Chromosomal Region 11p14.1 is Associated with Pharmacokinetics and Pharmacodynamics of Bisoprolol.

Purpose: Bisoprolol is a widely used beta-blocker in patients with cardiovascular diseases. As with other beta-blockers, there is variability in response to bisoprolol, but the underlying reasons for this have not been clearly elucidated. Our aim was to investigate genetic factors that affect bisoprolol pharmacokinetics (PK) and pharmacodynamics (PD), and potentially the clinical outcomes. Patients and Methods: Patients with non-ST elevation acute coronary syndrome were recruited prospectively on admission to hospital and followed up for up to 2 years. Patients from this cohort who were on treatment with bisoprolol, at any dose, had bisoprolol adherence data and a plasma sample, one month after discharge from index hospitalisation were included in the study. Individual bisoprolol clearance values were estimated using population pharmacokinetic modeling. Genome-wide association analysis after genotyping was undertaken using an Illumina HumanOmniExpressExome-8 v1.0 BeadChip array, while CYP2D6 copy number variations were determined by PCR techniques and phenotypes for CYP2D6 and CYP3A were inferred from the genotype. GWAS significant SNPs were analysed for heart rate response to bisoprolol in an independent cohort of hypertensive subjects. Results: Six hundred twenty-two patients on bisoprolol underwent both PK and genome wide analysis. The mean (IQR) of the estimated clearance in this population was 13.6 (10.0-18.0) L/h. Bisoprolol clearance was associated with rs11029955 (p=7.17×10-9) mapped to the region of coiled-coil domain containing 34 region (CCDC34) on chromosome 11, and with rs116702638 (p=2.54×10-8). Each copy of the minor allele of rs11029955 was associated with 2.2 L/h increase in clearance. In an independent cohort of hypertensive subjects, rs11029955 was associated with 24-hour heart rate response to 4-week treatment with bisoprolol (p= 9.3×10-5), but not with rs116702638. Conclusion: A novel locus on the chromosomal region 11p14.1 was associated with bisoprolol clearance in a real-world cohort of patients and was validated in independent cohort with a pharmacodynamic association.

Antibody-mediated delivery of LIGHT to the tumor boosts natural killer cells and delays tumor progression.

LIGHT is a member of the tumor necrosis factor superfamily, which has been claimed to mediate anti-tumor activity on the basis of cancer cures observed in immunocompetent mice bearing transgenic LIGHT-expressing tumors. The preclinical development of a LIGHT-based therapeutic has been hindered by the lack of functional stability exhibited by this protein. Here, we describe the cloning, expression, and characterization of five antibody-LIGHT fusion proteins, directed against the alternatively spliced extra domain A of fibronectin, a conserved tumor-associated antigen. Among the five tested formats, only the sequential fusion of the F8 antibody in single-chain diabody format, followed by the LIGHT homotrimer expressed as a single polypeptide, yielded a protein (termed "F8-LIGHT") that was not prone to aggregation. A quantitative biodistribution analysis in tumor-bearing mice, using radio-iodinated protein preparations, confirmed that F8-LIGHT was able to preferentially accumulate at the tumor site, with a tumor-to-blood ratio of ca. five to one 24 hours after intravenous administration. Tumor therapy experiments, performed in two murine tumor models (CT26 and WEHI-164), featuring different levels of lymphocyte infiltration into the neoplastic mass, revealed that F8-LIGHT could significantly reduce tumor-cell growth and was more potent than a similar fusion protein (KSF-LIGHT), directed against hen egg lysozyme and serving as negative control of irrelevant specificity in the mouse. At a mechanistic level, the activity of F8-LIGHT was mainly due to an intratumoral expansion of natural killer cells, whereas there was no evidence of expansion of CD8 + T cells, neither in the tumor, nor in draining lymph nodes. Abbreviations: CTLA-4: Cytotoxic T-lymphocytes-associated protein 4; EGFR: Epidermal growth factor receptor; HVEM: Herpesvirus entry mediator; IFNγ: Interferon-gamma; LIGHT: Lymphotoxin, exhibits inducible expression and competes with HSV glycoprotein D for binding to herpesvirus entry mediator, a receptor expressed on T lymphocytes; LTßR: Lymphotoxin beta receptor; NF-κB: Nuclear factor "kappa-light-chain-enhancer" of activated B cells; NK: Natural killer cells; PD-1: Programmed cell death protein 1; PD-L1: Programmed death-ligand 1; TNF: Tumor necrosis factor.

A Multi-Species Phenotypic Screening Assay for Leishmaniasis Drug Discovery Shows That Active Compounds Display a High Degree of Species-Specificity.

High genetic and phenotypic variability between Leishmania species and strains within species make the development of broad-spectrum antileishmanial drugs challenging. Thus, screening panels consisting of several diverse Leishmania species can be useful in enabling compound prioritization based on their spectrum of activity. In this study, a robust and reproducible high content assay was developed, and 1280 small molecules were simultaneously screened against clinically relevant cutaneous and visceral species: L. amazonensis, L. braziliensis, and L. donovani. The assay is based on THP-1 macrophages infected with stationary phase promastigotes and posterior evaluation of both compound antileishmanial activity and host cell toxicity. The profile of compound activity was species-specific, and out of 51 active compounds, only 14 presented broad-spectrum activity against the three species, with activities ranging from 52% to 100%. Notably, the compounds CB1954, Clomipramine, Maprotiline, Protriptyline, and ML-9 presented pan-leishmanial activity, with efficacy greater than 70%. The results highlight the reduced number of compound classes with pan-leishmanial activity that might be available from diversity libraries, emphasizing the need to screen active compounds against a panel of species and strains. The assay reported here can be adapted to virtually any Leishmania species without the need for genetic modification of parasites, providing the basis for the discovery of broad spectrum anti-leishmanial agents.

A Genome-wide Association Study of Circulating Levels of Atorvastatin and Its Major Metabolites.

Atorvastatin (ATV) is frequently prescribed and generally well  tolerated, but can lead to myotoxicity, especially at higher doses. A genome-wide association study of circulating levels of ATV, 2-hydroxy (2-OH) ATV, ATV lactone (ATV L), and 2-OH ATV L was performed in 590 patients who had been hospitalized with a non-ST elevation acute coronary syndrome 1 month earlier and were on high-dose ATV (80 mg or 40 mg daily). The UGT1A locus (lead single nucleotide polymorphism, rs887829) was strongly associated with both increased 2-OH ATV/ATV (P = 7.25 × 10-16 ) and 2-OH ATV L/ATV L (P = 3.95 × 10-15 ) metabolic ratios. Moreover, rs45446698, which tags CYP3A7*1C, was nominally associated with increased 2-OH ATV/ATV (P = 6.18 × 10-7 ), and SLCO1B1 rs4149056 with increased ATV (P = 2.21 × 10-6 ) and 2-OH ATV (P = 1.09 × 10-6 ) levels. In a subset of these patients whose levels of ATV and metabolites had also been measured at 12 months after hospitalization (n = 149), all of these associations remained, except for 2-OH ATV and rs4149056 (P = 0.057). Clinically, rs4149056 was associated with increased muscular symptoms (odds ratio (OR) 3.97; 95% confidence interval (CI) 1.29-12.27; P = 0.016) and ATV intolerance (OR 1.55; 95% CI 1.09-2.19; P = 0.014) in patients (n = 870) primarily discharged on high-dose ATV. In summary, both novel and recognized genetic associations have been identified with circulating levels of ATV and its major metabolites. Further study is warranted to determine the clinical utility of genotyping rs4149056 in patients on high-dose ATV.

Investigating the clinical factors and comedications associated with circulating levels of atorvastatin and its major metabolites in secondary prevention.

Aims The lipid-lowering drug, atorvastatin (ATV), is 1 of the most commonly prescribed medications worldwide. The aim of this study was to comprehensively investigate and characterise the clinical factors and comedications associated with circulating levels of ATV and its metabolites in secondary prevention clinical practice. Methods The plasma concentrations of ATV, 2-hydroxy (2-OH) ATV, ATV lactone (ATV L) and 2-OH ATV L were determined in patients 1 month after hospitalisation for a non-ST elevation acute coronary syndrome. Factors were identified using all subsets multivariable regression and model averaging with the Bayesian information criterion. Exploratory genotype-stratified analyses were conducted using ABCG2 rs2231142 (Q141K) and CYP2C19 metaboliser status to further investigate novel associations. Results A total of 571 patients were included; 534 and 37 were taking ATV 80 mg and 40 mg daily, respectively. Clinical factors associated with ATV and/or its metabolite levels included age, sex, body mass index and CYP3A inhibiting comedications. Smoking was newly associated with increased ATV lactonisation and reduced hydroxylation. Proton pump inhibitors (PPIs) and loop diuretics were newly associated with modestly increased levels of ATV (14% and 38%, respectively) and its metabolites. An interaction between PPIs and CYP2C19 metaboliser status on exposure to specific ATV analytes (e.g. interaction P = .0071 for 2-OH ATV L) was observed. Overall model R2 values were 0.14-0.24.ConclusionMultiple factors were associated with circulating ATV and metabolite levels, including novel associations with smoking and drug-drug(-gene) interactions involving PPIs and loop diuretics. Further investigations are needed to identify additional factors that influence ATV exposure.

A multi-species phenotypic screening assay for leishmaniasis drug discovery shows that active compounds display a high degree of species-specificity

High genetic and phenotypic variability between Leishmania species and strains within species make the development of broad-spectrum antileishmanial drugs challenging. Thus, screening panels consisting of several diverse Leishmania species can be useful in enabling compound prioritization based on their spectrum of activity. In this study, a robust and reproducible high content assay was developed, and 1280 small molecules were simultaneously screened against clinically relevant cutaneous and visceral species: L. amazonensis, L. braziliensis, and L. donovani. The assay is based on THP-1 macrophages infected with stationary phase promastigotes and posterior evaluation of both compound antileishmanial activity and host cell toxicity. The profile of compound activity was species-specific, and out of 51 active compounds, only 14 presented broad-spectrum activity against the three species, with activities ranging from 52% to 100%. Notably, the compounds CB1954, Clomipramine, Maprotiline, Protriptyline, and ML-9 presented pan-leishmanial activity, with efficacy greater than 70%. The results highlight the reduced number of compound classes with pan-leishmanial activity that might be available from diversity libraries, emphasizing the need to screen active compounds against a panel of species and strains. The assay reported here can be adapted to virtually any Leishmania species without the need for genetic modification of parasites, providing the basis for the discovery of broad spectrum anti-leishmanial agents.

A multi-species phenotypic screening assay for leishmaniasis drug discovery shows that active compounds display a high degree of species-specificity

High genetic and phenotypic variability between Leishmania species and strains within species make the development of broad-spectrum antileishmanial drugs challenging. Thus, screening panels consisting of several diverse Leishmania species can be useful in enabling compound prioritization based on their spectrum of activity. In this study, a robust and reproducible high content assay was developed, and 1280 small molecules were simultaneously screened against clinically relevant cutaneous and visceral species: L. amazonensis, L. braziliensis, and L. donovani. The assay is based on THP-1 macrophages infected with stationary phase promastigotes and posterior evaluation of both compound antileishmanial activity and host cell toxicity. The profile of compound activity was species-specific, and out of 51 active compounds, only 14 presented broad-spectrum activity against the three species, with activities ranging from 52% to 100%. Notably, the compounds CB1954, Clomipramine, Maprotiline, Protriptyline, and ML-9 presented pan-leishmanial activity, with efficacy greater than 70%. The results highlight the reduced number of compound classes with pan-leishmanial activity that might be available from diversity libraries, emphasizing the need to screen active compounds against a panel of species and strains. The assay reported here can be adapted to virtually any Leishmania species without the need for genetic modification of parasites, providing the basis for the discovery of broad spectrum anti-leishmanial agents.

Development, validation and application of a novel HPLC-MS/MS method for the quantification of atorvastatin, bisoprolol and clopidogrel in a large cardiovascular patient cohort.

Cardiovascular disease is a leading cause of morbidity, mortality, and healthcare expenditure worldwide. Importantly, there is interindividual variation in response to cardiovascular medications, leading to variable efficacy and adverse events. Therefore a rapid, selective, sensitive and reproducible multi-analyte HPLC-MS/MS assay for the quantification in human plasma of atorvastatin, its major metabolites 2-hydroxyatorvastatin, atorvastatin lactone and 2-hydroxyatorvastatin lactone, plus bisoprolol and clopidogrel-carboxylic acid has been developed, fully validated, and applied to a large patient study. Fifty microliter plasma samples were extracted with a simple protein precipitation procedure involving acetonitrile with acetic acid (0.1%, v/v). Chromatographic separation was via a 2.7 µm Halo C18 (50 × 2.1 mm ID, 90 Å) column and gradient elution at a flow rate of 500 µL/min consisting of a mobile phase of water (A) and acetonitrile (B), each containing 0.1% formic acid (v/v), over a 6.0 min run time. The six analytes and their corresponding six deuterated internal standards underwent positive ion electrospray ionisation and were detected with multiple reaction monitoring. The developed method was fully validated with acceptable selectivity, carryover, dilution integrity, and within-run and between-run accuracy and precision. Mean extraction recovery for the analytes was 92.7-108.5%, and internal standard-normalised matrix effects had acceptable precision (coefficients of variation 2.2-12.3%). Moreover, all analytes were stable under the tested conditions. Atorvastatin lactone to acid interconversion was assessed and recommendations for its minimisation are made. The validated assay was successfully applied to analyse 1279 samples from 1024 patients recruited to a cardiovascular secondary prevention prospective study.

Decoding resistant hypertension signalling pathways.

Resistant hypertension (RH) is a clinical condition in which the hypertensive patient has become resistant to drug therapy and is often associated with increased cardiovascular morbidity and mortality. Several signalling pathways have been studied and related to the development and progression of RH: modulation of sympathetic activity by leptin and aldosterone, primary aldosteronism, arterial stiffness, endothelial dysfunction and variations in the renin-angiotensin-aldosterone system (RAAS). miRNAs comprise a family of small non-coding RNAs that participate in the regulation of gene expression at post-transcriptional level. miRNAs are involved in the development of both cardiovascular damage and hypertension. Little is known of the molecular mechanisms that lead to development and progression of this condition. This review aims to cover the potential roles of miRNAs in the mechanisms associated with the development and consequences of RH, and explore the current state of the art of diagnostic and therapeutic tools based on miRNA approaches.

Exploiting the 2-Amino-1,3,4-thiadiazole Scaffold To Inhibit Trypanosoma brucei Pteridine Reductase in Support of Early-Stage Drug Discovery.

Pteridine reductase-1 (PTR1) is a promising drug target for the treatment of trypanosomiasis. We investigated the potential of a previously identified class of thiadiazole inhibitors of Leishmania major PTR1 for activity against Trypanosoma brucei (Tb). We solved crystal structures of several TbPTR1-inhibitor complexes to guide the structure-based design of new thiadiazole derivatives. Subsequent synthesis and enzyme- and cell-based assays confirm new, mid-micromolar inhibitors of TbPTR1 with low toxicity. In particular, compound 4m, a biphenyl-thiadiazole-2,5-diamine with IC50 = 16 µM, was able to potentiate the antitrypanosomal activity of the dihydrofolate reductase inhibitor methotrexate (MTX) with a 4.1-fold decrease of the EC50 value. In addition, the antiparasitic activity of the combination of 4m and MTX was reversed by addition of folic acid. By adopting an efficient hit discovery platform, we demonstrate, using the 2-amino-1,3,4-thiadiazole scaffold, how a promising tool for the development of anti-T. brucei agents can be obtained.

Matrix metalloproteinase-2 -735C/T polymorphism is associated with resistant hypertension in a specialized outpatient clinic in Brazil.

BACKGROUND: Matrix metalloproteinases (MMPs) are enzymes involved in cardiovascular (CV) remodeling and hypertension-mediated target organ damage (TOD). Genetic polymorphisms in matrix metalloproteinase 2 (MMP-2) gene [-1575G/A (rs243866); -1306C/T (rs243865); and -735C/T (rs2285053)] are associated with several CV conditions, however the relationship between MMP-2 polymorphisms and resistant hypertension (RH) is unknown. We evaluated whether these genetic single nucleotide polymorphisms (SNPs) in MMP-2 gene are associated with 1) MMP-2 and tissue inhibitor of metalloproteinase-2 (TIMP-2) levels in RH and mild to moderate hypertensive (HT) subjects, 2) left ventricular hypertrophy (LVH) and arterial stiffness and 3) the presence of RH. METHODS: One hundred and nineteen RH and 136 HT subjects were included in this cross-sectional study. Genotypes were determined by real-time PCR using TaqMan probes. Haplotypes were estimated using Bayesian method. RESULTS: The levels of MMP-2 and TIMP-2 were similar among genotypes and haplotypes for the three studied polymorphisms in HT and RH groups. RH showed higher frequency for GCC haplotype and lower frequency of GCT and ATC haplotypes (-1575G/A, -1306C/T and -735C/T, respectively) compared to HT (0.77 vs. 0.64; 0.09 vs. 0.17; 0.13 vs. 0.19, p=0.003 respectively). GCC haplotype was associated to RH apart from potential confounders (odds ratio (OR)=2.09; 95% confidence interval (CI)=1.20-3.64; p=0.01). In addition, CC genotype (OR=2.93; 95% CI=1.22-7.01; p=0.02) and C allele (OR=2.81; 95% CI=1.26-6.31; p=0.01) for -735C/T polymorphism were independently associated with RH. GCT haplotype was associated with reduced probability of having RH (OR=0.35; 95% CI=0.16-0.79; p=0.01). Finally, no relationship was found between studied MMP-2 SNPs and left ventricular hypertrophy and arterial stiffness in both groups. CONCLUSION: GCC haplotype carriers showed higher probability to have RH (odds ratio>1), while the GCT haplotype carriers showed lower probability to have RH, suggesting that the GCT haplotype may represent a protective genetic factor for the development of RH. These finds suggest that GCC and GCT haplotypes, and C allele and CC genotype of the -735C/T MMP-2 gene polymorphism may have a role in RH.

Chroman-4-One Derivatives Targeting Pteridine Reductase 1 and Showing Anti-Parasitic Activity.

Flavonoids have previously been identified as antiparasitic agents and pteridine reductase 1 (PTR1) inhibitors. Herein, we focus our attention on the chroman-4-one scaffold. Three chroman-4-one analogues (1-3) of previously published chromen-4-one derivatives were synthesized and biologically evaluated against parasitic enzymes (Trypanosoma brucei PTR1-TbPTR1 and Leishmania major-LmPTR1) and parasites (Trypanosoma brucei and Leishmania infantum). A crystal structure of TbPTR1 in complex with compound 1 and the first crystal structures of LmPTR1-flavanone complexes (compounds 1 and 3) were solved. The inhibitory activity of the chroman-4-one and chromen-4-one derivatives was explained by comparison of observed and predicted binding modes of the compounds. Compound 1 showed activity both against the targeted enzymes and the parasites with a selectivity index greater than 7 and a low toxicity. Our results provide a basis for further scaffold optimization and structure-based drug design aimed at the identification of potent anti-trypanosomatidic compounds targeting multiple PTR1 variants.

Methoxylated 2'-hydroxychalcones as antiparasitic hit compounds.

Chalcones display a broad spectrum of pharmacological activities. Herein, a series of 2'-hydroxy methoxylated chalcones was synthesized and evaluated towards Trypanosoma brucei, Trypanosoma cruzi and Leishmania infantum. Among the synthesized library, compounds 1, 3, 4, 7 and 8 were the most potent and selective anti-T. brucei compounds (EC50 = 1.3-4.2 µM, selectivity index >10-fold). Compound 4 showed the best early-tox and antiparasitic profile. The pharmacokinetic studies of compound 4 in BALB/c mice using hydroxypropil-ß-cyclodextrins formulation showed a 7.5 times increase in oral bioavailability.

Gene-Gene Interactions Among PRKCA, NOS3 and BDKRB2 Polymorphisms Affect the Antihypertensive Effects of Enalapril.

Protein kinase C (PKC) signalling is critically involved in the control of blood pressure. Angiotensin-converting enzyme inhibitors (ACEi) affect PKC expression and activity, which are partially associated with the responses to ACEi. We examined whether PRKCA (protein kinase C, alpha) polymorphisms (rs887797 C>T, rs1010544 T>C and rs16960228 G>A), or haplotypes, and gene-gene interactions within the ACEi pathway affect the antihypertensive responses in 104 hypertensive patients treated with enalapril as monotherapy. Patients were classified as poor responders (PR) or good responders (GR) to enalapril if their changes in mean arterial pressure were lower or higher than the median value, respectively. Multi-factor dimensionality reduction was used to characterize interactions among PRKCA, NOS3 (nitric oxide synthase 3) and BDKRB2 (bradykinin receptor B2) polymorphisms. The TC+CC genotypes for the rs1010544 polymorphism were more frequent in GR than in PR (p = 0.037). Conversely, the GA+AA genotypes for the rs16960228 polymorphism, and the CTA haplotype, were more frequent in PR than in GR (p = 0.040 and p = 0.008, respectively). Moreover, the GG genotype for the PRKCA rs16960228 polymorphism was associated with PR or GR depending on the genotypes for the rs2070744 (NOS3) and rs1799722 (BDKRB2) polymorphisms (p = 0.012). Our results suggest that PRKCA polymorphisms and gene-gene interactions within the ACEi pathway affect the antihypertensive responses to enalapril.

Chroman-4-one derivatives targeting pteridine reductase 1 and showing anti-parasitic activity

Flavonoids have previously been identified as antiparasitic agents and pteridine reductase 1 (PTR1) inhibitors. Herein, we focus our attention on the chroman-4-one scaffold. Three chroman-4-one analogues (1-3) of previously published chromen-4-one derivatives were synthesized and biologically evaluated against parasitic enzymes (Trypanosoma brucei PTR1-TbPTR1 and Leishmania major-LmPTR1) and parasites (Trypanosoma brucei and Leishmania infantum). A crystal structure of TbPTR1 in complex with compound 1 and the first crystal structures of LmPTR1-flavanone complexes (compounds 1 and 3) were solved. The inhibitory activity of the chroman-4-one and chromen-4-one derivatives was explained by comparison of observed and predicted binding modes of the compounds. Compound 1 showed activity both against the targeted enzymes and the parasites with a selectivity index greater than 7 and a low toxicity. Our results provide a basis for further scaffold optimization and structure-based drug design aimed at the identification of potent anti-trypanosomatidic compounds targeting multiple PTR1 variants.

Increased Circulating Tissue Inhibitor of Metalloproteinase-2 Is Associated With Resistant Hypertension.

Resistant hypertension (RH) is associated with organ damage and cardiovascular risk. Evidence suggests the involvement of matrix metalloproteinase 2 (MMP-2) and tissue inhibitor of metalloproteinase 2 (TIMP-2) in hypertension and in cardiovascular remodeling. The aim of this study was to assess the levels of MMP-2 and TIMP-2 in RH and its relation with organ damage, including arterial stiffness and cardiac hypertrophy. MMP-2 and TIMP-2 levels were compared among 19 patients with normotension (NT), 116 with nonresistant hypertension (HTN) and 116 patients with resistant HTN (RH). MMP-2 levels showed no differences among NT, HTN, and RH groups, while TIMP-2 levels were higher in RH compared with HTN and NT groups (90.0 [76.1-107.3] vs 70.1 [57.7-88.3] vs 54.7 [40.9-58.1] ng/mL, P<.01), respectively. MMP-2/TIMP-2 ratio was reduced in the RH group compared with the HTN and NT groups (2.7 [1.9-3.4] vs 3.3 [2.6-4.2] vs 4.9 [4.5-5.3], P<.01), respectively. No associations were found between MMP-2 levels, TIMP-2, and MMP-2/TIMP-2 ratio with cardiac hypertrophy and arterial stiffness in the RH and HTN groups. Finally, in a regression analysis, reduced MMP-2/TIMP-2 ratio and increased TIMP-2 levels were independently associated with RH. The present findings provide evidence that TIMP-2 is associated with RH and might be a possible biomarker for screening RH patients.

Endothelial nitric oxide synthase tagSNPs influence the effects of enalapril in essential hypertension.

The antihypertensive effects of angiotensin-converting enzyme inhibitors (ACEi) are associated with up-regulation of endothelial nitric oxide synthase (NOS3) activity. This mechanism may explain how polymorphisms in NOS3 gene affect the antihypertensive responses to ACEi. While clinically relevant NOS3 polymorphisms were previously shown to affect the antihypertensive responses to enalapril, no study has tested the hypothesis that NOS3 tagSNPs influence the antihypertensive effects of this drug. We examined whether the NOS3 tagSNPs rs3918226, rs3918188, and rs743506, and their haplotypes, affect the antihypertensive responses to enalapril in 101 patients with essential hypertension. Subjects were prospectively treated only with enalapril for 8 weeks. Genotypes were determined by Taqman(®) allele discrimination assay and real-time polymerase chain reaction (PCR) and haplotype frequencies were estimated. We compared the effects of NOS3 tagSNPs on changes in blood pressure after enalapril treatment. To confirm our findings, multiple linear regression analysis was performed adjusting for age, gender, ethnicity, and alcohol consumption. We found that hypertensive patients carrying the AA genotype for the tagSNP rs3918188 showed lower decreases in blood pressure in response to enalapril. Moreover, the TCA haplotype was associated with improved decreases in blood pressure in response to enalapril compared with the CAG haplotype. Adjustment for covariates in multiple linear regression analysis did not change these effects. In addition, when patients were stratified according to the dose of enalapril used, we found that the carries of the T allele for the functional tagSNP rs3918226 showed more intense decreases in blood pressure in response to enalapril 20 mg/day. Our findings suggest that NOS3 tagSNPs influence the effects of enalapril in essential hypertension.

Association of Mineralocorticoid Receptor Polymorphism I180V With Left Ventricular Hypertrophy in Resistant Hypertension.

OBJECTIVES: Genetic polymorphisms on mineralocorticoid receptor gene (NC3C2) are associated with variability of mineralocorticoid receptor (MR) function and cardiovascular implications. We sought to investigate whether I180V (rs5522) and MRc.-2G_C (rs2070951) polymorphisms in NR3C2 gene are associated with resistance to antihypertensive treatment and target-organ damage in resistant hypertensive (RHTN) patients. METHODS: One hundred and eighty-one RHTN and 122 mild to moderate hypertensive (HTN) patients were enrolled in this study. Genotypes were obtained by allelic discrimination assay using real-time polymerase chain reaction. We determined pulse wave velocity (PWV), microalbuminuria, and left ventricular mass index to assess target-organ damage. We compared clinical and laboratorial characteristics of AA vs. G carriers for rs5522 and AC vs. GG vs. CG for rs2070951. RESULTS: We did not found differences in allele, genotype, and haplotype frequencies for both polymorphisms between HTN and RHTN subjects. We found increased levels of aldosterone and ambulatory blood pressure (BP) in G carriers only for rs5522. Left ventricular hypertrophy (LVH) was more prevalent in G carriers than AA homozygous for rs5522 but not for rs2070951 in RHTN. On the other hand, microalbuminuria and PWV were similar among genotypes for both polymorphisms. No differences were observed between the haplotypes, except for higher aldosterone concentration in GG compared to AG and AC haplotypes. CONCLUSION: Our study suggests that rs5522 polymorphism might affect cardiac remodeling and aldosterone levels in RHTN subjects.

Vascular Damage in Resistant Hypertension: TNF-Alpha Inhibition Effects on Endothelial Cells.

Inflammatory cytokines have been associated with the pathophysiology of hypertension and target organ damage (TOD). Resistant hypertensive patients (RHTN) are characterized by poor blood pressure control and higher prevalence of TOD. This study evaluated the relationship between plasma levels of TNF-α and arterial stiffness (pulse wave velocity-PWV) in 32 RHTN and 19 normotensive subjects. Moreover, we investigated the effect of TNF-α inhibition on human endothelial cells (HUVECs) incubated with serum from RHTN and normotensive subjects. HUVECs containing serum obtained from normotensive (n = 8) and hypertensive (n = 8) individuals were treated with TNF-α inhibitor (infliximab). Cell suspensions were used for measurement of DNA fragmentation and reactive oxygen species (ROS) content. RHTN patients showed higher levels of TNF-α compared to normotensive subjects, as well as higher PWV. Positive correlation was found between TNF-α levels and PWV measures in the whole group. HUVECs incubated with serum from RHTN showed increased cell apoptosis and higher ROS content compared to normotensive subjects. Infliximab attenuated the apoptosis of HUVECs incubated with serum from RHTN, but no effect in ROS production was observed. Our findings suggest that TNF-α might mediate, at least in part, vascular damage in resistant hypertension.