Mice with collecting duct-specific deletion of endothelin-1 are hypertensive and have impaired Na excretion. Since endothelin-1 activates nitric oxide synthase (NOS) in collecting duct, we hypothesized that impaired renal NO production in knockout mice exacerbates the hypertensive state. Control and knockout mice were treated chronically with L-NAME, and arterial pressure (BP) and urinary nitrate/nitrite (NOx) excretion assessed. On a normal Na diet, knockout systolic BP was 18 mmHg greater than controls. L-NAME increased BP in control mice by 30 mmHg and 10 mmHg in CD ET-1 KO mice, thereby abolishing the difference in systolic BP between the groups. A high Na diet increased BP similarly in both groups. Urinary NOx excretion was lower in knockout mice than in controls on normal or high Na intake. In separate experiments, renal perfusion pressure was adjusted in anesthetized mice, and urinary NOx and Na excretion determined. Similar elevations of BP increased urinary Na and NOx excretion in control mice, but to a significantly lesser extent in knockouts. Isoform-specific NOS activity and expression were determined in renal inner medulla homogenates from control and knockout mice. NOS1 and NOS3 activities were lower in knockouts than in controls given normal or high Na diets. However, NOS1 or NOS3 protein expression were similar in both groups on normal or high Na intake. These data demonstrate that collecting duct-derived endothelin-1 is important in: 1) chronic L-NAME-induced hypertension; 2) full expression of pressure-dependent changes in sodium excretion; and 3) control of inner medullary NOS1 and NOS3 activity.
ET-1; nitric oxide; Na; natriuresis; diuresis; blood pressure
G protein-coupled receptor kinases (GRKs) are important regulatory proteins for many G protein-coupled receptors, but little is known about GRK4 pharmacogenetics. We hypothesized three nonsynonymous GRK4 SNPs, R65L (rs2960306), A142V (rs1024323) and A486V (rs1801058) would be associated with blood pressure response to atenolol, but not hydrochlorothiazide, and would be associated with long term cardiovascular outcomes (all cause, death, nonfatal myocardial infarction, nonfatal stroke) in participants treated with an atenolol-based versus verapamil-SR-based antihypertensive strategy. GRK4 SNPs were genotyped in 768 hypertensive participants from the Pharmacogenomic Evaluation of Antihypertensive Responses (PEAR) trial. In Caucasians and African Americans, increasing copies of the variant 65L-142V haplotype were associated with significantly reduced atenolol-induced diastolic blood pressure lowering (−9.1 ± 6.8 vs −6.8 ± 7.1 vs −5.3 ± 6.4 mmHg in participants with 0, 1 and 2 copies of 65L-142V respectively; p=0.0088). 1460 participants with hypertension and coronary artery disease from the INternational VErapamil SR / Trandolapril STudy (INVEST) were genotyped and variant alleles of all three GRK4 SNPs were associated with increased risk for adverse cardiovascular outcomes in an additive fashion, with 486V homozygotes reaching statistical significance (Odds ratio 2.29 [1.48–3.55], p=0.0002). These effects on adverse cardiovascular outcomes were independent of antihypertensive treatment. These results suggest the presence of GRK4 variant alleles may be important determinants of blood pressure response to atenolol and risk for adverse cardiovascular events. The associations with GRK4 variant alleles were stronger in patients who were also ADRB1 389R-homozygotes, suggesting a potential interaction between these two genes.
hypertension; GRK4; atenolol; beta-blocker; outcomes; ADRB1; pharmacogenetics
Aging is associated with increased retrograde and oscillatory shear in peripheral conduit arteries of humans. Although the mechanisms responsible for these age-related changes are not completely understood, augmented downstream α-adrenergic tone likely plays a significant role in this phenomenon. Therefore, in Protocol 1 brachial artery diameter and blood velocity were measured via Doppler ultrasound during 1) rest (control), 2) endogenous norepinephrine release via intra-arterial infusions of tyramine, and 3) α-adrenergic blockade via infusions of phentolamine in young healthy humans (n=12). Tyramine increased brachial artery retrograde (−4.0±1.4 to −9.5±1.4 s−1) and oscillatory shear (0.05±0.02 to 0.18±0.05 arbitrary units), whereas, phentolamine abolished retrograde and oscillatory shear (P<0.05). Additionally, in Protocol 2 we examined brachial artery shear patterns in young (n=12; 29±2 years) and older (n=13; 69±2 years) healthy adults during 1) rest (control), 2) sympathetic activation via lower body negative pressure, and 3) infusion of phentolamine. At rest older adults exhibited greater brachial artery retrograde and oscillatory shear (−9.9±2.7 s−1 and 0.11±0.03 arbitrary units, respectively) compared with younger adults (−3.1±1.0 s−1 and 0.05±0.02 arbitrary units, respectively; P<0.05 for both). Lower body negative pressure increased retrograde and oscillatory shear in young (P<0.05), but not older adults (P=0.85–0.97), such that differences between young and older were eliminated (P>0.05). During infusion of phentolamine, retrograde and oscillatory shear were abolished in young adults (P<0.05) and markedly reduced, yet still persistent, in older adults (P<0.01). Our data indicate that α-adrenergic vasoconstriction is a major contributor to age-related discrepancies in conduit artery shear rate patterns at rest.
α-adrenergic receptors; aging; shear rate; sympathetic activation
Hypertensive disorders occur in approximately 6–8% of all pregnancies and are a significant source of maternal and fetal morbidity. Little is known about the range of agents routinely used in practice. We used Medicaid claims from 2000–2007 to identify completed pregnancies. We included women who were Medicaid beneficiaries from at least 3 months prior to last menstrual period to 1 month post-delivery, and were successfully linked to infant records. Maternal exposure to antihypertensive medications was derived from Medicaid pharmacy claims files and duration of exposure was assigned based on the days supply dispensed. We identified 1,106,757 Medicaid patients in our cohort of whom 48,453 (4.4%) were exposed to antihypertensive medications during pregnancy. The prevalence of antihypertensive use increased from 3.5% to 4.9% during the study period. Antihypertensive medications users were older than non-users, more likely to be Caucasian or African-American, and more likely to have comorbid diabetes and/or renal disease. Overall, 1.9% of pregnant women were exposed during the 1st trimester, 1.7% during the 2nd trimester, and 3.2% during the 3rd trimester. The range of antihypertensive medications to which patients were exposed was highly heterogeneous and frequently included agents other than methyldopa or labetalol. ACE inhibitor exposure, which is contraindicated in late pregnancy, occurred in 928 (4.9%) antihypertensive medication users in the 2nd trimester and 383 (1.1%) in the 3rd trimester. Antihypertensive use during pregnancy is relatively common and increasing. The wide range of agents used during pregnancy includes medications considered contraindicated during pregnancy.
Hypertension; Pregnancy; Epidemiology; Antihypertensives
Previous studies have shown that preconditioning rats with a non-pressor dose of angiotensin II (AngII) sensitizes the pressor response produced by later treatment with a higher dose of AngII and that AngII and aldosterone (Aldo) can modulate each other’s pressor effects through actions involving the central nervous system (CNS). The current studies tested whether Aldo can cross-sensitize the pressor actions of AngII to enhance hypertension by employing an Induction-Delay-Expression (I-D-E) experimental design. Male rats were implanted for telemetered BP recording. During I, sub-pressor doses of either sc or icv Aldo were delivered for 1 week. Rats were then rested for 1 week (D) to assure that any exogenous Aldo was metabolized. After this, AngII was given sc for 2 weeks (E).
During I and D Aldo had no sustained effect on BP. However, during E AngII-induced hypertension was greater in the groups receiving sc or icv Aldo during I in comparison to those groups receiving vehicle. Central administration of mineralocorticoid receptor antagonist blocked sensitization. Brain tissue collected at the end of D and E showed increased mRNA expression of several renin-angiotensin-aldosterone system components in cardiovascular-related forebrain regions of cross-sensitized rats. Cultured subfornical organ neurons preincubated with Aldo displayed greater increases in [Ca2+]i after AngII, and there was greater Fra-like immunoreactivity present at the end of E in cardiovascular-related forebrain structures. Taken together, these results indicate that Aldo pretreatment cross-sensitizes the development of AngII-induced hypertension probably by mechanisms that involve the CNS.
Aldosterone; Angiotensin II; Sensitization; Blood Pressure; Brain Renin-Angiotensin-Aldosterone System Expression
A hallmark of hypertension is an increase in arterial myocyte voltage-dependent Ca2+ (CaV1.2) currents that induces pathological vasoconstriction. CaV1.2 channels are heteromeric complexes comprising a pore forming CaV1.2α1 with auxiliary α2δ and β subunits. Molecular mechanisms that elevate CaV1.2 currents during hypertension and the potential contribution of CaV1.2 auxiliary subunits are unclear. Here, we investigated the pathological significance of α2δ subunits in vasoconstriction associated with hypertension.
Age-dependent development of hypertension in spontaneously hypertensive rats (SHR) was associated with an unequal elevation in α2δ-1 and CaV1.2α1 mRNA and protein in cerebral artery myocytes, with α2δ-1 increasing more than CaV1.2α1. Other α2δ isoforms did not emerge in hypertension. Myocytes and arteries of hypertensive SHR displayed higher surface-localized α2δ-1 and CaV1.2α1 proteins, surface α2δ-1 to CaV1.2α1 ratio (α2δ-1:CaV1.2α1), CaV1.2 current-density and non-inactivating current, and pressure- and - depolarization-induced vasoconstriction than those of Wistar-Kyoto controls. Pregabalin, an α2δ-1 ligand, did not alter α2δ-1 or CaV1.2α1 total protein, but normalized α2δ-1 and CaV1.2α1 surface expression, surface α2δ-1:CaV1.2α1, CaV1.2 current-density and inactivation, and vasoconstriction in myocytes and arteries of hypertensive rats to control levels.
Genetic hypertension is associated with an elevation in α2δ-1 expression that promotes surface trafficking of CaV1.2 channels in cerebral artery myocytes. This leads to an increase in CaV1.2 current-density and a reduction in current inactivation that induces vasoconstriction. Data also suggest that α2δ-1 targeting is a novel strategy that may be used to reverse pathological CaV1.2 channel trafficking to induce cerebrovascular dilation in hypertension.
Calcium channels; Genetic Hypertension; Vasodilation; Vasoconstriction
Insomnia with objective short sleep duration appears to be a biologically more severe phenotype of the disorder. No longitudinal study to date has examined the association of this type of insomnia with incident hypertension using polysomnography. From a random, general population sample of 1741 adults of the Penn State Cohort, 1395 were followed-up after 7.5 years and 786 did not have hypertension at baseline. Hypertension was determined by a self-report of receiving treatment for high blood pressure. Chronic insomnia was defined as a complaint of insomnia lasting ≥ 1 year, while poor sleep was defined as moderate-to-severe sleep difficulties. All subjects underwent 8-hour polysomnography. Sleep disordered breathing (SDB) was defined as an obstructive apnea/hypopnea index ≥ 5. We used the median polysomnographic percent of sleep time to define short sleep duration (i.e., < 6 hours). We controlled for gender, race, age, caffeine, cigarettes, alcohol consumption, depression, SDB, diabetes, obesity, and blood pressure in our analyses. Compared to normal sleepers who slept ≥ 6 hours, the highest risk for incident hypertension was in chronic insomniacs with short sleep duration (OR= 3.8, 95% CI=1.6–9.0). The risk for incident hypertension in poor sleepers with short sleep duration was significantly increased but became marginally significant after controlling for obesity (OR= 1.6, 95% CI=0.9–2.8). Chronic insomnia with short sleep duration is associated with an increased risk for incident hypertension in a degree comparable to SDB. Objective short sleep duration in insomnia may serve as a useful predictor of the biological severity of the disorder.
Insomnia; Polysomnography; Hypertension; Incidence
Apolipoprotein E (apoE) is widely expressed in mammalian tissues and one of the important tissue-specific effects is the atheroprotection ascribed to macrophage-derived apoE in the arterial wall. However, underlying mechanisms are not well understood. In this study, using subcellular fractionation, confocal microscopy and co-immunoprecipitation, we demonstrate that macrophage-derived apoE is internalized by endothelial cells and impacts the subcellular distribution/interaction of caveolin-1 and eNOS. Addition of apoE disrupts the heteromeric complex formed between caveolin-1 and eNOS, and increases nitric oxide (NO) production. Sterol and oxysterol enhance endothelial caveolin-1/eNOS interaction and suppress NO production but these effects are reversed by apoE. Silencing endothelial caveolin-1 attenuates apoE induced NO production establishing the importance of Cav-1/eNOS interaction for the increment in endothelial NO for the apoE effect. Consistent with these observations, macrophage-derived apoE significantly improves vasodilation to acetylcholine in resistance arteries isolated from adipose tissue of obese humans. We conclude that macrophage-derived apoE enhances endothelial NO production by disrupting the inhibitory interaction of eNOS with caveolin-1. These results establish a novel mechanism by which apoE modulates endothelial cell function.
apolipoprotein E; endothelium; caveolin-1; macrophages; nitric oxide; nitric oxide synthase
Elevated blood pressure (BP) is a major risk factor for cardiovascular disease. Several studies have noted a consistent maternal effect on BP; consequently, mitochondrial DNA (mtDNA) variation has become an additional target of investigation of the missing BP heritability. Analyses of common mtDNA polymorphisms, however, have not found evidence of association with hypertension. To explore associations of relatively rare (frequency < 5%) mtDNA variants with BP, we identified uncommon/rare variants through sequencing the entire mitochondrial genome in 32 unrelated individuals with extreme-high BP in the Framingham Heart Study (FHS) and genotyped 40 mtSNPs in 7,219 FHS participants. The nonsynonymous mtSNP 5913G>A (Asp4Asn) in the cytochrome c oxidase subunit 1 of Complex IV demonstrated significant associations with BP and fasting blood glucose (FBG) levels. Individuals with the rare 5913A allele had, on average, 7 mm Hg higher systolic BP at baseline (Pempirical = 0.05) and 17 mg/dL higher mean FBG over 25 years of follow up (Pempirical = 0.009). Significant associations with FBG levels were also detected for nonsynonymous mtSNP 3316G>A (Ala4Thr) in the NADH dehydrogenase subunit 1 of Complex I. On average, individuals with rare allele 3316A had 17 and 25 mg/dL higher FBG at baseline (Pempirical = 0.01) and over 25 years of follow up (Pempirical = 0.007). Our findings provide the first evidence of putative association of variants in the mitochondrial genome with SBP and FBG in the general population. Replication in independent samples, however, is needed to confirm these putative associations.
Mitochondrial genome; Association study; Genetics; Hypertension; Diabetes
We have previously demonstrated that paracrine signaling occurs between medullary thick ascending limb (mTAL) and the contractile pericytes of outer-medullary vasa recta (VR) termed ‘tubular-vascular cross talk’. The aim of the current study was to determine whether tubular-vascular cross talk has a functional effect on vasoconstrictor responses to angiotensin II, and to determine whether this is altered in the Dahl salt-sensitive (SS) rat. Studies were performed on salt-resistant consomic SS.13BN and SS rats using a novel outer medullary tissue strip preparation in which freshly isolated VR within VR bundles were perfused either alone or in combination with nearby mTAL. In VR from SS.13BN rats, angiotensin II (1μM) increased VR bundle intracellular Ca2+ concentration ([Ca2+]VR) 19±9nM (n=8) and reduced focal diameter in perfused VR by (−20±7%;n=5). In the presence of nearby mTAL however, [Ca2+]VR (−9±8nM; n=8) and VR diameter (−1±4%, n=7) in SS.13BN rats was unchanged by angiotensin II. In contrast, in Dahl SS rats, angiotensin II resulted in rapid and sustained increase in [Ca2+]VR (89±48 n=7;50±24% n=8) and a reduction in VR diameter of (−17±7;n=7 and −11±4%;n=5) in both isolated VR and VR with nearby mTAL, respectively. In VR with mTAL from SS13BN rats, inhibiton of purinergic receptors resulted in an increase in [Ca2+]VR, indicating purinergic signaling buffers vasoconstriction. Importantly, our in vitro data were able to predict medullary blood flow responses to angiotensin II in SS and SS.13BN rats in vivo. We conclude that paracrine signaling from mTAL buffers angiotensin II vasoconstriction in Dahl salt-resistant SS.13BN rats but not SS rats.
HAEMODYMAICS; HYPERTENSION; BLOOD FLOW REGULATION; MICROCIRCULATION; NITRIC OXIDE; PURINERGIC EFFECT; IMAGING
We designed this study to explore to what extent the excess risk of cardiovascular events in diabetic individuals is attributable to hypertension. We retrospectively analyzed prospectively collected data from the Framingham Original and Offspring cohorts. Of the 1145 Framingham subjects newly diagnosed with diabetes who did not have a prior history of cardiovascular events, 663 (58%) had hypertension at the time diabetes was diagnosed. During 4154 person-years of follow-up, 125 died and 204 suffered a cardiovascular event. Framingham participants with hypertension at the time of diabetes diagnosis exhibited higher rates of all cause mortality (32 versus 20 per 1000 person years, p<0.001) and cardiovascular events (52 versus 31 per 1000 person years, p<0.001) compared with normotensive subjects with diabetes. After adjustment for demographic and clinical covariates, hypertension was associated with a 72% increase in the risk of all cause death and a 57% increase in the risk of any cardiovascular event in individuals with diabetes. The population attributable risk from hypertension in individuals with diabetes was 30% for all-cause death and 25% for any cardiovascular event (increasing to 44% and 41% respectively if the 110 normotensive subjects who developed hypertension during follow-up were excluded from the analysis). In comparison, after adjustment for concurrent hypertension, the population attributable risk from diabetes in Framingham subjects was 7% for all cause mortality and 9% for any CVD event. While diabetes is associated with increased risks of death and cardiovascular events in Framingham subjects, much of this excess risk is attributable to coexistent hypertension.
diabetes; hypertension; Framingham; population attributable risk
In the Eplerenone Post-Acute Myocardial Infarction Heart Failure Efficacy and Survival Study (n=6632), eplerenone-associated reduction in all-cause mortality was significantly greater in those with a history of hypertension (Hx-HTN). There were 4007 patients with Hx-HTN (eplerenone: n=1983) and 2625 patients without Hx-HTN (eplerenone: n=1336). Propensity scores for eplerenone use, separately calculated for patients with and without Hx-HTN, were used to assemble matched cohorts of 1838 and 1176 pairs of patients. In patients with Hx-HTN, all-cause mortality occurred in 18% of patients treated with placebo (rate, 1430/10 000 person-years) and 14% of patients treated with eplerenone (rate, 1058/10 000 person-years) during 2350 and 2457 years of follow-up, respectively (hazard ratio [HR]: 0.71; 95% CI: 0.59 to 0.85; P<0.0001). Composite end point of cardiovascular hospitalization or cardiovascular mortality occurred in 33% of placebo-treated patients (3029/10 000 person-years) and 28% of eplerenone-treated patients (2438/10 000 person-years) with Hx-HTN (HR: 0.82; 95% CI: 0.72 to 0.94; P=0.003). In patients without Hx-HTN, eplerenone reduced heart failure hospitalization (HR: 0.73; 95% CI: 0.55 to 0.97; P=0.028) but had no effect on mortality (HR: 0.91; 95% CI: 0.72 to 1.15; P=0.435) or on the composite end point (HR: 0.91; 95% CI: 0.76 to 1.10; P=0.331). Eplerenone should, therefore, be prescribed to all of the post–acute myocardial infarction patients with reduced left ventricular ejection fraction and heart failure regardless of Hx-HTN.
Eplerenone; hypertension; myocardial infarction; heart failure; morbidity; mortality
Prior studies report an association between vitamin D deficiency and hypertension, including the pregnancy-specific disorder, preeclampsia. Circulating vitamin D is almost entirely bound to vitamin D binding protein, which increases 2-fold during pregnancy, but previous studies have not examined vitamin D binding protein or free vitamin D levels. We performed a nested case-control study within the Massachusetts General Hospital Obstetrical Maternal Study (MOMS), measuring first trimester total 25-hydroxyvitamin D and vitamin D binding protein, and calculating free 25-hydroxyvitamin D levels. We compared these levels from pregnancies complicated by subsequent preeclampsia (cases, n=39) with those from normotensive pregnancies (controls, n=131). First trimester total 25-hydroxyvitamin D levels were similar in cases and controls (27.4 ±1.9 ng/ml vs. 28.8±0.80 ng/ml, p=0.435). Despite an association between higher first trimester blood pressures and subsequent preeclampsia, first trimester total 25-hydroxyvitamin D was not associated with first trimester systolic (r=0.11, p=0.16) or diastolic blood pressures (r=0.03, p=0.72). Although there was a trend toward increased risk of preeclampsia with 25-hydroxyvitamin D levels less than 15 ng/ml (OR 2.5, 95% CI 0.89-6.9), this was attenuated after adjustment for body mass index and other covariates (OR 1.35 95% CI 0.40-4.5). First trimester vitamin D binding protein and free 25-hydroxyvitamin D levels were similar in cases and controls and were not associated with first trimester blood pressures. These data suggest that first trimester total and free 25-hydroxyvitamin D levels are not independently associated with first trimester blood pressure or subsequent preeclampsia.
vitamin D; vitamin D Binding Protein; preeclampsia; pregnancy
Xanthine oxidoreductase (XOR) is an enzyme responsible for purine degradation, reactive oxygen species (ROS) production and adipogenesis. XOR gene disrupted (XOR−/−) mice demonstrate renal failure and early death within several months. The aim of this study was to elucidate the mechanism of renal damage in XOR−/− mice and to determine the physiological role of XOR in the kidney. Histological analysis revealed that renal tubular damage in XOR−/− mice was accompanied by deposition of crystals and lipid rich substances. Triglyceride content in renal homogenates was significantly increased in XOR−/− mice. The level of lipogenesis-related gene expression was comparable in XOR+/+ and XOR−/− mice, while the expression of adipogenesis-related gene expression was significantly elevated in XOR−/− mice. Urinary excretions of xanthine and hypoxanthine were markedly elevated in XOR−/− mice. Immunohistochemical analysis, Western blotting and real time RT-PCR revealed that various markers of fibrosis, inflammation, ischemia and oxidative stress were increased in XOR−/− mice. Finally, we demonstrate that primary renal epithelial cells from XOR−/− mice are more readily transformed to myofibroblasts, which is a marker of increased epithelial mesenchymal transition. These results suggest that XOR gene disruption induced the depletion of uric acid and the accumulation of triglyceride rich substances, xanthine and hypoxanthine in the renal tubules. We believe these changes contribute to a complex cellular milieu characterized by inflammation, tissue hypoxia and ROS production ultimately resulting in renal failure through increased renal interstitial fibrosis.
xanthine oxidoreductase; lipid; uric acid; xanthine; renal interstitial fibrosis; epithelial mesenchymal transition; oxidative stress
Neointimal Hyperplasia (NIH) can lead to restenosis after clinical vascular interventions. NIH results from complex, and poorly understood, interactions between signaling cascades in the extracellular matrix and disrupted endothelium which leads to vessel occlusion. Quantitative Trait Loci (QTLs) were previously reported on rat chromosomes 3 and 6 through linkage analysis of post-injury NIH in mid-iliac arterial sections. In the current study, substitution mapping validated the RNO3 NIH QTL but not the RNO6 NIH QTL. The SHR.BN3 congenic strain had a three-fold increase in %NIH compared to the parental SHR strain. A double congenic study of RNO3+RNO6 NIH QTL segments suggested less than additive effects of these two genomic regions. To test the hypothesis that changes in vessel dynamics account for the differences in NIH formation, we performed vascular reactivity studies in the BN, SHR, SHR.BN3 and SHR.BN6 strains. De-endothelialized left common carotid artery rings of the SHR.BN3 showed an increased vascular responsiveness when treated with serotonin or prostaglandin F2α, with significant differences in EC50 and Emax (p < 0.01) values compared to the SHR parental strain. Since both vascular reactivity and %NIH formation in the SHR.BN3 strain are significantly higher than the SHR strain, we postulate that these traits may be associated and are controlled by genetic elements on RNO3. In summary, these results confirm that the RNO3 NIH QTL carries the gene(s) contributing to post-injury NIH formation.
Neointimal Hyperplasia; QTL; Congenics; Rat; Vascular Reactivity
ambulatory blood pressure; cardiovascular events; dipper/non-dipper profiles; morning blood pressure surge; nocturnal hypertension
Inappropriate Na+ reabsorption by thick ascending limbs (THALs) induces hypertension. Nitric oxide (NO) produced by NO synthase type 3 (NOS3 or eNOS) inhibits NaCl reabsorption by THALs. Tumor necrosis factor alpha (TNF-α) decreases NOS3 expression in endothelial cells and contributes to increases in blood pressure. However, the effects of TNF-α on THAL NOS3 and the signaling cascade are unknown. TNF-α activates several signaling pathways including Rho/Rho kinase (ROCK) which is known to reduce NOS3 expression in endothelial cells. Therefore, we hypothesized that TNF-α decreases NOS3 expression via Rho/ROCK in rat THAL primary cultures. THAL cells were incubated with either vehicle or 1 nmol/L TNF-α for 24 hrs and NOS3 expression was measured by Western blot. TNF-α decreased NOS3 expression by 51±6% (p<0.002) and blunted stimulus-induced NO production. A 10-minutes treatment with TNF-α stimulated RhoA activity by 60±23% (p<0.04). Inhibition of Rho GTPase with 0.05 μg/mL C3 exoenzyme blocked TNF-α-induced reductions in NOS3 expression by 30±8% (p<0.02). Inhibition of ROCK with 10 μmol/L H-1152 blocked TNF-α-induced decreases in NOS3 expression by 66±15 % (p<0.001). Simultaneous inhibition of Rho and ROCK had no additive effect. Myosin light chain kinase, NO, protein kinase C, mitogen-activated kinase kinase, c-Jun amino terminal kinases and Rac-1 were also not involved in TNF-α-induced decreases in NOS3 expression. We conclude that TNF-α decreases NOS3 expression primarily via Rho/ROCK in rat THALs. These data suggest that some of the beneficial effects of ROCK inhibitors in hypertension could be due to the mitigation of TNF-α-induced reduction in NOS3 expression.
eNOS; hypertension; kidney; cytokines
Using flow-mediated vasodilation (FMD), reactive hyperemia (RH), and an acute oral antioxidant cocktail (AOC [Vitamin C, E and α-lipoic acid]), this study aimed to provide greater insight into altered vascular function and the role of oxidative stress in chronic heart failure patients with reduced ejection fraction (HFrEF) and at several time points beyond heart transplantation (HTx). A total of 61 age-matched subjects (12 healthy controls, 14 NYHA Class II and III HFrEF patients, and 35 HTx recipients (< 3 yrs post-HTx, 5-10 yrs post-HTx, and > 14 yrs post-HTx)) ingested either placebo (PL) or an AOC prior to FMD and RH testing of the brachial artery. Vascular function, as measured by FMD, was not different between the controls (6.8 ± 1.9 %), recent < 3 yrs post-HTx group (8.1 ± 1.2%), and the 5-10 yrs post-HTx group (5.5 ± 1.0%). However, PL FMD was lower in the HFrEF patients (4.5 ± 0.7%) and in the > 14 yrs post-HTx group (2.9 ± 0.8%). The AOC increased plasma ascorbate levels in all groups, but only increased FMD in the controls (PL 6.8 ± 1.9%; AOC 9.2 ± 1.0%) and > 14 yrs post-HTx recipients (PL 2.9 ± 0.8%; AOC 4.5 ± 1.3%). There were no differences in RH in any of the groups with PL or AOC. This cross-sectional study reveals that, compared to controls, vascular function is blunted in HFrEF patients, is similar soon after HTx, but is decreased with greater time post-HTx with free radicals implicated in this progression.
antioxidants; flow-mediated vasodilation; vascular health; blood flow
The normal IgG, a circulating antibody, is maintained at a constant level in humans. However, little is known if normal IgG has effects on the function of vascular endothelial cells. The purpose of this study was to investigate if IgG affects superoxide (O2-) generation and cell permeability in human aortic endothelial cells (HAECs) isolated from a hypertensive patient. The effect of normal human IgG on endothelial cell function was investigated in cultured HAECs isolated from a hypertensive patient who died of stroke. The results demonstrated, for the first time, that normal IgG attenuated the intracellular O2- level and decreased cell migration, cell permeability, and stress fiber formation in HAECs. IgG significantly decreased Rac1 activity and NADPH oxidase activity but upregulated MnSOD expression in HAECs, which may contribute to the IgG-induced decrease in O2- level. It is noted that AMPK was activated by IgG as evidenced by increased phosphorylation of AMPK (p-AMPK). Interestingly, inhibition of AMPK by an AMPK inhibitor (AI) abolished IgG-induced decreases in Rac1 and NADPH oxidase activities and IgG-induced increases in MnSOD expression, suggesting that AMPK is an important mediator of the IgG-induced regulation of these enzymes. Importantly, inhibition of AMPK activity also prevented the IgG-induced decrease in O2- levels, cell migration, cell permeability, and stress fiber formation. Therefore, normal human IgG may protect HAECs via activation of AMPK and subsequent decreases in intracellular O2-. These findings reveal a previously unidentified role of normal IgG in regulating AMPK and endothelial cell function.
IgG; superoxide; AMPK; NADPH oxidase; endothelial cell; permeability; migration
Estrogen receptor α (ERα) plays a key role in the adaptation of increased uterine blood flow in pregnancy. Chronic hypoxia is a common stress to maternal cardiovascular homeostasis and causes increased risk of preeclampsia. Studies in pregnant sheep demonstrated that hypoxia during gestation downregulated ERα gene expression in uterine arteries. The present study tested the hypothesis that hypoxia causes epigenetic repression of the ERα gene in uterine arteries via heightened promoter methylation. Ovine ERα promoter of 2035 bp spanning from −2000 to +35 of the transcription start site was cloned. No estrogen or HIF response elements were found at the promoter. Two transcription factor binding sites USF-15 and Sp1-520 containing CpG dinucleotides were identified, which had significant effects on the promoter activity. The USF element binds transcription factors USF1 and USF2, and the Sp1 element binds Sp1, as well as ERα through Sp1. Deletion of the Sp1 site abrogated 17β-estradiol-induced increase in the promoter activity. In normoxic control sheep, CpG methylation at the Sp1, but not USF, site was significantly decreased in uterine arteries of pregnant, as compared with nonpregnant animals. In pregnant sheep exposed to long-term high altitude hypoxia, CpG methylation at both Sp1 and USF sites in uterine arteries was significantly increased. Methylation inhibited transcription factor binding and the promoter activity. The results provide evidence of hypoxia causing heightened promoter methylation and resultant ERα gene repression in uterine arteries, and suggest new insights of molecular mechanisms linking gestational hypoxia to aberrant uteroplacental circulation and increased risk of preeclampsia.
Hypoxia; estrogen receptor; methylation; epigenetic modulation; uterine artery
The purpose of this study was to investigate the role of dipeptidyl peptidase IV in regulating the effects of two of its substrates, neuropeptide Y1-36 and peptide YY1-36, on proliferation of and collagen production by preglomerular vascular smooth muscle and glomerular mesangial cells from spontaneously hypertensive and normotensive rats. In cells from hypertensive rats, neuropeptide Y1-36 and peptide YY1-36 stimulated [3H]-thymidine incorporation (cell proliferation index), cell number and [3H]-proline incorporation (index of collagen synthesis); and sitagliptin (dipeptidyl peptidase IV inhibitor) significantly enhanced most of these effects. Neuropeptide Y3-36 and peptide YY3-36 (products of dipeptidyl peptidase IV) had little effect on [3H]-thymidine incorporation, and sitagliptin did not enhance the effects of either peptide. BIBP3226 (Y1 receptor antagonist) blocked the effects of neuropeptide Y1-36 and peptide YY1-36 on [3H]-thymidine incorporation in the absence and presence of sitagliptin. Neuropeptide Y1-36 and peptide YY1-36 stimulated [3H]-thymidine and [3H]-proline incorporation and cell number in cells from normotensive rats; however, the effects were weak and mostly not affected by sitagliptin. Real-time PCR and Western blotting showed similar dipeptidyl peptidase IV mRNA and protein levels in cells from hypertensive versus normotensive rats, with greater levels in smooth muscle versus mesangial cells. Both cell types converted peptide YY1-36 to peptide YY3-36 in a concentration-dependent manner that was attenuated by sitagliptin, and dipeptidyl peptidase IV activity was greater in smooth muscle versus mesangial cells. Conclusion: Dipeptidyl peptidase IV inhibitors might entail a risk of renal dysfunction due to abnormal proliferation of cells in the preglomerular microcirculation and glomeruli.
dipeptidyl peptidase IV; peptide YY1-36; neuropeptide Y1-36; preglomerular vascular smooth muscle cells; mesangial cells; spontaneously hypertensive rats; cell proliferation
A newly produced murine recombinant ACE2 was characterized in vivo and in vitro. The effects of available ACE2 inhibitors (MLN-4760 and two conformational variants of DX600 –linear and cyclic) were also examined. When murine ACE2 was given to mice for 4 weeks, a marked increase in serum ACE2 activity was sustainable. In acute studies, mouse ACE2 (1mg/kg) obliterated hypertension induced by angiotensin II infusion by rapidly decreasing plasma angiotensin II. These effects were blocked by MLN-4760 but not by either form of DX600. In vitro, conversion from angiotensin II to angiotensin-(1–7) by mouse ACE2 was blocked by MLN-4760 (10−6M) but not by either form of DX600 (10−5M). Quantitative analysis of multiple angiotensin peptides in plasma ex vivo revealed formation of angiotensin-(1–9) from angiotensin I by human but not by mouse ACE2. Both human and mouse ACE2 led to the dissipation of angiotensin II with formation of angiotensin-(1–7). By contrast, mouse ACE2-driven angiotensin-(1–7) formation from angiotensin II was blocked by MLN-4760 but not by either linear or cyclic DX600.
In conclusion, sustained elevations in serum ACE2 activity can be accomplished with murine ACE2 administration thereby providing a strategy for ACE2 amplification in chronic studies using rodent models of hypertension and cardiovascular disease. Human, but not mouse ACE2, degrades angiotensin I to form angiotensin-(1–9). There are also species differences regarding rodent and human ACE2 inhibition by known inhibitors such that MLN-4760 inhibits both human and mouse ACE2 whereas DX600 only blocks human ACE2 activity.
ACE2; Recombinant; Mouse; Human; Hypertension; Angiotensin; Inhibitor
Accumulating evidence suggests that increased visit-to-visit variability (VVV) of blood pressure is associated with stroke. No study has examined the association between VVV of blood pressure and stroke in postmenopausal women, and scarce data exists as to whether this relation is independent of the temporal trend of blood pressure. We examined the association of VVV of blood pressure with stroke in 58,228 postmenopausal women enrolled in the Women's Health Initiative. Duplicate blood pressure readings, which were averaged, were taken at baseline and at each annual visit. VVV was defined as the standard deviation about the participant's mean systolic blood pressure (SBP) across visits (SD), and about the participant's regression line with SBP regressed across visits (SDreg). Over a median follow-up of 5.4 years, 997 strokes occurred. In an adjusted model including mean SBP over time, the hazard ratios (95% CI) of stroke for higher quartiles of SD of SBP compared to the lowest quartile (referent) were 1.39 (1.03-1.89) for quartile 2, 1.52 (1.13-2.03) for quartile 3, and 1.72 (1.28-2.32) for quartile 4 (P trend<0.001). The relation was similar for SDreg of SBP quartiles in a model that additionally adjusted for the temporal trend in SBP (P trend<0.001). The associations did not differ by stroke type (ischemic vs. hemorrhagic). There was a significant interaction between mean SBP and SDreg on stroke with the strongest association seen below 120 mmHg. In postmenopausal women, greater VVV of SBP was associated with increased risk of stroke, particularly in the lowest range of mean SBP.
hypertension; blood pressure; stroke; postmenopause; women
Nitric oxide (NO) reduces NaCl absorption by thick ascending limbs (TALs) by inhibiting the Na/K/2Cl cotransporter (NKCC2). We have shown that NO-induced inhibition of Na transport is reduced in Dahl salt-sensitive rat (SS) TALs. Angiotensin II (Ang II) increases NO production in TALs via Ang II type 2 receptor (AT2R). It is unknown whether AT2Rs regulate TAL NaCl absorption and whether this effect is reduced in SS. We hypothesized that AT2R activation decreases TAL Na transport via NO, and this effect is blunted in SS. In the presence of AT1R antagonist losartan, AT2R activation with Ang II inhibited NKCC2 activity by 32 ± 7% (p < 0.03). AT2R antagonist PD-123319 (PD) abolished Ang II’s effect. Activation with the AT2R-selective agonist CGP42112A (CGP; 10 nmol/L) decreased NKCC2 activity by 29 ± 6% (p < 0.03). CGP’s effect on NKCC2 activity was blocked by PD and by NO synthase inhibitor L-NAME. In Dahl salt-resistant rat (SR) TALs, 1 nmol/L CGP decreased NKCC2 activity by 23 ± 4% (p < 0.01). In SS TALs, it had no effect. TAL AT2R mRNA did not differ in SS vs. SR. We conclude that: 1) TAL AT2R activation decreases Na absorption; 2) this effect is mediated by AT2R-induced stimulation of NO; 3) AT2R-induced reduction of NKCC2 activity is blunted in SS; and 4) defects in AT2R/NO signaling rather than decreased AT2R expression likely account for the blunted effect in SS TALs. Impaired AT2R-mediated signaling in TALs could contribute to the Na retention associated with salt-sensitive hypertension.
ion transport; angiotensin II; nitric oxide; AT2R; Dahl salt-sensitive