Journals RSS : Gourt

Adrenergic Cardiovascular Control Before and After Removal of Stimulatory {alpha}-1 Adrenoreceptor Antibodies [Letters to the Editor]

Blood Pressure Threshold for Abnormal Ocular Fundus Findings Is Lower Than Expected [Letters to the Editor]

How Is Slow Wave Sleep Related to Hypertension? [Letters to the Editor]

Response to How Is Slow Wave Sleep Related to Hypertension? [Letters to the Editor]

Tetrahydrobiopterin and Endothelial Nitric Oxide Synthase Uncoupling [Letters to the Editor]

Response to Tetrahydrobiopterin and Endothelial Nitric Oxide Synthase Uncoupling [Letters to the Editor]

Pin1 as a Protector of Vascular Endothelial Homeostasis [Letters to the Editor]

Response to Pin1 as a Protector of Vascular Endothelial Homeostasis [Letters to the Editor]

Reactive Oxygen Species and Angiotensin II Response in Human Omental Arteries: What About Tachyphylaxis? [Letters to the Editor]

Response to Reactive Oxygen Species and Angiotensin II Response in Human Omental Arteries: What About Tachyphylaxis? [Letters to the Editor]

Eplerenone Use in Primary Aldosteronism During Pregnancy [Letters to the Editor]

Correction [Correction]

Unusual Hypertensive Phenotypes: What Is Their Significance? [Brief Review]

Severe Hypertension With Large-Vessel Arteritis [Hypertension Grand Rounds]

Above Which Blood Pressure Level Does the Risk of Atrial Fibrillation Increase? [Editorial Commentaries]

Breast Cancer, Age, and Hypertension: A Complex Issue [Editorial Commentaries]

Preterm Birth: A Novel Risk Factor for Higher Blood Pressure in Later Life [Editorial Commentaries]

Placenta Messages to the Mother: Not Just Debris [Editorial Commentaries]

Interventional Approaches to Reduce Sympathetic Activity in Resistant Hypertension: To Ablate or Stimulate? [Editorial Commentaries]

Mutations of the Potassium Channel KCNJ5 Causing Aldosterone-Producing Adenomas: One or Two Hits? [Editorial Commentaries]

Upper Normal Blood Pressures Predict Incident Atrial Fibrillation in Healthy Middle-Aged Men: A 35-Year Follow-Up Study [Epidemiology/Population Science]

Hypertension is the most prevalent risk factor for incident atrial fibrillation (AF). Recently, even high normal blood pressures (BPs) have been established as predictive of AF in women. We aimed to study the long-term impact of upper normal BP on incident AF in a population-based study of middle-aged men. From 1972 to 1975, 2014 healthy Norwegian men were included in a prospective cardiovascular survey and underwent a comprehensive clinical examination including standardized BP measurements. During up to 35 years of follow-up, 270 men were documented with AF by scrutinizing all hospital discharges. Risk estimations for incident AF were analyzed in quartiles of BP using multivariate adjusted Cox proportional hazards. Men with baseline systolic BP ≥140 mm Hg and upper normal BP 128 to 138 mm Hg had 1.60-fold (95% CI 1.15–2.21) and 1.50-fold (1.10–2.03) risk of AF, respectively, compared with men with BP <128 mm Hg. Baseline diastolic BP ≥80 mm Hg increased the risk of incident AF 1.79-fold (95% CI 1.28–2.59) compared with diastolic BP <80 mm Hg. When adjusting for the occurrence of diabetes mellitus or cardiovascular diseases before an AF event, the results still maintained significance. Additional analyses, on average 7 years after baseline, including men still healthy, showed that sustained upper normal systolic BP remained a significant predictor of subsequent AF. In conclusion, upper normal blood pressures are long-term predictors of incident AF in initially healthy middle-aged men.

Comorbidity as a Mediator of Survival Disparity Between Younger and Older Women Diagnosed With Metastatic Breast Cancer [Epidemiology/Population Science]

The presence of comorbidity becomes increasingly important for its prognostic effect on survival in breast cancer patients with advancing age. This study aimed to evaluate the role of comorbidities including hypertension as a mediator of disparity in survival after metastasis diagnosis between younger (≤51 years) and older (>51 years) patients. A total of 553 patients 26–88 years of age with breast cancer metastasis diagnosis from 1 large urban practice were followed between January 1, 1999, and June 30, 2008. Comorbidity variables and survival were analyzed using Cox regression model. To assess comorbidity variables as a mediator of age-survival relationship, 2 approaches have been applied: (1) Baron Kenny approach and (2) alternative assessment to compute the percentage change in the hazard ratios (HRs). The median survival was 40 months, with 265 (47.9%) alive and 288 (52.1%) dead. Older patients had worse survival than younger patients (HR, 1.43; 95% confidence interval [CI], 1.11–1.84). Hypertension was related to survival (HR, 1.45; 95% CI, 1.12–1.89) when age and other covariates were controlled. The effect of age on survival was no longer significant after adjustment for hypertension (HR, 1.26; 95%, CI 0.97–1.65) or hypertension-augmented Charlson comorbidity score (HR, 1.24; 95% CI, 0.95–1.63). Hypertension-augmented Charlson comorbidity score or hypertension was a strong mediator of age-survival relationship among metastatic breast cancer patients, explaining survival disparity between younger and older patients by 44% and 40%, respectively. The study findings suggest that hypertension should be included in the comorbidity information for decision-making support programs.

Prognostic Value of the Variability in Home-Measured Blood Pressure and Heart Rate: The Finn-Home Study [Epidemiology/Population Science]

The objective of the study was to assess the prognostic value of variability in home-measured blood pressure (BP) and heart rate (HR) in a general population. We studied a representative sample of the Finnish adult population with 1866 study subjects aged 45–74 years. BP and HR self-measurements were performed on 7 consecutive days. The variabilities of BP and HR were defined as the SDs of morning minus evening, day-by-day, and first minus second measurements. The primary end point was incidence of a cardiovascular event. The secondary end point was total mortality. During a follow-up of 7.8 years, 179 subjects had experienced a cardiovascular event, and 130 subjects had died. In Cox proportional hazard models adjusted for age, sex, BP/HR, and other cardiovascular risk factors, morning-evening home BP variability (systolic/diastolic relative hazard: 1.04/1.10 [95% CI: 1.01–1.07/1.05–1.15] per 1-mm Hg increase in BP variability) and morning day-by-day home BP variability (relative hazard: 1.04/1.10 [95% CI: 1.00–1.07/1.04–1.16] per 1-mm Hg increase in BP variability) were predictive of cardiovascular events. Morning-evening home HR variability (relative hazard: 1.07 [95% CI: 1.02–1.12] per 1-bpm increase in HR variability) and morning day-by-day home HR variability (relative hazard: 1.11 [95% CI: 1.05–1.17] per 1-bpm increase in HR variability) were also independent predictors of cardiovascular events. Greater variabilities of morning home BP and HR are independent predictors of cardiovascular events. Because the variabilities of home BP and HR are easily acquired in conjunction with home BP and HR level, they should be used as the additive information in the assessment of cardiovascular risk.

Rate of Decline of Forced Vital Capacity Predicts Future Arterial Hypertension: The Coronary Artery Risk Development in Young Adults Study [Epidemiology/Population Science]

Lung function studies in middle-aged subjects predict cardiovascular disease mortality. We studied whether greater loss of forced vital capacity (FVC) early in life predicted incident hypertension. The sample was 3205 black and white men and women in the Coronary Artery Risk Development in Young Adults Study examined between 1985 and 1986 (Coronary Artery Risk Development in Young Adults year 0, ages 18–30 years) and 2005–2006 and who were not hypertensive by year 10. FVC was assessed at years 0, 2, 5, 10, and 20. Proportional hazard ratios and linear regression models predicted incident hypertension at years 15 or 20 (n=508) from the change in FVC (FVC at year 10 – peak FVC, where peak FVC was estimated as the maximum across years 0, 2, 5, and 10). Covariates included demographics, center, systolic blood pressure, FVC maximum, smoking, physical activity, asthma, and body mass index. Unadjusted cumulative incident hypertension was 25% in the lowest FVC loss quartile (Q1; median loss: 370 mL) compared with 12% cumulative incident hypertension in those who achieved peak FVC at year 10 (Q4). Minimally adjusted hazard ratio for Q1 versus Q4 was 2.21 (95% CI: 1.73–2.83), and this association remained significant in the fully adjusted model (1.37; 95% CI: 1.05–1.80). Decline in FVC from average age at peak (29.4 years) to 35 years old predicted incident hypertension between average ages 35 and 45 years. The findings may represent a common pathway that may link low normal FVC to cardiovascular disease morbidity and mortality.

Systematic Review and Meta-Analysis of Preterm Birth and Later Systolic Blood Pressure [Epidemiology/Population Science]

Lower birth weight because of fetal growth restriction is associated with higher blood pressure later in life, but the extent to which preterm birth (<37 completed weeks' gestation) or very low birth weight (<1500 g) predicts higher blood pressure is less clear. We performed a systematic review of 27 observational studies that compared the resting or ambulatory systolic blood pressure or diagnosis of hypertension among children, adolescents, and adults born preterm or very low birth weight with those born at term. We performed a meta-analysis with the subset of 10 studies that reported the resting systolic blood pressure difference in millimeters of mercury with 95% CIs or SEs. We assessed methodologic quality with a modified Newcastle-Ottawa Scale. The 10 studies were composed of 1342 preterm or very low birth weight and 1738 term participants from 8 countries. The mean gestational age at birth of the preterm participants was 30.2 weeks (range: 28.8–34.1 weeks), birth weight was 1280 g (range: 1098–1958 g), and age at systolic blood pressure measurement was 17.8 years (range: 6.3–22.4 years). Former preterm or very low birth weight infants had higher systolic blood pressure than term infants (pooled estimate: 2.5 mm Hg [95% CI: 1.7–3.3 mm Hg]). For the 5 highest quality studies, the systolic blood pressure difference was slightly greater, at 3.8 mm Hg (95% CI: 2.6–5.0 mm Hg). We conclude that infants who are born preterm or very low birth weight have modestly higher systolic blood pressure later in life and may be at increased risk for developing hypertension and its sequelae.

KCNJ5 Mutations in European Families With Nonglucocorticoid Remediable Familial Hyperaldosteronism [Genetics]

Primary aldosteronism is the most frequent cause of endocrine hypertension. Three forms of familial hyperaldosteronism (FH) have been described, named FH-I to -III. Recently, a mutation of KCNJ5 has been shown to be associated with FH-III, whereas the cause of FH-II is still unknown. In this study we searched for mutations in KCNJ5 in 46 patients from 21 families with FH, in which FH-I was excluded. We identified a new germline G151E mutation in 2 primary aldosteronism–affected subjects from an Italian family and 3 somatic mutations in aldosterone-producing adenomas, T158A described previously as a germline mutation associated with FH-III, and G151R and L168R both described as somatic mutations in aldosterone-producing adenoma. The phenotype of the family with the G151E mutation was remarkably milder compared with the previously described American family, in terms of both clinical and biochemical parameters. Furthermore, patients with somatic KCNJ5 mutations displayed a phenotype indistinguishable from that of sporadic primary aldosteronism. The functional characterization of the effects of the G151E mutation in vitro showed a profound alteration of the channel function, with loss of K+ selectivity, Na+ influx, and membrane depolarization. These alterations have been postulated to be responsible for voltage gate Ca2+ channel activation, increase in cytosolic calcium, and stimulation of aldosterone production and adrenal cell proliferation. In conclusion, we describe herein a new mutation in the KCNJ5 potassium channel associated with FH-III, responsible for marked alterations of channel function but associated with a mild clinical and hormonal phenotype.

Genome-Wide Profiling of Blood Pressure in Adults and Children [Genetics]

Hypertension is an important determinant of cardiovascular morbidity and mortality and has a substantial heritability, which is likely of polygenic origin. The aim of this study was to assess to what extent multiple common genetic variants contribute to blood pressure regulation in both adults and children and to assess overlap in variants between different age groups, using genome-wide profiling. Single nucleotide polymorphism sets were defined based on a meta-analysis of genome-wide association studies on systolic blood pressure and diastolic blood pressure performed by the Cohort for Heart and Aging Research in Genome Epidemiology (n=29 136), using different P value thresholds for selecting single nucleotide polymorphisms. Subsequently, genetic risk scores for systolic blood pressure and diastolic blood pressure were calculated in an independent adult population (n=2072) and a child population (n=1034). The explained variance of the genetic risk scores was evaluated using linear regression models, including sex, age, and body mass index. Genetic risk scores, including also many nongenome-wide significant single nucleotide polymorphisms, explained more of the variance than scores based only on very significant single nucleotide polymorphisms in adults and children. Genetic risk scores significantly explained ≤1.2% (P=9.6*10–8) of the variance in adult systolic blood pressure and 0.8% (P=0.004) in children. For diastolic blood pressure, the variance explained was similar in adults and children (1.7% [P=8.9*10–10] and 1.4% [P=3.3*10–5], respectively). These findings suggest the presence of many genetic loci with small effects on blood pressure regulation both in adults and children, indicating also a (partly) common polygenic regulation of blood pressure throughout different periods of life.

Genomewide Association Study Using a High-Density Single Nucleotide Polymorphism Array and Case-Control Design Identifies a Novel Essential Hypertension Susceptibility Locus in the Promoter Region of Endothelial NO Synthase [Genetics]

Essential hypertension is a multifactorial disorder and is the main risk factor for renal and cardiovascular complications. The research on the genetics of hypertension has been frustrated by the small predictive value of the discovered genetic variants. The HYPERGENES Project investigated associations between genetic variants and essential hypertension pursuing a 2-stage study by recruiting cases and controls from extensively characterized cohorts recruited over many years in different European regions. The discovery phase consisted of 1865 cases and 1750 controls genotyped with 1M Illumina array. Best hits were followed up in a validation panel of 1385 cases and 1246 controls that were genotyped with a custom array of 14 055 markers. We identified a new hypertension susceptibility locus (rs3918226) in the promoter region of the endothelial NO synthase gene (odds ratio: 1.54 [95% CI: 1.37–1.73]; combined P=2.58 · 10–13). A meta-analysis, using other in silico/de novo genotyping data for a total of 21 714 subjects, resulted in an overall odds ratio of 1.34 (95% CI: 1.25–1.44; P=1.032 · 10–14). The quantitative analysis on a population-based sample revealed an effect size of 1.91 (95% CI: 0.16–3.66) for systolic and 1.40 (95% CI: 0.25–2.55) for diastolic blood pressure. We identified in silico a potential binding site for ETS transcription factors directly next to rs3918226, suggesting a potential modulation of endothelial NO synthase expression. Biological evidence links endothelial NO synthase with hypertension, because it is a critical mediator of cardiovascular homeostasis and blood pressure control via vascular tone regulation. This finding supports the hypothesis that there may be a causal genetic variation at this locus.

Transcriptionally Active Syncytial Aggregates in the Maternal Circulation May Contribute to Circulating Soluble Fms-Like Tyrosine Kinase 1 in Preeclampsia [Pregnancy/Preeclampsia]

The cardinal manifestations of the pregnancy-specific disorder preeclampsia, new-onset hypertension, and proteinuria that resolve with placental delivery have been linked to an extracellular protein made by the placenta, soluble fms-like tyrosine kinase 1 (sFlt1), that injures the maternal vasculature. However, the mechanisms by which sFlt1, which is heavily matrix bound, gain access to the systemic circulation remain unclear. Here we report that the preeclamptic placenta's outermost layer, the syncytiotrophoblast, forms abundant "knots" that are enriched with sFlt1 protein. These syncytial knots easily detach from the syncytiotrophoblast, resulting in free, multinucleated aggregates (50–150 μm diameter) that are loaded with sFlt1 protein and mRNA, are metabolically active, and are capable of de novo gene transcription and translation. At least 25% of the measurable sFlt1 in the third-trimester maternal plasma is bound to circulating placental microparticles. We conclude that detachment of syncytial knots from the placenta results in free, transcriptionally active syncytial aggregates that represent an autonomous source of sFlt1 delivery into the maternal circulation. The process of syncytial knot formation, shedding of syncytial aggregates, and appearance of placental microparticles in the maternal circulation appears to be greatly accelerated in preeclampsia and may contribute to the maternal vascular injury that characterizes this disorder.

Hydroxysteroid (17-{beta}) Dehydrogenase 1 Is Dysregulated by Mir-210 and Mir-518c That Are Aberrantly Expressed in Preeclamptic Placentas: A Novel Marker for Predicting Preeclampsia [Pregnancy/Preeclampsia]

In this study, to search for novel preeclampsia (PE) biomarkers, we focused on microRNA expression and function in the human placenta complicated with PE. By comprehensive analyses of microRNA expression, we identified 22 microRNAs significantly upregulated in preeclamptic placentas, 5 of which were predicted in silico to commonly target the mRNA encoding hydroxysteroid (17-β) dehydrogenase 1 (HSD17B1), a steroidogenetic enzyme expressed predominantly in the placenta. In vivo HSD17B1 expression, at both the mRNA and protein levels, was significantly decreased in preeclamptic placentas. Of these microRNAs, miR-210 and miR-518c were experimentally validated to target HSD17B1 by luciferase assay, real-time PCR, and ELISA. Furthermore, we found that plasma HSD17B1 protein levels in preeclamptic pregnant women reflected the decrease of its placental expression. Moreover, a prospective cohort study of plasma HSD17B1 revealed a significant reduction of plasma HSD17B1 levels in pregnant women at 20 to 23 and 27 to 30 weeks of gestation before PE onset compared with those with normal pregnancies. The sensitivities/specificities for predicting PE at 20 to 23 and 27 to 30 weeks of gestation were 0.75/0.67 (cutoff value=21.9 ng/mL) and 0.88/0.51 (cutoff value=30.5 ng/mL), and the odds ratios were 6.09 (95% CI: 2.35–15.77) and 7.83 (95% CI: 1.70–36.14), respectively. We conclude that HSD17B1 is dysregulated by miR-210 and miR-518c that are aberrantly expressed in preeclamptic placenta and that reducing plasma level of HSD17B1 precedes the onset of PE and is a potential prognostic factor for PE.

Glycogen Phosphorylase Isoenzyme BB Plasma Concentration Is Elevated in Pregnancy and Preterm Preeclampsia [Pregnancy/Preeclampsia]

Glycogen phosphorylase is a key enzyme in glycogenolysis. Released with myocardial ischemia, blood concentration of glycogen phosphorylase isoenzyme BB (GPBB) is a marker of acute coronary syndromes. Pregnancy imposes metabolic stress, and preeclampsia is associated with cardiac complications. However, plasma GPBB concentration during pregnancy is unknown. This study was conducted to determine maternal plasma GPBB concentration in normal pregnancy and in preeclampsia. Plasma samples from 6 groups (n=396) were studied: nonpregnant and pregnant women with normal term delivery, term and preterm preeclampsia, and term and preterm small-for-gestational-age neonates. GPBB concentration was measured with a specific immunoassay. Placental tissues (n=45) obtained from pregnant women with preterm and term preeclampsia, spontaneous preterm delivery, and normal term delivery were analyzed for potential GPBB expression by immunoblotting. Median plasma GPBB concentration was higher in pregnant women than in nonpregnant women (38.7 versus 9.2 ng/mL; P<0.001), which remained significant after adjusting for age, race, and parity. Maternal plasma GPBB concentrations did not change throughout gestation. Cases of preterm (but not term) preeclampsia had higher median plasma GPBB concentrations than gestational age-matched normal pregnancy cases (72.6 versus 26.0 ng/mL; P=0.001). Small-for-gestational-age neonates did not affect plasma GPBB concentration. GPBB was detected in the placenta and was less abundant in preterm preeclampsia than in preterm delivery cases (P<0.01). There is physiological elevation of plasma GPBB concentration during pregnancy; an increase in maternal plasma GPBB is a novel phenotype of preterm preeclampsia. It is strongly suggested that these changes are attributed to GPBB of placental origin.

Increased Angiotensin II-Induced Hypertension and Inflammatory Cytokines in Mice Lacking Angiotensin-Converting Enzyme N Domain Activity [Renin-Angiotensin-Aldosterone System]

—Angiotensin-converting enzyme (ACE) is composed of the N- and C-terminal catalytic domains. To study the role of the ACE domains in the inflammatory response, N-knockout (KO) and C-KO mice, models lacking 1 of the 2 ACE domains, were analyzed during angiotensin II–induced hypertension. At 2 weeks, N-KO mice have systolic blood pressures that averaged 173±4.6 mm Hg, which is more than 25 mm Hg higher than the blood pressures observed in wild-type or C-KO mice (146±3.2 and 147±4.2 mm Hg). After 3 weeks, blood pressure differences between N-KO, C-KO, and wild-type were even more pronounced. Macrophages from N-KO mice have increased expression of tumor necrosis factor α after stimulation with either lipopolysaccharide (about 4-fold) or angiotensin II (about 2-fold), as compared with C-KO or wild-type mice. Inhibition of the enzyme prolyl oligopeptidase, responsible for the formation of acetyl-SerAspLysPro and other peptides, eliminated the blood pressure difference and the difference in tumor necrosis factor α expression between angiotensin II–treated N-KO and wild-type mice. However, this appears independent of acetyl-SerAspLysPro. These data establish significant differences in the inflammatory response as a function of ACE N- or C-domain catalytic activity. They also indicate a novel role of prolyl oligopeptidase in the cytokine regulation and in the blood pressure response to experimental hypertension.

Angiotensin Type 2 Receptor Agonist Compound 21 Reduces Vascular Injury and Myocardial Fibrosis in Stroke-Prone Spontaneously Hypertensive Rats [Renin-Angiotensin-Aldosterone System]

—Angiotensin type 2 receptor–mediated effects of angiotensin II appear to counteract many of the effects mediated via the angiotensin type 1 receptor. Compound 21 (C21), a selective angiotensin type 2 receptor agonist, has demonstrated beneficial effects on cardiac function after myocardial infarction in rodents. We hypothesized that C21 alone or in combination with an angiotensin type 1 receptor antagonist would blunt the development of hypertension and vascular damage in stroke-prone spontaneously hypertensive rats. Six-week–old stroke-prone spontaneously hypertensive rats received C21 (1 mg/kg per day), the angiotensin type 1 receptor antagonist losartan (10 mg/kg per day), C21 plus losartan, or vehicle PO for 6 weeks. Systolic blood pressure was lower in losartan and C21-losartan combination groups (P<0.001). Endothelium-dependent relaxation was enhanced (P<0.001) in the C21-losartan combination group at lower acetylcholine concentrations. C21 or C21-losartan combination reduced vascular stiffness, aortic medial and myocardial interstitial collagen content, and aortic fibronectin (P<0.05). C21 and losartan decreased the expression of 2 genes associated with cardiac hypertrophy, myosin heavy chain-β (myh7) by 30 to 50%, and α-skeletal muscle actin by 30% to 35% (P<0.05). C21-losartan combination caused an additional 40% reduction in myh7 compared with C21 (P<0.01). Aortic superoxide generation was reduced equally by the 3 treatments (P<0.001). Monocyte/macrophage infiltration in the aorta and kidney (P<0.001) and T-lymphocyte infiltration in the renal cortex (P<0.05) were lowered similarly by the 3 treatments. These data suggest that C21 alone or in combination with losartan may improve endothelial function and vascular composition and mechanics by reducing oxidative stress, collagen content, fibronectin, and inflammatory cell infiltration in stroke-prone spontaneously hypertensive rats.

Angiotensin-(1-9) Attenuates Cardiac Fibrosis in the Stroke-Prone Spontaneously Hypertensive Rat via the Angiotensin Type 2 Receptor [Renin-Angiotensin-Aldosterone System]

The renin-angiotensin system regulates cardiovascular physiology via angiotensin II engaging the angiotensin type 1 or type 2 receptors. Classic actions are type 1 receptor mediated, whereas the type 2 receptor may counteract type 1 receptor activity. Angiotensin-converting enzyme 2 metabolizes angiotensin II to angiotensin-(1-7) and angiotensin I to angiotensin-(1-9). Angiotensin-(1-7) antagonizes angiotensin II actions via the receptor Mas. Angiotensin-(1-9) was shown recently to block cardiomyocyte hypertrophy via the angiotensin type 2 receptor. Here, we investigated in vivo effects of angiotensin-(1-9) via the angiotensin type 2 receptor. Angiotensin-(1-9) (100 ng/kg per minute) with or without the angiotensin type 2 receptor antagonist PD123 319 (100 ng/kg per minute) or PD123 319 alone was infused via osmotic minipump for 4 weeks into stroke-prone spontaneously hypertensive rats. We measured blood pressure by radiotelemetry and cardiac structure and function by echocardiography. Angiotensin-(1-9) did not affect blood pressure or left ventricular mass index but reduced cardiac fibrosis by 50% (P<0.01) through modulating collagen I expression, reversed by PD123 319 coinfusion. In addition, angiotensin-(1-9) inhibited fibroblast proliferation in vitro in a PD123 319-sensitive manner. Aortic myography revealed that angiotensin-(1-9) significantly increased contraction to phenylephrine compared with controls after N-nitro-l-arginine methyl ester treatment, an effect abolished by PD123 319 coinfusion (area under the curve: angiotensin-(1-9) N-nitro-l-arginine methyl ester=98.9±11.8%; control+N-nitro-l-arginine methyl ester=74.0±10.4%; P<0.01), suggesting that angiotensin-(1-9) improved basal NO bioavailability in an angiotensin type 2 receptor–sensitive manner. In summary, angiotensin-(1-9) reduced cardiac fibrosis and altered aortic contraction via the angiotensin type 2 receptor supporting a direct role for angiotensin-(1-9) in the renin-angiotensin system.

Telmisartan Exerts Renoprotective Actions via Peroxisome Proliferator-Activated Receptor-{gamma}/Hepatocyte Growth Factor Pathway Independent of Angiotensin II Type 1 Receptor Blockade [Renin-Angiotensin-Aldosterone System]

Angiotensin (Ang) II type 1 receptor blockers have demonstrated beneficial effects beyond blood pressure control in the treatment of chronic kidney disease. There is clinical evidence that telmisartan is more effective than losartan in reducing proteinuria in hypertensive patients with diabetic nephropathy, because it is a partial agonist of peroxisome-proliferator activated receptor- (PPAR), as well as an Ang II type 1 receptor blocker (AMADEO Study [A comparison of telMisartan versus losArtan in hypertensive type 2 DiabEtic patients with Overt nephropathy]). In this study, we examined the role of PPAR activation in the renal protective actions of telmisartan using Ang II type 1 receptor–deficient mice. Renal injury was induced in Ang II type 1 receptor–deficient mice by producing unilateral ureteral obstruction, which exhibited severe renal interstitial fibrosis and inflammation. In these mice, telmisartan prevented hydronephrosis induced by unilateral ureteral obstruction more strongly than did losartan. Importantly, the prevention of renal atrophy and fibrosis by telmisartan was significantly attenuated by GW9662, a PPAR antagonist. Interestingly, the downstream effector of PPAR activation by telmisartan is hepatocyte growth factor (HGF), a well-known antifibrotic factor, because renal HGF expression was significantly increased by telmisartan, and a neutralizing antibody against HGF diminished the renal protective action of telmisartan. These beneficial changes by telmisartan were associated with a decrease in the expression of transforming growth factor-β1 and other proinflammatory and profibrotic cytokine genes through PPAR/HGF activation. Our findings provide evidence of organ protective actions of telmisartan through the PPAR/HGF pathway, independent of Ang II type 1 receptor blockade. Further development of the next generation of Ang II type 1 receptor blockers with added organ protective actions, such as PPAR activation, might provide new beneficial drugs to treat renal and cardiovascular diseases.

Sodium Depletion Enhances Renal Expression of (Pro)Renin Receptor via Cyclic GMP-Protein Kinase G Signaling Pathway [Renin-Angiotensin-Aldosterone System]

(Pro)renin receptor (PRR) is expressed in renal vasculature, glomeruli, and tubules. The physiological regulation of this receptor is not well established. We hypothesized that sodium depletion increases PRR expression through cGMP- protein kinase G (PKG) signaling pathway. Renal PRR expressions were evaluated in Sprague-Dawley rats on normal sodium or low-sodium diet (LS) and in cultured rat proximal tubular cells and mouse renal inner medullary collecting duct cells exposed to LS concentration. LS augmented PRR expression in renal glomeruli, proximal tubules, distal tubules, and collecting ducts. LS also increased cGMP production and PKG activity. In cells exposed to normal sodium, cGMP analog increased PKG activity and upregulated PRR expression. In cells exposed to LS, blockade of guanylyl cyclase with 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one decreased PKG activity and downregulated PRR expression. PKG inhibition decreased phosphatase protein phosphatase 2A activity; suppressed LS-mediated phosphorylation of extracellular signal–regulated kinase, c-Jun N-terminal kinase, c-Jun, and nuclear factor-B p65; and attenuated LS-mediated PRR upregulation. LS also enhanced DNA binding of cAMP response element binding protein 1 to cAMP response elements, nuclear factor-B p65 to nuclear factor-B elements, and c-Jun to activator protein 1 elements in PRR promoter in proximal tubular cells. We conclude that sodium depletion upregulates renal PRR expression via the cGMP-PKG signaling pathway by enhancing binding of cAMP response element binding protein 1, nuclear factor-B p65, and c-Jun to PRR promotor.

T Regulatory Lymphocytes Prevent Aldosterone-Induced Vascular Injury [Renin-Angiotensin-Aldosterone System]

Aldosterone mediates actions of the renin-angiotensin-aldosterone system inducing hypertension, oxidative stress, and vascular inflammation. Recently, we showed that angiotensin II–induced hypertension and vascular damage are mediated at least in part by macrophages and T-helper effector lymphocytes. Adoptive transfer of suppressor T-regulatory lymphocytes (Tregs) prevented angiotensin II action. We hypothesized that Treg adoptive transfer would blunt aldosterone-induced hypertension and vascular damage. Thirteen to 15-week–old male C57BL/6 mice were injected intravenously at 1-week intervals with 3x105 CD4+CD25+ cells (representing Treg) or control CD4+CD25– cells and then infused or not for 14 days with aldosterone (600 μg/kg per day, SC) while receiving 1% saline to drink. Aldosterone induced a small but sustained increase in blood pressure (P<0.001), decreased vasodilatory responses to acetylcholine by 66% (P<0.001), increased both media:lumen ratio (P<0.001) and media cross-sectional area of resistance arteries by 60% (P<0.05), and increased NADPH oxidase activity 2-fold in aorta (P<0.001), kidney and heart (P<0.05), and aortic superoxide production. As well, aldosterone enhanced aortic and renal cortex macrophage infiltration and aortic T-cell infiltration (all P<0.05), and tended to decrease Treg in the renal cortex. Treg adoptive transfer prevented all of the vascular and renal effects induced by aldosterone. Adoptive transfer of CD4+CD25– cells exacerbated aldosterone effects except endothelial dysfunction and increases in media:lumen ratio of resistance arteries. Thus, Tregs suppress aldosterone-mediated vascular injury, in part through effects on innate and adaptive immunity, suggesting that aldosterone-induced vascular damage could be prevented by an immunomodulatory approach.

Systemic and Renal-Specific Sympathoinhibition in Obesity Hypertension [Nervous System]

Chronic pressure-mediated baroreflex activation suppresses renal sympathetic nerve activity. Recent observations indicate that chronic electric activation of the carotid baroreflex produces sustained reductions in global sympathetic activity and arterial pressure. Thus, we investigated the effects of global and renal specific suppression of sympathetic activity in dogs with sympathetically mediated, obesity-induced hypertension by comparing the cardiovascular, renal, and neurohormonal responses to chronic baroreflex activation and bilateral surgical renal denervation. After control measurements, the diet was supplemented with beef fat, whereas sodium intake was held constant. After 4 weeks on the high-fat diet, when body weight had increased 50%, fat intake was reduced to a level that maintained this body weight. This weight increase was associated with an increase in mean arterial pressure from 100±2 to 117±3 mm Hg and heart rate from 86±3 to 130±4 bpm. The hypertension was associated with a marked increase in cumulative sodium balance despite an approximately 35% increase in glomerular filtration rate. The importance of increased tubular reabsorption to sodium retention was further reflected by 35% decrease in fractional sodium excretion. Subsequently, both chronic baroreflex activation (7 days) and renal denervation decreased plasma renin activity and abolished the hypertension. However, baroreflex activation also suppressed systemic sympathetic activity and tachycardia and reduced glomerular hyperfiltration while increasing fractional sodium excretion. In contrast, glomerular filtration rate increased further after renal denervation. Thus, by improving autonomic control of cardiac function and diminishing glomerular hyperfiltration, suppression of global sympathetic activity by baroreflex activation may have beneficial effects in obesity beyond simply attenuating hypertension.

Cyp2c44 Epoxygenase Is Essential for Preventing the Renal Sodium Absorption During Increasing Dietary Potassium Intake [Kidney]

The aim of this study is to test whether the Cyp2c44 epoxygenase-dependent metabolism of arachidonic acid prevents the hypertensive effect of a high K (HK) intake by inhibiting the epithelial sodium channel (ENaC) activity. A HK intake elevated Cyp2c44 mRNA expression and 11,12-epoxyeicosatrienoic acid levels in the cortical collecting duct in Cyp2c44(+/+) mice (wild-type [wt]). However, an HK intake failed to increase 11,12-epoxyeicosatrienoic acid formation in the cortical collecting ducts of Cyp2c44(–/–) mice. Moreover, increasing K intake enhanced arachidonic acid–induced inhibition of ENaC in the wt but not in Cyp2c44(–/–) mice. In contrast, 11,12-epoxyeicosatrienoic acid, a Cyp2c44 metabolite, inhibited ENaC in the wt and Cyp2c44(–/–) mice. The notion that Cyp2c44 is the epoxygenase responsible for mediating the inhibitory effects of arachidonic acid on ENaC is further suggested by the observation that inhibiting Cyp-epoxygenase increased the whole-cell Na currents in principal cells of wt but not in Cyp2c44(–/–) mice. Feeding mice with an HK diet raised the systemic blood pressures of Cyp2c44(–/–) mice but was without an effect on wt mice. Moreover, application of amiloride abolished the HK-induced hypertension in Cyp2c44(–/–) mice. The HK-induced hypertension of Cyp2c44(–/–) mice was accompanied by decreasing 24-hour urinary Na excretion and increasing the plasma Na concentration, and the effects were absent in wt mice. In contrast, disruption of the Cyp2c44 gene did not alter K excretion. We conclude that Cyp2c44 epoxygenase mediates the inhibitory effect of arachidonic acid on ENaC and that Cyp2c44 functions as an HK-inducible antihypertensive enzyme responsible for inhibiting ENaC activity and Na absorption in the aldosterone-sensitive distal nephron.

Cytochrome P450 1B1 Contributes to Renal Dysfunction and Damage Caused by Angiotensin II in Mice [Kidney]

Cytochrome P450 1B1 contributes to the development of angiotensin II–induced hypertension and associated cardiovascular pathophysiology. In view of the critical role of angiotensin II in the kidney, as well as in salt and water homeostasis, and blood pressure regulation, we determined the contribution of cytochrome P450 1B1 to renal dysfunction and injury associated with angiotensin II–induced hypertension in male Cyp1b1+/+ and Cyp1b1–/– mice. Angiotensin II infusion (700 ng/kg per minute) given by miniosmotic pumps for 13 and 28 days increased systolic blood pressure in Cyp1b1+/+ mice; this increase was significantly reduced in Cyp1b1–/– mice. Angiotensin II increased renal Cyp1b1 activity, vascular resistance, and reactivity to vasoconstrictor agents and caused endothelial dysfunction in Cyp1b1+/+ but not Cyp1b1–/– mice. Angiotensin II increased water consumption and urine output, decreased urine osmolality, increased urinary Na+ and K+ excretion, and caused proteinuria and albuminuria in Cyp1b1+/+ mice that was diminished in Cyp1b1–/– mice. Infusion of angiotensin II for 28 but not 13 days caused renal fibrosis, tubular damage, and inflammation in Cyp1b1+/+ mice, which was minimized in Cyp1b1–/– mice. Angiotensin II increased levels of 12- and 20-hydroxyeicosatetraenoic acids; reactive oxygen species; and activity of NADPH oxidase, extracellular signal-regulated kinase 1/2, p38 mitogen-activated protein kinase, and c-Src in the kidneys of Cyp1b1+/+ but not Cyp1b1–/– mice. These data suggest that increased thirst, renal dysfunction, and injury and inflammation associated with angiotensin II–induced hypertension in mice depend on cytochrome P450 1B1 activity, thus indicating that cytochrome P450 1B1 could serve as a novel target for treating renal disease and hypertension.

Sildenafil Prevents and Reverses Transverse-Tubule Remodeling and Ca2+ Handling Dysfunction in Right Ventricle Failure Induced by Pulmonary Artery Hypertension [Heart]

Right ventricular (RV) failure (RVF) is the main cause of death in patients with pulmonary artery hypertension (PAH). Sildenafil, a phosphodiesterase type 5 inhibitor, was approved recently for treatment of PAH patients. However, the mechanisms underlying RV contractile malfunction and the benefits of sildenafil on RV function are not well understood. We aimed to investigate the following: (1) the ultrastructural and excitation-contraction coupling alterations underlying PAH-induced RVF; (2) whether the ultrastructural changes are reversible; and (3) the mechanisms underlying the therapeutic benefits of sildenafil in PAH-RVF. We used a single injection of monocrotaline in Wistar rats to induce pulmonary vascular proliferation, which led to PAH and RVF. RV myocytes displayed severe transverse (T)-tubule loss and disorganization, as well as blunted and dys-synchronous sarcoplasmic reticulum Ca2+ release. Sildenafil prevented and reversed the monocrotaline-induced PAH and LV filling impairment. Early intervention with sildenafil prevented RV hypertrophy and the development of RVF, T-tubule remodeling, and Ca2+ handling dysfunction. Although late treatment with sildenafil did not reverse RV hypertrophy in animals with established RVF, RV systolic function was improved. Furthermore, late intervention partially reversed both the impairment of myocyte T-tubule integrity and Ca2+ handling protein and sarcoplasmic reticulum Ca2+ release function in monocrotaline-treated rats. In conclusion, PAH-induced increase in RV afterload causes severe T-tubule remodeling and Ca2+ handling dysfunction in RV myocytes, leading to RV contractile failure. Sildenafil prevents and partially reverses ultrastructural, molecular, and functional remodeling of failing RV myocytes. Reversal of pathological T-tubule remodeling, although incomplete, is achievable without the regression of RV hypertrophy.

Preface for the Joint Proceedings of the High Blood Pressure Research 2011 Scientific Sessions and the Inter-American Society of Hypertension Meeting [Preface]

Vascular Remodeling in Hypertension: Mechanisms and Treatment [Excellence Award in Hypertension Research]

Asymmetric Dimethylarginine and Reactive Oxygen Species: Unwelcome Twin Visitors to the Cardiovascular and Kidney Disease Tables [Excellence Award in Hypertension Research]

Plasma levels of asymmetric dimethylarginine or markers of reactive oxygen species are increased in subjects with risk factors for cardiovascular disease or chronic kidney disease. We tested the hypothesis that reactive oxygen species generate cellular asymmetric dimethylarginine that together cause endothelial dysfunction that underlies the risk of subsequent disease. Rat preglomerular vascular smooth muscle cells transfected with p22phox had increased NADPH oxidase activity, enhanced activity and expression of protein arginine methyltransferase, and reduced activity and protein expression of dimethylarginine dimethylaminotransferase and of cationic amino acid transferase 1 resulting in increased cellular levels of asymmetric dimethylarginine. Rats infused with angiotensin II had oxidative stress. The endothelial function of their mesenteric arterioles was changed from vasodilatation to vasoconstriction, accompanied by increased vascular asymmetric dimethylarginine. All of these changes were prevented by Tempol. In vivo silencing of dimethylarginine dimethylaminotransferase 1 increased plasma levels of asymmetric dimethylarginine, whereas silencing of dimethylarginine dimethylaminotransferase 2 impaired endothelial function. We suggest that initiation factors, such as angiotensin II, expressed in blood vessels or tissues of subjects with cardiovascular and kidney disease risk factors generate reactive oxygen species from NADPH oxidase that enhances cellular asymmetric dimethylarginine in an amplification loop. This leads to adverse changes in vascular and organ functions, as a consequence of reduced tissue levels of NO and increased reactive oxygen species. Thus, we conclude that reactive oxygen species and asymmetric dimethylarginine form a tightly coupled amplification system that translates cardiovascular/kidney risk into overt disease.

Council for High Blood Pressure Research/InterAmerican Society of Hypertension/International Society of Hypertension: First New Investigators Symposium at the High Blood Pressure Research 2011 Scientific Sessions [Editorial]

Appropriate Time Interval to Repeat Ambulatory Blood Pressure Monitoring in Patients With White-Coat Resistant Hypertension [Original Articles Part 2]

Resistant hypertension is defined as uncontrolled office blood pressure, despite the use of ≥3 antihypertensive drugs. Ambulatory blood pressure monitoring (ABPM) is mandatory to diagnose 2 different groups, those with true and white-coat resistant hypertension. Patients are found to change categories between controlled/uncontrolled ambulatory pressures without changing their office blood pressures. In this way, ABPM should be periodically repeated. The aim of this study was to evaluate the most appropriate time interval to repeat ABPM to assure sustained blood pressure control in patients with white-coat resistant hypertension. This prospective study enrolled 198 patients (69% women; mean age: 68.9±9.9 years) diagnosed as white-coat resistant hypertension on ABPM. Patients were submitted to a second confirmatory examination 3 months later and repeated twice at 6-month intervals. Statistical analyses included Bland-Altman repeatability coefficients and multivariate logistic regression. Mean office blood pressure was 163±20/84±17 mm Hg, and mean 24-hour blood pressure was 118±8/66±7 mm Hg. White-coat resistant hypertension diagnosis presented a moderate reproducibility and was confirmed in 144 patients after 3 months. In the third and fourth ABPMs, 74% and 79% of patients sustained the diagnosis. In multivariate regression, a daytime systolic blood pressure ≤115 mm Hg in the confirmatory ABPM triplicated the chance of white-coat resistant hypertension status persistence after 1 year. In conclusion, a confirmatory ABPM is necessary after 3 months of the first white-coat–resistant hypertension diagnosis, and the procedure should be repeated at 6-month intervals, except in patients with daytime systolic blood pressure ≤115 mm Hg, in whom it may be repeated annually.

Lack of Validation of a Same-Day Outpatient Protocol for Determination of Salt Sensitivity of Blood Pressure [Original Articles Part 2]

Salt sensitivity of blood pressure has been studied in humans with a 48-hour inpatient protocol of salt loading and depletion or with longer outpatient protocols using high- and low-salt diets. Results have been reproducible, but both methods are laborious and costly. A 6-hour protocol of intravenous salt loading and furosemide has been reported but never validated. We studied 14 normal volunteers (39±2 years old; 86% women and 21% black) with the inpatient and 6-hour protocols, separated by 30 days. Four subjects (29%) were salt sensitive in the inpatient protocol. They had higher systolic blood pressure, higher body mass index, and somewhat lower plasma renin activity than salt-resistant subjects. Baseline systolic blood pressure before both protocols was highly reproducible (r=0.90; P<0.0001; limits of agreement: +6.2 to –8.0 mm Hg), whereas the response to salt depletion was not (r=0.09). Three salt-sensitive and 4 salt-resistant subjects were misclassified by the short protocol. Three-hour natriuresis by furosemide in the short protocol (344±15 mmol) was not different from the 12-hour natriuresis in the inpatient protocol (357±19). However, stimulation of plasma renin activity and aldosterone was significantly less in the short (+0.10±0.07 ngAI/L/sec and –61±44 pmol/L) than in the inpatient protocol (+1.80±0.60 ngAI/L/sec and +256±88 pmol/L; P<0.03 for both). Activation of hormonal changes that regulate depressor responses to salt depletion may not have occurred with the 3-hour natriuresis of the short protocol. This methodology cannot be used to study salt sensitivity of blood pressure, because the phenotype, mechanisms, and prognosis of the latter have been defined with the inpatient protocol.

Cardiovascular Biomarker Midregional Proatrial Natriuretic Peptide During and After Preeclamptic Pregnancies [Original Articles Part 2]

Preeclampsia is associated with increased risk of cardiovascular disease. Midregional proatrial natriuretic peptide (MR-proANP), a precursor of the atrial natriuretic peptide, is a biomarker for cardiovascular disease. We obtained plasma from 184 pregnant women in gestational weeks 24 to 42 (normotensive pregnancies: n=77, preeclampsia: n=107), from 25 of these women at 5 to 8 years after index pregnancy (normotensive pregnancies: n=11, preeclampsia: n=14), and from 49 normotensive, nonpregnant women and analyzed them by immunoassay for MR-proANP. To investigate potential sources, placental and decidual atrial natriuretic peptide mRNA expression levels were analyzed by quantitative real-time PCR in 21 normotensive and 23 preeclamptic pregnancies, as well as in human heart and kidney samples. For further confirmation, we measured circulating MR-proANP and performed expression studies in a transgenic rat model for preeclampsia. MR-proANP was significantly elevated in maternal plasma in preeclampsia compared with normotensive pregnancies (135 versus 56 pmol/L; P<0.001). However, 5 to 8 years after pregnancy, there was no difference (formerly preeclamptic women versus formerly normotensive in pregnancy: 53 versus 49 pmol/L; P=0.5). Our preeclamptic rat model confirmed the acute MR-proANP differences between preeclamptic and normotensive pregnancies (10.9±1.9 versus 4.3±0.3 pmol/L; P=0.05). Atrial natriuretic peptide expression was high in the heart but negligible in the uteroplacental unit in both normotensive humans and rats, whereas expression in maternal and fetal hearts in the preeclamptic rats was significantly increased, compared with controls. MR-proANP is a serviceable biomarker in preeclampsia, both in humans and a rat model, probably reflecting cardiovascular hemodynamic stress.

Agonistic Autoantibodies as Vasodilators in Orthostatic Hypotension: A New Mechanism [Original Articles Part 2]

Agonistic autoantibodies to the β-adrenergic and muscarinic receptors are a novel investigative and therapeutic target for certain orthostatic disorders. We have identified the presence of autoantibodies to β2-adrenergic and/or M3 muscarinic receptors by ELISA in 75% (15 of 20) of patients with significant orthostatic hypotension. Purified serum IgG from all 20 of the patients and 10 healthy control subjects were examined in a receptor-transfected cell-based cAMP assay for β2 receptor activation and β-arrestin assay for M3 receptor activation. There was a significant increase in IgG-induced activation of β2 and M3 receptors in the patient group compared with controls. A dose response was observed for both IgG activation of β2 and M3 receptors and inhibition of their activation with the nonselective β blocker propranolol and muscarinic blocker atropine. The antibody effects on β2 and/or M3 (via production of NO) receptor-mediated vasodilation were studied in a rat cremaster resistance arteriole assay. Infusion of IgG from patients with documented β2 and/or M3 receptor agonistic activity produced a dose-dependent vasodilation. Sequential addition of the β-blocker propranolol and the NO synthase inhibitor NG-nitro-l-arginine methyl ester partially inhibited IgG-induced vasodilation (percentage of maximal dilatory response: from 57.7±10.4 to 35.3±4.6 and 24.3±5.8, respectively; P<0.01; n=3), indicating that antibody activation of vascular β2 and/or M3 receptors may contribute to systemic vasodilation. These data support the concept that circulating agonistic autoantibodies serve as vasodilators and may cause or exacerbate orthostatic hypotension.

Sex-Specific Influence of Angiotensin Type 2 Receptor Stimulation on Renal Function: A Novel Therapeutic Target for Hypertension [Original Articles Part 2]

The renin-angiotensin system is a powerful regulator of arterial pressure and body fluid volume. Increasing evidence suggests that the angiotensin type 2 receptor (AT2R), which mediates the vasodilatory and natriuretic actions of angiotensin peptides, is enhanced in females and may, therefore, represent an innovative therapeutic target. We investigated the therapeutic potential of direct AT2R stimulation on renal function in 11- to 12-week–old anesthetized male and female Sprague-Dawley rats. Renal blood flow was examined in response to a graded infusion of the highly selective, nonpeptide AT2R agonist, compound 21 (100, 200, and 300 ng/kg per minute), in the presence and absence of AT2R blockade (PD123319; 1 mg/kg per hour). Direct AT2R stimulation significantly increased renal blood flow in both males and females, without influencing arterial pressure. This was dose dependent in females only and occurred to a greater extent in females at the highest dose of compound 21 administered (males: 13.1±2.4% versus females: 23.0±3.2% change in renal blood flow at 300 ng/kg per minute versus baseline; P<0.01). In addition, AT2R stimulation significantly increased sodium and water excretion to a similar extent in males and females (PGroup=0.05 and 0.005). However, there was no significant change in glomerular filtration rate in either sex, suggesting that altered tubular function may be responsible for AT2R-induced natriuresis rather than hemodynamic effects. Taken together, this study provides evidence that direct AT2R stimulation produces vasodilatory and natriuretic effects in the male and female kidney. The AT2R may, therefore, represent a valuable therapeutic target for the treatment of renal and cardiovascular diseases in both men and women.

p47phox Is Required for Afferent Arteriolar Contractile Responses to Angiotensin II and Perfusion Pressure in Mice [Original Articles Part 2]

Myogenic and angiotensin contractions of afferent arterioles generate reactive oxygen species. Resistance vessels express neutrophil oxidase-2 and -4. Angiotensin II activates p47phox/neutrophil oxidase-2, whereas it downregulates NOX-4. Therefore, we tested the hypothesis that p47phox enhances afferent arteriolar angiotensin contractions. Angiotensin II infusion in p47phox +/+ but not –/– mice increased renal cortical NADPH oxidase activity (7±1–12±1 [P<0.01] versus 5±1–7±1 103 · RLU · min–1 · μg protein–1 [P value not significant]), mean arterial pressure (77±2–91±2 [P<0.005] versus 74±2–77±1 mm Hg [P value not significant]), and renal vascular resistance (7.5±0.4–10.1±0.7 [P<0.01] versus 7.9±0.4–8.3±0.4 mm Hg/mL · min–1 · gram kidney weight–1 [P value not significant]). Afferent arterioles from p47phox –/– mice had a lesser myogenic response (3.1±0.4 versus 1.4±0.2 dynes · cm–1 · mm Hg–1; P<0.02) and a lesser (P<0.05) contraction to 10–6 M angiotensin II (diameter change +/+: 9.3±0.2–3.4±0.6 μm versus –/–: 9.9±0.6–7.5±0.4 μm). Angiotensin and increased perfusion pressure generated significantly (P<0.05) more reactive oxygen species in p47phox +/+ than –/– arterioles. Angiotensin II infusion increased the maximum responsiveness of afferent arterioles from p47phox +/+ mice to 10–6 M angiotensin II yet decreased the response in p47phox –/– mice. The angiotensin infusion increased the sensitivity to angiotensin II only in p47phox +/+ mice. We conclude that p47phox is required to enhance renal NADPH oxidase activity and basal afferent arteriolar myogenic and angiotensin II contractions and to switch afferent arteriolar tachyphylaxis to sensitization to angiotensin during a prolonged angiotensin infusion. These effects likely contribute to hypertension and renal vasoconstriction during infusion of angiotensin II.

Chronic Intrarenal Insulin Replacement Reverses Diabetes Mellitus-Induced Natriuresis and Diuresis [Original Articles Part 2]

We showed recently that sustained natriuresis in type 1 diabetic dogs was attributed to the decrease in insulin rather than the hyperglycemia alone. The sodium-retaining action of insulin appeared to require hyperglycemia, and it completely reversed the diabetic natriuresis and diuresis. This study tested whether the sodium-retaining effect was attributed to direct intrarenal actions of insulin. Alloxan-treated dogs (D; n=7) were maintained normoglycemic using 24-h/d IV insulin replacement. After control measurements, IV insulin was decreased to begin a 6-day diabetic period. Blood glucose increased from 84±6 mg/dL to an average of 428 mg/dL on days 5 and 6, sodium excretion increased from 74±8 to 98±7 meq/d over the 6 days, and urine volume increased from 1645±83 to 2198±170 mL/d. Dir dogs (n=7) were subjected to the same diabetic regimen, but, in addition, insulin was infused continuously into the renal artery at 0.3 mU/kg per minute during the 6-day period. This did not affect plasma insulin. Blood glucose increased from 94±10 mg/dL to an average of 380 mg/dL on days 5 and 6, but sodium excretion averaged 76±5 and 69±8 meq/d during control and diabetes mellitus, respectively. The diuresis also was prevented. Glomerular filtration rate increased only in Dir dogs, and there was no change in mean arterial pressure in either group. This intrarenal insulin infusion had no effect on sodium or volume excretion in normal dogs. Intrarenal insulin replacement in diabetic dogs caused a sustained increase in tubular reabsorption that completely reversed diabetic natriuresis. Insulin plus glucose may work to prevent salt wasting in uncontrolled type 2 diabetes mellitus.

NADPH Oxidase and PKC Contribute to Increased Na Transport by the Thick Ascending Limb During Type 1 Diabetes [Original Articles Part 2]

—Type 1 diabetes triggers protein kinase C (PKC)-dependent NADPH oxidase activation in the renal medullary thick ascending limb (mTAL), resulting in accelerated superoxide production. As acute exposure to superoxide stimulates NaCl transport by the mTAL, we hypothesized that diabetes increases mTAL Na+ transport through PKC-dependent and NADPH oxidase–dependent mechanisms. An O2-sensitive fluoroprobe was used to measure O2 consumption by mTALs from rats with streptozotocin-induced diabetes and sham rats. In sham mTALs, total O2 consumption was evident as a 0.34±0.03 U change in normalized relative fluorescence (NRF)/min per mg protein. Ouabain (2 mmol/L) reduced O2 consumption by 69±4% and 500 μmol/L furosemide reduced O2 consumption by 58±8%. Total O2 consumption was accelerated in mTAL from diabetic rats (0.74±0.07 NRF/min/mg protein; P<0.05 versus sham), reflecting increases in ouabain- and furosemide-sensitive O2 consumption. NADPH oxidase inhibition (100 μmol/L apocynin) reduced furosemide-sensitive O2 consumption by mTAL from diabetic rats to values not different from sham. The PKC inhibitor calphostin C (1 μmol/L) or the PKCα/β inhibitor Gö6976 (1 μmol/L) decreased furosemide-sensitive O2 consumption in both groups, achieving values that did not differ between sham and diabetic. PKCβ inhibition had no effect in either group. Similar inhibitory patterns were evident with regard to ouabain-sensitive O2 consumption. We conclude that NADPH oxidase and PKC (primarily PKCα) contribute to an increase in O2 consumption by the mTAL during type 1 diabetes through effects on the ouabain-sensitive Na+-K+-ATPase and furosemide-sensitive Na+-K+-2Cl– cotransporter that are primarily responsible for active transport Na+ reabsorption by this nephron segment.

Mechanisms of Dopamine D1 and Angiotensin Type 2 Receptor Interaction in Natriuresis [Original Articles Part 2]

Renal dopamine D1–like receptors (D1Rs) and angiotensin type 2 receptors (AT2Rs) are important natriuretic receptors counterbalancing angiotensin type 1 receptor–mediated tubular sodium reabsorption. Here we explore the mechanisms of D1R and AT2R interactions in natriuresis. In uninephrectomized, sodium-loaded Sprague-Dawley rats, direct renal interstitial infusion of the highly selective D1R agonist fenoldopam induced a natriuretic response that was abolished by the AT2R-specific antagonist PD-123319 or by microtubule polymerization inhibitor nocodazole but not by actin polymerization inhibitor cytochalasin D. By confocal microscopy and immunoelectron microscopy, fenoldopam translocated AT2Rs from intracellular sites to the apical plasma membranes of renal proximal tubule cells, and this translocation was abolished by nocodazole. Because D1R activation induces natriuresis via an adenylyl cyclase/cAMP signaling pathway, we explored whether this pathway is responsible for AT2R recruitment and AT2R-mediated natriuresis. Renal interstitial coinfusion of the adenylyl cyclase activator forskolin and 3-isobutly-1-methylxanthine induced natriuresis that was abolished either by PD-123319 or nocodazole but was unaffected by specific the D1R antagonist SCH-23390. Coadministration of forskolin and 3-isobutly-1-methylxanthine also translocated AT2Rs to the apical plasma membranes of renal proximal tubule cells; this translocation was abolished by nocodazole but was unaffected by SCH-23390. The results demonstrate that D1R-induced natriuresis requires AT2R recruitment to the apical plasma membranes of renal proximal tubule cells in a microtubule-dependent manner involving an adenylyl cyclase/cAMP signaling pathway. These studies provide novel insights regarding the mechanisms whereby renal D1Rs and AT2Rs act in concert to promote sodium excretion in vivo.

Role of Renal DJ-1 in the Pathogenesis of Hypertension Associated With Increased Reactive Oxygen Species Production [Original Articles Part 2]

The D2 dopamine receptor (D2R) is important in the pathogenesis of essential hypertension. We have already reported that systemic deletion of the D2R gene in mice results in reactive oxygen species (ROS)-dependent hypertension, suggesting that the D2R has antioxidant effects. However, the mechanism of this effect is unknown. DJ-1 is a protein that has antioxidant properties. D2R and DJ-1 are expressed in the mouse kidney and colocalize and coimunoprecipitate in mouse renal proximal tubule cells. We hypothesized that D2Rs regulate renal ROS production in the kidney through regulation of DJ-1 expression or function. Heterozygous D2+/– mice have increased blood pressure, urinary 8-isoprostanes, and renal Nox 4 expression, but decreased renal DJ-1 expression. Silencing D2R expression in mouse renal proximal tubule cells increases ROS production and decreases the expression of DJ-1. Conversely, treatment of these cells with a D2R agonist increases DJ-1 expression and decreases Nox 4 expression and NADPH oxidase activity, effects that are partially blocked by a D2R antagonist. Silencing DJ-1 expression in mouse renal proximal tubule cells increases ROS production and Nox 4 expression. Selective renal DJ-1 silencing by the subcapsular infusion of DJ-1 siRNA in mice increases blood pressure, renal Nox4 expression, and NADPH oxidase activity. These results suggest that the inhibitory effects of D2R on renal ROS production are at least, in part, mediated by a positive regulation of DJ-1 expression/function and that DJ-1 may have a role in the prevention of hypertension associated with increased ROS production.

Angiotensin-(1-7) Deficiency and Baroreflex Impairment Precede the Antenatal Betamethasone Exposure-Induced Elevation in Blood Pressure [Original Articles Part 2]

Betamethasone is administered to accelerate lung development and improve survival of premature infants but may be associated with hypertension later in life. In a sheep model of fetal programming resulting from exposure at day 80 of gestation to Betamethasone (Beta-exposed), adult sheep at 6 to 9 months or 1.8 years of age have elevated mean arterial pressure (MAP) and attenuated spontaneous baroreflex sensitivity (sBRS) for control of heart rate compared to age-matched controls associated with imbalances in angiotensin (Ang) II vs Ang-(1-7) tone. At 6 weeks of age, evoked BRS is already low in the Beta-exposed animals. In this study, we assessed the potential contribution of the renin-angiotensin system to the impaired sBRS. Female lambs (6 weeks old) with Beta exposure in utero had similar MAP to control lambs (78±2 vs 77±2 mm Hg, n=4–5 per group), but lower sBRS (8±1 vs 16±3 ms/mm Hg; P<0.05) and impaired heart rate variability. Peripheral AT1 receptor blockade using candesartan lowered MAP in both groups (10 mm Hg) and improved sBRS and heart rate variability in Beta-exposed lambs to a level similar to control. AT7 receptor blockade by infusion of D-ala Ang-(1-7) (700 ng/kg/min for 45 minutes) reduced sBRS 46%±10% in Beta-exposed vs in control lambs (P<0.15) and increased MAP in both groups (6±2 mm Hg). Our data reveal that Beta exposure impairs sBRS and heart rate variability at a time point preceding the elevation in MAP via mechanisms involving an imbalance in the Ang II/Ang-(1-7) ratio consistent with a progressive loss in Ang-(1-7) function.

Sensitization of Slow Pressor Angiotensin II (Ang II)-Initiated Hypertension: Induction of Sensitization by Prior Ang II Treatment [Original Articles Part 2]

Sensitization involving the central nervous system has been studied in many conditions but has received little attention in investigation of the pathogenesis of hypertension. Our experiments were initiated to determine whether angiotensin II (Ang II)–induced hypertension can be sensitized by prior Ang II treatment and the role of the brain renin-angiotensin-aldosterone system (RAAS) in this process. To demonstrate Ang II–induced sensitization, we used an experimental design of induction-delay-expression. Male rats were implanted for telemetered blood pressure (BP) recording. During induction (I), low doses of subcutaneous or intracerebroventricular Ang II were delivered for 1 week, and then the rats were rested for 1 week (delay [D]) to ensure that any exogenous Ang II was metabolized. After this, a second higher dose of Ang II was given subcutaneously for 2 weeks (expression [E]). During I and D, the low doses of Ang II had no sustained effects on BP. However, during E, the Ang II–induced BP increase was greater in the groups that had received low doses of Ang II during I in comparison to the group receiving saline during I. Central angiotensin type 1 receptor antagonist delivery blocked this sensitization. Brain tissue collected at the end of D and E showed increased mRNA expression of several RAAS components in key forebrain regions of sensitized rats. Fos-related antigen–like immunoreactivity was also increased at the end of E in the sensitized forebrain. These results indicate that subpressor doses of Ang II act on the brain to sensitize the hypertensive response to subsequent Ang II and that sensitization is associated with altered expression of RAAS components in forebrain cardiovascular control structures.

Tonic Postganglionic Sympathetic Inhibition Induced by Afferent Renal Nerves? [Original Articles Part 2]

Other than efferent sympathetic innervation, the kidney has peptidergic afferent fibers expressing TRPV1 receptors and releasing substance P. We tested the hypothesis that stimulation of afferent renal nerve activity with the TRPV1 agonist capsaicin inhibits efferent renal sympathetic nerve activity tonically by a neurokinin 1 receptor–dependant mechanism. Anesthetized Sprague-Dawley rats were instrumented as follows: (1) arterial and venous catheters for recording of blood pressure and heart rate and drug administration; (2) left-sided renal arterial catheter for selective intrarenal administration of the TRPV1 agonist capsaicin (3.3, 6.6, 10, 33*10–7 m; 10 μL; after 15, 30, 45, and 60 minutes, respectively) to stimulate afferent renal nerve activity; (3) right-sided bipolar electrode for continuous renal sympathetic nerve recording; and (4) specialized renal pelvic and renal artery catheters to separate pelvic from intrarenal afferent activity. Before and after intrarenal capsaicin application, increasing intravenous doses of the neurokinin 1 receptor blocker RP67580 were given. Intrarenal capsaicin decreased integrated renal sympathetic activity from 65.4±13.0 mV*s (baseline) to 12.8±3.2 mV*s (minimum; P<0.01). This sustained renal sympathetic inhibition reached its minimum within 70 minutes and was not directly linked to the transient electric afferent response to be expected with intrarenal capsaicin. Suppressed renal sympathetic activity transiently but completely recovered after intravenous administration of the neurokinin 1 blocker (maximum: 120.3±19.4 mV*s; P<0.01). Intrarenal afferent activity could be unequivocally separated from pelvic afferent activity. For the first time we provide direct evidence that afferent intrarenal nerves provide a tonically acting sympathoinhibitory system, which seems to be rather mediated by neurokinin release acting via neurokinin 1 receptor pathways rather than by electric afferent effects on central sympathetic outflow.

Peroxisome Proliferator-Activated Receptor-{gamma} Regulates Inflammation and Renin-Angiotensin System Activity in the Hypothalamic Paraventricular Nucleus and Ameliorates Peripheral Manifestations of Heart Failure [Original Articles Part 2]

Activation of peroxisome proliferator-activated receptor (PPAR)-, a nuclear transcription factor, has been shown to inhibit the production of proinflammatory cytokines and, in peripheral tissues, to downregulate the renin-angiotensin system. PPAR- is expressed in key brain areas involved in cardiovascular and autonomic regulation. We hypothesized that activation of central PPAR- would reduce sympathetic excitation and ameliorate peripheral manifestations of heart failure (HF) by inhibiting central inflammation and brain renin-angiotensin system activity. Two weeks after coronary artery ligation, HF rats received an intracerebroventricular infusion of the PPAR- agonist pioglitazone or vehicle for another 2 weeks. PPAR- expression in the paraventricular nucleus of hypothalamus, an important cardiovascular region, was unchanged in HF compared with sham-operated rats. However, PPAR- DNA binding activity was reduced, nuclear factor-B activity was increased, and expression of proinflammatory cytokines and angiotensin II type-1 receptor was augmented in the HF rats. Mean blood pressure response to ganglionic blockade was greater; plasma norepinephrine levels, lung/body weight, right ventricle/body weight, and left ventricular end-diastolic pressure were increased; and maximal left ventricular dP/dt was decreased. All of these findings were ameliorated in HF rats treated with intracerebroventricular pioglitazone, which increased PPAR- expression and DNA binding activity in the paraventricular nucleus of hypothalamus. The results demonstrate that cardiovascular and autonomic mechanisms leading to heart failure after myocardial infarction can be modulated by activation of PPAR- in the brain. Central PPAR- may be a novel target for treatment of sympathetic excitation in myocardial infarction–induced HF.

Direct Angiotensin II Type 2 Receptor Stimulation in N{omega}-Nitro-L-Arginine-Methyl Ester-Induced Hypertension: The Effect on Pulse Wave Velocity and Aortic Remodeling [Original Articles Part 2]

Pulse wave velocity (PWV), a direct marker of arterial stiffness, is an independent cardiovascular risk factor. Although the angiotensin II type 1 receptor blockade belongs to major antihypertensive and cardioprotective therapies, less is known about the effects of long-term stimulation of the angiotensin II type 2 receptor. Previously, compound 21, a selective nonpeptide angiotensin II type 2 receptor agonist improved the outcome of myocardial infarction in rats along with anti-inflammatory properties. We investigated whether compound 21 alone or in combination with angiotensin II type 1 receptor blockade by olmesartan medoxomil could prevent PWV increase and aortic remodeling in N-nitro-l-arginine-methyl ester (l-NAME)–induced hypertension. Male adult Wistar rats (n=65) were randomly assigned to control, l-NAME, l-NAME+compound-21, l-NAME+olmesartan, and l-NAME+olmesartan+compound-21 groups and treated for 6 weeks. We observed that l-NAME hypertension was accompanied by enhanced PWV, increased wall thickness, and stiffness of the aorta, along with elevated hydroxyproline concentration. Olmesartan completely prevented hypertension, PWV and wall thickness increase, and the increase of aortic stiffness and partly prevented hydroxyproline accumulation. Compound 21 partly prevented all of these alterations, yet without concomitant prevention of blood pressure rise. Although the combination therapy with olmesartan and compound 21 led to blood pressure levels, PWV, and wall thickness comparable to olmesartan-alone–treated rats, only in the combination group was complete prevention of increased hydroxyproline deposition achieved, resulting in even more pronounced stiffness reduction. We conclude that chronic angiotensin II type 2 receptor stimulation prevented aortic stiffening and collagen accumulation without preventing hypertension in rats with inhibited NO synthase. These effects were additive to angiotensin II type 1 receptor blockade, yet without additional blood pressure–lowering effect, and they seem to be NO and blood pressure independent.

Roles of Interleukin 17 in Angiotensin II Type 1 Receptor-Mediated Insulin Resistance [Original Articles Part 2]

Interleukin 17 (IL-17) is known to contribute to the pathogenesis of hypertension, atherosclerosis, and adipocyte differentiation; however, the roles of IL-17 in glucose metabolism remain to be elucidated. Angiotensin II type 1 receptor blockers improve insulin resistance at least in part because of the amelioration of inflammation. Therefore, we examined the possible roles of IL-17 in the pathogenesis of insulin resistance in type 2 diabetes mellitus using a mouse model, KK-Ay, and angiotensin II type 1 receptor–mediated insulin resistance. KK-Ay mice were administered control-IgG2A or anti-IL-17 antibody 5 times at a dose of 100 μg every second day by IP injection. KK-Ay mice were administered telmisartan for 2 weeks. C57BL/6J mice treated with angiotensin II infusion for 2 weeks were administered telmisartan or hydralazine. Insulin resistance was evaluated by oral glucose tolerance test, insulin tolerance test, and uptake of 2-[3H]deoxy-d-glucose in peripheral tissues. Serum IL-17 concentration in KK-Ay mice was significantly higher than that in C57BL/6J mice. Treatment of KK-Ay mice with anti–IL-17 antibody significantly increased 2-[3H]deoxy-d-glucose uptake in skeletal muscle but not in white adipose tissue and attenuated the increase in blood glucose level after a glucose load. Blockade of IL-17 enhanced the expression of adipocyte differentiation markers and adiponectin. Treatment with telmisartan decreased serum IL-17 concentration in KK-Ay and ameliorated angiotensin II–induced insulin resistance with a decrease in serum IL-17 level in C57BL/6J. In conclusion, IL-17 could play an important role in the pathogenesis of angiotensin II type 1 receptor–induced insulin resistance.

Oxidative Stress Causes Mineralocorticoid Receptor Activation in Rat Cardiomyocytes: Role of Small GTPase Rac1 [Original Articles Part 2]

Overactivation of the mineralocorticoid receptor signaling is implicated in cardiovascular disease, including hypertensive heart disease. Oxidative stress is suggested to augment mineralocorticoid receptor signal transduction, but the precise mechanisms remain unclear. Mineralocorticoid receptor activity is regulated by multiple factors, in addition to plasma ligand levels. We previously identified Rac1 GTPase as a modulator of mineralocorticoid receptor activity. Here we show that oxidative stress induces mineralocorticoid receptor activation in a ligand-independent, Rac1-depenent manner in cardiomyocytes. Oxidant stress was induced in rat cultured cardiomyocytes (H9c2) by l-buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis. BSO depleted intracellular glutathione and concomitantly increased reactive oxygen species (199%; P<0.01). BSO significantly enhanced the corticosterone-induced, mineralocorticoid receptor–dependent luciferase reporter activity (186%; P<0.01) and basal luciferase activity without ligand stimulation. These effects were inhibited by the antioxidant N-acetylcysteine. The ligand independency of BSO action was indicated using a mutant mineralocorticoid receptor that does not bind ligands. With this mutant mineralocorticoid receptor, BSO-evoked mineralocorticoid receptor activation remained intact, whereas ligand-induced mineralocorticoid receptor activation was abolished. We next examined the involvement of Rac1. BSO increased active Rac1 in a redox-dependent fashion, and Rac inhibition suppressed the enhancing effect of BSO. Constitutively active Rac1, indeed, potentiated mineralocorticoid receptor transactivation. Furthermore, mineralocorticoid receptor transactivation by BSO was accompanied by enhanced nuclear accumulation of mineralocorticoid receptor. We conclude that alteration of redox state modulates mineralocorticoid receptor–dependent transcriptional activity via Rac1 in the heart. This redox-sensitive, ligand-independent mineralocorticoid receptor activation may contribute to the processes by which oxidant stress promotes cardiac injury.

Deletion of G Protein-Coupled Estrogen Receptor Increases Endothelial Vasoconstriction [Original Articles Part 2]

Endogenous estrogens mediate protective effects in the cardiovascular system, affecting both endothelium-dependent and endothelium-independent mechanisms. Previous studies have suggested that nonselective estrogen receptor agonists such as endogenous estrogens inhibit endothelium-dependent vasoconstriction; however, the role of estrogen receptors in this response has not yet been clarified. This study investigated whether the intracellular transmembrane G protein–coupled estrogen receptor (GPER) regulates vascular reactivity in mice. Effects of chronic deficiency (using mice lacking the GPER gene) and acute inhibition (using the GPER-selective antagonist G15) on endothelium-dependent and endothelium-independent vascular reactivity, and the effects of GPER deficiency on vascular gene expression and structure were investigated. We found that chronic GPER deficiency is associated with increased endothelial prostanoid-mediated vasoconstriction but has no effect on endothelial nitric oxide bioactivity, gene expression of endothelial nitric oxide synthase and thromboxane prostanoid (TP) receptor, or vascular structure. GPER deletion also increases TP receptor–mediated contraction. Acute GPER blockade enhances endothelium-dependent contractions and reduces endothelial nitric oxide bioactivity. Contractions in response to TP receptor activation are unaffected by G15. In conclusion, this study identifies GPER as the first estrogen receptor with inhibitory activity on endothelium-dependent contractility. These findings may be important for understanding and treating diseases associated with increased endothelial vasoconstrictor prostanoid activity such as hypertension and obesity.

Exercise Training Prevents the Microvascular Rarefaction in Hypertension Balancing Angiogenic and Apoptotic Factors: Role of MicroRNAs-16, -21, and -126 [Original Articles Part 2]

Aerobic exercise training (ET) lowers hypertension and improves patient outcomes in cardiovascular disease. The mechanisms of these effects are largely unknown. We hypothesized that ET modulates microRNAs (miRNAs) involved in vascularization. miRNA-16 regulates the expression of vascular endothelial growth factor and antiapoptotic protein Bcl-2. miRNA-21 targets Bcl-2. miRNA-126 functions by repressing regulators of the vascular endothelial growth factor pathway. We investigated whether miRNA-16, -21 and -126 are modulated in hypertension and by ET. Twelve-week–old male spontaneously hypertensive rats (SHRs; n=14) and Wistar Kyoto (WKY; n=14) rats were assigned to 4 groups: SHRs, trained SHRs (SHR-T), Wistar Kyoto rats, and trained Wistar Kyoto rats. ET consisted of 10 weeks of swimming. ET reduced blood pressure and heart rate in SHR-Ts. ET repaired the slow-to-fast fiber type transition in soleus muscle and the capillary rarefaction in SHR-Ts. Soleus miRNA-16 and -21 levels increased in SHRs paralleled with a decrease of 48% and 25% in vascular endothelial growth factor and Bcl-2 protein levels, respectively. Hypertension increased Bad and decreased Bcl-x and endothelial NO synthase levels and lowered p-Badser112:Bad ratio. ET in SHR-Ts reduced miRNA-16 and -21 levels and elevated vascular endothelial growth factor and Bcl-2 levels. ET restored soleus endothelial NO synthase levels plus proapoptotic and antiapoptotic mediators in SHR-Ts, indicating that the balance between angiogenic and apoptotic factors may prevent microvascular abnormalities in hypertension. miRNA-126 levels were reduced in SHRs with an increase of 51% in phosphoinositol-3 kinase regulatory subunit 2 expression but normalized in SHR-Ts. Our data show that ET promoted peripheral revascularization in hypertension, which could be associated with regulation of select miRNAs, suggesting a mechanism for its potential therapeutic application in vascular diseases.

Vascular Incorporation of Endothelial Colony-Forming Cells Is Essential for Functional Recovery of Murine Ischemic Tissue Following Cell Therapy [Integrative Biology/Experimental Medicine]

Objective— Cord blood–derived human endothelial colony-forming cells (ECFCs) bear a high proliferative capacity and potently enhance tissue neovascularization in vivo. Here, we investigated whether the leading mechanism for the functional improvement relates to their physical vascular incorporation or perivascular paracrine effects and whether the effects can be further enhanced by dual-cell–based therapy, including mesenchymal stem cells (MSCs). Methods and Results— ECFCs or MSCs were lentivirally transduced with thymidine kinase suicide gene driven by the endothelial-specific vascular endothelial growth factor 2 (kinase insert domain receptor) promoter and evaluated in a hindlimb ischemia model. ECFCs and MSCs enhanced neovascularization after ischemic events to a similar extent. Dual therapy using ECFCs and MSCs further enhanced neovascularization. Mechanistically, 3 weeks after induction of ischemia followed by cell therapy, ganciclovir-mediated elimination of kinase insert domain receptor+ cells completely reversed the therapeutic effect of ECFCs but not that of MSCs. Histological analysis revealed that ganciclovir effectively eliminated ECFCs incorporated into the vasculature. Conclusion— Endothelial-specific suicide gene technology demonstrates distinct mechanisms for ECFCs and MSCs, with complete abolishment of ECFC-mediated effects, whereas MSC-mediated effects remained unaffected. These data strengthen the notion that a dual-cell–based therapy represents a promising approach for vascular regeneration of ischemic tissue.

Targeting Angiogenesis as Treatment for Obesity [Editorial]

Humanizing the Problem of Transplant Vasculopathy [Editorials]

Endothelial-to-Mesenchymal Transition and MicroRNA-21: The Game Is On Again [Editorials]

Intraplaque Hemorrhage: An Imaging Marker for Atherosclerotic Plaque Destabilization? [Editorials]

Life After GWAS: Functional Genomics in Vascular Biology [ATVB in Focus: Life After GWAS: Functional Genomics in Vascular Biology]

Strategies Beyond Genome-Wide Association Studies for Atherosclerosis [ATVB in Focus: Life After GWAS: Functional Genomics in Vascular Biology]

Atherosclerotic diseases, including coronary artery disease (CAD) and myocardial infarction (MI), are the leading causes of death in the world. The genetic basis of CAD and MI, which are caused by multiple interacting endogenous and exogenous factors, has gained considerable interest in the last years as genome-wide association studies (GWASs) have identified many new susceptibility loci for CAD and MI, and the underlying genes provide new insights into the genetic architecture of these diseases. Here we summarize the recent findings from GWASs of atherosclerosis and discuss their functional and biological implications. We also discuss the different post-GWAS strategies that are currently used for refining the location of causal variants, understanding their role, and shedding light on molecular mechanisms explaining their association to CAD. We finally discuss potential clinical translations of GWAS findings for individual risk prediction, advanced clinical strategies, and personalized treatments.

The Use of High-Throughput Technologies to Investigate Vascular Inflammation and Atherosclerosis [ATVB in Focus: Life After GWAS: Functional Genomics in Vascular Biology]

The greatest challenge of scientific research is to understand the causes and consequences of disease. In recent years, great efforts have been devoted to unraveling the basic mechanisms of atherosclerosis (the underlying pathology of cardiovascular disease), which remains a major cause of morbidity and mortality worldwide. Because of the complex and multifactorial pathophysiology of cardiovascular disease, different research techniques have increasingly been combined to unravel genetic aspects, molecular pathways, and cellular functions involved in atherogenesis, vascular inflammation, and dyslipidemia to gain a multifaceted picture addressing this complexity. Thanks to the rapid evolution of high-throughput technologies, we are now able to generate large-scale data on the DNA, RNA, and protein levels. With the help of sophisticated computational tools, these data sets are integrated to enhance information extraction and are being increasingly used in a systems biology approach to model biological processes as interconnected and regulated networks. This review exemplifies the use of high-throughput technologies—such as genomics, transcriptomics, proteomics, and epigenomics—and systems biology to explore pathomechanisms of vascular inflammation and atherosclerosis.

Recent Studies of the Human Chromosome 9p21 Locus, Which Is Associated With Atherosclerosis in Human Populations [ATVB in Focus Life After GWAS: Functional Genomics in Vascular Biology]

The chromosome 9p21 (Chr9p21) locus was discovered in 2007 by independent genome-wide association studies for coronary artery disease. Since then, the locus has been replicated numerous times and can be considered the most robust genetic marker of coronary artery disease today. Subsequent work has shown associations of Chr9p21 with a number of additional cardiovascular disease traits, such as carotid artery plaque, stroke, aneurysms, peripheral artery disease, heart failure, and cardiovascular mortality, suggesting a more general role in vascular pathology. Importantly, Chr9p21 lacks associations with common cardiovascular risk factors, such as lipids and hypertension, indicating that the locus exerts its effect through a completely novel mechanism. One of the challenges is that the core haplotype block at Chr9p21 resides in a region of the genome devoid of protein-coding genes. Recent progress has been made by functional studies focusing on differential expression of antisense noncoding RNA in the INK4 locus (ANRIL), which is transcribed from the Chr9p21 locus, as well as neighboring protein-coding genes at the INK4/ARF locus. The emerging concept suggests that ANRIL might constitute a regulator of epigenetic modification and thus modulate cardiovascular risk. Here, we review the current clinical, mechanistic, and diagnostic implications of the Chr9p21 locus in cardiovascular disease.

Beyond Genome-Wide Association Studies: The Usefulness of Mouse Genetics in Understanding the Complex Etiology of Atherosclerosis [ATVB in Focus: Life After GWAS: Functional Genomics in Vascular Biology]

The development of population-based genome-wide association studies has led to the rapid identification of large numbers of genetic variants associated with coronary artery disease (CAD) and related traits. Together with large-scale gene-centric studies, at least 35 loci associated with CAD per se have been identified with replication. The majority of these associations are with common single-nucleotide polymorphisms exhibiting modest effects on relative risk. The modest nature of the effects, coupled with ethical/practical constraints associated with human sampling, makes it difficult to answer important questions beyond gene/locus localization and allele frequency via human genetic studies. Questions related to gene function, disease-causing mechanism(s), and effective interventions will likely require studies in model organisms. The use of the mouse model for further detailed studies of CAD-associated loci identified by genome-wide association studies is highlighted herein.

Use of Functional Genomics to Identify Candidate Genes Underlying Human Genetic Association Studies of Vascular Diseases [ATVB in Focus Life After GWAS: Functional Genomics in Vascular Biology]

Recent genome-wide association studies have identified hundreds of genetic loci as being associated with vascular diseases or traits and their risk factors. Many of the loci uncovered represent novel discoveries with no obvious candidate genes and molecular mechanisms, testifying to the complexity of vascular diseases. To understand the functional consequences of genetic variations and help pinpoint the underlying genes and mechanisms of common complex diseases, functional genomics that integrate genetic variations and intermediate molecular traits such as gene expression has been extensively studied in the past few years. This review summarizes the key concepts of functional genomics, the current state of the field, and its application in vascular diseases.

S100A8 and S100A9 in Cardiovascular Biology and Disease [Brief Review]

There is recent and widespread interest in the damage-associated molecular pattern molecules S100A8 and S100A9 in cardiovascular science. These proteins have a number of interesting features and functions. For example, S100A8 and S100A9 (S100A8/A9) have both intracellular and extracellular actions, they are abundantly expressed in inflammatory and autoimmune states, primarily by myeloid cells but also by other vascular cells, and they modulate inflammatory processes, in part through Toll-like receptor 4 and the receptor for advanced glycation end products. S100A8/A9 also have anti-inflammatory and immune regulatory actions. Furthermore, increased plasma levels of S100A8/A9 predict cardiovascular events in humans, and deletion of these proteins partly protects Apoe–/– mice from atherosclerosis. Understanding the roles of S100A8 and S100A9 in vascular cell types and the mechanisms whereby these proteins mediate their biological effects may offer new therapeutic strategies to prevent, treat, and predict cardiovascular diseases.

Reciprocal Metabolic Perturbations in the Adipose Tissue and Liver of GPIHBP1-Deficient Mice [Integrative Biology/Experimental Medicine]

Objective— Gpihbp1-deficient (Gpihbp1–/–) mice lack the ability to transport lipoprotein lipase to the capillary lumen, resulting in mislocalization of lipoprotein lipase within tissues, defective lipolysis of triglyceride-rich lipoproteins, and chylomicronemia. We asked whether GPIHBP1 deficiency and mislocalization of catalytically active lipoprotein lipase would alter the composition of triglycerides in adipose tissue or perturb the expression of lipid biosynthetic genes. We also asked whether perturbations in adipose tissue composition and gene expression, if they occur, would be accompanied by reciprocal metabolic changes in the liver. Methods and Results— The chylomicronemia in Gpihbp1–/– mice was associated with reduced levels of essential fatty acids in adipose tissue triglycerides and increased expression of lipid biosynthetic genes. The liver exhibited the opposite changes: increased levels of essential fatty acids in triglycerides and reduced expression of lipid biosynthetic genes. Conclusion— Defective lipolysis in Gpihbp1–/– mice causes reciprocal metabolic perturbations in adipose tissue and liver. In adipose tissue, the essential fatty acid content of triglycerides is reduced and lipid biosynthetic gene expression is increased, whereas the opposite changes occur in the liver.

Apolipoprotein B Secretion Is Regulated by Hepatic Triglyceride, and Not Insulin, in a Model of Increased Hepatic Insulin Signaling [Integrative Biology/Experimental Medicine]

Objective— States of insulin resistance, hyperinsulinemia, and hepatic steatosis are associated with increased secretion of triglycerides (TG) and apolipoprotein B (apoB), even though insulin targets apoB for degradation. We used hepatic-specific "phosphatase and tensin homologue deleted on chromosome 10" (Pten) knockout (hPten-ko) mice, with increased hepatic insulin signaling, to determine the relative roles of insulin signaling and hepatic TG in regulating apoB secretion. Methods and Results— TG and apoB secretion was elevated in hPten-ko mice. When hepatic TG was reduced by inhibition of diacylglycerol acyltransferase 1/diacylglycerol acyltransferase 2 or sterol regulatory element-binding protein-1c, both TG secretion and apoB secretion fell without changes in hepatic insulin signaling. Acute reconstitution of hPten reduced hepatic TG content, and both TG and apoB secretion fell within 4 days despite decreased hepatic insulin signaling. Acute depletion of hepatic Pten by adenoviral introduction of Cre into Pten floxed mice caused steatosis within 4 days, and secretion of both TG and apoB increased despite increased hepatic insulin signaling. Even when steatosis after acute Pten depletion was prevented by pretreatment with SREBP-1c antisense oligonucleotides, apoB secretion was not reduced after 4 days. Ex vivo results were in primary hepatocytes were similar. Conclusion— Either hepatic TG is the dominant regulator of apoB secretion or any inhibitory effects of hepatic insulin signaling on apoB secretion is very short-lived.

Accelerated Atherosclerosis in Apoe-/- Mice Heterozygous for the Insulin Receptor and the Insulin Receptor Substrate-1 [Integrative Biology/Experimental Medicine]

Objective— Prediabetic states are associated with accelerated atherosclerosis, but the availability of mouse models to study connections between these diseases has been limited. The aim of this study was to test the selective role of impaired insulin receptor/insulin receptor substrate-1 signaling on atherogenesis. Methods and Results— To address the effects of impaired insulin signaling associated with hyperinsulinemia on atherosclerosis in the absence of obesity and hyperglycemia, we generated insulin receptor (Insr)/insulin receptor substrate-1 (Insr1) double heterozygous apolipoprotein (Apoe)-knockout mice (Insr+/–Irs1+/–Apoe–/–) mice. Insr+/–Irs1+/–Apoe–/– mice fed a Western diet for 15 weeks showed elevated levels of fasting insulin compared to Insr+/+Irs1+/+Apoe–/– mice. There were no significant differences in glucose, triglyceride, HDL, VLDL, cholesterol levels or free fatty acid in the plasma of Insr+/–Irs1+/–Apoe–/– and Insr+/+Irs1+/+Apoe–/– mice. Atherosclerotic lesions were increased in male (brachiocephalic artery) and female (aortic tree) Insr+/–Irs1+/–Apoe–/– compared to Insr+/+Irs1+/+Apoe–/– mice. Bone marrow transfer experiments demonstrated that nonhematopoietic cells have to be Insr+/–Irs1+/– to accelerate atherosclerosis. Impaired insulin signaling resulted in decreased levels of vascular phospho-eNOS, attenuated endothelium-dependent vasorelaxation and elevated VCAM-1 expression in aortas of Insr+/–Irs1+/–Apoe–/– mice. In addition, phospho-ERK and vascular smooth muscle cell proliferation were significantly elevated in aortas of Insr+/–Irs1+/–Apoe–/– mice. Conclusion— These results demonstrate that defective insulin signaling is involved in accelerated atherosclerosis in Insr+/–Irs1+/–Apoe–/– mice by promoting vascular dysfunction and inflammation.

Aldosterone Induces Vascular Insulin Resistance by Increasing Insulin-Like Growth Factor-1 Receptor and Hybrid Receptor [Integrative Biology/Experimental Medicine]

Objective— We previously showed that aldosterone induces insulin resistance in rat vascular smooth muscle cells (VSMCs). Because insulin-like growth factor-1 receptor (IGF1R) affects insulin signaling, we hypothesized that aldosterone induces vascular insulin resistance and remodeling via upregulation of IGF1R and its hybrid insulin/insulin-like growth factor-1 receptor. Methods and Results— Hybrid receptor expression was measured by immunoprecipitation. Hypertrophy of VSMCs was evaluated by 3H-labeled leucine incorporation. Aldosterone (10 nmol/L) significantly increased protein and mRNA expression of IGF1R and hybrid receptor in VSMCs but did not affect insulin receptor expression. Mineralocorticoid receptor blockade with eplerenone inhibited aldosterone-induced increases in IGF1R and hybrid receptor. Aldosterone augmented insulin (100 nmol/L)-induced extracellular signal-regulated kinase 1/2 phosphorylation. Insulin-induced leucine incorporation and α-smooth muscle actin expression were also augmented by aldosterone in VSMCs. These aldosterone-induced changes were significantly attenuated by eplerenone or picropodophyllin, an IGF1R inhibitor. Chronic infusion of aldosterone (0.75 μg/hour) increased blood pressure and aggravated glucose metabolism in rats. Expression of hybrid receptor, azan-positive area, and oxidative stress in aorta was increased in aldosterone-infused rats. Spironolactone and tempol prevented these aldosterone-induced changes. Conclusion— Aldosterone induces vascular remodeling through IGF1R- and hybrid receptor–dependent vascular insulin resistance. Mineralocorticoid receptor blockade may attenuate angiopathy in hypertensive patients with hyperinsulinemia.

ApoE Suppresses Atherosclerosis by Reducing Lipid Accumulation in Circulating Monocytes and the Expression of Inflammatory Molecules on Monocytes and Vascular Endothelium [Integrative Biology/Experimental Medicine]

Objective— We investigated atheroprotective properties of apolipoprotein (apo) E beyond its ability to lower plasma cholesterol. We hypothesized that apoE reduces atherosclerosis by decreasing lipid accumulation in circulating monocytes and the inflammatory state of monocytes and the vascular endothelium. Methods and Results— We developed mice with spontaneous hyperlipidemia with and without plasma apoE. Hypomorphic apoE mice deficient in low-density lipoprotein receptor (Apoeh/hLdlr–/–) were compared to Apoe–/–Ldlr–/– mice. Despite 4-fold more plasma apoE than WT mice, Apoeh/hLdlr–/– mice displayed similar plasma cholesterol as Apoe–/– Ldlr–/– mice but developed 4-fold less atherosclerotic lesions by 5 months of age. The aortic arch of Apoeh/hLdlr–/– mice showed decreased endothelial expression of ICAM-1, PECAM-1, and JAM-A. In addition, Apoeh/hLdlr–/– mice had less circulating leukocytes and proinflammatory Ly6Chigh monocytes. These monocytes had decreased neutral lipid content and reduced surface expression of ICAM-1, VLA-4, and L-Selectin. Apoeh/hLdlr–/– mice displayed increased levels of apoA1-rich HDL that were potent in promoting cellular cholesterol efflux. Conclusions— Our findings suggest that apoE reduces atherosclerosis in the setting of hyperlipidemia by increasing plasma apoA1-HDL that likely contribute to reduce intracellular lipid accumulation and thereby the activation of circulating leukocytes and the vascular endothelium.

Interleukin-17A Deficiency Accelerates Unstable Atherosclerotic Plaque Formation in Apolipoprotein E-Deficient Mice [Integrative Biology/Experimental Medicine]

Objective— Interleukin(IL)-17A, an inflammatory cytokine, has been implicated in atherosclerosis, in which inflammatory cells within atherosclerotic plaques express IL-17A. However, its role in the development of atheroscelrosis remains to be controversial. Methods and Results— To directly examine the role of IL-17A in atherosclerosis, we generated apolipoprotein E (ApoE)/IL-17A double-deficient (ApoE–/–IL-17A–/–) mice. Mice were fed with high-fat diet (HFD) for either 8 or 16 weeks, both starting at ages of 6 to 8 weeks. We found that splenic CD4+ T-cells produced high amounts of IL-17A in ApoE–/– mice after HFD feeding for 8 weeks. Atherosclerosis was significantly accelerated in HFD-fed ApoE–/–IL-17A–/– mice compared with ApoE–/– mice. Splenic CD4+ T-cells of ApoE–/–IL-17A–/– mice after HFD feeding for 8 weeks, but not for 16 weeks, exhibited increased interferon gamma and decreased IL-5 production. Importantly, formation of vulnerable plaque as evidenced by reduced numbers of vascular smooth muscle cells and reduced type I collagen deposition in the plaque was detected in ApoE–/–IL-17A–/– mice after HFD feeding for 8 weeks. Conclusion— These results suggest that IL-17A regulates the early phase of atherosclerosis development after HFD feeding and plaque stability, at least partly if not all by modulating interferon gamma and IL-5 production from CD4+ T-cells.

Transsignaling of Interleukin-6 Crucially Contributes to Atherosclerosis in Mice [Integrative Biology/Experimental Medicine]

Objective— Transsignaling of interleukin (IL)-6 is a central pathway in the pathogenesis of disorders associated with chronic inflammation, such as Crohn disease, rheumatoid arthritis, and inflammatory colon cancer. Notably, IL-6 also represents an independent risk factor for coronary artery disease (CAD) in humans and is crucially involved in vascular inflammatory processes. Methods and Results— In the present study, we showed that treatment with a fusion protein of the natural IL-6 transsignaling inhibitor soluble glycoprotein 130 (sgp130) and IgG1-Fc (sgp130Fc) dramatically reduced atherosclerosis in hypercholesterolemic Ldlr–/– mice without affecting weight gain and serum lipid levels. Moreover, sgp130Fc treatment even led to a significant regression of advanced atherosclerosis. Mechanistically, endothelial activation and intimal smooth muscle cell infiltration were decreased in sgp130Fc-treated mice, resulting in a marked reduction of monocyte recruitment and subsequent atherosclerotic plaque progression. Of note, patients with CAD exhibited significantly lower plasma levels of endogenous sgp130, suggesting that a compromised counterbalancing of IL-6 transsignaling may contribute to atherogenesis in humans. Conclusion— These data clarify, for the first time, the critical involvement of, in particular, the transsignaling of IL-6 in CAD and warrant further investigation of sgp130Fc as a novel therapeutic for the treatment of CAD and related diseases.

Xanthine Oxidoreductase Is Involved in Macrophage Foam Cell Formation and Atherosclerosis Development [Integrative Biology/Experimental Medicine]

Objective— Hyperuricemia is common in patients with metabolic syndrome. We investigated the role of xanthine oxidoreductase (XOR) in atherosclerosis development, and the effects of the XOR inhibitor allopurinol on this process. Methods and Results— Oral administration of allopurinol to ApoE knockout mice markedly ameliorated lipid accumulation and calcification in the aorta and aortic root. In addition, allopurinol treatment or siRNA-mediated gene knockdown of XOR suppressed transformation of J774.1 murine macrophage cells, treated with acetylated LDL or very low density lipoprotein (VLDL) into foam cells. This inhibitory effect of allopurinol was also observed in primary cultured human macrophages. In contrast, overexpression of XOR promoted transformation of J774.1 cells into foam cells. Interestingly, SR-A1, SR-B1, SR-B II, and VLDL receptors in J774.1 cells were reduced by XOR knockdown, and increased by XOR overexpression. Conversely, expressions of ABCA1 and ABCG1 were increased by XOR knockdown and suppressed by XOR overexpression. Finally, productions of inflammatory cytokines accompanied by foam cell formation were also reduced by allopurinol administration. Conclusion— These results strongly suggest XOR activity and/or its expression level to contribute to macrophage foam cell formation. Thus, XOR inhibitors may be useful for preventing atherosclerosis.

Pharmacological Suppression of Hepcidin Increases Macrophage Cholesterol Efflux and Reduces Foam Cell Formation and Atherosclerosis [Integrative Biology/Experimental Medicine]

Objective— We recently reported that lowering of macrophage free intracellular iron increases expression of cholesterol efflux transporters ABCA1 and ABCG1 by reducing generation of reactive oxygen species. In this study, we explored whether reducing macrophage intracellular iron levels via pharmacological suppression of hepcidin can increase macrophage-specific expression of cholesterol efflux transporters and reduce atherosclerosis. Methods and Results— To suppress hepcidin, increase expression of the iron exporter ferroportin, and reduce macrophage intracellular iron, we used a small molecule inhibitor of bone morphogenetic protein (BMP) signaling, LDN 193189 (LDN). LDN (10 mg/kg IP b.i.d.) was administered to mice, and its effects on atherosclerosis, intracellular iron, oxidative stress, lipid efflux, and foam cell formation were measured in plaques and peritoneal macrophages. Long-term LDN administration to apolipoprotein E–/– mice increased ABCA1 immunoreactivity within intraplaque macrophages by 3.7-fold (n=8; P=0.03), reduced Oil Red O–positive lipid area by 50% (n=8; P=0.02), and decreased total plaque area by 43% (n=8; P=0.001). LDN suppressed liver hepcidin transcription and increased macrophage ferroportin, lowering intracellular iron and hydrogen peroxide production. LDN treatment increased macrophage ABCA1 and ABCG1 expression, significantly raised cholesterol efflux to ApoA-1, and decreased foam cell formation. All preceding LDN-induced effects on cholesterol efflux were reversed by exogenous hepcidin administration, suggesting modulation of intracellular iron levels within macrophages as the mechanism by which LDN triggers these effects. Conclusion— These data suggest that pharmacological manipulation of iron homeostasis may be a promising target to increase macrophage reverse cholesterol transport and limit atherosclerosis.

G Protein-Coupled Receptor Kinase-5 Attenuates Atherosclerosis by Regulating Receptor Tyrosine Kinases and 7-Transmembrane Receptors [Integrative Biology/Experimental Medicine]

Objective— G protein–coupled receptor kinase-5 (GRK5) is a widely expressed Ser/Thr kinase that regulates several atherogenic receptors and may activate or inhibit nuclear factor-B (NF-B). This study sought to determine whether and by what mechanisms GRK5 affects atherosclerosis. Methods and Results— Grk5–/–/Apoe–/– mice developed 50% greater aortic atherosclerosis than Apoe–/– mice and demonstrated greater proliferation of macrophages and smooth muscle cells (SMCs) in atherosclerotic lesions. In Apoe–/– mice, carotid interposition grafts from Grk5–/– mice demonstrated greater upregulation of cell adhesion molecules than grafts from wild-type mice and, subsequently, more atherosclerosis. By comparing Grk5–/– with wild-type cells, we found that GRK5 desensitized 2 key atherogenic receptor tyrosine kinases: the platelet-derived growth factor receptor-β in SMCs, by augmenting ubiquitination/degradation; and the colony-stimulating factor-1 receptor (CSF-1R) in macrophages, by reducing CSF-1-induced tyrosyl phosphorylation. GRK5 activity in monocytes also reduced migration promoted by the 7-transmembrane receptor for monocyte chemoattractant protein-1 CC chemokine receptor-2. Whereas GRK5 diminished NF-B-dependent gene expression in SMCs and endothelial cells, it had no effect on NF-B activity in macrophages. Conclusion— GRK5 attenuates atherosclerosis through multiple cell type-specific mechanisms, including reduction of SMC and endothelial cell NF-B activity and desensitization of receptor-specific signaling through the monocyte CC chemokine receptor-2, macrophage CSF-1R, and the SMC platelet-derived growth factor receptor-β.

Inhibitor of Differentiation-3 Mediates High Fat Diet-Induced Visceral Fat Expansion [Integrative Biology/Experimental Medicine]

Objective— Inhibitor of differentiation-3 (Id3) has been implicated in promoting angiogenesis, a key determinant of high-fat diet (HFD)-induced visceral adiposity. Yet the role of Id3 in HFD-induced angiogenesis and visceral adipose expansion is unknown. Methods and Results— Id3–/– mice demonstrated a significant attenuation of HFD-induced visceral fat depot expansion compared to wild type littermate controls. Importantly, unlike other Id proteins, loss of Id3 did not affect adipose depot size in young mice fed chow diet or differentiation of adipocytes in vitro or in vivo. Contrast enhanced ultrasound revealed a significant attenuation of visceral fat microvascular blood volume in HFD-fed mice null for Id3 compared to wild type controls. HFD induced Id3 and VEGFA expression in the visceral stromal vascular fraction and Id3–/– mice had significantly lower levels of VEGFA protein in visceral adipose tissue compared to wild type. Furthermore, HFD-induced VEGFA expression in visceral adipose tissue was completely abolished by loss of Id3. Consistent with this effect, Id3 abolished E12-mediated repression of VEGFA promoter activity. Conclusion— Results identify Id3 as an important regulator of HFD-induced visceral adipose VEGFA expression, microvascular blood volume, and depot expansion. Inhibition of Id3 may have potential as a therapeutic strategy to limit visceral adiposity.

Resolution of Mitochondrial Oxidative Stress Rescues Coronary Collateral Growth in Zucker Obese Fatty Rats [Integrative Biology/Experimental Medicine]

Objective— We have previously found abrogated ischemia-induced coronary collateral growth in Zucker obese fatty (ZOF) rats compared with Zucker lean (ZLN) rats. Because ZOF rats have structural abnormalities in their mitochondria suggesting dysfunction and also show increased production of O2macr;, we hypothesized that mitochondrial dysfunction caused by oxidative stress impairs coronary collateral growth in ZOF. Methods and Results— Increased levels of reactive oxygen species were observed in aortic endothelium and smooth muscle cells in ZOF rats compared with ZLN rats. Reactive oxygen species levels were decreased by the mitochondria-targeted antioxidants MitoQuinone (MQ) and MitoTempol (MT) as assessed by MitoSox Red and dihydroethidine staining. Lipid peroxides (a marker of oxidized lipids) were increased in ZOF by 47% compared with ZLN rats. The elevation in oxidative stress was accompanied by increased antioxidant enzymes, except glutathione peroxidase-1, and by increased uncoupling protein-2 in ZOF versus ZLN rats. In addition, elevated respiration rates were also observed in the obese compared with lean rats. Administration of MQ significantly normalized the metabolic profiles and reduced lipid peroxides in ZOF rats to the same level observed in lean rats. The protective effect of MQ also suppressed the induction of uncoupling protein-2 in the obese rats. Resolution of mitochondrial oxidative stress by MQ or MT restored coronary collateral growth to the same magnitude observed in ZLN rats in response to repetitive ischemia. Conclusion— We conclude that mitochondrial oxidative stress and dysfunction play a key role in disrupting coronary collateral growth in obesity and the metabolic syndrome, and elimination of the mitochondrial oxidative stress with MQ or MT rescues collateral growth.

Calpains Contribute to Vascular Repair in Rapidly Progressive Form of Glomerulonephritis: Potential Role of Their Externalization [Integrative Biology/Experimental Medicine]

Objective— Calpains, calcium-activated proteases, mediate the angiogenic signals of vascular endothelial growth factor. However, their involvement in vascular repair has not been investigated and the underlying mechanisms remain to be fully elucidated. Methods and Results— A rapidly progressive form of glomerulonephritis in wild type and transgenic mice expressing high levels of calpastatin, a calpain-specific inhibitor, was studied. Calpastatin transgene expression prevented the repair of peritubular capillaries and the recovery of renal function, limiting mouse survival. In vitro analysis detected a significant reduction of both intracellular and extracellular calpain activities in transgene expressing cells, whereas Western blotting revealed that proangiogenic factors vascular endothelial growth factor and norepinephrine increased calpain exteriorization. In vitro, extracellular calpains increased endothelial cell proliferation, migration and capillary tube formation. In vivo, delivery of nonpermeable extracellular calpastatin was sufficient to blunt angiogenesis and vascular repair. Endothelial cell response to extracellular calpains was associated with fibronectin cleavage, generating fibronectin fragments with proangiogenic capacity. In vivo, fibronectin cleavage was limited in the kidney of calpastatin transgenic mice with nephritis. Conclusion— This study demonstrates that externalized calpains participate in angiogenesis and vascular repair, partly by promoting fibronectin cleavage and thereby amplifying vascular endothelial growth factor efficiency. Thus, manipulation of calpain externalization may have therapeutic implications to control angiogenesis.

Effect of Ionizing Radiation Induced Damage of Endothelial Progenitor Cells in Vascular Regeneration [Integrative Biology/Experimental Medicine]

Objective— A number of studies have revealed that stress signaling and subsequent stress responses in stem/progenitor cells are responsible for attenuated regeneration or degenerative disease. Because ionizing radiation (IR), which sensitizes diverse types of stem cells, reportedly induces cardio-circulatory diseases, we hypothesized that IR-induced vascular abnormalities are associated with defects in endothelial progenitor cells (EPCs) that are responsible for vascular homeostasis. Methods and Results— We used an irradiated mouse model to mimic the IR effect on vasculogenesis. Mouse EPCs isolated from irradiated mice and human EPCs exposed to IR were used for functional analysis and gene expression study. Under IR exposure, EPCs were depleted, and their function for vasculogenesis in vitro and in vivo was significantly reduced. In such IR-mediated stress responses, upregulating p21Cip1 and downregulating vascular endothelial growth factor (VEGF) were mediated by p53 transcriptional activity. Conclusion— The results of the present study suggest that suppression of p53 would be clinically applicable to (1) minimize the functional defects in EPCs in order to prevent the onset of vascular diseases caused by radiation therapy or radiation exposure and also to (2) provide novel insight into the mechanisms of IR-induced vascular damage and a possible strategy to minimize vascular damage by IR.

Reperfusion Injury Intensifies the Adaptive Human T Cell Alloresponse in a Human-Mouse Chimeric Artery Model [Integrative Biology/Experimental Medicine]

Objective— Perioperative nonimmune injuries to an allograft can decrease graft survival. We have developed a model for studying this process using human materials. Methods and Results— Human artery segments were transplanted as infrarenal aortic interposition grafts into an immunodeficient mouse host, allowed to "heal in" for 30 days, and then retransplanted into a second mouse host. To induce a reperfusion injury, the healed-in artery segments were incubated for 3 hours under hypoxic conditions ex vivo before retransplantation. To induce immunologic rejection, the animals receiving the retransplanted artery segment were adoptively transferred with human peripheral blood mononuclear cells or purified T cells from a donor allogeneic to the artery 1 week before surgery. To compare rejection of injured versus healthy tissues, these manipulations were combined. Results were analyzed ex vivo by histology, morphometry, immunohistochemistry, and mRNA quantitation or in vivo by ultrasound. Our results showed that reperfusion injury, which otherwise heals with minimal sequelae, intensifies the degree of allogeneic T cell–mediated injury to human artery segments. Conclusion— We developed a new human-mouse chimeric model demonstrating interactions of reperfusion injury and alloimmunity using human cells and tissues that may be adapted to study other forms of nonimmune injury and other types of adaptive immune responses.

Transforming Growth Factor-{beta}-Induced Endothelial-to-Mesenchymal Transition Is Partly Mediated by MicroRNA-21 [Integrative Biology/Experimental Medicine]

Objective— MicroRNAs are a class of small ribonucleotides regulating gene/protein targets by transcript degradation or translational inhibition. Transforming growth factor-β (TGF-β) is involved in cardiac fibrosis partly by stimulation of endothelial-to-mesenchymal transition (EndMT). Here, we investigated whether microRNA (miR)-21, a microRNA enriched in fibroblasts and involved in general fibrosis, has a role in cardiac EndMT. Methods and Results— TGF-β treatment of endothelial cells significantly increased miR-21 expression and induced EndMT characterized by suppression of endothelial and increase of fibroblast markers. Overexpression of miR-21 alone also stimulated EndMT. Importantly, miR-21 blockade by transfection of specific microRNA inhibitors partly prevented TGF-β-induced EndMT. Mechanistically, miR-21 silenced phosphatase and tensin homolog in endothelial cells, resulting in activation of the Akt-pathway. Akt inhibition partly restored TGF-β-mediated loss of endothelial markers during EndMT. In vivo, pressure overload of the left ventricle led to increased expression of miR-21 in sorted cardiac endothelial cells, which displayed molecular and phenotypic signs of EndMT. This was attenuated by treatment of mice subjected to left ventricular pressure overload with an antagomir against miR-21. Conclusion— TGF-β-mediated EndMT is regulated at least in part by miR-21 via the phosphatase and tensin homolog/Akt pathway. In vivo, antifibrotic effects of miR-21 antagonism are partly mediated by blocking EndMT under stress conditions.

Dynamic Synchrotron Imaging of Diabetic Rat Coronary Microcirculation In Vivo [Integrative Biology/Experimental Medicine]

Objective— In diabetes, long-term micro- and macrovascular damage often underlies the functional decline in the cardiovascular system. However, it remains unclear whether early-stage diabetes is associated with in vivo functional impairment in the coronary microvasculature. Synchrotron imaging allows us to detect and quantify regional differences in resistance microvessel caliber in vivo, even under conditions of high heart rate. Methods and Results— Synchrotron cine-angiograms of the coronary vasculature were recorded using anesthetized Sprague-Dawley rats 3 weeks after treatment with vehicle or streptozotocin (diabetic). In the early diabetic state, in the presence of nitric oxide and prostacyclin, vessel diameters were smaller (P<0.01) and endothelium-dependent vessel recruitment was already depressed (P<0.05). Endothelium-dependent and -independent vasodilatory responses in individual coronary vessels were not different in vivo. Inhibition of NO and PGI2 production in diabetes uncovered early localized impairment in dilation. Diabetic animals displayed focal stenoses and segmental constrictions during nitric oxide synthase/cyclooxygenase blockade, which persisted during acetylcholine infusion (P<0.05), and a strong trend toward loss of visible microvessels. Conclusion— Synchrotron imaging provides a novel method to investigate coronary microvascular function in vivo at all levels of the arterial tree. Furthermore, we have shown that early-stage diabetes is associated with localized coronary microvascular endothelial dysfunction.

The Proteoglycan Syndecan 4 Regulates Transient Receptor Potential Canonical 6 Channels via RhoA/Rho-associated Protein Kinase Signaling [Integrative Biology/Experimental Medicine]

Objective— Syndecan 4 (Sdc4) modulates signal transduction and regulates activity of protein channels. Sdc4 is essential for the regulation of cellular permeability. We hypothesized that Sdc4 may regulate transient receptor potential canonical 6 (TRPC6) channels, a determinant of glomerular permeability, in a RhoA/Rho-associated protein kinase-dependent manner. Methods and Results— Sdc4 knockout (Sdc4–/–) mice showed increased glomerular filtration rate and ameliorated albuminuria under baseline conditions and after bovine serum albumin overload (each P<0.05). Using reverse transcription–polymerase chain reaction and immunoblotting, Sdc4–/– mice showed reduced TRPC6 mRNA by 79% and TRPC6 protein by 82% (each P<0.05). Sdc4–/– mice showed an increased RhoA activity by 87% and increased phosphorylation of ezrin in glomeruli by 48% (each P<0.05). Sdc4 knockdown in cultured podocytes reduced TRPC6 gene expression and reduced the association of TRPC6 with plasma membrane and TRPC6-mediated calcium influx and currents. Sdc4 knockdown inactivated negative regulatory protein Rho GTPase activating protein by 33%, accompanied by a 41% increase in RhoA activity and increased phosphorylation of ezrin (P<0.05). Conversely, overexpression of Sdc4 reduced RhoA activity and increased TRPC6 protein and TRPC6-mediated calcium influx and currents. Conclusion— Our results establish a previously unknown function of Sdc4 for regulation of TRPC6 channels and support the role of Sdc4 for the regulation of glomerular permeability.

Syndecan-1 Displays a Protective Role in Aortic Aneurysm Formation by Modulating T Cell-Mediated Responses [Integrative Biology/Experimental Medicine]

Objective— Chronic inflammation drives progressive and pathological remodeling inherent to formation of abdominal aortic aneurysm (AAA). Syndecan-1 (Sdc-1) is a cell surface heparan sulfate proteoglycan that displays the capacity to modulate inflammatory processes within the vascular wall. In the current investigation, the role of Sdc-1 in AAA formation was examined using 2 models of experimental aneurysm induction, angiotensin II infusion and elastase perfusion. Methods and Results— Sdc-1 deficiency exacerbated AAA formation in both experimental models and was associated with increased degradation of elastin, greater protease activity, and enhanced inflammatory cell recruitment into the aortic wall. Bone marrow transplantation studies indicated that deficiency of Sdc-1 in marrow-derived cells significantly contributed to AAA severity. Immunostaining revealed augmented Sdc-1 expression in a subset of AAA localized macrophages. We specifically characterized a higher percentage of CD4+ T cells in Sdc-1-deficient AAA, and antibody depletion studies established the active role of T cells in aneurysmal dilatation. Finally, we confirmed the ability of Sdc-1 macrophage to modulate the inflammatory chemokine environment. Conclusion— These investigations identify cross-talk between Sdc-1-expressing macrophages and AAA-localized CD4+ T cells, with Sdc-1 providing an important counterbalance to T-cell–driven inflammation in the vascular wall.

Regulation of Peroxisome Proliferator-Activated Receptor-{gamma} by Angiotensin II Via Transforming Growth Factor-{beta}1-Activated p38 Mitogen-Activated Protein Kinase in Aortic Smooth Muscle Cells [Cell Biology/Signaling]

Objective— Peroxisome proliferator–activated receptor- (PPAR) ligands attenuate angiotensin II (Ang II)–induced atherosclerosis through interactions with vascular smooth muscle cell (VSMC)–specific PPAR in hypercholesterolemic mice. Therefore, the purpose of this study was to determine the mechanism of Ang II–mediated intracellular regulation of PPAR in VSMCs. Methods and Results— Incubation of cultured mouse aortic VSMCs with Ang II for 24 hours reduced abundance of PPAR protein, mRNA, and transcriptional activity (P<0.001). This effect was attenuated by an angiotensin type 1 receptor antagonist, losartan. Ang II–induced PPAR reduction was dependent on stimulation of transforming growth factor (TGF)-β1 as demonstrated using either a neutralizing antibody or small interfering RNA (siRNA). Ang II–induced TGF-β1 secretion was dependent on epidermal growth factor receptor kinase activation through reactive oxygen species production. Inhibition of p38 mitogen-activated protein kinase by SB203580 or siRNA inhibited both Ang II– and TGF-β1–induced PPAR reduction. Blockade of TGF-β1 decreased p38 phosphorylation induced by Ang II. siRNA–mediated inhibition of histone deacetylase 3 attenuated p38-mediated reductions in PPAR abundance. Conclusion— These findings suggest that Ang II decreases PPAR abundance in cultured VSMCs via an angiotensin type 1 receptor–dependent secretion of TGF-β1 via phosphorylation of p38 mitogen-activated protein kinase and histone deacetylase 3.

Fatty Acid-Induced Nuclear Translocation of Heparanase Uncouples Glucose Metabolism in Endothelial Cells [Cell Biology/Signaling]

Objective— Heparanase is an endoglycosidase that specifically cleaves carbohydrate chains of heparan sulfate. We have recently reported that high fatty acid increased the nuclear content of endothelial heparanase. Here, we examined the mechanism and the consequences behind this nuclear translocation of heparanase. Methods and Results— Bovine coronary artery endothelial cells were grown to confluence and incubated with palmitic acid. Palmitic acid induced rapid nuclear accumulation of heparanase that was dependent on Bax activation and lysosome permeabilization. Heat shock protein 90 was an important mediator of palmitic acid-induced shuttling of heparanase to the nucleus. Nuclear heparanase promoted cleavage of heparan sulfate, a potent inhibitor of histone acetyltransferase activity and gene transcription. A TaqMan gene expression assay revealed an increase in genes related to glucose metabolism and inflammation. In addition, glycolysis was uncoupled from glucose oxidation, resulting in accumulation of lactate. Conclusion— The results presented in this study demonstrate that fatty acid can provoke lysosomal release of heparanase, its nuclear translocation, activation of genes controlling glucose metabolism, and accumulation of lactate. Given that lactate and inflammation have been implicated in the progression of atherosclerosis, our data may serve to reduce the associated cardiovascular complications seen during diabetes.

NADPH Oxidase 4 Mediates Monocyte Priming and Accelerated Chemotaxis Induced by Metabolic Stress [Cell Biology/Signaling]

Objective— Metabolic disorders increase monocyte chemoattractant protein-1 (MCP-1)-induced monocyte chemotaxis in mice. The goal of this study was to determine the molecular mechanisms responsible for the enhanced responsiveness of monocytes to chemoattractants induced by metabolic stress. Methods and Results— Chronic exposure of monocytes to diabetic conditions induced by human LDL plus high D-glucose concentrations (LDL+HG) promoted NADPH Oxidase 4 (Nox4) expression, increased intracellular H2O2 formation, stimulated protein S-glutathionylation, and increased chemotaxis in response to MCP-1, platelet-derived growth factor B, and RANTES. Both H2O2 added exogenously and overexpression of Nox4 mimicked LDL+HG-induced monocyte priming, whereas Nox4 knockdown protected monocytes against metabolic stress-induced priming and accelerated chemotaxis. Exposure of monocytes to LDL+HG promoted the S-glutathionylation of actin, decreased the F-actin/G-actin ratio, and increased actin remodeling in response to MCP-1. Preventing LDL+HG-induced protein S-glutathionylation by overexpressing glutaredoxin 1 prevented monocyte priming and normalized monocyte chemotaxis in response to MCP-1. Induction of hypercholesterolemia and hyperglycemia in C57BL/6 mice promoted Nox4 expression and protein S-glutathionylation in macrophages, and increased macrophage recruitment into MCP-1–loaded Matrigel plugs implanted subcutaneous in these mice. Conclusion— By increasing actin-S-glutathionylation and remodeling, metabolic stress primes monocytes for chemoattractant-induced transmigration and recruitment to sites of vascular injury. This Nox4-dependent process provides a novel mechanism through which metabolic disorders promote atherogenesis.

Factor VII-Activating Protease Promotes the Proteolysis and Inhibition of Tissue Factor Pathway Inhibitor [Cell Biology/Signaling]

Objective— Factor VII–activating protease (FSAP) activates both factor VII and pro-urokinase and inhibits platelet-derived growth factor-BB, thus regulating hemostasis- and remodeling-associated processes in the vasculature. A genetic variant of FSAP (Marburg I polymorphism) results in low enzymatic activity and is associated with an enhanced risk of carotid stenosis and stroke. We postulate that there are additional substrates for FSAP that will help to explain its role in vascular biology and have searched for such a substrate. Methods and Results— Using screening procedures to determine the influence of FSAP on various hemostasis-related processes on endothelial cells, we discovered that FSAP inhibited tissue factor pathway inhibitor (TFPI), a major anticoagulant secreted by these cells. Proteolytic degradation of TFPI by FSAP could also be demonstrated by Western blotting, and the exact cleavage sites were determined by N-terminal sequencing. The Marburg I variant of FSAP had a diminished ability to inhibit TFPI. A monoclonal antibody to FSAP that specifically inhibited FSAP binding to TFPI reversed the inhibitory effect of FSAP on TFPI. Conclusion— The identification of TFPI as a sensitive substrate for FSAP increases our understanding of its role in regulating hemostasis and proliferative remodeling events in the vasculature.

Rap1-Rac1 Circuits Potentiate Platelet Activation [Cell Biology/Signaling]

Objective— The goal of this study was to investigate the potential crosstalk between Rap1 and Rac1, 2 small GTPases central to platelet activation, particularly downstream of the collagen receptor GPVI. Methods and Results— We compared the activation response of platelets with impaired Rap signaling (double knock-out; deficient in both the guanine nucleotide exchange factor, CalDAG-GEFI, and the Gi-coupled receptor for ADP, P2Y12), to that of wild-type platelets treated with a small-molecule Rac inhibitor, EHT 1864 (wild-type /EHT). We found that Rac1 is sequentially activated downstream of Rap1 on stimulation via GPVI. In return, Rac1 provides important feedback for both CalDAG-GEFI– and P2Y12-dependent activation of Rap1. When analyzing platelet responses controlled by Rac1, we observed (1) impaired lamellipodia formation, clot retraction, and granule release in both double knock-out and EHT 1864-treated wild-type platelets; and (2) reduced calcium store release in EHT 1864-treated wild-type but not double knock-out platelets. Consistent with the latter finding, we identified 2 pools of Rac1, one activated immediately downstream of GPVI and 1 activated downstream of Rap1. Conclusion— We demonstrate important crosstalk between Rap1 and Rac1 downstream of GPVI. Whereas Rap1 signaling directly controls sustained Rac1 activation, Rac1 affects CalDAG-GEFI– and P2Y12-dependent Rap1 activation via its role in calcium mobilization and granule/ADP release, respectively.

Deleted in Malignant Brain Tumors 1 is Present in the Vascular Extracellular Matrix and Promotes Angiogenesis [Cell Biology/Signaling]

Objective— Deleted in malignant brain tumors 1 (DMBT1) belongs to the scavenger receptor cysteine-rich superfamily of proteins and is implicated in innate immunity, cell polarity, and differentiation. Here we studied the role of DMBT1 in endothelial cells. Methods and Results— DMBT1 was secreted into the extracellular matrix (ECM) by endothelial cells in vitro and in situ and the presence of DMBT1 in the ECM increased endothelial cell adherence. Endothelial cell-derived DMBT1 associated with galectin-3 (coprecipitation), and human recombinant DMBT1 bound EGF, vascular endothelial growth factor and Delta-like (Dll) 4 (specific ELISAs). Compared to cells from wild-type mice, endothelial cells from DMBT1–/– mice demonstrated reduced migration, proliferation, and tube formation. In vivo recovery from hindlimb ischemia was attenuated in DMBT1–/– animals as was vascular endothelial growth factor -induced endothelial sprouting from isolated aortic rings; the latter response could be rescued by the addition of recombinant DMBT1. The Notch pathway is involved in multiple aspects of vascular development, including arterial-venous differentiation and we found that endothelial cells from DMBT1–/– mice expressed more EphrinB2 than cells from wild-type mice. Levels of Dll1, Dll4, Hes1, Hey1, and EphB4, on the other hand, were decreased. Conclusion— Taken together, the results of this study indicate that DMBT1 functions as an important endothelium-derived ECM protein that is able to bind angiogenic factors and promote adhesion, migration, proliferation, and angiogenesis as well as vascular repair. Mechanistically, DMBT1 interacts with galectin-3 and modulates the Notch signaling pathway as well as the differential expression of ephrin-B2 and EphB4.

Urokinase-Type Plasminogen Activator Downregulates Paraoxonase 1 Expression in Hepatocytes by Stimulating Peroxisome Proliferator-Activated Receptor-{gamma} Nuclear Export [Cell Biology/Signaling]

Objective— The atherosclerotic lesion is characterized by lipid peroxide accumulation. Paraoxonase 1 (PON1) reduces atherosclerotic lesion oxidative stress, whereas urokinase-type plasminogen activator (uPA) increases oxidative stress in atherosclerotic lesions and contributes to the progression and complications of atherosclerosis. We hypothesized that uPA may promote oxidative stress in the arterial wall via modulation of PON1 activity. Because the liver is the main site for PON1 production, in the present study, we tested whether uPA influences PON1 expression in hepatocytes. Methods and Results— HuH7 hepatocytes were incubated in culture with increasing concentrations of uPA. uPA decreased PON1 gene expression and activity in a dose-dependent manner and accordingly suppressed PON1 secretion from hepatocytes. This effect required uPA/uPA receptor interaction. uPA downregulated PON1 gene expression via inactivation of peroxisome proliferator–activated receptor- (PPAR) activity, and this effect was dependent on uPA-mediated mitogen-activated protein kinase kinase activation. Mechanistic studies showed that uPA enhanced mitogen-activated protein kinase kinase–PPAR interaction, resulting in PPAR nuclear export to the cytosol. Conclusion— This study provides the first evidence that uPA interferes with PPAR transcriptional activity in hepatocytes, resulting in downregulation of PON1 expression and its secretion to the medium. This may explain, at least in part, the prooxidative effect of uPA in the vascular wall.

Apolipoprotein B-100-Containing Lipoprotein Metabolism in Subjects With Lipoprotein Lipase Gene Mutations [Clinical and Population Studies]

Objective— We investigated the impact of lipoprotein lipase (LPL) gene mutations on apolipoprotein B (apoB)-100 metabolism. Methods and Results— We studied 3 subjects with familial LPL deficiency; 14 subjects heterozygous for the LPL gene mutations Gly188Glu, Trp64Stop, and Ile194Thr; and 10 control subjects. Very-low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), and low-density lipoprotein (LDL)-apoB-100 kinetics were determined in the fed state using stable isotope methods and compartmental modeling. Compared with controls, familial LPL deficiency had markedly elevated plasma triglycerides and lower VLDL-apoB-100 fractional catabolic rate (FCR), IDL-apoB-100 FCR, VLDL-to-IDL conversion, and VLDL-apoB-100 production rate (P<0.01). Compared with controls, Gly188Glu had higher plasma triglyceride and VLDL- and IDL-apoB-100 concentrations and lower VLDL- and IDL-apoB-100 FCR (P<0.05). Plasma triglycerides were not different, but IDL-apoB-100 concentration and production rate and VLDL-to-IDL conversion were lower in Trp64Stop compared with controls (P<0.05). No differences between controls and Ile194Thr were observed. Conclusion— Our results confirm that hypertriglyceridemia is a key feature of familial LPL deficiency. This is due to impaired VLDL- and IDL-apoB-100 catabolism and VLDL-to-IDL conversion. Single-allele mutations of the LPL gene result in modest to elevated plasma triglycerides. The changes in plasma triglycerides and apoB-100 kinetics are attributable to the effects of the LPL genotype.

Arteriolar Function in Visceral Adipose Tissue Is Impaired in Human Obesity [Clinical and Population Studies]

Objective— The purpose of this study was to characterize the relationship between adipose tissue phenotype and depot-specific microvascular function in fat. Methods and Results— In 30 obese subjects (age 42±11 years, body mass index 46±11 kg/m2) undergoing bariatric surgery, we intraoperatively collected visceral and subcutaneous adipose tissue and characterized depot-specific adipose phenotypes. We assessed vasomotor function of the adipose microvasculature using videomicroscopy of small arterioles (75–250 μm) isolated from different fat compartments. Endothelium-dependent, acetylcholine-mediated vasodilation was severely impaired in visceral arterioles, compared to the subcutaneous depot (P<0.001 by ANOVA). Nonendothelium dependent responses to papaverine and nitroprusside were similar. Endothelial nitric oxide synthase inhibition with N-nitro-l-arginine methyl ester reduced subcutaneous vasodilation but had no effect on severely blunted visceral arteriolar responses. Visceral fat exhibited greater expression of proinflammatory, oxidative stress-related, hypoxia-induced, and proangiogenic genes; increased activated macrophage populations; and had a higher capacity for cytokine production ex vivo. Conclusion— Our findings provide clinical evidence that the visceral microenvironment may be intrinsically toxic to arterial health providing a potential mechanism by which visceral adiposity burden is linked to atherosclerotic vascular disease. Our findings also support the evolving concept that both adipose tissue quality and quantity may play significant roles in shaping cardiovascular phenotypes in human obesity.

Preventive Effects of Exenatide on Endothelial Dysfunction Induced by Ischemia-Reperfusion Injury via KATP Channels [Clinical and Population Studies]

Objective— The purpose of this study was to evaluate whether exenatide administration can prevent impairment in endothelium-dependent vasodilatation induced by ischemia-reperfusion (IR) injury and whether this effect is mediated by KATP channel opening. Methods and Results— In a double-blind, placebo-controlled, crossover design, 20 volunteers were randomly assigned to 2 groups: subcutaneous exenatide (10 μg) or placebo administration. At 30 minutes after the study drug administration, endothelium-dependent flow-mediated dilatation (FMD) of the radial artery was measured before and after IR (15 minutes of ischemia at the level of the brachial artery followed by 15 minutes of reperfusion) injury. Seven days later, both groups were crossed over and received the other treatment (ie, placebo or exenatide) and underwent the same protocol. Pre-IR radial artery diameter, FMD, and baseline radial artery diameter after IR injury were similar between 2 groups (P=no significant difference). After placebo administration, IR significantly blunted FMD (before IR: 12.0±6.23%; after IR: 4.6±3.57%, P=0.02). Exenatide prevented this impairment (FMD before IR: 15.0±7.14%; FMD after IR: 15.0±5.96%, P=no significant difference; P<0.001 compared with placebo). In a separate protocol, this protective effect was completely abolished by pretreatment with glibenclamide (glyburide, 5 mg), a blocker of KATP channels (n=7; FMD before IR: 12.0±2.2%; after IR: 3.2±2.1%, P<0.001). Conclusion— The present study demonstrates that subcutaneous exenatide protects IR-induced endothelial dysfunction through opening of KATP channels in human IR injury model.

Association of Testosterone Levels With Endothelial Function in Men: Results From a Population-Based Study [Clinical and Population Studies]

Objective— Because population-based data are lacking, we assessed the cross-sectional association between serum testosterone levels and endothelial function, as measured by flow-mediated dilation (FMD) and nitroglycerin-mediated dilation (NMD) of the brachial artery, in men from the population-based Study of Health in Pomerania. Methods and Results— Personal characteristics, including major cardiovascular confounders, were collected in 722 men, aged 25 to 85 years. Serum total testosterone and sexual hormone-binding globulin (SHBG) levels were determined by chemiluminescence immunoassays. Free testosterone levels were calculated according to the law of mass action. FMD and NMD measurements were performed using standardized ultrasound techniques. FMD and NMD values below the 20th percentile were considered decreased. Multivariable logistic regression analyses revealed an association for each decrement of total testosterone standard deviation (6.0 nmol/L) with decreased FMD after adjustment for potential confounders (odds ratio 1.30, 95% confidence interval 1.04–1.63; P=0.023). Multiple adjusted findings for free testosterone were similar (odds ratio 1.37, 95% confidence interval 1.06–1.76; P=0.016). There was no such association of SHBG levels with decreased FMD. Neither testosterone nor SHBG levels were significantly associated with decreased NMD. Conclusion— Lower serum total and free testosterone levels are associated with impaired endothelial function in this population-based sample of men.

Competing Risk of Atherosclerotic Risk Factors for Arterial and Venous Thrombosis in a General Population: The Tromso Study [Clinical and Population Studies]

Objective— To investigate and compare the impact of traditional atherosclerotic risk factors for the risk of arterial and venous thrombosis, taking into account competing risks. Methods and Results— In 1994–1995, 26,185 subjects were screened in the Tromsø study. Information on traditional atherosclerotic risk factors was obtained by physical examination, blood samples, and questionnaires. Subjects were followed to the first incident event of myocardial infarction (MI) or venous thromboembolism (VTE), or December 31, 2005. During a median of 10.8 years of follow-up, there were 1279 cases of incident MI and 341 VTE events. Advancing age and high body mass index were both associated with MI and VTE. Hazard ratio per decade of age was 2.34 (95% CI: 2.25–2.43) for MI and 1.87 (1.74–2.01) for VTE, and 3 kg/m2 increase in body mass index was associated with 1.16 (1.11–1.21) and 1.20 (1.12–1.29) increased risk of MI and VTE, respectively. Blood pressure, high levels of triglycerides and total cholesterol, low HDL cholesterol, self-reported diabetes, and smoking were all associated with increased risk of MI but not associated with VTE. Conclusion— Our findings imply that traditional atherosclerotic risk factors, such as smoking, hypertension, dyslipidemia, and diabetes mellitus are not shared by arterial and venous thrombosis.

Relationships Between Recent Intraplaque Hemorrhage and Stroke Risk Factors in Patients With Carotid Stenosis: The HIRISC Study [Clinical and Population Studies]

Objective— Intraplaque hemorrhage (IPH) is an emerging marker of plaque instability. However, little is known about the relationships between IPH and traditional risk factors and whether these relationships differ between symptomatic and asymptomatic disease. Methods and Results— Two hundred thirty-four patients with symptomatic (n=114) or asymptomatic (n=120) carotid stenosis underwent high-resolution plaque magnetic resonance imaging. Seventy-five patients had recent IPH (symptomatic, 33%; asymptomatic, 31%). In symptomatic stenosis, recent IPH was independently associated with degree of stenosis (odds ratio [OR]=4.21, 1.61–10.98 for North American Symptomatic Carotid Endarterectomy Trial >35%; OR=2.92, 1.18–7.24 for European Carotid Surgery Trial >60%), qualifying event (OR=4.13; 1.11–15.32 for stroke or hemispheric transient ischemic attack ≥1 hour versus transient ischemic attack <1 hour or ocular symptoms), time from ischemic event (OR=6.65, 1.56–28.35 for ≤2 weeks; OR=2.24, 0.87–5.81 for 2–12 weeks versus >12 weeks; P for trend=0.03). In asymptomatic stenosis, IPH was only associated with stenosis severity >70% by ECST (OR=6.65; 1.95–22.73) but not by the NASCET method. Conclusion— Our findings support the potential link between recent IPH and risk of ipsilateral stroke in symptomatic disease but also imply that prognostic studies should adjust for known stroke risk factors in multivariate analyses. In asymptomatic stenosis, the potential predictive value of recent IPH is less likely to be confounded by stroke risk factors.

Objectively Assessed Physical Activity, Sedentary Time, and Coronary Artery Calcification in Healthy Older Adults [Clinical and Population Studies]

Objective— Physical activity is related to lower risk of cardiovascular disease, but data relating to coronary lesions have been conflicting. These inconsistencies may in part be due to unreliable assessment of physical activity and limitations imposed by self-reported data. The purpose of this study was to determine the relationship between objectively measured physical activity and coronary artery calcium (CAC). Methods and Results— Participants were 443 healthy men and women (mean age=66±6 years), without history or objective signs of coronary heart disease, drawn from the Whitehall II epidemiological cohort. Physical activity was objectively measured using accelerometers worn during waking hours for 7 consecutive days (average daily wear time=889±68 minutes/day). CAC was measured in each participant using electron beam computed tomography and was quantified according to the Agatston scoring system. On average, 54.4% of the sample recorded at least 30 minutes/day of moderate to vigorous physical activity (MVPA). There was no association between MVPA and presence of detectable CAC. For the participants with detectable CAC (n=283) a weak inverse relationship between MVPA (minutes/day) and log Agatston score was observed (B=–0.008, 95% CI: –0.16 to 0.00, P=0.05), although the association was no longer present after adjustments for age, sex, and conventional risk factors. No associations were seen for light activity or sedentary time. Conclusion— Our results confirm no association between objectively assessed physical activity and CAC. Because CAC measures cannot identify more vulnerable lesions, additional studies are required to examine whether physical activity can promote plaque stability.

Genetic Variation in ABCG1 and Risk of Myocardial Infarction and Ischemic Heart Disease [Clinical and Population Studies]

Objective— ATP binding cassette transporter G1 (ABCG1) facilitates cholesterol efflux from macrophages to mature high-density lipoprotein particles. Whether genetic variation in ABCG1 affects risk of atherosclerosis in humans remains to be determined. Methods and Results— We resequenced the core promoter and coding regions of ABCG1 in 380 individuals from the general population. Next, we genotyped 10 237 individuals from the Copenhagen City Heart Study for the identified variants and determined the effect on lipid and lipoprotein levels and on risk of myocardial infarction (MI) and ischemic heart disease (IHD). g.–376C>T, g.-311T>A, and Ser630Leu predicted risk of MI in the Copenhagen City Heart Study, with hazard ratios of 2.2 (95% confidence interval: 1.2–4.3), 1.7 (1.0–2.9), and 7.5 (1.9–30), respectively. These results were confirmed for g.–376C>T in a case-control study comprising 4983 independently ascertained IHD cases and 7489 controls. Expression levels of ABCG1 mRNA were decreased by approximately 40% in g.–376C>T heterozygotes versus noncarriers (probability values: 0.005–0.009). Finally, in vitro specificity protein 1 (Sp1) bound specifically to a putative Sp1 binding site at position –382 to –373 in the ABCG1 promoter, and the presence of the –376 T allele reduced binding and transactivation of the promoter by Sp1. Conclusion— This is the first report of a functional variant in ABCG1 that associates with increased risk of MI and IHD in the general population.

Clinical and Genetic Association of Serum Ceruloplasmin With Cardiovascular Risk [Clinical and Population Studies]

Objective— Ceruloplasmin (Cp) is an acute-phase reactant that is increased in inflammatory diseases and in acute coronary syndromes. Cp has recently been shown to possess nitric oxide (NO) oxidase catalytic activity, but its impact on long-term cardiovascular outcomes in stable cardiac patients has not been explored. Methods and Results— We examined serum Cp levels and their relationship with incident major adverse cardiovascular events (MACE; death, myocardial infarction [MI], stroke) over 3-year follow-up in 4177 patients undergoing elective coronary angiography. We also carried out a genome-wide association study to identify the genetic determinants of serum Cp levels and evaluate their relationship to prevalent and incident cardiovascular risk. In our cohort (age 63±11 years, 66% male, 32% history of MI, 31% diabetes mellitus), mean Cp level was 24±6 mg/dL. Serum Cp level was associated with greater risk of MI at 3 years (hazard ratio [quartile 4 versus 1] 2.35, 95% confidence interval [CI] 1.79–3.09, P<0.001). After adjustment for traditional risk factors, high-sensitivity C-reactive protein, and creatinine clearance, Cp remained independently predictive of MACE (hazard ratio 1.55, 95% CI 1.10–2.17, P=0.012). A 2-stage genome-wide association study identified a locus on chromosome 3 over the CP gene that was significantly associated with Cp levels (lead single-nucleotide polymorphism rs13072552; P=1.90x10–11). However, this variant, which leads to modestly increased serum Cp levels (1.5–2 mg/dL per minor allele copy), was not associated with coronary artery disease or future risk of MACE. Conclusion— In stable cardiac patients, serum Cp provides independent risk prediction of long-term adverse cardiac events. Genetic variants at the CP locus that modestly affect serum Cp levels are not associated with prevalent or incident risk of coronary artery disease in this study population.

Soluble Vascular Adhesion Protein-1 Correlates With Cardiovascular Risk Factors and Early Atherosclerotic Manifestations [Clinical and Population Studies]

Objective— Vascular adhesion protein-1 is an endothelial enzyme that regulates leukocyte traffic and contributes to vascular damage in animal models. The relations of soluble vascular adhesion protein-1 (sVAP-1) with cardiovascular risk factors and markers of subclinical atherosclerosis at a population level have not been studied. Methods and Results— We developed a new high-throughput method and measured sVAP-1 activities in serum of 2183 persons (The Cardiovascular Risk in Young Finns Study). In women, sVAP-1 activity correlated indirectly with body mass index (r=–0.15, P<0.0001), triglycerides (r=–0.13, P<0.0001), C-reactive protein (r=–0.23; P<0.0001), and brachial artery flow-mediated vasodilatation (r=–0.076, P=0.0089) and directly with carotid plaques (r=0.066, P=0.023). None of these correlations was significant in men. In women, all these univariate correlations remained significant after adjustment for body mass index, and direct correlations with LDL-cholesterol (r=0.094, P=0.0014) and carotid intima-media thickness (r=0.075, P=0.010) became evident. In men, sVAP-1 activity associated directly with glucose (r=0.074, P=0.020), intima-media thickness (r=0.072, P=0.025), metabolic syndrome (P=0.016), and type 1 (P=0.0002) and type 2 (P<0.0001) diabetes. In multivariable analyses, sVAP-1 activity was an independent determinant of carotid intima-media thickness (P=0.0072) and plaques [odds ratio 1.71 (95% confidence interval 1.07–2.72, P=0.025] in women, but not in men. Conclusion— sVAP-1 activity correlates directly with intima-media thickness and carotid plaques in general population and may play a role in the pathophysiology of preclinical atherosclerosis.

Incidence of Coronary Events and Case Fatality Rate in Relation to Blood Lymphocyte and Neutrophil Counts [Clinical and Population Studies]

Objective— Elevated levels of blood leukocytes have been associated with acute coronary events (CEs), but data on leukocyte subclasses are limited. This study aimed to explore whether blood lymphocyte and neutrophil counts are associated with incidence of CEs and with fatal outcome in subjects who subsequently experienced a first CE. Methods and Results— Neutrophil and lymphocyte counts were measured in 27 419 subjects from the general population without a history of CEs, heart failure, or atrial fibrillation. Incidence of CEs was studied in relation to leukocyte counts during a mean follow-up of 13.6 years. Neutrophil but not lymphocyte counts were significantly associated with incidence of CEs. After adjustments for confounding factors, the hazard ratios (95% confidence interval) were 1.00 (reference), 1.07 (0.94–1.23), 1.09 (0.95–1.25), and 1.39 (1.22–1.59) for subjects with neutrophils in the first, second, third, and fourth (highest) sex-specific quartiles, respectively (P for trend <0.001). Of the 1965 subject who had a CE, 471 subjects died on the first day of the CE, in- or outside hospital. The proportions of subjects who died the first day were 19%, 21%, 25%, and 28%, respectively in the first, second, third, and fourth quartiles (P for trend <0.001). Conclusion— Increased neutrophil counts are associated with incidence of CEs and increased case-fatality rate after a CE.

Circadian Variations of Infarct Size in STEM1 [Letters to the Editor]

Response to Ibanez et al [Letters to the Editor]

In This Issue [In This Issue]

When Silence is Broken: Polycomb Group Proteins in Heart Development [Editorials]

Nur77turing Macrophages in Atherosclerosis [Editorials]

Endonuclease G: The Link Between Mitochondria and Cardiac Hypertrophy? [Commentaries on Cutting Edge Science]

James Willerson: Leading the Texans' Fight Against Heart Disease [Profiles in Cardiovascular Science]

Vascular Bioactivation of Nitroglycerin Is Catalyzed by Cytosolic Aldehyde Dehydrogenase-2 [Molecular Medicine]

Rationale: According to general view, aldehyde dehydrogenase-2 (ALDH2) catalyzes the high-affinity pathway of vascular nitroglycerin (GTN) bioactivation in smooth muscle mitochondria. Despite having wide implications to GTN pharmacology and raising many questions that are still unresolved, mitochondrial bioactivation of GTN in blood vessels is still lacking experimental support. Objective: In the present study, we investigated whether bioactivation of GTN is affected by the subcellular localization of ALDH2 using immortalized ALDH2-deficient aortic smooth muscle cells and mouse aortas with selective overexpression of the enzyme in either cytosol or mitochondria. Methods and Results: Quantitative Western blotting revealed that ALDH2 is mainly cytosolic in mouse aorta and human coronary arteries, with only approximately 15% (mouse) and approximately 5% (human) of the enzyme being localized in mitochondria. Infection of ALDH2-deficient aortic smooth muscle cells or isolated aortas with adenovirus containing ALDH2 cDNA with or without the mitochondrial signal peptide sequence led to selective expression of the protein in mitochondria and cytosol, respectively. Cytosolic overexpression of ALDH2 restored GTN-induced relaxation and GTN denitration to wild-type levels, whereas overexpression in mitochondria (6-fold vs wild-type) had no effect on relaxation. Overexpression of ALDH2 in the cytosol of ALDH2-deficient aortic smooth muscle cells led to a significant increase in GTN denitration and cyclic GMP accumulation, whereas mitochondrial overexpression had no effect. Conclusions: The data indicate that vascular bioactivation of GTN is catalyzed by cytosolic ALDH2. Mitochondrial GTN metabolism may contribute to oxidative stress-related adverse effects of nitrate therapy and the development of nitrate tolerance.

High-Mobility Group A1 Protein: A New Coregulator of Peroxisome Proliferator-Activated Receptor-{gamma}-Mediated Transrepression in the Vasculature [Molecular Medicine]

Rationale: The nuclear receptor peroxisome proliferator–activated receptor- (PPAR) is an important regulator of gene transcription in vascular cells and mediates the vascular protection observed with antidiabetic glitazones. Objective: To determine the molecular mechanism of ligand-dependent transrepression in vascular smooth muscle cells and their impact on the vascular protective actions of PPAR. Methods and Results: Here, we report a molecular pathway in vascular smooth muscle cells by which ligand-activated PPAR represses transcriptional activation of the matrix-degrading matrix metalloproteinase-9 (MMP-9) gene, a crucial mediator of vascular injury. PPAR-mediated transrepression of the MMP-9 gene was dependent on the presence of the high-mobility group A1 (HMGA1) protein, a gene highly expressed in vascular smooth muscle cells, newly identified by oligonucleotide array expression analysis. Transrepression of MMP-9 by PPAR and regulation by HMGA1 required PPAR SUMOylation at K367. This process was associated with formation of a complex between PPAR, HMGA1, and the SUMO E2 ligase Ubc9 (ubiquitin-like protein SUMO-1 conjugating enzyme). After PPAR ligand stimulation, HMGA1 and PPAR were recruited to the MMP-9 promoter, which facilitated binding of SMRT (silencing mediator of retinoic acid and thyroid hormone receptor), a nuclear corepressor involved in transrepression. The relevance of HMGA1 for vascular PPAR signaling was underlined by the complete absence of vascular protection through a PPAR ligand in HMGA1–/– mice after arterial wire injury. Conclusions: The present data suggest that ligand-dependent formation of HMGA1-Ubc9-PPAR complexes facilitates PPAR SUMOylation, which results in the prevention of SMRT corepressor clearance and induction of MMP-9 transrepression. These data provide new information on PPAR-dependent vascular transcriptional regulation and help us to understand the molecular consequences of therapeutic interventions with PPAR ligands in the vasculature.

Polycomb Repressive Complex 2 Regulates Normal Development of the Mouse Heart [Cellular Biology]

Rationale: Epigenetic marks are crucial for organogenesis, but their role in heart development is poorly understood. Polycomb repressive complex 2 (PRC2) trimethylates histone H3 at lysine 27, which establishes H3K27me3 repressive epigenetic marks that promote tissue-specific differentiation by silencing ectopic gene programs. Objective: We studied the function of PRC2 in murine heart development using a tissue-restricted conditional inactivation strategy. Methods and Results: Inactivation of the PRC2 subunit Ezh2 by Nkx2–5Cre (Ezh2NK) caused lethal congenital heart malformations, namely, compact myocardial hypoplasia, hypertrabeculation, and ventricular septal defect. Candidate and genome-wide RNA expression profiling and chromatin immunoprecipitation analyses of Ezh2NK heart identified genes directly repressed by EZH2. Among these were the potent cell cycle inhibitors Ink4a/b (inhibitors of cyclin-dependent kinase 4 A and B), the upregulation of which was associated with decreased cardiomyocyte proliferation in Ezh2NK. EZH2-repressed genes were enriched for transcriptional regulators of noncardiomyocyte expression programs such as Pax6, Isl1, and Six1. EZH2 was also required for proper spatiotemporal regulation of cardiac gene expression, because Hcn4, Mlc2a, and Bmp10 were inappropriately upregulated in ventricular RNA. PRC2 was also required later in heart development, as indicated by cardiomyocyte-restricted TNT-Cre inactivation of the PRC2 subunit Eed. However, Ezh2 inactivation by TNT-Cre did not cause an overt phenotype, likely because of functional redundancy with Ezh1. Thus, early Ezh2 inactivation by Nk2–5Cre caused later disruption of cardiomyocyte gene expression and heart development. Conclusions: Our study reveals a previously undescribed role of EZH2 in regulating heart formation and shows that perturbation of the epigenetic landscape early in cardiogenesis has sustained disruptive effects at later developmental stages.

NR4A1 (Nur77) Deletion Polarizes Macrophages Toward an Inflammatory Phenotype and Increases Atherosclerosis [Cellular Biology]

Rationale: NR4A1 (Nur77) is a nuclear receptor that is expressed in macrophages and within atherosclerotic lesions, yet its function in atherosclerosis is unknown. Objective: Nur77 regulates the development of monocytes, particularly patrolling Ly6C– monocytes that may be involved in resolution of inflammation. We sought to determine how absence of nuclear receptor subfamily 4, group A, member 1 (NR4A1) in hematopoietic cells affected atherosclerosis development. Methods and Results: Nur77–/– chimeric mice on a Ldlr–/– background showed a 3-fold increase in atherosclerosis development when fed a Western diet for 20 weeks, despite having a drastic reduction in Ly6C– patrolling monocytes. In a second model, mice deficient in both Nur77 and ApoE (ApoE–/–Nur77–/–) also showed increased atherosclerosis after 11 weeks of Western diet. Atherosclerosis was associated with a significant change in macrophage polarization toward a proinflammatory phenotype, with high expression of tumor necrosis factor-α and nitric oxide and low expression of Arginase-I. Moreover, we found increased expression of toll-like receptor 4 mRNA and protein in Nur77–/– macrophages as well as increased phosphorylation of the p65 subunit of NFB. Inhibition of NFB activity blocked excess activation of Nur77–/– macrophages. Conclusions: We conclude that the absence of Nur77 in monocytes and macrophages results in enhanced toll-like receptor signaling and polarization of macrophages toward a proinflammatory M1 phenotype. Despite having fewer monocytes, Nur77–/– mice developed significant atherosclerosis when fed a Western diet. These studies indicate that Nur77 is a novel target for modulating the inflammatory phenotype of monocytes and macrophages and may be important for regulation of atherogenesis.

Bone Marrow-Specific Deficiency of Nuclear Receptor Nur77 Enhances Atherosclerosis [Cellular Biology]

Rationale: Nuclear receptor Nur77, also known as NR4A1, TR3, or NGFI-B, is expressed in human atherosclerotic lesions in macrophages, endothelial cells, T cells and smooth muscle cells. Macrophages play a critical role in atherosclerosis and the function of Nur77 in lesion macrophages has not yet been investigated. Objective: This study aims to delineate the function of Nur77 in macrophages and to assess the effect of bone marrow–specific deficiency of Nur77 on atherosclerosis. Methods and Results: We investigated Nur77 in macrophage polarization using bone marrow-derived macrophages (BMM) from wild-type and Nur77-knockout (Nur77–/–) mice. Nur77–/– BMM exhibit changed expression of M2-specific markers and an inflammatory M1-phenotype with enhanced expression of interleukin-12, IFN, and SDF-1α and increased NO synthesis in (non)-stimulated Nur77–/– BMM cells. SDF-1α expression in nonstimulated Nur77–/– BMM is repressed by Nur77 and the chemoattractive activity of Nur77–/– BMM is abolished by SDF-1α inhibiting antibodies. Furthermore, Nur77–/– mice show enhanced thioglycollate-elicited migration of macrophages and B cells. The effect of bone marrow–specific deficiency of Nur77 on atherosclerosis was studied in low density lipoprotein receptor-deficient (Ldlr–/–) mice. Ldlr–/– mice with a Nur77–/–-deficient bone marrow transplant developed 2.1-fold larger atherosclerotic lesions than wild-type bone marrow–transplanted mice. These lesions contain more macrophages, T cells, smooth muscle cells and larger necrotic cores. SDF-1α expression is higher in lesions of Nur77–/–-transplanted mice, which may explain the observed aggravation of lesion formation. Conclusions: In conclusion, in bone marrow–derived cells the nuclear receptor Nur77 has an anti-inflammatory function, represses SDF-1α expression and inhibits atherosclerosis.

Integrin-Linked Kinase Regulates Vasomotor Function by Preventing Endothelial Nitric Oxide Synthase Uncoupling: Role in Atherosclerosis [Cellular Biology]

Rationale: Atherosclerotic lesions develop in regions of disturbed flow, whereas laminar flow protects from atherogenesis; however, the mechanisms involved are not completely elucidated. Integrins are mechanosensors of shear stress in endothelial cells, and integrin-linked kinase (ILK) is important for blood vessel integrity and cardiovascular development. Objectives: To explore the role of ILK in vascular function by studying conditionally ILK-deficient (cKO) mice and human atherosclerotic arteries. Results: ILK expression was detected in the endothelial cell layer of nonatherosclerotic vessels but was absent from the endothelium of atherosclerotic arteries. Live ultrasound imaging revealed that acetylcholine-mediated vasodilatation was impaired in cKO mice. These mice exhibited lowered agonist-induced nitric oxide synthase (NOS) activity and decreased cyclic guanosine monophosphate and nitrite production. ILK deletion caused endothelial NOS (eNOS) uncoupling, reflected in reduced tetrahydrobiopterin (BH4) levels, increased BH2 levels, decreased dihydrofolate reductase expression, and increased eNOS-dependent generation of superoxide accompanied by extensive vascular protein nitration. ILK reexpression prevented eNOS uncoupling in cKO cells, whereas superoxide formation was unaffected by ILK depletion in eNOS-KO cells, indicating eNOS as a primary source of superoxide anion. eNOS and ILK coimmunoprecipitated in aortic lysates from control animals, and eNOS-ILK–shock protein 90 interaction was detected in human normal mammary arteries but was absent from human atherosclerotic carotid arteries. eNOS-ILK interaction in endothelial cells was prevented by geldanamycin, suggesting heat shock protein 90 as a binding partner. Conclusions: Our results identify ILK as a regulatory partner of eNOS in vivo that prevents eNOS uncoupling, and suggest ILK as a therapeutic target for prevention of endothelial dysfunction related to shear stress–induced vascular diseases.

Endosomal Actin Remodeling by Coronin-1A Controls Lipoprotein Uptake and Degradation in Macrophages [Cellular Biology]

Rationale: The actin cytoskeleton has been implicated in the processing of atherogenic lipoproteins in macrophages. However, the functional role of actin and the regulatory proteins involved are unknown. Objective: Coronin-1A (Coro1A) was identified as a differentially expressed transcript in wild-type versus Niemann-Pick type C1 deficient macrophages exposed to acetylated low-density lipoproteins (AcLDL). We investigated whether Coro1A plays a role in the uptake or processing of modified lipoproteins in macrophages and if this is related to its actin regulatory functions. Methods and Results: In wild-type primary macrophages, filamentous actin transiently decorated AcLDL containing endosomes that also recruited Coro1A. This dynamic association of F-actin with endosomes was disturbed in Coro1A deficient macrophages. In Coro1A knockout macrophages the uptake of AcLDL was increased, rate of AcLDL delivery to lysosomes enhanced, and lipoprotein-derived cholesteryl ester hydrolysis accelerated. Overexpression of wild-type Coro1A normalized AcLDL uptake in Coro1A knockout macrophages while a Coro1A actin binding mutant did not. Furthermore, the effects of macrophage Coro1A silencing on endosomal actin association and AcLDL delivery to lysosomes resembled those of cofilin silencing. Conclusions: Coro1A controls actin association with endocytic organelles, thereby negatively regulating endo-lysosomal delivery, degradation of modified lipoproteins and cholesterol deposition in macrophages.

Thymosin Beta 4 Is Dispensable for Murine Cardiac Development and Function [Cellular Biology]

Rationale: Thymosin beta 4 (Tβ4) is a 43–amino acid factor encoded by an X-linked gene. Recent studies have suggested that Tβ4 is a key factor in cardiac development, growth, disease, epicardial integrity, and blood vessel formation. Cardiac-specific short hairpin (sh)RNA knockdown of tβ4 has been reported to result in embryonic lethality at E14.5–16.5, with severe cardiac and angiogenic defects. However, this shRNA tβ4-knockdown model did not completely abrogate Tβ4 expression. To completely ablate Tβ4 and to rule out the possibility of off-target effects associated with shRNA gene silencing, further studies of global or cardiac-specific knockouts are critical. Objective: We examined the role of Tβ4 in developing and adult heart through global and cardiac specific tβ4-knockout mouse models. Methods and Results: Global tβ4-knockout mice were born at mendelian ratios and exhibited normal heart and blood vessel formation. Furthermore, in adult global tβ4-knockout mice, cardiac function, capillary density, expression of key cardiac fetal and angiogenic genes, epicardial marker expression, and extracellular matrix deposition were indistinguishable from that of controls. Tissue-specific tβ4-deficient mice, generated by crossing tβ4-floxed mice to Nkx2.5-Cre and αMHC-Cre, were also found to have no phenotype. Conclusions: We conclude that Tβ4 is dispensable for embryonic viability, heart development, coronary vessel development, and adult myocardial function.

Inhibition of CaMKII Phosphorylation of RyR2 Prevents Induction of Atrial Fibrillation in FKBP12.6 Knockout Mice [Integrative Physiology]

Rationale: Abnormal calcium release from sarcoplasmic reticulum (SR) is considered an important trigger of atrial fibrillation (AF). Whereas increased Ca2+/calmodulin-dependent protein kinase II (CaMKII) activity has been proposed to contribute to SR leak and AF induction, downstream targets of CaMKII remain controversial. Objective: To test the hypothesis that inhibition of CaMKII-phosphorylated type-2 ryanodine receptors (RyR2) prevents AF initiation in FKBP12.6-deficient (–/–) mice. Methods and Results: Mice lacking RyR2-stabilizing subunit FKBP12.6 had a higher incidence of spontaneous and pacing-induced AF compared with wild-type mice. Atrial myocytes from FKBP12.6–/– mice exhibited spontaneous Ca2+ waves (SCaWs) leading to Na+/Ca2+-exchanger activation and delayed afterdepolarizations (DADs). Mutation S2814A in RyR2, which inhibits CaMKII phosphorylation, reduced Ca2+ spark frequency, SR Ca2+ leak, and DADs in atrial myocytes from FKBP12.6–/–:S2814A mice compared with FKBP12.6–/– mice. Moreover, FKBP12.6–/–:S2814A mice exhibited a reduced susceptibility to inducible AF, whereas FKBP12.6–/–:S2808A mice were not protected from AF. Conclusions: FKBP12.6 mice exhibit AF caused by SR Ca2+ leak, Na+/Ca2+-exchanger activation, and DADs, which promote triggered activity. Genetic inhibition of RyR2-S2814 phosphorylation prevents AF induction in FKBP12.6–/– mice by suppressing SR Ca2+ leak and DADs. These results suggest suppression of RyR2-S2814 phosphorylation as a potential anti-AF therapeutic target.

H2O2-Induced Dilation in Human Coronary Arterioles: Role of Protein Kinase G Dimerization and Large-Conductance Ca2+-Activated K+ Channel Activation [Clinical/Translational Research]

Rationale: Hydrogen peroxide (H2O2) serves as a key endothelium-derived hyperpolarizing factor mediating flow-induced dilation in human coronary arterioles (HCAs). The precise mechanisms by which H2O2 elicits smooth muscle hyperpolarization are not well understood. An important mode of action of H2O2 involves the oxidation of cysteine residues in its target proteins, including protein kinase G (PKG)-Iα, thereby modulating their activities. Objective: Here we hypothesize that H2O2 dilates HCAs through direct oxidation and activation of PKG-Iα leading to the opening of the large-conductance Ca2+-activated K+ (BKCa) channel and subsequent smooth muscle hyperpolarization. Methods and Results: Flow and H2O2 induced pressure gradient/concentration-dependent vasodilation in isolated endothelium-intact and -denuded HCAs, respectively. The dilation was largely abolished by iberiotoxin, a BKCa channel blocker. The PKG inhibitor Rp-8-Br-PET-cGMP also markedly inhibited flow- and H2O2-induced dilation, whereas the soluble guanylate cyclase inhibitor ODQ had no effect. Treatment of coronary smooth muscle cells (SMCs) with H2O2 elicited dose-dependent, reversible dimerization of PKG-Iα, and induced its translocation to the plasma membrane. Patch-clamp analysis identified a paxilline-sensitive single-channel K+ current with a unitary conductance of 246-pS in freshly isolated coronary SMCs. Addition of H2O2 into the bath solution significantly increased the probability of BKCa single-channel openings recorded from cell-attached patches, an effect that was blocked by the PKG-Iα inhibitor DT-2. H2O2 exhibited an attenuated stimulatory effect on BKCa channel open probability in inside-out membrane patches. Conclusions: H2O2 dilates HCAs through a novel mechanism involving protein dimerization and activation of PKG-Iα and subsequent opening of smooth muscle BKCa channels.

Introduction to the Series on MicroRNAs in the Cardiovascular System [Reviews]

Until recently, microRNAs (miRNAs) were considered to be relatively small players in biological systems by having a balancing function through moderate effects on gene expression levels. However, it has become appreciated that miRNAs are actually much more relevant during both development and disease, which is underscored by the attention they have been receiving. The goal of this thematic review series is to highlight current knowledge of miRNA function during cardiovascular development, their dysregulation under disease conditions and the disease modifying functions they have been shown to exert in the cardiovascular system. These reviews, in addition to discussing the recent advancements in using miRNAs as circulating biomarkers or therapeutic modalities, will hopefully be able to provide a strong basis for future research to further expand our insights into miRNA function in cardiovascular biology.

Circulating MicroRNAs: Novel Biomarkers and Extracellular Communicators in Cardiovascular Disease? [Reviews]

In the past few years, the crucial role of different micro-RNAs (miRNAs) in the cardiovascular system has been widely recognized. Recently, it was discovered that extracellular miRNAs circulate in the bloodstream and that such circulating miRNAs are remarkably stable. This has raised the possibility that miRNAs may be probed in the circulation and can serve as novel diagnostic markers. Although the precise cellular release mechanisms of miRNAs remain largely unknown, the first studies revealed that these circulating miRNAs may be delivered to recipient cells, where they can regulate translation of target genes. In this review, we will discuss the nature of the stability of miRNAs that circulate in the bloodstream and discuss the available evidence regarding the possible function of these circulating miRNAs in distant cell-to-cell communication. Furthermore, we summarize and discuss the usefulness of circulating miRNAs as biomarkers for a wide range of cardiovascular diseases such as myocardial infarction, heart failure, atherosclerosis, hypertension, and type 2 diabetes mellitus.

Developing MicroRNA Therapeutics [Reviews]

Rarely a new research area has gotten such an overwhelming amount of attention as have microRNAs. Although several basic questions regarding their biological principles still remain to be answered, many specific characteristics of microRNAs in combination with compelling therapeutic efficacy data and a clear involvement in human disease have triggered the biotechnology community to start exploring the possibilities of viewing microRNAs as therapeutic entities. This review serves to provide some general insight into some of the current microRNAs targets, how one goes from the initial bench discovery to actually developing a therapeutically useful modality, and will briefly summarize the current patent landscape and the companies that have started to explore microRNAs as the next drug target.

MicroRNAs in Vascular and Metabolic Disease [Reviews]

Recent findings demonstrated the importance of microRNAs (miRNAs) in the vasculature and the orchestration of lipid metabolism and glucose homeostasis. MiRNA networks represent an additional layer of regulation for gene expression that absorbs perturbations and ensures the robustness of biological systems. This function is very elegantly demonstrated in cholesterol metabolism where miRNAs reducing cellular cholesterol export are embedded in the very same genes that increase cholesterol synthesis. Often their alteration does not affect normal development but changes under stress conditions and in disease. A detailed understanding of the molecular and cellular mechanisms of miRNA-mediated effects on metabolism and vascular pathophysiology could pave the way for the development of novel diagnostic markers and therapeutic approaches. In the first part of this review, we summarize the role of miRNAs in vascular and metabolic diseases and explore potential confounding effects by platelet miRNAs in preclinical models of cardiovascular disease. In the second part, we discuss experimental strategies for miRNA target identification and the challenges in attributing miRNA effects to specific cell types and single targets.