Modulation of membrane type 1 matrix metalloproteinase by LPS and gamma interferon bound to extracellular matrix in intestinal crypt cells

Membrane type 1 matrix metalloproteinase (MT1-MMP) is an integral membrane protein that participates in the processing and degradation of cell surface proteins and the extracellular matrix (ECM). This enzyme regulates ECM turnover in wound repair, promotes cell migration and activates other MMPs, such as MMP-2, which is involved in angiogenesis, cell migration and tumoral metastasis. An increase in pro-inflammatory cytokine expression, such as gamma interferon (IFN-gamma), has been associated with chronic wounds in inflammatory bowel diseases. However, the extent to which cytokines modulate MT1-MMP has not been totally defined. In this report, the effects of the bacterial lipopolysaccharide (LPS) and ECM-bound IFN-gamma on MT1-MMP expression and MMP-2 activity were evaluated by Western blot, RT-PCR and zymography in isolated intestinal epithelial and cultured HT-29 cells. In the presence of LPS, ECM-bound IFN-gamma, but not soluble IFN-gamma, reduced the enterocyte MT1-MMP protein expression. In addition, the active form of MMP-2 was also decreased in the presence of both LPS and IFN-gamma, indicating that lower MMP-2 activity accompanied the decrease in MT1-MMP expression. These results suggest the possibility that endotoxin and ECM-bound IFN-gamma may affect matrix remodeling by modulating matrix metalloproteinase in enterocytes during wound healing.     

Expression of extracellular matrix metalloproteinase inducer (EMMPRIN) and its related extracellular matrix degrading enzymes in the endometrium during estrous cycle and early gestation in cattle

Background  

Extracellular matrix metalloproteinase inducer (EMMPRIN) regulates several biological functions involving the modulation of cell behaviors via cell-cell and cell-matrix interactions. According to its diverse functions, we hypothesized that EMMPRIN may play an important role in endometrial remodeling and establishment of pregnancy in cow.     

Localized mechanical stress induces time-dependent actin cytoskeletal remodeling and stiffening in cultured airway smooth muscle cells

Mechanical stress (MS) causes cytoskeletal (CSK) and phenotypic changes in cells. Such changes in airway smooth muscle (ASM) cells might contribute to the pathophysiology of asthma. We have shown that periodic mechanical strain applied to cultured ASM cells alters the structure and expression of CSK proteins and increases cell stiffness and contractility (Smith PG, Moreno R, and Ikebe M. Am J Physiol Lung Cell Mol Physiol 272: L20–L27, 1997; and Smith PG, Deng L, Fredberg JJ, and Maksym GN. Am J Physiol Lung Cell Mol Physiol 285: L456–L463, 2003). However, the mechanically induced CSK changes, altered cell function, and their time courses are not well understood. Here we applied MS to the CSK by magnetically oscillating ferrimagnetic beads bound to the CSK. We quantified CSK remodeling by measuring actin accumulation at the sites of applied MS using fluorescence microscopy. We also measured CSK stiffness using optical magnetic twisting cytometry. We found that, during MS of up to 120 min, the percentage of beads associated with actin structures increased with time. At 60 min, 68.1 ± 1.6% of the beads were associated with actin structures compared with only 6.7 ± 2.8% before MS and 38.4 ± 5.5% in time-matched controls (P < 0.05). Similarly, CSK stiffness increased more than twofold in response to the MS compared with time-matched controls. These changes were more pronounced than observed with contractile stimulation by 80 mM KCl or 10^–4 M acetylcholine. Together, these findings imply that MS is a potent stimulus to enhance stiffness and contractility of ASM cells through CSK remodeling, which may have important implications in airway narrowing and dilation in asthma. mechanical stress; actin cytoskeleton; stiffness; airway smooth muscle cell; optical magnetic twisting cytometry; airway constriction and dilation; asthma     

Human mast cells release metalloproteinase-9 on contact with activated T cells: juxtacrine regulation by TNF-alpha

Mast cells, essential effector cells in allergic inflammation, have been found to be activated in T cell-mediated inflammatory processes in accordance with their residence in close physical proximity to T cells. We have recently reported that mast cells release granule-associated mediators and TNF-alpha upon direct contact with activated T cells. This data suggested an unrecognized activation pathway, where mast cells may be activated during T cell-mediated inflammation. Herein, we show that this cell-cell contact results in the release of matrix metalloproteinase (MMP)-9 and the MMP inhibitor tissue inhibitor of metalloproteinase 1 from HMC-1 human mast cells or from mature peripheral blood-derived human mast cells. The expression and release of these mediators, as well as of beta-hexosaminidase and several cytokines, were also induced when mast cells were incubated with cell membranes isolated from activated, but not resting, T cells. Subcellular fractionation revealed that the mature form of MMP-9 cofractionated with histamine and tryptase, indicating its localization within the secretory granules. MMP-9 release was first detected at 6 h and peaked at 22 h of incubation with activated T cell membranes, while TNF-alpha release peaked after only 6 h. Anti-TNF-alpha mAb inhibited the T cell membrane-induced MMP-9 release, indicating a possible autocrine regulation of MMP release by mast cell TNF-alpha. This cascade of events, whereby mast cells are activated by T cells to release cytokines and MMP-9, which are known to be essential for leukocyte extravasation and recruitment to affected sites, points to an important immunoregulatory function of mast cells within the context of T cell-mediated inflammatory processes.     

Phenotypic characterization of a human synovial sarcoma cell line, SW982, and its response to dexamethasone

SW982 cells are characterized by expression of inflammatory cytokine and matrix metalloproteinase (MMP) genes and by their response to dexamethasone at different cell densities. They express genes encoding interleukin (IL)-1 beta; IL-6; transforming growth factor-beta; intercellular adhesion molecule-1; cycloxygenase (COX)-2; and MMPs, including MMP-1, MMP-2, MMP-13, and MT1-MMP; tissue inhibitor of metalloproteinase-2; and a disintegrin and metalloproteinase with thrombospondin motifs-4. Expression of all the genes examined was induced with 2 ng/ml IL-1 beta at low cell density. The cells, however, failed to express tumor necrosis factor-alpha, COX-1, and MMP-9, regardless of the presence of IL-1 beta. Dexamethasone significantly reduced IL-1 beta, IL-6, COX-2, and MMP-1 expression at high cell density. The results suggest that SW982 cells are a useful tool for studying the expression of inflammatory cytokine or MMP genes.     

JNK and PI3K differentially regulate MMP-2 and MT1-MMP mRNA and protein in response to actin cytoskeleton reorganization in endothelial cells

Increased production and activation of matrix metalloproteinase-2 (MMP-2) are critical events in skeletal muscle angiogenesis and are known to occur in response to mechanical stresses. We hypothesized that reorganization of the actin cytoskeleton would increase endothelial cell production and activation of MMP-2 and that this increase would require a MAPK-dependent signaling pathway in endothelial cells. The pharmacological actin depolymerization agent cytochalasin D increased expression of MMP-2 and membrane type 1-matrix metalloproteinase (MT1-MMP) mRNA, and this was reduced significantly in the presence of the JNK inhibitor SP600125. Activation of JNK by anisomycin was sufficient to induce expression of both MMP-2 and MT1-MMP mRNA in quiescent cells. Downregulation of c-Jun, a downstream target of JNK, with small interference (si)RNA inhibited MMP-2 expression in response to anisomycin. Inhibition of phosphoinositide 3-kinase (PI3K), but not JNK, significantly decreased the amount of active MMP-2 following cytochalasin D stimulation with a concurrent decrease in MT1-MMP protein. Physiological reorganization of actin occurs during VEGF stimulation. VEGF-induced MMP-2 protein production and activation, as well as MT1-MMP protein production, depended on PI3K activity. VEGF-induced MMP-2 mRNA expression was reduced by inhibition of JNK or by treatment with c-Jun siRNA. In summary, our results provide novel insight into the signaling cascades initiated in the early stages of angiogenesis through the reorganization of the actin cytoskeleton and demonstrate a critical role for JNK in regulating MMP-2 and MT1-MMP mRNA expression, whereas PI3K regulates protein levels of both MMP-2 and MT1-MMP. angiogenesis; mechanotransduction; vascular endothelial growth factor; c-Jun; phosphoinositide 3-kinase; membrane type 1-matrix metalloproteinase     

In vitro study of matrix metalloproteinase/tissue inhibitor of metalloproteinase production by mesenchymal stromal cells in response to inflammatory cytokines: the role of their migration in injured tissues

Background aimsThe transmigratory capacity of bone marrow (BM) mesenchymal stromal cells (MSC) through the endothelial cell barrier into various tissues and their differentiation potential makes them ideal candidates for cell therapy. Nevertheless, the mechanisms and agents promoting their migration are not fully understood. We evaluated the effects of several inflammatory cytokines on the migration of BM MSC and matrix metalloproteinase (MMP)/tissue inhibitor of metalloproteinase (TIMP) production.MethodsThe migratory potential of BM MSC was evaluated using a Boyden chamber coated with Matrigel^® in the presence and absence of stromal cell-derived (SDF)-1α, platelet-derived growth factor (PDGF)bb, insulin-like growth factor (IGF)-I and interleukin (IL)-6. The ability of inflammatory cytokines to induce MSC migration was tested in presence of their respective Ab or blocking peptide. We used immunofluorescence to check the expression of cytokine receptors, and MMP/TIMP production was analyzed at the protein (human cytokine array, enzyme-linked immunosorbent assay (ELISA), gelatine zymography and Western blot) and mRNA quantitative real-time polymerase chain reaction (qRT-PCR) levels.ResultsWe have demonstrated that inflammatory cytokines promote the migratory capacity of BM MSC according to the expression of their respective receptors. Higher migration through Matrigel was observed in response to IL-6 and PDGFbb. qRT-PCR and cytokine array revealed that migration was the result of the variable level of MMP/TIMP in response to inflammatory stimuli.ConclusionsOur observations suggest that chemokines and cytokines involved in the regulation of the immunity or inflammatory process promote the migration of MSC into BM or damaged tissues. One of the mechanisms used by MSC to promote their migration though the extracellular matrix is modulation of the production of MMP-1, MMP-2, MMP-13, TIMP-1 and TIMP-2.     

Cdc42 and RhoA have opposing roles in regulating membrane type 1-matrix metalloproteinase localization and matrix metalloproteinase-2 activation

Proteolysis of the basement membrane and interstitial matrix occurs early in the angiogenic process and requires matrix metalloproteinase (MMP) activity. Skeletal muscle microvascular endothelial cells exhibit robust actin stress fibers, low levels of membrane type 1 (MT1)-MMP expression, and minimal MMP-2 activation. Depolymerization of the actin cytoskeleton increases MT1-MMP expression and MMP-2 activation. Rho family GTPases are regulators of actin cytoskeleton dynamics, and their activity can be modulated in response to angiogenic stimuli such as vascular endothelial growth factor (VEGF). Therefore, we investigated their roles in MMP-2 and MT1-MMP production. Endothelial cells treated with H1152 [an inhibitor of Rho kinase (ROCK)] induced stress fiber depolymerization and an increase in cortical actin. Both MMP-2 and MT1-MMP mRNA increased, which translated into greater MMP-2 protein production and activation. ROCK inhibition rapidly increased cell surface localization of MT1-MMP and increased PI3K activity, which was required for MMP-2 activation. Constitutively active Cdc42 increased cortical actin polymerization, phosphatidylinositol 3-kinase activity, MT1-MMP cell surface localization, and MMP-2 activation similarly to inhibition of ROCK. Activation of Cdc42 was sufficient to decrease RhoA activity. Capillary sprout formation in a three-dimensional collagen matrix was increased in cultures treated with RhoAN19 or Cdc42QL and, conversely, decreased in cultures treated with dominant negative Cdc42N17. VEGF stimulation also induced activation of Cdc42 while inhibiting RhoA activity. Furthermore, VEGF-dependent activation of MMP-2 was reduced by inhibition of Cdc42. These results suggest that Cdc42 and RhoA have opposing roles in regulating cell surface localization of MT1-MMP and MMP-2 activation.     

Human airway epithelial cells in culture for studying the molecular mechanisms of the inflammatory response triggered by diesel exhaust particles

Epidemiological studies have shown that particulate air pollution is linked to the increase of morbidity and mortality due to respiratory diseases. Diesel exhaust particles (DEPs), which are the most important part of PM2.5 in Western European and Japanese urban areas, have been suspected. The mechanisms of proinflammatory response induced by DEPS were elucidated using a human epithelial cell line (16-HBE). It has been shown that DEPs can be phagocytosed by HBE cells, inducing the release of cytokines. MAP kinase pathways (i.e., ERK1/2 and P38) were triggered as well as the activation of the nuclear factor NF-κB. Reactive oxygen species (ROS) were strongly incriminated in this response because DEPs induce the increase of intracellular hydroperoxides and antioxidants inhibit the release of DEP-induced cytokines, the activation of MAP kinases and NF-κB. Organic compounds adsorbed on DEPs seemed to be involved in the response and the production of ROS. Moreover, we have demonstrated that DEPs can activate CYP1A1 in HBE cells. These experimental results give biological plausibility to the epidemiological findings. This revised version was published online in August 2006 with corrections to the Cover Date.     

Modulation of prostate cancer growth in bone microenvironments

Bone remains one of the major sites, and most lethal host organs, for prostate cancer metastasis. Prostate cell spread and establishment in bone depends on multiple reciprocal modifications of bone stromal and epithelial cancer cell behaviors. This review focuses on recent advances in the characterization of cell-cell and cell-matrix interplay, effects on cell growth, adhesion and invasion, and several therapeutic possibilities for co-targeting prostate cancer cells and bone stroma. We address the topic from three main perspectives: (1) the normal and aging bone stromal environment, (2) the "reactive" bone stromal environment, and (3) the cancerous prostate epithelial cells themselves. First, normal, and especially aging, bones provide uniquely rich and "fertile soil" for roaming cancer cells. The interactions between prostate cancer cells and insoluble extracellular matrices, soluble growth factors, and/or sex steroid hormones trigger bone remodeling, through increased osteoclastogenesis and furthur matrix metalloproteinase activity. Second, after cancer cell arrival and establishment in the bone, host stromal cells respond, becoming "reactive" in a process again involving extracellular matrix remodeling, together with growth factor and steroid receptor signaling this process ultimately enhances cancer cell migration, stromal transdifferentiation, and invasion of the cancer tissues by stromal, inflammatory, and immune-responsive cells. Third, prostate cancer cells also respond to supportive bone microenvironments, where soluble and matrix-associated molecules affect cancer cell growth and gene expression, especially altering cancer cell surface receptor and integrin-mediated cell signaling. We discuss both integrin cell-matrix and gap junctional cell-cell communication between cancer cells and their microenvironments during prostate cancer progression.     

Cystathionine- -synthase gene transfer and 3-deazaadenosine ameliorate inflammatory response in endothelial cells

Although elevated levels of homocysteine (Hcy) known as hyperhomocysteinemia (HHcy) are associated with increased inflammation and vascular remodeling, the mechanism of Hcy-mediated inflammation and vascular remodeling is unclear. The matrix metalloproteinases (MMPs) and adhesion molecules play an important role in vascular remodeling. We hypothesized that HHcy induces inflammation by increasing adhesion molecules and matrix protein expression. Endothelial cells were supplemented with high methionine, and Hcy accumulation was measured by HPLC. Nitric oxide (NO) bioavailability was detected by a NO probe. The protein expression was measured by Western blot analysis. MMP-9 activity was detected by gelatin-gel zymography. We demonstrated that methionine supplement promoted upregulation of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) through increased Hcy accumulation. In addition, increased synthesis of collagen type-1 was also observed. MMP-9 gene expression and protein activity were increased in methionine supplement groups. 3-Deazaadenosine (DZA), an adenosine analogue, prevented high methionine-induced ICAM-1 and VCAM-1 expression and collagen type-1 synthesis. Transfection of endothelial cells with cystathionine-β-synthase (CBS) gene construct, which converts Hcy to cystathionine, reduced Hcy accumulation in high methionine-fed cells. CBS gene transfection reduced the inflammatory response, as evident by attenuated ICAM-1 and VCAM-1 expression. Furthermore, collagen type-1 expression and MMP-9 activity were dramatically attenuated with CBS gene transfection. These results suggested that methionine supplement increased Hcy accumulation, which was associated with inflammatory response and matrix remodeling such as collagen type-1 synthesis and MMP-9 activity. However, in vitro DZA and CBS gene therapy successfully treated the HHcy-induced inflammatory reaction in the methionine metabolism pathway. extracellular matrix; matrix metalloproteinase-9; intercellular cell adhesion molecule-1; vascular cell adhesion molecule-1; collagen type-1; hyperhomocysteinemia     

Antimicrobial peptide KSL-W promotes gingival fibroblast healing properties in vitro

We investigated the effect of synthetic antimicrobial decapeptide KSL-W (KKVVFWVKFK) on normal human gingival fibroblast growth, migration, collagen gel contraction, and α-smooth muscle actin protein expression. Results show that in addition to promoting fibroblast adhesion by increasing F-actin production, peptide KSL-W promoted cell growth by increasing the S and G2/M cell cycle phases, and enhanced the secretion of metalloproteinase (MMP)-1 and MMP-2 by upregulating MMP inhibitors, such as tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2 in fibroblasts. An in vitro wound healing assay confirmed that peptide KSL-W promoted fibroblast migration and contraction of a collagen gel matrix. We also demonstrated a high expression of α-smooth muscle actin by gingival fibroblasts being exposed to KSL-W. This work shows that peptide KSL-W enhances gingival fibroblast growth, migration, and metalloproteinase secretion, and the expression of α-smooth muscle actin, thus promoting wound healing.     

Airway smooth muscle tone modulates mechanically induced cytoskeletal stiffening and remodeling.

The application of mechanical stresses to the airway smooth muscle (ASM) cell causes time-dependent cytoskeletal stiffening and remodeling (Deng L, Fairbank NJ, Fabry B, Smith PG, and Maksym GN. Am J Physiol Cell Physiol 287: C440-C448, 2004). We investigated here the extent to which these behaviors are modulated by the state of cell activation (tone). Localized mechanical stress was applied to the ASM cell in culture via oscillating beads (4.5 mum) that were tightly bound to the actin cytoskeleton (CSK). Tone was reduced from baseline level using a panel of relaxant agonists (10(-3) M dibutyryl cAMP, 10(-4) M forskolin, or 10(-6) M formoterol). To assess functional changes, we measured cell stiffness (G') using optical magnetic twisting cytometry, and to assess structural changes of the CSK we measured actin accumulation in the neighborhood of the bead. Applied mechanical stress caused a twofold increase in G' at 120 min. After cessation of applied stress, G' diminished only 24 +/- 6% (mean +/- SE) at 1 h, leaving substantial residual effects that were largely irreversible. However, applied stress-induced stiffening could be prevented by ablation of tone. Ablation of tone also inhibited the amount of actin accumulation induced by applied mechanical stress (P < 0.05). Thus the greater the contractile tone, the greater was applied stress-induced CSK stiffening and remodeling. As regards pathobiology of asthma, this suggests a maladaptive positive feedback in which tone potentiates ASM remodeling and stiffening that further increases stress and possibly leads to worsening airway function.     

N-cadherin cell-cell adhesion complexes are regulated by fibronectin matrix assembly

Fibronectin is a principal component of the extracellular matrix. Soluble fibronectin molecules are assembled into the extracellular matrix as insoluble, fibrillar strands via a cell-dependent process. In turn, the interaction of cells with the extracellular matrix form of fibronectin stimulates cell functions critical for tissue repair. Cross-talk between cell-cell and cell-extracellular matrix adhesion complexes is essential for the organization of cells into complex, functional tissue during embryonic development and tissue remodeling. Here, we demonstrate that fibronectin matrix assembly affects the organization, composition, and function of N-cadherin-based adherens junctions. Using fibronectin-null mouse embryonic myofibroblasts, we identified a novel quaternary complex composed of N-cadherin, β-catenin, tensin, and actin that exists in the absence of a fibronectin matrix. In the absence of fibronectin, homophilic N-cadherin ligation recruited both tensin and α5β1 integrins into nascent cell-cell adhesions. Initiation of fibronectin matrix assembly disrupted the association of tensin and actin with N-cadherin, released α5β1 integrins and tensin from cell-cell contacts, stimulated N-cadherin reorganization into thin cellular protrusions, and decreased N-cadherin adhesion. Fibronectin matrix assembly has been shown to recruit α5β1 integrins and tensin into fibrillar adhesions. Taken together, these studies suggest that tensin serves as a common cytoskeletal link for integrin- and cadherin-based adhesions and that the translocation of α5β1 integrins from cell-cell contacts into fibrillar adhesions during fibronectin matrix assembly is a novel mechanism by which cell-cell and cell-matrix adhesions are coordinated.     

Human mast cell-derived gelatinase B (matrix metalloproteinase-9) is regulated by inflammatory cytokines: role in cell migration

Mast cells are key effectors in the pathogenesis of inflammatory and tissue destructive diseases such as rheumatoid arthritis (RA). These cells contain specialized secretory granules loaded with bioactive molecules including cytokines, growth factors, and proteases that are released upon activation. This study investigated the regulation of matrix metalloproteinase MMP-9 (gelatinase B) in human mast cells by cytokines that are known to be involved in the pathogenesis of RA. Immunohistochemical staining of synovial tissue showed abundant expression of MMP-9 by synovial tissue mast cells in patients with RA but not in normal controls. The expression, activity, and production of MMP-9 in mast cells was confirmed by RT-PCR, zymography, and Western blotting using cord blood-derived human mast cells (CB-HMC). Treatment of CB-HMC with TNF-alpha significantly increased the expression of MMP-9 mRNA and up-regulated the activity of MMP-9 in a time- and dose-dependent manner. By contrast, IFN-gamma inhibited MMP-9 mRNA and protein expression. The cytokine-mediated regulation of MMP-9 was also apparent in the human mast cell line (HMC-1) and in mouse bone marrow-derived mast cells. Furthermore, TNF-alpha significantly increased the invasiveness of CB-HMC across Matrigel-coated membranes while the addition of IFN-gamma, rTIMP-1, or pharmacological MMP inhibitors significantly reduced this process. These observations suggest that MMP-9 is not a stored product in mast cells but these cells are capable of producing this enzyme under inflammatory conditions that may facilitate the migration of mast cell progenitors to sites of inflammation and may also contribute to local tissue damage.     

Cdc42 regulates extracellular matrix remodeling in three dimensions

Extracellular matrix (ECM) actively participates in normal cell regulation and in the process of tumor progression. The Rho GTPase Cdc42 has been shown to regulate cell-ECM interaction in conventional two-dimensional culture conditions by using dominant mutants of Cdc42 in immortalized cell lines that may introduce nonspecific effects. Here, we employ three-dimensional culture systems for conditional gene targeted primary mouse embryonic fibroblasts that better simulate the reciprocal and adaptive interactions between cells and surrounding matrix to define the role of Cdc42 signaling pathways in ECM organization. Cdc42 deficiency leads to a defect in global cell-matrix interactions reflected by a decrease in collagen gel contraction. The defect is associated with an altered cell-matrix interaction that is evident by morphologic changes and reduced focal adhesion complex formation. The matrix defect is also associated with a reduction in synthesis and activation of matrix metalloproteinase 9 (MMP9) and altered fibronectin deposition patterning. A Cdc42 mutant rescue experiment found that downstream of Cdc42, p21-activated kinase (PAK), but not Par6 or WASP, may be involved in regulating collagen gel contraction and fibronectin organization. Thus, in addition to the previously implicated roles in intracellular regulation of actin organization, proliferation, and vesicle trafficking, Cdc42 is essential in ECM remodeling in three dimensions.     

Effect of proinflammatory cytokines, tumor necrosis factor-alpha and interferon-gamma on epithelial barrier function and matrix metalloproteinase-9 in Madin Darby canine kidney cells.

BACKGROUND: Elevated matrix metalloproteinase-9 production during inflammation may be deleterious to epithelial barrier function. Therefore we examined the effect of proinflammatory cytokines on the expression and regulation of matrix metalloproteinase-9 in a model renal epithelial cell system. Tight junctions limit diffusion between compartments and permit directional transport of solutes. Impairment of these junctional complexes by proteolysis may contribute to renal failure through loss of barrier function. METHODS: The renal epithelial cell model, MDCK cells were employed to examine metalloproteinase activity and mRNA expression. Epithelial barrier function was determined using paracellular flux studies. RESULTS: We found that matrix metalloproteinase-9 expression (MMP-9) and activity is markedly elevated in response to tumor necrosis factor-alpha exposure through a mitogen-activated protein kinase dependent pathway. The MMP-9 is predominately secreted into the apical compartment and elevated MMP-9 expression correlates with impaired cell barrier function that was restored using a specific inhibitor of MMP activity. Addition of recombinant MMP-9 to the apical compartment of MDCK cultures significantly elevated paracellular flux rate. CONCLUSIONS: We provide direct evidence for a MMP-9-mediated mechanism that produces junctional disruption. Collectively, these findings support the hypothesis that impaired epithelial barrier function due to activation of tissue/matrix degrading mechanisms occurs in response to specific inflammatory cues.     

Defining the role of mesenchymal stromal cells on the regulation of matrix metalloproteinases in skeletal muscle cells

Recent studies indicate that mesenchymal stromal cell (MSC) transplantation improves healing of injured and diseased skeletal muscle, although the mechanisms of benefit are poorly understood. In the present study, we investigated whether MSCs and/or their trophic factors were able to regulate matrix metalloproteinase (MMP) expression and activity in different cells of the muscle tissue. MSCs in co-culture with C2C12 cells or their conditioned medium (MSC-CM) up-regulated MMP-2 and MMP-9 expression and function in the myoblastic cells; these effects were concomitant with the down-regulation of the tissue inhibitor of metalloproteinases (TIMP)-1 and -2 and with increased cell motility. In the single muscle fiber experiments, MSC-CM administration increased MMP-2/9 expression in Pax-7⁺ satellite cells and stimulated their mobilization, differentiation and fusion. The anti-fibrotic properties of MSC-CM involved also the regulation of MMPs by skeletal fibroblasts and the inhibition of their differentiation into myofibroblasts. The treatment with SB-3CT, a potent MMP inhibitor, prevented in these cells, the decrease of α-smooth actin and type-I collagen expression induced by MSC-CM, suggesting that MSC-CM could attenuate the fibrogenic response through mechanisms mediated by MMPs. Our results indicate that growth factors and cytokines released by these cells may modulate the fibrotic response and improve the endogenous mechanisms of muscle repair/regeneration.