Mesenchymal cells stimulate capillary morphogenesis via distinct proteolytic mechanisms

During angiogenesis, endothelial cells (ECs) degrade their surrounding extracellular matrix (ECM) to facilitate invasion. How interactions between ECs and other cells within their microenvironment facilitate this process is only partially understood. We have utilized a tractable 3D co-culture model to investigate the proteolytic mechanisms by which pre-committed or more highly committed mesenchymal cells stimulate capillary formation. On their own, ECs invade their surrounding matrix, but do not form capillaries. However, in the presence of either mesenchymal stem cells (MSCs) or fibroblasts, ECs form polarized, tubular structures that are intimately associated with mesenchymal cells. Further, ECs up-regulate gene expression of several extracellular proteases upon co-culture with either mesenchymal cell type. The administration of both broad spectrum and specific protease inhibitors demonstrated that MSC-stimulated capillary formation relied solely on membrane-type matrix metalloproteinases (MT-MMPs) while fibroblast-mediated sprouting proceeded independent of MMP inhibition unless the plasminogen activator/plasmin axis was inhibited in concert. While other studies have established a role for the ECM itself in dictating proteolysis and matrix degradation during capillary morphogenesis, the present study illustrates that heterotypic cellular interactions within the microenvironment can direct the proteolytic mechanisms required for capillary formation.     

Caveolin‐1 mediates tissue plasminogen activator‐induced MMP‐9 up‐regulation in cultured brain microvascular endothelial cells

Thrombolysis with tissue plasminogen activator (tPA) increases matrix metalloproteinase‐9 (MMP‐9) activity in the ischemic brain, which exacerbates blood‐brain barrier injury and increases the risk of symptomatic cerebral hemorrhage. The mechanism through which tPA enhances MMP‐9 activity is not well understood. Here we report an important role of caveolin‐1 in mediating tPA‐induced MMP‐9 synthesis. Brain microvascular endothelial cell line bEnd3 cells were incubated with 5 or 20 μg/ml tPA for 24 hrs before analyzing MMP‐9 levels in the conditioned media and cellular extracts by gelatin zymography. tPA at a dose of 20 μg/mL tPA, but not 5 μg/mL, significantly increased MMP‐9 level in cultured media while decreasing it in cellular extracts. Concurrently, tPA treatment induced a 2.3‐fold increase of caveolin‐1 protein levels in endothelial cells. Interestingly, knockdown of Cav‐1 with siRNA inhibited tPA‐induced MMP‐9 mRNA up‐regulation and MMP‐9 increase in the conditioned media, but did not affect MMP‐9 decrease in cellular extracts. These results suggest that caveolin‐1 critically contributes to tPA‐mediated MMP‐9 up‐regulation, but may not facilitate MMP‐9 secretion in endothelial cells.

     

Nicotine treatment induces expression of matrix metalloproteinases in human osteoblastic Saos-2 cells

Tobacco smoking is an important risk factor for the development of severe periodontitis.Recently,we showed that nicotine affected mineralized nodule formation,and that nicotine andlipopolysaccharide stimulated the formation of osteoclast-like cells by increasing production of macrophagecolony-stimulating factor (M-CSF) and prostaglandin E_2 (PGE_2) by human osteoblastic Saos-2 cells.In thepresent study,we examined the effects of nicotine on the expression of matrix metalloproteinases (MMPs),tissue inhibitors of matrix metalloproteinases (TIMPs),the plasminogen activation system including thecomponent of tissue-type plasminogen activator (tPA),urokinase-type PA (uPA),and PA inhibitor type 1(PAI-1),α7 nicotine receptor,and c-fos.We also examined the effect of the nicotine antagonist D-tubocurarineon nicotine-induced expression of MMP-1.Gene expression was examined using real-time polymerase chainreaction (PCR) to estimate mRNA levels.In addition,expression of the MMP,TIMP,uPA,tPA,and PAI-1proteins was determined by Western blotting analysis.Nicotine treatment caused expression of MMP-1,2,3,and 13,but not MMP-14,to increase significantly after 5 or 10 d of culture;MMP-14 expression did notchange through day 14.Enhancement of MMP-1 expression by nicotine treatment was eliminated bysimultaneous treatment with D-tubocurarine.In the presence of nicotine,expression of uPA,PAI-1,orTIMP-1,2,3,or 4 did not change over 14 d of culture,whereas expression of tPA increased significantly byday 7.Nicotine also increased expression of the α7 nicotine receptor and c-fos genes.These results suggestthat nicotine stimulates bone matrix turnover by increasing production of tPA and MMP-1,2,3,and 13,thereby tipping the balance between bone matrix formation and resorption toward the latter process.     

Matrix metalloproteinase‐3 in the central nervous system: a look on the bright side

Matrix metalloproteinases (MMPs) are a large family of proteases involved in many cell‐matrix and cell‐cell signalling processes through activation, inactivation or release of extracellular matrix (ECM) and non‐ECM molecules, such as growth factors and receptors. Uncontrolled MMP activities underlie the pathophysiology of many disorders. Also matrix metalloproteinase‐3 (MMP‐3) or stromelysin‐1 contributes to several pathologies, such as cancer, asthma and rheumatoid arthritis, and has also been associated with neurodegenerative diseases like Alzheimer's disease, Parkinson's disease and multiple sclerosis. However, based on defined MMP spatiotemporal expression patterns, the identification of novel candidate molecular targets and in vitro and in vivo studies, a beneficial role for MMPs in CNS physiology and recovery is emerging. The main purpose of this review is to shed light on the recently identified roles of MMP‐3 in normal brain development and in plasticity and regeneration after CNS injury and disease. As such, MMP‐3 is correlated with neuronal migration and neurite outgrowth and guidance in the developing CNS and contributes to synaptic plasticity and learning in the adult CNS. Moreover, a strict spatiotemporal MMP‐3 up‐regulation in the injured or diseased CNS might support remyelination and neuroprotection, as well as genesis and migration of stem cells in the damaged brain.     

Matrix metalloproteinase-9 regulates survival of neurons in newborn hippocampus

The number of neurons in the adult rodent brain is strongly influenced by events in early postnatal life that eliminate approximately half of the neurons. Recently, we reported that neurotrophins induced survival of neonatal rat hippocampal neurons by promoting neural activity and activation of the Ser/Thr kinase, Akt. The survival of neurons also depended on integrin signaling, but a role for the extracellular matrix (ECM) in this mechanism was yet to be explored. Here, we show that levels of the matrix metalloproteinase-9 (MMP9) decrease, and the level of the ECM protein laminin increases in rat hippocampus during the period of neuronal death. Hippocampi from MMP9 null mice showed higher levels of laminin expression than wild type at P1 and no further increase at P10. In vitro, the matrix metalloproteinase inhibitor FN-439 promoted survival of neurons in a laminin-integrin β1-dependent manner. Blocking laminin signaling attenuated activation of Akt by depolarization. In vivo, injecting FN-439 into the neonatal hippocampus increased the level of laminin and promoted neuronal survival through an integrin-dependent mechanism. These results show signals from the ECM are not simply permissive but rather actively regulated, and they interact with neuronal activity to control the number of hippocampal neurons. This work is the first to report a role for MMP9 in regulating neuronal survival through the developmental process that establishes the functional brain.     

Lost in disruption: Role of proteases in glioma invasion and progression

A characteristic feature of malignant glial tumors (gliomas) is their tendency to diffusely infiltrate the nervous system preventing their complete surgical resection. Proteases play a decisive role in this malignant process, either by degradation of brain extracellular matrix (ECM) components, adhesion molecules, or by regulating the activity of growth and chemotactic factors. Secreted matrix metalloproteinases (MMPs) and ADAMTS proteases (ADAMs with thrombospondin motifs) cleave different ECM components like the proteoglycans (lecticans) aggrecan, versican, neurocan and brevican with selective preferences; they are further regulated by endogenous inhibitors and activating metallo- and serine proteases. Cell surface proteases of the ADAM family (A Disintegrin And Metalloproteinase), but also serine proteases regulate the activity of growth factors and chemokines that act as autocrine / paracrine stimulators within gliomas. Thus, proteases play a decisive role for the spread and growth of gliomas and are prominent targets for their therapy.     

CHOP is involved in neuronal apoptosis induced by neurotrophic factor deprivation

Neurotrophic factors are essential for the survival of neurons. We found that the endoplasmic reticulum (ER) stress-C/EBP homologues protein (CHOP) pathway to be activated during neurotrophic factor deprivation-induced apoptosis in PC12 neuronal cells and in primary cultured neurons, and this apoptosis was suppressed in the neurons from chop(-/-) mice. In addition, we found that CHOP is expressed in the subventricular zone (SVZ) and striatum of the young adult mouse brain. The number of apoptotic cells in the SVZ decreased in chop(-/-) mice. These results indicate that the ER stress-CHOP pathway plays a role in neuronal apoptosis during the development of the brain.     

Absence of tissue inhibitor of metalloproteinases 3 disrupts alveologenesis in the mouse

Tissue inhibitors of metalloproteinases (TIMPs) regulate extracellular matrix (ECM) degradation by matrix metalloproteinases (MMPs) throughout lung development. We examined lungs from TIMP3 null mice and found significant air space enlargement compared with wild type (WT) animals during a time course spanning early alveologenesis (post‐partum days 1, 5, 9 and 14). Trichrome staining revealed a similar pattern of collagen distribution in the walls of nascent alveoli; however, the alveolar walls of TIMP3 mutant mice appeared to be thinner than controls. Assessment of MMP2 and MMP9 activities by gelatin zymography demonstrated a significant elevation in the active form of MMP2 at post‐partum days 1 and 5. Treatment of null pregnant dams with a broad spectrum synthetic metalloproteinase inhibitor, GM6001, on embryonic day 16.5 enhanced the formation of primitive alveoli during the saccular stage of lung development as evidenced by a partial, but significant, rescue of alveolar size in post‐partum day 1 animals. We propose that increased MMP activity in the absence of TIMP3 enhances ECM proteolysis, upsetting proper formation of primitive alveolar septa during the saccular stage of alveologenesis. Therefore, TIMP3 indirectly regulates alveolar formation in the mouse. To our knowledge, ours is the first study to demonstrate that in utero manipulation of the TIMP/MMP proteolytic axis, to specifically inhibit proteolysis, significantly affects lung development.     

Role of matrix metalloproteinase and tissue inhibitor of MMP in methamphetamine-induced behavioral sensitization and reward: implications for dopamine receptor down-regulation and dopamine release

Matrix metalloproteinases (MMPs) and its inhibitors (TIMPs) function to remodel the pericellular environment. We have demonstrated that methamphetamine (METH)-induced behavioral sensitization and reward were markedly attenuated in MMP-2- and MMP-9 deficient [MMP-2-(-/-) and MMP-9-(-/-)] mice compared with those in wild-type mice, suggesting that METH-induced expression of MMP-2 and MMP-9 in the brain plays a role in the development of METH-induced sensitization and reward. In the present study, we investigated the changes in TIMP-2 expression in the brain after repeated METH treatment. Furthermore, we studied a role of MMP/TIMP system in METH-induced behavioral changes and dopamine neurotransmission. Repeated METH treatment induced behavioral sensitization, which was accompanied by an increase in TIMP-2 expression. Antisense TIMP-2 oligonucleotide (TIMP-AS) treatment enhanced the sensitization, which was associated with the potentiation of METH-induced dopamine release in the nucleus accumbens (NAc). On the other hand, MMP-2/-9 inhibitors blocked the METH-induced behavioral sensitization and conditioned place preference, a measure of the rewarding effect, and reduced the METH-increased dopamine release in the NAc. Dopamine receptor agonist-stimulated [(35)S]GTPgammaS binding was reduced in the frontal cortex of sensitized rats. TIMP-AS treatment potentiated, while MMP-2/-9 inhibitor attenuated, the reduction of dopamine D2 receptor agonist-stimulated [(35)S]GTPgammaS binding. Repeated METH treatment also reduced dopamine D2 receptor agonist-stimulated [(35)S]GTPgammaS binding in wild-type mice, but such changes were significantly attenuated in MMP-2-(-/-) and MMP-9-(-/-) mice. These results suggest that the MMP/TIMP system is involved in METH-induced behavioral sensitization and reward, by regulating dopamine release and receptor signaling.     

Direct Interaction of CASK/LIN-2 and Syndecan Heparan Sulfate Proteoglycan and Their Overlapping Distribution in Neuronal Synapses

CASK, the rat homolog of a gene (LIN-2) required for vulval differentiation in Caenorhabditis elegans, is expressed in mammalian brain, but its function in neurons is unknown. CASK is distributed in a punctate somatodendritic pattern in neurons. By immunogold EM, CASK protein is concentrated in synapses, but is also present at nonsynaptic membranes and in intracellular compartments. This immunolocalization is consistent with biochemical studies showing the presence of CASK in soluble and synaptosomal membrane fractions and its enrichment in postsynaptic density fractions of rat brain. By yeast two-hybrid screening, a specific interaction was identified between the PDZ domain of CASK and the COOH terminal tail of syndecan-2, a cell surface heparan sulfate proteoglycan (HSPG). The interaction was confirmed by coimmunoprecipitation from heterologous cells. In brain, syndecan-2 localizes specifically at synaptic junctions where it shows overlapping distribution with CASK, consistent with an interaction between these proteins in synapses. Cell surface HSPGs can bind to extracellular matrix proteins, and are required for the action of various heparin-binding polypeptide growth/differentiation factors. The synaptic localization of CASK and syndecan suggests a potential role for these proteins in adhesion and signaling at neuronal synapses.     

Matrix metalloproteinases regulate mesonephric cell migration in developing XY gonads which correlates with the inhibition of tissue inhibitor of metalloproteinase-3 by Sry

In the mouse, the sex determining gene Sry, on the Y chromosome, controls testis differentiation during embryogenesis. Following Sry expression, indifferent XY gonads increase their size relative to XX gonads and form cord-like structures with the adjacent mesonephros, providing XY gonad somatic cells. This mesonephric cell migration is known to depend on Sry, but the molecular mechanism of mesonephric cell migration remains unknown. In this study, it was shown that cells expressing Sry induced proliferation of mesonephric cells migrating into male gonads, and inhibited expression of the tissue inhibitor of metalloproteinases (TIMP)-3 gene, which is the endogenous inhibitor of matrix metalloproteinases (MMP). In addition, the mesonephric cell migration was blocked by a chemically synthesized inhibitor of MMP in a gonad/mesonephros organ co-culture system with enhanced green fluorescent protein transgenic embryos. The findings indicate that MMP may play a critical role in mesonephric cell migration, and the function of MMP may be regulated by a Sry-TIMP-3 cascade. These findings are an important clue for the elucidation of testicular formation in developing gonads.     

Acetylcholine induces neurite outgrowth and modulates matrix metalloproteinase 2 and 9

The matrix metalloproteinases (MMPs), responsible for the degradation of extracellular matrix (ECM) proteins, may regulate brain cellular functions. Choline acetyltransferase (ChAT) transfected murine neuroblastoma cell line N18TG2, that synthesize acetylcholine and show enhancement of several neurospecific markers (i.e., sinapsin I, voltage gated Na(+) channels, high affinity choline uptake) and fiber outgrowth, were studied for the MMP regulation during neuronal differentiation. Zymography of N18TG2 culture medium revealed no gelatinolytic activity, whereas after carbachol treatment of cells both MMP-9 and activated MMP-2 forms were detected. ChAT-transfected clone culture medium contains three MMP forms at 230, 92, and 66kDa. Carbachol treatment increased MMP-2 and MMP-9 gene expression in N18TG2 cells and higher levels for both genes were also observed in ChAT transfected cells. The data are consistent with the hypothesis that acetylcholine brings about the activation of an autocrine loop modulating MMP expression.     

Increased intranuclear matrix metalloproteinase activity in neurons interferes with oxidative DNA repair in focal cerebral ischemia

Increased matrix metalloproteinase (MMP) activity is implicated in proteolysis of extracellular matrix in ischemic stroke. We recently observed intranuclear MMP activity in ischemic brain neurons at early reperfusion, suggesting a possible role in nuclear matrix proteolysis. Nuclear proteins, poly-ADP-ribose polymerase-1 (PARP-1) and X-ray cross-complementary factor 1 (XRCC1), as well as DNA repair enzymes, are important in DNA fragmentation and cell apoptosis. We hypothesized that intranuclear MMP activity facilitates oxidative injury in neurons during early ischemic insult by cleaving PARP-1 and XRCC1, interfering with DNA repair. We induced a 90-min middle cerebral artery occlusion in rats. Increase activity of MMP-2 and -9, detected in the ischemic neuronal nuclei at 3 h, was associated with DNA fragmentation at 24 and 48 h reperfusion. The intranuclear MMPs cleaved PARP-1. Treatment of the rats with a broad-spectrum MMP inhibitor, BB1101, significantly attenuated ischemia-induced PARP-1 cleavage, increasing its activity. Degradation of XRCC1 caused by ischemic insult in rat brain was also significantly attenuated by BB1101. We found elevation of oxidized DNA, apurinic/apyrimidinic sites, and 8-hydroxy-2'-deoxyguanosine, in ischemic brain cells at 3 h reperfusion. BB1101 markedly attenuated the early increase of oxidized DNA. Using tissue from stroke patients, we found increased intranuclear MMP expression. Our data suggest that intranuclear MMP activity cleaves PARP-1 and XRCC1, interfering with oxidative DNA repair. This novel role for MMPs could contribute to neuronal apoptosis in ischemic injuries.     

Circulating biomarkers of tissue remodelling in pulmonary hypertension

Objective: Besides persisting high pulmonary arterial pressure and increased pulmonary vascular resistance, remodelling of pulmonary tissues and subsequently the right heart are the key pathomechanisms of pulmonary hypertension (PH). Extracellular matrix maintenance in this context plays a central role.

Methods: We tested the hypothesis that plasma concentration of matrix metalloproteinase (MMP)-2, tissue inhibitor of matrix metalloproteinases (TIMP)-4 and tenascin C (TNC) might be useful as biomarkers for assessing the severity of PH. Therefore, the concentrations of MMP-2, TIMP-4, TNC and N-terminal b-type natriuretic peptide (NT-proBNP) of 36 PH patients were compared with those of 44 age- and gender-matched healthy volunteers. Additionally, lung function, 6-min walk distance and right heart function were assessed.

Results: In PH patients, significantly elevated plasma levels of MMP-2, TIMP-4, TNC and NT-proBNP were detected. In particular, TIMP-4 was significantly increased in patients with higher NYHA classification, and in patients with severe right ventricular hypertrophy.

Conclusion: Monitoring of plasma TIMP-4 and to a lesser extent of MMP-2 and TNC levels in PH patients might help to assess the beneficial effects of PH pharmacotherapy on tissue remodelling.     

Flavanone and 2'-OH flavanone inhibit metastasis of lung cancer cells via down-regulation of proteinases activities and MAPK pathway

Flavanones richly exist in citrus and have been well characterized to have various bioactive properties. However, the anti-metastasis properties of flavanones remain unclear. The anti-metastatic effects of six flavanones including flavanone, 2'-OH flavanone, 4'-OH flavanone, 6-OH flavanone, naringin, and naringenin were investigated in lung cancer cells. Despite little influence on cell viability, flavanone and 2'-OH flavanone markedly inhibited the invasion, motility, and cell-matrix adhesion of A549 cells. This was associated with a reduced expression of matrix metalloproteinase (MMP)-2 and urokinase-type plasminogen activator (u-PA) in treated cells. Treatment with flavanone and 2'-OH flavanone also potently attenuated the phosphorylations of extracellular signal-regulated kinase 1/2 (ERK 1/2) and p38(MAPK), as well as the activations of NF-kappaB and AP-1. The reduced expressions of MMP-2 and u-PA, as well as inhibition of cell invasion were obtained in the cultures treated with U0126 (ERK 1/2 inhibitor) and SB203580 (p38(MAPK) inhibitor). Thus, the inhibitory effects of flavanone and 2'-OH flavanone on the expression of MMP-2 and u-PA may be at least partly through inactivation of ERK 1/2 and p38(MAPK) signaling pathways. Finally, oral administration of flavanone and 2'-OH flavanone were evidenced by its inhibition on the metastasis of A549 cells and Lewis lung carcinoma (LLC) cells in vivo. In conclusion, flavanone and 2'-OH flavanone perturb the invasion and metastasis of lung cancer cells, thereby constituting an adjuvant treatment for metastasis control.     

Activation of the P2X7 receptor induces migration of glial cells by inducing cathepsin B degradation of tissue inhibitor of metalloproteinase 1

The P2X₇ receptor is an ion‐gated channel, which is activated by high extracellular concentrations of adenosine triphosphate (ATP). Activation of P2X₇ receptors has been shown to induce neuroinflammatory changes associated with several neurological conditions. The matrix metalloproteinases (MMPs) are a family of endopeptidases that have several functions including degradation of the extracellular matrix, cell migration and modulation of bioactive molecules. The actions of MMPs are prevented by a family of protease inhibitors called tissue inhibitors of metalloproteinases (TIMPs). In this study, we show that ATP‐treated glial cultures from neonatal C57BL/6 mice release and increase MMP‐9 activity, which is coupled with a decrease in release of TIMP‐1 and an increase in activated cathepsin B within the extracellular space. This process occurs independently of NLRP3‐inflammasome formation. Treatment with a P2X₇ receptor antagonist prevents ATP‐induced MMP‐9 activity, inhibition of active cathepsin B release and allows for TIMP‐1 to be released from the cell. We have shown that cathepsin B degrades TIMP‐1, and inhibition of cathepsin B allows for release of TIMP‐1 and inhibits MMP‐9 activity. We also present data that indicate that ATP or cell damage induces glial cell migration, which is inhibited by P2X₇ antagonism, depletion of MMP‐9 or inhibition of cathepsin B.     

Aggrecan,link protein and tenascin-R are essential components of the perineuronal net to protect neurons against iron-induced oxidative stress

In Alzheimer''s disease (AD), different types of neurons and different brain areas show differential patterns of vulnerability towards neurofibrillary degeneration, which provides the basis for a highly predictive profile of disease progression throughout the brain that now is widely accepted for neuropathological staging. In previous studies we could demonstrate that in AD cortical and subcortical neurons are constantly less frequently affected by neurofibrillary degeneration if they are enwrapped by a specialized form of the hyaluronan-based extracellular matrix (ECM), the so called ‘perineuronal net'' (PN). PNs are basically composed of large aggregating chondroitin sulphate proteoglycans connected to a hyaluronan backbone, stabilized by link proteins and cross-linked via tenascin-R (TN-R). Under experimental conditions in mice, PN-ensheathed neurons are better protected against iron-induced neurodegeneration than neurons without PN. Still, it remains unclear whether these neuroprotective effects are directly mediated by the PNs or are associated with some other mechanism in these neurons unrelated to PNs. To identify molecular components that essentially mediate the neuroprotective aspect on PN-ensheathed neurons, we comparatively analysed neuronal degeneration induced by a single injection of FeCl₃ on four different mice knockout strains, each being deficient for a different component of PNs. Aggrecan, link protein and TN-R were identified to be essential for the neuroprotective properties of PN, whereas the contribution of brevican was negligible. Our findings indicate that the protection of PN-ensheathed neurons is directly mediated by the net structure and that both the high negative charge and the correct interaction of net components are essential for their neuroprotective function.     

Expression of catalytically active matrix metalloproteinase‐1 in dermal fibroblasts induces collagen fragmentation and functional alterations that resemble aged human skin

Increased expression of matrix metalloproteinase‐1 (MMP‐1) and reduced production of type I collagen by dermal fibroblasts are prominent features of aged human skin. We have proposed that MMP‐1‐mediated collagen fibril fragmentation is a key driver of age‐related decline of skin function. To investigate this hypothesis, we constructed, characterized, and expressed constitutively active MMP‐1 mutant (MMP‐1 V94G) in adult human skin in organ culture and fibroblasts in three‐dimensional collagen lattice cultures. Expression of MMP‐1 V94G in young skin in organ culture caused fragmentation and ultrastructural alterations of collagen fibrils similar to those observed in aged human skin in vivo. Expression of MMP‐1 V94G in dermal fibroblasts cultured in three‐dimensional collagen lattices caused substantial collagen fragmentation, which was markedly reduced by MMP‐1 siRNA‐mediated knockdown or MMP inhibitor MMI270. Importantly, fibroblasts cultured in MMP‐1 V94G‐fragmented collagen lattices displayed many alterations observed in fibroblasts in aged human skin, including reduced cytoplasmic area, disassembled actin cytoskeleton, impaired TGF‐β pathway, and reduced collagen production. These results support the concept that MMP‐1‐mediated fragmentation of dermal collagen fibrils alters the morphology and function of dermal fibroblasts and provide a foundation for understanding specific mechanisms that link collagen fibril fragmentation to age‐related decline of fibroblast function.