Morphologic evaluation and expression of matrix metalloproteinases-2 and 9 and nitric oxide during experimental periodontal disease in rat

The immunopathologic and inflammatory mechanisms involved in periodontal disease (PD) include the participation of host resident, inflammatory cells and chemical mediators. Metalloproteinases (MMPs) and nitric oxide (NO) play essential role in extracellular matrix turnover of periodontal tissue destruction. In this study, by means of RT-PCR through semi-quantitative densitometric scanning methods, the expression of MMPs -2 and -9 and inducible NO synthase (iNOS) was temporally and spatially investigated during the destructive mechanisms of experimentally induced PD in rats. Samples from different periods were microscopically analyzed and compared with the contralateral side (control). Our results showed significant expression of MMP-9 and iNOS in tissues affected by PD, as compared with controls, three days after PD induction, simultaneously with the beginning of alveolar bone loss. At 7 days post induction, only the MMP-9 mRNA presented a significantly higher expression, as compared with the respective controls. Thus, in the rat ligature-induced PD, MMP-9 and iNOS might importantly participate in the early stages of the disease, including inflammatory cell migration, tissue destruction and alveolar bone resorption. Also, we may suggest that the exuberant presence of PMNs may be related to the important expression of iNOS and MMP-9 found at 3 days post induction.

Increased activities of cardiac matrix metalloproteinases matrix metalloproteinase (MMP)-2 and MMP-9 are associated with mortality during the acute phase of experimental Trypanosoma cruzi infection

The strong inflammatory reaction that occurs in the heart during the acute phase of Trypanosoma cruzi infection is modulated by cytokines and chemokines produced by leukocytes and cardiomyocytes. Matrix metalloproteinases (MMPs) have recently emerged as modulators of cardiovascular inflammation. In the present study we investigated the role of MMP-2 and MMP-9 in T. cruzi-induced myocarditis, by use of immunohistochemical analysis, gelatin zymography, enzyme-linked immunosorbent assay, and real-time polymerase chain reaction to analyze the cardiac tissues of T. cruzi-infected C57BL/6 mice. Increased transcripts levels, immunoreactivity, and enzymatic activity for MMP-2 and MMP-9 were observed by day 14 after infection. Mice treated with an MMP inhibitor showed significantly decreased heart inflammation, delayed peak in parasitemia, and improved survival rates, compared with the control group. Reduced levels of cardiac tumor necrosis factor-alpha, interferon-gamma, serum nitrite, and serum nitrate were also observed in the treated group. These results suggest an important role for MMPs in the induction of T. cruzi-induced acute myocarditis.

Increases in circulating matrix metalloproteinase-9 levels following fibrinolysis for acute pulmonary embolism

Introduction: Fibrinolyis is one of the first line therapies in high risk pulmonary embolism (PE) according to current guidelines. Previous studies showed that brinolytic therapy with tPA (tissue plasminogen activator, or alteplase) upregulates the concentrations of matrix metalloproteinases (MMPs) and contributes to hemorrhagic transformation after cardioembolic stroke. However, no previous study has described the circulating MMPs levels following fibrinolysis for acute PE. Materials and Methods: We serially measured the circulating levels of MMPs (MMP-9 and MMP-2) and their endogenous inhibitors, the tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 in alteplase and in streptokinase-treated patients with acute PE by gelatin zymography and by enzyme-linked immunosorbent assays, respectively. Results: We found that therapy of PE streptokinase or with alteplase is associated increased pro-MMP-9, but not MMP-2, concentrations for up to 24 hours, whereas no significant changes were found in TIMP-1 or TIMP-2 concentrations. This alteration returned to normal 3 to 5 days after thrombolysis. This is the first study reporting on MMPs alterations following fibrinolysis for acute PE. Conclusions: We found transient increases in circulating pro-MMP-9 levels following fibrinolysis for acute PE. Our findings support the hypothesis that increased MMP-9 levels may underlie the risk of intracerebral hemorrhage or other bleeding complication of thrombolysis for acute PE, and the use of MMP inhibitors may decrease such risk. (C) 2010 Elsevier Ltd. All rights reserved.

Matrix Metalloproteinase Inhibition Improves Cardiac Dysfunction and Remodeling in 2-Kidney, 1-Clip Hypertension

Background: Enhanced cardiac matrix metalloproteinase activity (MMPs) has been associated with ventricular remodeling and cardiac dysfunction. It is unknown whether MMPs contribute to systolic/diastolic dysfunction and compensatory remodeling in 2-kidney, 1-clip (2K1C) hypertensive rats. To test this hypothesis, we used 2K1C rats after 2 weeks of surgery treated or not with a nonspecific inhibitor of MMPs (doxycycline). Methods and Results: We found that blood pressure and +/-dP/dt increased in 2K1C rats compared with sham groups, and these parameters were attenuated by doxycycline treatment (P < .05). Doxycycline also reversed cardiac hypertrophy observed in 2K1C rats (P < .05). Hypertensive rats showed increased MMP-2 levels in zymograms and in the tissue by immunofluorescence (P < .05) compared with sham groups. Increased total gelatinolytic activity was observed in untreated 2K1C rats when compared with sham groups (P < .05). Doxycycline decreased total gelatinolytic activity in 2K1C rats to control levels (P < .05). Conclusion: An imbalance in gelatinolytic activity, with increased MMP-2 levels and activity underlies the development of morphological and functional alterations found in the compensatory hypertrophy observed in 2K1C hearts. Because function and structure were restored by doxycycline, the inhibition of MMPs or their modulation may provide beneficial effects for therapeutic intervention in cardiac hypertrophy. (J Cardiac Fail 2010;16:599-608)

Deregulation of LOXL1 and HTRA1 Gene Expression in Endometriosis

Endometriosis is a gynecologic disease characterized by the presence of endometrial tissue outside the uterine cavity. Although 15% of the female population in reproductive age is affected by endometriosis, its pathogenesis remains unclear. According to the most accepted pathogenesis hypothesis, endometrial fragments from the menstrual phase are transported through the uterine tubes to the peritoneal cavity, where they undergo implantation and growth, invading adjacent tissues. However, the establishment of the disease requires that endometrial cells present molecular characteristics favoring the onset and progression of ectopic implantation. In this investigation, we analyzed the differential gene expression profiles of peritoneal and ovarian endometriotic lesions compared to the endometrial tissue of nonaffected women using rapid subtraction hybridization (RaSH). In our study, this method was applied to samples of endometriotic lesions from affected women and to biopsies of endometrium of healthy women without endometriosis, where we could identify 126 deregulated genes. To evaluate the expression of genes found by RaSH method, we measured LOXL1, HTRA1, and SPARC genes by real-time polymerase chain reaction. Significant different expression was obtained for HTRA1 and LOXL1, upregulated in the ectopic endometrium, suggesting that these genes are involved in the physiopathology of endometriosis and may favor the viability of endometrial cells at ectopic sites.

Matrix metalloproteinase (MMP)-9 genotypes and haplotypes in preeclampsia and gestational hypertension

Background: Abnormal production of matrix metalloproteinases (MMPs), especially MMP-9, may play a role in hypertensive disorders of pregnancy. These alterations may result from functional genetic polymorphisms in the promoter region of MMP-9 gene, which are known to change MMP-9 expression. We examined whether 2 MMP-9 polymorphisms (C(-) (1562) T and (CA)n) and haplotypes are associated with preeclampsia and/or gestational hypertension. Methods: We studied 476 pregnant women: 176 healthy pregnant (HP), 146 pregnant with gestational hypertension (GH), and 154 pregnant with preeclampsia (PE). Genomic DNA was extracted from whole blood and genotypes for C(- 1562) T and (CA)n polymorphisms were determined by PCR-RFLP. Haplotype frequencies were inferred using the PHASE ver. 2.1 program. Results: For the g.-90(CA)13-25 polymorphism, no significant differences were found in genotype and allele distributions when PE or GH groups were compared with HP group. However, the CT genotype and T allele for g.-1562C>T polymorphism were more commonly found in GH subjects compared with the HP group (both P<0.05). Conversely, we found no differences in genotypes or allele distributions for the g.-1562C>T polymorphism when the PE and the HP groups were compared. No significant differences were found in overall distributions of haplotype frequencies when the GH or the PE group was compared with the HP group. Conclusions: The C(- 1562) T polymorphism in MMP-9 gene is associated with gestational hypertension, but not with preeclampsia. These findings may help to explain the higher plasma MMP-9 levels previously reported in GH compared with HP. (C) 2010 Elsevier B.V. All rights reserved.

Imbalanced circulating matrix metalloproteinases in polycystic ovary syndrome

Altered levels of matrix metalloproteinases (MMPs) may reflect relevant pathogenetic mechanisms of disease conditions. The objective of this study was to compare the plasma levels of MMPs and tissue inhibitors of MMPs (TIMPs) in polycystic ovary syndrome (PCOS) patients with those found in healthy ovulatory controls and to examine whether the levels of these biomarkers are associated with clinical and biochemical features of this syndrome. Sixty-five healthy ovulatory subjects (controls) and 80 patients with PCOS were include in this study. MMP-2, MMP-8, MMP-9, TIMP-1, TIMP-2 concentrations were measured in plasma samples by gelatin zymography or enzyme-linked immunoassays. MMP-2, MMP-8, MMP-9, and TIMP-1 levels were similar in PCOS patients and in healthy controls (P > 0.05). PCOS patients had lower plasma TIMP-2 levels than healthy controls (P < 0.05). We found higher MMP-2/TIMP-2 and MMP-9/TIMP-1 ratios in PCOS patients than in healthy controls (all P < 0.05). Testosterone levels correlated positively with the MMP-9/TIMP-1 ratio and negatively with TIMP-2 levels (r = 0.26, P < 0.01 and r = -0.21, P = 0.02, respectively). In addition, only testosterone was an independent predictor of TIMP-2 levels (estimate = -0.35, P = 0.04) and the MMP-9/TIMP-1 ratio (estimate = 0.01, P = 0.04). We found evidence indicating that the balance between MMPs and TIMPs in women with PCOS is altered, probably due to androgen excess found in these women.

Comparative assessment of matrix metalloproteinase (MMP)-2 and MMP-9, and their inhibitors, tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2 in preeclampsia and gestational hypertension

Objectives: To compare the circulating levels of matrix metalloproteinase (MMP)-2, MMP-9, tissue inhibitors of metalloproteinase (TIMP)-1, TIMP-2, and the MMP-9/TIMP-1 and MMP-2/TIMP-2 ratios in preeclampsia and gestational hypertension with those found in normotensive pregnancies. Design and methods: We studied 83 pregnant women (30 healthy pregnant women with uncomplicated pregnancies, 26 with gestational hypertension, and 27 with preeclampsia) and 30 healthy nonpregnant women in a cross-sectional study. MMP and TIMP concentrations were measured in plasma samples by gelatin zymography and ELISA, respectively. Results: We found higher plasma pro-MMP-9 levels, and higher pro-MMP-9/TIMP-1 ratios in women with gestational hypertension (95%-CI: 1.031 to 2.357, and 0.012 to 0.031, respectively), but not with preeclampsia, compared with those found in normotensive pregnant women (95%-CI: 0.810 to 1.350, and 0.006 to 0.013, respectively; both P<0.05). We found no significant differences in pro-MMP-2 levels (P>0.05). Conclusions: The higher net MMP-9 (but not MMP-2) activity in gestational hypertension compared with normotensive pregnancy suggests that MMP-9 plays a role in the pathophysiology of gestational hypertension. Conversely, the lack of such alterations in preeclampsia is consistent with the notion that different pathophysiological mechanisms are involved in these hypertensive disorders. (C) 2008 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Matrix metalloproteinase-9 genotypes and haplotypes are associated with multiple sclerosis and with the degree of disability of the disease

Multiple sclerosis (MS) is an autoimmune disease causing severe neurological disability. This study was carried out in order to determine whether the MMP-9 C(-1562)T and (CA)(13-25) polymorphisms are associated with MS. A total of 165 patients (92 whites/73 mulattos) and 191 controls (96 whites/95 mulattos) were enrolled in the study. While no difference in C(-1562)T polymorphism was observed between MS and healthy subjects, (CA)(n) genotypes and alleles were associated with MS. Moreover, the haplotypes are not associated with MS but seem to be relevant to the clinical status of MS. Thus the (CA)(n) polymorphism may contribute to MS susceptibility, but C(-1562)T and (CA)(n) haplotypes may modulate disease severity. (c) 2009 Elsevier B.V. All rights reserved.

Proposal for a Trigonometric Method to Evaluate the Abduction Angle of the Lower Limbs in Neonates

It is difficult to precisely measure articular arc movement in newborns using a goniometer. This article proposes an objective method based on trigonometry for the evaluation of lower limb abduction. With the newborn aligned in the dorsal decubitus position, 2 points are marked at the level of the medial malleolus, one on the sagittal line and the other at the end of the abduction. Using the right-sided line between these 2 points and a line from the medial malleolus to the reference point at the anterior superior iliac spine or umbilical scar, an isosceles triangle is drawn, and half of the inferential abduction angle is obtained by calculating the sine. Twenty healthy full-term newborns comprise the study cohort. Intersubject and intrasubject variability among the abduction angle values (mean [SD], 37 degrees [4]degrees) is low. This method is advantageous because the measurement is precise and because the sine can be used without approximation.

Evaluation of erythrocyte dysmorphism by light microscopy with lowering of the condenser lens: A simple and efficient method

Aim: To demonstrate that the evaluation of erythrocyte dysmorphism by light microscopy with lowering of the condenser lens (LMLC) is useful to identify patients with a haematuria of glomerular or non-glomerular origin. Methods: A comparative double-blind study between phase contrast microscopy (PCM) and LMLC is reported to evaluate the efficacy of these techniques. Urine samples of 39 patients followed up for 9 months were analyzed, and classified as glomerular and non-glomerular haematuria. The different microscopic techniques were compared using receiver-operator curve (ROC) analysis and area under curve (AUC). Reproducibility was assessed by coefficient of variation (CV). Results: Specific cut-offs were set for each method according to their best rate of specificity and sensitivity as follows: 30% for phase contrast microscopy and 40% for standard LMLC, reaching in the first method the rate of 95% and 100% of sensitivity and specificity, respectively, and in the second method the rate of 90% and 100% of sensitivity and specificity, respectively. In ROC analysis, AUC for PCM was 0.99 and AUC for LMLC was 0.96. The CV was very similar in glomerular haematuria group for PCM (35%) and LMLC (35.3%). Conclusion: LMLC proved to be effective in contributing to the direction of investigation of haematuria, toward the nephrological or urological side. This method can substitute PCM when this equipment is not available.

Protective effect of Calendula officinalis extract against UVB-induced oxidative stress in skin: Evaluation of reduced glutathione levels and matrix metalloproteinase secretion

Background and purpose: Calendula officinalis flowers have long been employed time in folk therapy, and more than 35 properties have been attributed to decoctions and tinctures from the flowers. The main uses are as remedies for burns (including sunburns), bruises and cutaneous and internal inflammatory diseases of several origins. The recommended doses are a function both of the type and severity of the condition to be treated and the individual condition of each patient. Therefore, the present study investigated the potential use of Calendula officinalis extract to prevent UV irradiation-induced oxidative stress in skin. Methods: Firstly, the physico-chemical composition of marigold extract(ME) (hydroalcoholic extract)was assessed and the in vitro antioxidant efficacy was determined using different methodologies. Secondly, the cytotoxicity was evaluated in L929 and HepG2 cells with the MTT assay. Finally, the in vivo protective effect of ME against UVB-induced oxidative stress in the skin of hairless mice was evaluated by determining reduced glutathione (GSH) levels and monitoring the secretion/activity of metalloproteinases. Results and conclusions: The polyphenol, flavonoid, rutin and narcissin contents found in ME were 28.6 mg/g, 18.8 mg/g, 1.6 mg/g and 12.2 mg/g, respectively and evaluation of the in vitro antioxidant activity demonstrated a dose-dependent effect of ME against different radicals. Cytoxicity experiments demonstrated that ME was not cytotoxic for L929 and HepG2 cells at concentrations less than or equal to of 15 mg/mL However, concentrations greater than or equal to 30 mg/mL, toxic effects were observed. Finally, oral treatment of hairless mice with 150 and 300 mg/kg of ME maintained GSH levels close to non-irradiated control mice. In addition, this extract affects the activity/secretion of matrix metalloproteinases 2 and 9 (MMP-2 and -9) stimulated by exposure to UVB irradiation. However, additional studies are required to have a complete understanding of the protective effects of ME for skin. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Detection of Auditory Cortex Activity by fMRI Using a Dependent Component Analysis

Functional MRI (fMRI) data often have low signal-to-noise-ratio (SNR) and are contaminated by strong interference from other physiological sources. A promising tool for extracting signals, even under low SNR conditions, is blind source separation (BSS), or independent component analysis (ICA). BSS is based on the assumption that the detected signals are a mixture of a number of independent source signals that are linearly combined via an unknown mixing matrix. BSS seeks to determine the mixing matrix to recover the source signals based on principles of statistical independence. In most cases, extraction of all sources is unnecessary; instead, a priori information can be applied to extract only the signal of interest. Herein we propose an algorithm based on a variation of ICA, called Dependent Component Analysis (DCA), where the signal of interest is extracted using a time delay obtained from an autocorrelation analysis. We applied such method to inspect functional Magnetic Resonance Imaging (fMRI) data, aiming to find the hemodynamic response that follows neuronal activation from an auditory stimulation, in human subjects. The method localized a significant signal modulation in cortical regions corresponding to the primary auditory cortex. The results obtained by DCA were also compared to those of the General Linear Model (GLM), which is the most widely used method to analyze fMRI datasets.

mRNA expression of matrix metalloproteinases (MMPs) 2 and 9 and tissue inhibitor of matrix metalloproteinases (TIMPs) 1 and 2 in childhood acute lymphoblastic leukemia: Potential role of TIMP1 as an adverse prognostic factor

This study evaluates the mRNA expression profile of genes TIMP1, TIMP2, MMP2 and MMP9 in diagnostic bone marrow samples from 134 consecutive ALL children by real-time quantitative PCR. A significant association was observed between higher expression levels of MMP9 and low risk group and absence of extramedullary infiltration and higher expression levels of TIMP2 and MMP2 with T-ALL. TIMP1 gene expression values higher than the median were associated with a significantly lower 5-year event free-survival in univariable (P = 0.04) and multivariable analysis (P = 0.01). Our data address new information in the complex interaction of the migration/adhesion genes and childhood ALL. (c) 2009 Elsevier Ltd. All rights reserved.

Consistent Alterations of Circulating Matrix Metalloproteinases Levels in Untreated Hypertensives and in Spontaneously Hypertensive Rats: A Relevant Pharmacological Target

Inconsistent matrix metalloproteinases (MMPs) levels have been reported in hypertension, with higher, similar and lower MMPs levels reported in hypertensives compared with normotensives. Differences between studies may reflect lack of control of drug effects, accompanying diseases and pre-analytical issues. We compared MMP-2, MMP-8 and MMP-9 levels in 38 untreated hypertensive patients (with no other diseases) with those found in 33 normotensive controls. We also studied endogenous MMPs inhibitors (TIMP-1, TIMP-2 and alpha-2-macroglobulin-A2M). Additionally, we assessed MMPs and A2M levels in spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats. We hypothesized that similar MMPs/endogenous inhibitors` profiles would be found in this animal model of hypertension and in clinical hypertension. MMPs, TIMPs and A2M were measured in plasma samples with commercially available ELISA and gelatin zymography. We found unaltered MMP-2, MMP-8, TIMP-1, TIMP-2 and A2M levels in hypertension. However, hypertensives had higher MMP-9 levels and MMP-9/A2M ratios than normotensives. Moreover, while we found similar MMP-2 and A2M levels in SHR and WKY rats, we found higher MMP-9 levels and MMP-9/A2M ratios in SHR versus WKY rats. These findings show consistent abnormal net plasma MMP-9 (but not MMP-2) activity in clinical and experimental hypertension. These parallel alterations in clinical hypertension and in SHR suggest an important role for MMPs in hypertension. While MMPs may be a relevant pharmacological target, antihypertensive drugs that down-regulate MMPs may offer advantages in the management of this disease.