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 metalloproteinases, tissue inhibitors of metalloproteinases, and growth factors regulate the aggressiveness and proliferative activity of keratocystic odontogenic tumors

Objective. The objective of this preliminary study was to evaluate the expression of matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs) and growth factors in keratocystic odontogenic tumors (KOTs). Study Design. The expression of MMPs, TIMPs, growth factors, and the extracellular signal-regulated kinase (ERK) 1/2 signaling pathway were assessed by immunohistochemistry in 15 cases of KOT and 4 cases of calcifying cystic odontogenic tumor (CCOT). Results. KOT samples expressed significantly higher amounts of MMPs, TIMPs, growth factors, epidermal growth factor receptor (EGFR), and ERK compared with CCOT samples, with the exception of MMP-2 and TIMP-1. Conclusions. MMP-9, TIMP-2, EGF and transforming growth factor alpha act together and likely regulate the proliferation and aggressiveness of KOT. ERK-1/2 serves as the transducer of signals generated by these proteins, which signal through the common receptor, EGFR. This process may be related to the increased proliferation and aggressiveness observed in KOT. (Oral Surg Oral Med Oral Pathol Oral Radiol 2012;114:487-496)

Matrix Metalloproteinase (MMP)-2 Genetic Variants Modify the Circulating MMP-2 Levels in End-Stage Kidney Disease

Background: Matrix metalloproteinases (MMPs) play important roles in the pathophysiology of renal diseases, and imbalanced MMP-2 and its endogenous inhibitor (the tissue inhibitor of metalloproteinases-2; TIMP-2) are implicated in the vascular alterations of end-stage kidney disease (ESKD) patients. We have examined whether MMP-2 gene polymorphisms and haplotypes modify MMP-2 and TIMP-2 levels in ESKD patients as well as the effects of hemodialysis on the concentrations of these biomarkers. Methods: We determined MMP-2 and TIMP-2 plasma levels by gelatin zymography and ELISA, respectively, in 98 ESKD patients and in 38 healthy controls. Genotypes for two relevant MMP-2 polymorphisms (C-T-1306 and C-T-735 in the promoter region) were determined by TaqMan (R) allele discrimination assay and real-time polymerase chain reaction. The software program PHASE 2.1 was used to estimate the haplotype frequencies. Results: We found increased plasma MMP-2 and TIMP-2 levels in ESKD patients compared to controls (p<0.05), and hemodialysis decreased MMP-2 (but not TIMP-2) levels (p<0.05). The T allele for the C-T-735 polymorphism and the C-T haplotype were associated with higher MMP-2 (but not TIMP-2) levels (p<0.05), whereas the C-T-1306 had no effects. Hemodialysis decreased MMP-2 (but not TIMP-2) levels independently of MMP-2 genotypes or haplotypes (p<0.05). Conclusions: MMP-2 genotypes or haplotypes modify MMP-2 levels in ESKD patients, and may help to identify patients with increased MMP-2 activity in plasma. Hemodialysis reduces MMP-2 levels independently of MMP-2 genetic variants. Copyright (C) 2012 S. Karger AG, Basel

Time course involvement of matrix metalloproteinases in the vascular alterations of renovascular hypertension

Increased vascular matrix metalloproteinases (MMPs) levels play a role in late phases of hypertensive vascular remodeling. However, no previous study has examined the time course of MMPs in the various phases of two-kidney, one-clip hypertension (2K1C). We examined structural vascular changes, collagen and elastin content, vascular oxidative stress, and MMPs levels/activities during the development of 2K1C hypertension. Plasma angiotensin converting enzyme (ACE) activity was measured to assess renin-angiotensin system activation. Sham or 2K1C hypertensive rats were studied after 2, 4, 6, and 10 weeks of hypertension. Systolic blood pressure (SBP) was monitored weekly. Morphometry of structural changes in the aortic wall was studied in hematoxylin/eosin, orcein and picrosirius red sections. Aortic NADPH activity and superoxide production was evaluated. Aortic gelatinolytic activity was determined by in situ zymography, and MMP-2, MMP-14, and tissue inhibitor of MMPs (TIMP)-2 levels were determined by gelatin zymography, immunofluorescence and immunohistochemistry. 2K1C hypertension was associated with increased ACE activity, which decreased to normal after 10 weeks. We found increased aortic collagen and elastin content in the early phase of hypertension, which were associated with vascular hypertrophy, increased vascular MMP-2 and MMP-14 (but not TIMP-2) levels, and increased gelatinolytic activity, possibly as a result of increased vascular NADPH oxidase activity and oxidative stress. These results indicate that vascular remodeling of renovascular hypertension is an early process associated with early increases in MMPs activities, enhanced matrix deposition and oxidative stress. Using antioxidants or MMPs inhibitors in the early phase of hypertension may prevent the vascular alterations of hypertension. (C) 2012 Elsevier B.V. All rights reserved.

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

Circulating matrix metalloproteinases and their endogenous inhibitors in patients with erectile dysfunction

Erectile dysfunction (ED) may reflect vascular alterations associated with imbalanced matrix metalloproteinases (MMPs) activities. However, no previous study has compared MMPs levels in ED patients with those found in healthy subjects. We measured the circulating MMP-2, MMP-9, TIMP-1 and TIMP-2 levels in ED patients, with or without diabetes mellitus (DM), and in healthy controls. We studied 28 healthy men (control group), 35 men with ED (ED group), and 33 men with ED and DM (ED/DM group). MMP-2, MMP-9, TIMP-1 and TIMP-2 plasma levels were measured by enzyme-linked immunosorbent assay and zymography. We found no differences in MMP-9 levels (P>0.05) among groups. However, while patients in the ED group had similar TIMP-1 levels compared with those found in the control group, we found higher TIMP-1 levels and lower MMP-9/TIMP-1 ratios in the ED/DM group compared with controls (P<0.05). While both groups of patients (ED and ED/DM) had slightly lower MMP-2 levels compared with controls (P<0.05), we found no differences in TIMP-2 levels among the study groups (P>0.05), and no differences in MMP-2/TIMP-2 ratios (P>0.05). We found evidence indicating lack of significant alterations in circulating net MMP-9 and MMP-2 activities in patients with ED, and lower net MMP-9 activity in diabetic patients with ED. International Journal of Impotence Research (2012) 24, 38-43; doi:10.1038/ijir.2011.44; published online 15 September 2011

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.

An inhibitor of leukotriene synthesis affects vasopressin secretion following osmotic stimulus in rats

Previous studies revealed the presence of LTC4 synthase in paraventricular vasopressinergic neurons, suggesting a role for leukotrienes (LTs) in certain neuroendocrine system functions. Our aim was to study the effect of an inhibitor of LT synthesis in the release of arginine vasopressin (AVP) following an osmotic stimulus in rats. Male Wistar rats received an intra-cerebroventricular injection of 2 mu l of the LT synthesis inhibitor MK-886 (1, 2, or 4 mu g/kg), or vehicle (DMSO 5%), 1 h before an intraperitoneal injection of hypertonic saline (NaCl 2 M) or isotonic saline (NaCl 0.01 M) in a volume corresponding to 1% of body weight. Thirty minutes after the osmotic stimulus, the animals were decapitated and blood was collected for determining hematocrit, plasma osmolality and plasma AVP levels. As expected, the injection of hypertonic saline significantly increased (P<0.05) the hematocrit, plasma osmolality and plasma AVP levels. While inhibiting LT synthesis by central administration of MK-886 did not cause any additional increase in hematocrit or osmolality, plasma AVP levels were augmented (P<0.05). We conclude that central leukotrienes may have a modulatory role in AVP secretion following an osmotic stimulus, this deserving future studies. (C) 2012 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.

Functional matrix metalloproteinase (MMP)-9 genetic variants modify the effects of hemodialysis on circulating MMP-9 levels

Background: Altered levels of matrix metalloproteinases (MMPs) and their inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), are involved in cardiovascular alterations associated with end stage kidney disease (ESKD). Genetic polymorphisms in MMP-9 gene affect MMP-9 levels. We examined how MMP-9 polymorphisms and haplotypes affect the changes in plasma MMP-9 and TIMP-1 levels found in patients with ESKD undergoing hemodialysis. Methods: We studied 94 ESKD patients undergoing hemodialysis for at least 3 months. MMP-9 and TIMP-1 were measured by ELISA in plasma from blood samples collected before and after a session of hemodialysis. Genotypes for three MMP-9 polymorphisms (C-1562T, rs3918242; -90 (CA)(14-24), rs2234681; and Q279R, rs17576) were determined by Taqman (R) Allele Discrimination Assay and real-time polymerase chain reaction. Haplotype frequencies were determined with the software program PHASE 2.1. Results: Hemodialysis increased MMP-9 and TIMP-1 levels (P<0.05). Genotypes had no effects on baseline MMP-9 and TIMP-1 levels (P>0.05). Hemodialysis increased MMP-9 and TIMP-1 levels in subjects with the CC (but not CT or TT) genotype for the C-1562T polymorphism (P<0.05), and increased MMP-9 levels in subjects with the QQ (but not QR or RR) genotype for the Q279R polymorphism (P<0.05), whereas the CA(n)(14-24) polymorphism had no major effects. While MMP-9 haplotypes had no effects on baseline MMP-9 levels (P>0.05), hemodialysis increased MMP-9 levels and MMP-9/TIMP-1 ratios in subjects carrying the CLQ haplotype (P = 0.0012 and P = 0.0045, respectively). Conclusion: ESKD patients with the QQ genotype for the Q279R polymorphism or with the CLQ haplotype are exposed to more severe increases in MMP-9 levels after hemodialysis. Such patients may benefit from the use of MMP inhibitors. (C) 2012 Elsevier B.V. All rights reserved.

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

Association between matrix metalloproteinase (MMP)-2 polymorphisms and MMP-2 levels in hypertensive disorders of pregnancy

We examined whether two functional polymorphisms (g.-1306 C> T and g.-735 C>T) in matrix metalloproteinase (MMP)-2 gene are associated with preeclampsia (PE) or gestational hypertension (GH), and whether they modify MMP-2 or tissue inhibitor of metalloproteinase (TIMP)-2 plasma concentrations in these hypertensive disorders of pregnancy. We studied 130 healthy pregnant (HP), 130 pregnant with GH, and 133 pregnant with PE. Genomic DNA was extracted from whole blood and genotypes for g.-1306 C>T and g.-735 C>T polymorphisms were determined by Real Time-PCR, using Taqman allele discrimination assays. Haplotypes were inferred using the PHASE program. Plasma MMP-2 and TIMP-2 concentrations were measured by ELISA. The main findings were that pregnant with PE have higher plasma MMP-2 and TIMP-2 concentrations than HP (P<0.05), although the MMP-2/TIMP-2 ratios were similar (P>0.05). Moreover, pregnant with GH have elevated plasma MMP-2 levels and MMP-2/TIMP-2 ratios compared to HP (P<0.05). While MMP-2 genotypes and haplotypes are not linked with hypertensive disorders of pregnancy, MMP-2 genotypes and haplotypes are associated with significant alterations in plasma MMP-2 and TIMP-2 concentrations in preeclampsia (P<0.05). Our findings may help to understand the relevance of MMP-2 and its genetic polymorphisms to the pathophysiology of hypertensive disorders of pregnancy. It is possible that patients with PE and the MMP-2 haplotype combining the C and T alleles for the g.-1306 C>T and g.-735 C>T polymorphisms may benefit from the use of MMPs inhibitors such as doxycycline. However, this possibility remains to be determined. (C) 2012 Elsevier Inc. All rights reserved.

Evaluation of plasmatic MMP-8, MMP-9, TIMP-1 and MPO levels in obese and lean women

Objectives: To compare the plasma concentrations of matrix metalloproteinase (MMP)-9, tissue inhibitor of MMP (TIMP)-1, MMP-8, and myeloperoxidase (MPO) for obese and lean women. Design and methods: We recruited 30 lean and 36 obese women without comorbidities. The MMP-9, TIMP-1, and MMP-8 levels were measured using enzyme-linked immunosorbent assay (ELISA). MPO activity was assessed by a colorimetric assay. Results: Obese women had higher MMP-9 levels and MMP-9:TIMP-1 ratios than lean women. Conversely, the MMP-8 levels and MMP-8:TIMP-1 ratios in the obese women were significantly lower than those in the lean women despite neutrophil activation, which was assessed by MPO activity., Conclusion: We observed that MMP-9 and MMP-8 had distinct profiles, which suggested that these 2 enzymes play different roles in obesity. (C) 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Cyclosporin A causes impairment of the ventral prostate tissue structure of Wistar rats

Cyclosporin A (CsA) is an immunosuppressive drug widely used in medicine to reduce the immune system activity and, therefore, the risk of organ rejection after transplantation. However, many side effects can be related to its use, such as, reduction in serum testosterone levels due to damage of the testis structure and, consequently, male infertility. The present study aims to evaluate the effects of chronic CsA administration on the ventral prostate tissue ( 1 5 mg/kg per d, for 56 days). Stereological, morphometrical, morphological and ultrastructural observations were employed. The plasmatic testosterone and glucose levels were measured. An androgen receptor (AR) immunohistochemical method was applied on ventral prostate sections. Apoptosis was detected with the terminal deoxynucleotidyl transferase dUTP nick end labeling technique. CsA treatment caused reduction in plasmatic testosterone levels and an increase in glycemia. The volume of all ventral prostate tissue components (lumen, epithelium and muscular and nonmuscular stroma) and ventral prostate weight were reduced in the CsA-treated group. Light and transmission electron microscopy confirmed epithelium atrophy of treated animals. There was no alteration of AR expression or apoptotic index. CsA chronic treatment in the therapeutic doses caused damage to prostate tissue of adult Wistar rats, probably due to increase in the glucose levels and reduction in the plasmatic testosterone levels.

Evaluation of the mutagenic activity of chrysin, a flavonoid inhibitor of the aromatization process

Chrysin is one of the natural flavonoids present in plants, and large amounts are present in honey and propolis. In addition to anticancer, antioxidation, and anti-inflammatory activities, chrysin has also been reported to be an inhibitor of aromatase, an enzyme converting testosterone into estrogen. The present study evaluated the mutagenicity of this flavonoid using micronucleus (MN) with HepG2 cells and Salmonella. Cell survival after exposure to different concentrations of chrysin was also determined using sulforhodamine B (SRB) colorimetric assay in HepG2 cells and the influence of this flavonoid on growth of cells in relation to the cell cycle and apoptosis. TheMN test showed that from 1 to 15 mu M of this flavonoid mutagenic activity was noted in HepG2 cells. The Salmonella assay demonstrated a positive response to the TA100 Salmonella strain in the presence or absence of S9, suggesting that this compound acted on DNA, inducing base pair substitution before or after metabolism via cytochrome P-450. The SRB assay illustrated that chrysin promoted growth inhibition of HepG2 cells in both periods studied (24 and 48 h). After 24 h of exposure it was noted that the most significant results were obtained with a concentration of 50 mu M, resulting in 83% inhibition and SubG0 percentage of 12%. After 48 h of incubation cell proliferation inhibition rates (97% at 50 mu M) were significantly higher. Our results showed that chrysin is a mutagenic and cytotoxic compound in cultured human HepG2 cells and Salmonella typhimurium. Although it is widely accepted that flavonoids are substances beneficial to health, one must evaluate the risk versus benefit relationship and concentrations of these substances to which an individual may be exposed.