The epithelial barrier regulates the movement of ions, macromolecules, immune cells and pathogens. The objective of this study was to investigate the role of the matrix metalloproteinase (MMP)-9 in the degradation of tight junction protein during infection with rat nematode lungworm Angiostrongylus cantonensis. The results showed that phosphorylation of IκB and NF-κB was increased in mice with eosinophilic meningoencephalitis. Treatment with MG132 reduced the phosphorylation of NF-κB and the activity of MMP-9, indicating upregulation of MMP-9 through the NF-κB signaling pathway. Claudin-5 was reduced in the brain but elevated in the cerebrospinal fluid (CSF), implying that A. cantonensis infection caused tight junction breakdown and led to claudin-5 release into the CSF. Degradation of claudin-5 coincided with alteration of the blood-CSF barrier permeability and treatment with the MMP inhibitor GM6001 attenuated the degradation of claudin-5. These results suggested that degradation of claudin-5 was caused by MMP-9 in angiostrongyliasis meningoencephalitis. Claudin-5 could be used for the pathophysiologic evaluation of the blood-CSF barrier breakdown and tight junction disruption after infection with A. cantonensis.
Brain edema in acute liver failure (ALF) remains lethal. The role of vasogenic mechanisms of brain edema has not been explored. We previously demonstrated that matrix metalloproteinase-9 (MMP-9) contributes to the pathogenesis of brain edema. Here, we show that MMP-9 mediates disruptions in tight junction proteins in vitro and in brains of mice with ALF. We transfected murine brain endothelial cells with MMP-9 cDNA using pc DNA3.1 (+)/Myc-His A expression vector. Tissue inhibitor of matrix metalloproteinases (TIMP-1) cDNA transfection or GM6001 was used to inhibit MMP-9. ALF was induced in mice with azoxymethane. Endogenous overexpression of MMP-9 in brain endothelial cells resulted in significant degradation of tight junction proteins occludin and claudin-5. The alterations in tight junction proteins correlated with increased permeability to FITC-dextran molecules. The degradation of tight junction proteins and the increased permeability were reversed by TIMP-1 and GM6001. Similar results were found when MMP-9 was exogenously added to brain EC. We also found that tight junction proteins degradation was reversed with GM6001 in brains of mice with ALF.
Tight junction proteins are significantly perturbed in brains of mice with ALF. These data corroborate the important role of MMP-9 in the vasogenic mechanism of brain edema in ALF.
acute liver failure; blood brain barrier permeability
We hypothesized that neutrophils and their secreted factors mediate breakdown of the integrity of the outer blood-retina-barrier by degrading the apical tight junctions of the retinal pigment epithelium (RPE). The effect of activated neutrophils or neutrophil cell lysate on apparent permeability of bovine RPE-Choroid explants was evaluated by measuring [3H] mannitol flux in a modified Ussing chamber. The expression of matrix metalloproteinase- (MMP-) 9 in murine peritoneal neutrophils, and the effects of neutrophils on RPE tight-junction protein expression were assessed by confocal microscopy and western blot. Our results revealed that basolateral incubation of explants with neutrophils decreased occludin and ZO-1 expression at 1 and 3 hours and increased the permeability of bovine RPE-Choroid explants by >3-fold (P < .05). Similarly, basolateral incubation of explants with neutrophil lysate decreased ZO-1 expression at 1 and 3 hours (P < .05) and increased permeability of explants by 75%. Further, we found that neutrophils prominently express MMP-9 and that incubation of explants with neutrophils in the presence of anti-MMP-9 antibody inhibited the increase in permeability. These data suggest that neutrophil-derived MMP-9 may play an important role in disrupting the integrity of the outer blood-retina barrier.
To study the effects of -gingerol, a ginger phytochemical, on tight junction (TJ) molecules, we investigated TJ tightening and signal transduction pathways in human pancreatic duct cell-derived cancer cell line PANC-1. The following methods were utilized: MTT assay to determine cytotoxicity; zymography to examine matrix metalloproteinase (MMP) activities; transepithelial electrical resistance (TER) and paracellular flux for TJ measurement; RT-PCR and immunoblotting for proteins related to TJ and invasion; and EMSA for NF-κB activity in PANC-1 cells. Results revealed that TER significantly increased and claudin 4 and MMP-9 decreased compared to those of the control. TJ protein levels, including zonula occludens (ZO-) 1, occludin, and E-cadherin, increased in -gingerol-treated cells, which correlated with a decrease in paracellular flux and MMP activity. Furthermore, NF-κB/Snail nuclear translocation was suppressed via downregulation of the extracellular signal-regulated kinase (ERK) pathway in response to -gingerol treatment. Moreover, treatment with U0126, an ERK inhibitor, completely blocked NF-κB activity. In conclusion, these findings demonstrate that -gingerol regulates TJ-related proteins and suppresses invasion and metastasis through NF-κB/Snail inhibition via inhibition of the ERK pathway. Therefore, -gingerol may suppress the invasive activity of PANC-1 cells.
Blood-brain barrier (BBB) dysfunction caused by activation of matrix metalloproteinases (MMPs) is a pathological feature in vascular/neurological disease. Here, we describe the mechanisms of BBB dysfunction and neuroinflammation as a result of MMP-3/9 activation and disruption of VEGF-A/VEGFR-2 interaction, impairing effective angiogenesis.
Methods and Results
We investigate the hypothesis in human brain endothelial cells and animal model of chronic alcohol ingestion. Proteome array analysis, zymography, immunofluorescence and Western blotting techniques detected the activation, expression and levels of MMP-3 and MMP-9. We found that degradation of VEGFR-2 and BBB proteins viz, occludin, claudin-5, and ZO-1 by MMP-3/9 causes rupture of capillary endothelium and BBB leakiness. Impairment of BBB integrity was demonstrated by increased permeability of dye tracers and Fluo-3/calcein-AM labeled monocytes adhesion or infiltration, and decrease in trans-endothelial electrical resistance. Alcohol-induced degradation of endothelial VEGFR-2 by MMP-3/9 led to a subsequent elevation of cellular/serum VEGF-A level. The decrease in VEGFR-2 with subsequent increase in VEGF-A level led to apoptosis and neuroinflammation via the activation of caspase-1 and IL-1β release. The use of MMPs, VEGFR-2 and caspase-1 inhibitors helped to dissect the underlying mechanisms.
Alcohol-induced MMPs activation is a key mechanism for dysfunction of BBB via degradation of VEGFR-2 protein and activation of caspase-1 or IL-1β release. Targeting VEGF-induced MMP-3/9 activation can be a novel preventive approach to vascular inflammatory disease in alcohol abuse.
Alcohol; blood-brain barrier; vascular inflammation; VEGF/VEGFR-2; matrix metalloproteinase
Disruption of the blood-brain barrier (BBB) is a hallmark event in the pathophysiology of bacterial meningitis. Several inflammatory mediators, such as tumor necrosis factor alpha (TNF-α), nitric oxide and matrix metalloproteinases (MMPs), contribute to this disruption. Here we show that infection of human brain microvascular endothelial cells (HBMEC) with Neisseria meningitidis induced an increase of permeability at prolonged time of infection. This was paralleled by an increase in MMP-8 activity in supernatants collected from infected cells. A detailed analysis revealed that MMP-8 was involved in the proteolytic cleavage of the tight junction protein occludin, resulting in its disappearance from the cell periphery and cleavage to a lower-sized 50-kDa protein in infected HBMEC. Abrogation of MMP-8 activity by specific inhibitors as well as transfection with MMP-8 siRNA abolished production of the cleavage fragment and occludin remained attached to the cell periphery. In addition, MMP-8 affected cell adherence to the underlying matrix. A similar temporal relationship was observed for MMP activity and cell detachment. Injury of the HBMEC monolayer suggested the requirement of direct cell contact because no detachment was observed when bacteria were placed above a transwell membrane or when bacterial supernatant was directly added to cells. Inhibition of MMP-8 partially prevented detachment of infected HBMEC and restored BBB permeability. Together, we established that MMP-8 activity plays a crucial role in disassembly of cell junction components and cell adhesion during meningococcal infection.
A crucial step in the pathogenesis of bacterial meningitis is the disturbance of cerebral microvascular endothelial function, resulting in blood-brain barrier (BBB) breakdown. Matrix metalloproteinases (MMPs) have been implicated in BBB damage in bacterial meningitis in several studies. MMPs are a family of zinc-dependent endopeptidases that catalyze the proteolysis of extracellular matrix proteins, but can also cleave a range of other molecules, including cell adhesion molecules. In this study we showed that brain endothelial cells produced MMPs—in particular MMP-8—upon infection with Neisseria meningitidis, a bacterium that causes meningitis and septic shock. We found that MMP-8 was then involved in disruption of the tight junction protein occludin. In addition to the effect of MMP-8 on the tight junction component, MMP-8 activity also accounted for brain endothelial cell detachment that occurred during prolonged time of infection with N. meningitidis. When we inhibited MMP-8 activity, occludin disruption was completely abolished and cell detachment could be partially prevented, which resulted in restored BBB permeability. Our data reveal a molecular mechanism of cellular dysfunction during meningococcal meningitis that enhances our understanding how MMPs affect cerebral endothelial function and that can aid in our understanding and prevention of this disease.
Background. Insulin directly changes the sheep pleural electrophysiology. The aim of this study was to investigate whether insulin induces similar effects in human pleura, to clarify insulin receptor's involvement, and to demonstrate if glibenclamide (hypoglycemic agent) reverses this effect.
Methods. Human parietal pleural specimens were mounted in Ussing chambers. Solutions containing insulin or glibenclamide and insulin with anti-insulin antibody, anti-insulin receptor antibody, and glibenclamide were used. The transmesothelial resistance (RTM) was determined. Immunohistochemistry for the presence of Insulin Receptors (IRa, IRb) was also performed. Results. Insulin increased RTM within 1st min (P = .016), when added mesothelially which was inhibited by the anti-insulin and anti-insulin receptor antibodies. Glibenclamide also eliminated the insulin-induced changes. Immunohistochemistry verified the presence of IRa and IRb.
Conclusion. Insulin induces electrochemical changes in humans as in sheep via interaction with its receptor. This effect is abolished by glibenclamide.
Altered expression of Twist, matrix metalloproteinase (MMP)-2 and MMP-9 proteins has been identified in various types of human cancers. However, the correlation between Twist and these gelatinases in breast cancer remains unclear. In this study, immunohistochemical analysis of Twist, MMP-2 and MMP-9 expression was performed on tissue microarrays from 200 breast cancer cases. The association of Twist and gelatinase expression with clinicopathological factors and patient survival was analyzed. Altered expression of Twist, MMP-2 and MMP-9 proteins was observed in breast cancer tissue. The positive rates of Twist, MMP-2 and MMP-9 protein expression were 75.5, 97.0 and 96.0%, respectively. Increased expression of Twist was positively correlated with the status of axillary lymph node metastasis and higher tumor-node-metastasis (TNM) stage (P<0.01). Moreover, increased expression of Twist was correlated with poor overall survival (OS) and post-operative relapse-free survival (RFS), compared with those for the patients with reduced expression levels of Twist (P<0.05, P<0.01). The expression of MMP-2 and MMP-9 was positively correlated with Twist expression (P<0.001). Our results indicate that Twist may play an important role in the invasion, metastasis and prognosis of breast cancer. Additionally, our results suggest that Twist may be a regulator of gelatinases (MMP-2 and MMP-9).
breast neoplasms; pathology; gelatinases; matrix metalloproteinase 2; matrix metalloproteinase 9; immunohistochemistry
Estrogen modulates tight junctional resistance through estrogen receptor-α-mediated remodeling of occludin. The objective of the study was to understand the mechanisms involved. Experiments using human normal vaginal-cervical epithelial cells showed that human normal vaginal-cervical epithelial cells secrete constitutively matrix-metalloproteinase-7 (MMP-7) into the luminal solution and that MMP-7 is necessary and sufficient to produce estrogen decrease of tight junctional resistance and remodeling of occludin. Treatment with estrogen stimulated activation of the pro-MMP-7 intracellularly and augmented secretion of the activated MMP-7 form. Steady-state levels of MMP-7 mRNA and protein were not affected by estrogen. Estrogen modulated phosphorylation of the MMP-7, but the changes were most likely secondary to changes in cellular MMP-7 mass. Estrogen increased coimmunoreactivity of MMP-7 with the Golgi protein GPP130. Tunicamycin and brefeldin-A had no effect on cellular MMP-7 but monensin (inhibitor of Golgi traffic) blocked estrogen effects, suggesting estrogen site of action is at the Golgi system. Estrogen increased generalized secretory activity, including of luminal exocytosis of polycarbohydrates. However, estrogen increased coimmunoreactivity of MMP-7 with synaptosomal-associated protein of 25 kDa in apical membranes, suggesting soluble N-ethylmaleimide sensitive fusion factor attachment protein receptor-facilitated exocytosis of MMP-7. Treatment with the vesicular-ATPase inhibitor bafilomycin A1 inhibited activation of MMP-7. These data suggest that estrogen up-regulates activation of the MMP-7 intracellularly, at the level of Golgi, and augments secretion of activated MMP-7 through soluble N-ethylmaleimide sensitive fusion factor attachment protein receptor-dependent exocytosis. On the other hand, estrogen acidification of the luminal solution would tend to alkalinize exocytotic vesicles and may lead to decreased activation of the MMP-7. These mechanisms acting in concert could be important for regulation and control of estrogen modulation of paracellular permeability in vivo.
Matrix metalloproteinases (MMPs) constitute a large family of enzymes with specificity for the various proteins of the extracellular matrix which are implicated in tissue remodeling processes and chronic inflammatory conditions. To investigate the role of MMPs in immunity to mycobacterial infections, we incubated murine peritoneal macrophages with viable Mycobacterium bovis BCG or Mycobacterium tuberculosis H37Rv and assayed MMP activity in the supernatants by zymography. Resting macrophages secreted only small amounts of MMP-9 (gelatinase B), but secretion increased dramatically in a dose-dependent manner in response to either BCG or M. tuberculosis in vitro. Incubation with mycobacteria also induced increased MMP-2 (gelatinase A) activity. Neutralization of tumor necrosis alpha (TNF-α), and to a lesser extent interleukin 18 (IL-18), substantially reduced MMP production in response to mycobacteria. Exogenous addition of TNF-α or IL-18 induced macrophages to express MMPs, even in the absence of bacteria. The immunoregulatory cytokines gamma interferon (IFN-γ), IL-4, and IL-10 all suppressed BCG-induced MMP production, but through different mechanisms. IFN-γ treatment increased macrophage secretion of TNF-α but still reduced their MMP activity. Conversely, IL-4 and IL-10 seemed to act by reducing the amount of TNF-α available to the macrophages. Finally, infection of BALB/c or severe combined immunodeficiency (SCID) mice with either BCG or M. tuberculosis induced substantial increases in MMP-9 activity in infected tissues. In conclusion, we show that mycobacterial infection induces MMP-9 activity both in vitro and in vivo and that this is regulated by TNF-α, IL-18, and IFN-γ. These findings indicate a possible contribution of MMPs to tissue remodeling processes that occur in mycobacterial infections.
West Nile virus (WNV) encephalitis is characterized by neuroinflammation, neuronal loss and blood–brain barrier (BBB) disruption. However, the mechanisms associated with the BBB disruption are unclear. Complex interactions between the tight junction proteins (TJP) and the adherens junction proteins (AJP) of the brain microvascular endothelial cells are responsible for maintaining the BBB integrity. Herein, we characterized the relationship between the BBB disruption and expression kinetics of key TJP, AJP and matrix metalloproteinases (MMPs) in the mice brain. A dramatic increase in the BBB permeability and extravasation of IgG was observed at later time points of the central nervous system (CNS) infection and did not precede virus–CNS entry. WNV-infected mice exhibited significant reduction in the protein levels of the TJP ZO-1, claudin-1, occludin and JAM-A, and AJP β-catenin and vascular endothelial cadherin, which correlated with increased levels of MMP-1, -3 and -9 and infiltrated leukocytes in the brain. Further, intracranial inoculation of WNV also demonstrated increased extravasation of IgG in the brain, suggesting the role of virus replication in the CNS in BBB disruption. These data suggest that altered expression of junction proteins is a pathological event associated with WNV infection and may explain the molecular basis of BBB disruption. We propose that WNV initially enters CNS without altering the BBB integrity and later virus replication in the brain initiates BBB disruption, allowing enhanced infiltration of immune cells and contribute to virus neuroinvasion via the ‘Trojan-horse’ route. These data further implicate roles of multiple MMPs in the BBB disruption and strategies to interrupt this process may influence the WNV disease outcome.
The aim of this study was to evaluate the physicochemical properties of the pleural mesothelial barrier and of the biological markers that facilitate or eliminate the passage of molecules through the pleura.
Methods and Material:
Pleural fluid samples from sixty-five patients with heart failure were analyzed. The biological markers studied were lactate dehydrogenase (LDH), adenosine deaminase (ADA), interleukin-6 (IL-6), C-reactive protein (CRP), tumor necrosis factor-α (TNF-α), carcinoembryonic antigen (CEA), copper/zinc superoxide dismutase (CuZnSOD), matrix metalloproteinase-2 (MMP-2), -3 (MMP-3), -7(MMP-7), -8 (MMP-8) and -9 (MMP-9). Based on the pleural fluid/serum ratio, these molecules were divided into three groups: a) the LDH-like group with a pleural fluid/serum ratio between 0,4 and 0,8 (LDH, CEA, CuZnSOD, ADA, CRP, MMP-8), b) molecules with a pleural fluid/serum ratio less than 0,4 (MMP-7 and MMP-9) and c) molecules with a pleural fluid/serum ratio equal or above 1 (TNF-α, IL-6, MMP-2 and MMP-3).
No correlation between the molecular radius and the pleural fluid to serum ratio of the above biological markers was found.
The molecular size is not a major determinant for the passage of molecules through the mesothelial barrier. Several other factors may influence the transport of the above molecules to pleural cavity, such as their charge and shape.
Biological markers; mesothelial barrier; pleural fluid/ serum ratio; transudates; lactate dehydrogenase; tumor necrosis factor.
Mucopolysaccharidoses (MPS) represent a heterogeneous group of hereditary disorders, characterized by accumulation of glycosaminoglycans within the lysosomes. The objective of this study was to elucidate the expression and activity of matrix metalloproteinases (MMPs) in the serum of pediatric patients with MPS. Serum gelatinase activity was assessed by gelatin zymography and the concentration of circulating MMP-2, MMP-9, and of tissue inhibitors of MMPs (TIMP)-1 and TIMP-2 was measured by ELISA in the serum of seven patients with MPS (five with MPS III, 1 with MPS II and 1 with MPS VI), and healthy age- and sex-matched participants. Serum activity and protein levels of MMP-9 were significantly reduced whereas of MMP-2 were significantly increased in patients with MPS III, as compared to controls. There were no significant alterations in serum protein levels of TIMP-1 and TIMP-2 in patients with MPS III, as compared to controls. In MPS II, proMMP-2 activity and protein levels of MMP-2 were significantly increased, as compared to control. In MPS VI, enzyme replacement therapy reduced the activity and protein levels of MMP-9 up to 4 months after the initiation of treatment. The reported alterations in the expression of MMPs in the serum of patients with MPS suggest that these molecules may be used as potential biomarkers for the diagnosis, follow-up and response to therapy in patients with MPS.
Objective: Pleural effusion is common problem, but the rapid and reliable diagnosis for specific pathogenic effusions are lacking. This study aimed to identify the diagnosis based on clinical variables to differentiate pleural tuberculous exudates from other pleural effusions. We also investigated the role of renin-angiotensin system (RAS) and matrix metalloproteinase (MMPs) in the pathogenesis of pleural exudates.
Experimental design: The major components in RAS and extracellular matrix metabolism, including angiotensin converting enzyme (ACE), ACE2, MMP-2 and MMP-9 activities, were measured and compared in the patients with transudative (n = 45) and exudative (n = 80) effusions. The exudative effusions were come from the patients with tuberculosis (n = 20), pneumonia (n = 32), and adenocarcinoma (n = 28).
Results: Increased ACE and equivalent ACE2 activities, resulting in a significantly increased ACE/ACE2 ratio in exudates, were detected compared to these values in transudates. MMP-9 activity in exudates was significantly higher than that in transudates. The significant correlation between ACE and ACE2 activity that was found in transudates was not found in exudates. Advanced analyses showed significantly increased ACE and MMP-9 activities, and decreased ACE2 activity in tuberculous pleural effusions compared with those in pneumonia and adenocarcinoma effusions. The results indicate that increased ACE and MMP-9 activities found in the exudates were mainly contributed from a higher level of both enzyme activities in the tuberculous pleural effusions.
Conclusion: Interplay between ACE and ACE2, essential functions in the RAS, and abnormal regulation of MMP-9 probably play a pivotal role in the development of exudative effusions. Moreover, the ACE/ACE2 ratio combined with MMP-9 activity in pleural fluid may be potential biomarkers for diagnosing tuberculous pleurisy.
angiotensin converting enzyme; angiotensin converting enzyme 2; exudative effusion; matrix metalloproteinase-9; tuberculous effusion.
Blood–brain barrier (BBB) disruption, resulting from loss of tight junctions (TJ) and activation of matrix metalloproteinases (MMPs), is associated with edema formation in ischemic stroke. Cerebral edema develops in a phasic manner and consists of both vasogenic and cytotoxic components. Although it is contingent on several independent mechanisms, involving hypoxic and inflammatory responses, the single effect of prolonged hypoxia on BBB integrity in vivo was not addressed so far. Exposing mice to normobaric hypoxia (8% oxygen for 48 h) led to a significant increase in vascular permeability associated with diminished expression of the TJ protein occludin. Immunofluorescence studies revealed that hypoxia resulted in disrupted continuity of occludin and zonula occludens-1 (Zo-1) staining with significant gap formation. Hypoxia increased gelatinolytic activity specifically in vascular structures and gel zymography identified MMP-9 as enzymatic source. Treatment with an MMP inhibitor reduced vascular leakage and attenuated disorganization of TJ. Inhibition of vascular endothelial growth factor (VEGF) attenuated vascular leakage and MMP-9 activation induced by hypoxia. In conclusion, our data suggest that hypoxia-induced edema formation is mediated by MMP-9-dependent TJ rearrangement by a mechanism involving VEGF. Therefore, inhibition of MMP-9 might provide the basis for therapeutic strategies to treat brain edema.
blood–brain barrier; hypoxia; in vivo; matrix metalloproteinase; occludin; vascular permeability
Claudin-4 is a member of the claudin family, a large family of transmembrane proteins that are essential in the formation and maintenance of tight junctions. Matrix metal-loproteinase (MMP)-2 and -9 degrade type IV collagen of the extracellular matrix and basal membranes. Claudin-4 activates MMP-2, indicating that claudin-mediated increased cancer cell invasion may result from the activation of MMP proteins. In the present study, we used immunohistochemistry to examine the expression levels of claudin-4, MMP-2 and MMP-9 in 189 gastric cancer samples, and analyzed their correlation with tumor invasion, clinicopathologic parameters and clinical outcome. The relationship between claudin-4 expression and MMP-2 and -9 expression was also investigated. The expression of claudin-4 was found to be significantly higher in gastric cancer cases with advanced depth of wall invasion, lymph node metastasis, lymphatic invasion and higher TNM stage. Further analysis revealed claudin-4 expression to be significantly correlated with the expression of MMP-2 and -9. Kaplan-Meier survival analysis indicated that MMP-9 expression was correlated with poor prognosis. These results suggest that claudin-4 expression is associated with tumor invasion and with MMP-2 and -9 expression in gastric cancer. Additionally, MMP-9 expression was demonstrated to serve as a prognostic factor in patients with gastric cancer.
claudin-4; matrix metalloproteinase-2; matrix metalloproteinase-9; gastric cancer
The objective of the current study was to determine if altered regulation of matrix metalloproteinases (MMPs) may predispose to extracellular matrix degradation, facilitating arterial calcification in chronic kidney disease (CKD) using a progressive model of CKD-MBD, the Cy/+ rat.
Sera were collected from normal or CKD rats at various times and MMP-2 and MMP-9 levels determined by ELISA or zymography. Aorta tissue was harvested at sacrifice for RT-PCR and immunostaining. Calcification of aorta rings was assessed with MMP inhibitors.
There was an increase in MMP-2, MMP-9, TIMP-1, and RUNX-2 expression in the aorta with progressive CKD, and increased MMP-2 activity in the serum. Immunostaining revealed increased expression of MMP-2 and MMP-9 in areas of aorta calcification. There was also an upregulation of MMP-2 and MMP-9 in vascular smooth muscle cells (VSMC) from CKD rats. MMP inhibitors decreased calcification of aorta rings from normal and CKD rats. High phosphorus increased MMP-2 and MMP-9 expressions in VSMC from normal rats but not from CKD rats.
MMP-2 and MMP-9 expression and activity are increased with progressive CKD, and blockade of MMP activity can inhibit arterial calcification. These data suggest degradation of the extracellular matrix is a critical step in the pathogenesis of arterial calcification in CKD.
Matrix metalloproteinase; Gelatinase; Vascular calcification; Chronic kidney disease
OBJECTIVE—To define the pattern of mRNA expression of all human matrix metalloproteinases (MMPs) described to date in rheumatoid arthritis (RA) and traumatic synovial membrane, in order to differentiate between a physiological tissue remodelling pattern and that associated with inflammatory tissue destruction.
METHODS—Analysis of SwissProt protein and EMBL/GenBank nucleotide sequence banks, protein sequence alignment, reverse transcriptase-polymerase chain reaction and nucleotide sequencing were used.
RESULTS—MMP-2 (gelatinase A), MMP-3 (stromelysin-1), MMP-11 (stromelysin-3) and MMP-19 were constitutively expressed. MMP-1 (fibroblast type collagenase), MMP-9 (gelatinase B) and MMP-14 (MT1-MMP) were expressed in all RA, but only in 55-80% of trauma samples. MMP-13 (collagenase-3) and MMP-15 (MT2-MMP) were expressed exclusively in RA (80-90% of the samples). MMP-20 (enamelysin) was absent and MMP-8 (collagenase-2) was rarely found in RA or trauma. All other MMPs (-7, -10, -12, -16, -17) had an intermediate pattern of expression.
CONCLUSIONS—Some MMPs without interstitial collagenase activity seem to have a constitutive pattern of expression and probably participate in physiological synovial tissue remodelling. Some MMPs are exclusively associated to RA synovitis, for example, MMP-13, which preferentially degrades type II collagen and aggrecan, and MMP-15, which activates proMMP-2 and proMMP-13 and is involved in tumour necrosis factor α processing. This clear cut rheumatoid/inflammatory MMP profile, more complex than has been previously appreciated, may facilitate inflammatory tissue destruction in RA.
House dust mite (HDM) allergens are important factors in the increasing prevalence of asthma. The lung epithelium forms a barrier that allergens must cross before they can cause sensitization. However, the mechanisms involved are unknown. Here we show that the cysteine proteinase allergen Der p 1 from fecal pellets of the HDM Dermatophagoides pteronyssinus causes disruption of intercellular tight junctions (TJs), which are the principal components of the epithelial paracellular permeability barrier. In confluent airway epithelial cells, Der p 1 led to cleavage of the TJ adhesion protein occludin. Cleavage was attenuated by antipain, but not by inhibitors of serine, aspartic, or matrix metalloproteinases. Putative Der p 1 cleavage sites were found in peptides from an extracellular domain of occludin and in the TJ adhesion protein claudin-1. TJ breakdown nonspecifically increased epithelial permeability, allowing Der p 1 to cross the epithelial barrier. Thus, transepithelial movement of Der p 1 to dendritic antigen-presenting cells via the paracellular pathway may be promoted by the allergen’s own proteolytic activity. These results suggest that opening of TJs by environmental proteinases may be the initial step in the development of asthma to a variety of allergens.
PURPOSE: It is proposed that the sclera is a metabolically active and pharmacologically responsive tissue. These studies were undertaken to determine whether prostaglandin exposure can enhance scleral permeability to high-molecular-weight substances. METHODS: Topical prostaglandin F2 alpha (PGF2 alpha) was administered to monkeys to determine if this altered the amount of scleral matrix metalloproteinases (MMPs). Experiments also were performed to determine whether the prostaglandin F (FP) receptor and gene transcripts are expressed in normal human sclera. Permeability of organ-cultured human sclera following prostaglandin exposure then was studied and the amount of MMP released into the medium measured. Finally, the permeability of human sclera to basic fibroblast growth factor (FGF-2) was determined following prostaglandin exposure. RESULTS: Topical prostaglandin administration that reduced scleral collagen also increased scleral MMP-1, MMP-2, and MMP-3 by 63 +/- 35%, 267 +/- 210%, and 729 +/- 500%, respectively. FP receptor protein was localized in scleral fibroblasts, and FP receptor gene transcript was identified in sclera. Exposure to prostaglandin F2 alpha, 17-phenyltrinor, PGF2 alpha, or latanoprost acid increased scleral permeability by up to 124%, 183%, or 213%, respectively. In these cultures, MMP-1, MMP-2, and MMP-3 were increased by up to 37%, 267%, and 96%, respectively. Finally, transscleral absorption of FGF-2 was increased by up to 126% with scleral exposure to latanoprost. CONCLUSIONS: These studies demonstrate that the sclera is metabolically active and pharmacologically responsive to prostaglandins. Further, they demonstrate the feasibility of cotreatment with prostaglandin to enhance transscleral delivery of peptides, such as growth factors and high-molecular-weight substances, to the posterior segment of the eye.
Matrix metalloproteinases (MMPs) and their endogenous inhibitors TIMPs (tissue inhibitors of MMPs), are two protein families that work together to remodel the extracellular matrix (ECM). TIMPs serve not only to inhibit MMP activity, but also aid in the activation of MMPs that are secreted as inactive zymogens. Xenopus laevis metamorphosis is an ideal model for studying MMP and TIMP expression levels because all tissues are remodeled under the control of one molecule, thyroid hormone. Here, using RT-PCR analysis, we examine the metamorphic RNA levels of two membrane-type MMPs (MT1-MMP, MT3-MMP), two TIMPs (TIMP-2, TIMP-3) and a potent gelatinase (Gel-A) that can be activated by the combinatory activity of a MT-MMP and a TIMP. In the metamorphic tail and intestine the RNA levels of TIMP-2 and MT1-MMP mirror each other, and closely resemble that of Gel-A as all three are elevated during periods of cell death and proliferation. Conversely, MT3-MMP and TIMP-3 do not have similar RNA level patterns nor do they mimic the RNA levels of the other genes examined. Intriguingly, TIMP-3, which has been shown to have anti-apoptotic activity, is found at low levels in tissues during periods of apoptosis.
Brain edema remains a challenging obstacle in the management of acute liver failure (ALF). Cytotoxic mechanisms associated with brain edema have been well recognized, but evidence for vasogenic mechanisms in the pathogenesis of brain edema in ALF has been lacking. Recent reports have not only shown a role of matrix metalloproteinase-9 in the pathogenesis of brain edema in experimental ALF but have also found significant alterations in the tight junction elements including occludin and claudin-5, suggesting a vasogenic injury in the blood-brain barrier (BBB) integrity. This article reviews and explores the role of the paracellular tight junction proteins in the increased selective BBB permeability that leads to brain edema in ALF.
Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes that degrade various components of the extracellular matrix (ECM). Members of the MMP family include collagenases, gelatinases, stromelysins, matrilysins and membrane-type MMPs. ProMMPs are cleaved into active forms that promote degradation of ECM proteins. Also, recent evidence suggests direct or indirect effects of MMPs on ion channels in the endothelium and vascular smooth muscle, and on other mechanisms of vascular relaxation/contraction. Endogenous tissue inhibitors of metalloproteinases (TIMPs) reduce excessive proteolytic ECM degradation by MMPs. The balance between MMPs and TIMPs plays a major role in vascular remodeling, angiogenesis, and the uterine and systemic vasodilation during normal pregnancy. An imbalance in the MMPs/TIMPs activity ratio may underlie the pathogenesis of vascular diseases such as abdominal aortic aneurysm, varicose veins, hypertension and preeclampsia. Downregulation of MMPs using genetic manipulations of endogenous TIMPs, or synthetic pharmacological inhibitors such as BB-94 (Batimastat) and doxycycline, and Ro-28-2653, a more specific inhibitor of gelatinases and membrane type 1-MMP, could be beneficial in reducing the MMP-mediated vascular dysfunction and the progressive vessel wall damage associated with vascular disease.
MMP; TIMP; blood vessels; extracellular matrix; aneurysm; varicose veins
Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) remodel the pericellular environment by regulating the cleavage of extracellular matrix proteins, cell surface components, neurotransmitter receptors, and growth factors that mediate cell adhesion, synaptogenesis, synaptic plasticity, and long-term potentiation. Interestingly, increased MMP activity and dysregulation of the balance between MMPs and TIMPs have also been implicated in various pathologic conditions. In this paper, we discuss various animal models that suggest that the activation of the gelatinases MMP-2 and MMP-9 is involved in pathogenesis of drug dependence, Alzheimer's disease, and epilepsy.
This study illustrates a novel proapoptotic role of MMP-9 in the development of diabetic retinopathy and shows that MMP-9 activation is downstream of a small molecular weight G-protein, H-Ras.
Diabetes activates a small molecular weight G-protein, H-Ras, in the retina and its capillary cells, and H-Ras activation is implicated in the apoptosis of retinal capillary cells. Matrix metalloproteinase (MMP)-9 is regulated by H-Ras, and in diabetes its activation is associated with increased vascular permeability. The goal of this study was to investigate the role of sustained activation of MMP-9 in the pathogenesis of diabetic retinopathy and to illustrate the mechanism through which it is upregulated in diabetes.
Retinal MMP-9 activation and its tissue inhibitor, TIMP-1, were quantified in streptozotocin-induced diabetic rats. Inhibition of H-Ras by simvastatin on diabetes-induced activation of H-Ras was evaluated. The mechanism by which diabetes regulates retinal MMP-9 was confirmed by determining the effect of genetic or pharmacologic regulation of H-Ras on its activation in retinal endothelial cells.
In rats, MMP-9 was activated and expression of TIMP-1 was decreased in the retina and its microvasculature at both 2 months and 12 months of diabetes. In retinal endothelial cells, high glucose activated MMP-9, and inhibition of its activation (by pharmacologic inhibitor or siRNA) ameliorated accelerated apoptosis. Inhibition of H-Ras, both in diabetic rats (simvastatin) and in isolated endothelial cells (H-Ras siRNA), abrogated the activation of MMP-9 and prevented the reduction of TIMP-1.
Hyperglycemia-induced activation of MMP-9 accelerates apoptosis of retinal capillary cells, a phenomenon that predicts the development of diabetic retinopathy, and the activation of MMP-9 is downstream of H-Ras. Characterizing the role of MMP-9 in the development of diabetic retinopathy will help explore novel molecular targets for future pharmacological interventions.