Journals RSS : Gourt

[In Memoriam] Juan Carlos Romero, MD: 1937-2008

[Letters to the Editor] Depression and Blood Pressure Control: All Antidepressants Are not the Same

[Letters to the Editor] Response to Depression and Blood Pressure Control: All Antidepressants Are not the Same

[Brief Reviews] Vascular Aging: A Tale of EVA and ADAM in Cardiovascular Risk Assessment and Prevention

[Letters to the Editor] Is Combined L- and T-Channel Blockade Better Than L-Channel Blockade in Therapy?

[Letters to the Editor] Response to Is Combined L- and T-Channel Blockade Better than L-Channel Blockade in Therapy?

[Letters to the Editor] Implementation of Pay for Performance Policy in England

[Letters to the Editor] Meta-Analysis of the Effects of Treating Blood Pressure on Cardiovascular Outcomes of Dialysis Patients

[Letters to the Editor] Response to Meta-Analysis of the Effects of Treating Blood Pressure on Cardiovascular Outcomes of Dialysis Patients

[Letters to the Editor] Sympathetic Activity and Clinical Outcome in Dialysis Patients

[Letters to the Editor] Response to Sympathetic Activity and Clinical Outcome in Dialysis Patients

[Letters to the Editor] Combination Treatment to Prevent Atherosclerosis

[Brief Reviews] Postmenopausal Hypertension: Mechanisms and Therapy

[Letters to the Editor] Response to Combination Treatment to Prevent Atherosclerosis

[Letters to the Editor] Method of Blood Collection May Explain the Suppression of Plasma Renin Concentration in Prorenin Transgenic Mice

[Letters to the Editor] Response to Method of Blood Collection May Explain the Suppression of Plasma Renin Concentration in Prorenin Transgenic Mice

[Editorial Commentaries] Triple Fixed-Dose Combination Therapy: Back to the Past

[Editorial Commentaries] Does It Matter When Drugs Are Taken?

[Editorial Commentaries] The Kidney: Both Culprit and Victim

[Editorial Commentaries] Hypertension: Are We {beta}ARKing Up the Right Tree?

[Editorial Commentaries] Treatment of Chronic Proteinuric Kidney Disease: What Next?

[Original Articles] Triple Antihypertensive Therapy With Amlodipine, Valsartan, and Hydrochlorothiazide: A Randomized Clinical Trial

Many patients with hypertension require ≥3 agents to achieve target blood pressure (BP). The efficacy/safety of the dual combinations of valsartan (Val)/hydrochlorothiazide (HCTZ) and amlodipine (Aml)/Val in hypertension are well established. This randomized, double-blind study evaluated the efficacy/safety of triple therapy with Aml/Val/HCTZ for moderate or severe hypertension (mean sitting systolic BP: ≥145 mm Hg; mean sitting diastolic BP: ≥100 mm Hg). The study included a single-blind, placebo run-in period, followed by double-blind treatment for 8 weeks; patients were randomly assigned to 1 of 4 groups titrated to Aml/Val/HCTZ 10/320/25 mg, Val/HCTZ 320/25 mg, Aml/Val 10/320 mg, or Aml/HCTZ 10/25 mg once daily. Dual-therapy recipients received half of the target doses of both agents for the first 2 weeks, titrating to target doses during week 3. Those on triple therapy received Val/HCTZ 160.0/12.5 mg during week 1, Aml/Val/HCTZ 5.0/160.0/12.5 mg during week 2, and target doses of all 3 of the agents during week 3. Of the 4285 patients enrolled, 2271 were randomly assigned to treatment, and 2060 completed the study. Triple therapy was significantly superior to all of the dual therapies in reducing mean sitting systolic BP and mean sitting diastolic BP from baseline to end point (all P<0.0001). Significantly more patients on triple therapy achieved overall BP control (<140/90 mm Hg; P<0.0001) and systolic and diastolic control (P≤0.0002) compared with each dual therapy. Aml/Val/HCTZ was well tolerated. The benefits of triple therapy over dual therapy were observed regardless of age, sex, race, ethnicity, or baseline mean sitting systolic BP. In conclusion, this study demonstrates the efficacy/safety of treating moderate and severe hypertension with Aml/Val/HCTZ 10/320/25 mg.

[Original Articles] Chronotherapy With the Angiotensin-Converting Enzyme Inhibitor Ramipril in Essential Hypertension: Improved Blood Pressure Control With Bedtime Dosing

Clinical studies have demonstrated a different effect on blood pressure of some angiotensin-converting enzyme inhibitors when administered in the morning versus the evening. Their administration at bedtime resulted in a higher effect on nighttime blood pressure as compared with morning dosing. This study investigated the administration time-dependent effects of ramipril on ambulatory blood pressure. We studied 115 untreated hypertensive patients, 46.7±11.2 years of age, randomly assigned to receive ramipril (5 mg/d) as a monotherapy either on awakening or at bedtime. Blood pressure was measured for 48 hours before and after 6 weeks of treatment. The blood pressure reduction during diurnal activity was similar for both treatment times. Bedtime administration of ramipril, however, was significantly more efficient than morning administration in reducing asleep blood pressure. The awake:asleep blood pressure ratio was decreased after ramipril on awakening but significantly increased toward a more dipping pattern after bedtime dosing. The proportion of patients with controlled ambulatory blood pressure increased from 43% to 65% (P=0.019) with bedtime treatment. Nocturnal blood pressure regulation is significantly better achieved at bedtime as compared with morning administration of ramipril, without any loss in efficacy during diurnal active hours. This might be clinically important, because nighttime blood pressure has been shown to be a more relevant marker of cardiovascular risk than diurnal mean values. The change in the dose-response curve, increased proportion of controlled patients, and improved efficacy on nighttime blood pressure with administration of ramipril at bedtime should be taken into account when prescribing this angiotensin-converting enzyme inhibitor for treatment of essential hypertension.

[Original Articles] Blood Pressure Control Among Persons Without and With Chronic Kidney Disease: US Trends and Risk Factors 1999-2006

Recent guidelines recommending more aggressive blood pressure control in patients with chronic kidney disease have unknown impact. We assessed trends in and predictors of blood pressure control in 8829 adult National Health and Nutrition Examination Survey 1999–2006 participants with hypertension (self-report, measured blood pressure, or use of antihypertensive medications), without (n=7178) and with (n=1651) chronic kidney disease. Uncontrolled blood pressure was defined as follows: general definition, systolic blood pressure ≥140 mm Hg and diastolic blood pressure ≥90 mm Hg, and disease-specific definition, systolic blood pressure ≥130 mm Hg and diastolic blood pressure ≥85 mm Hg (1999–2002) and systolic blood pressure ≥130 mm Hg and diastolic blood pressure ≥80 mm Hg (2003–2006) for those with chronic kidney disease (estimated glomerular filtration rate: <60 mL/min per 1.73 m2) or diabetes mellitus (self-report). Proportions with uncontrolled blood pressure in 1999–2006 were greater in those with chronic kidney disease versus those without chronic kidney disease (51.5% versus 48.7% [general definition: P=0.122] and 68.8% versus 51.7% [disease-specific definition: P<0.001]). In those with chronic kidney disease, there were significant decreases in uncontrolled blood pressure over time (55.9% to 47.8% [general definition: P=0.011]). With adjustment for demographic, socioeconomic, and clinical variables, older age (P<0.001) and lack of antihypertensive treatment (P<0.001) were associated with uncontrolled blood pressure, regardless of chronic kidney disease status; nonwhite race (P=0.002) was associated in those without chronic kidney disease, whereas female sex (P=0.030) was associated in those with chronic kidney disease. Multiple medications (P<0.001) and angiotensin-converting enzyme inhibitors/angiotensin II receptor blockers (P=0.001) were associated with less uncontrolled blood pressure. Although some improvement has occurred over time, uncontrolled blood pressure remains highly prevalent, especially in subjects with chronic kidney disease and in nonwhites, older persons, and women. Therapy appears suboptimal.

[Original Articles] Blood Pressure Components and Changes in Relation to White Matter Lesions: A 32-Year Prospective Population Study

This study aimed to examine the long-term effect of high blood pressure (systolic blood pressure, diastolic blood pressure, pulse pressure, and mean arterial pressure) on white matter lesions and to study changes in different blood pressure components in relation to white matter lesions. A representative population of women was examined in 1968 and re-examined in 1974, 1980, 1992, and 2000. The presence and severity of white matter lesions on computed tomography were rated by a visual rating scale in 1992 and 2000 in 539 women. Systolic and diastolic blood pressures were measured at all of the examinations. We found that presence and severity of white matter lesions in 1992/2000 were associated with higher diastolic blood pressure and mean arterial pressure at each examination but not with systolic blood pressure and pulse pressure. Odds ratios (95% CIs) for the presence of white matter lesions per 10-mm Hg increase in diastolic pressure were 1.4 (1.0 to 1.9) in 1968, 1.3 (1.0 to 1.8) in 1974, 1.4 (1.1 to 1.9) in 1980, and 1.3 (1.0 to 1.6) in 1992 after adjustment for confounders. The presence of white matter lesions was also associated with a 24-year increase in diastolic pressure (>10 mm Hg), systolic pressure (>40 mm Hg), pulse pressure (>24 mm Hg), and mean arterial pressure (>6 mm Hg; odds ratios [95% CIs]: 2.6 [1.3 to 5.1] for diastolic pressure; 2.0 [1.2 to 3.4] for systolic pressure; 1.8 [1.1 to 2.7] for pulse pressure; and 2.2 [1.4 to 3.4] for mean arterial pressure). Our findings suggest that lowering high diastolic blood pressure and preventing large increases in systolic and diastolic blood pressures may have a protective effect on white matter lesions.

[Original Articles] Antihypertensive Medication Use During Pregnancy and the Risk of Cardiovascular Malformations

We used data from the National Birth Defects Prevention Study, a population-based, case-control study, to examine whether previously reported associations between antihypertensive medications and cardiovascular malformations could be confirmed and to explore whether new associations might be identified. Cases (n=5021) were ascertained through birth defects surveillance systems from 1997 through 2003 in 10 US states. Controls (n=4796) were live births without birth defects selected randomly from birth certificates or hospital discharge listings in the same geographic regions. Logistic regression was used to examine the relationship between antihypertensive medication treatment and the occurrence of cardiovascular malformations while controlling for confounding variables. First-trimester treatment with antihypertensive medication was associated with pulmonary valve stenosis (odds ratio [OR]: 2.6; 95% CI: 1.3 to 5.4), Ebstein malformation (crude OR: 11.4; exact 95% CI: 2.8 to 34.1), coarctation of the aorta (OR: 3.0; 95% CI: 1.3 to 6.6), and secundum atrial septal defects (OR: 2.4; 95% CI: 1.3 to 4.4). Treatment initiated after the first trimester was associated with pulmonary valve stenosis (OR: 2.4; 95% CI: 1.1 to 5.4), perimembranous ventricular septal defects (OR: 2.3; 95% CI: 1.2 to 4.6), and secundum atrial septal defects (OR: 2.4; 95% CI: 1.3 to 4.4). Untreated hypertension was associated with Ebstein malformation (OR: 2.1; 95% CI: 1.0 to 4.3) and secundum atrial septal defects (OR: 1.3; 95% CI: 1.0 to 1.6). Antihypertensive medication use and/or the underlying hypertension might increase the risk of having an infant with specific left and right obstructive and septal defects. Additional studies with adequate power will be needed to confirm these findings.

[Original Articles] G Protein-Coupled Receptor Kinase 2 Expression and Activity Are Associated With Blood Pressure in Black Americans

Hypertension occurs with higher prevalence and morbidity in black Americans compared with other groups. Alterations in the signal transduction pathways of 7-transmembrane spanning receptors are found in hypertensive patients. G protein–coupled receptor kinases (GRKs) play an important role in regulating this receptor signaling. The 2 most abundantly expressed GRKs in the cardiovascular system are GRK2 and GRK5, and each has unique substrates. Understanding changes in expression may give us insight into activated receptors in the pathophysiological progression of hypertension. In heart failure and white hypertensives, increased GRK2 expression arises because of neurohormonal stimulation of particular receptors. GRK2 subsequently desensitizes specific receptors, including β-adrenergic receptors. In blood pressure control, β-adrenergic receptor desensitization could lead to increased blood pressure. GRK2 and GRK5 mRNA were evaluated in lymphocytes of black Americans via quantitative real-time PCR. GRK2 mRNA expression directly correlated with systolic blood pressure and norepinephrine levels. GRK2 was elevated >30% among those with systolic blood pressure ≥130 mm Hg. No significant correlation between GRK5 mRNA expression and blood pressure or catecholamines was observed. Diabetic status, age, sex, and body mass index were also compared with GRK2 expression using univariate and multivariate analyses. GRK2 protein expression was elevated 2-fold in subjects with higher blood pressure, and GRK activity was increased >40%. Our data suggest that GRK2, but not GRK5, is correlated with increasing blood pressure in black Americans. Understanding the receptors stimulated by increased neurohormonal activation may give insight into the pathophysiology of hypertension in this at-risk population.

[Original Articles] Hypertension Prevalence, Awareness, Treatment, and Control in Mozambique: Urban/Rural Gap During Epidemiological Transition

The prediction of cardiovascular risk profile trends in low-income countries and timely action to modulate their transitions are among the greatest global health challenges. In 2005 we evaluated a nationally representative sample of the Mozambican population (n=3323; 25 to 64 years old) following the Stepwise Approach to Chronic Disease Risk Factor Surveillance. Prevalence of hypertension (systolic blood pressure ≥140 mm Hg and/or diastolic blood pressure ≥90 mm Hg and/or antihypertensive drug therapy), awareness (having been informed of the hypertensive status by a health professional in the previous year), treatment among the aware (use of antihypertensive medication in the previous fortnight), and control among those treated (blood pressure <140/90 mm Hg) were 33.1% (women: 31.2%; men: 35.7%), 14.8% (women: 18.4%; men: 10.6%), 51.9% (women: 61.1%; men: 33.3%), and 39.9% (women: 42.9%; men: 28.7%), respectively. Urban/rural comparisons are presented as age- and education-adjusted odds ratios (ORs) and 95% CIs. Among women, hypertension (OR: 2.0; 95% CI: 1.2 to 3.0) and awareness (OR: 4.3; 95% CI: 1.9 to 9.5) were more frequent in urban areas. No urban/rural differences were observed in men (hypertension: OR: 1.3, 95% CI: 0.9 to 2.0; awareness: OR: 1.5, 95% CI: 0.5 to 4.7). Treatment prevalence was not significantly different across urban/rural settings (women: OR: 1.4, 95% CI: 0.5 to 4.4; men: OR: 0.3, 95% CI: 0.1 to 1.4). Control was less frequent in urban women (OR: 0.2; 95% CI: 0.0 to 1.0) and more frequent in urban men (OR: 78.1; 95% CI: 2.2 to 2716.6). Our results illustrate the changing paradigms of "diseases of affluence" and the dynamic character of epidemiological transition. The urban/rural differences across sexes support a trend toward smaller differences, emphasizing the need for strategies to improve prevention, correct diagnosis, and access to effective treatment.

[Original Articles] Does Greater Adiposity Increase Blood Pressure and Hypertension Risk?: Mendelian Randomization Using the FTO/MC4R Genotype

Elevated blood pressure increases the risk of experiencing cardiovascular events like myocardial infarction and stroke. Current observational data suggest that body mass index may have a causal role in the etiology of hypertension, but this may be influenced by confounding and reverse causation. Through the use of instrumental variable methods, we aim to estimate the strength of the unconfounded and unbiased association between body mass index/adiposity and blood pressure. We explore these issues in the Copenhagen General Population Study. We used instrumental variable methods to obtain estimates of the causal association between body mass index and blood pressure. This was performed using both rs9939609 (FTO) and rs17782313 (MC4R) genotypes as instruments for body mass index. Avoiding the epidemiological problems of confounding, bias, and reverse causation, we confirmed observational associations between body mass index and blood pressure. In analyses including those taking antihypertensive drugs, but for whom appropriate adjustment had been made, systolic blood pressure was seen to increase by 3.85 mm Hg (95% CI: 1.88 to 5.83 mm Hg) for each 10% increase in body mass index (P=0.0002), with diastolic blood pressure showing an increase of 1.79 mm Hg (95% CI: 0.68 to 2.90 mm Hg) for each 10% increase in body mass index (P=0.002). Observed associations are large and illustrate the considerable benefits in terms of reductions in blood pressure–related morbidity that could be achieved through a reduction in body mass index.

[Original Articles] Low Cardiorespiratory Fitness Levels and Elevated Blood Pressure: What Is the Contribution of Visceral Adiposity?

Individuals with poor cardiorespiratory fitness have higher blood pressure than fit individuals. Individuals with low fitness levels also tend to be characterized by higher visceral adiposity compared with physically fit individuals. We tested the hypothesis that the relationship between low fitness and elevated blood pressure could be related, at least in part, to the higher level of visceral adipose tissue often found among unfit individuals. This study included 407 asymptomatic, nondiabetic participants. Visceral adipose tissue was assessed by computed tomography, and fitness was measured by a progressive submaximal physical working capacity test. Participants in the highest visceral adipose tissue tertile showed the highest systolic and diastolic blood pressures, whereas participants in the highest fitness tertile had the lowest blood pressure values (P<0.001). When participants were classified into fitness tertiles and then subdivided on the basis of visceral adipose tissue (high versus low), participants with a high visceral adipose tissue had higher systolic and diastolic blood pressure values (P=0.01), independent of their fitness category. Linear regression analyses showed that age and visceral adipose tissue, but not fitness, predicted systolic blood pressure (r2=0.11 [P<0.001], 0.12 [P<0.001], and 0.01 [P value nonsignificant], for age, visceral adipose tissue, and fitness, respectively) and diastolic blood pressure (r2=0.17 [P<0.001], 0.14 [P<0.001], and 0.01 [P value nonsignificant], for age, visceral adipose tissue, and fitness, respectively). Individuals with high visceral adipose tissue levels have higher blood pressure, independent of their fitness. Visceral adipose tissue may represent an important clinical target in the management of elevated blood pressure.

[Original Articles] Prolactin and Preclinical Atherosclerosis in Menopausal Women With Cardiovascular Risk Factors

Hyperprolactinemia has been associated with endothelial dysfunction and an adverse cardiovascular risk profile, possibly as a result of the vasoconstrictive properties of prolactin. In this cross-sectional study, we examined the hypothesis that prolactin contributes to the increased cardiovascular risk occurring in early menopause by studying apparently healthy women without hyperprolactinemia. Prolactin serum levels were measured by immunoassay in 76 women aged 54.4±4.9 years in menopause for 4.9±2.8 years, and possible correlations with traditional cardiovascular risk factors and surrogate markers of preclinical atherosclerosis, arterial stiffening, and endothelial and microcirculatory function were examined. Positive correlations between prolactin serum levels and arterial blood pressure, but no other traditional risk factors, were found. Prolactin also correlated with central aortic systolic (r=0.337; P=0.002) and diastolic (r=0.272; P=0.012) blood pressures and pulse wave velocity (r=0.264; P=0.02), a marker of aortic stiffness, but not with endothelial or microcirculatory function or carotid intima-media thickness. By multivariate regression analysis, prolactin levels determined, independent of traditional risk factors, both blood pressures and aortic stiffness. Notably, prolactin correlated with European Society of Cardiology HeartScore (r=0.364; P=0.002), a composite index that predicts 10-year cardiovascular mortality. Prolactin levels >8.0 ng/mL had 100% sensitivity to predict a high peripheral blood pressure. Prolactin may play a role in accelerated arteriosclerosis in early menopause by affecting central/peripheral blood pressure and arterial stiffness. In contrast, no correlation was observed with other risk factors or surrogate markers of atherosclerosis. Prospective studies to assess whether prolactin is an additional hormone increasing cardiovascular risk are warranted.

[Original Articles] Circulating and Placental Growth-Differentiation Factor 15 in Preeclampsia and in Pregnancy Complicated by Diabetes Mellitus

Growth-differentiation factor 15 (GDF-15), a stress-responsive transforming growth factor-β–related cytokine, is emerging as a new risk marker in patients with cardiovascular disease. We explored GDF-15 in preeclampsia and in diabetic pregnancies, because these conditions are associated with augmented risk for cardiovascular disease, both in mother and in offspring. Plasma from pregnant women (n=267; controls: n=59, preeclampsia: n=85, diabetes mellitus: n=112, and superimposed preeclampsia in diabetes mellitus: n=11), fetal plasma (n=72), and amniotic fluid (n=99) were analyzed by immunoassay for GDF-15. Placental GDF-15 mRNA and protein expression levels were analyzed by quantitative real-time PCR and immunoblots in 78 and 18 pregnancies, respectively. Conditioned media from preeclamptic (n=6) and control (n=6) villous placenta explants were analyzed by immunoassay for GDF-15. Median maternal GDF-15 concentration was elevated in those with diabetes mellitus, as compared with controls (91 549 versus 79 875 ng/L; P=0.02). Median GDF-15 concentration was higher in patients with preeclampsia than in controls in term maternal blood samples (127 061 versus 80 319 ng/L; P<0.001). In the fetal circulation and amniotic fluid, GDF-15 was elevated in preeclampsia and superimposed preeclampsia in diabetes mellitus, as compared with controls. GDF-15 placental mRNA expression was elevated in preeclampsia, as compared with controls (P=0.002). Placenta immunoblots confirmed a single GDF-15 protein band, and a time-dependent increase in GDF-15 protein was detected in the conditioned media. Our study is the first to show that GDF-15 is dysregulated, both in preeclampsia and in diabetic pregnancies. The mechanisms and diagnostic implications of these findings remain to be explored.

[Original Articles] Blood Pressure-Independent Reduction in Proteinuria and Arterial Stiffness After Acute Endothelin-A Receptor Antagonism in Chronic Kidney Disease

Endothelin 1 is implicated in the development and progression of chronic kidney disease and associated cardiovascular disease. We, therefore, studied the effects of selective endothelin-A receptor antagonism with BQ-123 on key independent surrogate markers of cardiovascular risk (blood pressure, proteinuria and renal hemodynamics, arterial stiffness, and endothelial function) in patients with nondiabetic chronic kidney disease. In a double-blind, randomized crossover study, 22 subjects with proteinuric chronic kidney disease received, on 2 separate occasions, placebo or BQ-123. Ten of these subjects also received nifedipine (10 mg) as an active control for the antihypertensive effect of BQ-123. Blood pressure, pulse wave velocity, flow-mediated dilation, renal blood flow, and glomerular filtration rate were monitored after drug dosing. BQ-123 reduced blood pressure (mean arterial pressure: –7±1%; P<0.001 versus placebo) and increased renal blood flow (17±4%; P<0.01 versus placebo). Glomerular filtration rate remained unchanged. Proteinuria (–26±4%; P<0.01 versus placebo) and pulse wave velocity (–5±1%; P<0.001 versus placebo) fell after BQ-123, but flow-mediated dilation did not change. Nifedipine matched the blood pressure and renal blood flow changes seen with BQ-123. Nevertheless, BQ-123 reduced proteinuria (–38±3% versus 26±11%; P<0.001) and pulse wave velocity (–9±1% versus –3±1%; P<0.001) to a greater extent than nifedipine. Selective endothelin-A receptor antagonism reduced blood pressure, proteinuria, and arterial stiffness on top of standard treatment in renal patients. Furthermore, these studies suggest that the reduction in proteinuria and arterial stiffness is partly independent of blood pressure. If maintained longer term, selective endothelin-A receptor antagonism may confer cardiovascular and renal benefits in patients with chronic kidney disease.

[Original Articles] Enhanced Distal Nephron Sodium Reabsorption in Chronic Angiotensin II-Infused Mice

Chronic angiotensin II (Ang II) infusions enhance urinary excretion of angiotensinogen, suggesting augmentation of distal nephron sodium reabsorption. To assess whether chronic Ang II infusions (15 ng/min for 2 weeks) enhance distal nephron sodium reabsorption, we compared sodium excretion before and after blockade of the 2 main distal nephron sodium transporters by IV amiloride (5 mg/kg of body weight) plus bendroflumethiazide (12 mg/kg of body weight) in male C57/BL6 anesthetized control mice (n=10) and in chronic Ang II–infused mice (n=8). Chronic Ang II infusions increased systolic blood pressure to 141±6 mm Hg compared with 106±4 mm Hg in control mice. After anesthesia, mean arterial pressure averaged 97±4 mm Hg in chronic Ang II–infused mice compared with 94±3 mm Hg in control mice, allowing comparison of renal function at similar arterial pressures. Ang II–infused mice had lower urinary sodium excretion (0.16±0.04 versus 0.30±0.05 µEq/min; P<0.05), higher distal sodium reabsorption (1.74±0.18 versus 1.12±0.18 µEq/min; P<0.05), and higher fractional reabsorption of distal sodium delivery (91.1±1.8% versus 77.9±4.3%; P<0.05) than control mice. Urinary Ang II concentrations, measured during distal blockade, were greater in Ang II–infused mice (1235.0±277.2 versus 468.9±146.9 fmol/mL; P<0.05). In chronic Ang II–infused mice treated with spironolactone (n=5), fractional reabsorption of distal sodium delivery was similarly augmented as in chronic Ang II–infused mice (94.6±1.7%; P<0.01). These data provide in vivo evidence that there is enhanced distal sodium reabsorption dependent on sodium channel and Na+-Cl– cotransporter activity and increased urinary Ang II concentrations in mice infused chronically with Ang II.

[Original Articles] Age-Related Differences in the Sympathetic-Hemodynamic Balance in Men

As humans age, the tonic level of activity in sympathetic vasoconstrictor nerves increases and may contribute to age-related increases in blood pressure. In previous studies in normotensive young men with varying levels of resting sympathetic nerve activity, we observed a balance among factors contributing to blood pressure regulation, such that higher sympathetic activity was associated with lower cardiac output and lesser vascular responsiveness to -adrenergic agonists, which limited the impact of high sympathetic activity on blood pressure. In the present study, we tested the hypothesis that older normotensive men would exhibit a similar balance among these variables (sympathetic nerve activity, cardiac output, and -adrenergic responsiveness) but that this balance would be shifted toward higher sympathetic nerve activity values. We measured muscle sympathetic nerve activity, cardiac output, arterial pressure, and forearm vasoconstrictor responses in 17 older men and compared these with previous data collected in 14 younger men. Muscle sympathetic activity (burst incidence) was positively related to diastolic blood pressure in the older men (r=0.49; P=0.05); this relationship was not observed in young men. In addition, there was no relationship between cardiac output and muscle sympathetic activity (r=0.29; P>0.05) or between muscle sympathetic activity and vasoconstrictor responses in the older men (eg, norepinephrine: r=–0.21; P>0.05). Although our older subjects were normotensive, the relationship between muscle sympathetic nerve activity and diastolic blood pressure and the lack of "balance" among the other variables suggest that these changes with aging may contribute to the risk of sympathetically mediated hypertension in older humans.

[Original Articles] Eutrophic Remodeling of Small Arteries in Type 1 Diabetes Mellitus Is Enabled by Metabolic Control: A 10-Year Follow-Up Study

Type 2 diabetes mellitus profoundly changes small artery remodeling in response to hypertension. Abnormal increases of both wall thickness and lumen diameter are associated with an increased mortality. Changes to small artery structure in response to blood pressure (BP) in patients with type 1 diabetes mellitus have never been examined. In 1997, 17 patients with type 1 diabetes mellitus and 9 control subjects underwent in vitro assessment of gluteal-fat small arteries using pressure myography. Patients with BP <140/90 mm Hg (systolic BP: 119±3 mm Hg; n=12) had normal-resistance artery structure. However, patients with BP >140/90 mm Hg (systolic BP: 152±5 mm Hg; n=5) demonstrated vascular hypertrophic remodeling with a significant increase in the medial cross-sectional area and wall thickness. In 2008, 8 of the original 17 diabetic patients returned for a repeat assessment. All 8 of the patients had significantly improved cholesterol (2008: 154±9 mg/dL versus 1997: 191±9 mg/dL; P=0.01) and low-density lipoprotein cholesterol (2008: 79±8 mg/dL versus 1997: 122±9 mg/dL; P=0.003) but higher BPs (systolic BP: 2008: 136±3 mm Hg versus 1997: 119±6 mm Hg; P=0.03). Glycemia was improved (2008: 7.9±0.3% versus 1997: 8.9±0.6%; P=0.17), but not significantly so. In the small arteries studied, there were significant increases in medial wall thickness and wall:lumen ratio, but cross-sectional area was unchanged, indicating eutrophic remodeling. Collectively, these findings suggest that, with poor metabolic control, small arteries from patients with type 1 diabetes mellitus show hypertrophic growth in response to elevated BP, similar to that seen in type 2 diabetes mellitus. However, metabolic improvements enable eutrophic remodeling to occur in response to an increase in BP. This has only been observed previously in patients without diabetes mellitus.

[Original Articles] Atorvastatin Prevents Angiotensin II-Induced Vascular Remodeling and Oxidative Stress

Angiotensin II (Ang II) modulates vasomotor tone, cell growth, and extracellular matrix deposition. This study analyzed the effect of atorvastatin in the possible alterations induced by Ang II on structure and mechanics of mesenteric resistance arteries and the signaling mechanisms involved. Wistar rats were infused with Ang II (100 ng/kg per day, SC minipumps, 2 weeks) with or without atorvastatin (5 mg/kg per day). Ang II increased blood pressure and plasmatic malondialdehyde levels. Compared with controls, mesenteric resistance arteries from Ang II–treated rats showed the following: (1) decreased lumen diameter; (2) increased wall/lumen; (3) decreased number of adventitial, smooth muscle, and endothelial cells; (4) increased stiffness; (5) increased collagen deposition; and (6) diminished fenestrae area and number in the internal elastic lamina. Atorvastatin did not alter blood pressure but reversed all of the structural and mechanical alterations of mesenteric arteries, including collagen and elastin alterations. In mesenteric resistance arteries, Ang II increased vascular O2·– production and diminished endothelial NO synthase and CuZn/superoxide dismutase but did not modify extracellular-superoxide dismutase expression. Atorvastatin improved plasmatic and vascular oxidative stress, normalized endothelial NO synthase and CuZn/superoxide dismutase expression, and increased extracellular-superoxide dismutase expression, showing antioxidant properties. Atorvastatin also diminished extracellular signal–regulated kinase 1/2 activation caused by Ang II in these vessels, indicating an interaction with Ang II–induced intracellular responses. In vascular smooth muscle cells, collagen type I release mediated by Ang II was reduced by different antioxidants and statins. Moreover, atorvastatin downregulated the Ang II–induced NADPH oxidase subunit, Nox1, expression. Our results suggest that statins might exert beneficial effects on hypertension-induced vascular remodeling by improving vascular structure, extracellular matrix alterations, and vascular stiffness. These effects might be mediated by their antioxidant properties.

[Original Articles] Tumor Necrosis Factor-{alpha} Mediates Hemolysis-Induced Vasoconstriction and the Cerebral Vasospasm Evoked by Subarachnoid Hemorrhage

Hypertension can lead to subarachnoid hemorrhage and eventually to cerebral vasospasm. It has been suggested that the latter could be the result of oxidative stress and an inflammatory response evoked by subarachnoid hemorrhage. Because an unavoidable consequence of hemorrhage is lysis of red blood cells, we first tested the hypothesis on carotid arteries that the proinflammatory cytokine tumor necrosis factor- contributes to vascular oxidative stress evoked by hemolysis. We observed that hemolysis induces a significant increase in tumor necrosis factor- both in blood and in vascular tissues, where it provokes Rac-1/NADPH oxidase–mediated oxidative stress and vasoconstriction. Furthermore, we extended our observations to cerebral vessels, demonstrating that tumor necrosis factor- triggered this mechanism on the basilar artery. Finally, in an in vivo model of subarachnoid hemorrhage obtained by the administration of hemolyzed blood in the cisterna magna, we demonstrated, by high-resolution ultrasound analysis, that tumor necrosis factor- inhibition prevented and resolved acute cerebral vasoconstriction. Moreover, tumor necrosis factor- inhibition rescued the hemolysis-induced brain injury, evaluated with the method of 2,3,5-triphenyltetrazolium chloride and by the histological analysis of pyknotic nuclei. In conclusion, our results demonstrate that tumor necrosis factor- plays a crucial role in the onset of hemolysis-induced vascular injury and can be used as a novel target of the therapeutic strategy against cerebral vasospasm.

[Original Articles] {beta}2-Adrenoceptor Antagonist ICI 118,551 Decreases Pulmonary Vascular Tone in Mice via a Gi/o Protein/Nitric Oxide-Coupled Pathway

β2-Adrenoceptors are important modulators of vascular tone, particularly in the pulmonary circulation. Because neurohormonal activation occurs in pulmonary arterial hypertension, we have investigated the effect of different adrenergic vasoactive substances on tone regulation in large and small pulmonary arteries, as well as in systemic vessels of mice. We found that the β2-adrenoceptor antagonist ICI 118,551 (ICI) evoked a decrease of vascular tone in large pulmonary arteries and reduced the sensitivity of pulmonary arteries toward different contracting agents, eg, norepinephrine, serotonin, or endothelin. ICI proved to act specifically on pulmonary vessels, because it shifted the dose-response curve of norepinephrine to the right in pulmonary arteries, whereas there was no effect in the aorta. Pharmacological experiments proved that the right shift of the norepinephrine dose-response curve by ICI was mediated via a β2-adrenoceptor/Gi/o protein-dependent pathway enhancing NO production in the endothelium; these results were corroborated in β-adrenoceptor and endothelial NO synthase knockout mice where ICI had no effect. ICI increased vascular lumen diameter in lung sections and reduced pulmonary arterial pressure under normoxia and under hypoxia in the isolated perfused lung model. These effects were found to be physiologically relevant, because ICI specifically decreased pulmonary but not systemic blood pressure in vivo. Thus, the β2-adrenoceptor antagonist ICI is a pulmonary arterial-specific vasorelaxant and, therefore, a potentially interesting novel therapeutic agent for the treatment of pulmonary arterial hypertension.

[Original Articles] Chymase Plays an Important Role in Left Ventricular Remodeling Induced by Intermittent Hypoxia in Mice

Intermittent hypoxia caused by sleep apnea is associated with cardiovascular disease. Chymase has been reported to play an important role in the development of cardiovascular disease, but it is unclear whether chymase is involved in the pathogenesis of left ventricular remodeling induced by intermittent hypoxia. The aim of this study was to evaluate the effect of a novel chymase inhibitor (NK3201) on hypoxia-induced left ventricular remodeling in mice. Male C57BL/6J mice (9 weeks old) were exposed to intermittent hypoxia or normoxia and were treated with NK3201 (10 mg/kg per day) or the vehicle for 10 days. Left ventricular systolic pressure showed no significant differences among all of the experimental groups. Exposure to intermittent hypoxia increased left ventricular chymase activity and angiotensin II expression, which were both suppressed by treatment with NK3201. Intermittent hypoxia also increased the mean cardiomyocyte diameter, perivascular fibrosis, expression of inflammatory cytokines, oxidative stress, and NADPH-dependent superoxide production in the left ventricular myocardium. These changes were all suppressed by NK3201 treatment. Therefore, chymase might play an important role in intermittent hypoxia-induced left ventricular remodeling, which is independent of the systemic blood pressure.

[Original Articles] Cooperative Activation of Npr1 Gene Transcription and Expression by Interaction of Ets-1 and p300

The objective of the present study was to gain insight into the cooperative roles of Ets-1 and p300 in transcriptional regulation and expression of the Npr1 gene (coding for guanylyl cyclase-A/natriuretic peptide receptor-A). Overexpression of Ets-1 and p300 in mouse mesangial cells increased Npr1 promoter activity by 12-fold, natriuretic peptide receptor-A mRNA levels by 5-fold, and ANP-dependent intracellular accumulation of cGMP by 26-fold. Knockdown of Ets-1 and p300 expression by small interfering RNA inhibited Npr1 gene transcription by 90%. Sequential chromatin immunoprecipitation assay demonstrated a direct physical association between p300 and Ets-1 on binding to the Npr1 promoter, suggesting that a physical interaction between Ets-1 and p300 is important to enhance Npr1 gene transcription. Mutant p300 lacking histone acetyltransferase activity did not show a functional effect with Ets-1, suggesting that histone acetyltransferase activity of p300 is required for the cooperative interaction in modulating Npr1 gene transcription. Overexpression of wild-type adenovirus E1A significantly decreased the Npr1 promoter activity by 40%, whereas mutant E1A, which is incapable of binding to p300, did not show any effect. The results indicate that Npr1 gene transcription is critically controlled by histone acetyltransferase p300 and Ets-1. The present findings should yield important insights into the molecular signaling governing Npr1 gene transcription, an important regulator in the control of hypertension and cardiovascular events.

[2009 ATVB Abstracts] Arteriosclerosis, Thrombosis, and Vascular Biology Annual Conference 2009

[Brief Reviews] Cardiovascular Molecular Imaging

[Brief Reviews] Molecular Imaging in Atherosclerosis, Thrombosis, and Vascular Inflammation

Appreciation of the molecular and cellular processes of atherosclerosis, thrombosis, and vascular inflammation has identified new targets for imaging. The common goals of molecular imaging approaches are to accelerate and refine diagnosis, provide insights that reveal disease diversity, guide specific therapies, and monitor the effects of those therapies. Here we undertake a comparative analysis of imaging modalities that have been used in this disease area. We consider the elements of contrast agents, emphasizing how an understanding of the biology of atherosclerosis and its complications can inform optimal design. We address the potential and limitations of current contrast approaches in respect of translation to clinically usable agents and speculate on future applications.

[Brief Reviews] Nanotechnology in Medical Imaging: Probe Design and Applications

Nanoparticles have become more and more prevalent in reports of novel contrast agents, especially for molecular imaging, the detection of cellular processes. The advantages of nanoparticles include their potency to generate contrast, the ease of integrating multiple properties, lengthy circulation times, and the possibility to include high payloads. As the chemistry of nanoparticles has improved over the past years, more sophisticated examples of nano-sized contrast agents have been reported, such as paramagnetic, macrophage targeted quantum dots or vβ3-targeted, MRI visible microemulsions that also carry a drug to suppress angiogenesis. The use of these particles is producing greater knowledge of disease processes and the effects of therapy. Along with their excellent properties, nanoparticles may produce significant toxicity, which must be minimized for (clinical) application. In this review we discuss the different factors that are considered when designing a nanoparticle probe and highlight some of the most advanced examples.

[Brief Reviews] Iron Oxide Particles for Atheroma Imaging

The selection of patients for vascular interventions has been solely based on luminal stenosis and symptomatology. However, histological data from both the coronary and carotid vasculature suggest that other plaque features such as inflammation may be more important in predicting future thromboembolic events. Ultrasmall superparamagnetic iron oxide (USPIO) contrast agents have been used for noninvasive MRI assessment of atherosclerotic plaque inflammation in humans. It has reached the stage of development to have been recently used in an interventional drug study to not only assess inflammatory progression but also select patients at high risk. This article reviews the basic science behind the use of USPIO contrast agents in atheroma MR imaging, experimental work in animals, and how this has led to the emergence of this promising targeted imaging platform for assessment of high risk carotid atherosclerosis in humans.

[Brief Reviews] Inflammation Imaging in Atherosclerosis

Inflammation is important at many stages of atherosclerotic plaque development. We highlight several imaging modalities that can quantify the degree of plaque inflammation noninvasively. Imaging of this type might allow testing of novel antiatherosclerosis drugs, identification of patients at risk of plaque rupture, and deeper insight into the biology of the disease. The imaging modalities are discussed in relation to their potential use in these areas.

[Brief Reviews] Optical and Multimodality Molecular Imaging: Insights Into Atherosclerosis

Imaging approaches that visualize molecular targets rather than anatomic structures aim to illuminate vital molecular and cellular aspects of atherosclerosis biology in vivo. Several such molecular imaging strategies stand ready for rapid clinical application. This review describes the growing role of in vivo optical molecular imaging in atherosclerosis and highlights its ability to visualize atheroma inflammation, calcification, and angiogenesis. In addition, we discuss advances in multimodality probes, both in the context of multimodal imaging as well as multifunctional, or "theranostic," nanoparticles. This review highlights particular molecular imaging strategies that possess strong potential for clinical translation.

[Brief Reviews] In Vivo Imaging of Stem Cells and Beta Cells Using Direct Cell Labeling and Reporter Gene Methods

Cellular transplantation therapy offers a means to stimulate cardiovascular repair either by direct (graft-induced) or indirect (host-induced) tissue regeneration or angiogenesis. Typically, autologous or donor cells of specific subpopulations are expanded exogenously before administration to enrich the cells most likely to participate in tissue repair. In animal models of cardiovascular disease, the fate of these exogenous cells can be determined using histopathology. Recently, methods to label cells with contrast agents or transduce cells with reporter genes to produce imaging beacons has enabled the serial and dynamic assessment of the survival, fate, and engraftment of these cells with noninvasive imaging. Although cell tracking methods for cardiovascular applications have been most studied in stem or progenitor cells, research in tracking of whole islet transplants and particularly insulin producing beta cells has implications to the cardiovascular community attributable to the vascular changes associated with diabetes mellitus. In this review article, we will explore some of the state-of-the art methods for stem, progenitor, and beta cell tracking.

[Brief Reviews] Molecular Imaging of Macrophage Cell Death for the Assessment of Plaque Vulnerability

The ability to identify atherosclerotic plaques that are prone to rupture, also called vulnerable plaques, may provide a major step forward in the recognition of patients that have a high risk of developing acute myocardial infarction. Current clinical risk profiling algorithms, such as the Framingham and Procam risk scores, have reasonable predictive value in the assessment of the 10 year risk. These clinical risk profiling scores typically classify patients into low risk (10-year risk, less than 5%), intermediate risk (5% to 20% risk), and high risk (greater than 20%). The challenge to imagers is to identify the risk that is beyond 2% yearly risk. Molecular imaging may help identify plaque inflammation and apoptosis of inflammatory cells, which are obligatory components of the plaque instability. These processes offer specific biological targets that can potentially be exploited to obtain biological information on atherosclerosis development in the individual patient.

[Integrative Physiology/Experimental Medicine] Disruption of SEMA4D Ameliorates Platelet Hypersensitivity in Dyslipidemia and Confers Protection Against the Development of Atherosclerosis

Objective— In dyslipidemic states, platelets become hyperreactive, secreting molecules that promote atherosclerosis. We have shown that the semaphorin family member, sema4D (CD100), is expressed on the surface of platelets and proposed that its role includes promoting thrombus growth by binding to nearby platelets and endothelial cells, both of which express sema4D receptors. Here we tested the hypothesis that deleting sema4D will attenuate the adverse consequences of dyslipidemia on platelets and the vessel wall. Methods and Results— Platelet function and atherosclerotic lesion formation were measured in LDLR(–/–) and sema4D(–/–)LDLR(–/–) mice after 6 months on a high-fat diet. All of the mice developed the dyslipidemia expected on this diet in the absence of functional LDL receptors. However, when compared to LDLR(–/–) mice, sema4D(–/–) LDLR(–/–) mice had reduced lipid deposition in the descending aorta, a 6-fold decrease in the frequency of arterial occlusion and a reduction to near wild-type levels in the accumulation of platelets after injury. These differences were retained ex vivo, with a marked decrease in platelet accumulation on collagen under flow and in platelet aggregation. Conclusions— These results show that loss of sema4D expression reduces the platelet hyperactivity otherwise found in dyslipidemia, and confers protection against the development of atherosclerosis.

[Integrative Physiology/Experimental Medicine] Inactivation of the Adenosine A2A Receptor Protects Apolipoprotein E-Deficient Mice From Atherosclerosis

Background— Atherosclerosis is a chronic inflammatory disease of the arterial vessel wall. The A2A receptor (A2AR) plays a central role in many antiinflammatory effects of adenosine. However, the role of A2AR in atherosclerosis is not clear. Methods and Results— The knockout of A2AR in apolipoprotein E–deficient (Apoe–/–/A2AR–/–) mice led to an increase in body weight and levels of blood cholesterol and proinflammatory cytokines, as well as the inflammation status of atherosclerotic lesions. Unexpectedly, Apoe–/–/A2AR–/– mice developed smaller lesions, as did chimeric Apoe–/– mice lacking A2AR in bone marrow–derived cells (BMDCs). The lesions of those mice exhibited a low density of foam cells and the homing ability of A2AR-deficient monocytes did not change. Increased foam cell apoptosis was detected in atherosclerotic lesions of Apoe–/–/A2AR–/– mice. In the absence of A2AR, macrophages incubated with oxidized LDL or in vivo–formed foam cells also exhibited increased apoptosis. A2AR deficiency in foam cells resulted in an increase in p38 mitogen–activated protein kinase (MAPK) activity. Inhibition of p38 phosphorylation abrogated the increased apoptosis of A2AR-deficient foam cells. Conclusion— Inactivation of A2AR, especially in BMDCs, inhibits the formation of atherosclerotic leisons, suggesting that A2AR inactivation may be useful for the treatment of atherosclerosis.

[Integrative Physiology/Experimental Medicine] Core2 1-6-N-Glucosaminyltransferase-I Deficiency Protects Injured Arteries From Neointima Formation in ApoE-Deficient Mice

Objective— Core2 1 to 6-N-glucosaminyltransferase-I (C2GlcNAcT-I) plays an important role in optimizing the binding functions of several selectin ligands, including P-selectin glycoprotein ligand. We used apolipoprotein E (ApoE)-deficient atherosclerotic mice to investigate the role of C2GlcNAcT-I in platelet and leukocyte interactions with injured arterial walls, in endothelial regeneration at injured sites, and in the formation of arterial neointima. Methods and Results— Arterial neointima induced by wire injury was smaller in C2GlcNAcT-I-deficient apoE–/– mice than in control apoE–/– mice (a 79% reduction in size). Compared to controls, apoE–/– mice deficient in C2GlcNAcT-I also demonstrated less leukocyte adhesion on activated platelets in microflow chambers (a 75% reduction), and accumulation of leukocytes at injured areas of mouse carotid arteries was eliminated. Additionally, endothelial regeneration in injured lumenal areas was substantially faster in C2GlcNAcT-I-deficient apoE–/– mice than in control apoE–/– mice. Endothelial regeneration was associated with reduced accumulation of platelet factor 4 (PF4) at injured sites. PF4 deficiency accelerated endothelial regeneration and protected mice from neointima formation after arterial injury. Conclusions— C2GlcNAcT-I deficiency suppresses injury-induced arterial neointima formation, and this effect is attributable to decreased leukocyte recruitment to injured vascular walls and increased endothelial regeneration. Both C2GlcNAcT-I and PF4 are promising targets for the treatment of arterial restenosis.

[Integrative Physiology/Experimental Medicine] Insulin Increases Reendothelialization and Inhibits Cell Migration and Neointimal Growth After Arterial Injury

Objective— Insulin has both growth-promoting and protective vascular effects in vitro, however the predominant effect in vivo is unclear. We investigated the effects of insulin in vivo on neointimal growth after arterial injury. Methods and Results— Rats were given subcutaneous control (C) or insulin implants (3U/d;I) 3 days before arterial (carotid or aortic) balloon catheter injury. Normoglycemia was maintained by oral glucose and, after surgery, by intraperitoneal glucose infusion (saline in C). Insulin decreased intimal area (P<0.01) but did not change intimal cell proliferation or apoptosis. However, insulin inhibited cell migration into the intima (P<0.01) and increased expression of smooth muscle cell (SMC) differentiation markers (P<0.05). Insulin also increased reendothelialization (P<0.01) and the number of circulating progenitor cells (P<0.05). Conclusions— These results are the first demonstration that insulin has a protective effect on both SMC and endothelium in vivo, resulting in inhibition of neointimal growth after vessel injury.

[Integrative Physiology/Experimental Medicine] PECAM-1 Is Necessary for Flow-Induced Vascular Remodeling

Objective— Vascular remodeling is a physiological process that occurs in response to long-term changes in hemodynamic conditions, but may also contribute to the pathophysiology of intima-media thickening (IMT) and vascular disease. Shear stress detection by the endothelium is thought to be an important determinant of vascular remodeling. Previous work showed that platelet endothelial cell adhesion molecule-1 (PECAM-1) is a component of a mechanosensory complex that mediates endothelial cell (EC) responses to shear stress. Methods and Results— We tested the hypothesis that PECAM-1 contributes to vascular remodeling by analyzing the response to partial carotid artery ligation in PECAM-1 knockout mice and wild-type littermates. PECAM-1 deficiency resulted in impaired vascular remodeling and significantly reduced IMT in areas of low flow. Inward remodeling was associated with PECAM-1-dependent NFB activation, surface adhesion molecule expression, and leukocyte infiltration as well as Akt activation and vascular cell proliferation. Conclusions— PECAM-1 plays a crucial role in the activation of the NFB and Akt pathways and inflammatory cell accumulation during vascular remodeling and IMT. Elucidation of some of the signals that drive vascular remodeling represent pharmacologically tractable targets for the treatment of restenosis after balloon angioplasty or stent placement.

[Integrative Physiology/Experimental Medicine] Fibronectin Is an Important Regulator of Flow-Induced Vascular Remodeling

Objective— Fibronectin is an important regulator of cell migration, differentiation, growth, and survival. Our data show that fibronectin also plays an important role in regulating extracellular matrix (ECM) remodeling. Fibronectin circulates in the plasma and is also deposited into the ECM by a cell dependent process. To determine whether fibronectin affects vascular remodeling in vivo, we asked whether the fibronectin polymerization inhibitor, pUR4, inhibits intima-media thickening, and prevents excess ECM deposition in arteries using a mouse model of vascular remodeling. Methods and Results— To induce vascular remodeling, partial ligation of the left external and internal carotid arteries was performed in mice. pUR4 and the control peptide were applied periadventitially in pluronic gel immediately after surgery. Animals were euthanized 7 or 14 days after surgery. Morphometric analysis demonstrated that the pUR4 fibronectin inhibitor reduced carotid intima (63%), media (27%), and adventitial thickening (40%) compared to the control peptide (III-11C). Treatment with pUR4 also resulted in a dramatic decrease in leukocyte infiltration into the vessel wall (80%), decreased ICAM-1 and VCAM-1 levels, inhibited cell proliferation (60% to 70%), and reduced fibronectin and collagen I accumulation in the vessel wall. In addition, the fibronectin inhibitor prevented SMC phenotypic modulation, as evidenced by the maintenance of smooth muscle (SM) -actin and SM myosin heavy chain levels in medial cells. Conclusions— These data are the first to demonstrate that fibronectin plays an important role in regulating the vascular remodeling response. Collectively, these data suggest a therapeutic benefit of periadventitial pUR4 in reducing pathological vascular remodeling.

[Integrative Physiology/Experimental Medicine] Reduced Collagen Biosynthesis Is the Hallmark of Cerebral Aneurysm: Contribution of Interleukin-1{beta} and Nuclear Factor-{kappa}B

Background— Reduced extracellular matrix is a prominent feature of cerebral aneurysms (CAs). We previously reported excessive ECM degradation in CA walls. In the present study, we examined collagen biosynthesis in CA walls and the molecular mechanisms underlying it in CA progression. Methods and Results— RT-PCR and immunohistochemistry showed reduced expression of procollagen type I, III, and lysyl oxidase (LOX) in CA walls. Treatment with the LOX inhibitor β-aminopropionitrile resulted in enhanced progression of CA. Expression of procollagen type I, III, and LOX was inhibited by interleukin-1β (IL-1β) in cultured rat aortic smooth muscle cells (RASMCs) in vitro. Nuclear factor -B (NF-B) was activated in IL-1β-stimulated RASMCs, and treatment with NF-B decoy oligodeoxynucleotides (ODN) restored reduced expression of procollagen type I, III, and LOX in vitro. NF-B decoy ODNs ameliorated the expression of procollagen type I, III, and LOX in CA walls in vivo. Conclusions— Collagen biosynthesis was significantly inhibited at the transcriptional level and in the posttranscriptional enzymatic modification in CA walls through upregulated expression of IL-1β and the NF-B pathway. Reduced collagen biosynthesis may contribute to CA progression, and inhibition of this process may lead to the prevention of the progression and rupture of CAs.

[Integrative Physiology/Experimental Medicine] Activated Protein C Protects Against Myocardial Ischemia/ Reperfusion Injury via Inhibition of Apoptosis and Inflammation

Objective— In spite of major advances in reperfusion therapy for patients presenting with acute coronary syndrome, long-term morbidity is still substantial. A limitation of initial treatment of myocardial ischemia is the lack of prevention of ischemia/reperfusion (I/R) injury. Activated protein C (APC), a crucial mediator in the coagulation process, plays a prominent role in the crosstalk between coagulation and inflammation and provides cytoprotective effects via inhibition of apoptosis and inflammation in several human and animal studies. Methods and Results— APC was administered in an animal model for myocardial I/R. APC largely inhibited early myocardial I/R injury after varying reperfusion times, an effect that was absent on administration of heparin, a nonspecific anticoagulant agent. The protective effects of APC were absent in case of absence or blockade of protease activated receptor-1 (PAR-1), indicating a critical role for PAR-1 in this process. Furthermore, we showed a strong antiapoptotic effect of APC in the early phase of reperfusion combined with an antiinflammatory effect at an early stage (IL-6), as well as at a later stage (leukocyte infiltration). Conclusions— APC exerts strong protective effects on early myocardial I/R injury, primarily via inhibition of apoptosis and inflammation, which are regulated via PAR-1.

[Integrative Physiology/Experimental Medicine] Preconditioning by Mitochondrial Reactive Oxygen Species Improves the Proangiogenic Potential of Adipose-Derived Cells-Based Therapy

Objective— Transplantation of adipose-derived stroma cells (ADSCs) stimulates neovascularization after experimental ischemic injury. ADSC proangiogenic potential is likely mediated by their ability to differentiate into endothelial cells and produce a wide array of angiogenic and antiapoptotic factors. Mitochondrial reactive oxygen species (ROS) have been shown to control ADSC differentiation. We therefore hypothesized that mitochondrial ROS production may change the ADSC proangiogenic properties. Methods and Results— The use of pharmacological strategies (mitochondrial inhibitors, antimycin, and rotenone, with or without antioxidants) allowed us to specifically and precisely modulate mitochondrial ROS generation in ADSCs. We showed that transient stimulation of mitochondrial ROS generation in ADSCs before their injection in ischemic hindlimb strongly improved revascularization and the number of ADSC-derived CD31-positive cells in ischemic area. Mitochondrial ROS generation increased the secretion of the proangiogenic and antiapoptotic factors, VEGF and HGF, but did not affect ADSC ability to differentiate into endothelial cells, in vitro. Moreover, mitochondrial ROS-induced ADSC preconditioning greatly protect ADSCs against oxidative stress-induced cell death. Conclusion— Our study demonstrates that in vitro preconditioning by moderate mitochondrial ROS generation strongly increases in vivo ADSC proangiogenic properties and emphasizes the crucial role of mitochondrial ROS in ADSC fate.

[Cell Biology/Signaling] Induction and Isolation of Vascular Cells From Human Induced Pluripotent Stem Cells--Brief Report

Objective— Induced pluripotent stem (iPS) cells are a novel stem cell population derived from human adult somatic cells through reprogramming using a defined set of transcription factors. Our aim was to determine the features of the directed differentiation of human iPS cells into vascular endothelial cells (ECs) and mural cells (MCs), and to compare that process with human embryonic stem (hES) cells. Methods and Results— We previously established a system for differentiating hES cells into vascular cells. We applied this system to human iPS cells and examined their directed differentiation. After differentiation, TRA1–60– Flk1+ cells emerged and divided into VE-cadherin-positive and -negative populations. The former were also positive for CD34, CD31, and eNOS and were consistent with ECs. The latter differentiated into MCs, which expressed smooth muscle -actin and calponin after further differentiation. The efficiency of the differentiation was comparable to that of human ES cells. Conclusions— We succeeded in inducing and isolating human vascular cells from iPS cells and indicate that the properties of human iPS cell differentiation into vascular cells are nearly identical to those of hES cells. This work will contribute to our understanding of human vascular differentiation/development and to the development of vascular regenerative medicine.

[Cell Biology/Signaling] Notch Signaling Induces Osteogenic Differentiation and Mineralization of Vascular Smooth Muscle Cells: Role of Msx2 Gene Induction via Notch-RBP-Jk Signaling

Objective— Vascular calcification is closely correlated with cardiovascular morbidity and mortality. Here, we demonstrate the role of Notch signaling in osteogenic differentiation and mineralization of vascular smooth muscle cells (SMCs). Methods and Results— The Msx2 gene, a key regulator of osteogenesis, was highly induced by coculture with Notch ligand-expressing cells or overexpression of Notch intracellular domains (NICDs) in human aortic SMCs (HASMCs). Furthermore, the Notch1 intracellular domain (N1-ICD) overexpression markedly upregulated alkaline phosphatase (ALP) activity and matrix mineralization of HASMCs. A knockdown experiment with a small interfering RNA confirmed that Msx2 mediated N1-ICD–induced osteogenic conversion of HASMCs. Interestingly, Msx2 induction by N1-ICD was independent of bone morphogenetic protein–2 (BMP-2), an osteogenic morphogen upstream of Msx2. The transcriptional activity of the Msx2 promoter was significantly enhanced by N1-ICD overexpression. The RBP-Jk binding element within the Msx2 promoter was critical to Notch-induced Msx2 gene expression. Correspondingly, N1-ICD overexpression did not induce the Msx2 expression in RBP-Jk–deficient fibroblasts. Immunohistochemistry of human carotid artery specimens revealed localization of Notch1, Jagged1 and Msx2 to fibrocalcific atherosclerotic plaques. Conclusion— These results imply a new mechanism for osteogenic differentiation of vascular SMCs in which Notch/RBP-Jk signaling directly induces Msx2 gene expression and suggest its crucial role in mediating vascular calcification.

[Cell Biology/Signaling] Sonic Hedgehog Induces Notch Target Gene Expression in Vascular Smooth Muscle Cells via VEGF-A

Objective— Notch, VEGF, and components of the Hedgehog (Hh) signaling pathway have been implicated in vascular morphogenesis. The role of Notch in mediating hedgehog control of adult vascular smooth muscle cell (SMC) growth and survival remains unexplored. Methods and Results— In cultured SMCs, activation of Hh signaling with recombinant rShh (3.5 µg/mL) or plasmid encoded Shh increased Ptc1 expression, enhanced SMC growth and survival and promoted Hairy-related transcription factor (Hrt) expression while concomitantly increasing VEGF-A levels. These effects were significantly reversed after Hh inhibition with cyclopamine. Shh-induced stimulation of Hrt-3 mRNA and SMC growth and survival was attenuated after inhibition of Notch-mediated CBF-1/RBP-Jk-dependent signaling with RPMS-1 while siRNA knockdown of Hrt-3 inhibited SMC growth and survival. Recombinant VEGF-A increased Hrt-3 mRNA levels while siRNA knockdown abolished rShh stimulated VEGF-A expression while concomitantly inhibiting Shh-induced increases in Hrt-3 mRNA levels, proliferating cell nuclear antigen (PCNA), and Notch 1 IC expression, respectively. Hedgehog components were expressed within intimal SMCs of murine carotid arteries after vascular injury concomitant with a significant increase in mRNA for Ptc1, Gli2, VEGF-A, Notch 1, and Hrts. Conclusion— Hedgehog promotes a coordinate regulation of Notch target genes in adult SMCs via VEGF-A.

[Cell Biology/Signaling] Rab GTPase Regulation of VEGFR2 Trafficking and Signaling in Endothelial Cells

Objective— Vascular endothelial growth factor receptor 2 (VEGFR2) is a receptor tyrosine kinase that regulates vascular physiology. However, mechanism(s) by which VEGFR2 signaling and trafficking is coordinated are not clear. Here, we have tested endocytic Rab GTPases for regulation of VEGFR2 trafficking and signaling linked to endothelial cell migration. Methods and Results— Quiescent VEGFR2 displays endosomal localization and colocalization with the Rab5a GTPase, an early endosome fusion regulator. Expression of GTP or GDP-bound Rab5a mutants block activated VEGFR2 trafficking and degradation. Manipulation of Rab7a GTPase activity associated with late endosomes using overexpression of wild-type or mutant proteins blocks activated VEGFR2 trafficking and degradation. Depletion of Rab7a decreased VEGFR2 Y1175 phosphorylation but increased p42/44 (pERK1/2) MAPK phosphorylation. Endothelial cell migration was increased by Rab5a depletion but decreased by Rab7a depletion. Conclusions— Rab5a and Rab7a regulate VEGFR2 trafficking toward early and late endosomes. Our data suggest that VEGFR2-mediated regulation of endothelial function is dependent on different but specific Rab-mediated GTP hydrolysis activity required for endosomal trafficking.

[Cell Biology/Signaling] Stimulation of Cell Surface F1-ATPase Activity by Apolipoprotein A-I Inhibits Endothelial Cell Apoptosis and Promotes Proliferation

Objectives— Several findings argue for a protective effect of high-density lipoproteins (HDLs) against endothelial dysfunction. The molecular mechanisms underlying this protective effect are not fully understood, although recent works suggest that the actions of HDL on the endothelium are initiated by multiple interactions between HDLs (lipid or protein moiety) and cell surface receptors. We previously showed that the mitochondrial related F1-ATPase is a cell surface receptor for HDLs and their main atheroprotective apolipoprotein (apoA-I). Herein we test the hypothesis that the cell surface F1-ATPase may contribute to the ability of apoA-I and HDLs to maintain endothelial cell survival. Methods and Results— Cell imaging and binding assays confirmed the presence of the F1-ATPase at the surface of human umbilical vein endothelial cells (HUVECs) and its ability to bind apoA-I. Cell surface F1-ATPase activity (ATP hydrolysis into ADP) was stimulated by apoA-I and was inhibited by its specific inhibitor IF1-H49K. Furthermore the antiapoptotic and proliferative effects of apoA-I on HUVECs were totally blocked by the F1-ATPase ligands IF1-H49K, angiostatin and anti-βF1-ATPase antibody, independently of the scavenger receptor SR-BI and ABCA1. Conclusion— This study suggests an important contribution of cell surface F1-ATPase to apoA-I-mediated endothelial cell survival, which may contribute to the atheroprotective functions of apoA-I.

[Cell Biology/Signaling] COX-2 Limits Prostanoid Production in Activated HUVECs and Is a Source of PGH2 for Transcellular Metabolism to PGE2 by Tumor Cells

Objective— Inducible expression of cyclooxygenase-2 (COX-2) and terminal prostaglandin synthases (tPGS) has been mainly analyzed in tumor, stromal, and inflammatory cells, and little is known about the regulation of prostanoid biosynthesis by endothelial cells. Here we characterize the profile of prostanoids produced by activated HUVECs and analyze the expression and activities of tPGS. Methods and Results— Enzyme immunoassays indicated increased endothelial prostanoid production after proangiogenic stimulation, but without parallel upregulation of tPGS. Endothelial prostanoid production instead depended on the induction of COX-2 and was abolished by COX-2 silencing or pharmacological inhibition. COX-2 is functionally coupled to prostacyclin and thromboxane synthases in HUVECs, but these cells show no detectable PGE2 synthase (PGES) activity. Endothelial PGE2 production is partly mediated by nonenzymatic decomposition of COX-2-derived PGH2, but endothelial-produced PGH2 can also be metabolized enzymatically by microsomal PGES-1 in cocultured tumor cells. Conclusions— Our findings identify a novel transcellular metabolism of PGE2 between the endothelial and tumor compartments. Given the role of PGE2 as a mediator of COX-2 proangiogenic effects, transcellular metabolism of endothelial-derived PGH2 is a potential target for treatment of pathological angiogenesis.

[Cell Biology/Signaling] Augmentation of Megakaryocyte Expression of Fc{gamma}RIIa by Interferon {gamma}

Objective— The purpose of this study was to identify factors that alter expression of FcRIIa by megakaryocytes. Methods and Results— Effects of selected cytokines and growth factors on megakaryocyte expression of FcRIIa were assessed with phorbol 12-myristate 13-acetate (PMA)-differentiated human erythroleukemia (HEL) cells and with thrombopoietin-differentiated CD34 stem cells and compared with those obtained with myelocytic cell lines and a monocytic cell lines. Expression of FcRIIa was quantified with the use of Western blots and real-time reverse transcriptase-polymerase chain reaction. Megakaryocyte differentiation was identified by expression of CD41, CD42, and von Willebrand factor with the use of flow cytometry. Interferon (IFN) increased protein expression of FcRIIa by HEL cells and CD34 stem cells after megakaryocyte differentiation (maximal 4-fold, P<0.001 for each). IFN did not increase expression of FcRIIa by undifferentiated HEL and CD34 cells. Expression of FcRIIa mRNA was increased (2-fold, P<0.001) in megakaryocyte-differentiated HEL cells. IFN did not increase expression of FcRIIa by undifferentiated myelocytic or monocytic cell lines. Conclusions— IFN appears to selectively increase expression of FcRIIa by cells exhibiting characteristics of megakaryocytes. This effect of IFN may contribute to greater platelet expression of FcRIIa in patients with atherosclerotic vascular disease.

[Clinical and Population Studies] Serum Resistin Concentrations and Risk of New Onset Heart Failure in Older Persons: The Health, Aging, and Body Composition (Health ABC) Study

Objective— Resistin is associated with inflammation and insulin resistance and exerts direct effects on myocardial cells including hypertrophy and altered contraction. We investigated the association of serum resistin concentrations with risk for incident heart failure (HF) in humans. Methods and Results— We studied 2902 older persons without prevalent HF (age, 73.6±2.9 years; 48.1% men; 58.8% white) enrolled in the Health, Aging, and Body Composition (Health ABC) Study. Correlation between baseline serum resistin concentrations (20.3±10.0 ng/mL) and clinical variables, biochemistry panel, markers of inflammation and insulin resistance, adipocytokines, and measures of adiposity was weak (all rho <0.25). During a median follow-up of 9.4 years, 341 participants (11.8%) developed HF. Resistin was strongly associated with risk for incident HF in Cox proportional hazards models controlling for clinical variables, biomarkers, and measures of adiposity (HR, 1.15 per 10.0 ng/mL in adjusted model; 95% CI, 1.05 to 1.27; P=0.003). Results were comparable across sex, race, diabetes mellitus, and prevalent and incident coronary heart disease subgroups. In participants with available left ventricular ejection fraction at HF diagnosis (265 of 341; 77.7%), association of resistin with HF risk was comparable for cases with reduced versus preserved ejection fraction. Conclusions— Serum resistin concentrations are independently associated with risk for incident HF in older persons.

[Editorials] Ten Good Years

[Corrections] Correction

[Editorials] Calcineurin Finds a New Partner in the L-Type Ca2+ Channel

[Editorials] Limits to Growth of Native Collateral Vessels: Just One Mouse CLIC Away From Unlimited Collateral Perfusion?

[Reports] Avoidance of Transient Cardiomyopathy in Cardiomyocyte-Targeted Tamoxifen-Induced MerCreMer Gene Deletion Models

Cardiac myocyte targeted MerCreMer transgenic mice expressing tamoxifen-inducible Cre driven by the -myosin heavy chain promoter are increasingly used to control gene expression in the adult heart. Here, we show tamoxifen-mediated MerCreMer (MCM) nuclear translocation can induce severe transient dilated cardiomyopathy in mice with or without loxP transgenes. The cardiomyopathy is accompanied by marked reduction of energy/metabolism and calcium-handling gene expression (eg, PGC1-, peroxisome proliferator-activated , SERCA2A), all fully normalized with recovery. MCM-negative/flox-positive controls display no dysfunction with tamoxifen. Nuclear Cre translocation and equally effective gene knockdown without cardiomyopathy is achievable with raloxifene, suggesting toxicity is not simply from Cre. Careful attention to controls, reduced tamoxifen dosing and/or use of raloxifene is advised with this model.

[Molecular Medicine] Peroxisome Proliferator-Activated Receptor {delta} Regulates Extracellular Matrix and Apoptosis of Vascular Smooth Muscle Cells Through the Activation of Transforming Growth Factor-{beta}1/Smad3

Homeostasis of the extracellular matrix and apoptosis of vascular smooth muscle cells (VSMCs) are key components in the regulation of the stability of atherosclerotic plaques. Here, we demonstrate that peroxisome proliferator-activated receptor (PPAR) regulates extracellular matrix synthesis and degradation through transforming growth factor-β1 and its effector, Smad3. Activation of PPAR strongly amplified the expression of types I and III collagen, fibronectin, elastin, and TIMP-3 (tissue inhibitor of metalloproteinases 3), but not of TIMP-1, matrix metalloproteinase-2 or -9. The effect of PPAR on the expression of type III collagen was dually regulated by the direct binding of PPAR and Smad3 to a direct repeat-1 site and a Smad-binding element, respectively, in the type III collagen gene promoter. The activation of PPAR attenuated apoptotic cell death in VSMCs induced by oxidized low-density lipoprotein, and similar antiapoptotic effects were observed on treatment of cells with exogenous type I and/or III collagen. Administration of a PPAR ligand GW501516 to mice also suppressed elastase-induced cell death of aortic VSMCs. These results suggest that PPAR-induced upregulation of extracellular matrix proteins exerts an antiapoptotic effect, thereby maintaining the stability of atherosclerotic plaques. Specific ligands of PPAR may aid in the therapeutic intervention of atherosclerosis by improving plaque stability and patient prognosis.

[Molecular Medicine] Activating Transcription Factor 3 Constitutes a Negative Feedback Mechanism That Attenuates Saturated Fatty Acid/Toll-Like Receptor 4 Signaling and Macrophage Activation in Obese Adipose Tissue

Obese adipose tissue is markedly infiltrated by macrophages, suggesting that they may participate in the inflammatory pathways that are activated in obese adipose tissue. Evidence has suggested that saturated fatty acids released via adipocyte lipolysis serve as a naturally occurring ligand that stimulates Toll-like receptor (TLR)4 signaling, thereby inducing the inflammatory responses in macrophages in obese adipose tissue. Through a combination of cDNA microarray analyses of saturated fatty acid-stimulated macrophages in vitro and obese adipose tissue in vivo, here we identified activating transcription factor (ATF)3, a member of the ATF/cAMP response element-binding protein family of basic leucine zipper-type transcription factors, as a target gene of saturated fatty acids/TLR4 signaling in macrophages in obese adipose tissue. Importantly, ATF3, when induced by saturated fatty acids, can transcriptionally repress tumor necrosis factor- production in macrophages in vitro. Chromatin immunoprecipitation assay revealed that ATF3 is recruited to the region containing the activator protein-1 site of the endogenous tumor necrosis factor- promoter. Furthermore, transgenic overexpression of ATF3 specifically in macrophages results in the marked attenuation of proinflammatory M1 macrophage activation in the adipose tissue from genetically obese KKAy mice fed high-fat diet. This study provides evidence that ATF3, which is induced in obese adipose tissue, acts as a transcriptional repressor of saturated fatty acids/TLR4 signaling, thereby revealing the negative feedback mechanism that attenuates obesity-induced macrophage activation. Our data also suggest that activation of ATF3 in macrophages offers a novel therapeutic strategy to prevent or treat obesity-induced adipose tissue inflammation.

[Molecular Medicine] Nitric Oxide-Independent Vasodilator Rescues Heme-Oxidized Soluble Guanylate Cyclase From Proteasomal Degradation

Nitric oxide (NO) is an essential vasodilator. In vascular diseases, oxidative stress attenuates NO signaling by both chemical scavenging of free NO and oxidation and downregulation of its major intracellular receptor, the β heterodimeric heme-containing soluble guanylate cyclase (sGC). Oxidation can also induce loss of the heme of sGC, as well as the responsiveness of sGC to NO. sGC activators such as BAY 58-2667 bind to oxidized/heme-free sGC and reactivate the enzyme to exert disease-specific vasodilation. Here, we show that oxidation-induced downregulation of sGC protein extends to isolated blood vessels. Mechanistically, degradation was triggered through sGC ubiquitination and proteasomal degradation. The heme-binding site ligand BAY 58-2667 prevented sGC ubiquitination and stabilized both and β subunits. Collectively, our data establish oxidation–ubiquitination of sGC as a modulator of NO/cGMP signaling and point to a new mechanism of action for sGC activating vasodilators by stabilizing their receptor, oxidized/heme-free sGC.

[Cellular Biology] Angiotensin II-Mediated Adaptive and Maladaptive Remodeling of Cardiomyocyte Excitation-Contraction Coupling

Cardiac hypertrophy is associated with alterations in cardiomyocyte excitation–contraction coupling (ECC) and Ca2+ handling. Chronic elevation of plasma angiotensin II (Ang II) is a major determinant in the pathogenesis of cardiac hypertrophy and congestive heart failure. However, the molecular mechanisms by which the direct actions of Ang II on cardiomyocytes contribute to ECC remodeling are not precisely known. This question was addressed using cardiac myocytes isolated from transgenic (TG1306/1R [TG]) mice exhibiting cardiac specific overexpression of angiotensinogen, which develop Ang II–mediated cardiac hypertrophy in the absence of hemodynamic overload. Electrophysiological techniques, photolysis of caged Ca2+ and confocal Ca2+ imaging were used to examine ECC remodeling at early (20 weeks of age) and late (60 weeks of age) time points during the development of cardiac dysfunction. In young TG mice, increased cardiac Ang II levels induced a hypertrophic response in cardiomyocyte, which was accompanied by an adaptive change of Ca2+ signaling, specifically an upregulation of the Na+/Ca2+ exchanger–mediated Ca2+ transport. In contrast, maladaptation was evident in older TG mice, as suggested by reduced sarcoplasmic reticulum Ca2+ content resulting from a shift in the ratio of plasmalemmal Ca2+ removal and sarcoplasmic reticulum Ca2+ uptake. This was associated with a conserved ECC gain, consistent with a state of hypersensitivity in Ca2+-induced Ca2+ release. Together, our data suggest that chronic elevation of cardiac Ang II levels significantly alters cardiomyocyte ECC in the long term, and thereby contractility, independently of hemodynamic overload and arterial hypertension.

[Cellular Biology] Physical and Functional Interaction Between Calcineurin and the Cardiac L-Type Ca2+ Channel

The L-type Ca2+ channel (LTCC) is the major mediator of Ca2+ influx in cardiomyocytes, leading to both mechanical contraction and activation of signaling cascades. Among these Ca2+-activated cascades is calcineurin, a protein phosphatase that promotes hypertrophic growth of the heart. Coimmunoprecipitations from heart extracts and pulldowns using heterologously expressed proteins provided evidence for direct binding of calcineurin at both the N and C termini of 11.2. At the C terminus, calcineurin bound specifically at amino acids 1943 to 1971, adjacent to a well-characterized protein kinase (PK)A/PKC/PKG phospho-acceptor site Ser1928. In vitro assays demonstrated that calcineurin can dephosphorylate 11.2. Channel function was increased in voltage-clamp recordings of ICa,L from cultured cardiomyocytes expressing constitutively active calcineurin, consistent with previous observations in cardiac hypertrophy in vivo. Conversely, acute suppression of calcineurin pharmacologically or with specific peptides decreased ICa,L. These data reveal direct physical interaction between the LTCC and calcineurin in heart. Furthermore, they demonstrate that calcineurin induces robust increases in ICa,L and highlight calcineurin as a key modulator of pathological electrical remodeling in cardiac hypertrophy.

[Cellular Biology] Gene Expression Profiling of the Forming Atrioventricular Node Using a Novel Tbx3-Based Node-Specific Transgenic Reporter

The atrioventricular (AV) node is a recurrent source of potentially life-threatening arrhythmias. Nevertheless, limited data are available on its developmental control or molecular phenotype. We used a novel AV nodal myocardium–specific reporter mouse to gain insight into the gene programs determining the formation and phenotype of the developing AV node. In this reporter, green fluorescent protein (GFP) expression was driven by a 160-kbp bacterial artificial chromosome with Tbx3 and flanking sequences. GFP was selectively active in the AV canal of embryos and AV node of adults, whereas the Tbx3-positive AV bundle and sinus node were devoid of GFP, demonstrating that distinct regulatory sequences and pathways control expression in the components of the conduction system. Fluorescent AV nodal and complementary Nppa-positive chamber myocardial cell populations of embryonic day 10.5 embryos and of embryonic day 17.5 fetuses were purified using fluorescence-activated cell sorting, and their expression profiles were assessed by genome-wide microarray analysis, providing valuable information concerning their molecular identities. We constructed a comprehensive list of sodium, calcium, and potassium channel genes specific for developing nodal or chamber myocardium. Furthermore, the data revealed that the AV node and the chamber (working) myocardium phenotypes diverge during development but that the functional gene classes characterizing both subtypes are maintained. One of the repertoires identified in the AV node–specific gene profiles consists of multiple neurotrophic factors and semaphorins, not yet appreciated to play a role in nodal development, revealing shared characteristics between nodal and nervous system development.

[Integrative Physiology] The I{kappa}B Kinase {beta}/Nuclear Factor {kappa}B Signaling Pathway Protects the Heart From Hemodynamic Stress Mediated by the Regulation of Manganese Superoxide Dismutase Expression

Cardiomyocyte death plays an important role in the pathogenesis of heart failure. The nuclear factor (NF)-B signaling pathway regulates cell death, however, the effect of NF-B pathway on cell death can vary in different cells or stimuli. The purpose of the present study was to clarify the in vivo role of the NF-B pathway in response to pressure overload. First, we subjected C57Bl6/J mice to pressure overload by means of transverse aortic constriction (TAC) and examined the activity of the NF-B pathway in response to pressure overload. IB kinase (IKK) and NF-B were activated after TAC. Then, we investigated the role of the activation using cardiac-specific IKKβ-deficient mice (CKO). CKO displayed normal global cardiac structure and function compared with control littermates. We subjected CKO and control mice to pressure overload. One week after TAC, CKO showed cardiac dilation, dysfunction, and lung congestion, which are characteristics of heart failure. The number of apoptotic cells in the hearts of CKO mice increased significantly after TAC. The levels of manganese superoxide dismutase mRNA and protein expression in CKO after TAC were significantly attenuated compared with control mice. The levels of oxidative stress and c-Jun N-terminal kinase (JNK) activation in CKO after TAC were significantly greater than those in control mice. Isoproterenol-induced cell death of isolated adult CKO cardiomyocytes was inhibited by treatment with either a manganese superoxide dismutase mimetic or a JNK inhibitor. Thus, the IKKβ/NF-B signaling pathway plays a protective role in cardiomyocytes because of the attenuation of oxidative stress and JNK activation in a setting of acute pressure overload.

[Integrative Physiology] Cardiac Muscle Ring Finger-1 Increases Susceptibility to Heart Failure In Vivo

Muscle ring finger (MuRF)1 is a muscle-specific protein implicated in the regulation of cardiac myocyte size and contractility. MuRF2, a closely related family member, redundantly interacts with protein substrates and heterodimerizes with MuRF1. Mice lacking either MuRF1 or MuRF2 are phenotypically normal, whereas mice lacking both proteins develop a spontaneous cardiac and skeletal muscle hypertrophy, indicating cooperative control of muscle mass by MuRF1 and MuRF2. To identify the unique role that MuRF1 plays in regulating cardiac hypertrophy in vivo, we created transgenic mice expressing increased amounts of cardiac MuRF1. Adult MuRF1 transgenic (Tg+) hearts exhibited a nonprogressive thinning of the left ventricular wall and a concomitant decrease in cardiac function. Experimental induction of cardiac hypertrophy by transaortic constriction (TAC) induced rapid failure of MuRF1 Tg+ hearts. Microarray analysis identified that the levels of genes associated with metabolism (and in particular mitochondrial processes) were significantly altered in MuRF1 Tg+ hearts, both at baseline and during the development of cardiac hypertrophy. Surprisingly, ATP levels in MuRF1 Tg+ mice did not differ from wild-type mice despite the depressed contractility following TAC. In comparing the level and activity of creatine kinase (CK) between wild-type and MuRF1 Tg+ hearts, we found that mCK and CK-M/B protein levels were unaffected in MuRF1 Tg+ hearts; however, total CK activity was significantly inhibited. We conclude that increased expression of cardiac MuRF1 results in a broad disruption of primary metabolic functions, including alterations in CK activity that leads to increased susceptibility to heart failure following TAC. This study demonstrates for the first time a role for MuRF1 in the regulation of cardiac energetics in vivo.

[Integrative Physiology] Chloride Intracellular Channel-4 Is a Determinant of Native Collateral Formation in Skeletal Muscle and Brain

The capacity of the collateral circulation to lessen injury in occlusive vascular disease depends on the density and caliber of native (preexisting) collaterals, as well as their ability to outwardly remodel in ischemia. Native collateral conductance varies widely among healthy individuals, yet little is known about what specifies collateral formation. Chloride intracellular channel (CLIC)4 protein is required for endothelial cell hollowing, a process necessary for vessel formation during embryogenesis and ischemia. Whether CLIC4 has other physiological roles in vascular biology is uncertain. We studied collateral formation and remodeling in mice deficient in CLIC1 and CLIC4. Vascular responses to femoral artery ligation were similar in Clic1–/– and wild-type mice. In contrast, immediately after ligation perfusion dropped more in Clic4–/– than wild-type mice, suggesting fewer preexisting collaterals, a finding confirmed by angiography, greater ischemia, and worse recovery of perfusion; however, collateral remodeling was unaffected. Likewise, native cerebral collateral density in Clic4–/– (but not Clic1–/–) mice was reduced, resulting in severe infarctions. This was associated with impaired perinatal formation and stabilization of nascent collaterals. Clic4 hemizygous mice had intermediate deficits in the above parameters, suggesting a gene-dose effect. Ischemia augmented CLIC1 and CLIC4 expression similarly in wild-type mice. However, CLIC1 increased 3-fold more in Clic4–/– mice, suggesting compensation. Despite greater ischemia in Clic4–/– mice, hypoxia-inducible factor-1, vascular endothelial growth factor (VEGF) and angiopoietin-2 increased less compared to wild-type, suggesting CLIC4 exerts influences upstream of hypoxia-inducible factor-1-VEGF signaling. Hence, CLIC4 represents the second gene that, along with VEGF shown by us previously, specifies native collateral formation.

[Integrative Physiology] MIF Deficiency Reduces Chronic Inflammation in White Adipose Tissue and Impairs the Development of Insulin Resistance, Glucose Intolerance, and Associated Atherosclerotic Disease

Chronic inflammation in white adipose tissue (WAT) is positively associated with obesity, insulin resistance (IR) and the development of type 2 diabetes. The proinflammatory cytokine MIF (macrophage migration inhibitory factor) is an essential, upstream component of the inflammatory cascade. This study examines whether MIF is required for the development of obesity, IR, glucose intolerance, and atherosclerosis in the LDL receptor-deficient (Ldlr–/–) mouse model of disease. Ldlr–/– mice develop IR and glucose intolerance within 15 weeks, whereas Mif–/–Ldlr–/– littermates are protected. MIF deficiency does not affect obesity and lipid risk factors but specifically reduces inflammation in WAT and liver, as reflected by lower plasma serum amyloid A and fibrinogen levels at baseline and under inflammatory conditions. Conversely, MIF stimulates the in vivo expression of human C-reactive protein, an inflammation marker and risk factor of IR and cardiovascular disease. In WAT, MIF deficiency reduces nuclear c-Jun levels and improves insulin sensitivity; MIF deficiency also reduces macrophage accumulation in WAT and blunts the expression of two proteins that regulate macrophage infiltration (intercellular adhesion molecule-1, CD44). Mechanistic parallels to WAT were observed in aorta, where the absence of MIF reduces monocyte adhesion, macrophage lesion content, and atherosclerotic lesion size. These data highlight the physiological importance of chronic inflammation in development of IR and atherosclerosis and suggest that MIF is a potential therapeutic target for reducing the inflammatory component of metabolic and cardiovascular disorders.