Acute doxorubicin-induced cardiotoxicity is associated with matrix metalloproteinase-2 alterations in rats

Background: Doxorubicin can cause cardiotoxicity. Matrix metalloproteinases (MMP) are responsible for degrading extracellular matrix components which play a role in ventricular dilation. Increased MMP activity occurs after chronic doxorubicin treatment. In this study we evaluated in vivo and in vitro cardiac function in rats with acute doxorubicin treatment, and examined myocardial MMP and inflammatory activation, and gene expression of proteins involved in myocyte calcium transients. Methods: Wistar rats were injected with doxorubicin (Doxo, 20 mg/kg) or saline (Control). Echocardiogram was performed 48 h after treatment. Myocardial function was assessed in vitro in Langendorff preparation. Results: In left ventricle, doxorubicin impaired fractional shortening (Control 0.59 +/- 0.07; Doxo 0.51 +/- 0.05; p < 0.001), and increased isovolumetric relaxation time (Control 20.3 +/- 4.3; Doxo 24.7 +/- 4.2 ms; p = 0.007) and myocardial passive stiffness. MMP-2 activity, evaluated by zymography, was increased in Doxo (Control 141338 +/- 8924; Doxo 188874 +/- 7652 arbitrary units; p < 0.001). There were no changes in TNF-alpha, INF-gamma, IL-10, and ICAM-1 myocardial levels. Expression of phospholamban, Serca-2a, and ryanodine receptor did not differ between groups. Conclusion: Acute doxorubicin administration induces in vivo left ventricular dysfunction and in vitro increased myocardial passive stiffness in rats. Cardiac dysfunction is related to myocardial MMP-2 activation. Increased inflammatory stimulation or changed expression of the proteins involved in intracellular calcium transients is not involved in acute cardiac dysfunction.

Unravelling the reaction mechanism of matrix metalloproteinase 3 using QM/MM calculations

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Matrix metalloproteinase gene polymorphisms and oral cancer

Since oral squamous cell carcinoma (OSCC) is the most prevalent malignant cancer in the oral cavity, several researches have been performed to study the role of important enzymes in this disease. Among them, the matrix metalloproteinases (MMPs) are highlighted, due to the fact that they are proteinases responsible to degrade many extra-cellular matrix components, making possible the invasion of neoplasic cells. Important tools in cancer prognosis have been utilized aiming to correlate high levels of MMPs and OSCC, such as immunohistochemical, zymographic and mRNA detection methods. However, these techniques are usually applied after cancer detection, characterizing a curative but not a preventive medicine. Trying to make interventions before the development of the disease and making possible the identification of people at high risk and, analysis of modifications in MMP genes has been a chance for modern medicine. Recently, polymorphisms in MMP genes have been related to different neoplasias, including OSCC. Despite investigation is beginning, MMP gene polymorphisms seems to have a promising future in oral cancer research and some of the present results have shown that there are MMP polymorphisms related to an increased risk for developing oral cancer. Key words:Oral cancer, polymorphism, matrix metalloproteinase.

Expression of soluble and functional full-length human matrix metalloproteinase-2 in Escherichia coli

Characterization of the matrix metalloproteinase-2 (MMP-2) substrates and understanding of its function remain difficult because up to date preparations containing minor amounts of other eukaryotic proteins that are co-purified with MMP-2 are still used. In this work, the expression of a soluble and functional full-length recombinant human MMP-2 (rhMMP-2) in the cytoplasm of Escherichia coli is reported, and the purification of this metalloproteinase is described. Culture of this bacterium at 18 degrees C culminated in maintenance of the soluble and functional rhMMP-2 in the soluble fraction of the E. coli lysate and its purification by affinity with gelatin-sepharose yielded approximately 0.12 mg/L of medium. Western Blotting and zymographic analysis revealed that the most abundant form was the 72-kDa MMP-2, but some gelatinolytic bands corresponding to proteins with lower molecular weight were also detected. The obtained rhMMP-2 was demonstrated to be functional in a gelatinolytic fluorimetric assay, suggesting that the purified rhMMP-2 was correctly folded. The method described here involves fewer steps, is less expensive, and is less prone to contamination with other proteinases and MMP inhibitors as compared to expression of rhMMP-2 in eukaryotic tissue culture. This protocol will facilitate the use of the full-length rhMMP-2 expressed in bacteria and will certainly help researchers to acquire new knowledge about the substrates and biological activities of this important proteinase. (C) 2011 Elsevier B.V. All rights reserved.

Functional Polymorphism Located in MMP-9 Gene Promoter Is Strongly Associated with Obesity

The adipose tissue expansion is accompanied by remodeling of extracellular matrix performed by matrix metalloproteinases (MMPs). Higher plasma and tissue MMP-9 levels are found in obese; therefore, we evaluated if the functional C-1562T polymorphism (rs3918242) located in promoter region of the MMP-9 gene is associated with obesity in women. We studied 112 lean and 114 obese women. Plasma MMP-9 and tissue inhibitor of MMP-9 (TIMP)-1 were measured using enzyme-linked immunosorbent assay. We found different genotype frequencies between lean and obese women (p = 0.008), prevailing T-allele in obese (2.3-fold). However, although obese women present higher levels of plasma MMP-9, lack of modulation by the polymorphism was found (all p > 0.05). Our findings suggest that C-1562T polymorphism may contribute to pathogenetic mechanisms involved in the development of obesity in women.

Comprehensive Evaluation of the Effects of Enalapril on Matrix Metalloproteinases Levels in Hypertension

Angiotensin-converting enzyme inhibitors (ACEi) may downregulate matrix metalloproteinases (MMPs). We examined whether enalapril affects MMP-2, MMP-8, and MMP-9 levels and activity, and their endogenous inhibitors (tissue inhibitors of MMPs, TIMP-1 and TIMP-2) levels in hypertensive patients. Moreover, we assessed the effects of enalaprilat on MMP-9 and TIMP-1 secretion by human endothelial cells (HUVECs). Thirty-eight hypertensive patients received enalapril for 8 weeks and were compared with thirty-eight normotensive controls. Blood samples were collected at baseline and after treatment. Plasma ACE activity was determined by a fluorimetric assay. Plasma MMP-2, MMP-8, MMP-9, TIMP-1, and TIMP-2 were measured by ELISA and gelatin zymography. A fluorogenic peptide cleavage assay was used to measure MMP activity. HUVECs cells were stimulated by phorbol-12-myristate-13-acetate (PMA) and the effects of enalaprilat (10(-10) to 10(-6) M) on MMP-9 and TIMP-1 levels were determined. Enalapril decreased blood pressure and ACE activity in hypertensive patients (P < 0.05), but had no effects on plasma MMP-2, MMP-8, MMP-9, TIMP-1, and TIMP-2 levels, or MMP activity. Enalaprilat had no effects on PMA-induced increases in MMP-9 and TIMP-1 secretion by HUVECs or on MMP activity. We show consistent evidence, both in vivo and in vitro, that enalapril does not affect MMPs and TIMPs levels in hypertensive patients.

Increased expression of MMP-9 and IL-8 are correlated with poor prognosis of Bladder Cancer

Background: Extracellular matrix homeostasis is strictly maintained by a coordinated balance between the expression of metalloproteinases (MMPs) and their inhibitors. The purpose of this study was to investigate whether the expression of MMP-9, MMP-2 and its specific inhibitors, are expressed in a reproducible, specific pattern and if the profiles are related to prognosis in Bladder Cancer (BC). Methods: MMP-9, MMP-2 and its specific inhibitors expression levels were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) in fresh-frozen malignant tissue collected from 40 patients with BC submitted to transurethral resection of bladder. The control group consisted of normal bladder tissue from five patients who had undergone retropubic prostatectomy to treat benign prostatic hyperplasia. Results: MMP-9 was overexpressed in 59.0 % of patients, and MMP-2, TIMP-1, TIMP-2, MMP-14, RECK and IL-8 was underexpressed in most of the patients. Regarding prognostic parameters we observed that high-grade tumors exhibited significantly higher levels of MMP-9 and IL-8 (p = 0.012, p = 0.003). Invasive tumors (pT1-pT2) had higher expression levels of MMP-9 than superficial tumors (pTa) (p = 0.026). The same was noted for IL-8 that was more expressed by invasive tumors (p = 0.015, p = 0.048). Most importantly tumor recurrence was related with higher levels of both MMP-9 (p = 0.003) and IL-8 (p = 0.005). Conclusion: We have demonstrated that the overexpression of MMP-9 and higher expression of IL-8 are related to unfavorable prognostic factors of urothelial bladder cancer and tumor recurrence and may be useful in the follow up of the patients.

Rho-kinase inhibition attenuates airway responsiveness, inflammation, matrix remodeling, and oxidative stress activation induced by chronic inflammation

Possa SS, Charafeddine HT, Righetti RF, da Silva PA, Almeida-Reis R, Saraiva-Romanholo BM, Perini A, Prado CM, Leick-Maldonado EA, Martins MA, Tiberio ID. Rho-kinase inhibition attenuates airway responsiveness, inflammation, matrix remodeling, and oxidative stress activation induced by chronic inflammation. Am J Physiol Lung Cell Mol Physiol 303: L939-L952, 2012. First published September 21, 2012; doi:10.1152/ajplung.00034.2012.-Several studies have demonstrated the importance of Rho-kinase in the modulation of smooth muscle contraction, airway hyperresponsiveness, and inflammation. However, the effects of repeated treatment with a specific inhibitor of this pathway have not been previously investigated. We evaluated the effects of repeated treatment with Y-27632, a highly selective Rho-kinase inhibitor, on airway hyperresponsiveness, oxidative stress activation, extracellular matrix remodeling, eosinophilic inflammation, and cytokine expression in an animal model of chronic airway inflammation. Guinea pigs were subjected to seven ovalbumin or saline exposures. The treatment with Y-27632 (1 mM) started at the fifth inhalation. Seventy-two hours after the seventh inhalation, the animals' pulmonary mechanics were evaluated, and exhaled nitric oxide (E-NO) was collected. The lungs were removed, and histological analysis was performed using morphometry. Treatment with Y-27632 in sensitized animals reduced E-NO concentrations, maximal responses of resistance, elastance of the respiratory system, eosinophil counts, collagen and elastic fiber contents, the numbers of cells positive for IL-2, IL-4, IL-5, IL-13, inducible nitric oxide synthase, matrix metalloproteinase-9, tissue inhibitor of metalloproteinase-1, transforming growth factor-beta, NF-kappa B, IFN-gamma, and 8-iso-prostaglandin F2 alpha contents compared with the untreated group (P < 0.05). We observed positive correlations among the functional responses and inflammation, remodeling, and oxidative stress pathway activation markers evaluated. In conclusion, Rho-kinase pathway activation contributes to the potentiation of the hyperresponsiveness, inflammation, the extracellular matrix remodeling process, and oxidative stress activation. These results suggest that Rho-kinase inhibitors represent potential pharmacological tools for the control of asthma.

Epistasis among eNOS, MMP-9 and VEGF maternal genotypes in hypertensive disorders of pregnancy

Polymorphisms of the endothelial nitric oxide synthase (eNOS), matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor (VEGF) genes were shown to be associated with hypertensive disorders of pregnancy. However, epistasis is suggested to be an important component of the genetic susceptibility to preeclampsia (PE). The aim of this study was to characterize the interactions among these genes in PE and gestational hypertension (GH). Seven clinically relevant polymorphisms of eNOS (T-786C, rs2070744, a variable number of tandem repeats in intron 4 and Glu298Asp, rs1799983), MMP-9 (C-1562T, rs3918242 and -90(CA)(13-25), rs2234681) and VEGF (C-2578A, rs699947 and G-634C, rs2010963) were genotyped by TaqMan allelic discrimination assays or PCR and fragment separation by electrophoresis in 122 patients with PE, 107 patients with GH and a control group of 102 normotensive pregnant (NP) women. A robust multifactor dimensionality reduction analysis was used to characterize gene-gene interactions. Although no significant genotype combinations were observed for the comparison between the GH and NP groups (P>0.05), the combination of MMP-9-1562CC with VEGF-634GG was more frequent in NP women than in women with PE (P<0.05). Moreover, the combination of MMP-9-1562CC with VEGF-634CC or MMP-9-1562CT with VEGF-634CC or-634GG was more frequent in women with PE than in NP women (P<0.05). These results are obscured when single polymorphisms in these genes are considered and suggest that specific genotype combinations of MMP-9 and VEGF contribute to PE susceptibility. Hypertension Research (2012) 35, 917-921; doi:10.1038/hr.2012.60; published online 10 May 2012

Effect of dental tissue conditioners and matrix metalloproteinase inhibitors on type I collagen microstructure analyzed by Fourier transform infrared spectroscopy

This study aimed to evaluate the chemical interaction of collagen with some substances usually applied in dental treatments to increase the durability of adhesive restorations to dentin. Initially, the similarity between human dentin collagen and type I collagen obtained from commercial bovine membranes of Achilles deep tendon was compared by the Attenuated Total Reflectance technique of Fourier Transform Infrared (ATR-FTIR) spectroscopy. Finally, the effects of application of 35% phosphoric acid, 0.1M ethylenediaminetetraacetic acid (EDTA), 2% chlorhexidine, and 6.5% proanthocyanidin solution on microstructure of collagen and in the integrity of its triple helix were also evaluated by ATR-FTIR. It was observed that the commercial type I collagen can be used as an efficient substitute for demineralized human dentin in studies that use spectroscopy analysis. The 35% phosphoric acid significantly altered the organic content of amides, proline and hydroxyproline of type I collagen. The surface treatment with 0.1M EDTA, 2% chlorhexidine, or 6.5% proanthocyanidin did not promote deleterious structural changes to the collagen triple helix. The application of 6.5% proanthocyanidin on collagen promoted hydrogen bond formation. (c) 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2012.

Assessment of MMP-9, TIMP-1, and COX-2 in normal tissue and in advanced symptomatic and asymptomatic carotid plaques

Abstract Background Mature carotid plaques are complex structures, and their histological classification is challenging. The carotid plaques of asymptomatic and symptomatic patients could exhibit identical histological components. Objectives To investigate whether matrix metalloproteinase 9 (MMP-9), tissue inhibitor of MMP (TIMP), and cyclooxygenase-2 (COX-2) have different expression levels in advanced symptomatic carotid plaques, asymptomatic carotid plaques, and normal tissue. Methods Thirty patients admitted for carotid endarterectomy were selected. Each patient was assigned preoperatively to one of two groups: group I consisted of symptomatic patients (n = 16, 12 males, mean age 66.7 ± 6.8 years), and group II consisted of asymptomatic patients (n = 14, 8 males, mean age 67.6 ± 6.81 years). Nine normal carotid arteries were used as control. Tissue specimens were analyzed for fibromuscular, lipid and calcium contents. The expressions of MMP-9, TIMP-1 and COX-2 in each plaque were quantified. Results Fifty-eight percent of all carotid plaques were classified as Type VI according to the American Heart Association Committee on Vascular Lesions. The control carotid arteries all were classified as Type III. The median percentage of fibromuscular tissue was significantly greater in group II compared to group I (p < 0.05). The median percentage of lipid tissue had a tendency to be greater in group I than in group II (p = 0.057). The percentages of calcification were similar among the two groups. MMP-9 protein expression levels were significantly higher in group II and in the control group when compared with group I (p < 0.001). TIMP-1 expression levels were significantly higher in the control group and in group II when compared to group I, with statistical difference between control group and group I (p = 0.010). COX-2 expression levels did not differ among groups. There was no statistical correlation between MMP-9, COX-2, and TIMP-1 levels and fibrous tissue. Conclusions MMP-9 and TIMP-1 are present in all stages of atherosclerotic plaque progression, from normal tissue to advanced lesions. When sections of a plaque are analyzed without preselection, MMP-9 concentration is higher in normal tissues and asymptomatic surgical specimens than in symptomatic specimens, and TIMP-1 concentration is higher in normal tissue than in symptomatic specimens.

Immunohistochemical evaluation of MMP-2, MMP-9 and CD31/microvascular density in squamous cell carcinomas of the floor of the mouth

The aim of this study was to evaluate the immunoexpression of MMP-2, MMP-9 and CD31/microvascular density in squamous cell carcinomas of the floor of the mouth and to correlate the results with demographic, survival, clinical (TNM staging) and histopathological variables (tumor grade, perineural invasion, embolization and bone invasion). Data from medical records and diagnoses of 41 patients were reviewed. Histological sections were subjected to immunostaining using primary antibodies for human MMP-2, MMP-9 and CD31 and streptavidin-biotin-immunoperoxidase system. Histomorphometric analyses quantified positivity for MMPs (20 fields per slide, 100?points grade, ×200) and for CD31 (microvessels <50?µm in the area of the highest vascularization, 5 fields per slide, 100?points grade, ×400). Statistical design was composed by non-parametric Mann-Whitney U test (investigating the association between numerical variables and immunostainings), chi-square frequency test (in contingency tables), Fisher's exact test (when at least one expected frequency was less than 5 in 2×2 tables), Kaplan-Meier method (estimated probabilities of overall survival) and Iogrank test (comparison of survival curves), all with a significance level of 5%. There was a statistically significant correlation between immunostaining for MMP-2 and lymph node metastasis. Factors associated negatively with survival were N stage, histopathological grade, perineural invasion and immunostaining for MMP-9. There was no significant association between immunoexpression of CD31 and the other variables. The intensity of immunostaining for MMP-2 can be indicative of metastasis in lymph nodes and for MMP-9 of a lower probability of survival

Ein transgenes Mausmodell zur Untersuchung des Einflusses von Matrix Metalloproteinase-2 und -14 auf die Leber

MMP-2 and MMP-14 process extracellular matrix proteins,cytokines, growth factors and adhesion molecules to generatefragments with enhanced or reduced biological activity.In this study, a vectorsystem was developed for theconditional expression of MMP-2 and MMP-14 in the liver oftransgenic mice. For this vectorsystem the murine albuminpromotor was chosen together with the cre/lox system toachieve an inducible MMP-expression in the liver.Only one of the MMP-14 transgenic lines expressed highamounts of active MMP-14 protein after recombination of thelox-P sites. In these mice MMP-14 was able to activate MMP-2and MMP-13 in vivo. However, none of the livers of MMP-14overexpressing mice showed no differences in liverweight,amount of extracellular matrixproteins and rate ofproliferation, apoptosis and tumor-induction when comparedto the liver of wildtype mice.On the other hand overexpression of MMP-2 was embryoniclethal in all MMP-2 transgenic lines. After crossing theMMP-2 transgenic mice with cre deleter mice, a cre mediatedrecombination could be shown at day 6.5 post coitum (pc).Some of the double transgenic embryos of one of thetransgenic lines had severe deformations of the head,especially of the telencephalon and the mesencephalon.It could be shown in this study that disregulation of MMP-2in early embryonic development is lethal but anoverexpression of MMP-14 has no influence on the embryonicdevelopment or the homeostasis of the adult liver.With this conditional vectorsystem it is to possible studythe influnce of MMP-2 and MMP-14 on fibrogenesis,regeneration and tumorgenesis in the liver of mice.

Molecular characterization of the immune modulatory function of matrix metalloproteinase-7, a factor in the tumour micromilieu

Matrix metalloproteinases are the components of the tumour microenvironment which play a crucial role in tumour progression. Matrix metalloproteinase-7 (MMP-7) is expressed in a variety of tumours and the expression is associated with an aggressive malignant phenotype and poor prognosis. A role for MMP-7 in the immune escape of tumours has been postulated, but the mechanisms are not clearly understood. The present study was focused on identifying physiological inactivators of MMP-7 and also to unravel the mechanisms involved in MMP-7 mediated immune escape. This study shows that human leukocyte elastase (HLE), secreted by polymorphonuclear leukocytes cleaves MMP-7 in the catalytic domain as revealed by N-terminal sequencing. Further analysis demonstrates that the activity of MMP-7 was drastically decreased after HLE treatment in a time and dose dependent manner. MMP-7 induces apoptosis resistance in tumour cells by cleaving CD95 and CD95L. The effect of HLE on MMP-7 mediated apoptosis resistance was analysed. In vitro stimulation of apoptosis by anti-Apo-1 (anti-CD95 antibody) and the chemotherapeutic drug doxorubicin is reduced by MMP-7. Also tumour specific cytotoxic T cells do not effectively kill tumour cells in the presence of MMP-7. This study revealed that HLE abrogates the negative effect of MMP-7 on apoptosis induced by CD95 stimulation, doxorubicin or cytotoxic T cells and restores apoptosis sensitivity of tumour cells. To gain insight into the possible immune modulatory functions of MMP-7, experiments were performed to identify new immune relevant substrates. The human T cell line, Jurkat, was selected for these studies. Hsc70 which is involved in uncoating of clathrin vesicles was found in the supernatants of the MMP-7 treated cells indicating a modulatory role of MMP-7 on endocytosis. Further studies demonstrated that MMP-7 leads to decreased clathrin staining in HEK293, HepG2, Jurkat, CD4+ T cells and dendritic cells. Results also show MMP-7 treatment increased surface expression of cytotoxic T lymphocyte associated protein-4 (CTLA-4) which accumulated due to inhibition of the clathrin mediated internalization in CD4+CD25+ cells.

Herpes-simplex virus encephalitis is characterized by an early MMP-9 increase and collagen type IV degradation

Cerebrovascular complications including cerebral edema, raised intracranial pressure and hemorrhage contribute to the high mortality and morbidity of herpes-simplex virus encephalitis (HSE). We examined changes of collagen type IV, the major constituent of the neurovascular matrix, together with expression and localization of matrix-degrading enzymes during the development of acute HSE. In an experimental model of focal HSE, we found that early, symptomatic HSE (3 days after infection) and acute, fully developed HSE (7 days after infection) are associated with significantly raised levels of matrix-metalloproteinase-9 (MMP-9) (both P<0.05). In situ zymography of brain sections revealed that the increase of MMP-9 was restricted to the cerebral vasculature in early HSE and further expanded towards the perivascular space and adjacent tissue in acute HSE. Around the cerebral vasculature, we observed that MMP-9 activity was insufficiently counterbalanced by its endogenous tissue inhibitor of MMP (TIMP) TIMP-1, resulting in loss of collagen type IV. Our findings suggest that MMP-9 is involved in the evolution of HSE by causing damage to the cerebral vasculature. The degradation of the neurovascular matrix in HSE facilitates the development of cerebrovascular complications and may represent a target for novel adjuvant treatment strategies.