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Computation methods that predict the binding of peptides to MHC-I are important tools for screening and identifying immunogenic antigens and have the potential to accelerate vaccine and drug development. However, most available tools are sequence-based and optimized only for peptides containing the twenty canonical amino acids. This omits a large number of peptides containing non-canonical amino acids (NCAA), or residues that undergo varied post-translational modifications such as glycosylation or phosphorylation. These modifications fundamentally alter peptide immunogenicity. Similarly, existing structure-based methods are biased towards canonical peptide backbone structures, which may or may not be preserved when NCAAs are present. Rosetta FlexPepDock ab-initio is a structure-based computational protocol able to evaluate peptide-receptor interaction where no prior information of the peptide backbone is known. We benchmarked FlexPepDock ab-initio for docking canonical peptides to MHC-I, and illustrate for the first time the method's ability to accurately model MHC-I bound epitopes containing NCAAs. FlexPepDock ab-initio protocol was able to recapitulate near-native structures (≤1.5Å) in the top lowest-energy models for 20 out of 25 cases in our initial benchmark. Using known experimental binding affinities of twenty peptides derived from an influenza-derived peptide, we showed that FlexPepDock protocol is able to predict relative binding affinity as Rosetta energies correlate well with experimental values (r = 0.59, p = 0.006). ROC analysis revealed 80% true positive and a 40% false positive rate, with a prediction power of 93%. Finally, we demonstrate the protocol's ability to accurately recapitulate HLA-A*02:01 bound phosphopeptide backbone structures and relative binding affinity changes, the theoretical structure of the lymphocytic choriomeningitis derived glycosylated peptide GP392 bound to MHC-I H-2Db, and isolevuglandin-adducted peptides. The ability to use non-canonical amino acids in the Rosetta FlexPepDock protocol may provide useful insight into critical amino acid positions where the post-translational modification modulates immunologic responses., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2022 Bloodworth et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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Salt-sensing mechanisms in hypertension involving the kidney, vasculature, and central nervous system have been well studied; however, recent studies suggest that immune cells can sense sodium (Na + ). Antigen-presenting cells (APCs) including dendritic cells critically modulate inflammation by activating T cells and producing cytokines. We recently found that Na + enters dendritic cells through amiloride-sensitive channels including the α and γ subunits of the epithelial sodium channel (ENaC) and mediates nicotinamide adenine dinucleotide phosphate oxidase-dependent formation of immunogenic IsoLG (isolevuglandin)-protein adducts leading to inflammation and hypertension. Here, we describe a novel pathway in which the salt-sensing kinase SGK1 (serum/glucocorticoid kinase 1) in APCs mediates salt-induced expression and assembly of ENaC-α and ENaC-γ and promotes salt-sensitive hypertension by activation of the nicotinamide adenine dinucleotide phosphate oxidase and formation of IsoLG-protein adducts. Mice lacking SGK1 in CD11c + cells were protected from renal inflammation, endothelial dysfunction, and developed blunted hypertension during the high salt feeding phase of the N-Nitro-L-arginine methyl ester hydrochloride/high salt model of salt-sensitive hypertension. CD11c + APCs treated with high salt exhibited increased expression of ENaC-γ which coimmunoprecipitated with ENaC-α. This was associated with increased activation and expression of various nicotinamide adenine dinucleotide phosphate oxidase subunits. Genetic deletion or pharmacological inhibition of SGK1 in CD11c + cells prevented the high salt-induced expression of ENaC and nicotinamide adenine dinucleotide phosphate oxidase. These studies indicate that expression of SGK1 in CD11c + APCs contributes to the pathogenesis of salt-sensitive hypertension.

Excess dietary salt contributes to inflammation and hypertension via poorly understood mechanisms. Antigen presenting cells including dendritic cells (DCs) play a key role in regulating intestinal immune homeostasis in part by surveying the gut epithelial surface for pathogens. Previously, we found that highly reactive γ-ketoaldehydes or isolevuglandins (IsoLGs) accumulate in DCs and act as neoantigens, promoting an autoimmune-like state and hypertension. We hypothesized that excess dietary salt alters the gut microbiome leading to hypertension and this is associated with increased immunogenic IsoLG-adduct formation in myeloid antigen presenting cells. To test this hypothesis, we performed fecal microbiome analysis and measured blood pressure of healthy human volunteers with salt intake above or below the American Heart Association recommendations. We also performed 16S rRNA analysis on cecal samples of mice fed normal or high salt diets. In humans and mice, high salt intake was associated with changes in the gut microbiome reflecting an increase in Firmicutes, Proteobacteria and genus Prevotella bacteria. These alterations were associated with higher blood pressure in humans and predisposed mice to vascular inflammation and hypertension in response to a sub-pressor dose of angiotensin II. Mice fed a high salt diet exhibited increased intestinal inflammation including the mesenteric arterial arcade and aorta, with a marked increase in the B7 ligand CD86 and formation of IsoLG-protein adducts in CD11c+ myeloid cells. Adoptive transfer of fecal material from conventionally housed high salt-fed mice to germ-free mice predisposed them to increased intestinal inflammation and hypertension. These findings provide novel insight into the mechanisms underlying inflammation and hypertension associated with excess dietary salt and may lead to interventions targeting the microbiome to prevent and treat this important disease.

T and B cells have been implicated in hypertension, but the mechanisms by which they produce a coordinated response is unknown. T follicular helper (Tfh) cells that produce interleukin 21 (IL21) promote germinal center (GC) B cell responses leading to immunoglobulin (Ig) production. Here we investigate the role of IL21 and Tfh cells in hypertension. In response to angiotensin (Ang) II-induced hypertension, T cell IL21 production is increased, and Il21-/- mice develop blunted hypertension, attenuated vascular end-organ damage, and decreased interleukin 17A (IL17A) and interferon gamma production. Tfh-like cells and GC B cells accumulate in the aorta and plasma IgG1 is increased in hypertensive WT but not Il21-/-mice. Furthermore, Tfh cell deficient mice develop blunted hypertension and vascular hypertrophy in response to Ang II infusion. Importantly, IL21 neutralization reduces blood pressure (BP) and reverses endothelial dysfunction and vascular inflammation. Moreover, recombinant IL21 impairs endothelium-dependent relaxation ex vivo and decreases nitric oxide production from cultured endothelial cells. Finally, we show in humans that peripheral blood T cell production of IL21 correlates with systolic BP and IL17A production. These data suggest that IL21 may be a novel therapeutic target for the treatment of hypertension and its micro- and macrovascular complications.

Excess dietary salt intake contributes to inflammation and plays a vital role in the development of hypertension. We previously found that antigen-presenting dendritic cells (DCs) can sense elevated extracellular sodium leading to the activation of the NADPH oxidase and formation of isolevuglandin (IsoLG)-protein adducts. These IsoLG-protein adducts react with self-proteins and promote an autoimmune-like state and hypertension. We have developed and optimized state-of-the-art methods to study DC function in hypertension. Here, we provide a detailed protocol for isolation, in vitro treatment with elevated sodium, and adoptive transfer of murine splenic CD11c + cells into recipient mice to study their role in hypertension.

We sought to investigate whether the polymorphisms rs243865 (-1306C>T); rs243866 (-1575G>A) and rs2285053 (-735C>T) in metalloproteinases 2 - MMP-2 gene and rs17576 (Q279R), rs17577 (Q668R) and rs3918242 (-1562C>T) in MMP-9 gene are associated with clinical outcomes in obese resistant hypertensive (RH) subjects. One hundred and twenty RH were enrolled in this cross-sectional study and divided into obese (n=63) and non-obese (n=57) according to body mass index. Genotypes were determined by real-time PCR using TaqMan probes. We determined pulse wave velocity (PWV), microalbuminuria and left ventricular mass index (LVMI) to assess TODs. Obese and non-obese RH had similar allele, genotype and haplotype distributions for all polymorphisms assessed but obese RH subjects carrying the low frequency allele for SNPs in MMP-2 gene had higher ambulatory diastolic blood pressure. Also, PWV and LVMI were higher in subjects carrying the low frequency allele for SNPs in MMP-2 gene. Regarding MMP-9 gene, office diastolic BP levels were higher in the AA genotype individuals compared to the G allele group for rs17576 polymorphism, while the opposite was found regarding the microalbuminuria level. Independent multiple linear regression analyses revealed that both A allele for rs243865 and T allele for rs243866 in MMP-2 gene were associated with ambulatory diastolic levels in obese RH subjects, apart from potential confounders. Our study suggests that rs243866/rs243865 in the MMP-2 gene are related to BP levels in obese RH subjects, although TODs present in this population seem to be dependent of a combination of other factors besides the genetic polymorphisms., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Sodium accumulates in the interstitium and promotes inflammation through poorly defined mechanisms. We describe a pathway by which sodium enters dendritic cells (DCs) through amiloride-sensitive channels including the alpha and gamma subunits of the epithelial sodium channel and the sodium hydrogen exchanger 1. This leads to calcium influx via the sodium calcium exchanger, activation of protein kinase C (PKC), phosphorylation of p47 phox , and association of p47 phox with gp91 phox . The assembled NADPH oxidase produces superoxide with subsequent formation of immunogenic isolevuglandin (IsoLG)-protein adducts. DCs activated by excess sodium produce increased interleukin-1β (IL-1β) and promote T cell production of cytokines IL-17A and interferon gamma (IFN-γ). When adoptively transferred into naive mice, these DCs prime hypertension in response to a sub-pressor dose of angiotensin II. These findings provide a mechanistic link between salt, inflammation, and hypertension involving increased oxidative stress and IsoLG production in DCs., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2017. Published by Elsevier B.V.)

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., (©2016 Wiley Periodicals, Inc.)

Rationale: Accumulating evidence supports a role of adaptive immunity and particularly T cells in the pathogenesis of hypertension. Formation of memory T cells, which requires the costimulatory molecule CD70 on antigen-presenting cells, is a cardinal feature of adaptive immunity., Objective: To test the hypothesis that CD70 and immunologic memory contribute to the blood pressure elevation and renal dysfunction mediated by repeated hypertensive challenges., Methods and Results: We imposed repeated hypertensive challenges using either N(ω)-nitro-L-arginine methyl ester hydrochloride (L-NAME)/high salt or repeated angiotensin II stimulation in mice. During these challenges effector memory T cells (T(EM)) accumulated in the kidney and bone marrow. In the L-NAME/high-salt model, memory T cells of the kidney were predominant sources of interferon-γ and interleukin-17A, known to contribute to hypertension. L-NAME/high salt increased macrophage and dendritic cell surface expression of CD70 by 3- to 5-fold. Mice lacking CD70 did not accumulate T(EM) cells and did not develop hypertension to either high salt or the second angiotensin II challenge and were protected against renal damage. Bone marrow-residing T(EM) cells proliferated and redistributed to the kidney in response to repeated salt feeding. Adoptively transferred T(EM) cells from hypertensive mice homed to the bone marrow and spleen and expanded on salt feeding of the recipient mice., Conclusions: Our findings illustrate a previously undefined role of CD70 and long-lived T(EM) cells in the development of blood pressure elevation and end-organ damage that occur on delayed exposure to mild hypertensive stimuli. Interventions to prevent repeated hypertensive surges could attenuate formation of hypertension-specific T(EM) cells., (© 2016 American Heart Association, Inc.)

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&#95;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., (© American Journal of Hypertension, Ltd 2015. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Vascular oxidative injury accompanies many common conditions associated with hypertension. In the present study, we employed mouse models with excessive vascular production of ROS (tg(sm/p22phox) mice, which overexpress the NADPH oxidase subunit p22(phox) in smooth muscle, and mice with vascular-specific deletion of extracellular SOD) and have shown that these animals develop vascular collagen deposition, aortic stiffening, renal dysfunction, and hypertension with age. T cells from tg(sm/p22phox) mice produced high levels of IL-17A and IFN-γ. Crossing tg(sm/p22phox) mice with lymphocyte-deficient Rag1(-/-) mice eliminated vascular inflammation, aortic stiffening, renal dysfunction, and hypertension; however, adoptive transfer of T cells restored these processes. Isoketal-protein adducts, which are immunogenic, were increased in aortas, DCs, and macrophages of tg(sm/p22phox) mice. Autologous pulsing with tg(sm/p22phox) aortic homogenates promoted DCs of tg(sm/p22phox) mice to stimulate T cell proliferation and production of IFN-γ, IL-17A, and TNF-α. Treatment with the superoxide scavenger tempol or the isoketal scavenger 2-hydroxybenzylamine (2-HOBA) normalized blood pressure; prevented vascular inflammation, aortic stiffening, and hypertension; and prevented DC and T cell activation. Moreover, in human aortas, the aortic content of isoketal adducts correlated with fibrosis and inflammation severity. Together, these results define a pathway linking vascular oxidant stress to immune activation and aortic stiffening and provide insight into the systemic inflammation encountered in common vascular diseases.

Resistant hypertension (RHTN) is a multifactorial and polygenic disease, frequently associated with obesity. Low plasma adiponectin levels, a hormone produced by the adipose tissue, were associated with RHTN. Single nucleotide polymorphisms (SNPs) -11377C/G (rs266729) and +276G/T (rs1501299) in ADIPOQ (adiponectin gene) were associated with hypertension. This study evaluated the association between two SNPs (-11377C/G and +276G/T) and adiponectin levels in RHTN. This study comprised 109 patients with RHTN genotyped for both polymorphisms. A cross-sectional study was designed to compare features of CC homozygous versus G allele carriers for -11377C/G and GG homozygous versus T allele carriers for +276G/T. Office and ambulatory BP measurements were similar among genotypes subgroups in both SNPs as well as the markers of target organ damage (arterial stiffness, left ventricular mass index and microalbuminuria). Adiponectin concentrations were significantly higher in CC compared to G carrier for -11377C/G (CC:7.0 (4.0-10.2) versus G allele:5.5 (2.5-7.9), p = 0.04) and lower in GG compared to T carrier for +276G/T (GG:5.3 (2.3-7.7) versus T allele:7.1 (3.6-10.5), p = 0.04). Adjusting for systolic ambulatory BP, body mass index, age, gender, race and presence of type 2 diabetes, multiple linear regression analyses revealed that the minor alleles G (β-coefficient= -0.14, SE=0.07, p = 0.03) and T (β-coefficient=0.12, SE=0.06, p = 0.04) were independent predictors of adiponectin. The -11377C/G and +276G/T SNPs in ADIPOQ were associated with adiponectin levels in RHTN individuals., (© 2014 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).)

Resistant hypertension (RH) is defined as uncontrolled blood pressure (BP) despite the use of ≥3 anti-hypertensive drugs, or controlled requiring use of ≥4 drugs. Recently, a new definition for an extreme phenotype of RH (uncontrolled BP using at least five drugs) has emerged-the refractory hypertension (RfH). Although characteristics of RH are well established, little is known about this newly described subgroup. For this work, 116 subjects with RH were enrolled from a specialized clinic and divided into RH (n = 80) and RfH (n = 36). Subjects were submitted to echocardiography, 24-hour ambulatory BP measurement and biochemical analyses. Logistic regression analysis demonstrated that: (1) white-coat effect (odds ratio [OR], 3.23; 95% confidence interval [CI], 1.12-9.27; P = .03), (2) black race (OR, 6.67; 95% CI, 1.99-16.16; P < .001), and (3) left ventricular mass index (OR, 1.02; 95% CI, 1.01-1.03; P = .04) were independent predictors of refractoriness. In conclusion, RfH and RH present different patient characteristics, and these phenotypic aspects can be useful for better understanding this harder-to-treat subgroup., (Copyright © 2015 American Society of Hypertension. Published by Elsevier Inc. All rights reserved.)

Background: Hypertension is the most prevalent and significant modifiable risk factor for coronary heart disease. A portion of patients with uncontrolled hypertension are considered to have resistant hypertension (RHTN). Myocardial ischemia incidence increases along with blood pressure (BP) levels. However, the prevalence of myocardial ischemia in patients with RHTN, as well as the factors associated with it, is unknown., Methods: We enrolled 129 patients with true RHTN regularly followed in our specialty hypertension clinic and evaluated then by resting and dipyridamole pharmacological stress myocardial perfusion scintigraphy. Patients were then divided into 2 groups: those with (I-RHTN; n = 36) and those without (NI-RHTN; n = 93) myocardial ischemia. Echocardiography, 24-hour ambulatory BP monitoring (ABPM), and flow mediated dilation (FMD) were also evaluated., Results: Thirty six (28%) patients had myocardial ischemia. There was no difference between groups regarding age, sex, biochemical parameters, office, and 24-hour ABPM levels. Patients in the I-RHTN group were more likely diabetic (31% vs. 11%; P < 0.05) and obese (75% vs. 40%; P < 0.001). Adjusting for age and body mass index, multiple logistic regression showed that diabetes (odds ratio (OR) = 6.5; 95% confidence interval (CI) = 1.06-40.14; P = 0.04), FMD (OR = 0.18; 95% CI = 0.07-0.41; P < 0.001), heart rate (OR = 1.23; 95% CI = 1.11-1.36; P < 0.001), left ventricular mass index (OR = 1.02; 95% CI = 1.01-1.04; P = 0.04), and microalbuminuria (OR = 1.02; 95% CI = 1.01-1.04; P = 0.002) were independent predictors of ischemia., Conclusions: In conclusion, there is a high prevalence of myocardial ischemia in patients with RHTN. Increased microalbuminuria, heart rate, endothelial dysfunction, and left ventricular mass can be useful to guide the investigation for myocardial ischemia in these high risk patients., (© American Journal of Hypertension, Ltd 2014. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: Increased levels of inflammatory biomarkers such as interleukin-6 (IL-6), 10 (IL-10), 1β (IL-1β), tumor necrosis factor-α (TNF-α) high-sensitivity C-reactive protein (hs-CRP) are associated with arterial stiffness in hypertension. Indeed, resistant hypertension (RHTN) leads to unfavorable prognosis attributed to poor blood pressure (BP) control and target organ damage. This study evaluated the potential impact of inflammatory biomarkers on arterial stiffness in RHTN., Methods: In this cross-sectional study, 32 RHTN, 20 mild hypertensive (HTN) and 20 normotensive (NT) patients were subjected to office BP and arterial stiffness measurements assessed by pulse wave velocity (PWV). Inflammatory biomarkers were measured in plasma samples., Results: PWV was increased in RHTN compared with HTN and NT (p < 0.05). TNF-α levels were significantly higher in RHTN and HTN than NT patients. No differences in IL-6 levels were observed. RHTN patients had a higher frequency of subjects with increased levels of IL-10 and IL-1β compared with HTN and NT patients. Finally, IL-1β was independently associated with PWV (p < 0.001; R(2) = 0.5; β = 0.077)., Conclusion: RHTN subjects have higher levels of inflammatory cytokines (TNF-α, IL-1β and IL-10) as well as increased arterial stiffness, and detectable IL-1β levels are associated arterial stiffness. These findings suggest that inflammation plays a possible role in the pathophysiology of RHTN.

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.

Background: Impaired endothelial function and arterial stiffness are associated with hypertension and are important risk factors for cardiovascular events. Reactive oxygen species reduce nitric oxide bioavailability and have a pivotal role in endothelial function. Resistant hypertension (RHTN) is characterized by blood pressure (BP) above goal (140/90mmHg) in spite of the concurrent use of ≥3 antihypertensive drugs of different classes. This study evaluated the association between 8-isoprostane levels, an oxidative stress marker, endothelial function and arterial stiffness, in RHTN., Methods: Ninety-four RHTN and 55 well-controlled hypertensive (HT) patients were included. Plasma 8-isoprostane levels were determined by ELISA. Also, flow-mediated dilation (FMD) and pulse wave velocity (PWV) were evaluated to determine endothelial function and arterial stiffness, respectively., Results: Levels of 8-isoprostane were markedly higher in RHTN compared to HT patients (22.5±11.2 vs. 17.3±9.8pg/ml, p<0.05, respectively). A significant inverse correlation was observed between FMD and 8-isoprostane (r=-0.35, p=0.001) in RHTN. Finally, multiple logistic regression revealed that 8-isoprostane was a significant predictor of endothelial dysfunction (FMD≤median) in RHTN group., Conclusion: RHTN showed markedly higher oxidative stress measured by 8-isoprostane, compared to HT patients. Taken together, our findings suggest the involvement of oxidative stress in endothelial function in RHTN., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Resistant hypertension (RHTN) includes patients with controlled blood pressure (BP) (CRHTN) and uncontrolled BP (UCRHTN). In fact, RHTN patients are more likely to have target organ damage (TOD), and resistin, leptin and adiponectin may affect BP control in these subjects. We assessed the relationship between adipokines levels and arterial stiffness, left ventricular hypertrophy (LVH) and microalbuminuria (MA). This cross-sectional study included CRHTN (n=51) and UCRHTN (n=38) patients for evaluating body mass index, ambulatory blood pressure monitoring, plasma adiponectin, leptin and resistin concentrations, pulse wave velocity (PWV), MA and echocardiography. Leptin and resistin levels were higher in UCRHTN, whereas adiponectin levels were lower in this same subgroup. Similarly, arterial stiffness, LVH and MA were higher in UCRHTN subgroup. Adiponectin levels negatively correlated with PWV (r=-0.42, P<0.01), and MA (r=-0.48, P<0.01) only in UCRHTN. Leptin was positively correlated with PWV (r=0.37, P=0.02) in UCRHTN subgroup, whereas resistin was not correlated with TOD in both subgroups. Adiponectin is associated with arterial stiffness and renal injury in UCRHTN patients, whereas leptin is associated with arterial stiffness in the same subgroup. Taken together, our results showed that those adipokines may contribute to vascular and renal damage in UCRHTN patients.

Interindividual variability in plasma aldosterone levels comprises environmental and genetic sources. Increased aldosterone levels have been associated with higher risk of hypertension and target-organ damage related to hypertension. Aldosterone excess and intravascular volume expansion are implicated in pathophysiology of resistant hypertension (RH). We sought to investigate whether -344 C/T polymorphism (rs1799998) in aldosterone synthase gene (CYP11B2) is associated with plasma aldosterone levels in patients with resistant hypertension. Sixty-two patients with resistant hypertension were enrolled in this cross-sectional study. Genotypes were obtained by allelic discrimination assay using real time polymerase chain reaction. Multivariable linear regression was used to identify whether TT genotype was a predictor of aldosterone levels. No differences in clinical and laboratorial parameters were found among genotype groups. We found an additive effect of the T allele on plasma aldosterone concentration in RH. Also, there was higher aldosterone levels in TT homozygous under use of spironolactone compared with C carriers and compared with TT subjects who was not under use of spironolactone. TT genotype and the use of spironolactone were significant predictors of aldosterone levels in RH subjects. Plasma aldosterone concentration is significantly associated with -344 C/T CYP11B2 polymorphism and with the treatment with spironolactone in resistant hypertensive subjects., (Copyright © 2014 American Society of Hypertension. Published by Elsevier Inc. All rights reserved.)

Purpose: Left ventricular hypertrophy and diastolic dysfunction (LVDD) remain highly frequent markers of cardiac damage and risk of progression to symptomatic heart failure, especially in resistant hypertension (RHTN). We have previously demonstrated that administration of sildenafil in hypertensive rats improves LVDD, restoring phosphodiesterase type 5 (PDE-5) inhibition in cardiac myocytes., Methods: We hypothesized that the long-acting PDE-5 inhibitor tadalafil may be clinically useful in improving LVDD in RHTN independently of blood pressure (BP) reduction. A single blinded, placebo-controlled, crossover study enrolled 19 patients with both RHTN and LVDD. Firstly, subjects received tadalafil (20 mg) for 14 days and after a 2-week washout period, they received placebo orally for 14 days. Patients were evaluated by office BP and ambulatory BP monitoring (ABPM), endothelial function (FMD), echocardiography, plasma brain natriuretic peptide (BNP-32), cyclic guanosine monophosphate (cGMP) and nitrite levels., Results: No significant differences were detected in BP measurements. Remarkably, at least four echocardiographic parameters related with diastolic function improved accompanied by decrease in BNP-32 in tadalafil use. Although increasing cGMP, tadalafil did not change endothelial function or nitrites. There were no changes in those parameters after placebo., Conclusion: The current findings suggest that tadalafil improves LV relaxation through direct effects PDE-5-mediated in the cardiomyocytes with potential benefit as an adjunct to treat symptomatic subjects with LVDD such as RHTN patients.

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.

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.

Leptin and aldosterone have been associated with the pathophysiological mechanisms of hypertension. However, despite studies showing the association of leptin with intima-media thickness, arterial distensibility and sympathetic nerve activation, the relationship between leptin and blood pressure (BP) in resistant hypertension (RHTN) is unknown. We aimed to assess the correlation of plasma leptin and aldosterone levels with BP in uncontrolled controlled RHTN (UCRHTN) and CRHTN patients. Plasma leptin and aldosterone levels, office BP, ambulatory BP monitoring and heart rate were measured in 41 UCRHTN, 39 CRHTN and 31 well-controlled HTN patients. No differences were observed between the three groups regarding gender, body mass index and age. The UCRHTN group had increased leptin when compared with CRHTN and well-controlled HTN patients (38.2±21.4, 19.6±8.7 and 20.94±13.9 ng ml(-1), respectively; P<0.05). Aldosterone levels values were also statistically different when comparing RHTN, CRHTN and well-controlled HTN patients (9.6±3.8, 8.1±5.0 and 8.0±4.7 ng dl(-1), respectively; P<0.05). As expected, UCRHTN patients had higher heart rate values compared with CRHTN and well-controlled HTN patients (86.2±7.2, 83.5±6.7 and 83.4±8.5, respectively; P<0.05). Plasma leptin positively correlated with systolic (SBP) and diastolic BP (DBP), and aldosterone (r=0.43, 0.35 and 0.47, respectively; all P<0.05) in UCRHTN, but neither in the CRHTN nor in the HTN group. Simple linear regression showed that SBP, DBP and aldosterone may be predicted by leptin (r(2)=0.16, 0.15 and 0.19, respectively; all P<0.05) only in the UCRHTN subgroup. In conclusion, UCRHTN patients have higher circulating leptin levels associated with increased plasma aldosterone and BP levels when compared with CRHTN and HTN subjects.