inhibitor of metalloproteinases, Neurodegenerative diseases

26

Tuberculosis (TB), a chronic infectious disease of global importance, is facing the emergence of drug-resistant strains with few new drugs to treat the infection. Pulmonary cavitation, the hallmark of established disease, is associated with very high bacillary burden. Cavitation may lead to delayed sputum culture conversion, emergence of drug resistance, and transmission of the infection. The host immunological reaction to M. tuberculosis is implicated in driving the development of TB cavities. TB is characterized by a matrix degrading phenotype in which the activity of proteolytic matrix metalloproteinases (MMPs) is relatively unopposed by the specific tissue inhibitors of metalloproteinases (TIMPs). Proteases, in particular MMPs, secreted from monocyte-derived cells, neutrophils and stromal cells, are involved in both cell recruitment and tissue damage and may cause cavitation. MMP activity is augmented by pro-inflammatory chemokines and cytokines, is tightly regulated by complex signalling paths and causes matrix destruction. MMP concentrations are elevated in human TB and are closely associated with clinical and radiological markers of lung tissue destruction. Immunomodulatory therapies targeting MMPs in preclinical and clinical trials are potential adjuncts to TB treatment. Strategies targeting patients with cavitatory TB have the potential to improve cure rates and reduce disease transmission.

Concepts: Medicine, Epidemiology, Infectious disease, Lung, Infection, Tuberculosis, Matrix metalloproteinase, Tissue inhibitor of metalloproteinases

23

The rate of intervertebral disc degeneration (IVDD) is influenced by environmental factors. Extracellular matrix (ECM) destruction and apoptosis of intervertebral disc cells are major characteristics of IVDD. ECM degradation is closely linked to up-regulation of matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMP). This study aimed to elucidate the molecular biological changes during IVDD under conditions of weightlessness and hypergravity.

Concepts: DNA, Gene, Molecular biology, Extracellular matrix, Cell biology, Matrix metalloproteinase, Biomolecule, Tissue inhibitor of metalloproteinases

22

Mesenchymal stem cells (MSCs) are stromal cells that display self-renewal and multipotent differentiation capacity. The repertoire of mature cells generated ranges but is not restricted to: fat, bone and cartilage. Their potential importance for both cell therapy and maintenance of in vivo homeostasis is indisputable. Nonetheless, both their in vivo identity and use in cell therapy remain elusive. A drawback generated by this fact is that little is known about the MSC niche and how it impacts differentiation and homeostasis maintenance. Hence, the roles played by the extracellular matrix (ECM) and its main regulators namely: the Matrix Metalloproteinases (MMPs) and their counteracting inhibitors (TIMPs and RECK) upon stem cells differentiation are only now beginning to be unveiled. Here, we will focus on mesenchymal stem cells and review the main mechanisms involved in adipo, chondro and osteogenesis, discussing how the extracellular matrix can impact not only lineage commitment, but, also, their survival and potentiality. This review critically analyzes recent work in the field in an effort towards a better understanding of the roles of Matrix Metalloproteinases and their inhibitors in the above-cited events.

Concepts: Extracellular matrix, Stem cell, Mesenchymal stem cell, Bone marrow, Stem cells, Cartilage, Matrix metalloproteinase, Tissue inhibitor of metalloproteinases