Matrix metalloproteinase-9 of tubular and macrophage origin contributes to the pathogenesis of renal fibrosis via macrophage recruitment through osteopontin cleavage

A pro-fibrotic role of matrix metalloproteinase-9 (MMP-9) in tubular cell epithelial-mesenchymal transition (EMT) is well established in renal fibrosis; however studies from our group and others have demonstrated some previously unrecognized complexity of MMP-9 that has been overlooked in renal fibrosis. Therefore, the aim of this study was to determine the expression pattern, origin and the exact mechanism underlying the contribution of MMP-9 to unilateral ureteral obstruction (UUO), a well-established model of renal fibrosis via MMP-9 inhibition. Renal MMP-9 expression in BALB/c mice with UUO was examined on day 1, 3, 5, 7, 9, 11 and 14. To inhibit MMP-9 activity, MMP-2/9 inhibitor or MMP-9-neutralizing antibody was administered daily for 4 consecutive days from day 0-3, 6-9 or 10-13 and tissues harvested at day 14. In UUO, there was a bi-phasic early- and late-stage upregulation of MMP-9 activity. Interestingly, tubular epithelial cells (TECs) were the predominant source of MMP-9 during early stage, whereas TECs, macrophages and myofibroblasts produced MMP-9 during late-stage UUO. Early- and late-stage inhibition of MMP-9 in UUO mice significantly reduced tubular cell EMT and renal fibrosis. Moreover, MMP-9 inhibition caused a significant reduction in MMP-9-cleaved osteopontin and macrophage infiltration in UUO kidney. Our in vitro study showed MMP-9-cleaved osteopontin enhanced macrophage transwell migration and MMP-9 of both primary TEC and macrophage induced tubular cell EMT. In summary, our result suggests that MMP-9 of both TEC and macrophage origin may directly or indirectly contribute to the pathogenesis of renal fibrosis via osteopontin cleavage, which, in turn further recruit macrophage and induce tubular cell EMT. Our study also highlights the time dependency of its expression and the potential of stage-specific inhibition strategy against renal fibrosis.

Children with stable asthma have reduced airway matrix metalloproteinase-9 and matrix metalloproteinase-9/tissue inhibitor of metalloproteinase-1 ratio

Background Childhood asthma is characterized by inflammation of the airways. Structural changes of the airway wall may also be seen in some children early in the course of the disease. Matrix metalloproteinases (MMPs) are key mediators in the metabolism of the extracellular matrix (ECM). Objective To investigate the balance of MMP-8, MMP-9 and tissue inhibitor of metalloproteinases (TIMP)-1 in the airways of children with asthma. Methods One hundred and twenty-four children undergoing elective surgical procedures also underwent non-bronchoscopic bronchoalveolar lavage (BAL). MMP-8, MMP-9 and TIMP-1 were measured by ELISA. Results There was a significant reduction in MMP-9 in atopic asthmatic children (n=31) compared with normal children (n=30) [median difference: 0.57 ng/mL (95% confidence interval: 0.18–1.1 ng/mL)]. The ratio of MMP-9 to TIMP-1 was also reduced in asthmatic children. Levels of all three proteins were significantly correlated to each other and to the relative proportions of particular inflammatory cells in BAL fluid (BALF). Both MMP-8 and MMP-9 were moderately strongly correlated to the percentage neutrophil count (r=0.40 and 0.47, respectively, P

Salbutamol up-regulates matrix metalloproteinase-9 in the alveolar space in the acute respiratory distress syndrome.

Objectives: Acute respiratory distress syndrome (ARDS) is characterized by alveolar-capillary barrier damage. Matrix metalloproteinases (MMPs) are implicated in the pathogenesis of ARDS. In the Beta Agonists in Acute Lung Injury Trial, intravenous salbutamol reduced extravascular lung water (EVLW) in patients with ARDS at day 4 but not inflammatory cytokines or neutrophil recruitment. We hypothesized that salbutamol reduces MMP activity in ARDS.

Methods: MMP-1/-2/-3/-7/-8/-9/-12/-13 was measured in supernatants of distal lung epithelial cells, type II alveolar cells, and bronchoalveolar lavage (BAL) fluid from patients in the Beta Agonists in Acute Lung Injury study by multiplex bead array and tissue inhibitors of metalloproteinases (TIMPs)-1/-2 by enzyme-linked immunosorbent assay. MMP-9 protein and activity levels were further measured by gelatin zymography and fluorokine assay.

Measurements and Main Results: BAL fluid MMP-1/-2/-3 declined by day 4, whereas total MMP-9 tended to increase. Unexpectedly, salbutamol augmented MMP-9 activity. Salbutamol induced 33.7- and 13.2-fold upregulation in total and lipocalin-associated MMP-9, respectively at day 4, compared with 2.0- and 1.3-fold increase in the placebo group, p < 0.03. Salbutamol did not affect BAL fluid TIMP-1/-2. Net active MMP-9 was higher in the salbutamol group (4222 pg/mL, interquartile range: 513-7551) at day 4 compared with placebo (151 pg/mL, 124-2108), p = 0.012. Subjects with an increase in BAL fluid MMP-9 during the 4-day period had lower EVLW measurements than those in whom MMP-9 fell (10 vs. 17 mL/kg, p = 0.004): change in lung water correlated inversely with change in MMP-9, r = -.54, p = 0.0296. Salbutamol up-regulated MMP-9 and down-regulated TIMP-1/-2 secretion in vitro by distal lung epithelial cells. Inhibition of MMP-9 activity in cultures of type II alveolar epithelial cells reduced wound healing.

Conclusions: Salbutamol specifically up-regulates MMP-9 in vitro and in vivo in patients with ARDS. Up-regulated MMP-9 is associated with a reduction in EVLW. MMP-9 activity is required for alveolar epithelial wound healing in vitro. Data suggest MMP-9 may have a previously unrecognized beneficial role in reducing pulmonary edema in ARDS by improving alveolar epithelial healing.

Characterization of the zebrafish matrix metalloproteinase 9 gene and its developmental expression pattern

Members of the matrix metalloproteinase (MMP) family are important for the remodeling of the extracellular matrix in a number of biological processes including a variety of immune responses. Two members of the family, MMP2 and MMP9, are highly expressed in specific myeloid cell populations in which they play a role in the innate immune response. To further expand the repertoire of molecular reagents available to study zebrafish myeloid cell development, the matrix metalloproteinase 9 (mmp9) gene from this organism has been identified and characterized. The encoded protein is 680 amino acids with high homology to known MMP9 proteins, particularly those of other teleost fish. Maternal transcripts of mmp9 are deposited in the oocyte and dispersed throughout the early embryo. These are replaced by specific zygotic transcripts in the notochord from 12 h post fertilization (hpf) and also transiently in the anterior mesoderm from 14 to 16 h post fertilization. From 24 h post fertilization, mmp9 expression was detected in a population of circulating white blood cells that are distinct from macrophages, and which migrate to the site of trauma, and so likely represent zebrafish heterophils. In the adult, mmp9 expression was most prominent in the splenic cords, a site occupied by mature myeloid cells in other species. These results suggest that mmp9 will serve as a useful marker of mature myeloid cells in the zebrafish.

Matrix Metalloproteinase-9 reduces islet amyloid formation by degrading islet amyloid polypeptide

Deposition of islet amyloid polypeptide (IAPP) as amyloid is a pathological hallmark of the islet in type 2 diabetes, which is toxic to β-cells. We previously showed that the enzyme neprilysin reduces islet amyloid deposition and thereby reduces β-cell apoptosis, by inhibiting fibril formation. Two other enzymes, matrix metalloproteinase (MMP)-2 and MMP-9, are extracellular gelatinases capable of degrading another amyloidogenic peptide, Aβ, the constituent of amyloid deposits in Alzheimer disease. We therefore investigated whether MMP-2 and MMP-9 play a role in reducing islet amyloid deposition. MMP-2 and MMP-9 mRNA were present in mouse islets but only MMP-9 activity was detectable. In an islet culture model where human IAPP (hIAPP) transgenic mouse islets develop amyloid but nontransgenic islets do not, a broad spectrum MMP inhibitor (GM6001) and an MMP-2/9 inhibitor increased amyloid formation and the resultant β-cell apoptosis. In contrast, a specific MMP-2 inhibitor had no effect on either amyloid deposition or β-cell apoptosis. Mass spectrometry demonstrated that MMP-9 degraded amyloidogenic hIAPP but not nonamyloidogenic mouse IAPP. Thus, MMP-9 constitutes an endogenous islet protease that limits islet amyloid deposition and its toxic effects via degradation of hIAPP. Because islet MMP-9 mRNA levels are decreased in type 2 diabetic subjects, islet MMP-9 activity may also be decreased in human type 2 diabetes, thereby contributing to increased islet amyloid deposition and β-cell loss. Approaches to increase islet MMP-9 activity could reduce or prevent amyloid deposition and its toxic effects in type 2 diabetes.

Matrix metalloproteinase-9 expression in pterygium

OBJETIVO: Avaliar a expressão da metaloprotease de matriz (MMP)-9 nos pterígios. MÉTODOS: Foi realizado na Faculdade de Medicina de Botucatu estudo prospectivo, aleatório, com o intuito de avaliar a expressão da metaloprotease de matriz na cápsula de Tenon normal e de pterígios primários e recidivados, usando o método da imuno-histoquímica e o sistema computadorizado de análise de imagem. Os resultados foram avaliados estatisticamente. RESULTADOS: A expressão da metaloprotease de matriz foi semelhante na cápsula de Tenon normal e nos pterígios primários e recidivados. CONCLUSÃO: A expressão da metaloprotease de matriz na cápsula de Tenon normal e nos pterígios primários ou recidivados é semelhante, o que nos leva a concluir que esta metaloprotease de matriz não esteja envolvida na gênese ou na recidiva do pterígio.

Exenatide and sitagliptin decrease interleukin 1β, matrix metalloproteinase 9, and nitric oxide synthase 2 gene expression but does not reduce alveolar bone loss in rats with periodontitis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Genetic Variants in Matrix Metalloproteinase-9 Gene Modify Metalloproteinase-9 Levels in Black Subjects

Altered matrix metalloproteinases (MMPs) levels are involved in cardiovascular diseases and increased MMP-9 levels enhance the cardiovascular risk in apparently healthy subjects. We investigated the effects of MMP-9 gene polymorphisms and haplotypes on the circulating MMP-9 levels in healthy black subjects and the effects of an MMP-2 polymorphism on the plasma MMP-2 concentrations. We studied 190 healthy subjects, nonsmokers, self-reported as blacks (18-63 years). Genotypes for the MMP-2 C-1306T polymorphism and the MMP-9 C-1562T, 90(CA)(14-24) and Q279R polymorphisms (rs243865, rs3918242, rs2234681, and rs17576, respectively) were determined by TaqMan (R) Allele Discrimination assay and real-time polymerase chain reaction or restriction fragment length polymorphism. Alleles for the 90(CA)(14-24) polymorphism were grouped as low (L) when there were < 21 and high (H) when there were >= 21 CA repeats. The plasma levels of MMP-2 and MMP-9 were determined by gelatin zymography. The software PHASE 2.1 was used to estimate the haplotypes frequencies. Although we found no effects of the MMP-9 C-1562T or the Q279R polymorphisms on MMP-9 levels, higher MMP-9 levels were associated with the HH genotype for the -90(CA)(14-24) polymorphism compared with the HL or LL genotypes. Lower MMP-9 levels were found in carriers of the CRL haplotype (combining the C, R, and L alleles for the MMP-9 polymorphisms) compared with the CRH haplotype. Consistent with this finding, the CRL haplotype was more commonly found in subjects with low MMP-9 levels. The MMP-2 C-1306T polymorphism had no effects on the plasma MMP-2 levels. Our results show that MMP-9 genetic variations modify MMP-9 levels in black subjects and may offer biochemical evidence implicating MMP-9 in the pathogenesis of cardiovascular diseases in blacks.

Specific matrix metalloproteinase 9 (MMP-9) haplotype affect the circulating MMP-9 levels in women with migraine

We investigated whether three relevant polymorphisms (C-1562T, microsatellite - 90(CA)(14-24), and Q279R) in the MMP-9 gene, or MMP-9 haplotypes, are associated with migraine and affect MMP-9 and tissue inhibitor of MMPs (TIMP)-1 levels in patients with migraine. We studied 102 healthy women (controls) and 187 women with migraine (141 without aura - MWA, and 46 with aura - MA). Patients with MWA had higher plasma MMP-9 concentrations than patients with MA. Patients with MA had the highest TIMP-1 and lowest MMP-9/TIMP-1 ratios. The MMP-9 "C L Q" haplotype was associated with higher plasma MMP-9 concentrations in migraine patients. (C) 2012 Elsevier B.V. All rights reserved.

Imbalanced matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 activities in patients with thromboangiitis obliterans

The pathogenic mechanisms of thromboangiitis obliterans (TAO) are not entirely known and the imbalance of matrix metalloproteinases (MMPs) plays a role in vascular diseases. We evaluated the MMP-2 and MMP-9 circulating levels and their endogenous tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) in TAO patients with clinical manifestations. The study included 20 TAO patients (n = 10 female, n = 10 male) aged 38-59 years under clinical follow-up. The patients were classified into two groups: (1) TAO former smokers (n = 11) and (2) TAO active smokers (n = 9); the control group included normal volunteer non-smokers (n = 10) and active smokers without peripheral artery disease (n = 10). Patient plasma samples were used to analyze MMP-2 and MMP-9 levels using zymography, and TIMP-1 and TIMP-2 concentrations were determined by enzyme-linked immunosorbent assays. The analysis of MMP-2/TIMP-2 and MMP-9/TIMP-1 ratios (which were used as indices of net MMP-2 and MMP-9 activity, respectively) showed significantly higher MMP-9/TIMP-1 ratios in TAO patients (p < 0.05). We found no significant differences in MMP-2/TIMP-2 ratios (p > 0.05). We found higher MMP-9 levels and decreased levels of TIMP-1 in the TAO groups (active smokers and former smokers), especially in active smokers compared with the other groups (all p < 0.05). MMP-2 and TIMP-2 were not significantly different in patients with TAO as compared to the control group (p > 0.05). In conclusion, our results showed increased MMP-9 and reduced TIMP-1 activity in TAO patients, especially in active smokers compared with non-TAO patients. These data suggest that smoke compounds could activate MMP-9 production or inhibit TIMP-1 activity.

Matrix metalloproteinase-9 polymorphisms affect plasma MMP-9 levels and antihypertensive therapy responsiveness in hypertensive disorders of pregnancy

Abnormal matrix metalloproteinase (MMP)-9 levels may have a role in hypertensive disorders of pregnancy. We examined whether MMP-9 genetic polymorphisms (g.-1562C>T and g.-90(CA)(13-25)) modify plasma MMP-9 and tissue inhibitor of metalloproteinase (TIMP)-1 levels and the responses to antihypertensive therapy in 214 patients with preeclampsia (PE), 185 patients with gestational hypertension (GH) and a control group of 214 healthy pregnant (HP). Alleles for the g.-90(CA)(13-25) polymorphism were grouped L (low) (<21 CA repeats) or H (high) (>= 21 CA repeats). Plasma MMP-9 and TIMP-1 concentrations were measured by enzyme-linked immunosorbent assay. Plasma MMP-9 concentrations were not affected by genotypes or haplotypes in HP and PE groups, except for the g.-90(CA)(13-25) polymorphism: GH patients with the LH genotype for this polymorphism have higher MMP-9 levels than those with other genotypes. The T allele for the g.-1562C>T polymorphism and the H4 haplotype (combining T and H alleles) are associated with GH and lack of responsiveness to antihypertensive therapy in GH. The H2 haplotype (combining C and H alleles) was associated with lack of responsiveness to antihypertensive therapy in PE, but not in GH. In conclusion, our results show that MMP-9 genetic variants are associated with GH and suggest that MMP-9 haplotypes affect the responsiveness to antihypertensive therapy in hypertensive disorders of pregnancy. The Pharmacogenomics Journal (2012) 12, 489-498; doi: 10.1038/tpj.2011.31; published online 19 July 2011

Matrix metalloproteinase-9 genetic variations affect MMP-9 levels in obese children

Objective: Matrix metalloproteinase-9 (MMP-9) is involved in the atherosclerotic process and functional polymorphisms in the MMP-9 gene affect MMP-9 expression/activity, and are associated with cardiovascular diseases. However, no study has tested the hypothesis that functional MMP-9 polymorphisms could affect MMP-9 levels in obese children. We investigated whether three MMP-9 gene polymorphisms (C-1562T (rs3918242), 90(CA)((14-24)) (rs2234681) and Q279R (rs17576)), or haplotypes, affect MMP-9 levels in obese children. Methods: We studied 175 healthy control children and 127 obese children. Plasma MMP-9, tissue inhibitor of MMPs (TIMP)-1 and adiponectin concentrations were measured using enzyme-linked immunosorbent assay. Results: We found similar MMP-9 genotypes, allelic and haplotypes distributions in the two study groups (P > 0.05). However, we found lower plasma MMP-9 concentrations in obese subjects carrying the CC or the QQ genotypes for the C-1562T and the Q279R polymorphisms, respectively, in obese children compared with children with the other genotypes, or with non-obese children with the same genotypes (all P < 0.05). Moreover, we found lower MMP-9 levels and lower MMP-9/TIMP-1 ratios (which reflect net MMP-9 activity) in obese children carrying the H2 haplotype (which combines the C, H and Q alleles for the three polymorphisms, respectively) when compared with obese children carrying the other haplotypes, or with non-obese children carrying the same haplotype (P < 0.05). Conclusions: Our findings show that MMP-9 genotypes and haplotypes affect MMP-9 levels in obese children and adolescents, and suggest that genetic factors may modify relevant pathogenetic mechanisms involved in the development of cardiovascular complications associated with obesity in childhood. International Journal of Obesity (2012) 36, 69-75; doi:10.1038/ijo.2011.169; published online 16 August 2011

Regulation der Phorbolester-induzierten Matrix-Metalloproteinase-9-Expression in humanen Brustkrebszelllinien durch Stickstoffmonoxid

Das Hauptziel dieser Arbeit war die Identifizierung der Regulationsebenen auf denen die TPA-induzierte Matrix-Metalloproteinase-9 (MMP-9) durch das nitrose Gas Stickstoffmonoxid (NO) in MCF-7-Zellen verändert wird. Dabei konnte sowohl mit Hilfe der Zymographie als auch mit einem MMP-9-Aktivitäts-ELISA gezeigt werden, dass die extrazellulären MMP-9-Spiegel durch die Behandlung der Zellen mit NO reduziert werden. Gleichzeitig zeigte sich auch eine durch NO bedingte Abnahme der intrazellulären MMP-9-Spiegel, wie mit Hilfe von Western-Blot-Analyse nachgewiesen werden konnte. Experimente mit dem Proteasominhibitor Lactacystin und dem Proteinsynthesehemmstoff Cycloheximid ließen darüber hinaus eine NO-bedingte Veränderung der MMP-9-Proteinstabilität ausschließen. Im Gegensatz dazu konnte mittels der metabolischen Markierung mit radioaktiv markiertem Methionin und Cystein gezeigt werden, dass die Proteinneusynthese der MMP-9 durch eine Behandlung der Zellen mit NO stark beeinträchtigt wird. In Übereinstimmung mit diesen Daten finden sich reduzierte MMP-9-mRNA-Spiegel auch in der polysomalen Zellfraktion von MCF-7-Zellen. Wie mit Hilfe des Transkriptionshemmstoffes Actinomycin D und durch Reportergenstudien mit hybriden MMP-9-Promotorkonstrukten gezeigt werden konnte, ist die NO-induzierte Reduktion der MMP-9-mRNA-Spiegel nicht auf eine Verringerung der MMP-9-mRNA-Stabilität zurückzuführen. Reportergenstudien mit einem 670bp langen Promotorfragment des 5’flankierenden Bereichs des humanen MMP-9-Gens zeigten jedoch auf, dass der hemmende Effekt des NOs zum Teil auf eine NO-vermittelte Abnahme der TPA-induzierten MMP-9-Promotoraktivität zurückgeführt werden kann. Demzufolge wurde in den nachfolgenden Experimenten nach den für die MMP-9-Expression notwendigen und von NO modulierten Transkriptionsfaktoren in MCF-7-Zellen gesucht. Anhand von Western-Blot-Analysen und Gelshiftanalysen konnte gezeigt werden, dass die Aktivität des Transkriptionsfaktors AP-1 in MCF-7-Zellen durch NO gehemmt wird, während weder die Expressionspiegel noch die Bindungsaffinität der Transkriptionsfaktoren NFκB und Sp1 durch die NO-Behandlung verändert sind. Weiterhin konnte unter Verwendung von pharmakologischen Inhibitoren der MAPK-Signalwege mit Hilfe der Western-Blot-Analyse nachgewiesen werden, dass MAPK-vermittelte Signalwege zwar für die Induktion der MMP-9-Expression essenziell sind, diese jedoch nicht von NO beeinflusst sind. Im Unterschied hierzu konnte mit Hilfe eines PKC-Aktivitätsassays gezeigt werden, dass die Gesamtaktivität von PKCs nach Behandlung von MCF-7-Zellen mit NO signifikant gehemmt ist. Zusammenfassend zeigen diese Untersuchungen, dass die NO-vermittelte Hemmung der TPA-induzierten MMP-9-Expression in MCF-7-Zellen im Wesentlichen auf eine NO-abhängige Reduktion der Protein-Kinase-C-Aktivität und einer daraus resultierenden Aktivitätshemmung des Transkriptionsfaktors AP-1 zurückgeführt werden kann.