proMMP-2 Levels Research Articles

Slit2 exerts anti-inflammatory actions in human placenta and is decreased with maternal obesity.

Obese pregnancies are characterised by increased inflammation. Members of the Slit/Roundabout (Robo) family are key regulators of the inflammatory response. The aim of this study was to determine the effect of (i) pre-existing maternal obesity on Slit-Robo expression in human placenta and (ii) Slit2 knockdown by siRNA in primary trophoblast cells on markers of inflammation. The expression of Slit-Robo protiens was assessed in human placenta from lean (n=15) and obese (n=16) patients by qRT-PCR and Western blotting. Primary trophoblast cells were used to determine the effect of pro-inflammatory mediators on Slit2 expression, and the effect of Slit2 siRNA on pro-inflammatory mediators. While there was no change in Slit3, Robo1 or Robo4 expression, Slit2 expression was significantly lower in obese placenta compared to lean placenta. Human primary trophoblast cells treated with pro-inflammatory mediators IL-1β, TNF-α and LPS significantly decreased Slit2 expression. Slit2 silencing by siRNA augmented IL-6 expression and secretion in cells stimulated with TNF-α, LPS and TNF-α; IL-8 gene expression and/or release in cells stimulated with IL-1β and LPS; TNF-α gene expression and secretion in cells stimulated with LPS; and MMP-9 gene expression and pro MMP-9 levels in cells stimulated with TNF-α. The anti-inflammatory effects of Slit2 in human placenta is a novel finding, and suggests that inflammatory mediators, which are increased with obesity, downregulates Slit2 to enhance placental inflammation. Given the central role of pro-inflammatory cytokines in placental nutrient transport, our findings suggest Slit2 may play a role in fetal growth and development.

Do salivary and serum collagenases have a role in an association between obstructive sleep apnea syndrome and periodontal disease? A preliminary case–control study

ObjectivesDespite increasing evidence for an association of obstructive sleep apnea syndrome (OSAS) and periodontal disease, the pathophysiological linking mechanisms remain unclear. This study aims to evaluate the salivary and serum matrix metalloproteinase-2, -8, -9 (MMP-2, -8, -9), tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), myeloperoxidase (MPO), neutrophil elastase (NE), neutrophil gelatinase-associated lipocalin (NGAL), as well as degree of activation of MMP-2, -9 of patients with and without OSAS. DesignA total of 50 individuals were included in the study. There were 13, 17 and 20 individuals, respectively in the control (non-OSAS) group, mild-to-moderate OSAS and severe OSAS groups. Saliva, serum samples and clinical periodontal parameters were collected. Biofluid samples were analysed by immunofluorometric assay (IFMA), enzyme-linked immunosorbent assay (ELISA), western immunoblotting and gelatine zymography. Statistical analyses were performed using D’Agostino–Pearson omnibus normality test, Kruskal–Wallis test and Spearman rho rank correlation analysis. ResultsThere were no statistically significant differences in clinical periodontal parameters between the study groups. Salivary NE and proMMP-2 levels were significantly lower in the OSAS groups than the control group (p<0.05). Serum proMMP-9 concentration and the degree of MMP-9 activation in saliva were significantly lower in the severe OSAS group than the control group (p<0.05). There were significant correlations between salivary and serum proMMP-9 and -2 concentrations (p<0.05). Serum proMMP-2, NE and salivary proMMP-9 and -2 negatively correlated with indicators of OSAS severity (p<0.05). ConclusionsThe present findings do not support a pathophysiological link between the severity of OSAS and clinical periodontal status via neutrophil enzymes or MMPs.

Activation of adenosine A2B receptor impairs properties of trophoblast cells and involves mitogen-activated protein (MAP) kinase signaling

IntroductionShallow trophoblast invasion of the maternal spiral arteries contributes to impaired placental perfusion and is hypothesized to be involved in the pathophysiology of preeclampsia. Hypoxia is a potent stimulus for the release of adenosine. MethodsWe investigated the effects of hypoxia and A2B adenosine receptor signaling on migration, invasion, proteolytic activity of matrix metalloproteinase (MMP)-2, expression of MMP-2 and vascular endothelial growth factor (VEGF) mRNA, and production of human chorionic gonadotropin (hCG) in trophoblast cells (HTR-8/SVneo, BeWo). ResultsThe adenosine A2B receptor agonist 5-N-ethylcarboxamidoadenosine (NECA) reduced trophoblast (HTR-8/SVneo and BeWo) migration at 2%, 8% and 21% O2 compared to untreated control cells. A2B adenosine receptor stimulation decreased phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and stress-activated protein kinase/Jun-amino-terminal kinase (SAPK/JNK) at all three O2 concentrations. ProMMP-2 activity, MMP-2 mRNA levels and hCG levels were markedly decreased after A2B adenosine receptor activation in trophoblast cells. Adenosine receptor A2B stimulation decreased VEGF expression at 2% and 8% O2 but led to increased levels at 21% O2. ConclusionsThese data indicate A2B receptor activation blunts trophoblast migration possibly as a result of reduced activation of the MAPK signaling pathway and lower proMMP-2 levels. These data suggest a role for adenosine receptor A2B in placental development and possibly in the pathophysiology of preeclampsia.

Low intravitreal angiopoietin‐2 and VEGF levels in vitrectomized diabetic patients with simvastatin treatment

To investigate the intravitreal levels of potent vasoactive, angiogenic and extracellular matrix remodelling factors in the diabetic patients with simvastatin treatment. This is an institutional, prospective, observational case-control study. Type-1 and type-2 diabetic patients on lipophilic simvastatin (N = 14) compared with patients without statin medication (N = 50). Vitreous samples were subjected to protein measurements of angiopoietin (Ang)-1 and Ang-2, erythropoietin (EPO), transforming growth factor (TGF)-β1, vascular endothelial growth factor (VEGF) by ELISA and matrix metalloproteinase (MMP)-2 and MMP-9 by gelatin zymography. Intravitreal levels of Ang-2 (p = 0.029), VEGF (p = 0.001) and proMMP-9 (p = 0.015) were lower in simvastatin-treated than in non-statin-treated controls. In diabetics with macular oedema (DME), intravitreal Ang-2 (p = 0.006) and VEGF (p = 0.002) levels were lower in simvastatin-treated patients compared with non-statin-treated controls. In those patients with proliferative diabetic retinopathy (PDR), intravitreal Ang-2 (p = 0.002), TGF-β1 (p = 0.037), VEGF (p = 0.001) and pro- and totalMMP-9 (p = 0.004 and p = 0.007) levels were lower when receiving simvastatin medication. In diabetic patients with DME or PDR, the intravitreal levels of permeability and proangiogenic factors Ang-2 and VEGF were lower in simvastatin-treated than in those without statin medication. Moreover, the levels of MMP-9 and TGF-β1, factors involved in the breakdown of basement membrane and fibroproliferation, were lower in patients with PDR having simvastatin medication. When acetylsalicylic acid was combined with simvastatin treatment, the intraocular levels of Ang-2 and VEGF were significantly lower than in diabetics treated with simvastatin alone. These data provide a novel insight into the potential protective mechanisms underlying simvastatin medication in patients with diabetic retinopathy complications.

Rho-Kinase Inhibition Attenuates Airway Inflammation In An Experimental Asthma Model In Sickle Cell Mice

IntroductionAsthma in sickle cell disease is known to be associated with increased morbidity and an elevated rate of sickle cell complications, such as acute chest syndrome, stroke, vaso-occlusive episodes and early mortality. Experimental asthma increases eosinophil and collagen deposition in the lungs of SCD mice, induces profound increases in pulmonary inflammation, shifts in TH1 and TH2 cytokine production, and airway resistance. A small GTPase, Rho, and its target molecule, Rho-kinase (ROCK), play an important role in cell functions, including contractility, chemotaxis, adhesion, and migration, where the Rho/ROCK-mediated pathway facilitates infiltration of inflammatory cells both in vitro and in vivo. This study was designed to determine whether the Rho/Rho-kinase pathways are involved in eosinophil recruitment and inflammation. To investigate the role of Rho/ROCK in the pathogenesis of SCD asthma, we investigated the effect of fasudil, a specific inhibitor of Rho-kinase, on acute allergic inflammation in SCD mice model. MethodsBerkeley SCD mouse bone marrow was transplanted into lethally-irradiated C57BL/6 animals to generate age- and gender-matched genetically identical cohorts of SCD mice. Female SCD mice were sensitized and challenged with ovalbumin (OVA). OVA-challenge mice were treated intraperitoneally with fasudil (10 mg/kg), 1 h before each OVA-challenge. At 48 h after OVA challenged total cell counts, differential cell counts, cytokines, and chemokine levels were measured by ELISA in bronchoalveolar lavage fluid (BALF), and the mRNA expressions of ROCK-1, ROCK-2, IL-6, MMP-8, MMP-9, MMP-12, TIMP-1, TIMP-2 were measured in lungs by RT-PCR. ResultsIntranasal challenge of OVA in SCD mice induced airway inflammation after 48h, characterized by increases in total leukocytes numbers (WBC) that were almost exclusively accounted for by eosinophils, when compared with non-sensitized mice (WBC: 12.8 ±1.5 vs 5.5 ± 0.68; eosinophils: 6.67 ± 1.2 vs 0.01 ± 0.01, p<0.05, respectively). When fasudil was administer to OVA-challenged SC mice, increased numbers of total cells and eosinophils in the BALF were significantly attenuated (WBC: 6.8 ± 0.6; eosinophils: 1.14 ± 0.33, p<0.05). The numbers of neutrophils were also reduced in animals treated with fasudil (0.84 ± 0.13 vs 2.32 ± 0.34, p<0.05). However, mononuclear cells were not affected by fasudil treatment. ROCK-1 and ROCK-2 mRNA expressions in mice of the asthmatic group (1.2 ± 0.18 and 0.84 ± 0.10, respectively) were similar than those of the non-sensitized group (1.1±0.05 and 0.97±0.04, respectively). When fasudil was administered, the expressions of ROCK-1 and ROCK-2 mRNA did not alter in the asthmatic group (0.96 ± 0.12 and 0.98 ± 0.15, respectively). MMP-2, MMP-8, MMP-9, MMP-12 and TIMP-1 gene expressions were not significantly different between SCD asthmatic mice and SCD non-sensitized mice. Increased IL-6 mRNA levels were detected in the lungs at 48h after OVA challenge (0.83 ± 0.09 vs 0.4 ± 0.1, p<0.05). Lung mRNA expressions of MMPs, TIMP-1 and cytokines were not significantly reduced in SCD mice treated with fasudil. OVA challenge in sensitized SCD mice induced IL-4, IL-5, IL-6, eotaxin, RANTES, MCP-1, TIMP-1 release into BALF (OVA-sensitized: 11.1 ± 3.2, 31.8 ± 7.7, 20.4 ± 4.0, 8.8 ± 2.2, 11.7 ± 1.8, 46.6 ± 7.1, 2239 ± 424.7, respectively vs OVA-nonsensitized:1.1 ± 0.6; 1.4 ± 0.6, 4.1 ± 2.0, 2.3 ± 1.5; 6.9 ± 1.9; 4.6 ± 0.97; 294.3 ± 45.6 pg/ml, p<0.05). Fasudil reduced levels of IL-5, L-6, RANTES, MCP-1, proMMP-9 and TIMP-1 in BALF of SCD mice after OVA challenges (-46.4 fold, -62.5 fold, -41.4 fold, -48.6 fold, -53.6 fold, p<0.05, respectively). ConclusionAdministration of fasudil inhibits eosinophil recruitment in response to allergen challenge. The effects of this agent may be mediated by suppressing the production of chemokine and cytokines related to the pathophysiology of bronchial asthma, such as IL-5, RANTES and MCP-1. In addition, the Rho-kinase inhibitor reduced levels of proteins involved in the degraded the extracelular matrix, such as MMP-9, which directly or indirectly promoted the progression of pulmonary inflammatory responses. Our findings provide evidence that inhibition of the Rho/Rho-kinase pathway may be beneficial for pulmonary complications in sickle cell disease. Financial SupportFAPESP/CNPq/INCTS Disclosures:No relevant conflicts of interest to declare.

The Effect of Celecoxib Administration on the Healing and Neovascularization of Colonic Anastomosis in Rats

ABSTRACTBackground: The aim of this experimental study was to investigate whether the perioperative administration of the selective cyclooxygenase-2 inhibitor celecoxib affects the angiogenetic process and the healing of colonic anastomoses. Methods: Seventy-two male Wistar rats underwent colonic resection and anastomosis. Celecoxib (10 mg/kg/day—celecoxib group) or placebo (control group) was administered perioperatively. Rats of both groups were sacrificed on either the third or the seventh postoperative day and bursting pressures of the anastomoses were measured. Gelatine-degrading matrix metalloproteinases (MMPs) were identified with gelatine zymography, and proMMP-2 and vascular endothelial growth factor (VEGF) levels from both anastomotic site and tissue adjacent to the anastomosis were evaluated. Histologic evaluation of microvessels was performed by immunohistochemistry using an anti-CD34 monoclonal antibody. Results: Celecoxib did not significantly decrease anastomotic bursting pressures. Gelatin zymography revealed the presence of MMP-2, proMMP-2, and proMMP-9. MMP concentration was higher at the anastomotic tissue as compared with tissue distant to the anastomosis. Celecoxib resulted in a significant reduction in proMMP-2 levels at the anastomosis at both third and seventh postoperative day. VEGF levels from the anastomotic tissue were also found lower in the celecoxib group. Histological examination showed a celecoxib-induced reduction of newly formed CD34-stained vessels. Conclusions: Although the perioperative administration of celecoxib resulted in suppression of angiogenesis in the newly formed anastomoses, bursting pressures remained unaffected and subsequently safety was not compromised.

Heat stress effects on the cumulus cells surrounding the bovine oocyte during maturation: altered matrix metallopeptidase 9 and progesterone production

When the effects of heat stress are detrimental during maturation, cumulus cells are intimately associated with the oocyte. To determine the extent to which heat stress affects these cells, in this study, transcriptome profiles of the cumulus that surrounded control and heat-stressed oocytes (41 °C during the first 12 h only and then shifted back to 38.5 °C) during in vitro maturation (IVM) were compared using Affymetrix bovine microarrays. The comparison of cumulus-derived profiles revealed a number of transcripts whose levels were increased (n=11) or decreased (n=13) ≥ twofold after heat stress exposure (P<0.01), sufficient to reduce the development of blastocysts by 46.4%. In a separate study, quantitative PCR (qPCR) was used to confirm heat-induced differences in the relative abundances of the transcripts of five different genes (caveolin 1, matrix metallopeptidase 9, FSH receptor, Indian hedgehog homolog, and inducible nitric oxide synthase). Heat stress exposure resulted in >1.7-fold decrease in the protein levels of latent matrix metallopeptidase 9 (proMMP9). Heat-induced reductions in transcript levels were noted at 6 h IVM with reductions in proMMP9 protein levels at 18 h IVM (P=0.0002). Independent of temperature, proMMP9 levels at 24 h IVM were positively correlated with the development rate of blastocysts (R²=0.36; P=0.002). The production of progesterone increased during maturation; heat-induced increases were evident by 12 h IVM (P=0.002). Both MMP9 and progesterone are associated with the developmental competence of the oocyte; thus, it seems plausible for some of the negative consequences of heat stress on the cumulus-oocyte complex to be mediated through heat-induced perturbations occurring in the surrounding cumulus.

Abstract 4931: Inhibitor of DNA binding 4 (Id4) suppresses MMP2 mediated invasion of glioblastoma-derived cells by direct inactivation of twist-1 function.

Abstract Invasion of normal tissues by high grade glioma is a key aspect of increased morbidity and mortality associated with these tumors. Malignant glioma comprise over 70% of primary brain tumors with a median survival ranging from 12 to 15 months for patients with glioblastoma multiforme (GBM), the highest grade of glial tumors. Inhibitor of DNA binding (Id) genes are highly expressed in development and tumorigenesis and some members (Id1, Id2 and Id3) have been linked to increased tumor invasion. In sharp contrast to these members of the Id gene family, we found that Id4 expression in GBM-derived cells decreased invasion in in vitro assays. Results of gain-of-function and loss-of-function experiments indicated that Id4 expression decreased GBM invasion and suppressed a gene, matrix metallo-protease 2 (MMP-2), which encodes a proteolytic enzyme key for the destruction of glial tumor extracellular matrix. Western blot and zymography analysis demonstrated that conditioned media from Id4 expressing cells had low levels of pro-MMP2 and cleaved MMP2. An antibody that is known to inactivate MMP-2 was used to demonstrate that MMP2 accumulated in media conditioned by Id4 deficient cells was sufficient to rescue the invasive activity lost in cells expressing high levels of Id4. These findings confirmed that MMP2 suppression by Id4 is responsible for the decrease in invasion observed in Id4 expressing glioma cells. Twist-1 is a well described mediator of invasion. Co-IP and co-localization studies demonstrated an interaction suggesting a direct inhibition of Twist-1 by Id4. Other data indicate that Id4 also blocks Twist-1 induced transcription of MMP2. Importantly, Twist-1 overexpression can rescue the invasion defect induced by Id4 expression, providing further support that the Id4/Twist-1 interaction mediates the function of Id4 in suppressing GBM invasion. Analysis of The Cancer Genome Atlas (TCGA) data indicated a significantly better survival and prognosis for GBM patients with high Id4 expression (Z&amp;gt;1) suggesting that Id4 could have an important role in the survival of GBM patients. Our data provide strong evidence demonstrating that Id4 suppresses GBM invasion by inhibiting Twist-1 induced expression of MMP2. To our knowledge, this is the first report of an Id protein acting as an inhibitor of invasion rather than mediating the pro-invasive functions of the other Id-gene family members. Citation Format: Gilbert J. Rahme, Mark A. Israel. Inhibitor of DNA binding 4 (Id4) suppresses MMP2 mediated invasion of glioblastoma-derived cells by direct inactivation of twist-1 function. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4931. doi:10.1158/1538-7445.AM2013-4931

Taurine inhibits increased MMP-2 expression in a model of oxidative stress induced by glutathione depletion in rabbit heart

Matrix metalloproteinase enzymes (MMPs) activated by oxidative stress are involved in the pathogenesis of cardiovascular diseases. Glutathione (GSH) plays an important protective role against oxidatively induced damage in mammalian tissues. We investigated the possible role of gelatinases and the effect of the semiessential amino acid 2-aminoethanesulfonic acid (taurine) in oxidatively induced damage by GSH depletion in rabbit cardiac tissues. Rabbits were treated with buthionine sulfoximine (BSO), an effective GSH-depleting compound. BSO treatment significantly reduced GSH and increased MDA (malondialdehyde) levels. BSO treatment caused significant increase in proMMP-2 levels. MMP-9 (pro and active) expressions were not found in either treated- or untreated heart tissues. TIMP-1(endogenous inhibitor of MMP-9) and MT-MMP1 (endogenous activator of MMP-2) were not affected by BSO. Immunoscoring showed that MMP-2 expression significantly increased in hearts from BSO treated group but MMP-9 antibody did not show any significant positive immunostaining from all groups. Type I procollagen and total collagen did not significantly alter in heart tissues from all treatment groups. Taurine restored the increased MDA and the diminished GSH levels by BSO treatment. Pro MMP-2 expression was prevented by taurine. These results suggest that MMP-2 is a major gelanitase in rabbit hearts under oxidative stress and pharmacological inhibition of MMP-2 activation by taurine could represent a useful strategy for the prevention and/or treatment of different cardiovascular disorders.

Effects of Plasmodium falciparum-infected erythrocytes on matrix metalloproteinase-9 regulation in human microvascular endothelial cells

ObjectiveTo investigate the regulation of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in human microvascular endothelium (HMEC-1) exposed to erythrocytes infected by different strains of Plasmodium falciparum (P. falciparum). MethodsHMEC-1 cells were co-incubated for 72 h with erythrocytes infected by late stage trophozoite of D10 (chloroquine-sensitive) or W2 (chloroquine-resistant) P. falciparum strains. Cell supernatants were then collected and the levels of pro- or active gelatinases MMP-9 and MMP-2 were evaluated by gelatin zymography and densitometry. The release of pro-MMP-9, MMP-3, MMP-1 and TIMP-1 proteins was analyzed by western blotting and densitometry. ResultsInfected erythrocytes induced de novo proMMP-9 and MMP-9 release. Neither basal levels of proMMP-2 were altered, nor active MMP-2 was found. MMP-3 and MMP-1 secretion was significantly enhanced, whereas basal TIMP-1 was unaffected. All effects were similar for both strains. ConclusionsP. falciparum parasites, either chloroquine-sensitive or -resistant, induce the release of active MMP-9 protein from human microvascular endothelium, by impairing balances between proMMP-9 and its inhibitor, and by enhancing the levels of its activators. This work provides new evidence on MMP involvement in malaria, pointing at MMP-9 as a possible target in adjuvant therapy.

Ang-2 upregulation correlates with increased levels of MMP-9, VEGF, EPO and TGFβ1 in diabetic eyes undergoing vitrectomy

Angiogenesis in diabetic retinopathy (DR) is a multifactorial process regulated by hypoxia-induced growth factors and inflammatory cytokines. In addition to the angiogenic switch, the proteolytic processing and altered synthesis of the extracellular matrix are critical steps in this disease. This study was performed to evaluate the levels of matrix metalloproteinase-2 and matrix metalloproteinase-9 (MMP-2 and MMP-9), angiopoietin-1 and angiopoietin-2 (Ang-1 and Ang-2), vascular endothelial growth factor (VEGF), erythropoietin (EPO) and transforming growth factor-β1 (totalTGFβ1) in the vitreous of diabetic eyes undergoing vitrectomy compared with control eyes operated because of macular hole or pucker. Prospective consecutive controlled observational study performed in the unit of vitreoretinal surgery in Finland during the years 2006-2008. Vitreous samples were collected before the start of the conventional 3-ppp vitrectomy. Vitreous MMP-2 and MMP-9, Ang-1 and Ang-2, VEGF, EPO and TGFβ1 concentrations were measured from 69 patients with Type 1 or 2 diabetes and 40 controls. Comparison of eyes with DR with controls revealed that the mean vitreous concentrations of proMMP-2 (p = 0.0015), totalMMP-2 (p = 0.0011), proMMP-9 (p = 0.00001), totalMMP-9 (p < 0.00001), Ang-2 (p < 0.00001), VEGF (p < 0.00001), EPO (p < 0.00001) and totalTGFβ1 (p = 0.000026) were significantly higher in the former group. A multivariate logistic regression analysis suggested intravitreal Ang-2 concentration being the key marker of PDR (p = 0.00025) (OR = 1507.9). The main new finding is that the intravitreal concentrations of Ang-2 correlated significantly with MMP-9, VEGF, EPO and TGFβ1 levels in diabetic eyes undergoing vitrectomy. Thus, these factors could promote retinal angiogenesis synergistically.

Detection and analysis of MMP-2 and MMP-9 in seminal plasma

BackgroundThe aim of this study was to explore the association between semen parameters and matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) expression in human seminal plasma samples. MethodsConventional semen analysis was performed on semen samples (n=64). MMP-2 and MMP-9 expression in seminal plasma was determined using gelatin zymography. ResultsCompared to semen samples with a normal sperm count, semen samples with a low sperm count (≤ 19×106/ml) showed reduced sperm viability, a reduced percentage of Grade A sperm, a reduced percentage of morphologically-normal sperm, and lower proMMP-9 and MMP-9 but higher proMMP-2 and MMP-2 levels (P<0.05). There were correlations between MMP-2 and MMP-9 expression and the percentage of Grade A sperm and morphologically-normal sperm (P<0.05). ConclusionsMMP-2 and MMP-9 are both present in human semen, and low-concentration semen samples have increased MMP-2 and decreased MMP-9. We also observed relationships between MMP-2, MMP-9 and seminal parameters.

The Role of Extracellular Signal-Related Kinase During Abdominal Aortic Aneurysm Formation

It is hypothesized that activation of extracellular signal-related kinase (ERK) is critical in activating matrix metalloproteinases (MMPs) during abdominal aortic aneurysm (AAA) formation. C57BL/6 male mice underwent either elastase or heat-inactivated elastase aortic perfusion (n = 9 per group). Mouse aortic smooth muscle cells were transfected with ERK-1 and 2 siRNA along with or without elastase treatment. Mouse and human aortic tissue were analyzed by Western blots, zymograms, and immunohistochemistry, and statistical analysis was done using Graphpad and Image J softwares. Western blot and immunohistochemistry documented increased phospho-mitogen-activated protein kinase kinase-1/2 (pMEK-1/2; 153%, p = 0.270 by Western) and pERK (171%, p = 0.004 by Western blot) in the elastase perfused aortas. Male ERK-1(-/-) mice underwent elastase perfusion, and aortic diameter was determined at day 14. ERK-1(-/-) mice failed to develop AAA, and histologic analysis depicted intact collagen and elastin fibers in the aortas. Zymography of aortas of elastase-treated ERK-1(-/-) mice showed lower levels of proMMP2 (p < 0.005) and active MMP2 (p < 0.0001), as well as proMMP9 (p = 0.037) compared with C57BL/6 mice. siRNA transfection of ERK-1 and -2 significantly reduced formation of pro- and active MMP2 (p < 0.01 for both isoforms) in aortic smooth muscle cells treated with elastase in vitro. Human AAA tissue had significantly elevated levels of pMEK-1/2 (150%, p = 0.014) and pERK (159%, p = 0.013) compared with control tissues. The MAPK (mitogen-activated protein kinase)/ERK pathway is an important modulator of MMPs during AAA formation. Targeting the ERK pathway by reagents that inhibit either the expression or phosphorylation of ERK isoforms could be a potential therapy to prevent AAA formation.

Sperm quality following 4 days of cooled storage: impact on fertility

degrade protein components of extracellular matrix and basement membranes, and are thought to be involved in pre-fertilization events. The purpose of this study was to compare the levels of MMP-2 and MMP-9 in different fractions of SP, and to evaluate the correlation of the MMP levels with fertility. During the breeding season, semenwas collected from 43 stallions (20 Standardbreds, 17 Finnhorses, 4 warmblood riding horses, 2 ponies; ages 4-23 years) residing at nine different stud farms in Finland. The breeding history and current use of the stallions was variable, with the stallions serving 2 to 150 (median 14) mares during the season. Semen jets were collected as 1-4 fractions using an open-ended artificial vagina (AV) or a modified closed AV. After semen collection, the gel was removed and the volume of each fraction was measured. A sample representing the whole ejaculate was formed by combining 10% of the volume of each fraction. The samples were centrifuged at 4000 x g (15 min), and the supernatant was filtered using 0.45 mm filters. The SP samples were stored frozen in 1-mL aliquots in -75 C until analyzed. The levels of the pro-form of MMP-2, active MMP-2 and total MMP-9 were evaluated using gelatin zymography and densitometry. Fertility data were provided by the stud farms and the Finnish trotting and breeding association (Suomen Hippos). The first cycle pregnancy rate was calculated from all of the reported matings in the year in question for the stallions included in the study, and all mares that foaled or aborted were considered pregnant. Pro-MMP-2, active MMP-2 and total MMP-9 were detected in stallion seminal plasma, with the highest levels found in the sperm-rich fraction. In all fractions, the levels of proMMP-2 were significantly higher than the levels of active MMP-2 and total MMP-9, which did not differ. The levels of the different types of MMP were not correlated with the first cycle pregnancy rate. Pro-MMP-2, active MMP-2 and MMP-9 are present in all fractions of the stallion's ejaculate, with the highest levels in the sperm-rich fraction. Despite a possible role in the fertilization process, the levels of MMP-2 and MMP-9 were not correlated with fertility in this study on a limited number of animals.

Canine cerebral leishmaniasis: Potential role of matrix metalloproteinase-2 in the development of neurological disease

Matrix metalloproteinases (MMPs) are a group of calcium- and zinc-dependent endopeptidases that are involved in maintaining the extracellular matrix. MMP-2 and MMP-9 are thought to be related to the disruption of the blood–brain-barrier (BBB) by their ability to cleave type IV collagen, the main component of the basal membrane. To establish the presence of MMP-2 and MMP-9 in the pathogenesis of canine cerebral leishmaniasis, we examined the levels of these metalloproteinases in the cerebrospinal fluid (CSF) and serum of dogs with visceral leishmaniasis and neurological symptoms (n=16) and in the CSF and serum of uninfected healthy dogs (n=10) using zymography. In the CSF of dogs with cerebral leishmaniasis there was a massive presence of active MMP-2, whereas only the levels of both proMMP-2 and proMMP-9 were elevated in the serum. Although the detected MMP activity in the CSF might merely be related to CNS inflammation, these enzymes may also play a collaborative role in the disease progression. Both MMP-2 and MMP-9 are known to target critical constituents of the BBB, and once activated, they may promote cerebral barrier breakdown, allowing the entrance of inflammatory cells and proteins within the nervous system milieu.

Constitutively active lysophosphatidic acid receptor-1 enhances the induction of matrix metalloproteinase-2

Lysophosphatidic acid (LPA) is a simple phospholipid which interacts with at least six G protein-coupled transmembrane LPA receptors (LPA1–LPA6). In rat neuroblastoma B103 cells, we have recently reported that each LPA receptor indicates the different cellular functions, including cell motility, invasion and tumorigenicity. Especially, mutated and constitutively active LPA1 enhanced these cellular effects in B103 cells. In the present study, to better understand a role of mutated LPA1 underlying progression of cancer cells, we measured the expression and activity levels of matrix metalloproteinases (MMPs) in constitutively active mutant Lpar1-expressing B103 cells (lpa1Δ-1), compared with each wild-type LPA receptor-expressing cells. LPA receptor-unexpressing cells were also used as control. In quantitative real time RT-PCR analysis, the expressions of Mmp-9 were detected at the same levels in all cells. By contrast, Mmp-2 expressions of lpa1Δ-1 were significantly higher than those of other cells. In gelatin zymography, proMmp-9 was observed at the same levels in all cells. Interestingly, markedly high levels of proMmp-2 and Mmp-2 were detected in lpa1Δ-1 cells, whereas no activation was in other cells. The increased expression and activity of Mmp-2 in lpa1Δ-1 cells were suppressed by the pretreatment with a Gq protein inhibitor. These results suggest that mutated LPA1 may involve in the enhancement of Mmp-2 expression and activation in rat neuroblastoma cells.

Increased JNK in Males Compared with Females in a Rodent Model of Abdominal Aortic Aneurysm

In humans, there is a 4:1 male:female ratio in the incidence of abdominal aortic aneurysms (AAAs). c-Jun-N-terminal kinase (JNK) is an important upstream regulator of several enzymes involved in AAA formation, including the matrix metalloproteinases (MMPs). The purpose of this study was to determine if there is a gender difference between males and females in JNK during AAA formation. Male and female C57/B6 mice underwent aortic perfusion with elastase or heat inactivated elastase with aortas harvested at d 3 and 14 for phenotype determination, RT-PCR, Western blot, and zymography. Additionally, in vitro experiments using siRNA were conducted to define JNK regulation of matrix metalloproteinases (MMPs). A t-test was used to compare between groups. Males formed larger AAAs at d 14 compared with females (P < 0.001), with significantly higher levels of JNK1 protein, proMMP9, proMMP2, and active MMP2. At d 3, males had more JNK1 mRNA, protein, and MMP activity. Knockdown of JNK 1 or 2 in vitro decreased MMP activity, while knockdown of JNK 1 and 2 together blocked all MMP activity. Alterations in JNK between genders is partially responsible for the differential rates of experimental AAA formation, likely through differential regulation of MMPs.

Lamellar leukocyte infiltration and involvement of IL-6 during oligofructose-induced equine laminitis development

Laminitis is known to involve deregulation of proteases and destruction of the lamellar basement membrane with the host inflammatory response also playing a role. Leukocyte infiltration has been well characterized in the black walnut model of laminitis induction, but not in carbohydrate induced models. Increased gene expression of multiple cytokines, including IL-6, has also been implicated in laminitis development. Using real time PCR, immunohistochemistry and zymography methods, we characterize leukocyte infiltration and IL-6 gene expression in oligofructose (OF) induced laminitis. As well, we use two in vitro models to investigate a role for IL-6 in protease regulation. Laminitis was induced in normal standardbred horses ( n = 5) by alimentary OF dosing and lamellar biopsies were obtained throughout the 48 h experimental period. Lamellar explants and keratinocytes were also isolated from clinically normal horses for in vitro experiments. We found infiltration of calprotectin-positive leukocytes (monocytes and neutrophils) at 18–24 h post oligofructose dosing, while IL-6 gene expression was increased as early as 12 h post dosing. Additionally, while we found that IL-6 did not cause significant BM damage in vitro, it did result in increased secreted proMMP-9 levels from lamellar explants. Thus, we find that leukocyte infiltration does occur during oligofructose-induced laminitis development, however, IL-6 gene expression in the lamellae may precede leukocyte infiltration. Additionally, we show IL-6 plays a role in increasing the level of proMMP-9 in vivo in a manner that does not involve keratinocytes.

Azithromycin decreases MMP-9 expression in the airways of lung transplant recipients

The neomacrolide antibiotic azithromycin is known to have an anti-inflammatory effect and is increasingly being used in the treatment of chronic inflammatory pulmonary diseases. We investigated whether azithromycin influenced matrix remodeling.Matrix metalloproteinase (MMP)-9 protein levels were measured by ELISA in bronchoalveolar lavaga fluid in 10 stable patients and in 10 lung transplant patients suffering from nCLAD/NRAD. MMP-9 was measured via ELISA before and after 3 to 6months of azithromycin therapy. We further elaborated on the role of MMP-9 by performing gelatin-zymography and gelatinolytic activity assays. Differential and total cell counts on BAL were performed in all cases.The nCLAD/NRAD patients showed higher airway neutrophilia (p<0.0001), ELISA MMP-9 (p<0.0001), zymography proMMP-9 (p<0.0001), activated MMP-9 (p=0.0003) and gelatinolytic activity (p=0.0002) compared to the control group. Airway neutrophilia in the nCLAD/NRAD group significantly decreased after 3–6months of treatment with azithromycin (p=0.0020). This was associated with a decrease in ELISA MMP-9 levels (p=0.0059), in activated MMP-9 shown on zymography (p=0.016) and in gelatinolytic activity (p=0.031). Remarkably, proMMP-9 levels were not altered by azithromycin. Although azithromycin significantly reduced ELISA MMP-9 levels and gelatinolytic activity in transplant patients, these levels remained higher compared to control patients (p=0.0011 and p=0.043).Neutrophil counts, activated MMP-9 and gelatinolytic activity levels in nCLAD/NRAD decreased after azithromycin treatment, but some remained elevated compared to control patients. This illustrates that treatment with azithromycin did not completely restore chronic inflammation in the airways and suggested that preventive therapy may yield added value to curative therapy.

Disturbed Matrix Metalloproteinase Activity of Bruch's Membrane in Age-Related Macular Degeneration

To evaluate the potential role of the matrix metalloproteinase (MMP) system of Bruch's membrane in the pathology of age-related macular degeneration. Free and bound pools of gelatinase activity in Bruch's membrane-choroid preparations were isolated by phosphate-buffered saline (PBS) and sodium dodecyl sulfate (SDS) extraction, respectively. Individual MMP species were separated by gelatin-substrate zymography and the levels were quantified by densitometric techniques. Altogether, 13 control (age range, 71-99 years) and 6 AMD (age range, 71-95 years) donor eyes were used. All the gelatinase components normally present in control samples were also present in AMD tissue without any significant differences in their molecular masses. Total levels (bound plus free) of active MMP2 and -9 were significantly reduced in AMD donors (P < 0.05). The decrease in active MMP2 may be attributable to a similar reduction in the level of free pro-MMP2, the precursor to the active form. Reduction in active MMP9 occurred despite a nearly 3.5-fold increase in free pro-MMP9. The high-molecular-mass gelatinases denoted by HMW1 and -2 and comprising homo- and heteropolymers of pro-MMP2 and -9 were also raised in AMD (P < 0.05). The sequestration of free pro-MMP2 and -9 by these high-molecular-mass complexes may further contribute to reduced rates of activation of MMPs. The reduction in the levels of activated MMP2 and -9 may be responsible for impaired matrix degradation of Bruch's membrane, leading to the pathology associated with AMD. The degradation pathway is therefore a viable therapeutic target for future intervention.