Increased activity of MMP-2 in hypertensive obese children is associated with hypoadiponectinemia.

OBJECTIVE: To compare the circulating levels of MMP-2 and TIMP-2 and the MMP-2/TIMP-2 ratio in control, obese, and obese hypertensive children and adolescents and to assess whether hypoadiponectinemia is associated with MMP-2 and TIMP-2 levels and MMP-2/TIMP-2 ratios. METHODS: Studies were carried out with 53 control, 73 obese, and 29 obese hypertensive children and adolescents in this cross-sectional study. Adiponectin and TIMP-2 concentrations were measured by ELISA. MMP-2 concentrations were measured by gelatin zymography. Multiple linear regression analysis was carried out to assess the effects of adiponectin on MMP-2 and TIMP-2 levels and MMP-2/TIMP-2 ratios. RESULTS: The obese hypertensive group had the lowest adiponectin levels among groups (P < 0.05) while obese subjects had lower adiponectin levels than control subjects (P < 0.05). Obese hypertensive subjects also had higher circulating MMP-2 concentrations than obese subjects (P < 0.05) and had the highest MMP-2/TIMP-2 ratios among groups (P < 0.05). Moreover, obese/hypertensive subjects had the lowest TIMP-2 levels among groups (P < 0.05). A multiple linear regression analysis showed that MMP-2 levels and MMP-2/TIMP-2 ratios are negatively associated with adiponectin levels (P = 0.034 and P = 0.011, respectively) while TIMP-2 levels is positively associated with adiponectin levels (P = 0.013). CONCLUSIONS: Increased activity of MMP-2 (MMP-2/TIMP-2 ratio) and reduced TIMP-2 levels may play an important role in clinical hypertension of childhood obesity. Additionally, hypoadiponectinemia may contribute to increased activity of MMP-2 in obese/hypertensive children and adolescents.

Effect of metabolic syndrome risk factors and MMP-2 genetic variations on circulating MMP-2 levels in childhood obesity.

Matrix metalloproteinase-2 is involved in the development of the adipose tissue, and associated with cardiovascular diseases. Metabolic risk factors (MRFs) and functional polymorphisms in the MMP-2 gene may affect its expression and activity. We investigated whether traditional MRFs and two MMP-2 gene polymorphisms (C(-1306)T; rs243865, and C(-735)T; rs2285053) affect circulating MMP-2 levels in children and adolescents, and whether MMP-2 polymorphisms and/or haplotype are associated with susceptibility to childhood obesity. We studied 114 healthy controls, 43 obese, and 83 obese with ≥ 3 MRFs children and adolescents. Genotypes were determined by Taqman allele discrimination assay and real-time PCR. Plasma MMP-2 was measured using zymography. We found positive correlations between MMP-2 concentrations and mean blood pressure in all children and adolescents group (r = 0.132; P < 0.05) and in obese children and adolescents (r = 0.247; P < 0.01). We found that the CC genotype for the C(-1306)T polymorphism was more common in subjects with higher MMP-2 concentrations in controls (P = 0.003) and in the obese group (P = 0.013). The CT genotype (OR = 0.40; P < 0.01) and the T allele (OR = 0.48; P < 0.01) for the C(-735)T polymorphism were less common in obese children and adolescents than in controls. The haplotypes distribution did not show significant differences between control and obese (P > 0.05). Ours findings show that blood pressure is associated with circulating MMP-2 concentrations, and that the CC genotype for the C(-1306)T polymorphism was more common subjects (controls and obese) with higher MMP-2 concentrations, whereas the CT genotype and the T allele for the C(-735)T polymorphism are less common in obesity.

eNOS polymorphism associated with metabolic syndrome in children and adolescents.

We investigated whether genetic polymorphisms in the endothelial nitric oxide (eNOS) gene (T(786)C in the promoter region, Glu298Asp in exon 7, and 4b/4a in intron 4) or eNOS haplotypes are associated with metabolic syndrome (MetS) in obese children and adolescents. We studied 242 subjects: 108 healthy (controls), 64 normotensive obese, and 70 obese children and adolescents with MetS. Genotypes were determined by Taqman(®) allele discrimination assay and real-time polymerase chain reaction (PCR), and PCR followed by fragment separation by electrophoresis. We compared the distribution of eNOS genotypes, alleles, and haplotypes in the three groups of subjects. The CC genotype for the T(786)C polymorphism was more common in the MetS group than in the control group (OR = 3.27; CI 1.81-9.07; P < 0.05). However, we found no significant differences in the distribution of eNOS haplotypes (P > 0.00625; P for significance after correction for multiple comparisons). Our findings suggest that while eNOS haplotypes are not relevant, the CC genotype for the T(786)C polymorphism is associated with MetS in obese children and adolescents. Further studies examining interactions of eNOS haplotypes with environmental factors and other genetic markers are warranted.

Vascular endothelial growth factor haplotypes associated with childhood obesity.

Expansion of adipose tissue in obesity is associated with angiogenesis and adipose tissue mass depends on neovascularization. Vascular endothelial growth factor (VEGF) is the main angiogenic factor in the adipose tissue, and VEGF expression is tightly regulated at both transcriptional and translational levels. However, no previous study has tested the hypothesis that genetic polymorphisms in the VEGF gene could affect susceptibility to obesity. To test this hypothesis, we compared the distribution of genotypes and haplotypes including three VEGF genetic polymorphisms in obese children and adolescents with those found in healthy controls. We studied 172 healthy children and adolescents and 113 obese children and adolescents. Genotypes of three clinically relevant VEGF polymorphisms in the promoter region (C-2578A, G-1154A, and G-634C) of the VEGF gene were determined by TaqMan allele discrimination assay and real-time polymerase chain reaction. VEGF haplotypes were inferred using Haplo.stats and PHASE 2.1 programs. We found no differences in the distributions of VEGF genotypes and alleles (p > 0.05). However, the CAG haplotype was more frequent in the obese group than in the control group (4% versus 0%, respectively, in white subjects; p = 0.008; odds ratio = 10.148 (95% confidence interval: 1.098-93.788). Our findings suggest that VEGF haplotypes affect susceptibility to obesity in children and adolescents.

Identification of periodontal pathogens and severity of periodontitis in patients with and without chronic kidney disease.

OBJECTIVE: In this study of patients with chronic periodontitis (CP), the severity of the disease and the main periodontal pathogens identified in patients with chronic kidney disease (CKD) were compared with those detected in individuals without systemic disease. DESIGN: Nineteen patients with CP without evidence of systemic disease (control group), 25 patients with CP and CKD who were in the pre-dialysis stages (pre-dialysis group), and 22 patients with CP and CKD who were on renal replacement therapy (RRT group) were examined. The severity of CP was based on the investigation of probing depth (PD) and clinical attachment level (CAL). The definition and stage of CKD were based on the criteria proposed by the Kidney Disease Outcomes Quality Initiative of the National Kidney Foundation. Glomerular filtration rate (GFR) was estimated using the equation of Modification of Diet in Renal Disease and the identification of microorganisms in subgingival plaque was performed using polymerase chain reaction (PCR). RESULTS: Candida albicans, Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola were more common in patients who were on RRT and pre-dialysis than in control subjects. CP was more severe in patients with CKD. A strong association was observed between the frequency of C. albicans (P = 0.056), P.gingivalis (P = 0.008), T. denticola (P = 0.013) and CAL, when CKD patients were compared with the control group. CONCLUSION: CP is more severe and is associated with increased frequency of C. albicans, P. gingivalis, T. forsythia, and T. denticola in patients with CKD.

Assessment of matrix metalloproteinase (MMP)-2, MMP-8, MMP-9, and their inhibitors, the tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2 in obese children and adolescents.

OBJECTIVES: To compare the circulating levels of matrix metalloproteinase (MMP)-8, pro-MMP-2, pro-MMP-9, and total MMP-9, their endogenous inhibitors, the tissue inhibitors of metalloproteinases (TIMP)-1 and TIMP-2, and the MMP-8/TIMP-1, MMP-9/TIMP-1, and MMP-2/TIMP-2 ratios in normotensive obese children and adolescents with those found in non obese children and adolescents. DESIGN AND METHODS: We studied 40 obese and 40 non obese (controls) children and adolescents in this cross-sectional study. MMP and TIMP concentrations were measured in plasma samples by gelatin zymography and ELISA. RESULTS: Obese children and adolescents had higher circulating MMP-8 concentrations, lower plasma TIMP-1 concentrations, and higher MMP-8/TIMP-1 ratios than non obese controls (P<0.05). We found no differences in pro-MMP-9 or total MMP-9 levels, or in MMP-9/TIMP-1 ratios between groups (P>0.05). While we found no significant differences in pro-MMP-2 levels (P>0.05) obese subjects had higher TIMP-2 concentrations and lower pro-MMP-2/TIMP-2 ratios (P<0.05) than non obese controls. CONCLUSIONS: In conclusion, we found evidence indicating higher net MMP-8 (but not MMP-9 and MMP-2) activity in childhood obesity. The increased MMP-8 levels found in obese children suggest a possibly relevant pathophysiological mechanism that may be involved in the increase of cardiovascular risk associated with childhood obesity.

Anti-inflammatory effects of atorvastatin: modulation by the T-786C polymorphism in the endothelial nitric oxide synthase gene.

Statins produce cholesterol-independent, anti-inflammatory effects, which result at least in part from increased endothelial nitric oxide production. These effects may be modulated by polymorphisms in the endothelial nitric oxide synthase (eNOS) gene. Here, we examined whether the T-786C polymorphism of eNOS gene affects the concentrations of markers of atherosclerosis and inflammation (sCD40L, sVCAM-1, sICAM-1, sP-selectin, MCP-1, high sensitivity (hs)-CRP, MMP-2, MMP-9, and TIMP-1). We also studied whether atorvastatin-induced anti-inflammatory effects are modulated by this polymorphism. Healthy male volunteers (N=200), Caucasians, non-smokers, were genotyped for the T-786C polymorphism by restriction fragment length polymorphism. Subjects with TT or CC genotype received placebo for 14 days followed by 14 days of treatment with atorvastatin, 10mg/day p.o. The concentrations of inflammatory markers were measured with ELISA kits or by gelatin zymography. Serum cholesterol and LDL-cholesterol were significantly reduced after atorvastatin treatment in both genotype groups (P<0.05). No significant differences between genotype groups were found in the concentrations of the inflammatory markers after placebo. However, atorvastatin significantly reduced the concentrations of sCD40L, sVCAM-1, sP-selectin and MMP-9 in subjects with CC (but not TT) genotype (P<0.05). While atorvastatin decreased hs-CRP levels in both genotype groups (P<0.05), no significant effects were found on the concentrations of sICAM-1, MCP-1, pro-MMP-9, pro-MMP-2 and TIMP-1. These results suggest no effects for the T-786C polymorphism on the concentrations of inflammatory markers. However, this polymorphism modulates the anti-inflammatory effects of atorvastatin. These findings may be relevant for the primary prevention of cardiovascular events in subjects with CC genotype, who may be at increased cardiovascular risk and could benefit from treatment with statins.

Protective effects of atorvastatin in rat models of acute pulmonary embolism: involvement of matrix metalloproteinase-9.

OBJECTIVE: Matrix metalloproteinases (MMPs) have been implicated in the pathophysiology of acute pulmonary embolism (APE)-induced pulmonary hypertension. Here, we evaluate the effects of atorvastatin pretreatment on APE-induced pulmonary hypertension, 24-hr mortality rate, and changes in plasma and lung MMP-2 and MMP-9 activities. DESIGN: Controlled animal study. SETTING: University research laboratory. SUBJECTS: Male Wistar rats. INTERVENTIONS: Rats received atorvastatin (30 mg/kg/day orally) or tap water for 2 wks. In study 1, we examined whether atorvastatin affected APE-induced pulmonary hypertension by using a rat isolated lung perfusion model of APE. In study 2, we examined whether atorvastatin affects the survival rate after APE, which was induced by rapid intravenous injection of 14 mg/kg of a suspension of microspheres (or saline) into the tail vein. MEASUREMENTS AND MAIN RESULTS: Plasma nitrite/nitrate concentrations were measured by chemiluminescence. Pretreatment with atorvastatin was associated with 49% higher nitrite/nitrate levels compared with controls (p < .05). In study 1, whereas APE increased mean pulmonary artery pressure (MPAP) by 13.0 +/- 1.6 mm Hg in perfused lungs isolated from rats pretreated with water, pretreatment with atorvastatin attenuated by 27% the increases in MPAP after APE. In study 2, pretreatment with atorvastatin was associated with a significant increase in 24-hr survival rate after APE, which was 48% in embolized rats pretreated with water and 64% in rats pretreated with atorvastatin (p < .05). Gelatin zymography of lung and plasma MMP-2 and MMP-9 was performed. Lungs and plasma from embolized rats showed higher levels of both pro- and activated forms of MMP-9 compared with those from nonembolized animals (all p < .05). However, pretreatment with atorvastatin attenuated by 32% the increases in lung-activated MMP-9 levels after APE (p < .05). CONCLUSIONS: These results suggest that pretreatment with atorvastatin attenuates APE-induced pulmonary hypertension and increases 24-hr survival rate by mechanisms that result in attenuated increases in lung activated MMP-9 after APE.

Farmacogenética: princípios, aplicações e perspectivas / Pharmacogenetic: principles, aplications and perspectives

RESUMO: As respostas às drogas são influenciadas por múltiplos fatores, incluindo-se estado de saúde, influências ambientais e características genéticas. A farmacogenética é uma área da farmacologia clínica que estuda como diferenças genéticas entre indivíduos podem afetar as respostas às drogas. Neste sentido, polimorfismos genéticos em enzimas metabolizadoras, transportadores ou receptores contribuem para as variações nas respostas a medicamentos. Esta revisão objetiva introduzir alguns princípios, aplicações clínicas e perspectivas da farmacogenética, enfatizando alguns importantes achados clínicos e aplicações que podem contribuir para a melhoria da terapêutica.

L-arginine attenuates acute pulmonary embolism-induced increases in lung matrix metalloproteinase-2 and matrix metalloproteinase-9.

STUDY OBJECTIVES: To evaluate the effects of L-arginine on acute pulmonary embolism (APE)-induced pulmonary hypertension and increases in lung matrix metalloproteinase (MMP)-2 and MMP-9 activities. DESIGN: Prospective trial. SETTING: University laboratory. INTERVENTIONS: Using an isolated lung perfusion rat model of APE, we examined whether L-arginine (0, 0.5, 3, and 10 mmol/L; five to seven rats per group) attenuates the pulmonary hypertension induced by the injection of 6.6 mg/kg of 300 microm microspheres into the pulmonary artery. In a second series of experiments (6 to 11 rats per group), we investigated whether nonselective inhibition of nitric oxide (NO) synthases with N(G)-nitro-L-arginine methyl ester (L-NAME; 4 mmol/L) decreases the effects produced by L-arginine. Lung MMP-2 and MMP-9 activities were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis gelatin zymography. RESULTS: L-arginine at 0.5, 3, and 10 mmol/L attenuated APE-induced pulmonary hypertension by 25 to 42% (all p < 0.05). The protective effect of L-arginine was completely reversed by inhibition of NO synthesis with L-NAME. APE was associated with increased lung MMP-2 and MMP-9 activities (both p < 0.05). While L-arginine at 0.5 mmol/L produced no effect on MMPs, L-arginine 3 at mmol/L and 10 mmol/L attenuated the increases in MMP-2 and MMP-9 activities after APE (both p < 0.05). CONCLUSIONS: L-arginine attenuates APE-induced pulmonary hypertension through mechanisms involving increased NO synthesis and maybe attenuation of lung MMP-2 and MMP-9 activities.

Endothelial nitric oxide synthase gene haplotypes associated with circulating concentrations of nitric oxide products in healthy men.

OBJECTIVES: Controversy exists regarding the effects of polymorphisms in the endothelial nitric oxide synthase (eNOS) gene on nitrites/nitrates (NOx) plasma concentrations. In this study we compared the distribution of haplotypes involving three relevant eNOS polymorphisms (T-786C in the promoter region; b/a in intron 4, and Glu298Asp in exon 7) in healthy subjects with low and high circulating NOx levels. METHODS: We studied 154 healthy subjects (fasting, white males, who were non-smokers, 18-60 years of age, and not taking any medication). Genomic DNA was isolated from blood samples and genotypes were determined by PCR and restriction fragment length digestion. Circulating NOx was determined by chemiluminescence. RESULTS: Haplotype frequencies were compared in two groups of subjects: those with the 30 lowest NOx levels (group L) and those with the 30 highest NOx levels (group H). NOx levels in group L and H were 24.2+/-4.5 microM and 80.9+/-8.9 microM, respectively. Genotype frequencies for the three polymorphisms were not different when the two groups were compared (all P>0.05, chi-squared test). However, the haplotype including the alleles C (promoter), 4b (intron 4), and Glu (exon 7) was significantly more common in group L (16%) than in group H (4%) (P=0.0047). The frequencies of the remaining haplotypes were not different among group L and H. CONCLUSIONS: While eNOS genotypes are not significantly associated with changes in the circulating NOx concentrations, the specific eNOS haplotype that includes the 'C', '4b', and 'Glu' alleles is associated with lower circulating NOx concentrations.

The effect of sildenafil on pulmonary embolism-induced oxidative stress and pulmonary hypertension.

Acute pulmonary embolism (APE) is a major cause of pulmonary hypertension and death. We examined the effects of sildenafil on the hemodynamic changes caused by APE in anesthetized dogs. Sham-operated dogs (n = 3) received only saline. APE was induced by stepwise IV injections of 300 mum microspheres in amounts adjusted to increase mean pulmonary artery pressures by 20 mm Hg. Hemodynamic evaluation was performed at baseline, after APE was induced, and then after sildenafil 0.25 mg/kg (n = 8), or sildenafil 1 mg/kg + 0.3 mg . kg(-1) . h(-1) (n = 8) or saline (n = 9) infusions were started. Similar experiments were conducted to examine the effects of sildenafil in rat isolated perfused lung preparation. Plasma thiobarbituric acid reactive species were also determined in both studies to measure oxidative stress. Both doses of sildenafil reduced mean pulmonary artery pressures in dogs by approximately 8 to 16 mm Hg (both P < 0.05) and attenuated the increase in oxidative stress after APE. Mean arterial blood pressure remained unaltered after both doses of sildenafil. Sildenafil produced similar effects after APE in rat isolated perfused lung preparation. These findings indicate that IV sildenafil can selectively attenuate the increases in mean pulmonary artery pressures after APE, possibly through antioxidant mechanisms.

Consistent interethnic differences in the distribution of clinically relevant endothelial nitric oxide synthase genetic polymorphisms.

A maldistribution of endothelial nitric oxide synthase (eNOS) genetic variants may explain differences in NO-mediated effects and response to drugs among black and white subjects. While interethnic differences in the distribution of eNOS genetic variants exist in the American population, it is not known whether such interethnic differences exist in other populations. To test this possibility, we examined the distribution of genetic variants of three clinically relevant eNOS polymorphisms (T(-786)C in the promoter, the VNTR in intron 4, and the Glu298Asp variant in exon 7) in 136 black and 154 white subjects from a Brazilian population, which is very heterogeneous. We also estimated the haplotype frequency and evaluated associations between these variants. The Asp298 variant was more common in whites (32.8%) than in blacks (15.1%) (P < 0.004). Similarly, the C(-786) variant was more common in whites (41.9%) than in blacks (19.5%) (P < 0.0004). However, the 4a variant was more common in blacks (32.0%) than in whites (17.9%) (P < 0.003). The most common predicted haplotype in both ethnic groups combined only wild-type variants. While the second most common haplotype in blacks includes the variant 4a and the wild-type variants for the remaining polymorphisms, the second most common haplotype in whites includes the variants Asp298 and C(-786) and the wild-type variant for polymorphism in intron 4. The marked interethnic differences that we found in Brazilians are very similar to those previously reported in Americans. These findings strongly suggest a consistent difference in the distribution of eNOS genetic variants in blacks compared with whites and indicate that the interethnic differences do not vary with geographic origin.

eNOS genotype is without effect on circulating nitrite/nitrate level in healthy male population.

INTRODUCTION: Nitric oxide (NO) plays an important role in the regulation of the cardiovascular system. It is produced by endothelial nitric oxide synthase (eNOS), which exhibits genetic polymorphisms. Although the clinically relevant polymorphism T(+IhI-786)C reduces eNOS-promoter activity, it is not clear whether circulating nitrite/nitrate (NOx) are affected by this polymorphism. MATERIALS AND METHODS: We addressed this issue by studying a homogeneous group of 200 healthy subjects (males, Caucasians, nonsmokers, 18+IBM-56 years of age, and not taking any medication). Genotypes were determined by restriction fragment length polymorphism and circulating NOx were determined by chemiluminescence. RESULTS: We found nonsignificant effects of the T(+IhI-786)C polymorphism on circulating NOx (mean+ALE-S.D.=52.2+ALE-21.4, 49.0+ALE-17.8, and 45.9+ALE-16.8 +A7w-mol/L for genotypes +IBw-TT,+IB0 +IBw-TC,+IB0 and +IBw-CC,+IB0 respectively) and on total plasma cholesterol concentrations (both P>.05). No correlation was found between circulating NOx and total plasma cholesterol concentrations (P>.05). CONCLUSIONS: Our study provides strong evidence that the T(+IhI-786)C polymorphism does not affect plasma NOx concentrations, which are believed to reflect endogenous production of NO. Therefore, our results suggest that this polymorphism does not affect endogenous NO production.