Prevalence of clinical forms of Chagas disease: a systematic review and meta-analysis - data from the RAISE study.

Background: There is a lack of up-to-date estimates about the prevalence of Chagas disease (ChD) clinical presentations and, therefore, we aimed to assess the prevalence of clinical forms of ChD among seropositive adults, pooling available data. Methods: A systematic review was conducted in Medline, Embase, Biblioteca Virtual em Saúde and Cochrane databases looking for studies published from 1990 to August 2023, which investigated the prevalence of ChD clinical forms among seropositive adults, including: (i) indeterminate phase, (ii) chronic Chagas cardiomyopathy (CCM), (iii) digestive and (iv) mixed (CCM + digestive) forms. Pooled estimates and 95% confidence intervals (CI) were calculated using random-effects models. Studies quality and risk of bias was assessed with the Leboeuf-Yde and Lauritsen tool. Heterogeneity was assessed with the I2 statistic. The study was registered in the PROSPERO database (CRD42022354237). Findings: 1246 articles were selected for screening and 73 studies were included in the final analysis (17,132 patients, 44% men). Most studies were conducted with outpatients (n = 50), followed by population-based studies (n = 15). The pooled prevalence of the ChD clinical forms was: indeterminate 42.6% (95% CI: 36.9-48.6), CCM 42.7% (95% CI: 37.3-48.3), digestive 17.7% (95% CI: 14.9-20.9), and mixed 10.2% (95% CI: 7.9-13.2). In population-based studies, prevalence was lower for CCM (31.2%, 95% CI: 24.4-38.9) and higher for indeterminate (47.2%, 95% CI: 39.0-55.5) form. In meta-regression, age was inversely associated with the prevalence of indeterminate (ß = -0.05, P < 0.001) form, and directly associated with CCM (ß = 0.06, P < 0.001) and digestive (ß = 0.02, P < 0.001) forms. Heterogeneity was overall high. Interpretation: Compared to previous publications, our pooled estimates show a higher prevalence of CCM among ChD seropositive patients, but similar rates of the digestive form. Funding: This study was funded by the World Heart Federation, through a research collaboration with Novartis Pharma AG.

The Burden of Chagas Disease in the Contemporary World: The RAISE Study.

Chagas disease (ChD), a Neglected Tropical Disease, has witnessed a transformative epidemiological landscape characterized by a trend of reduction in prevalence, shifting modes of transmission, urbanization, and globalization. Historically a vector-borne disease in rural areas of Latin America, effective control measures have reduced the incidence in many countries, leading to a demographic shift where most affected individuals are now adults. However, challenges persist in regions like the Gran Chaco, and emerging oral transmission in the Amazon basin adds complexity. Urbanization and migration from rural to urban areas and to non-endemic countries, especially in Europe and the US, have redefined the disease's reach. These changing patterns contribute to uncertainties in estimating ChD prevalence, exacerbated by the lack of recent data, scarcity of surveys, and reliance on outdated models. Besides, ChD's lifelong natural history, marked by acute and chronic phases, introduces complexities in diagnosis, particularly in non-endemic regions where healthcare provider awareness is low. The temporal dissociation of infection and clinical manifestations, coupled with underreporting, has rendered ChD invisible in health statistics. Deaths attributed to ChD cardiomyopathy often go unrecognized, camouflaged under alternative causes. Understanding these challenges, the RAISE project aims to reassess the burden of ChD and ChD cardiomyopathy. The project is a collaborative effort of the World Heart Federation, Novartis Global Health, the University of Washington's Institute for Health Metrics and Evaluation, and a team of specialists coordinated by Brazil's Federal University of Minas Gerais. Employing a multidimensional strategy, the project seeks to refine estimates of ChD-related deaths, conduct systematic reviews on seroprevalence and prevalence of clinical forms, enhance existing modeling frameworks, and calculate the global economic burden, considering healthcare expenditures and service access. The RAISE project aspires to bridge knowledge gaps, raise awareness, and inform evidence-based health policies and research initiatives, positioning ChD prominently on the global health agenda.

MDR-1 C3435T polymorphism may affect blood pressure in resistant hypertensive patients independently of its effects on aldosterone release.

Aldosterone increases plasma volume and may be involved with resistant hypertension. P-glycoprotein is a transporter involved in the distribution and disposition of aldosterone, and is encoded by the MDR-1 gene. MDR-1 has functional polymorphisms that may affect P-glycoprotein expression. We hypothesized that the C(3435)T polymorphism in MDR-1 could be associated with resistant hypertension and with changes in hypertension-related parameters. We studied 105 healthy volunteers, 137 hypertensive patients responsive to treatment, and 83 resistant hypertensive patients. While we found no association of C(3435)T genotypes with resistance to treatment (p = 0.31), C allele was associated with hypertension (p = 0.03). Furthermore, the CC genotype was associated with higher systolic blood pressure (p < 0.01 for both daytime and nighttime, respectively) and diastolic blood pressure (p < 0.01 for both daytime and nighttime, respectively). This effect was probably independent of aldosterone, as we found no differences in aldosterone plasma levels, nor in pulse wave velocity (PVW) between the genotypes groups (p = 0.77 and p = 0.48, respectively). Our results show an association of C(3435)T with hypertension and with blood pressure levels in resistant hypertensive subjects.

Hypoadiponectinemia and aldosterone excess are associated with lack of blood pressure control in subjects with resistant hypertension.

Obesity, arterial stiffness and high aldosterone levels can interact to cause resistant hypertension (RHTN). Lower adiponectin (APN) levels may be significantly associated with hypertension. However, the importance of hypoadiponectinemia as a complicating factor in the lack of blood pressure (BP) control in individuals with RHTN has not been demonstrated. Ninety-six RHTN patients were classified into uncontrolled (UCRHTN, n = 44) and controlled (CRHTN, n = 52) subgroups. Their APN and aldosterone levels, office and ambulatory BP (ABPM) measurements, endothelium-dependent brachial artery responses (flow-mediated dilation (FMD)), left ventricular mass index (LVMI) and pulse wave velocity (PWV) were evaluated. The UCRHTN subgroup had increased aldosterone levels, as well as higher LVMI and PWV. In addition, lower APN levels and impaired FMD response were found in this subgroup. The brachial and ABPM pulse pressures were inversely associated with the APN levels (r = -0.45, P = 0.002; r = -0.33, P = 0.03, respectively), as were the aldosterone levels and the PWV (r = -0.38, P = 0.01; r = -0.36, P = 0.02, respectively) in UCRHTN patients. The PWV was only significantly influenced by the APN level in the UCRHTN subgroup in the multivariate regression analysis. None of the correlations mentioned above were observed in the CRHTN subgroup. Hypoadiponectinemia and high aldosterone levels may therefore be implicated in resistance to antihypertensive therapy related to arterial stiffness.

Acute cardiac and hemodynamic effects of sildenafil on resistant hypertension.

PURPOSE: Failure to control blood pressure (BP) despite the use of three or more drugs characterizes resistant hypertension (RHTN). Impaired endothelial function is associated with this condition and phosphodiesterase-5 inhibitors (PDE5i)-inhibiting cGMP breakdown-reduce BP in RHTN patients. We hypothesized that acute administration of PDE5i could ameliorate hemodynamic, endothelial parameters and left ventricular diastolic function (LVDF) in RHTN patients. Also, an exploratory analysis was performed to assess the influence of the T-786C endothelial NO synthase polymorphism on those responses. METHODS: Subjects (n = 26) underwent a 6-month clinical screening for RHTN diagnosis. Increasing doses of oral sildenafil were given at 30 min intervals (37.5, 50 and 100 mg) while continuous non-invasive hemodynamic measures were assessed. LVDF, flow mediated dilation (FMD), nitrite and cGMP levels were also determined. RESULTS: Mean arterial pressure and total peripheral resistance decreased in all patients (84.17 ± 21.04 to 75 ± 17.21 mmHg; 1149 ± 459.7 to 1037 ± 340 dyn.s/cm(-5), respectively). Likewise, sildenafil improved diastolic dysfunction parameters (Left atrial volume: 25 ± 5.8 to 20 ± 4.4; IVRT: 104 ± 19.33 to 88 ± 15.22; E/e' septal: 9.7 ± 3.8 to 7.9 ± 2.9; E/e' lateral: 7.7 ± 3.4 to 6.4 ± 3.2). No statistical changes were found in FMD, nitrite and cGMP with PDE5i. CONCLUSION: Our data suggest PDE5i acutely improves diastolic function and hemodynamic profile in RHTN subjects, despite unchanging endothelial dysfunction.

Leptina e aldosterona na atividade simpática na hipertensão resistente, com ou sem Diabetes Tipo 2 / Leptin and aldosterone in sympathetic activity in resistant hypertension with or without type 2 diabetes

FUNDAMENTO: A descoberta da leptina como um estimulador da atividade simpática trouxe uma nova perspectiva para os mecanismos fisiopatológicos da obesidade-hipertensão. OBJETIVO: Avaliamos a relação entre a atividade simpática aumentada e as concentrações plasmáticas de leptina e aldosterona em Hipertensos Resistentes (HR), comparando os grupos com e sem Diabetes Tipo 2 (DT2). MÉTODOS: Vinte e cinco pacientes HR foram avaliados por eletrocardiografia ambulatorial para análise da Variabilidade da Frequência Cardíaca (VFC) nos domínios do tempo e frequência, os quais foram estratificados em dois períodos: 24 horas e período Diurno (D), compreendendo as medidas entre 14 e 18h (domínio do tempo) e uma hora às 15h (domínio da frequência). RESULTADOS: O grupo DT2 (n = 10) apresentou maiores concentrações de aldosterona e leptina que o grupo não DT2 (n = 15) (26,0 ± 11,5 vs. 16,9 ± 7,0 ng/dL - p = 0,021; 81,368.7 ± 47,086.1 vs. 41,228.1 ± 24,523.1 pg/mL - p = 0,048, respectivamente). Houve correlação entre aldosterona e VFC no domínio da frequência em ambos os grupos. Não-DT2 apresentaram a aldosterona correlacionada com D baixa frequência em unidades normalizadas (BFnu) (r = 0,6 [0,12 - 0,85] p = 0,018) e D alta frequência em unidades normalizadas (AFnu) (r = -0,6 [-0,85 - -0,12] p = 0,018). No grupo com diabetes, a aldosterona correlacionou-se com DBFnu (r = 0,72 [0,16 - 0,93] p = 0,019) e DAFnu (r = -0,72 [-0,93 - -0,16] p = 0,019). Apesar da importância da leptina na atividade simpática aumentada na hipertensão, não houve correlação com VFC. CONCLUSÃO: A aldosterona parece estimular a atividade simpática em HR com ou sem DT2. Essa informação combinada com a eficácia clínica dos bloqueadores de receptor mineralocorticoide em HR pode reforçar a aldosterona como alvo terapêutico relevantes em HR. (Arq Bras Cardiol. 2012; [online].ahead print, PP.0-0).

BACKGROUND: The finding of adipocyte-derived hormone leptin as an overstimulator of sympathetic activity brought a new perspective to the pathophysiological mechanisms of obesity-hypertension. OBJECTIVES: As aldosterone also increases sympathetic activity, we aimed to assess the relationship between sympathetic overactivity and plasma leptin and aldosterone levels in resistant hypertension (RHTN), comparing the groups with and without T2D. METHODS: Twenty-five RHTN patients underwent ambulatory electrocardiography to analyze heart rate variability (HRV) in time and frequency domains, which were stratified into two periods: 24 hours and daytime (DT), comprising the records between 2:00 p.m to 6:00 p.m (time domain) and one hour at 3:00 p.m (frequency domain). RESULTS: T2D group (n=10) had higher serum aldosterone and plasma leptin levels than the non-T2D (n=15) (26.0±11.5 vs. 16.9±7.0 ng/dL - p=0.021; 81.368.7±47.086.1 vs 41.228.1±24.523.1 pg/mL - p=0.048, respectively). Both groups had aldosterone correlated with HRV in frequency domain. Non-T2D had aldosterone correlated with DT low frequency in normalized units (LF nu) (r=0.6 [0.12-0.85] p=0.018) and DT high frequency in normalized units (HF nu) (r=-0.6 [-0.85- -0.12] p=0.018). Type-2-diabetes group had aldosterone correlated with DT LF nu (r=0.72 [0.16-0.93] p=0.019) and DT HF nu (r=-0.72 [-0.93- -0.16] p=0.019). However, despite of the importance of leptin in sympathetic overactivity in hypertension, leptin did not correlate with HRV. CONCLUSION: Aldosterone seems to overdrive sympathetic activity in RHTN with and without T2D. This information combined with the clinical efficacy of mineralocorticoid receptor blocker in RHTN may reinforce that aldosterone is a major player to be a therapeutic target in RHTN. (Arq Bras Cardiol. 2012; [online].ahead print, PP.0-0).

Leptin and aldosterone in sympathetic activity in resistant hypertension with or without type 2 diabetes.

BACKGROUND: The finding of adipocyte-derived hormone leptin as an overstimulator of sympathetic activity brought a new perspective to the pathophysiological mechanisms of obesity-hypertension. OBJECTIVES: As aldosterone also increases sympathetic activity, we aimed to assess the relationship between sympathetic overactivity and plasma leptin and aldosterone levels in resistant hypertension (RHTN), comparing the groups with and without T2D. METHODS: Twenty-five RHTN patients underwent ambulatory electrocardiography to analyze heart rate variability (HRV) in time and frequency domains, which were stratified into two periods: 24 hours and daytime (DT), comprising the records between 2:00 p.m to 6:00 p.m (time domain) and one hour at 3:00 p.m (frequency domain). RESULTS: T2D group (n=10) had higher serum aldosterone and plasma leptin levels than the non-T2D (n=15) (26.0 ± 11.5 vs. 16.9 ± 7.0 ng/dL - p=0.021; 81.368.7 ± 47.086.1 vs 41.228.1 ± 24.523.1 pg/mL - p=0.048, respectively). Both groups had aldosterone correlated with HRV in frequency domain. Non-T2D had aldosterone correlated with DT low frequency in normalized units (LF nu) (r=0.6 [0.12-0.85] p=0.018) and DT high frequency in normalized units (HF nu) (r=-0.6 [-0.85- -0.12] p=0.018). Type-2-diabetes group had aldosterone correlated with DT LF nu (r=0.72 [0.16-0.93] p=0.019) and DT HF nu (r=-0.72 [-0.93- -0.16] p=0.019). However, despite of the importance of leptin in sympathetic overactivity in hypertension, leptin did not correlate with HRV. CONCLUSION: Aldosterone seems to overdrive sympathetic activity in RHTN with and without T2D. This information combined with the clinical efficacy of mineralocorticoid receptor blocker in RHTN may reinforce that aldosterone is a major player to be a therapeutic target in RHTN.

Sildenafil preserves diastolic relaxation after reduction by L-NAME and increases phosphodiesterase-5 in the intercalated discs of cardiac myocytes and arterioles.

OBJECTIVES: We investigated the influence of sildenafil on cardiac contractility and diastolic relaxation and examined the distribution of phosphodiesterase-5 in the hearts of hypertensive rats that were treated with by NG-nitro-L-arginine methyl ester (L-NAME). METHODS: Male Wistar rats were treated with L-NAME and/or sildenafil for eight weeks. The Langendorff method was used to examine the effects of sildenafil on cardiac contractility and diastolic relaxation. The presence and location of phosphodiesterase-5 and phosphodiesterase-3 were assessed by immunohistochemistry, and cGMP plasma levels were measured by ELISA. RESULTS: In isolated hearts, sildenafil prevented the reduction of diastolic relaxation (dP/dt) that was induced by L-NAME. In addition, phosphodiesterase-5 immunoreactivity was localized in the intercalated discs between the myocardial cells. The staining intensity was reduced by L-NAME, and sildenafil treatment abolished this reduction. Consistent with these results, the plasma levels of cGMP were decreased in the L-NAME-treated rats but not in rats that were treated with L-NAME + sildenafil. CONCLUSION: The sildenafil-induced attenuation of the deleterious hemodynamic and cardiac morphological effects of L-NAME in cardiac myocytes is mediated (at least in part) by the inhibition of phosphodiesterase-5.

Gene variation in resistant hypertension: multilocus analysis of the angiotensin 1-converting enzyme, angiotensinogen, and endothelial nitric oxide synthase genes.

Resistant hypertension, a complex multifactorial hypertensive disease, is triggered by genetic and environmental factors and involves multiple physiological pathways. Single genetic variants may not reveal significant associations with resistant hypertension because their effects may be dependent on gene-gene or gene-environment interactions. We examined the interaction of angiotensin I-converting enzyme (ACE), angiotensinogen (AGT), and endothelial nitric oxide synthase (NOS3) polymorphisms with environmental factors (gender, age, body mass index, glycemia, total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, triglycerides, estimated glomerular filtration rate, and urinary sodium excretion) in 70 resistant, 80 well-controlled hypertensive patients, and 70 normotensive controls. All subjects were genotyped for ACE insertion/deletion (rs1799752); AGT M235T (rs699), and NOS3 Glu298Asp (rs 1799983). Multifactorial associations were tested using two statistical methods: the traditional parametric method (adjusted logistic regression analysis) and gene-gene and gene-environment interactions evaluated by multifactor dimensionality reduction analyses. While adjusted logistic regression found no significant association between the studied polymorphisms and controlled or resistant hypertension, the multifactor dimensionality reduction analyses showed that carriers of the AGT 235T allele were at increased risk for resistant hypertension, especially if they were older than 50 years. The AGT 235T allele constituted an independent risk factor for resistant hypertension.

Relationship of autonomic imbalance and circadian disruption with obesity and type 2 diabetes in resistant hypertensive patients.

BACKGROUND: Hypertension, diabetes and obesity are not isolated findings, but a series of interacting interactive physiologic derangements. Taking into account genetic background and lifestyle behavior, AI (autonomic imbalance) could be a common root for RHTN (resistant hypertension) or RHTN plus type 2 diabetes (T2D) comorbidity development. Moreover, circadian disruption can lead to metabolic and vasomotor impairments such as obesity, insulin resistance and resistant hypertension. In order to better understand the triggered emergence of obesity and T2D comorbidity in resistant hypertension, we investigated the pattern of autonomic activity in the circadian rhythm in RHTN with and without type 2 diabetes (T2D), and its relationship with serum adiponectin concentration. METHODS: Twenty five RHTN patients (15 non-T2D and 10 T2D, 15 males, 10 females; age range 34 to 70 years) were evaluated using the following parameters: BMI (body mass index), biochemical analysis, serum adiponectinemia, echocardiogram and ambulatory electrocardiograph heart rate variability (HRV) in time and frequency domains stratified into three periods: 24 hour, day time and night time. RESULTS: Both groups demonstrated similar characteristics despite of the laboratory analysis concerning T2D like fasting glucose, HbA1c levels and hypertriglyceridemia. Both groups also revealed disruption of the circadian rhythm: inverted sympathetic and parasympathetic tones during day (parasympathetic > sympathetic tone) and night periods (sympathetic > parasympathetic tone). T2D group had increased BMI and serum triglyceride levels (mean 33.7 ± 4.0 vs 26.6 ± 3.7 kg/m² - p = 0.00; 254.8 ± 226.4 vs 108.6 ± 48.7 mg/dL - p = 0.04), lower levels of adiponectin (6729.7 ± 3381.5 vs 10911.5 ± 5554.0 ng/mL - p = 0.04) and greater autonomic imbalance evaluated by HRV parameters in time domain compared to non-T2D RHTN patients. Total patients had HRV correlated positively with serum adiponectin (r = 0.37 [95% CI -0.04 - 1.00] p = 0.03), negatively with HbA1c levels (r = -0.58 [95% CI -1.00 - -0.3] p = 0.00) and also adiponectin correlated negatively with HbA1c levels (r = -0.40 [95% CI -1.00 - -0.07] p = 0.02). CONCLUSION: Type 2 diabetes comorbidity is associated with greater autonomic imbalance, lower adiponectin levels and greater BMI in RHTN patients. Similar circadian disruption was also found in both groups indicating the importance of lifestyle behavior in the genesis of RHTN.

Sildenafil preserves diastolic relaxation after reduction by L-NAME and increases phosphodiesterase-5 in the intercalated discs of cardiac myocytes and arterioles

OBJECTIVES: We investigated the influence of sildenafil on cardiac contractility and diastolic relaxation and examined the distribution of phosphodiesterase-5 in the hearts of hypertensive rats that were treated with by NG-nitro-L-arginine methyl ester (L-NAME). METHODS: Male Wistar rats were treated with L-NAME and/or sildenafil for eight weeks. The Langendorff method was used to examine the effects of sildenafil on cardiac contractility and diastolic relaxation. The presence and location of phosphodiesterase-5 and phosphodiesterase-3 were assessed by immunohistochemistry, and cGMP plasma levels were measured by ELISA. RESULTS: In isolated hearts, sildenafil prevented the reduction of diastolic relaxation (dP/dt) that was induced by L-NAME. In addition, phosphodiesterase-5 immunoreactivity was localized in the intercalated discs between the myocardial cells. The staining intensity was reduced by L-NAME, and sildenafil treatment abolished this reduction. Consistent with these results, the plasma levels of cGMP were decreased in the L-NAME-treated rats but not in rats that were treated with L-NAME + sildenafil. CONCLUSION: The sildenafil-induced attenuation of the deleterious hemodynamic and cardiac morphological effects of L-NAME in cardiac myocytes is mediated (at least in part) by the inhibition of phosphodiesterase-5.

Vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (NOS3) polymorphisms are associated with high relapse risk in childhood acute lymphoblastic leukemia (ALL).

BACKGROUND: Angiogenesis has been shown as an important process in hematological malignancies. It consists in endothelial proliferation, migration, and tube formation following pro-angiogenic factors releasing, specially the vascular endothelial growth factor (VEGF), which angiogenic effect seems to be dependent on nitric oxide (NO). We examined the association among functional polymorphisms in these two angiogenesis related genes: VEGF (-2578C>A, -1154G>A, and -634G>C) and NOS3 (-786T>C, intron 4 b>a, and Glu298Asp) with prognosis of childhood acute lymphoblastic leukemia (ALL). METHODS: The genotypes were determined and haplotypes estimated in 105 ALL patients that were divided in 2 groups: high risk (HR) and low risk of relapse (LR) patients. In addition, event-free survival curves according to genotypes were assessed. RESULTS: The group HR compared to the LR showed a higher frequency of the alleles -2578C and -634C and the haplotype CGC for VEGF (0.72 vs. 0.51, p<0.008; 0.47 vs. 0.26, p<0.008; and 42.1 vs. 14.5, p<0.006; respectively) and a lower frequency of the haplotype CbGlu (0.4 vs. 8.8,p<0.006), for NOS3. CONCLUSION: Polymorphisms of VEGF and NOS3 genes are associated with high risk of relapse, therefore may have a prognostic impact in childhood ALL.

Lercanidipine reduces matrix metalloproteinase-2 activity and reverses vascular dysfunction in renovascular hypertensive rats.

Increased expression/activity of matrix metalloproteinases (MMPs), especially MMP-2, plays a role in the vascular alterations induced by hypertension, and increased oxidative stress is a major factor activating MMPs. Here, we hypothesized that lercanidipine, a calcium channel blocker, could attenuate the increases in oxidative stress and MMP-2 expression/activity in the two-kidney, one-clip (2K-1C) hypertensive rats. Sham-operated or 2K-1C hypertension rats were treated with lercanidipine 2.5 mg/kg/day (or vehicle) starting three weeks after hypertension was induced. Systolic blood pressure was monitored weekly. After five weeks of treatment, aortic rings were isolated to assess endothelium-dependent and independent relaxations. Quantitative morphometry of structural changes in the aortic wall were studied in hematoxylin/eosin sections. Aortic MMP-2 levels were determined by gelatin zymography. Aortic MMP-2/tissue inhibitor of metalloproteinases (TIMP)-2 mRNA levels were determined by quantitative real-time RT-PCR. Plasma thiobarbituric acid reactive substances concentrations were determined using a fluorometric method. Lercanidipine attenuated 2K-1C hypertension (224+/-12 versus 183+/-11 mm Hg in 2K-1C rats and 2K-1C + Lercandipine rats, respectively; P<0.01) and prevented the reduction in endothelium-dependent vasorelaxation found in 2K-1C rats. Increased MMP-2 and Pro-MMP-2 levels were found in the aortas of 2K-1C rats (all P<0.05). Lercandipine attenuated 2K-1C-induced increases in MMP-2 by more than 60% and blunted 2K-1C-induced increases in oxidative stress (both P<0.001). While hypertension-induced significant aortic wall hypertrophy and approximately 9-fold increases in the ratio of MMP-2/TIMP-2 mRNA expression (both P<0.05), lercandipine did not affect these changes. These results suggest that lercanidipine produces antihypertensive effects and reverses the endothelial dysfunction associated with 2K-1C hypertension, probably through mechanisms involving antioxidant effects leading to lower MMP-2 activation.

Inverse relationship between markers of nitric oxide formation and plasma matrix metalloproteinase-9 levels in healthy volunteers.

BACKGROUND: Nitric oxide (NO) is a major regulator of cardiovascular homeostasis and has anti-atherogenic properties. Reduced NO formation is associated with endothelial dysfunction and with cardiovascular risk factors. Although NO downregulates the expression and activity of the pro-atherogenic enzyme matrix metalloproteinase-9 (MMP-9), no previous clinical study has examined whether endogenous NO formation is inversely associated with the circulating levels of pro-MMP-9, which are associated with cardiovascular events. We examined this hypothesis in 175 healthy male subjects who were non-smokers. METHODS: To assess NO bioavailability, the plasma concentrations of nitrite, nitrate, and cGMP were determined using an ozone-based chemiluminescence assay and an enzyme immunoassay. Pro-MMP-9 and pro-MMP-2 levels were measured in plasma samples by gelatin zymography. RESULTS: We found significant negative correlations between pro-MMP-9 levels and plasma nitrite (P=0.035, rs= -0.159), nitrate (P=0.040, rs= -0.158), and cGMP (P=0.011, rs= -0.189) concentrations. However, no significant correlations were found between pro-MMP-2 levels and the plasma concentrations of markers of NO bioavailability (all P>0.05). CONCLUSIONS: There is an inverse relationship between markers of NO formation and plasma MMP-9 levels. This finding may shed some light on the possible mechanisms involved in the increased cardiovascular risk of apparently healthy subjects with low NO bioavailability or high circulating levels of pro-MMP-9.

Functional polymorphisms in the promoter of the matrix metalloproteinase-9 (MMP-9) gene are not linked with significant plasma MMP-9 variations in healthy subjects.

BACKGROUND: Matrix metalloproteinase-9 (MMP-9) is involved in the degradation of the extracellular matrix during physiological and pathological processes. Two functional polymorphisms [C(-1562)T and microsatellite (CA)13-25] in the promoter region of the MMP-9 gene have been associated with several diseases. The aim of this study was to examine whether these MMP-9 polymorphisms and haplotypes are linked with plasma MMP-9 variations in healthy subjects. METHODS: We studied 177 healthy male white volunteers (age range 20-55 years) who were non-smokers and not taking any medication. Genomic DNA was extracted from whole blood and genotypes for the C(-1562)T and the microsatellite (CA)n polymorphisms were determined. MMP-9 levels were measured in plasma samples by gelatin zymography. RESULTS: The frequency of the alleles C and T for the C(-1562)T polymorphism were 90% and 10%, respectively. The frequency of the alleles with less than 21 CA repeats (L) and with 21 repeats or higher (H) were 47% and 53%, respectively. We found no differences in plasma MMP-9 levels among the genotype groups or among different haplotypes (all p>0.05). CONCLUSIONS: These findings suggest that functional polymorphisms in the promoter of the MMP-9 gene are not linked with significant plasma MMP-9 variations in healthy subjects.

Rapid separation of serum does not avoid artificially higher matrix metalloproteinase (MMP)-9 levels in serum versus plasma.

OBJECTIVES: To examine whether the time between blood drawing and centrifugation (TBDC) affects the levels of matrix metalloproteinase (MMP)-9 and MMP-2 levels in serum and in plasma samples, and to assess whether there is correlation between MMP-9 and MMP-2 levels in serum and plasma samples. DESIGN AND METHODS: Serum and plasma samples (N=8) were separated from venous blood collected into citrate, heparin, and EDTA tubes, which were either centrifuged immediately or after 5, 10, 20, or 30 min after blood drawing. We assessed the correlation between MMP-9/MMP-2 in serum and citrate, heparin, and plasma samples (N=20), which were assayed for gelatine zymography of MMP-2 and MMP-9. RESULTS: MMPs are released by platelets or leukocytes during platelet activation or sampling process, thus leading to artificially higher MMP-9 levels in serum compared with citrate, heparin, or EDTA plasma samples, independently of TBDC. Citrate and heparin plasma samples had the lowest Pro-MMP-9 and MMP-9 levels, which correlated with each other. Pro-MMP-9 levels in serum correlated with Pro-MMP-9 levels in EDTA or citrate plasma, but not with heparin plasma. While no significant correlations were found between MMP-9 levels in serum and those found in plasma samples, the total MMP-9 levels (Pro-MMP-9+MMP-9) in serum and in plasma samples correlated with each other. No significant differences were found in pro-MMP-2 levels. CONCLUSIONS: These results suggest that the circulating levels of MMP-9 should be assessed in citrate or heparin plasma samples, but not in serum samples because of artificially higher MMP-9 levels in serum, independently of TBDC, and because they do not correlate with the MMP-9 levels in plasma samples.

Genetic polymorphism of matrix metalloproteinase (MMP)-9 does not affect plasma MMP-9 activity in healthy subjects.

BACKGROUND: Plasma MMP-9 levels have been shown to predict cardiovascular risk, and a functional substitution C to T at position -1562 in the promoter region of the MMP-9 gene has been associated with the severity of cardiovascular diseases. We examined the association between the C(-1562)T polymorphism and MMP-9 activity in healthy subjects. METHODS: We studied 200 healthy male white volunteers (age range: 20-55 y) who were nonsmokers and were not taking medicines. Genomic DNA was extracted and genotypes for the C(-1562)T polymorphism were determined by PCR and restriction fragment length digestion. Plasma was assayed for pro-MMP-9 and MMP-9 activities by gelatin zymography. RESULTS: The frequency of the alleles "C" and "T" were 90% and 10%, respectively. Because of the relatively low frequency of the TT genotype, we combined both TT and CT genotypes together (CT+TT group) and compared with the CC genotype group. We found no differences in pro-MM9 and MMP-9 activity levels among the genotype groups (both P>0.05). CONCLUSIONS: While the present study indicates lack of effect for the C(-1562)T polymorphism on MMP-9 activity in plasma, it is possible that the C(-1562)T polymorphism contributes to an increased cardiovascular risk under conditions of induced MMP-9 expression.