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American Journal of Physiology. Lung... Mar 2003High-volume mechanical ventilation leads to ventilator-induced lung injury. This type of lung injury is accompanied by an increased release and activation of matrix...
High-volume mechanical ventilation leads to ventilator-induced lung injury. This type of lung injury is accompanied by an increased release and activation of matrix metalloproteinases (MMPs). To investigate the mechanism leading to the increased MMP release, we systematically studied the effect of mechanical stretch on human microvascular endothelial cells isolated from the lung. We exposed cells grown on collagen 1 BioFlex plates to sinusoidal cyclic stretch at 0.5 Hz using the Flexercell system with 17-18% elongation of cells. After 4 days of cell stretching, conditioned media and cell lysate were collected and analyzed by gelatin, casein, and reverse zymograms as well as Western blotting. RT-PCR of mRNA extracted from stretched cells was performed. Our results show that 1) cyclic stretch led to increased release and activation of MMP-2 and MMP-1; 2) the activation of MMP-2 was accompanied by an increase in membrane type-1 MMP (MT1-MMP) and inhibited by a hydroxamic acid-derived inhibitor of MMPs (Prinomastat, AG3340); and 3) the MMP-2 release and activation were preceded by an increase in production of extracellular MMP inducer (EMMPRIN). These results suggest that cyclic mechanical stretch leads to MMP-2 activation through an MT1-MMP mechanism. EMMPRIN may play an important role in the release and activation of MMPs during lung injury.
Topics: Antigens, CD; Antigens, Neoplasm; Antineoplastic Agents; Basigin; Blotting, Western; Cell Line; Culture Media, Conditioned; Endothelium, Vascular; Enzyme Activation; Enzyme Induction; Enzyme Inhibitors; Humans; Lung; Matrix Metalloproteinase 1; Matrix Metalloproteinase 2; Matrix Metalloproteinase 3; Matrix Metalloproteinase Inhibitors; Matrix Metalloproteinases, Membrane-Associated; Membrane Glycoproteins; Metalloendopeptidases; Organic Chemicals; Periodicity; RNA, Messenger; Reverse Transcriptase Polymerase Chain Reaction; Stress, Mechanical; Tissue Inhibitor of Metalloproteinase-2
PubMed: 12456388
DOI: 10.1152/ajplung.00290.2002 -
Drug Metabolism and Pharmacokinetics 2014The natural variant of the cytochrome P450 enzyme CYP2D6.1, CYP2D6.17, is most common in African populations, has three amino acid substitutions (T107I, R296C, and...
The natural variant of the cytochrome P450 enzyme CYP2D6.1, CYP2D6.17, is most common in African populations, has three amino acid substitutions (T107I, R296C, and S486T) compared to the wild-type, and is known to have a different ligand preference from CYP2D6.1. It is becoming increasingly important to understand differences in the metabolism of medicines in different ethnic groups in order to assess the relevance of clinical data from different countries. This study investigated differences in the inhibition profiles of drugs for CYP2D6 with respect to gene polymorphisms. Firstly, we used computer docking with six drugs to several CYP2D6.1 structures, sampled from the trajectory of MD simulations, and calculated MM-GB/SA scores representing binding free energies. We then used regression analysis to predict the potency with which drugs inhibited CYP2D6.1 based on MM-GB/SA scores. The pKi-values obtained were in good agreement with experimental values measured for the six drugs (r(2) = 0.81). We carried out the same analysis for CYP2D6.17 and the pKi-values calculated were also in good agreement with experimental values (r(2) = 0.92). Finally, we were able to successfully explain the different abilities of CYP2D6.1 and CYP2D6.17 to metabolize drugs in different ethnic groups with reference to their 3D-structures.
Topics: Cocaine; Computer Simulation; Cytochrome P-450 CYP2D6; Cytochrome P-450 CYP2D6 Inhibitors; Enzyme Inhibitors; Fluoxetine; Imipramine; Molecular Docking Simulation; Molecular Dynamics Simulation; Organic Chemicals; Polymorphism, Genetic; Protein Conformation; Quinidine; Regression Analysis; Stereoisomerism; Thioridazine
PubMed: 23857029
DOI: 10.2133/dmpk.dmpk-13-rg-044 -
Journal of Virology Mar 2002The release of neurotoxins by activated brain macrophages or microglia is one mechanism proposed to contribute to the development of neurological disease following...
The release of neurotoxins by activated brain macrophages or microglia is one mechanism proposed to contribute to the development of neurological disease following infection by lentiviruses, including feline immunodeficiency virus (FIV). Since molecular diversity in the lentiviral envelope gene influences the expression of host molecules implicated in neuronal injury, the role of the envelope sequence in FIV neuropathogenesis was investigated by using the neurovirulent FIV strain V1CSF, the nonneurovirulent strain Petaluma, and a chimera (FIVCh) containing the V1CSF envelope gene in a Petaluma background. All three viruses replicated in primary feline macrophages with equal efficiency, but conditioned medium from V1CSF- or FIVCh-infected cells was significantly more neurotoxic than medium from Petaluma-infected cultures (P < 0.001) and could be attenuated in a dose-dependent manner by treatment with either the matrix metalloproteinase (MMP) inhibitor prinomastat (PMT) or function-blocking antibodies to MMP-2. Although FIV sequences were detectable by PCR in brain tissue from neonatal cats infected with each of the viral strains, immunohistochemistry revealed increased astrogliosis and macrophage activation in the brains of V1CSF- and FIVCh-infected cats relative to the other groups, together with elevated markers of neuronal stress that included morphological changes and increased c-fos immunoreactivity. Similarly, MMP-2, but not MMP-9, mRNA and protein expression was increased in brain tissues of V1CSF- and FIVCh-infected cats relative to Petaluma-infected animals (P < 0.01). Infection with V1CSF or FIVCh was also associated with greater CD4(+) cell depletion (P < 0.001) and neurodevelopmental delays (P < 0.005), than in Petaluma-infected animals; these deficits improved following PMT therapy. These findings indicated that diversity in the envelope gene sequence influenced the neurovirulence exhibited by FIV both in vitro and in vivo, possibly through a mechanism involving the differential induction of MMP-2.
Topics: Animals; Animals, Newborn; Brain; Cats; Central Nervous System Viral Diseases; Female; Gene Expression Regulation, Viral; Genes, env; Humans; Immunodeficiency Virus, Feline; Infant, Newborn; Lentivirus Infections; Macrophages; Matrix Metalloproteinases; Mice; Neurons; Pregnancy; Tumor Cells, Cultured; Viral Envelope Proteins; Virulence
PubMed: 11861828
DOI: 10.1128/jvi.76.6.2622-2633.2002 -
The Journal of Biological Chemistry Mar 2002Recently, we have shown that membrane type 1 matrix metalloproteinase (MT1-MMP) exhibits integrin convertase activity. Similar to furin-like proprotein convertases,...
Processing of integrin alpha(v) subunit by membrane type 1 matrix metalloproteinase stimulates migration of breast carcinoma cells on vitronectin and enhances tyrosine phosphorylation of focal adhesion kinase.
Recently, we have shown that membrane type 1 matrix metalloproteinase (MT1-MMP) exhibits integrin convertase activity. Similar to furin-like proprotein convertases, MT1-MMP directly processes a single chain precursor of alpha(v) integrin subunit (pro-alpha(v)) into the heavy and light alpha-chains connected by a disulfide bridge. To evaluate functionality of MT1-MMP-processed integrins, we examined breast carcinoma MCF7 cells co-expressing alpha(v)beta(3) integrin with either the wild type or mutant MT1-MMP in a variety of migration and adhesion tests. Specific inhibitors of proprotein convertases and MMP were employed in our cell system to attenuate the individual pathways of pro-alpha(v) maturation. We present evidence that MT1-MMP cleavage of pro-alpha(v) in the cells did not affect RGD-ligand binding of the resulting alpha(v)beta(3) integrin but enhanced outside-in signal transduction through a focal adhesion kinase pathway. Enhanced tyrosine phosphorylation of focal adhesion kinase in cells co-expressing MT1-MMP and alpha(v)beta(3) integrin contributed to efficient adhesion and, especially, migration of cells on vitronectin, a ligand of alpha(v)beta(3) integrin. These mechanisms underscore the significance of a coordinated interplay between MT1-MMP and alpha(v)beta(3) integrin in tumor cells and identify downstream signaling pathways resulting from their interactions. Regulation of integrin maturation and functionality may be an important role of MT1-MMP in tumor cells.
Topics: Antineoplastic Agents; Binding Sites; Blotting, Western; Cell Adhesion; Cell Movement; Disulfides; Dose-Response Relationship, Drug; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Humans; Ligands; Matrix Metalloproteinase 1; Oligopeptides; Organic Chemicals; Phosphorylation; Precipitin Tests; Protein Binding; Protein-Tyrosine Kinases; Receptors, Vitronectin; Time Factors; Transfection; Tumor Cells, Cultured; Tyrosine; Vitronectin
PubMed: 11724803
DOI: 10.1074/jbc.M110269200 -
The Journal of Biological Chemistry Nov 2007Human diabetes mellitus (IDDM; type I diabetes) is a T cell-mediated disease that is closely modeled in non-obese diabetic (NOD) mice. The pathogenesis of IDDM involves...
Human diabetes mellitus (IDDM; type I diabetes) is a T cell-mediated disease that is closely modeled in non-obese diabetic (NOD) mice. The pathogenesis of IDDM involves the transmigration of autoimmune T cells into the pancreatic islets and the subsequent destruction of insulin-producing beta cells. Therapeutic interventions leading to beta cell regeneration and the reversal of established IDDM are exceedingly limited. We report here that specific inhibition of T cell intra-islet transmigration by using a small molecule proteinase inhibitor restores beta cell functionality, increases insulin-producing beta cell mass, and alleviates the severity of IDDM in acutely diabetic NOD mice. As a result, acutely diabetic NOD mice do not require insulin injections for survival for a significant time period, thus providing a promising clue to effect IDDM reversal in humans. The extensive morphometric analyses and the measurements of both the C-peptide blood levels and the proinsulin mRNA levels in the islets support our conclusions. Diabetes transfer experiments suggest that the inhibitor specifically represses the T cell transmigration and homing processes as opposed to causing immunosuppression. Overall, our data provide a rationale for the pharmacological control of the T cell transmigration step in human IDDM.
Topics: Animals; C-Peptide; Diabetes Mellitus, Type 1; Glucose; Immunosuppressive Agents; Insulin; Insulin-Secreting Cells; Matrix Metalloproteinase 14; Matrix Metalloproteinase Inhibitors; Mice; Mice, Inbred C57BL; Mice, Inbred NOD; Organic Chemicals; RNA, Messenger; T-Lymphocytes
PubMed: 17761671
DOI: 10.1074/jbc.M705348200 -
The Journal of Biological Chemistry Jul 2005We have discovered that clinically tested inhibitors of matrix metalloproteinases can control the functional activity of T cell membrane type-1 matrix metalloproteinase...
We have discovered that clinically tested inhibitors of matrix metalloproteinases can control the functional activity of T cell membrane type-1 matrix metalloproteinase (MT1-MMP) and the onset of disease in a rodent model of type 1 diabetes in non-obese diabetic mice. We determined that MT1-MMP proteolysis of the T cell surface CD44 adhesion receptor affects the homing of T cells into the pancreas. We also determined that both the induction of the intrinsic T cell MT1-MMP activity and the shedding of cellular CD44 follow the adhesion of insulin-specific, CD8-positive, Kd-restricted T cells to the matrix. Conversely, inhibition of these events by AG3340 (a potent hydroxamate inhibitor that was widely used in clinical trials in cancer patents) impedes the transmigration of diabetogenic T cells into the pancreas and protects non-obese diabetic mice from diabetes onset. Overall, our studies have divulged a previously unknown function of MT1-MMP and identified a promising novel drug target in type I diabetes.
Topics: Animals; Antibodies, Monoclonal; Blotting, Western; CD8-Positive T-Lymphocytes; Catalytic Domain; Cell Separation; Enzyme Inhibitors; Flow Cytometry; Hyaluronan Receptors; Matrix Metalloproteinase 14; Matrix Metalloproteinase 2; Matrix Metalloproteinases, Membrane-Associated; Metalloendopeptidases; Mice; Mice, Inbred NOD; Microscopy, Fluorescence; Organic Chemicals; Pancreas; T-Lymphocytes; Time Factors
PubMed: 15944163
DOI: 10.1074/jbc.M506016200 -
PloS One Mar 2011Aquaporin-4 (AQP4) constitutes the principal water channel in the brain and is clustered at the perivascular astrocyte endfeet. This specific distribution of AQP4 plays...
BACKGROUND
Aquaporin-4 (AQP4) constitutes the principal water channel in the brain and is clustered at the perivascular astrocyte endfeet. This specific distribution of AQP4 plays a major role in maintaining water homeostasis in the brain. A growing body of evidence points to a role of the dystroglycan complex and its interaction with perivascular laminin in the clustering of AQP4 at perivascular astrocyte endfeet. Indeed, mice lacking components of this complex or in which laminin-dystroglycan interaction is disrupted show a delayed onset of brain edema due to a redistribution of AQP4 away from astrocyte endfeet. It is therefore important to identify inhibitory drugs of laminin-dependent AQP4 clustering which may prevent or reduce brain edema.
METHODOLOGY/PRINCIPAL FINDINGS
In the present study we used primary rat astrocyte cultures to screen a library of >3,500 chemicals and identified 6 drugs that inhibit the laminin-induced clustering of dystroglycan and AQP4. Detailed analysis of the inhibitory drug, chloranil, revealed that its inhibition of the clustering is due to the metalloproteinase-2-mediated ß-dystroglycan shedding and subsequent loss of laminin interaction with dystroglycan. Furthermore, chemical variants of chloranil induced a similar effect on ß-dystroglycan and this was prevented by the antioxidant N-acetylcysteine.
CONCLUSION/SIGNIFICANCE
These findings reveal the mechanism of action of chloranil in preventing the laminin-induced clustering of dystroglycan and AQP4 and validate the use of high-throughput screening as a tool to identify drugs that modulate AQP4 clustering and that could be tested in models of brain edema.
Topics: Animals; Aquaporin 4; Astrocytes; Cell Survival; Cells, Cultured; Chloranil; Dystroglycans; Flunarizine; Gelatin; High-Throughput Screening Assays; Humans; Laminin; Mice; Microscopy; Organic Chemicals; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Reproducibility of Results; Small Molecule Libraries; Time Factors; Tissue Inhibitor of Metalloproteinases
PubMed: 21408176
DOI: 10.1371/journal.pone.0017559