Differential gene and protein expression in abluminal sprouting and intraluminal splitting forms of angiogenesis

In adult skeletal muscle, abluminal sprouting or longitudinal splitting of capillaries can be initiated separately by muscle overload and elevated microcirculation shear stress respectively. In the present study, gene and protein expression patterns associated with the different forms of angiogenesis were examined using a targeted gene array (Superarray), validated by quantitative RT (reverse transcription)-PCR and immunoblots. Sprouting angiogenesis induced large changes in expression levels in genes associated with extracellular matrix remodelling, such as MMP-2 (matrix metalloproteinase-2), TIMP (tissue inhibitor of metalloproteinases), SPARC (secreted protein, acidic and rich in cysteine) and thrombospondin. Changes in neuropilin, midkine and restin levels, which may underpin changes in endothelial morphology, were seen during splitting angiogenesis. Up-regulation of VEGF (vascular endothelial growth factor), Flk-1, angiopoietin-2 and PECAM-1 (platelet/endothelial cell adhesion molecule-1) was seen in both forms of angiogenesis, representing a common angiogenic response of endothelial cells. In conclusion, the present study demonstrates that general angiogenic signals from growth factors can be influenced by the local microenvironment resulting in differing forms of capillary growth to produce a co-ordinated expansion of the vascular bed.

Temporal expression of inflammatory mediators in brain basilar artery vasculitis and cerebrospinal fluid of rabbits with coccidioidal meningitis

Strokes due to transmural vasculitis associated with coccidioidal meningitis result in significant morbidity and mortality. The immunological and inflammatory processes responsible are poorly understood. To determine the inflammatory mediators, i.e. cytokines, chemokines, iNOS, matrix metalloproteinase-9 (MMP-9), that possibly contribute to vasculitis, temporal mRNA expression in brain basilar artery samples and MMP-9 protein in the CSF of male NZW rabbits infected intracisternally with 6.5 x 10(4) arthroconidia of Coccidioides immitis were assessed. Five infected and 3 sham-injected rabbits at each time point were euthanized 4, 9, 14 and 20 days post infection. All infected rabbits had neurological abnormalities and severe vasculitis in the basilar arteries on days 9-20. In basilar arteries of infected animals versus controls, mRNAs encoding for IL-6, iNOS, IFN-gamma, IL-2, MCP-1, IL-1beta, IL-10, TNF-alpha, CCR-1, MMP-9, TGF-beta, as well as MMP-9 protein in CSF, were found to be significantly up-regulated. Thus, this study identified inflammatory mediators associated with CNS vasculitis and meningitis due to C. immitis infection. Assessment of the individual contribution of each mediator to vasculitis may offer novel approaches to the treatment of coccidioidal CNS infection. This study also provides unique methodology for immunology studies in a rabbit model.

Metzincins, including matrix metalloproteinases and meprin, in kidney transplantation

Chronic allograft nephropathy, including chronic rejection, remains one of the major causes of renal allograft failure. Amongst other mediators, metzincins, such as matrix metalloproteinases (MMP), direct extracellular matrix metabolism and cell proliferation. Thus, we hypothesized, that these proteolytic enzymes are differentially regulated in chronic renal transplant rejection in rats and in human renal allograft nephropathy. Our studies demonstrated on the experimental level and in humans an overall up-regulation of MMP, tissue inhibitors of metalloproteinases (TIMP) and related enzymes as a result of rejection processes. Thus, metzincins may represent novel markers and therapeutic targets with respect to renal allograft rejection.

Platelet activation induces metalloproteinase-dependent GP VI cleavage to down-regulate platelet reactivity to collagen

Glycoprotein (GP) VI, the primary collagen receptor on platelets, has been shown to have variable expression, possibly as a consequence of immune modulation. The present study was designed to determine the mechanism by which GP VI clearance occurs. We found that direct activation of GP VI both by a GP VI-specific antibody and by GP VI ligands (collagen and convulxin) reduced binding of biotinylated convulxin to the stimulated platelets. Analysis of immunoblots of platelets and supernatants showed that the stimulated platelets contained less GP VI, while the soluble fraction contained a 57-kDa cleavage product. Stimulation of platelets with PAR-1 agonists (TRAP peptide and thrombin) also caused GP VI cleavage, although the amount of GP VI loss was less than that observed with direct GP VI ligands. The metalloproteinase (MMP) inhibitors GM6001 and TAPI prevented both the clearance of GP VI from the platelet surface and the appearance of the soluble cleavage product. Induction of GP VI cleavage caused specific down-regulation of collagen-induced platelet aggregation, providing a mechanism for the modulation of platelet responsiveness to this important platelet agonist.

Airway remodelling in children with cystic fibrosis

BACKGROUND: The relationship between airway structural changes and inflammation is unclear in early cystic fibrosis (CF) lung disease. A study was undertaken to determine changes in airway remodelling in children with CF compared with appropriate disease and healthy controls. METHODS: Bronchoalveolar lavage and endobronchial biopsy were performed in a cross-sectional study of 43 children with CF (aged 0.3-16.8 years), 7 children with primary ciliary dyskinesia (PCD), 26 with chronic respiratory symptoms (CRS) investigated for recurrent infection and/or cough and 7 control children with no lower airway symptoms. Inflammatory cells, cytokines, proteases and matrix constituents were measured in bronchoalveolar lavage fluid (BALF). Reticular basement membrane (RBM) thickness was measured on biopsy specimens using light microscopy. RESULTS: Increased concentrations of elastin, glycosaminoglycans and collagen were found in BALF from children with CF compared with the CRS group and controls, each correlating positively with age, neutrophil count and proteases (elastase activity and matrix metalloproteinase-9 (MMP-9) concentration). There were significant negative correlations between certain of these and pulmonary function (forced expiratory volume in 1 s) in the CF group (elastin: r = -0.45, p<0.05; MMP-9:TIMP-1 ratio: r = -0.47, p<0.05). Median RBM thickness was greater in the CF group than in the controls (5.9 microm vs 4.0 microm, p<0.01) and correlated positively with levels of transforming growth factor-beta(1) (TGF-beta(1); r = 0.53, p = 0.01), although not with other inflammatory markers or pulmonary function. CONCLUSIONS: This study provides evidence for two forms of airway remodelling in children with CF: (1) matrix breakdown, related to inflammation, proteolysis and impaired pulmonary function, and (2) RBM thickening, related to TGF-beta(1) concentration but independent of other markers of inflammation.

Essential role of choline for pneumococcal virulence in an experimental model of meningitis

Objectives: The goal of the present study was to elucidate the contribution of the newly recognized virulence factor choline to the pathogenesis of Streptococcus pneumoniae in an animal model of meningitis. Results: The choline containing strain D39Cho(-) and its isogenic choline-free derivative D39Cho(-)licA64 -each expressing the capsule polysaccharide 2 - were introduced intracisternally at an inoculum size of 10(3) CFU into 11 days old Wistar rats. During the first 8 h post infection both strains multiplied and stimulated a similar immune response that involved expression of high levels of proinflammatory cytokines, the matrix metalloproteinase 9 (MMP-9), IL-10, and the influx of white blood cells into the CSF. Virtually identical immune response was also elicited by intracisternal inoculation of 10(7) CFU equivalents of either choline-containing or choline-free cell walls. At sampling times past 8 h strain D39Cho(-) continued to replicate accompanied by an intense inflammatory response and strong granulocytic pleiocytosis. Animals infected with D39Cho(-) died within 20 h and histopathology revealed brain damage in the cerebral cortex and hippocampus. In contrast, the initial immune response generated by the choline-free strain D39Cho(-)licA64 began to decline after the first 8 h accompanied by elimination of the bacteria from the CSF in parallel with a strong WBC response peaking at 8 h after infection. All animals survived and there was no evidence for brain damage. Conclusion: Choline in the cell wall is essential for pneumococci to remain highly virulent and survive within the host and establish pneumococcal meningitis.

Matrix metalloproteinases contribute to brain damage in experimental pneumococcal meningitis

The present study was performed to evaluate the role of matrix metalloproteinases (MMP) in the pathogenesis of the inflammatory reaction and the development of neuronal injury in a rat model of bacterial meningitis. mRNA encoding specific MMPs (MMP-3, MMP-7, MMP-8, and MMP-9) and the inflammatory cytokine tumor necrosis factor alpha (TNF-alpha) were significantly (P < 0.04) upregulated, compared to the beta-actin housekeeping gene, in cortical homogenates at 20 h after infection. In parallel, concentrations of MMP-9 and TNF-alpha in cerebrospinal fluid (CSF) were significantly increased in rats with bacterial meningitis compared to uninfected animals (P = 0.002) and showed a close correlation (r = 0.76; P < 0. 001). Treatment with a hydroxamic acid-type MMP inhibitor (GM6001; 65 mg/kg intraperitoneally every 12 h) beginning at the time of infection significantly lowered the MMP-9 (P < 0.02) and TNF-alpha (P < 0.02) levels in CSF. Histopathology at 25.5 +/- 5.7 h after infection showed neuronal injury (median [range], 3.5% [0 to 17.5%] of the cortex), which was significantly (P < 0.01) reduced to 0% (0 to 10.8%) by GM6001. This is the first report to demonstrate that MMPs contribute to the development of neuronal injury in bacterial meningitis and that inhibition of MMPs may be an effective approach to prevent brain damage as a consequence of the disease.

No role of matrixmetalloproteinase-3 genetic promoter polymorphism 1171 as a risk factor for cirrhosis in alcoholic liver disease

BACKGROUND: As only a minority of alcoholics develop cirrhosis, polymorphic genes, whose products are involved in fibrosis development were suggested to confer individual susceptibility. We tested whether a functional promoter polymorphism in the gene encoding matrix metalloproteinase-3 (MMP-3; 1171 5A/6A) was associated liver cirrhosis in alcoholics. METHODS: Independent cohorts from the UK and Germany were studied. (i) UK cohort: 320 alcoholic cirrhotics and 183 heavy drinkers without liver damage and (ii) German cohort: 149 alcoholic cirrhotics, 220 alcoholic cirrhotics who underwent liver transplantation and 151 alcoholics without liver disease. Patients were genotyped for MMP-3 variants by restriction fragment length polymorphism, single strand confirmation polymorphism, and direct sequencing. In addition, MMP-3 transcript levels were correlated with MMP-3 genotype in normal liver tissues. RESULTS: Matrix metalloproteinase-3 genotype and allele distribution in all 1023 alcoholic patients were in Hardy-Weinberg equilibrium. No significant differences in MMP-3 genotype and allele frequencies were observed either between alcoholics with or without cirrhosis. There were no differences in hepatic mRNA transcription levels according to MMP-3 genotype. CONCLUSIONS: Matrix metalloproteinase-3 1171 promoter polymorphism plays no role in the genetic predisposition for liver cirrhosis in alcoholics. Stringently designed candidate gene association studies are required to exclude chance observations.

Proteomic identification of in vivo substrates for matrix metalloproteinases 2 and 9 reveals a mechanism for resolution of inflammation.

Clearance of allergic inflammatory cells from the lung through matrix metalloproteinases (MMPs) is necessary to prevent lethal asphyxiation, but mechanistic insight into this essential homeostatic process is lacking. In this study, we have used a proteomics approach to determine how MMPs promote egression of lung inflammatory cells through the airway. MMP2- and MMP9-dependent cleavage of individual Th2 chemokines modulated their chemotactic activity; however, the net effect of complementing bronchoalveolar lavage fluid of allergen-challenged MMP2(-/-)/MMP9(-/-) mice with active MMP2 and MMP9 was to markedly enhance its overall chemotactic activity. In the bronchoalveolar fluid of MMP2(-/-)/MMP9(-/-) allergic mice, we identified several chemotactic molecules that possessed putative MMP2 and MMP9 cleavage sites and were present as higher molecular mass species. In vitro cleavage assays and mass spectroscopy confirmed that three of the identified proteins, Ym1, S100A8, and S100A9, were substrates of MMP2, MMP9, or both. Function-blocking Abs to S100 proteins significantly altered allergic inflammatory cell migration into the alveolar space. Thus, an important effect of MMPs is to differentially modify chemotactic bioactivity through proteolytic processing of proteins present in the airway. These findings provide a molecular mechanism to explain the enhanced clearance of lung inflammatory cells through the airway and reveal a novel approach to target new therapies for asthma.

Functional studies of insulin-like growth factor binding protein 2 pathway in glioma progression

Comparison of gene expressing profiles between gliomas with different grades revealed frequent overexpression of insulin-like growth factor binding protein 2 (IGFBP2) in glioblastomas (GBM), in which uncontrolled cell proliferation, angiogenesis, invasion and anti-apoptosis are hallmarks. Using the glia-specific gene transfer transgenic mouse and the stable LN229(BP2) GBM cell lines, we found that IGFBP2 by itself cannot transform cells in vitro and in vivo. IGFBP2 had growth inhibitory effects on mouse primary neural progenitors, but overexpression of IGFBP2 had no effect on GBM cells. ^ Although IGFBP2 does not initiate gliomagenesis, using tissue array technology, we observed strong correlation between IGFBP2 overexpression and VEGF up-regulation in human diffuse gliomas. Furthermore, overexpression of IGFBP2 in GBM cells not only enhanced VEGF expression but also increased the malignant potential of U87 MG cells in our angiogenesis xenograft animal model. ^ In parallel to these studies, using established stable SNB19 GBM cells that overexpress IGFBP2, we found that IGFBP2 significantly increased invasion by induction of matrix metalloproteinase-2 (MMP-2) as well as other invasion related genes, providing evidence that IGFBP2 contributes to glioma progression in part by enhancing MMP-2 gene transcription and in turn tumor cell invasion. ^ Finally, we found that primary filial cells infected with an anti-sense IGFBP2 construct have markedly increased sensitivity to γ irradiation and reduced Akt activation. On the other hand, SNB19(BP2) stable lines have consistently increased levels of Akt and NFkB activation, suggesting that one possible mechanism for anti-apoptosic function of IGFBP2 is through the activation of Akt and NFkB. Beside this, what is especially interesting is the finding that Akt protein was cleaved and inactivated during apoptosis by caspases, and IGFBP2 can prevent Akt cleavage, revealing another possible mechanism through it IGFBP2 exhibit strong antiapoptotic effects. Our data showed that IGFBP2 is a specific substrate for caspase-3, raising the possibility that IGFBP2 may inhibit apoptosis by a suicide mechanism. ^ In summary, using cellular, genomics, and molecular approaches, this thesis documented the potential roles of IGFBP2 in glioma progression. Our findings shed light on an important biological aspect of glioma progression and may provide new insights useful for the design of novel mechanism-based therapies for GBM. ^

The role and mechanism of supressor of cytokine signaling 1 in brain metastasis

Brain metastasis, which occurs in 40%-60% of patients with advanced melanoma, has led directly to death in the majority of cases. Unfortunately, little is known about the biological and molecular basis of melanoma brain metastases. In our previous study, we developed a model to study human melanoma brain metastasis and found that Stat3 activity was increased in human brain metastatic melanoma cells when compared with that in cutaneous melanoma cells. The increased activation of Stat3 is also responsible for affecting melanoma angiogenesis in vivo and melanoma cell invasion in vitro and significantly affecting the expression of bFGF, VEGF, and MMP-2 in vivo and in vitro. Interestingly, a member of a new family of cytokine-inducible inhibitors of signal transduction, termed suppressors of cytokine signaling 1 (SOCS1) was found to negatively regulate the Janus kinase signal transducer and activator of transcription (Jak/STAT) signaling cascade. Here we report that restoration of SOCS1 expression by transfecting of SOCS1-expressing vector effectively inhibited melanoma brain metastasis through inhibiting Stat3 activation and further affecting melanoma angiogenesis and melanoma cell invasion in vitro, and significantly affected the expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinase-2 (MMP-2) in vitro and in vivo. In addition, we used cDNA array to compare mRNA expression in the SOCS1-transfected and vector-transfected cell lines and found some genes are tightly correlated to the restoration of SOCS1. One of them is Caveolin-1 (Cav-1). Cav-1 was reported to function as a tumor suppressor gene by several groups. Finally, the Cav-1 expression is up-regulated in SOCS1-overexpressing cell line. Further study found the regulation of Cav-1 by SOCS1 occurs through inhibiting Stat3 activation. Activated Stat3 binds directly to Cav-1 promoter and the Cav-1 promoter within -575bp is essential for active Stat3 binding. My studies reveal that Stat3 activation and SOCS1 expression play important roles in melanoma metastases. Moreover, the expression between SOCS1, Stat3 and Cav-1 forms a feedback regulation loop. ^

Batimastat, a potent matrix mealloproteinase inhibitor, exhibits an unexpected mode of binding.

Matrix metalloproteinase enzymes have been implicated in degenerative processes like tumor cell invasion, metastasis, and arthritis. Specific metalloproteinase inhibitors have been used to block tumor cell proliferation. We have examined the interaction of batimastat (BB-94) with a metalloproteinase [atrolysin C (Ht-d), EC 3.4.24.42] active site at 2.0-angstroms resolution (R = 16.8%). The title structure exhibits an unexpected binding geometry, with the thiophene ring deeply inserted into the primary specificity site. This unprecedented binding geometry dramatizes the significance of the cavernous primary specificity site, pointing the way for the design of a new generation of potential antitumor drugs.

Régulation de l'expression de PPARγ dans l'arthrose

L’arthrose (OA) est une maladie dégénérative très répondue touchant les articulations. Elle est caractérisée par la destruction progressive du cartilage articulaire, l’inflammation de la membrane synoviale et le remodelage de l’os sous chondral. L’étiologie de cette maladie n’est pas encore bien définie. Plusieurs études ont été menées pour élucider les mécanismes moléculaires et cellulaires impliqués dans le développement de l’OA. Les effets protecteurs du récepteur activé par les proliférateurs de peroxysomes gamma (PPARγ) dans l'OA sont bien documentés. Il a été démontré que PPARγ possède des propriétés anti-inflammatoires et anti-cataboliques. Aussi, plusieurs stimuli ont été impliqués dans la régulation de l’expression de PPARγ dans différents types cellulaires. Cependant, les mécanismes exacts responsables de cette régulation ainsi que le profil de l’expression de ce récepteur au cours de la progression de l’OA ne sont pas bien connus. Dans la première partie de nos travaux, nous avons essayé d’élucider les mécanismes impliqués dans l’altération de l’expression de PPARγ dans cette maladie. Nos résultats ont confirmé l’implication de l’interleukine-1β (IL-1β), une cytokine pro-inflammatoire, dans la réduction de l’expression de PPARγ au niveau des chondrocytes du cartilage articulaire. Cet effet coïncide avec l'induction de l’expression du facteur de transcription à réponse précoce de type 1 (Egr-1). En plus, la diminution de l'expression de PPARγ a été associée au recrutement d'Egr-1 et la réduction concomitante de la liaison de Sp1 au niveau du promoteur de PPARγ. Dans la deuxième partie de nos travaux, nous avons évalué le profil d’expression de ce récepteur dans le cartilage au cours de la progression de cette maladie. Le cochon d’inde avec OA spontanée et le chien avec OA induite par rupture du ligament croisé antérieur (ACLT) deux modèles animaux d’OA ont été utilisés pour suivre l’expression des trois isoformes de PPARs : PPAR alpha (α), PPAR béta (β) et PPAR gamma (γ) ainsi que la prostaglandine D synthase hématopoïétique (H-PGDS) et la prostaglandine D synthase de type lipocaline (L-PGDS) deux enzymes impliquées dans la production de l’agoniste naturel de PPARγ, la 15-Deoxy-delta(12,14)-prostaglandine J(2) (15d-PGJ2). Nos résultats ont démontré des changements dans l’expression de PPARγ et la L-PGDS. En revanche, l’expression de PPARα, PPARβ et H-PGDS est restée stable au fil du temps. La diminution de l’expression de PPARγ dans le cartilage articulaire semble contribuer au développement de l’OA dans les deux modèles animaux. En effet, le traitement des chondrocytes par de siRNA dirigé contre PPARγ a favorisé la production des médiateurs arthrosiques tels que l'oxyde nitrique (NO) et la métalloprotéase matricielle de type 13 (MMP-13), confirmant ainsi le rôle anti-arthrosique de ce récepteur. Contrairement à ce dernier, le niveau d'expression de la L-PGDS a augmenté au cours de la progression de cette maladie. La surexpression de la L-PGDS au niveau des chondrocytes humains a été associée à la diminution de la production de ces médiateurs arthrosiques, suggérant son implication dans un processus de tentative de réparation. En conclusion, l’ensemble de nos résultats suggèrent que la modulation du niveau d’expression de PPARγ, de la L-PGDS et d’Egr-1 au niveau du cartilage articulaire pourrait constituer une voie thérapeutique potentielle dans le traitement de l’OA et probablement d’autres formes d'arthrite.

Equine laminitis: glucose deprivation and MMP activation induce dermo-epidermal separation in vitro

Reasons for performing study: Acute laminitis is characterised by hoof lamellar dermal-epidermal separation at the basement membrane (BM) zone. Hoof lamellar explants cultured in vitro can also be made to separate at the basement membrane zone and investigating how this occurs may give insight into the poorly understood pathophysiology of laminitis. Objectives: To investigate why glucose deprivation and metalloproteinase (MMP) activation in cultured lamellar explants leads to dermo-epidermal separation. Methods: Explants, cultured without glucose or with the MMP activator p-amino-phenol-mercuric acetate (APMA), were subjected to tension and processed for transmission electron microscopy (TEM). Results: Without glucose, or with APMA, explants under tension separated at the dermo-epidermal junction. This in vitro separation occurred via 2 different ultrastructural processes. Lack of glucose reduced hemidesmosomes (HDs) numbers until they disappeared and the basal cell cytoskeleton collapsed. Anchoring filaments (AFs), connecting the basal cell plasmalemma to the BM, were unaffected although they failed under tension. APMA activation of constituent lamellar MMPs did not affect HDs but caused AFs to disappear, also leading to dermo-epidermal separation under tension. Conclusions: Natural laminitis may occur in situations where glucose uptake by lamellar basal cells is compromised (e.g. equine Cushing's disease, obesity, hyperlipaemia, ischaemia and septicaemia) or when lamellar MMPs are activated (alimentary carbohydrate overload). Potential relevance: Therapies designed to facilitate peripheral glucose uptake and inhibit lamellar MMP activation may prevent or ameliorate laminitis.

Functional analysis of the a-defensin disulfide array in mouse cryptdin-4

The alpha-defensin antimicrobial peptide family is defined by a unique tridisulfide array. To test whether this invariant structural feature determines alpha-defensin bactericidal activity, mouse cryptdin-4 (Crp4) tertiary structure was disrupted by pairs of site-directed Ala for Cys substitutions. In a series of Crp4 disulfide variants whose cysteine connectivities were confirmed using NMR spectroscopy and mass spectrometry, mutagenesis did not induce loss of function. To the contrary, the in vitro bactericidal activities of several Crp4 disulfide variants were equivalent to or greater than those of native Crp4. Mouse Paneth cell alpha-defensins require the proteolytic activation of precursors by matrix metalloproteinase-7 (MMP-7), prompting an analysis of the relative sensitivities of native and mutant Crp4 and proCrp4 molecules to degradation by MMP-7. Although native Crp4 and the alpha-defensin moiety of proCrp4 resisted proteolysis completely, all disulfide variants were degraded extensively by MMP-7. Crp4 bactericidal activity was eliminated by MMP-7 cleavage. Thus, rather than determining alpha-defensin bactericidal activity, the Crp4 disulfide arrangement confers essential protection from degradation by this critical activating proteinase.