Search

Background: Obese individuals have disturbed blood pressure (BP) rhythmicity and relative natriuretic peptide (NP) deficiency. The relationship of diurnal variation in NP levels and 24h BP rhythm is not known. Furthermore, mechanisms behind difference in circulating NPs in healthy obese and lean individuals has not been explored. We conducted a prospective clinical trial to evaluate 1) the diurnal rhythmicity of NPs and its relationship with 24-hour BP rhythm between healthy lean and obese individuals, and 2) elucidate mechanism behind NP deficiency in obese. Methods: Healthy, normotensive, lean (BMI:18.5-25 kg/m 2 ) and obese (BMI:30-45 kg/m 2 ) individuals aged 18-40 years, underwent 24-hour standardized inpatient protocol involving ambulatory BP monitoring (ABPM), controlled light intensity, and 10 blood draws, following 5-days of standardized diet. NP gene expression was evaluated in a cohort of 37 healthy donor heart tissues obtained from the MAGNet repository. Results: Among 52 participants screened, a total of 40 participants (18 lean; 22 obese) were enrolled. Diurnal variation in MRproANP levels was seen in both lean and obese individuals (p for rhythmicity=0.001). The mesor of the NP rhythm was 15% (8.5-21.6%) lower in obese. The diurnal variation in MRproANP was in antiphase with diurnal variation of systolic BP (pFigure ). Obese participants had lower 24h renin levels (p=0.06) and higher nocturnal sodium excretion (p=0.08). Among obese, there was lower expression of NP production genes ( NPPA, NPPB) (p, and higher expression of clearance gene ( NPR3 ) (p Conclusions: In a mechanistic human trial, we elucidate key neurohormonal differences in rhythm and evidence of poor salt handling in obese. Decreased production and increased clearance may contribute to the NP deficiency in obese. Targeting the diurnal NP-BP rhythm axis may reduce the cardiovascular risk burden, specifically in obese individuals.

Background: Aged Dahl salt sensitive rats (DSS) rats represent a model of vascular dementia, i.e., they develop central arterial stiffness (CAS), hypertension, and cognitive decline. DSS rats have compromised renin-angiotensin system (RAS) which is activated with age and contributes to hypertension. This study examined whether angiotensin converting enzyme (ACE) inhibitor, lisinopril, affects CAS and cognitive functions in aged male DSS (n=26). Methods & Results: Following initial measurements at 16-mo of age (baseline; BL), DSS were administered lisinopril through their water (LISI; 15mg/kg/day, n=11) or control treatment (n=15) for 2-mo. Pulse wave velocity (PWV), a marker of CAS, and systolic blood pressure (SBP) were measured at BL and at 18-mo of age. Open field test (OFT) to assess anxiety-like behavior and Morris water maze (MWM) to assess spatial memory were performed at 18-mo, then aortae and hearts were collected and weighed. Aortic wall collagen abundance was estimated by histochemistry. Statistical analyses were performed by 2-way ANOVA mixed effects model and t-test. The data are presented as mean ± SEM. At 18-mo of age, control animals had high SBP similar to BL (187±4 vs. 184±7 mmHg), while LISI-treated DSS had lower SBP by 39±4 mmHg vs. BL (p Conclusion: Initiation of anti-hypertensive treatment in old age was effective in reducing aortic fibrosis and lowering SBP and PWV in DSS rats. Longer treatment may be needed to improve cognitive function in this model of vascular dementia. Supported by the NIH/NIA Intramural Research Program

Hypertension is recognized as one of the largest contributors to the global burden of disease. Unfortunately, for the majority of patients, hypertension is poorly controlled due, in part, to poor medical compliance resulting from the need to take one or more drugs, often several times daily. An ideal therapy would be administered a single time but yield long-term blood pressure control. Under the appropriate condition, we hypothesize that this modality could potentially lead to a cure for hypertension. In this study, we investigated whether that sustained, and possibly life-long, reductions in blood pressure could be achieved via Crispr-Cas9 mediated disruption of a key gene in the RAAS, Angiotensinogen (AGT). In vitro, we demonstrated that expression of Crispr-Cas9 system in the BRL 3A rat liver cell line led to a significant reduction in AGT protein levels (75% reduction, N=3, P

Rationale: Pulmonary arterial hypertension (PAH) is a cardiopulmonary disorder characterized by elevation of pulmonary arterial (PA) pressure and premature death. PA smooth muscle cells (PASMCs) from PAH patients present a cancer-like hyperproliferative and apoptosis-resistant phenotype contributing to remodeling of distal PAs. Although epigenetic alterations contribute to PAH development, one important challenge is defining which genes are the drivers . A growing body of literature points to the role of an epigenetic factor called G9a in cancer pathogenesis. Indeed, G9a is a histone methyltransferase overexpressed in many cancers promoting cell proliferation and survival. Given the similarities between PAH and cancer, it is of interest to determine whether G9a is implicated in PAH. We thus hypothesized that G9a inhibition reduces the pro-proliferative and apoptosis resistance phenotype of PAH-PASMCs. Methods and Results: Using Western blot (WB) and immunofluorescence (IF), we showed that G9a is overexpressed in distal PAs and isolated PASMCs from PAH patients (n= 6-14, p Conclusion: We showed for the first time that G9a is overexpressed in PAH contributing to the pro-proliferative and anti-apoptotic phenotype of PAH-PASMCs. Current experiments aim to determine whether G9a inhibition provides therapeutic benefits in PAH.

Objective: Aging-associated downregulation of Klotho is partly due to epigenetic upregulation of H3K27me3 in the kidney. The purpose of this study is to investigate the potential role of KDM6a, a demethylase of histone 3 lysine (K) 27 trimethylation (H3K27me3), in the regulation of blood pressure and explore the related molecular pathways that may influence the kidney function during aging. Methods and Results: Wild type and Ksp-Cre/KDM6a-floxed mice were divided into vehicle control and tamoxifen (10 mg/kg/mouse/7days) treated cohorts (WT mice: KDM6a floxed or Ksp-Cre only and Ksp-Cre/KDM6a -loxed mice). Blood pressure (BP) was measured by tail cuff method and confirmed by carotid artery cannulation at the end of study. Deletion of the KDM6a gene (KDM6a-cKO) significantly increased BP at day 7. BP remained elevated throughout the study. We found that the H3K27me3 level was significantly increased in the kidneys of KDM6a-cKO mice compared to control mice confirming that KDM6a is an important H3K27 demethylase in the control of methylation levels in the kidney. Elevation of BP was likely due to upregulation of the Na + :K + :2Cl - co-transporter (NKCC2) in the thick ascending limb of Henle's loop and NaCl co-transporter (NCC) in the distal convoluted tubule confirmed by qPCR and immunohistochemistry. Accordingly, we found that the urine sodium level was decreased in KDM6a-cKO mice compared to KDM6a-con mice. We showed that expression of aquaporin 2 (AQP2) was increased in the kidney of KDM6a-cKO mice, suggesting that AQP2 may also contribute to increased BP through modulation of body water homeostasis. Furthermore, we demonstrated that expression of Klotho was downregulated by 50%, which blocked FGFR1/Klotho/ERK signaling in the kidney of KDM6a-cKO mice. Notably, expression of aging markers including p53, p21, and p16 was significantly increased in the kidney of KDM6a-cKO mice, indicating that deletion of KDM6a in the renal tubule results in kidney aging. Conclusion: KDM6A is essential to the maintenance of normal kidney function and blood pressure. Renal-specific KDM6A knockout-induced hypertension is likely attributed to increased H3K27me3 levels and the resultant dowregulation of Klotho gene expression which impairs the FGFR1/Klotho/ERK signaling.

Hypertension is a major cause of cardiovascular morbidity and mortality, despite the availability of antihypertensive drugs with different targets and mechanisms of action. There is an unmet need for antihypertensive drugs with novel mechanisms of action to better control hypertension and reduce cardiovascular morbidity and mortality. Here, we provide evidence that pharmacological inhibition of TMEM16A (transmembrane member 16A or anoctamin-1), a Ca 2+ -activated Cl - channel expressed in vascular smooth muscle cells, reduces in vitro vascular smooth muscle contraction and decreases blood pressure in spontaneously hypertensive rats (SHR). We recently identified by high-throughput screening and subsequent medicinal chemistry, small molecule TMEM16A inhibitor TM inh -23 (2-bromodifluoroacetylamino-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-carboxylic acid o-tolylamide) that inhibits TMEM16A current fully, with IC 50 ~ 30 nM. TM inh -23 pretreatment blocked maximum in vitro vascular smooth muscle contractions induced by a thromboxane mimetic (U46619) in rat mesenteric arteries by 90%. Intraperitoneal (ip) administration of TM inh -23 to rodents at 10 mg/kg produced sustained serum concentrations of >10 μM for >4 hours. BP measurements by tail-cuff and telemetry showed a maximum ~45 mmHg reduction in SBP in spontaneously hypertensive rats (SHR) after a single dose (10 mg/kg, ip) TM inh -23 administration compared to vehicle treatment, with BP gradually returning to baseline values within 6-8 hours after TM inh -23 treatment. Minimal effect on BP (less than 10 mmHg decrease in SBP) was seen in wild-type rats and mice with TM inh -23 treatment (10 mg/kg, ip). Chronic 5-day treatment of SHR with TM inh -23 (10 mg/kg, ip, twice daily) caused sustained decreases (~20-25 mmHg) in daily average SBP, DBP and MAP during the treatment period. TM inh -23 action was reversible, with BP returning to baseline (~170/115 mmHg) by 3 days after discontinuation of treatment. These studies provide validation for TMEM16A as a target for antihypertensive therapy, and demonstrate the proof-of-concept for efficacy of TM inh -23 as an antihypertensive with a novel mechanism of action.

Hypertension has now been considered as one of the risk factors of Alzheimer's disease (AD), due to its contribution to the dysfunction of cerebrovascular system. To investigate its neuronal contribution, hypertension was induced in C57BL/6j male mice by either systemic infusion of Ang-II (600 ng/kg/min, s.c., 14 days) or DOCA-salt treatment (1 mg/g, s.c., 21 days), then markers for neuronal function were measured via qRT-PCR. In the hippocampus, Ang-II treatment significantly down-regulated the mRNA levels of BDNF (brain-derived neurotrophic factor) and DLG4 (discs large homolog 4, encoding PSD95), while DOCA-salt treatment only down-regulated BDNF expression (P2 ) re-synthesis, was found to be markedly down-regulated in the hippocampus of both hypertension models (P1 R plays pivotal role in the maintenance of neurogenic hypertension, and here we hypothesized that activation of AT 1 R could also exacerbate hypertension-induced reduction in neuronal function. In mice with DOCA-salt hypertension, the function of cortical neurons was shown to be improved by selective deletion of neuronal AT 1a R, as evidenced by significantly higher mRNA levels of BDNF and PI4KIIIβ, compared to the controls (P1 R in AD, 5хFAD mice were bred with mice with neuronal AT 1 R deletion (AT1NKO). AD-associated reduction of ACE2 protein, mainly in neurons, was found to be slightly ameliorated in the prefrontal cortex of 5хFAD-AT1NKO, compared to the age/sex-matched 5хFAD, showing by immunocytochemistry (24610 ±4182 vs. 13420 ±3720 AFU, n=6 slices). Although the detailed mechanism is still unknown, our data suggest that, neuron-expressing AT 1 R could participate in the development of hypertension-associated cognitive impairment and AD, independently of vascular AT 1 R.

Hypertension (HTN) is associated with reduced fertility in men. Although numerous studies report that HTN disrupts hormonal balance in men, less is known about the direct effect of HTN on testes and how HTN influences testicular inflammation and lymphatics. We hypothesized that HTN increases testicular lymphatic vessel density and this is associated with immune cell infiltration and inflammation. Male mice (8 weeks old) were made hypertensive by providing them with L-arginine methyl ester hydrochloride (L-NAME) (0.5 mg/mL) in the drinking water for 3 weeks and control mice received tap water. Testes of hypertensive mice had a significant increase in gene expression of the lymphatic vessel markers Lyve-1 (17.7 ± 2.1 fold; p

MicroRNA miR-29 promotes endothelial function in human arterioles in part by targeting LYPLA1 and increasing nitric oxide production. Endothelial dysfunction is common in cardiovascular diseases such as hypertension and diabetic microvascular complications. In addition, miR-29 is a master inhibitor of extracellular matrix gene expression, which may attenuate fibrosis but could also weaken tissue structure. The goal of this study was to develop an effective miR-29 therapeutic for multiple cardiovascular diseases using mouse models. Substantial accumulation of miR-29b and effective knockdown of Lypla1 in mouse tissues were achieved using a chitosan-packaged, chemically modified miR-29b mimic (miR-29b-CH) injected systemically at 200 μg/kg body weight. miR-29b-CH, injected once every three days, significantly attenuated angiotensin II-induced hypertension over two weeks (mean arterial pressure of 128 ± 5 vs 149 ± 5 mmHg, N = 7 Scr-CH, 8 miR-29b-CH p

Lifestyle modifications, a mainstay of hypertension (HTN) therapy, include recommendations for regular physical activity. However, fitness is not typically assessed. In the HTN clinic at Seattle Children’s, we conduct the 6-minute walk test (6MWT) in children > 6 yrs referred for HTN. We sought to assess whether children referred for HTN underperform on the 6MWT. Methods: We performed a retrospective chart review of patients seen from 9/1/18 - 9/30/19 who completed an ambulatory blood pressure monitor (ABPM) and 6MWT. Chart review included ABPM results, age, sex, BMI z-score, self-reported physical activity (RepPA) levels, rating of perceived exertion (RPE) and comorbid conditions. Exclusion criteria included lack of ABPM, 6MWT or compromised exercise capacity. ABPM was classified according to the 2014 AHA guidelines and 6MWT was compared to normative datasets. Fisher’s Exact test was used to compare the distributions of distance from mean values for the 6MWT and ANOVA was used to compare mean BMI z-score and RPE among groups. Linear regression assessed 6MWT with HTN status, adjusted for age, sex, and BMI z-score. Results: Demographics and clinical measures are summarized in Table 1. RepPA, 6MWT and RPE did not differ significantly. Most children underachieved regardless of group; the distance walked was > 2 SD below mean in 31 of 52 (60%). BMI z-score was significantly higher in the abnormal ABPM group. Linear regression showed no effect of HTN status. Conclusion: Most children underperformed on the 6MWT and results did not differ based on HTN status. This study suggests the 6MWT is useful to assess fitness in this population and serves as baseline for comparison.

The recently deorphanized G protein couple receptor 83 (GPR83) is expressed in proximal and distal convoluted tubules of the mouse kidney and in human renal proximal tubule cells (hRPTCs). Peptides derived from the cleavage of the precursor neuroendocrine hormone pro-SAAS (pro serine, alanine, alanine, serine), in particular PEN (proline, glutamic acid, and asparagine), have been proposed as the endogenous ligands of GPR83 but their function in the kidney is unknown. GPR83 is constitutively active; silencing (siRNA, 20 nM, 72h) its expression in hRPTCs decreases the phosphorylation of ERK1/2, p38, p90RSK and AKT (43%, 52%, 65%, and 25% respectively, n=4/group, PGpr83 gene in mice increased systolic blood pressure (SBP). High salt diet increased Gpr83 transcription 2-fold (P+ excretion and renal NHE3 expression (P+ /K + -ATPase expression, suggesting that PEN is only a partial ligand of GPR83. By contrast, the renal specific siRNA-mediated silencing of pro-SAAS in mice increased SBP (130±4 vs control wild-type 100±1 mmHg, n=3/group; P+ excretion (P+ /K + -ATPase (P

Uncontrolled hypertension is the leading cause of spontaneous ICH and elevated SBP is a strong predictor of poor outcomes in ICH patients. An animal model of the chronically hypertensive, metabolically dysfunctional and pro-inflammatory state of the ICH patient population is needed to advance clinically-relevant ICH research. Transgenic (mRen2)27 rats overexpress the murine Ren2 gene and develop hypertension and metabolic syndrome by 12-14 weeks of age. Indices of diastolic and systolic cardiac function are in decline in (mRen2)27 males by 30 weeks of age, but are preserved in females at this age. To establish whether (mRen2)27 rats exhibit sensorimotor dysfunction as compared with control Sprague Dawley (SD) rats, male and female (mRen2)27 rats underwent corner turn (CT), vibrissae evoked forelimb placing (VEF), cage wire hang (CWH), and open-field (OF) testing for five consecutive weeks starting at 20-weeks of age (n=16; female=8; each strain). Despite the significant between-strain difference in SBP [189 ± 3 mmHg in (mRen2)27 and 119 ± 3 mmHg in SD; p (Cardiovascular Sci. and Hypertension & Vasc. Res. Ctrs; Neurosurgery)

Hypertension (HTN) is a complex disease influenced by heritable genetic elements and environmental interactions. Dietary salt is among the most influential modifiable factors contributing to increased blood pressure (BP). It is well established that men and women develop BP impairment in different patterns and a recent emphasis has been placed on identifying mechanisms leading to the differences observed between the sexes in HTN development. The current work reported here builds on an extensive genetic mapping experiment which sought to identify genetic determinants of salt sensitive (SS) HTN using the Dahl SS rat. BTG anti-proliferation factor 2 ( Btg2 ) was previously isolated by our group using congenic breeding experiments which identified it as a female causative SS HTN candidate gene. In the current study, Btg2 was mutated using TALEN targeted gene disruption on the SSBN congenic rat background. The Btg2 mutated rats exhibited impaired BP and proteinuria responses to a high salt diet compared to wild type littermates. Differences in body weight, mutant pup viability, skeletal morphology, and adult nephron density suggest a potential role for Btg2 in developmental signaling pathways. Subsequent cell cycle gene expression assessment provides several additional signaling pathways that Btg2 may function through during salt handling in the kidney. The expression analysis also indicates several upstream targets that can be explored to further isolate therapeutic approaches for SS HTN.

Among ~25% of patients with essential hypertension (EH), circulating endogenous ouabain (EO) and aldosterone (Aldo) are typically coelevated and their BP is especially salt-sensitive (SS). We probed for exaggerated natriuresis in a large cohort (712) of hypertensive patients that underwent an acute 2hr (T 120 ) load with saline (0.9%/2 L). Results: Using a quartiles analysis of sodium excretion, the 4th quartile subgroups showed higher SBP and DBP at T 120 , and at 2 hrs recovery (T 240 ). Fractional sodium excretion (FE Na) was significantly higher during loading and recovery in the 4 th quartile subgroup. Plasma Aldo was suppressed after saline in all subgroups as expected. SS patients showed an increased EO. In the 4 th quartiles EO was elevated at baseline and after saline (see figure), p Manova > 0.001 and the urinary Na/K ratio output was higher. Further, increases in plasma Na + were greater in the 4 th quartile group (1.91 ± 0.15 vs 0.83 ± 0.13 mM, p

Background: RNAi therapeutics targeting angiotensinogen (AGT) lower blood pressure in animal models of essential hypertension, but whether this treatment is beneficial in hypertensive chronic kidney disease (CKD) is yet unknown. Our aim was to assess the effectiveness of a small interfering RNA (siRNA) targeting hepatic AGT in a rat model of hypertensive CKD. Methods: Sprague Dawley rats (n=7-12 per group) were subjected to 5/6th nephrectomy, allowed to recover for four weeks, and subsequently subjected to one of five treatments: (1) vehicle, (2) AGT siRNA (10-30 mg/kg, s.c. every 2 weeks), (3) AGT siRNA + losartan (30 mg/kg/d), (4) losartan (30 mg/kg/d), or (5) losartan + captopril (30 and 6 mg/kg/d). Mean arterial pressure (MAP) was measured by radiotelemetry for 4 weeks, glomerular filtration rate (GFR) was measured by transcutaneous FITC-sinistrin clearance. Results: AGT siRNA and AGT siRNA + losartan reduced plasma AGT by 96.5 ± 1.4% and 97.0 ± 2.3%, respectively. Over the course of 4 weeks, proteinuria increased with vehicle and this increase was abrogated by all treatments (Δ proteinuria at 4 weeks versus baseline: +0.17 [0.12, 0.23], +0.02 [-0.04, 0.05], -0.01 [-0.07, 0.06], -0.05 [-0.12, 0.00], -0.01 [-0.06, 0.11] for vehicle, AGT siRNA, AGT siRNA + losartan, losartan, losartan + captopril, P < 0.05 for all groups compared with vehicle). Similarly, heart weight was equally reduced by all treatments except vehicle. No intervention affected GFR. Over the course of 4 weeks, MAP increased (from a baseline of 154 ± 18 mmHg) during vehicle treatment and this rise was prevented by AGT siRNA (+13 ± 15 mmHg vs. -2 ± 14 mmHg, P > 0.05 for difference between groups). Combination treatment with AGT siRNA + losartan, losartan + captopril, and losartan alone caused a similar and marked reduction in MAP (-17 ± 14 mmHg, -21 ± 17 mmHg, and -33 ± 20 mmHg, P < 0.05 versus vehicle for all; differences between groups not significant). Conclusion: In experimental CKD, AGT siRNA monotherapy appears to reduce proteinuria and heart weight in a blood pressure-independent fashion, to a similar degree as losartan or combination treatment. Thus, an siRNA targeting hepatic AGT may be a potential therapeutic approach for providing renal and cardioprotection in CKD.

The rise in opioid use underscores the importance to better understand the direct and indirect effects of opioids on renal function and blood pressure. Although opioid use is associated with predictors of cardiovascular diseases, these drugs are common analgesics for hypertensive patients. We hypothesize that stimulation of opioid receptors (ORs) leads to elevated intracellular calcium level in podocytes ultimately leading to cell apoptosis, development of albuminuria and consequent progression of hypertension. Live calcium imaging experiments on freshly isolated glomeruli from rat and human kidneys, as well as human immortalized podocyte cell line, was performed to test the effect of specific ORs agonists. Following experiments assessed the effect of opioid signaling on the development of hypertension and kidney function in Dahl salt-sensitive (SS) rats, which were fed a 0.4% (LS) or 8% (HS) NaCl diets for 14 days with or without a daily i.v. bolus infusion of BRL52537, a potent and selective kappa-OR agonist. Stimulation of kappa-ORs, but not mu-ORs or delta-ORs, mediated calcium influx in podocytes through activation of TRPC6 channels. The effect of BRL52537 was completely abolished when we used the 0 mM calcium media or when a TRPC6 channel inhibitor (SAR7334) was applied. Triggering the kappa-OR/TRPC6 pathway induced podocyte cell shape changes via actin cytoskeleton remodeling. In vivo studies revealed that rats chronically treated with BRL52537 exhibited augmented blood pressure (MAP was 179 ± 15 vs. 151 ± 11 mmHg), albuminuria, and elevation in podocyte calcium. Western blot analysis revealed elevated levels of nephrin in urine samples and pro-caspase-3 in renal cortex. Moreover, TRPC6 expression was elevated under hypertensive conditions and further promoted pathological increase in calcium influx in response to kappa-OR stimulation. Summarizing the data, the opioid-induced increase in the calcium flux in podocytes is expected to contribute to kidney injury leading to progression of salt-induced hypertension. These data demonstrate that the kappa-OR/TRPC6 signaling pathway directly influences podocyte calcium handling, provoking the development of kidney injury in the opioid treated hypertensive cohort.

Rho-related BTB domain containing protein 1 (RhoBTB1) is a transcriptional target of peroxisome proliferator activated receptor γ (PPARγ), but its physiological and molecular function in blood pressure (BP) control remains unclear. We showed that aortic RhoBTB1 expression was decreased in mice expressing vascular smooth muscle (VSM) specific dominant negative PPARγ mutation (S-P467L). Genetic complementation of VSM RhoBTB1 reversed the hypertension, vascular dysfunction, and arterial stiffness in S-P467L mice; and suppressed the elevated enzymatic activity of phosphodiesterase 5 (PDE5) in S-P467L mice. In HEK293 cells, RhoBTB1 increases PDE5 ubiquitination in a Cullin-3-dependent manner. We concluded that VSMC RhoBTB1 mediates the anti-hypertensive effect of PPARγ. RhoBTB1 expression was decreased in aorta from mice treated with angiotensin II (AngII, 490ng/kg/min, 2-weeks). We hypothesized that restoration of VSM RhoBTB1 would reverse established AngII-mediated hypertension, vascular dysfunction, and arterial stiffness. Transgenic mice expressing inducible VSM-specific RhoBTB1 (S-RhoBTB1) were treated with AngII (490ng/kg/min, 6-weeks) and the transgene was activated by Tamoxifen (Tx) during week 3. ISM-Cre mice expressing VSM Cre-recombinase were similarly treated as controls. AngII equally increased BP, as measured by radiotelemetry in both ISM-Cre and S-RhoBTB1 mice. The vasodilatory and vasoconstrictor responses in the carotid artery were not different in AngII-treated S-RhoBTB1 and ISM-Cre mice. RhoBTB1 did not affect pulse wave velocity (PWV), a measure of arterial stiffness, in mice without AngII (ISM-Cre vs S-RhoBTB1: 2.64±0.08 vs 2.48±0.06 mm/ms, p>0.05, n=6); and AngII increased PWV similarly in both strains before Tx (ISM-Cre vs S-RhoBTB1: 3.54±0.22 vs 3.52±0.18 mm/ms, p>0.05, n=7-9). PWV of S-RhoBTB1 mice started to decrease after the first week whereas it continued to rise in ISM-Cre mice. By the second week of Tx, VSMC RhoBTB1 expression significantly attenuated AngII induced arterial stiffness (ISM-Cre vs S-RhoBTB1: 4.21±0.37 vs 3.17±0.16 mm/ms, p=0.02, n=7-9). These data suggest a mechanism where RhoBTB1 improves vascular compliance independently from BP and is protective against arterial stiffness.

Introduction: Saccharina japonica (SJ) is one of brown algae. The extract of SJ (SJE) is often used for soup stock in Japanese cuisine. We recently observed the decreases in blood pressure (BP) by intake of SJ or SJE diet in 2-kidney, 1-clip renovascular hypertensive (2K1C) rats. SJ is rich in aspartate (Asp), which is the umami taste of amino acid. The intake of Asp was reported to reduce BP in Dahl salt sensitive rats. As well, the potassium Asp (AspK) intake was reported to improve BP in hypertensive patients. 70% (70 of 100) of amino acids such as Asp are eluted into the SJE from SJ. Therefore, we hypothesized that Asp in SJE contributes to the mechanism of BP reduced by dietary SJE in 2K1C rats. In this study, we observed BP in 2K1C rats fed a diet containing Asp or AspK and investigated whether those diets alleviate hypertension to the same extent of the diet containing SJE. Methods: Male Sprague-Dawley rats (6 wks) were treated with sham operation (SHAM) or clipping the left renal artery (2K1C). After surgery, the rats fed a control diet (C), a diet containing Asp, AspK or SJE for 6 weeks. The content of SJE was set to be the same as that in our previous study. The amount of Asp in diet was set to be the same as that in our previous study which showed the antihypertensive effect of SJ. The amount of AspK in diet was set to be equimolar to Asp. The systolic BP (SBP) was measured by a tail-cuff method every week. At the end of the procedure, mean arterial BP (MAP) was measured in each rat under anesthesia. Results: Six weeks after the surgery, 2K1C-C significantly increased in SBP compared with SHAM-C (126±2 vs 159±4 mmHg, p Conclusions: Asp and AspK may attenuate hypertension in 2K1C renovascular hypertension as much as SJE which contains similar quantities of Asp. It is possible that Asp and/or AspK eluted into SJE contributes to the mechanism of alleviating hypertension by SJE in 2K1C.

The present study used global ( Nhe3 -/- ), kidney-selective (tg Nhe3 -/- ), and proximal tubule-specific Na + /H + exchanger 3 (NHE3)-deficient mice (PT- Nhe3 -/- ) to test the hypothesis that NHE3 is required for the full development of angiotensin II (Ang II)-induced hypertension in mice. Four groups of adult male, age-matched wild-type (WT), global Nhe3 -/- , kidney-selective tg Nhe3 -/- and proximal tubule-specific Nhe3 -/- mice were infused with: a) saline; b) Ang II (10 pmol/min, i.v.); Ang II via an osmotic minipump for 2 weeks (1.5 mg/kg/day, i.p.); or treated with Ang II and losartan concurrently for 2 weeks (20 mg/kg/day, p.o.). Under basal conditions, global Nhe3 -/- , kidney-selective tg Nhe3 -/- and proximal tubule-specific Nhe3 -/- mice all showed significantly lower systolic, diastolic, and mean arterial pressure than wild-type mice (~15 ± 3 mmHg, P Nhe3 -/- and kidney-selective tg Nhe3 -/- mice was associated with abnormal intestinal structures, diarrhea, increased 24 h fecal Na + excretion, and salt wasting ( P + excretion between wild-type and PT- Nhe3 -/- mice. PT- Nhe3 -/- mice showed significant diuretic and natriuretic responses compared with wild-type mice ( P P Nhe3 -/- , kidney-selective tg Nhe3 -/- , and PT- Nhe3 -/- mice ( P Nhe3 -/- , tgNhe3 -/- , and PT- Nhe3 -/- mice, compared with wild-type mice ( P Nhe3 -/- , tg Nhe3 -/- , and PT- Nhe3 -/- mice (p

Salt-sensitive (SS) hypertension is accompanied with severe cardiorenal complications. In this condition, elevated blood pressure (BP) resulting from high salt intake can be partially associated with a lower level of the atrial natriuretic peptide (ANP). In plasma, ANP is quickly degraded by neprilysin; therefore, pharmacological inhibition of this metalloprotease (f.i. with sacubitril) can increase ANP level. We have shown earlier that lower dose of sacubitril in combination with valsartan (75 μg/day) has beneficial effects on renal function in SS hypertension. The goal of this study was to evaluate the effects of sacubitril administered at a higher dose on renal damage of Dahl SS rats. Sacubitril (125 μg/day) or vehicle were administered to male Dahl SS rats (Charles River) via s.c. osmotic pumps. To induce hypertension, both groups were fed a purified 4% NaCl diet (HS, Dyets Inc) for 21 days. Glomerular filtration rate (GFR, FITC-inulin) and BP (tail cuff) values were obtained before and after the HS challenge. Data was analyzed with 1-way ANOVA. When compared to vehicle treated rats, 125 μg/day of sacubitril insignificantly increased systolic BP measured at the end of the 21-day HS challenge; no changes in GFR, heart weight, plasma electrolytes, BUN and creatinine were observed. However, sacubitril caused kidney hypertrophy (two kidneys to body weight: 8.8±0.4 vs 10.4±0.4 mg/g, p=0.04,), but did not affect renal medullary of cortical fibrosis. We also observed aggravated glomerular lesions in the sacubitril-treated animals compared to controls (glomerular injury score: 1.6±0.02 vs 0.9±0.08 au, p=0.034), and increased formation of protein casts (1.8±0.3 vs 1.3±0.2 au, p=0.034). Thus, in Dahl SS rats, administration of sacubitril at 125 μg/day had adverse effects on renal disease progression, especially glomerular damage and protein casts formation. In our prior study we showed that the combination of sacubitril at 75 μg/day with the angiotensin receptor blocker (ARB), valsartan, is effective to alleviate these outcomes. It is likely that the inhibition of neprilysin by sacubitril results in accumulation of Ang II and/or bradykinin and/or RAAS activation, which masks the beneficial effects of ANP level increase.

Preterm infants (born prior to 37 weeks (wks) gestation) are susceptible to hypoxia, which predisposes to hypertension in later life. Underdeveloped organs, including the kidney, prevent preterm infants from effectively regulating blood volume and O 2 delivery. Since rat nephrogenesis completes ~ postnatal day (PND) 8, we hypothesized that exposure to hypoxia before nephrogenesis is complete will promote hypertension in adulthood. Male and female Sprague Dawley pups were randomized to Ctrl (room air) or intermittent hypoxia (IH) at PND 1 (n=6/group). IH pups were exposed to ~10% O 2 three times a day, 10 minutes/session, from PND 1-8. O 2 saturation was measured at PND 6. Mean arterial pressure (MAP) was measured via telemetry from ~14 – 16 wks of age. To determine the MAP response to a cardiovascular challenge, osmotic minipumps containing angiotensin (Ang) II (400 ng/kg/min) were implanted at 15 wks of age. IH pups had lower O 2 saturation vs ctrl (P O2 Int . = 0.04). IH animals had higher DC-MAP vs ctrl (P O2 = 0.02). IH decreased blood oxygen, suggesting a global decrease in oxygen delivery to organs, similar to what is seen with hypoxia. Perinatal IH alone did not increase MAP. However, this exposure did increase MAP in response to Ang II. While both males and females exposed to perinatal hypoxia had higher Ang II-induced hypertension vs ctrls in the dark cycle, this effect was preserved only in males in the light cycle. This suggests males are more susceptible to blood pressure effects of perinatal IH.

Background: There is a large increase in uterine arterial blood flow during normal pregnancy. Structural and cellular adjustments occur in the uterine vasculature during pregnancy to accommodate this increased blood flow through a process is known as ‘vascular remodeling’. The etiology of preeclampsia involves aberrant placentation and vascular remodeling leading to reduced uteroplacental perfusion. However, the underlying source of the deficient vascular remodeling and the subsequent development of preeclampsia remains to be fully understood. Piezo 1 channels have been shown to be highly expressed in vascular smooth muscle cells of small-diameter arteries and play a role in the structural remodeling of the arteries. Studies have also shown that Piezo 1 is present in uterine arteries and it’s not exclusive to the endothelial cells. Hypothesis: This study tests the hypothesis that reduced Piezo 1 activity contributes to decreased uterine vascular relaxation in hypertensive pregnant rats. Methods: Hypertension was induced by treating the pregnant rats with synthetic CpG ODN (ODN 2395) via three intraperitoneal injections (100μg/rats) while the normotensive controls were treated with saline (vehicle) on the 14 th , 17th and 18 th days of pregnancy. Mean arterial pressure (MAP) was measured. In vitro vascular reactivity of uterine arterial (UA) ring segments were evaluated using isometric wire myograph system. Rings were pre-contracted with 3μM phenylephrine (PE), concentration responses of to Yoda1; a pharmacological agonist of Piezo 1 channel were compared. Statistical analysis was performed using nonlinear regression and Students’ t-test. Results: Our results show that MAP was greater in rats treated with ODN2395 vs untreated rats (112 ± 1 vs 90 ± 1 p =0.0004). Concentration-dependent relaxation responses to Yoda1 were greater in UAs of untreated rats compared to those treated with ODN2395 (EC50 0.06571 ± 0.09781 vs. 0.5774 ± 0.1187 p =0.0018). Conclusion: These results suggest that the reduced vasodilation in pregnancy-associated hypertension may be due to a reduced Piezo 1 channel activity.

Cleavage of the extra-cellular domain of the (pro)renin receptor (PRR) yields a soluble fragment (sPRR), which maybe involved in mediating hypertension. We recently developed a novel mouse model with mutation in the cleavage site of the PRR using CRISPR/Cas9 such that sPRR is not generated and showed that absence of sPRR attenuated angiotensin-II induced hypertension and kidney damage. In this study, we examined if sPRR alters blood pressure (BP) in angiotensin-II independent hypertension using deoxycorticosterone acetate (DOCA)-salt treatment. Mutant sPRR mice and littermate controls were treated with DOCA (50 mg/kg) and high Na + diet for 3 weeks. BP was monitored by radio-telemetry and metabolic balance studies performed at the end of the study (Day 17-18). Compared to controls, male mutant sPRR mice had markedly lower plasma sPRR levels (control: 21.5 ± 2.5 vs mutant 0.2 ± 0.03 ng/ml) and baseline BP (systolic control: 122 ± 3 vs mutant 114 ± 3; diastolic control: 94 ±5 vs mutant 82 ± 3 mm Hg). BP remained low in mutant sPRR mice relative to controls following 12 days of DOCA-salt treatment (systolic control: 141 ± 2 vs mutant 132 ± 5; diastolic control: 110 ± 4 vs mutant 95 ± 5 mm Hg). Mutant sPRR mice had lower body weight but similar food intake and urinary albumin excretion compared to controls (Table 1). Mutant sPRR mice had lower urine volume, water intake and urinary K + but not Na + excretion. No differences in renal histology were noted between control and mutant sPRR mice. Loss of sPRR attenuates DOCA-salt mediated hypertension. The mechanisms by which sPRR might regulate BP and water/Na + homeostasis in DOCA-salt hypertension are currently being investigated.

Objective: We hypothesized that renal denervation (RDN) attenuates the progression of aorto-caval fistula (ACF)-induced heart failure (HF) and the development of renal dysfunction in Ren-2 transgenic rats (TGR), a model of ANG II-dependent hypertension. Methods: RDN (severing renal nerve fibers, followed by phenol application) was done one week after creation of ACF; the follow-up period was 70 days. The animals studied were: 1) Sham-operated TGR (initial n = 8); 2) Sham-operated TGR + RDN (n = 9); 3) ACF TGR (n = 31); 4) ACF TGR + RDN (n = 34). In separate groups (n = 8 in each), three weeks after induction of ACF, renal artery blood flow (RBF, Transonic probe) responses were determined to intrarenal ANG II (2 and 8 ng), norepinephrine (NE) (20 and 40 ng) and acetylcholine (Ach) (10 and 40 ng). Results: Untreated ACF TGR began to die 3 weeks after induction of ACF and the final survival rate was 10% (3 from 31). RDN substantially improved the final survival rate to 50% (17 from 34). Basal RBF was significantly lower in ACF TGR than in sham-operated TGR (6.9 ± 0.4 vs. 10.1 ± 0.5 ml.min -1 .g -1 , p Conclusion: The results show that RDN significantly improved survival rate in ACF TGR, however this beneficial effect was not associated with improvement of reduced RBF or with attenuation of exaggerated renal vascular responsiveness to ANG II.

A single dose of small interfering ribonucleic acids (siRNA) targeting angiotensinogen (AGT) provides a strong and sustained antihypertensive effect, lasting weeks to months in preclinical models. It is not known whether conventional vasopressors are effective to reverse RNAi-mediated blood pressure lowering. Here, we investigated whether conventional vasopressors can elevate arterial pressure under conditions of AGT depletion. Spontaneously hypertensive rats on a low-salt diet (

Introduction: Both innate and adaptive immune cells (such as T lymphocytes) have been shown to play a role in hypertension and vascular injury. Recently, we demonstrated that a small subpopulation of T cells considered "innate-like", expressing the γδ T-cell receptor (TCR) rather than the much more frequent αβ TCR, plays a key role in hypertension and vascular injury. The number and activation of γδ T cells were increased after 7 days of angiotensin (Ang) II infusion, and absence of γδ T cells prevented the development of hypertension, endothelial dysfunction of resistance arteries, and activation of CD4 + and CD8 + T cells in mice infused with Ang II for 14 days. γδ T cells can be subdivided according to the TCR variant (V) subtype that is generally specific for a tissue. A subpopulation of γδ T cells in the lungs and skin that are Vγ6 + and produce interleukin (IL)-17A was shown to respond promptly to pneumococcal infection and skin inflammation. However, γδ T cell Vγ subtypes involved in hypertension are still unknown. We hypothesized that γδ T cells involved in hypertension are Vγ6 + . Methods: Eleven- to 13-week old C57BL/6J male mice were infused or not with Ang II (490 ng /kg /min, sc) for 14 days (n = 5-14), and γδ T cell Vγ subtypes were profiled by flow cytometry in the spleen, mesenteric lymph nodes (MLNs), thoracic aortic (TA), perivascular adipose tissue (PVAT) and mesenteric artery (MA) PVAT. Results: In spleen and MLNs the most abundant γδ T cell Vγ subtypes were Vγ1,2 + and Vγ4 + followed by Vγ6 + , Vγ5 + and Vγ7 + . In TA PVAT, the most abundant γδ T cell Vγ subtype was Vγ6 + followed by Vγ4 + , Vγ1,2 + , Vγ5 + and Vγ7 + . In MA PVAT, the most abundant γδ T cell Vγ subtype was Vγ6 + followed by Vγ4 + , Vγ7 + , Vγ5 + and Vγ1,2 + . Ang II infusion increased the frequency of Vγ6 + γδ T cells in the spleen (% of CD3 + T cells: 0.77±0.09 vs. 0.5±0.02, P P P =0.07). The Vγ6 + γδ T cell frequency in MLNs was unaffected by Ang II infusion. Conclusion: A differential distribution of γδ T cell Vγ subtypes was observed in lymphoid organs compared to PVAT. Vγ6 + γδ T cells may play a role in hypertension. Targeting Vγ6 + γδ T cells could be a therapeutic approach to reduce inflammation in hypertension.

Deoxycorticosterone acetate (DOCA)-salt is suggested to increase blood pressure via selective upregulation of the brain renin-angiotensin system (RAS), while suppressing the circulating RAS. Yet, we have observed parallel downregulation of plasma and brain renin in mice. Here, we quantified brainstem angiotensinogen (AGT) and angiotensin (Ang) levels in rats exposed to DOCA (200mg; 60-day release) and 0.9% NaCl as drinking water for 7 weeks. To determine the contribution of AGT (liver vs. kidney/brain), Ang II type 1 receptors (AT1R) and blood pressure to tissue Ang II content, rats received vehicle, liver-targeted AGT siRNA (10 mg/kg fortnightly; s.c.), valsartan (31 mg/kg/day; s.c.) or spironolactone (80 mg/kg/day; s.c.) during the final 3 weeks, the latter fully normalizing blood pressure (n=3-9 per group). Plasma renin and AGT were determined by enzyme-kinetic assay, tissue AGT by western blotting, and Ang I and II by LC-MS/MS. Plasma renin, AGT, Ang I and II in rats not exposed to DOCA-salt were 24±8ng Ang I/mL per h, 731±51nM, 123±38pM and 109±43pM. AGT was present in liver, kidney and brainstem. Kidney Ang I and II were 540±135and 510±97fmol/g, while brainstem Ang I and II were undetectable in all and 50% of the rats, respectively (

Introduction: Pulmonary arterial hypertension (PAH) is characterized by pulmonary arterial occlusion leading to increased pulmonary arterial pressure. Recently, a growing body of evidence demonstrated a robust increase in oxidized lipids, including 15-hydroxyeicosatetraenoic acids (15HETE), in the lungs and plasma of PAH patients and animal models of pulmonary hypertension (PH). Nonetheless, the the causal role of 15HETE in promoting PH development remains elusive. In order to investigate this mechanism, we fed wild type mice with a diet rich in 15HETE and examine whether ApoA-I mimetic peptide can rescue PH induced by 15HETE. Methods: Wild type male mice (C57BL/6) were fed for 3 weeks with regular chow (n=16-21 mice/group), 15HETE (5μg/day), 15HETE diet supplemented with either empty vector or 6F for the last week of 15HETE diet. PH development was assessed every week via serial echocardiography. Right ventricular systolic pressure (RVSP) was measured via heart catheterization. RV hypertrophy index (RV/[IVS+LV]) was measured. Lung morphology and lipid accumulation were assessed using H&E and Oil red O staining. Results: Echocardiography revealed the first sign of PH as early as one week after starting 15HETE diet and a significant decrease in the pulmonary arterial acceleration time (PAAT) after 2 weeks of treatment (16.6±1.9 vs. 20.6±1.4 msec, p Conclusion: Our data demonstrates that APOA-I mimetic peptide 6F is able to rescue pre-exisiting PH induced by 15-HETE diet in wild type mice.

Introduction: Increasing evidences describe that Brain-derived neurotrophic factor (BDNF) is neuroprotective against cerebrovascular injuries and we previously revealed BDNF preservation on cerebral vessels was associated with inhibition of stroke onset in hypertensive rats. As BDNF was reported to increase blood pressure (BP) in hypertension, however, the upregulation might show conflicting results regarding stroke onset. In this study, we pursued the role of BDNF on stroke onset induced by high-salt diet (HSD) in hypertensive rats. Methods: In experiment 1, spontaneously hypertensive stroke-prone rats (SHRSP) were fed an 8% (n=22) or 0.3% (n=8) sodium diet from 11 weeks of age through 28 days. In experiment 2, SHRSP were treated with continuous intracerebroventricular injection of 2.1μg/day BDNF (n=10) or the vehicle (PBS; n=10) and fed an 8% sodium diet from 11 weeks of age through 24 days. Results and Conclusions: In experiment 1, stroke onset (95%) and mortality (55%) were frequently seen in the rats with HSD, whereas the rats with normal-salt diet were healthy. The expression level of BNDF was upregulated in the cerebral hemisphere and the increase were seen in reactive astrocytes at somatosensory cortex, whereas it was downregulated in endothelial cells. In experiment 2, in comparison with vehicle group, SHRSP with BDNF showed significant increase of incidence of stroke signs (60% vs. 89%), mortality (20% vs. 67%), BP at 2 weeks (214±6 vs. 244±12 mmHg), heart rate (366±20 vs. 457±17 beat/min) and decrease of body weight at 2 week (253±6 vs. 212±11 g), symptom score (5.6±0.3 vs. 2.8±0.8), and gastrocnemius muscle/BW (57±1 vs. 53±1 mg/g). Conclusion Despite the consistent observations of protective roles of BDNF in endothelial cells against the stroke, the present study demonstrated that endogenous BDNF was produced in reactive astrocytes in relation to stroke onset and additive application of exogenous BDNF by continuous intracerebroventricular injection enhanced the onset via elevation of BP in hypertensive rats. As BDNF is a potential candidate treatment not only for post-stroke but also stroke inhibition, we suggest that hypertensive patients need care for the elevation of BP in the clinical trials with BDNF.

Introduction: Renovascular hypertension is the leading cause of secondary hypertension and is also involved in the physiopathogenesis of resistant hypertension. The activation of toll-like receptors 9 (TLR9) was demonstrated to contribute to the increase in arterial pressure (AP) in spontaneously hypertensive rats. We hypothesize that the TLR9 are also activated in two-kidney one- clip (2K1C) hypertensive mice, eliciting cardiac and vascular dysfunction; therefore, contributing to the increase in AP. Methods and Results: C57BL (WT) and TLR9 knockout (TLR9_KO) mice were anesthetized with isoflurane and submitted to 2K1C hypertension by placing a silver clip (0.12 mm) around the left renal artery. After 4 weeks, the cardiac function of mice was evaluated by echocardiography in anesthetized subjects. Following, the direct AP measurement was carried out and the third-order mesenteric resistance arteries (MRA) were removed for vascular function evaluation. All data were compared to Sham-operated WT mice. The AP was remarkably elevated in 2K1C WT mice (133±2 vs 93 ± 4 mmHg), whereas this increase was partially prevented by the absence of the TLR9 (114±5 mmHg). 2K1C hypertension caused cardiac dysfunction in WT mice, displaying decreased ejection fraction (42.6±3.1 vs 52.7±1.5%), fractional shortening (10.1±0.7 vs 13.7±0.9%), stroke volume (25.9±2.2 vs 37.9±1.9 μL) and cardiac output (9.2±0.8 vs 14.6±0.8 mL/min); however, the 2K1K hypertension did not affect the cardiac function in TLR9_KO mice: ejection fraction (54.5±1.6%), fractional shortening (14.5±1.1%), stroke volume (43.4±4.6 μL) and cardiac output (15.2±1.6 mL/min). Vascular dysfunction, characterized by increased contractile response to phenylephrine (Emax: 167.2±2.2 vs 125.9±3.5%) and reduced relaxation to acetylcholine (Emax: 69.3±1.6 vs 87.6±1.4%) and sodium nitroprusside (pD2: 7.3±0.07 vs 7.9±0.05), was observed in 2K1C WT mice, but not in 2K1C TLR9_KO mice (Emax: 127.7±2.9%, 88.5±2.0% and pD2: 7.78±0.06, to phenylephrine, acetylcholine and sodium nitroprusside). Conclusions: TLR9 is involved in both cardiac and vascular dysfunctions of 2K1C mice and also in the increase of AP of this experimental model.

Introduction: Corin, a serine protease converts pro-atrial natriuretic peptide (pro-ANP) and pro-brain natriuretic peptide (pro-BNP) to the mature respective peptides. There is evidence that the pregnant uterus produces ANP, where it promotes spiral artery remodelling, essential for adequate blood supply to the developing conceptus. Aim: Here we examine corin and PCSK6, a key enzyme in the conversion of pro-corin to corin, in the placenta of hyperinsulinemic dams (HD) in comparison to normal pregnant dams (NPD). Materials and Methods: Female Wistar rats were rendered hyperinsulinemic by subcutaneous insulin pellet, mated and followed to day 21 of pregnancy. Both groups were then sacrificed and their placentas were dissected together with the implantation site designated mesometrial triangle (MT). Placentas and MT were sectioned from the maternal through the fetal surface to the base of the MT. For western blot (WB) the placentas and MT were separated, snap frozen in liquid nitrogen and stored at –80°C. RNA expression of corin was carried out by in-situ hybridization in 5 NPD and 6 HD with corin probe (RNAscope® Assay 2.5 Detection Reagent, Cat No. 507231) and image analysis was performed. Results: As previously described, HD (n=20) developed hypertension as compared with in NPD (n=16). Moreover, HD exhibited lower placental and foetal weights. Corin and PCSK6 mRNA and immunoreactive peptide were detected in the mesometrial tissues. Corin abundance as determined by western blot was significantly decreased in the placenta/ mesometrial triangle by ~40% (Pin-situ hybridization for corin mRNA revealed a 50% (P Summary and Conclusion: Corin, PCSK6 and ANP are all expressed in the placenta of NPD but declined in dams with chronic hyperinsulinemia, suggesting a role of corin/PCSK6 pathway in the prevention of pregnancy-induced hypertension and intrauterine growth restriction, in this setting.

The G protein coupled receptor 83 (Gpr83) is an orphan receptor that has been associated with essential hypertension. Gpr83 is expressed in the kidney but its function is unknown. We found that Gpr83 is expressed in mouse renal proximal and distal convoluted tubules, as well as in human renal proximal tubule cells (hRPTCs). In C57Bl/6J mice on normal salt diet, the lack of one (Gpr83+/-) or both Gpr83 (Gpr83-/-) alleles resulted in an increase in systolic blood pressure (SBP, ~20 mm Hg (PGpr83 gene down-regulation in mouse and human renal proximal tubule cells decreased about 50% the expression of nuclear factor of activated T cells 5 (NFAT5). The renal expressions of NFAT5 and the NFAT5 targets UT-A3, Tau T and BGT1 were increased by a high salt diet in salt resistant mice. The neuropeptide PEN (PEN), a ligand of Gpr83, is also expressed in hRPTCs (0.43±0.03 mRNA PEN/mRNA GAPDH). We found that Gpr83 stimulation with PEN (1 μM, 3 hr) decreased the transcription of NFAT5 (0.59±0.05 vs 1.0±0.2fold, P

Objective: Inflammation drives cardiovascular disease, anti-inflammatory approaches may be beneficial. Short-chain fatty acids (SCFA) are bacterial metabolites with anti-inflammatory properties affecting host immune homeostasis including regulatory T cells (Treg). We investigated effects of the SCFA propionate (administered in drinking water, NaCl as control) in two mouse models, namely hypertensive heart disease (wild-type NMRI (WT), angiotensin (Ang)II infusion 1.44 mg/kg/d s.c. for 14 days) and atherosclerosis (Apolipoprotein E knockout (ApoE), AngII infusion 0.72 mg/kg/d s.c. for 28 days), respectively. Results: Propionate attenuated cardiac hypertrophy and fibrosis in both models significantly. Susceptibility to cardiac ventricular arrhythmias was significantly reduced in propionate-treated WT mice. Aortic atherosclerotic lesion area was significantly reduced in propionate-treated ApoE (27.6±8 vs. 7.9±2.4%). Treatment reduced splenic effector memory (CD4+ CD44+ CD62L-) T cell frequencies (WT: 30.5±4.6 vs. 19.1±1.6; ApoE: 41.1±3.1 vs. 32.7±1.4%) and splenic Th17 cells (WT: 1.0±0.2 vs. 0.6±0.1; ApoE: 1.3±0.1 vs. 0.9±0.1%) in both models, indicating beneficial effects on systemic inflammation. Similarly, propionate reduced cardiac immune cell infiltration (CD4+, CD8+, F4/80+) in WT mice. Propionate improved vascular dysfunction and moderately reduced blood pressure in both models. Organ-protective actions of propionate (cardiac inflammation and fibrosis) were abrogated in Treg-depleted (antiCD25-treated) AngII-infused WT mice, suggesting a central role for Treg. To verify our findings in a human cohort, we re-analyzed clinical and metagenomic data from a recent randomized controlled trial investigating the effect of a 90-day synbiotic intervention in 84 subjects with metabolic syndrome including healthy controls. Interestingly, in synbiotic-treated subjects an increased capacity for SFCA production was significantly correlated to blood pressure reduction. Conclusion: Data underscore the importance of SCFA for cardiovascular health and suggest that lifestyle modifications leading to augmented SCFA production could be a beneficial non-pharmacological add-on strategy for cardiovascular disease.

Fundus images from the left eyes of 440 subjects aged 50-59 years enrolled in the Northern Ireland Cohort of Longitudinal Ageing were analyzed. Subjects were categorized as normotensive or hypertensive, according to thresholds on systolic/diastolic blood pressure measurement (140/90 mm Hg) averaged over two sitting measurements in a clinical setting. A fully automatic system to analyze each image used conventional and deep neural network machine learning techniques to locate retinal landmarks and detect, classify and measure retinal vessels. From this data, a measure of the arteriolar-venular ratio (AVR) in the peripheral retina was calculated. Semi-automatic analysis was also performed. Results are presented in Table 1. Subjects had mean age of 54.6 ± 2.9 years; 56.1% (247 of 440) females, with 34.3% (151 of 440) subjects categorized as hypertensive. Narrower arterioles and smaller AVR were observed in subjects with hypertension. This was also observed in fully-automated analysis, however 4% (17 of 440) subjects failed to be processed by the system. In fully-automated analysis the area under a receiver operator characteristic curve of AVR for hypertensive status was 0.69 (95% CI, 0.63 to 0.74). Table 1 - Results for semi-automated and automated analysis of retinal vessel parameters. *p Automated measurement of AVR in ultra-widefield fundus imaging was associated with hypertension. With further development, such as evaluation against diagnosis of hypertension obtained from ambulatory blood pressure monitoring clinics, this system could become a test for undiagnosed hypertension in people attending routine eye health check-ups.

Background: Transgenic mouse with tamoxifen-inducible endothelium-restricted human ET-1 overexpression (ieET-1) exhibited blood pressure (BP) elevation three weeks (short-term) and three months after induction (long-term). Vascular injury was observed only after long-term exposure to endothelial ET-1 overexpression. It is unknown whether short or long-term exposure to ET-1 overexpression results in gene dysregulation. We aimed to identify differentially expressed (DE) microRNAs (miRs) and genes in mesenteric arteries of ieET-1 mice after short- and long-term induction of human ET-1 overexpression. Methods and Results: Ten to 12-week old male ieET-1 mice and control ieCre mice expressing a tamoxifen-inducible Cre recombinase under the control of endothelium-specific Tie2 promoter, were treated with tamoxifen (1 mg/kg/day, s.c.) for 5 days and sacrificed 16 days or 3 month later. RNA was extracted from mesenteric arteries of ieCre and ieET-1 mice and used for small and total RNA-sequencing using Illumina HiSeq-2500. EdgeR was used for differential expression analysis (false discovery rate Khdrbs3 (KH domain containing, RNA binding, signal transduction associated 3), which was up-regulated after both short- and long-term exposure to endothelial ET-1 overexpression, was validated by reverse transcription-quantitative PCR (RT-qPCR). We demonstrated a correlation between RNA-sequencing and RT-qPCR data for short- and long-term groups (r=0.8, P Khdrbs3 was 2 times more in fibroblasts than in smooth muscle cells and endothelial cells (2.37±0.28 vs. 1.11±0.17 and 1.12±0.12, P Conclusions: The results showed that short- and long-term exposure to endothelial ET-1 overexpression up-regulated Khdrbs3 in mesenteric arteries. In vitro study revealed that this gene was expressed to a greater level in fibroblasts than other vascular cells. However, the role of Khdrbs3 in ET-1-induced vascular injury remains to be determined.

Endogenously produced asymmetrically methylated forms of L-arginine (ADMA) competitively inhibit all three isoforms of nitric oxide synthase enzymes and therefore have the potential to exert significant cardiovascular effects. Elevated circulating ADMA concentrations have been reported in a number of cardiovascular disease states, including preeclampsia, suggesting that impaired ADMA metabolism may contribute to pathology. In order to test the causal relationship between ADMA concentrations and hemodynamics in pregnancy we employed radiotelemetric measurement of hemodynamic function in mice with either global or fetal-specific deletion of the predominant ADMA metabolising enzyme dimethylarginine dimethylaminohydrolase 1 (DDAH1). Global deletion of DDAH1 in non-pregnant female mice caused an elevation in circulating ADMA concentrations (2.851±0.289 vs 1.216±0.072 μM p-/- mice showed no further elevation in ADMA and normal pregnancy related changes in blood pressure were observed. However, during the third trimester ADMA levels in DDAH1 -/- mice increased further and this was associated with increased concentrations of sEng and sFlt1 (2288±301.58 vs 1181.35 ± 161.83 pg/ml p-/- mice. Our data indicate that elevated ADMA is sufficient to increase levels of sFLlt1 and sEng and disrupt maternal hemodynamics. The fetus is a significant source of ADMA in late pregnancy suggesting that dysfunctional fetal ADMA metabolism is sufficient to impair maternal hemodynamic function.

Objectives: Despite the increased incidence and worse outcomes of cardiovascular diseases among African-Americans compared to the general population, the contribution of sickle cell trait (AS) to this racial disparity remains unknown. We aimed to delineate the impact of AS on the pathogenesis of cardiovascular diseases alone, and in the setting of other risk factors. Methods: We compared multiple cardiovascular endpoints between AS and wild-type genotype (AA) using the humanized Townes sickle cell mouse model. To determine whether AS modifies a pre-existing cardiovascular risk factor, diabetes was induced in a cohort of mice using streptozotocin (STZ). Hind limb ischemia model was employed to evaluate collateral vessel formation, and perfusion recovery was characterized with LASER Doppler perfusion imaging (LDPI). The running wheel assay assessed spontaneous motor function after ischemia. Angiotensin II (Ang II) and L-NAME were used to evaluate hypertensive response. Experimental stroke was induced by unilateral common carotid artery ligation and daily 15 min exposure to 7.5% oxygen for 3 days, and the infarct size was quantified as the ratio of the infarcted area to the total area of the uninjured contralateral hemisphere. PCSK9 gain-of-function mutation in the setting of Ang II and high-fat diet was used to evaluate atherosclerosis development. Results: 4 groups of mice were studied: AA, AS, AA-STZ and AS-STZ. The formation of collateral vessels was equivalent between the AA and AS (day 28, AA: 80 ± 6%, AS: 85 ± 5%, p=0.8), and in the setting of diabetes (39 ± 5% AA-STZ; 36 ± 4% AS-STZ). Systolic blood pressure at baseline (110 ± 4 mmHg vs 112 ± 5 mmHg), and in response to Ang II (148 ± 4 mmHg vs 142 ± 5 mmHg) and L-NAME (135 ± 3mmHg vs 138 ± 5 mmHg) were equivalent. Mortality from stroke was equivalent between AA and AS. After 1 month of PCKS9 gain-of-function mutation/high-fat diet, AA and AS demonstrated similar lipid profiles. Mortality and infarct volume after stroke induction were identical between AA and AS. Conclusions: All major indices of cardiovascular outcomes were similar between AA and AS, even in the presence of diabetes. These various experimental models suggest that the sickle trait likely does not contribute significantly to the poor cardiovascular outcomes seen in the African-American population.

Background and Purpose: Deletion of Klotho leads to hypertension. The purpose of this study is to investigate if renal epithelial sodium channel (ENaC) contributes to klotho deficiency-induced hypertension. Methods and Results: We generated kidney-specific conditional klotho null (KspKL -/- ) mice and found out that renal alpha subunit of ENaC is significantly upregulated compared to wild type (WT) mice. Blood pressure measured by telemetry demonstrated systolic hypertension and elevated pulse pressure suggesting vascular dysfunction in KspKL -/- . Pulse wave velocity, a marker of arterial stiffening was significantly increased in KspKL -/- mice. Amiloride, an ENaC inhibitor, completely prevented systolic hypertension and arterial stiffening in KspKL -/- mice. Ex vivo vascular relaxing responses to acetylcholine were diminished in mesenteric arteries in KspKL -/- group, indicating that klotho deficiency causes vascular endothelial dysfunction. Interestingly, treatment with amiloride abolished Klotho deficiency-induced vascular endothelial dysfunction. We found that urinary sodium excretion (U Na V) was significantly impaired in KspKL -/- group. Na retention was also associated with a decrease in glomerular filtration rate (GFR), suggesting impaired renal function in KspKL -/- mice. Treatment with amiloride prevented sodium retention and improved GFR in KspKL -/- mice. Total renal nitric oxide bioavailabililty and eNOS activity were significantly decreased in KspKl -/- mice which was ameliorated by amiloride treatment. Histological and morphometric analysis showed glomerular and tubular damage in KspKL -/- , which was improved by amiloride treatment. Western blotting analysis demonstrated a significant decrease in the αγ subunits of ENaC in the kidney cortex following treatment with amiloride. Conclusion: This study demonstrates for the first time that kidney-specific depletion of the klotho gene causes impairment in Na-excretion and hypertension by affecting ENaCα levels. ENaC may be a promising therapeutic target for klotho gene deficiency-induced renal and vascular dysfunction and hypertension.

Small vessel disease and/or atherosclerosis produce microvascular and parenchymal inflammation in white matter and results in vascular cognitive impairment (VCI). We have performed bilateral carotid artery stenosis in hypertensive rats (SHR) to better understand disease pathology, targets for intervention and markers. Hypothesis: Complex cognitive deficits and diffuse fiber tract changes relevant to human VCI can be quantified and validated for future use. Methodology: We performed a series of behavioral assays to test declarative memory and executive functioning in stenosis compared to sham surgery SHR. Behavioral assays included T-maze decision making and alternation, novel object recognition (NOR) and active place avoidance (APA). MRI (DTI, DWI, Arterial Spin Labeling; ASL) and FDG-PET imaging was done in Corpus Callosum (CC). Histology-immunohistochemistry included measurements of microglia (Iba-1), astrocytes (GFAP) and Luxol fast blue (for myelin) in CC. Results: Stenosis resulted in consistent executive function decision making (T-maze) deficits (p Conclusion: We have successfully modeled the behavioral, imaging and histologic profile of human VCI in the rat. Currently pre/mature oligodendrocyte changes are being evaluated. This approach provides future opportunities to localize forebrain white matter changes using MR imaging parameters as markers for monitoring VCI demyelination/pathology and intervention.

The peculiarities of cardiac natriuretic peptide secretion have been investigated during ontogenesis using the hypertonic disease model in ISIAH rat line (with inherited stress induced arterial hypertension). The qualitative and quantitative ultrastructural investigations of right atrium myocardium revealed that the cardiac hormonal synthetic and secretory activities in ISIAH rats are higher as compared with normotensive even-aged rats from control group. Secretory hyperactivity of atrial myocytes in ISIAH rats during early ontogeny precedes the manifestations of hereditary hypertension. Natriuretic peptides present the hypotensive circuit of hemodynamic regulation during the whole ontogeny and the complementary chain in hypertension development.

Hypertension is associated with immune system activation and inflammation. Renal infiltration of both innate and adaptive immune cells contributes to injury, dysfunction, and increased blood pressure. Activated immune cells that exit blood vessels into the interstitium then travel through lymphatic vessels to draining lymph nodes where they signal to other immune cells to increase the immune response. It is unknown how renal lymphatic vessels change in the context of hypertension, immune system activation, inflammation, and injury. We hypothesized that renal macrophage infiltration, inflammation, and injury would significantly increase lymphangiogenesis in various strains of rats. SHR rats that exhibit hypertension and renal injury (SHR-A3 strain) had significantly increased numbers of renal lymphatic vessels at 40 weeks of age compared to WKY controls (total of 3 fields of view: 52 ± 1 vs. 28 ± 1; p

Aim: We hypothesize that challenges to sodium homeostasis differentially activate chemo- vs. mechanosensitive afferent renal nerves to evoke sympathoinhibition, sodium homeostasis and normotension in the Sprague-Dawley (SD) rat. Methods: Conscious SD rats, post sham (S) or afferent renal nerve ablation (Renal-CAP; capsaicin 33 mM) underwent IV volume expansion (VE; 5% BW) or IV sodium loading (1M NaCl Infusion – constant infusion volume) and HR, MAP, natriuresis and PVN neuronal activation (c-Fos expression) were assessed (N=4/gp). Naïve SD rats were fed a 0.6% (NS) or 4% NaCl (HS) diet for 21 days and afferent renal nerve activity was assessed as norepinephrine (NE) (1250 pmol) and NaCl-evoked (450mM) substance P (SP) release in a renal pelvic assay (N=4/gp). Radiotelemetered SD rats post S or Renal-CAP immediately prior a 0.6% (NS) or 4% NaCl (HS) diet underwent continuous MAP monitoring. On day-21 plasma and renal NE content was assessed (N=5/group). Results: Renal-CAP attenuated the natriuretic and PVN parvocellular responses to IV VE (peak UNaV [μeq/min]; S 43±4 vs Renal-CAP 26±6, P Conclusion: The mechanosensitive afferent renal nerves mediate acute natriuresis and blood pressure regulation via activation of PVN sympathoinhibitory neurons. During HS intake the afferent renal nerves counter the development of salt-sensitive hypertension via a mechanism involving increased mechano but not chemosensitive afferent nerve responsiveness to potentiate sympathoinhibition.

Aim: We hypothesize that excess norepinephrine (NE) modulates NCC activity via an α1 adrenoceptor pathway to drive the development of salt-sensitive hypertension (HTN). Methods: Male Sprague-Dawley (SD) rats receiving a continuous s.c. saline or NE (600ng/min) infusion and naïve Dahl Salt-Sensitive (DSS) rats were fed a 0.6% (NS) or 8% NaCl (HS) diet for 14 or 21 days respectively (N=4/gp). On day 14 (SD) or 21 (DSS) MAP and NCC activity (peak natriuresis to iv hydrochlorothiazide (HCTZ; 2mg/kg) infusion) and expression (via immunoblotting) was assessed. Additional groups of NE infused SD and DSS rats received a propranolol (9.9mg/kg/day; s.c.) or prazosin (2.5mg/kg/day; oral) and a NS or HS diet for 14 or 21 days. Results: SD rats exhibit HS evoked suppression of NCC expression and activity. In contrast, NE infused SD rats and DSS rats exhibit HTN and fail to suppress NCC expression and activity during HS-intake. β-adrenoceptor antagonism (confirmed pharmacologically) reduced MAP in NE infused SD and DSS rats, but failed to decrease NCC activity or expression. In contrast α1-adreoceptor antagonism (confirmed pharmacologically) abolished the salt-sensitive component of HTN and restored dietary sodium evoked suppression of NCC activity and expression in NE infused SD rats and DSS rats. Conclusion: Our data suggests NE activates α, but not β, adrenoceptors to prevent dietary sodium evoked suppression of NCC activity and the development of salt-sensitive hypertension. The PATHWAY-2 Trial reported a primary role of sodium retention in resistant HTN suggesting α1-adreoceptor antagonism represents a new therapeutic approach for resistant and sympathetically mediated HTN.