Journals RSS : Gourt

Home Blood Pressure Measurements Will or Will Not Replace 24-Hour Ambulatory Blood Pressure Measurement [Letters to the Editor]

Response to Home Blood Pressure Measurements Will or Will Not Replace 24-Hour Ambulatory Blood Pressure Measurement [Letters to the Editor]

Flawed Measurement of Brachial Tonometry for Calculating Aortic Pressure? [Letters to the Editor]

Response to Flawed Measurement of Brachial Tonometry for Calculating Aortic Pressure? [Letters to the Editor]

Long-Term Risk in Subjects With White-Coat Hypertension [Letters to the Editor]

Response to Long-Term Risk in Subjects With White-Coat Hypertension [Letters to the Editor]

A Novel Measurement Index for Antihypertensive Medication Burden and Its Use [Letters to the Editor]

Determination of Travel Distance for Noninvasive Measurement of Pulse Wave Velocity: Case Closed? [Letters to the Editor]

Correction [Corrections]

Thomas G. Pickering: 1940-2009 [In Memoriam]

Noninvasive Assessment of Subclinical Atherosclerosis in Children and Adolescents: Recommendations for Standard Assessment for Clinical Research: A Scientific Statement From the American Heart Association [AHA Scientific Statement]

Deterioration in endothelial function and arterial stiffness are early events in the development of cardiovascular diseases. In adults, noninvasive measures of atherosclerosis have become established as valid and reliable tools for refining cardiovascular risk to target individuals who need early intervention. With limited pediatric data, the use of these techniques in children and adolescents largely has been reserved for research purposes. Therefore, this scientific statement was written to (1) review the current literature on the noninvasive assessment of atherosclerosis in children and adolescents, (2) make recommendations for the standardization of these tools for research, and (3) stimulate further research with a goal of developing valid and reliable techniques with normative data for noninvasive clinical evaluation of atherosclerosis in pediatric patients. Precise and reliable noninvasive tests for atherosclerosis in youth will improve our ability to estimate future risk for heart attack and stroke. Currently, large longitudinal studies of cardiovascular risk factors in youth, such as the Bogalusa and Muscatine studies, lack sufficient adult subjects experiencing hard outcomes, such as heart attack and stroke, to produce meaningful risk scores like those developed from Framingham data.

The Choice of Thiazide Diuretics: Why Chlorthalidone May Replace Hydrochlorothiazide [Editorials]

Does Prehypertension Represent an Increased Risk for Incident Hypertension and Adverse Cardiovascular Outcome? [Editorial Commentaries]

Newly Reported Hypertension After Military Combat Deployment: Research Implications From a Biopsychosocial Perspective [Editorial Commentaries]

Wave Intensity Analysis and Central Blood Pressure [Editorial Commentaries]

Exogenous Ghrelin on Nitric Oxide-Endothelin 1 Imbalance in Metabolic Syndrome: Can We Kill 2 Birds With 1 Stone? [Editorial Commentaries]

Yes, No, Maybe So: ENaC Proteins as Mediators of Renal Myogenic Constriction [Editorial Commentaries]

A New PIXel in the Puzzle: How Increased Vascular Pressure Induces Oxidative Stress [Editorial Commentaries]

Newly Reported Hypertension After Military Combat Deployment in a Large Population-Based Study [Original Articles]

High-stress situations, such as combat deployments, are a potential risk factor for hypertension. Although stress is postulated to increase blood pressure, the underlying role of stress on hypertension is not well established. We sought to determine the relations between combat deployment–induced stress and hypertension. The Millennium Cohort baseline questionnaire (2001–2003) was completed by 77 047 US active-duty and Reserve/National Guard members. Follow-up was completed by 55 021 responders 3 years later (2004–2006). Multivariable logistic regression was used to estimate the 3-year risk of newly reported hypertension, adjusting for general and mental health, demographics, and occupational and behavioral characteristics. After applying exclusion criteria, our analyses included 36 061 service members. Subanalyses of deployers included 8829 participants. Newly reported hypertension was identified in 6.9% of the cohort between baseline and follow-up, many of whom had deployed on military operations in support of the conflicts in Iraq and Afghanistan. After adjusting, deployers who experienced no combat exposures were less likely to report hypertension than nondeployers (odds ratio: 0.77; 95% CI: 0.67 to 0.89). Among deployers, those reporting multiple combat exposures were 1.33 times more likely to report hypertension compared with noncombat deployers (95% CI: 1.07 to 1.65). Although military deployers, in general, had a lower incidence of hypertension than nondeployers, deployment with multiple stressful combat exposures appeared to be a unique risk factor for newly reported hypertension.

Cardiovascular and Metabolic Predictors of Progression of Prehypertension Into Hypertension: The Strong Heart Study [Original Articles]

Prehypertension (defined by the Seventh Report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure) frequently evolves to hypertension (HTN) and increases cardiovascular risk. It is unclear whether metabolic and/or cardiac characteristics favor development of HTN in prehypertensive subjects. We evaluated baseline anthropometric, laboratory, and echocardiographic characteristics of 625 untreated prehypertensive participants in the Strong Heart Study, without prevalent cardiovascular disease (63% women; 22% with diabetes mellitus; mean age: 59±7 years) to identify predictors of the 4-year incidence of HTN. Diabetes mellitus was assessed by American Diabetic Association criteria, and a diabetes-specific definition of HTN was used. Four-year incidence of HTN was 38%. Incident HTN was independently predicted by baseline systolic blood pressure (odds ratio [OR]: 1.60 per 10 mm Hg; 95% CI: 1.30 to 2.00; P<0.0001), waist circumference (OR: 1.10 per 10 cm; 95% CI: 1.01 to 1.30; P=0.04), and diabetes mellitus (OR: 2.73; 95% CI=1.77 to 4.21; P<0.0001), with no significant effect for age, sex, hemoglobin A1c, homeostatic model assessment index, C-reactive protein, fibrinogen, low-density lipoprotein and high-density lipoprotein cholesterol, triglycerides, plasma creatinine, or urine albumin:creatinine ratio. Higher left ventricular mass index (OR: 1.15 per 5 g/m2.7; 95% CI: 1.01 to 1.25; P=0.03) or stroke volume index (OR: 1.25 per 5 mL/m2.04; 95% CI: 1.10 to 1.50; P=0.03) was also identified, together with baseline systolic blood pressure and the presence of diabetes mellitus, as an independent predictor of incident HTN, without an additional predictive contribution from other anthropometric, metabolic, or echocardiographic parameters (all P>0.10). Thus, progression to HTN in 38% of Strong Heart Study prehypertensive participants could be predicted by higher left ventricular mass and stroke volume in addition to baseline systolic blood pressure and prevalent diabetes mellitus.

Environmental Mercury Exposure and Blood Pressure Among Nunavik Inuit Adults [Original Articles]

Epidemiological evidence suggests a negative impact of methylmercury on the cardiovascular system, but findings regarding the effect on blood pressure (BP) are not consistent. We aimed to study the impact of mercury levels on BP among Nunavik Inuit adults. The health survey Qanuippitaa? was conducted in Nunavik (northern Quebec, Canada), and data were obtained from 732 Inuit ≥18 years of age. Anthropometric blood samples, as well as systolic BP and diastolic BP, were assessed. Pulse pressure (systolic BP–diastolic BP) was calculated. Mercury blood concentration was used as a biomarker of recent exposure. Simple relations between mercury and BP parameters were studied by using the Pearson correlation, whereas multiple regressions were performed to control for confounders. Mean age of the participants was 34.3 years (95% CI: 33.6 to 34.9 years). Systolic BP, diastolic BP, and pulse pressure means were 117 mm Hg (95% CI: 116 to 118 mm Hg), 73 mm Hg (95% CI: 72 to 74 mm Hg), and 43 mm Hg (95% CI: 42 to 44 mm Hg), respectively. Mercury mean was 50.2 nmol/L. In multivariable analyses, mercury was associated with systolic BP (β=2.14; P=0.0004), whereas the association with diastolic BP was near the significance level (β=0.96; P=0.069). In conclusion, mercury is associated with increasing BP and pulse pressure among Nunavik Inuit adults after considering the effect of fish nutrients (n-3 fatty acids and selenium) and other confounders.

Early Progression of the Autonomic Dysfunction Observed in Pediatric Type 1 Diabetes Mellitus [Original Articles]

To focus on early cardiac and vascular autonomic dysfunction that might complicate type 1 diabetes mellitus in children, we planned an observational, cross-sectional study in a population of 93 young patients, under insulin treatment, subdivided in 2 age subgroups (children: 11.5±0.4 years; adolescents: 19.3±0.2 years). Time and frequency domain analysis of RR interval and systolic arterial pressure variability provided quantitative indices of the sympatho-vagal balance regulating the heart period, of the gain of cardiac baroreflex, and of the sympathetic vasomotor control. Sixty-eight children of comparable age served as a reference group. At rest, systolic arterial pressure and the power of its low-frequency component were greater in patients than in controls, particularly in children (14.0±2.3 versus 3.1±0.3 mm Hg2). Moreover, baroreflex gain was significantly reduced in both subgroups of patients. Standing induced similar changes in the autonomic profiles of controls and patients. A repeat study after 1 year showed a progression in low-frequency oscillations of arterial pressure and a shift toward low frequency in RR variability. Data in young patients with type 1 diabetes mellitus show a significant increase in arterial pressure, a reduced gain of the baroreflex regulation of the heart period, and an increase of the low-frequency component of systolic arterial pressure variability, suggestive of simultaneous impairment of vagal cardiac control and increases of sympathetic vasomotor regulation. A repeat study after 1 year shows a further increase of sympathetic cardiac and vascular modulation, suggesting early progression of the autonomic dysfunction.

Ghrelin Restores the Endothelin 1/Nitric Oxide Balance in Patients With Obesity-Related Metabolic Syndrome [Original Articles]

Obesity is associated with endothelial dysfunction related to decreased NO bioavailability, increased endothelin 1 vasoconstrictor activity, and decreased circulating ghrelin. Therefore, we tested whether exogenous ghrelin may have benefits to improve the balance between endothelin 1 and NO in patients with obesity-related metabolic syndrome. Vasoactive actions of endothelin 1 and NO were assessed in 8 patients with metabolic syndrome and 8 matched controls by evaluating forearm blood flow responses (strain-gauge plethysmography) to intra-arterial infusion of BQ-123 (endothelin A receptor antagonist; 10 nmol/min), followed by NG-monomethyl-l-arginine (NO synthase inhibitor; 4 µmol/min), before and after infusion of ghrelin (200 ng/min). In the absence of ghrelin, the vasodilator response to BQ-123 was greater in patients than in controls (P<0.001), whereas infusion of NG-monomethyl-l-arginine induced smaller vasoconstriction in patients than in controls (P=0.006). Importantly, exogenous ghrelin decreased the vasodilator response to BQ-123 (P=0.007 versus saline) and enhanced the magnitude of changes in forearm blood flow induced by NG-monomethyl-l-arginine (P=0.003) in patients but not in controls (both P>0.05). The favorable effect of ghrelin on endothelin A–dependent vasoconstriction was likely related to the stimulation of NO production, because no change in the vascular effect of BQ-123 was observed after ghrelin (P=0.44) in 5 patients with metabolic syndrome during continuous infusion of the NO donor sodium nitroprusside (0.2 µg/min). In patients with metabolic syndrome, ghrelin has benefits to normalize the balance between vasoconstrictor (endothelin 1) and vasodilating (NO) mediators, thus suggesting that this peptide has important peripheral actions to preserve vascular homeostasis in humans.

Leptin Impairs Cardiovagal Baroreflex Function at the Level of the Solitary Tract Nucleus [Original Articles]

Circulating leptin is elevated in some forms of obesity-related hypertension, associated with impaired baroreflex function. Leptin receptors are present on vagal afferent fibers and neurons within the solitary tract nucleus, providing an anatomic distribution consistent with baroreflex modulation. Although solitary tract nucleus microinjection of 144 fmol/60 nL of leptin had no significant effect on baroreflex sensitivity for control of the heart rate in urethane/chloralose-anesthetized Sprague-Dawley rats, 500 fmol of leptin impaired baroreflex sensitivity for bradycardia in response to increases in pressure (1.15±0.04 versus 0.52±0.12 ms/mm Hg; P<0.01). Transgenic ASrAOGEN rats with low brain angiotensinogen have an upregulation of the leptin receptor and p85 mRNA in the dorsal medulla relative to Sprague-Dawley rats. Consistent with these observations, the response to leptin was enhanced in ASrAOGEN rats, because both the 144-fmol (1.46±0.08 versus 0.75±0.10 ms/mm Hg; P<0.001) and 500-fmol (1.36±0.32 versus 0.44±0.06 ms/mm Hg; P<0.05) leptin microinjections impaired baroreflex sensitivity. At these doses, leptin microinjection had no effect on resting pressure, heart rate, or the tachycardic response to decreases in pressure in Sprague-Dawley or ASrAOGEN rats. Thus, exogenous leptin at sites within the solitary tract nucleus impairs the baroreflex sensitivity for bradycardia induced by increases in arterial pressure, consistent with a permissive role in mediating increases in arterial pressure. Baroreflex inhibition was enhanced in animals with evidence of increased leptin receptor and relevant signaling pathway mRNA.

Atorvastatin Treatment Is Associated With Less Augmentation of the Carotid Pressure Waveform in Hypertension: A Substudy of the Anglo-Scandinavian Cardiac Outcome Trial (ASCOT) [Original Articles]

Hydroxymethylglutaryl-CoA reductase inhibitors (statins) reduce cardiovascular events in hypertensive subjects, but their effect on carotid BP, pressure augmentation, and wave reflection is unknown. We compared the effect of atorvastatin with placebo in a substudy of the lipid-lowering arm of the Anglo-Scandinavian Cardiac Outcomes Trial (ASCOT-LLA). Hypertensive patients (n=142; age=43 to 79 years; 127 male) with total cholesterol ≤6.5 mmol/L were randomized to atorvastatin 10 mg or placebo. Carotid BP and flow velocity were measured by tonometry and Doppler ultrasound. Augmentation index (carotid AIx) was calculated, and waveforms were separated into backward and forward components by wave intensity analysis. Brachial BP was similar in atorvastatin and placebo groups. Carotid AIx and augmentation pressure were significantly less in patients randomized to atorvastatin (mean [SD]: 21.7 [12.1] versus 25.9 [10.3] %; P=0.027 and 10.2 [6.5] versus 13.1 [6.6] mm Hg; P=0.016, respectively), and atorvastatin treatment was associated with significantly less wave reflection from the body. Carotid systolic BP was slightly lower in the atorvastatin group, but there was a statistically significant interaction between lipid-lowering and antihypertensive regimen with lower carotid systolic BP in patients randomized to amlodipine-based therapy and atorvastatin. Carotid wave velocity, timings of waves, and wave intensities did not differ significantly between atorvastatin and placebo groups. Atorvastatin treatment is associated with less augmentation of the carotid BP waveform and less wave reflection from the body. This could contribute to the reduction in risk of cardiovascular events by statins.

Effect of Cardiorespiratory Fitness on Vascular Regulation and Oxidative Stress in Postmenopausal Women [Original Articles]

Increasing evidence exists suggesting an important role for oxidative stress in the pathogenesis and progression of hypertension in women via a decrease of NO production after menopause. Regular physical training has been shown to upregulate antioxidant enzymatic systems, which may slow down the usual increase of oxidative stress in postmenopausal women. The aims of this study were to determine the impact of fitness status on enzymatic antioxidant efficiency, oxidative stress, and NO production and to determine the associations among oxidative stress, enzymatic antioxidant and NO production, mean arterial blood pressure (MABP), and cerebrovascular conductance (CVC) in postmenopausal women (n=40; 50 to 90 years old). Physical fitness, physical activity, resting MABP, and CVC were measured. End product of NO, lipid peroxidation (malondialdehyde and 8-iso-prostaglandin F2), DNA oxidation (8-hydroxy-2'-deoxyguanosine), protein nitration (nitrotyrosine), antioxidant glutathione peroxidase, and catalase activities were measured in plasma. We identified significant negative associations between oxidative stress and indices of physical fitness (malondialdehyde: r=–0.33, P<0.05; 8-iso-prostaglandin F2: r=–0.39, P<0.05; 8-hydroxy-2'-deoxyguanosine: r=–0.35, P<0.05) and physical activity (malondialdehyde: r=–0.30, P<0.05; 8-iso-prostaglandin F2: r=–0.41, P<0.01; 8-hydroxy-2'-deoxyguanosine: r=–0.39, P<0.05). Conversely, glutathione peroxidase was positively correlated with fitness level (r=0.55; P<0.01). Finally, MABP and CVC were significantly associated with 8-hydroxy-2'-deoxyguanosine (MABP: r=0.36, P<0.05; CVC: r=–0.36, P<0.05), nitrotyrosine (MABP: r=0.39, P<0.05; CVC: r=–0.32, P<0.05), and the end product of NO (MABP: r=–0.57, P<0.01; CVC: r=0.44, P<0.01). These findings demonstrate that, after menopause, fitness level and regular physical activity mediate against oxidative stress by maintaining antioxidant enzyme efficiency. Furthermore, these results suggest that oxidative stress and NO production modulate MABP and CVC.

Arterial Wave Reflections During the Menstrual Cycle of Healthy Women: A Reproducibility Study [Original Articles]

Increased wave reflection is an independent factor associated with cardiovascular diseases, risk, and mortality. The influence of the menstrual cycle on wave reflections and particularly on the reproducibility of their measurement has never been examined. The aim of the present study was to examine the reproducibility and variability of wave reflection indices in premenopausal healthy women during their menstrual cycle. Thirty-two women were examined at 3 phases of their menstrual cycle: days 1 to 2 (menstrual phase), days 6 to 14 (late follicular), and days 4 to 7 after ovulation (early luteal phase). Applanation tonometry of the radial artery and aortic pulse wave analysis were performed for the calculation of augmentation pressure, augmentation index, and timing of reflected waves. Reproducibility of these measures was evaluated by intraclass correlation coefficient and Bland-Altman analysis, whereas ANOVA was performed to assess their variability during the menstrual cycle. The SD of augmentation index differences between repeated measurements within the menstrual cycle ranged from 7.6% to 9.9%. Bland-Altman analysis indicated no evidence of systemic bias and no trend for the reproducibility of measurements to vary with their underlying mean value. Intraclass correlation coefficient indicated a moderate reproducibility of augmentation index and augmentation pressure (>0.80) and a rather low reproducibility for timing of reflected waves (0.43). Mean augmentation pressure, augmentation index, and timing of reflected waves did not vary significantly during the menstrual cycle (ANOVA). Measurement of wave reflections at the same phase of the menstrual cycle or statistical adjustment could be suggested for optimal study design and data interpretation.

Pressure-Induced Vascular Oxidative Stress Is Mediated Through Activation of Integrin-Linked Kinase 1/{beta}PIX/Rac-1 Pathway [Original Articles]

High blood pressure induces a mechanical stress on vascular walls and evokes oxidative stress and vascular dysfunction. The aim of this study was to characterize the intracellular signaling causing vascular oxidative stress in response to pressure. In carotid arteries subjected to high pressure levels, we observed not only an impaired vasorelaxation, increased superoxide production, and NADPH oxidase activity, but also a concomitant activation of Rac-1, a small G protein. Selective inhibition of Rac-1, with an adenovirus carrying a dominant-negative Rac-1 mutant, significantly reduced NADPH oxidase activity and oxidative stress and, more importantly, rescued vascular function in carotid arteries at high pressure. The analysis of molecular events associated with mechanotransduction demonstrated at high pressure levels an overexpression of integrin-linked kinase 1 and its recruitment to plasma membrane interacting with paxillin. The inhibition of integrin-linked kinase 1 by small interfering RNA impaired Rac-1 activation and rescued oxidative stress–induced vascular dysfunction in response to high pressure. Finally, we showed that βPIX, a guanine-nucleotide exchange factor, is the intermediate molecule recruited by integrin-linked kinase 1, converging the intracellular signaling toward Rac-1–mediated oxidative vascular dysfunction during pressure overload. Our data demonstrate that biomechanical stress evoked by high blood pressure triggers an integrin-linked kinase 1/βPIX/Rac-1 signaling, thus generating oxidative vascular dysfunction.

Hepatocyte gp130 Deficiency Reduces Vascular Remodeling After Carotid Artery Ligation [Original Articles]

Inflammation and vascular remodeling are hallmarks of atherosclerosis, hypertension, and restenosis after angioplasty. Here we investigated the role of the hepatocyte gp130-dependent systemic acute phase response on vascular remodeling after carotid artery ligation. Mice with a hepatocyte-specific gp130 knockout on an apolipoprotein E–/– background (gp130–) were compared with control mice (gp130flox). Vascular remodeling was induced by permanent ligation of the left common carotid artery. This, in turn, activated the systemic acute phase reaction in gp130flox mice, as measured by serum amyloid A plasma levels, which was completely abrogated in gp130– mice (P<0.05). Morphometric analysis of the carotid artery revealed severe neointima formation and media thickening 28 days after ligation in gp130flox mice, which was suppressed in gp130– mice (P<0.01). Serial sections from gp130– carotid segments showed significantly less smooth muscle cell (SMC) proliferation and monocyte recruitment (P<0.01). To evaluate the impact of the gp130-dependent systemic acute phase response on SMCs, hepatocytes from gp130flox and gp130– mice were stimulated with interleukin 6. Interleukin 6–induced secretion of serum amyloid A was completely abolished in gp130– hepatocytes (P<0.01). Moreover, when stimulated with supernatants from gp130– hepatocytes, SMCs showed significantly less migration and proliferation compared with supernatants from gp130flox hepatocytes (P<0.01). Recombinant serum amyloid A induced SMC migration and proliferation (P<0.05) and serum amyloid A injection after carotid artery ligation restored vascular remodeling in gp130– mice (P<0.01). These results imply a critical role for the gp130-dependent systemic acute phase response for vascular inflammation and SMC migration, as well as proliferation, and, subsequently, for vascular remodeling.

Sildenafil Increases Endothelial Progenitor Cell Function and Improves Ischemia-Induced Neovascularization in Hypercholesterolemic Apolipoprotein E-Deficient Mice [Original Articles]

Hypercholesterolemia is associated with impaired neovascularization in response to ischemia. Potential mechanisms include defective NO bioactivity and a reduction in the number/function of endothelial progenitor cells (EPCs). Here we tested the hypothesis that sildenafil, a phosphodiesterase 5 inhibitor that increases NO-driven cGMP levels, could stimulate EPC function and improve ischemia-induced neovascularization in hypercholesterolemic conditions. Apolipoprotein E–deficient (ApoE–/–) mice were treated (or not treated) with sildenafil (40 mg/kg per day in water), and hindlimb ischemia was surgically induced by femoral artery removal. Sildenafil treatment led to an improved blood flow recovery, an increased capillary density, and a reduction of oxidative stress levels in ischemic muscles at day 7 after surgery. Sildenafil therapy is associated with an increased activation of angiogenic transduction pathways, including Akt, p44/42 mitogen-activated protein kinase, and p38. In vitro, sildenafil increases cellular migration and tubule formation of mature endothelial cells (human umbilical vascular endothelial cells) in a cGMP-dependent manner. In vivo, ApoE–/– mice treated with sildenafil exhibit a significant increase in the number of bone marrow–derived EPCs. Moreover, the angiogenic activities of EPCs (migration and adhesion) are significantly improved in ApoE–/– mice treated with sildenafil. In summary, this study demonstrates that sildenafil treatment is associated with improved ischemia-induced neovascularization in hypercholesterolemic ApoE–/– mice. The mechanisms involve beneficial effects on angiogenic transduction pathways together with an increase in the number and the functional activity of EPCs. Sildenafil could constitute a novel therapeutic strategy to reduce tissue ischemia in atherosclerotic diseases.

Hypertension Correlates With Lenticulostriate Arteries Visualized by 7T Magnetic Resonance Angiography [Original Articles]

Hypertension, a major risk factor for stroke, is associated with altered arterial anatomy and function; however, the limited resolution of current imaging techniques has restricted the in vivo study of microvascular changes in the brain. In this report, we quantitatively examined the lenticulostriate arteries in hypertensive patients using ultrahigh-field 7T MRI. We compared the number of stems and branches, curvature, and tortuosity of the lenticulostriate arteries by 3D time-of-flight magnetic resonance angiography among 20 hypertensive patients (mean age: 46.6±9.1 years) and 20 age-matched healthy subjects (mean age: 47.7±8.1 years). The average numbers of stems and branches in hypertensive patients were significantly less than those of healthy subjects (P<0.002). However, this difference was abolished in older volunteers (>45 years old), whereas the difference between young hypertensive patients (≤45 years old) and age-matched healthy controls was augmented by 55% for stems and 91% for branches (P=0.001). In comparison, there were no differences in the average curvature and tortuosity of the lenticulostriate arteries and no significant difference when corrected for smoking (P=0.064). In conclusion, our results showed that there was a substantial difference in the lenticulostriate arteries of hypertensive patients compared with healthy individuals when observed in vivo by ultrahigh-resolution 7T magnetic resonance angiography, and the difference was considerable in young subjects.

Renal Impairment of Pure Autonomic Failure [Original Articles]

Supine hypertension is difficult to manage in patients with pure autonomic failure (PAF), because treatment can worsen orthostatic hypotension. Supine hypertension in PAF has been associated with left ventricular hypertrophy, but end organ damage in the kidney has not been assessed. We reviewed hemodynamic and laboratory data of 64 male patients with PAF who were 69±11 (mean±SD) years old. Systolic blood pressure fell by 67±40 mm Hg within 10 minutes of standing, with an inappropriately low 13±11-bpm increase in heart rate. Plasma norepinephrine levels were below normal (0.62±0.32 nmol/L supine and 1.28±1.25 nmol/L standing). A control data set of 75 men (67±12 years) was obtained from a deidentified version of the Vanderbilt University Medical Center electronic medical chart database. Compared with controls, PAF patients had lower hemoglobin (8.3±0.9 versus 9.3±0.8 mmol/L; P<0.001), packed cell volume (0.40±0.04 versus 0.45±0.04; P<0.001), and red blood cell count (4.4±0.5x1012 versus 5.0±0.5x1012 cells/L; P<0.001). Serum creatinine and blood urea nitrogen levels were elevated in patients. Forty-eight percent of patients with PAF had supine hypertension (supine systolic blood pressure: ≥150 mm Hg). Serum creatinine was higher in patients with supine hypertension (133±44 versus 106±27 µmol/L; P=0.021) and estimated glomerular filtration rate was lower (57±22 versus 70±20 mL/min per 1.73 m2; P=0.022) compared with patients who did not have supine hypertension. These findings may indicate that renal function is diminished in PAF in association with supine hypertension.

Effect of Epithelial Sodium Channel Blockade on the Myogenic Response of Rat Juxtamedullary Afferent Arterioles [Original Articles]

The mechanotransduction mechanism underlying the myogenic response is poorly understood, but evidence implicates participation of epithelial sodium channel (ENaC)-like proteins. Therefore, the role of ENaC on the afferent arteriolar myogenic response was investigated in vitro using the blood-perfused juxtamedullary nephron technique. Papillectomy was used to isolate myogenic influences by eliminating tubuloglomerular feedback signals. Autoregulatory responses were assessed by manipulating perfusion pressure in 30-mm Hg steps. Under control conditions, arteriolar diameter increased by 15% from 13.0±1.3 to 14.7±1.2 µm (P<0.05) after reducing perfusion pressure from 100 to 70 mm Hg. Diameter decreased to 11.3±1.1 and 10.6±1.0 µm after increasing pressure to 130 and 160 mm Hg (88±1 and 81±2% of control diameter, P<0.05), respectively. Pressure-mediated autoregulatory responses were significantly inhibited by superfusion of 10 µmol/L amiloride (102±2, 97±4, and 94±3% of control diameter), or 10 µmol/L benzamil (106±5, 100±3, and 103±3% of control diameter), and when perfusing with blood containing 5 µmol/L amiloride (106±2, 97±4, and 97±4% of control diameter). Vasoconstrictor responses to 55 mmol/L KCl were preserved as diameters decreased by 67±4, 55±8, and 60±4% in afferent arterioles superfused with amiloride or benzamil, and perfused with amiloride, respectively. These responses were similar to responses obtained from control afferent arterioles (64±6%, P>0.05). Immunofluorescence revealed expression of the , β, and subunits of ENaC in freshly isolated preglomerular microvascular smooth muscle cells. These results demonstrate that selective ENaC inhibitors attenuate afferent arteriolar myogenic responses and suggest that ENaC may function as mechanosensitive ion channels initiating pressure-dependent myogenic responses in rat juxtamedullary afferent arterioles.

Caveolin-1 and Dopamine-Mediated Internalization of NaKATPase in Human Renal Proximal Tubule Cells [Original Articles]

In moderate sodium-replete states, dopamine 1–like receptors (D1R/D5R) are responsible for regulating >50% of renal sodium excretion. This is partly mediated by internalization and inactivation of NaKATPase, when associated with adapter protein 2. We used dopaminergic stimulation via fenoldopam (D1-like receptor agonist) to study the interaction among D1-like receptors, caveolin-1 (CAV1), and the G protein–coupled receptor kinase type 4 in cultured human renal proximal tubule cells (RPTCs). We compared 2 groups of RPTCs, 1 of cell lines that were isolated from normal subjects (nRPTCs) and a second group of cell lines that have D1-like receptors that are uncoupled (uncoupled RPTCs) from adenylyl cyclase second messengers. In nRPTCs, fenoldopam increased the plasma membrane expression of D1R (10.0-fold) and CAV1 (1.3-fold) and markedly decreased G protein–coupled receptor kinase type 4 by 94±8%; no effects were seen in uncoupled RPTCs. Fenoldopam also increased the association of adapter protein 2 and NaKATPase by 53±9% in nRPTCs but not in uncoupled RPTCs. When CAV1 expression was reduced by 86.0±8.5% using small interfering RNA, restimulation of the D1-like receptors with fenoldopam in nRPTCs resulted in only a 7±9% increase in association between adapter protein 2 and NaKATPase. Basal CAV1 expression and association with G protein–coupled receptor kinase type 4 was decreased in uncoupled RPTCs (58±5% decrease in association) relative to nRPTCs. We conclude that the scaffolding protein CAV1 is necessary for the association of D1-like receptors with G protein–coupled receptor kinase type 4 and the adapter protein 2–associated reduction in plasma membrane NaKATPase.

Intrarenal Dopamine Attenuates Deoxycorticosterone Acetate/High Salt-Induced Blood Pressure Elevation in Part Through Activation of a Medullary Cyclooxygenase 2 Pathway [Original Articles]

Locally produced dopamine in the renal proximal tubule inhibits salt and fluid reabsorption, and a dysfunctional intrarenal dopaminergic system has been reported in essential hypertension and experimental hypertension models. Using catechol-O-methyl-transferase knockout (COMT–/–) mice, which have increased renal dopamine because of deletion of the major renal dopamine-metabolizing enzyme, we investigated the effect of intrarenal dopamine on the development of hypertension in the deoxycorticosterone acetate/high-salt (DOCA/HS) model. DOCA/HS led to significant increases in systolic blood pressure in wild-type mice (from 115±2 to 153±4 mm Hg), which was significantly attenuated in COMT–/– mice (from 114±2 to 135±3 mm Hg). In DOCA/HS COMT–/– mice, the D1-like receptor antagonist SCH-23390 increased systolic blood pressure (156±2 mm Hg). DOCA/HS COMT–/– mice also exhibited more urinary sodium excretion (COMT–/– versus wild-type: 3038±430 versus 659±102 µmol/L per 24 hours; P<0.01). Furthermore, DOCA/HS-induced renal oxidative stress was significantly attenuated in COMT–/– mice. COX-2–derived prostaglandins in the renal medulla promote sodium excretion, and dopamine stimulates medullary prostaglandin production. Renal medullary COX-2 expression and urinary prostaglandin E2 excretion were significantly higher in COMT–/– than in wild-type mice after DOCA/HS treatment. In DOCA/HS-treated COMT–/– mice, the COX-2 inhibitor SC-58236 reduced urinary sodium and prostaglandin E2 excretion and increased systolic blood pressure (153±2 mm Hg). These studies indicate that an activated renal dopaminergic system attenuates the development of hypertension, at least in large part through activating medullary COX-2 expression/activity, and also decreases oxidative stress resulting from DOCA/HS.

Regression of Left Ventricular Mass by Antihypertensive Treatment: A Meta-Analysis of Randomized Comparative Studies [Original Articles]

Blood pressure–lowering therapy reduces left ventricular mass, but the question of whether differences exist among drug classes has not been fully resolved. Our aim was to compare the effects of diuretics, β-blockers, calcium channel blockers, angiotensin-converting enzyme inhibitors, and angiotensin receptor blockers on left ventricular mass regression in patients with hypertension on the basis of prospective, randomized comparative studies. We performed meta-analyses, involving pooled pairwise comparisons of the drug classes and of each class versus other classes statistically combined, and meta-regression analyses to identify the determinants of the regression. The 75 relevant publications involved 84 pairwise comparisons and 6001 patients. Regression of left ventricular mass was significantly less (P=0.01) with β-blockers (9.8%) than with angiotensin receptor blockers (12.5%), but none of the other analyzable pairwise comparisons between drug classes revealed significant differences (P>0.10). In addition, β-blockers showed less regression than the other 4 classes statistically combined (P<0.01), and regression was more pronounced with angiotensin receptor blockers versus the others (P<0.01). In multivariable meta-regression analysis on all of the treatment arms, β-blocker treatment was a significant and negative predictor of the regression (–3.6%; P<0.01), but this was not the case for the other drug classes, including angiotensin receptor blockers. In conclusion, β-blockers show less regression of left ventricular mass, whereas angiotensin receptor blockers may induce larger regression. The inferiority of β-blockers appears to be more convincing than the superiority of angiotensin receptor blockers.

Influence of Altered Blood Rheology on Ventricular-Vascular Response to Exercise [Original Articles]

Blood (or plasma) rheology is related to cardiovascular risk. Mechanisms of this association are unclear but may be partially related to impaired left ventricular (LV) function and increased central blood pressure (BP) during light activity. This study aimed to test these hypotheses. Twenty patients (14 men; aged 61±12 years) with polycythemia rubra vera (n=16) or hemochromatosis (n=4) were studied at rest and during exercise at 50% of maximal heart rate before and after venesection (500 mL; volume replaced with saline) to elicit an acute decrease in plasma viscosity at stable BP. Controls (n=20) underwent the same protocol with 25-mL venesection. Central BP and augmentation index were determined by tonometry. Resting LV systolic (peak longitudinal systolic strain rate and strain) and diastolic functions were determined by tissue-Doppler echocardiography. Venesection with blood volume replacement decreased viscosity (1.46±0.10 to 1.41±0.11 centipoise), protein, and hemoglobin (P<0.05 for all) and increased strain rate and strain (P<0.001 for both) in patients but not in controls (P>0.10 for all). There was no change in LV diastolic function (P>0.12 for all). Exercise augmentation index in patients was reduced after venesection (24±12% to 17±9%; P=0.001) despite no significant change in other BP variables. Hemodynamics (resting or exercise) were not significantly changed in controls. Exercise central systolic BP correlated with triglycerides (r=0.59; P<0.001). However, neither exercise hemodynamic changes nor LV functional changes correlated with any biochemical changes after venesection (P>0.05). We conclude that an acute change in blood rheology improves ventricular-vascular interaction by enhanced LV systolic function and reduced light-exercise central BP.

Augmentation Index, Left Ventricular Contractility, and Wave Reflection [Original Articles]

Augmentation index (AIx), a correlate of mortality, is thought to be influenced by left ventricular contractility and wave reflections. However, the relationship of AIx with left ventricular contractility changes has never been assessed, and the wave reflection theory has recently been questioned. This study sought to examine arterial waveform changes in response to reduced "wave reflection" and increased left ventricular contractility induced by dobutamine. Simultaneous radial tonometry (for AIx) and tissue Doppler echocardiography (for peak longitudinal systolic strain rate [SR] as an analogue of left ventricular contractility) were recorded at rest and peak dobutamine-induced stress in 50 patients (41 men; aged 62±10 years). From baseline to peak stress there was an increase in heart rate (70±11 to 127±17 bpm; P<0.001) and SR (–0.88±0.23 to –1.81±0.43 1/s; P<0.001), whereas AIx decreased (27±9% to –7±15%; P<0.001). There was also a greater increase in the systolic (compared with diastolic) pressure-time integral relative to cardiac cycle length (3.2±1.9 versus 1.8±1.1 mm Hg; P<0.001), indicating that wave reflection was not shifted into diastole as per the current belief. AIx was significantly associated with ejection duration (r=0.88), heart rate (r=–0.81), and SR (r=0.72; P<0.001 for all). However, when SR was heart rate corrected, there was no significant association with AIx (r=0.18; P=0.11). The strongest independent correlate of AIx was ejection duration, accounting for 78% variance (β=0.88; model R2=0.77; P<0.001). Neither SR (β=0.12; P=0.18) nor heart rate–corrected SR (β=0.02; P=0.72) was associated with AIx. We conclude that AIx is determined by chronotropic rather than inotropic effects, as well as factors other than wave reflection.

Genetic Silencing of Nox2 and Nox4 Reveals Differential Roles of These NADPH Oxidase Homologues in the Vasopressor and Dipsogenic Effects of Brain Angiotensin II [Original Articles]

The renin-angiotensin system exerts a tremendous influence over fluid balance and arterial pressure. Angiotensin II (Ang-II), the effector peptide of the renin-angiotensin system, acts in the central nervous system to regulate neurohumoral outflow and thirst. Dysregulation of Ang-II signaling in the central nervous system is implicated in cardiovascular diseases; however, the mechanisms remain poorly understood. Recently we established that NADPH oxidase (Nox)–derived superoxide acting in the forebrain subfornical organ is critical in the physiological responses to central Ang-II. In addition, we have found that Nox2 and Nox4 are the most abundantly expressed Nox homologues within Ang-II–sensitive sites in the forebrain. To dissect out the functional importance and unique roles of these Nox enzymes in the pressor and dipsogenic effects of central Ang-II, we developed adenoviral vectors expressing small interfering RNA to selectively silence Nox2 or Nox4 expression in the subfornical organ. Our results demonstrate that both Nox2 and Nox4 are required for the full vasopressor effects of brain Ang-II but that only Nox2 is coupled to the Ang-II–induced water intake response. These studies establish the importance of both Nox2- and Nox4-containing NADPH oxidases in the actions of Ang-II in the central nervous system and are the first to reveal differential involvement of these Nox enzymes in the various physiological effects of central Ang-II.

Prenatal Dexamethasone Exposure Does Not Alter Blood Pressure and Nephron Number in the Young Adult Marmoset Monkey [Original Articles]

The influence of prenatal factors on the development of arterial hypertension has gained considerable interest in recent years. Prenatal dexamethasone exposure was found to induce hypertension and to alter nephron number and size in rodents and sheep. However, it is not clear whether these findings are applicable to nonhuman primates. Thus, we examined the effects of prenatal dexamethasone treatment on blood pressure (BP) and nephron number in marmoset monkeys. Fifty-two marmosets were allotted to 3 groups according to the gestational stage during which their mothers were exposed to oral 5-mg/kg dexamethasone for 7 days (gestation period: 20 weeks): (1) the early dexamethasone group at week 7; (2) the late dexamethasone group at week 13; and (3) the control group. BP was determined by telemetric (n=12) or cuff measurements (n=30), along with cystatin C, proteinuria, and body weight. All of the animals were euthanized at the age of 24 months, and glomerular number and volume were determined. Prenatal exposure to dexamethasone did not lead to a significant difference between the groups with regard to BP, kidney morphology and function, or body weight. BP correlated significantly with body weight, relative kidney weight, and mean glomerular volume and the body weight with the glomerular volume regardless of dexamethasone treatment. In conclusion, prenatal exposure to dexamethasone in marmosets does not, in contrast to other mammals studied, result in hypertension or changes in kidney morphology. Our data support the role of body weight as a predictor of elevated glomerular volume and BP development rather than prenatal dexamethasone exposure.

Prenatal Cocaine Exposure Causes Sex-Dependent Impairment in the Myogenic Reactivity of Coronary Arteries in Adult Offspring [Original Articles]

Cocaine abuse is a significant problem among pregnant women. The present study tested the hypothesis that prenatal cocaine exposure impairs myogenic reactivity of coronary arteries in adult offspring. Pregnant rats received cocaine (30 mg kg–1 day–1) or saline from days 15 to 21 of gestational age, and experiments were conducted in 3-month-old offspring. In pressurized coronary septal arteries, the diameter and vessel wall intracellular Ca2+ concentrations were measured simultaneously in the same tissue as a function of intraluminal pressure. Cocaine did not affect KCl-induced contractions of coronary arteries in either males or females but decreased the distensibility in male vessels. In male offspring, cocaine treatment resulted in a significant decease in pressure-dependent myogenic contractions. Inhibition of eNOS with NG-nitro-l-arginine did not alter the myogenic response in either saline control or cocaine-treated animals. In females, cocaine caused a significant increase in pressure-dependent myogenic contractions. NG-nitro-l-arginine did not affect the myogenic response in the control animals but blocked the cocaine-mediated effect. In both males and females, the pressure-induced increases in vessel wall Ca2+ concentrations were not significantly different between cocaine and saline groups. The ratio of changes in the diameter to Ca2+ concentrations in the pressurized arteries was significantly less in male but greater in female offspring after cocaine treatment. The results suggest that prenatal cocaine exposure causes reprogramming of coronary myogenic tone via changes in the Ca2+ sensitivity in a sex-dependent manner, leading to an increased risk of dysfunction of coronary autoregulation in adult offspring.

Effect of Recombinant Placental Growth Factor 2 on Hypertension Induced by Full-Length Mouse Soluble fms-Like Tyrosine Kinase 1 Adenoviral Vector in Pregnant Mice [Original Articles]

The first aim of our study was to develop a pregnant mouse model for preeclampsia using adenoviral vector containing mouse full-length soluble fms-like tyrosine kinase 1 (sFlt-1) but not truncated sFlt-1. The second aim was to evaluate effects of recombinant mouse (rm) vascular endothelial growth factor (VEGF) and rm placental growth factor (PlGF) on a preeclampsia model induced by adenoviral vector containing mouse full-length sFlt-1. We injected adenoviral vector containing mouse full-length sFlt-1 on day 8.5 or 9.5 of gestation into pregnant Institute of Cancer Research mice, resulting in hypertension, proteinuria, and similar glomerular histological changes as those seen in human preeclamptic women with glomerular endotheliosis on day 16.5 or 17.5 of gestation. The preeclampsia models were treated with 100 µg/kg of rmVEGF164 (n=5), 100 µg/kg of rmPlGF-2 (n=5), or vehicle (n=7) twice a day for 2 days IP. The rmVEGF164 treatment significantly decreased the mean blood pressure on day 16.5 or 17.5 of gestation compared with the vehicle treatment (85±4 versus 97±2 mm Hg; P=0.018). The rmPlGF-2 treatment also significantly decreased the mean blood pressure on day 16.5 or 17.5 of gestation compared with the vehicle treatment (86±3 versus 97±2 mm Hg; P=0.018). However, proteinuria was not affected by either rmVEGF164 or rmPlGF-2. In conclusion, we, for the first time, created a mouse preeclampsia model using mouse full-length sFlt-1. VEGF and PlGF may be promising for ameliorating hypertension in women with preeclampsia. Additional study of PlGF as a potential drug for preeclampsia is warranted.

Time-Dependent Effects of Low-Dose Aspirin on Plasma Renin Activity, Aldosterone, Cortisol, and Catecholamines [Original Articles]

Studies have shown that aspirin may decrease blood pressure when given at bedtime but not when administered on awakening. However, until now, a biologically plausible mechanism of this striking phenomenon was not revealed. We investigated the effect of 100 mg of aspirin administered at bedtime compared with administration on awakening on plasma renin activity and aldosterone levels over 24 hours and excretion of cortisol and catecholamines in 24-hour urine samples. A randomized, placebo-controlled, double-blind, crossover trial was performed in 16 grade 1 hypertensive subjects. During 2 periods of 2 weeks separated by a 4-week washout period, participants used aspirin both at morning and at night, which was blinded with placebo. After both periods, subjects were admitted for 24 hours to measure the aforementioned parameters. Aspirin intake at bedtime compared with on awakening reduced average (24-hour) plasma renin activity by 0.08 µg/L per hour (95% CI: 0.03 to 0.13 µg/L per hour; P=0.003) without affecting aldosterone levels (95% CI: –0.01 to 0.01 nmol/L; P=0.93). Cortisol excretion in 24-hour urine was 52 nmol/24 hours (95% CI: 5 to 99 nmol/24 hours; P=0.05) lower, and dopamine and norepinephrine excretions were 0.25 µmol/24 hours (95% CI: 0.01 to 0.48 µmol/24 hours; P=0.04) and 0.22 µmol/24 hours (95% CI: –0.03 to 0.46 µmol/24 hours; P=0.02) lower in patients treated with bedtime aspirin. In conclusion, aspirin taken at bedtime compared with on awakening significantly diminished 24-hour plasma renin activity and excretion of cortisol, dopamine, and norepinephrine in 24-hour urine. Decreased activity of these pressor systems forms a biologically plausible explanation for the finding that aspirin at night may reduce blood pressure, whereas aspirin at morning does not.

Mediterranean-Style Diet Effect on the Structural Properties of the Erythrocyte Cell Membrane of Hypertensive Patients: The Prevencion con Dieta Mediterranea Study [Original Articles]

A currently ongoing randomized trial has revealed that the Mediterranean diet, rich in virgin olive oil or nuts, reduces systolic blood pressure in high-risk cardiovascular patients. Here, we present a structural substudy to assess the effect of a Mediterranean-style diet supplemented with nuts or virgin olive oil on erythrocyte membrane properties in 36 hypertensive participants after 1 year of intervention. Erythrocyte membrane lipid composition, structural properties of reconstituted erythrocyte membranes, and serum concentrations of inflammatory markers are reported. After the intervention, the membrane cholesterol content decreased, whereas that of phospholipids increased in all of the dietary groups; the diminishing cholesterol:phospholipid ratio could be associated with an increase in the membrane fluidity. Moreover, reconstituted membranes from the nuts and virgin olive oil groups showed a higher propensity to form a nonlamellar inverted hexagonal phase structure that was related to an increase in phosphatidylethanolamine lipid class. These data suggest that the Mediterranean-style diet affects the lipid metabolism that is altered in hypertensive patients, influencing the structural membrane properties. The erythrocyte membrane modulation described provides insight in the structural bases underlying the beneficial effect of a Mediterranean-style diet in hypertensive subjects.

Inhibition of 20-Hydroxyeicosatetraenoic Acid Synthesis Using Specific Plant Lignans: In Vitro and Human Studies [Original Articles]

Sesamin, the major lignan found in sesame, has been shown to increase vitamin E levels by inhibiting its metabolism via the cytochrome P450 isozyme CYP4F2. CYP4F2 and CYP4A11 are the predominant human isoforms that synthesize 20-hydroxyeicosatetraenoic acid (20-HETE) from arachidonic acid. Considerable evidence suggests that 20-HETE may play a role in the pathogenesis of hypertension. We hypothesized that sesamin could be an inhibitor of 20-HETE synthesis. This study investigated the effects of sesamin on 20-HETE synthesis in vitro and the effect of sesame supplementation on plasma and urinary 20-HETE concentrations in humans. Human microsomes were used to investigate the potency and selectivity of sesamin inhibition of 20-HETE synthesis. Sesamin inhibited human renal and liver microsome 20-HETE synthesis with IC50 <20 µmol/L. It was selective toward CYP4F2 (IC50: 1.9 µmol/L) and had reduced activity toward CYP4A11 (IC50: >150 µmol/L), as well as cytochrome P epoxygenation of arachidonic acid (IC50: >50 µmol/L). In a randomized, controlled crossover trial, overweight men and women (n=33) consumed 25 g/d of sesame (50 mg/d of sesame lignan) or an isocaloric matched control for 5 weeks each. Relative to control, sesame supplementation resulted in a 28% decrease in plasma and a 32% decrease in urinary 20-HETE (P<0.001). Urinary sodium, potassium, and blood pressure were not affected. This study demonstrates for the first time that sesame supplementation in humans reduces the plasma and urinary levels of 20-HETE, likely via inhibition of CYP4F2 by sesame lignans. These results suggest that sesame lignans could be used for the investigation of potential roles of 20-HETE in humans.

Potassium Inhibits Dietary Salt-Induced Transforming Growth Factor-{beta} Production [Original Articles]

Human and animal studies demonstrate an untoward effect of excess dietary NaCl (salt) intake on cardiovascular function and life span. The endothelium in particular augments the production of transforming growth factor (TGF)-β, a fibrogenic growth factor, in response to excess dietary salt intake. This study explored the initiating mechanism that regulates salt-induced endothelial cell production of TGF-β. Male Sprague-Dawley rats were given diets containing different amounts of NaCl and potassium for 4 days. A bioassay for TGF-β demonstrated increased (35.2%) amounts of active TGF-β in the medium of aortic ring segments from rats on the high-salt diet compared with rats maintained on a 0.3% NaCl diet. Inhibition of the large-conductance, calcium-activated potassium channel inhibited dietary salt-induced vascular production of TGF-β but did not affect production of TGF-β by ring segments from rats on the low-salt diet. Immunohistochemical and Western analyses demonstrated the subunit of the calcium-activated potassium channel in endothelial cells. Increasing medium [K+] inhibited production of dietary salt-induced vascular production levels of total and active TGF-β but did not alter TGF-β production by aortic rings from rats on the 0.3% NaCl diet. Increasing dietary potassium content decreased urinary active TGF-β in animals receiving the high-salt diet but did not change urinary active TGF-β in animals receiving the low-salt diet. The findings demonstrated an interesting interaction between the dietary intake of potassium and excess NaCl and further showed the fundamental role of the endothelial calcium-activated potassium channel in the vascular response to excess salt intake.

Abstracts From the 14th Annual Meeting of the ECCR [Abstracts From the 14th Annual Meeting of the European Council for Cardiovascular Research (ECCR)]

Tyrosine Sulfation of Leukocyte Adhesion Molecules and Chemokine Receptors Promotes Atherosclerosis [Editorials]

Dimorphisms in the Membrane-Spanning Domain of EPCR Impact Systemic Coagulation [Editorials]

The Discovery of Cellular Immunity in the Atherosclerotic Plaque [History of Discovery]

It is now generally accepted that atherosclerosis is an inflammatory/immune disease triggered by LDL accumulation in the artery wall. When discovering T cells and the molecular components of a cellular immune response, we proposed that atherosclerosis is an inflammatory process with an autoimmune component. This notion was met with general skepticism but has gained support from experimental and clinical studies. Here we describe some of the early studies that helped developing this concept.

The Molecular Mechanisms of HDL and Associated Vesicular Trafficking Mechanisms to Mediate Cellular Lipid Homeostasis [History of Discovery]

HDL functions mainly as a cholesterol scavenger, facilitating transport of cholesterol to the liver for conversion to bile acids and secretion into the bile for elimination or recycling in the enterohepatic bile acid cycle. Because of its major function in cholesterol clearance, HDL is in general considered to be atheroprotective. From cell cholesterol can be removed by efflux especially to apoA-I and HDL as extracellular acceptors which transport the cholesterol to the liver for excretion. This process is called reverse cholesterol transport. In this context the ATP binding cassette transporter protein ABCA1 facilitates cellular cholesterol and phospholipid release to apoA-I-containing HDL precursors. In addition ABCA1 plays a role in vesicular lipid transport mechanisms required for HDL particle formation. In general to maintain intracellular lipid homeostasis, sterols and associated lipids move between cellular compartments by vesicular and nonvesicular pathways. However, cholesterol sorting on vesicle formation is poorly understood. This review summarizes the current knowledge of the molecular mechanisms of HDL and associated vesicular trafficking mechanisms to mediate cellular lipid homeostasis.

Adipose Tissue-Derived Stem Cells: Characterization and Potential for Cardiovascular Repair [Brief Reviews]

Experimental studies have shown that cardiac transfer of unfractionated or partially purified bone marrow cells, as well as stem cells and progenitor cells derived from the bone marrow or peripheral blood, can enhance functional recovery after an acute myocardial infarction. However, the relatively low abundance, small tissue volume, difficult accessibility, and disease-related malfunction of bone marrow-derived stem cells hamper their clinical usefulness. Numerous studies have provided evidence that stromal cells derived from the adipose tissue (adipose tissue-derived stromal cells [ADSCs]) contain a population of adult multipotent mesenchymal stem cells and endothelial progenitor cells that can differentiate into several lineages, including endothelial cells, smooth muscle cells, and cardiomyocytes. The similarities between stem cells extracted from the bone marrow and the adipose tissue suggest the potential for the adipose tissue to act as an alternative, and perhaps preferable, cell source for repairing damaged tissues, such as the ischemic or infarcted heart. We have here reviewed the medical literature describing molecular and functional characterization, differentiation, potential role, and results obtained so far using ADSCs in tissue repair, with a particular focus on the role for ADSCs in cardiovascular repair and regeneration.

Lack of Tyrosylprotein Sulfotransferase Activity in Hematopoietic Cells Drastically Attenuates Atherosclerosis in Ldlr-/- Mice [Integrative Physiology/Experimental Medicine]

Objective— Leukocyte recruitment is a major contributor in the development of atherosclerosis and requires a variety of proteins such as adhesion molecules, chemokines, and chemokine receptors. Several key molecular players implicated in this process are expressed on monocytes and require protein-tyrosine sulfation for optimal function in vitro, including human CCR2, CCR5, CX3CR1, and PSGL-1. We therefore hypothesized that protein-tyrosine sulfation in hematopoietic cells plays an important role in the development of atherosclerosis. Methods and Results— Lethally-irradiated Ldlr–/– mice were rescued with hematopoietic progenitors lacking tyrosylprotein sulfotransferase (TPST) activity attributable to deletion of the Tpst1 and Tpst2 genes. TPST deficient progenitors efficiently reconstituted hematopoiesis in Ldlr–/– recipients and transplantation had no effect on plasma lipids on a standard or atherogenic diet. However, we observed a substantial reduction in the size of atherosclerotic lesions and the number of macrophages in lesions from hyperlipidemic Ldlr–/– recipients transplanted with TPST deficient progenitors compared to wild-type progenitors. We also document for the first time that murine Psgl-1 and Cx3cr1 are tyrosine-sulfated. Conclusions— These data demonstrate that protein-tyrosine sulfation is an important contributor to monocytes/macrophage recruitment and/or retention in a mouse model of atherosclerosis.

Level of Macrophage uPA Expression Is an Important Determinant of Atherosclerotic Lesion Growth in Apoe-/- Mice [Integrative Physiology/Experimental Medicine]

Objective— Enhanced plasminogen activation, mediated by overexpression of urokinase-type plasminogen activator (uPA), accelerates atherosclerosis in apolipoprotein E–null mice. However, the mechanisms through which uPA acts remain unclear. In addition, although elevated uPA expression can accelerate murine atherosclerosis, there is not yet any evidence that decreased uPA expression would retard atherosclerosis. Methods and Results— We used a bone marrow transplant (BMT) approach and apolipoprotein E–deficient (Apoe–/–) mice to investigate cellular mechanisms of uPA-accelerated atherosclerosis, aortic dilation, and sudden death. We also used BMT to determine whether postnatal loss of uPA expression in macrophages retards atherosclerosis. BMT from uPA-overexpressing mice yielded recipients with macrophage-specific uPA overexpression; whereas BMT from uPA knockout mice yielded recipients with macrophage-specific loss of uPA expression. Recipients of uPA-overexpressing BM acquired all the vascular phenotypes (accelerated atherosclerosis, aortic medial destruction and dilation, severe coronary stenoses) as well as the sudden death phenotype of uPA-overexpressing mice. Moreover, fat-fed 37-week-old recipients of uPA-null BM had significantly less atherosclerosis than recipients of uPA wild-type marrow (40% less aortic surface lesion area; P=0.03). Conclusions— The level of uPA expression by macrophages—over a broad range—is an important determinant of atherosclerotic lesion growth in Apoe–/– mice.

Moderately Decreased Cholesterol Absorption Rates Are Associated With a Large Atheroprotective Effect [Integrative Physiology/Experimental Medicine]

Objective— Human treatment with ezetimibe results in a moderate 50% to 54% decrease in cholesterol absorption and a 15% to 20% decrease in plasma LDL-cholesterol levels; nevertheless, the efficacy of ezetimibe therapy has been recently challenged by the ENHANCE trial. We examined the efficacy of a moderate decrease in cholesterol absorption in preventing atherosclerosis formation in the mouse. Methods and Results— Congenic 14DKK animals, consisting of a castaneus (CASA/Rk) chromosome 14 interval introgressed onto the C57BL/6J background, displayed a moderate decrease in cholesterol absorption rates. The effect of moderately decreased absorption on atherosclerosis formation was determined in 14DKK apolipoprotein E knockouts (14DKK-apoEKO). When compared to chow diet–fed control apoEKO mice, congenic 14DKK-apoEKO displayed a moderate 41% decrease in cholesterol absorption rates, 30% to 37% decrease in plasma cholesterol levels, and a 70% decrease in atherosclerosis formation. Studies on cholesterol efflux and reverse cholesterol transport (RCT) from 14DKK bone marrow–derived macrophages rejected a 14DKK interval-dependent atheroprotective effects that operate in macrophages. In contrast, 14DKK-apoEKO congenics were characterized by a 60% increase in RCT from peripheral tissue macrophages. Conclusions— These studies strongly suggest that moderately decreased cholesterol absorption rates result in a large atheroprotective effect attributable to a decrease in plasma cholesterol levels and an increase in RCT from peripheral tissue macrophages.

Heat Shock Protein 27 Protects Against Atherogenesis via an Estrogen-Dependent Mechanism: Role of Selective Estrogen Receptor Beta Modulation [Integrative Physiology/Experimental Medicine]

Objective— We recently identified HSP27 as an atheroprotective protein that acts extracellularly to prevent foam cell formation and atherogenesis in female but not male mice, where serum levels of HSP27 were increased and inversely correlated with degree of lesion burden. In the current study we sought to determine whether estrogens are required for the observed atheroprotective benefits of HSP27 as well as its extracellular release. Methods and Results— In vitro estrogens prompted the release of HSP27 from macrophages in an ERβ specific manner that involved exosomal trafficking. Ovariectomy nullified the previously recognized attenuation in aortic lesion area in HSP27o/eapoE–/– mice compared to apoE–/– mice. Supplementation with 17β-estradiol resulted in a >15x increase in uterine weight and attenuation of atherogenesis in all mice, although HSP27o/eapoE–/– had 34% less lesion burden compared to apoE–/– mice. Mice treated with the ERβ-specific agonist, DPN had no effect on uterine weight but a 28% decrease in aortic lesion area in HSP27o/eapoE–/– compared to apoE–/– mice. HSP27 serum levels showed a similar gradual increase with E2 and DPN replacement treatment but did not change in untreated mice. Conclusions— The extracellular release of and atheroprotection provided by HSP27 is estrogen dependent.

Laminar Shear Stress Regulates Endothelial Kinin B1 Receptor Expression and Function: Potential Implication in Atherogenesis [Integrative Physiology/Experimental Medicine]

Objective— The proinflammatory phenotype induced by low laminar shear stress (LSS) is implicated in atherogenesis. The kinin B1 receptor (B1R), known to be induced by inflammatory stimuli, exerts many proinflammatory effects including vasodilatation and leukocyte recruitment. We investigated whether low LSS is a stimulus for endothelial B1R expression and function. Methods and Results— Human and mouse atherosclerotic plaques expressed high level of B1R mRNA and protein. In addition, B1R expression was upregulated in the aortic arch (low LSS region) of ApoE–/– mice fed a high-fat diet compared to vascular regions of high LSS and animals fed normal chow. Of interest, a greater expression of B1R was noticed in endothelial cells from regions of low LSS in aortic arch of ApoE–/– mice. B1R was also upregulated in human umbilical vein endothelial cells (HUVECs) exposed to low LSS (0 to 2 dyn/cm2) compared to physiological LSS (6 to 10 dyn/cm2): an effect similarly evident in murine vascular tissue perfused ex vivo. Functionally, B1R activation increased prostaglandin and CXCL5 expression in cells exposed to low, but not physiological, LSS. IL-1β and ox-LDL induced B1R expression and function in HUVECs, a response substantially enhanced under low LSS conditions and inhibited by blockade of NFB activation. Conclusion— Herein, we show that LSS is a major determinant of functional B1R expression in endothelium. Furthermore, whereas physiological high LSS is a powerful repressor of this inflammatory receptor, low LSS at sites of atheroma is associated with substantial upregulation, identifying this receptor as a potential therapeutic target.

Simvastatin Inhibits Angiotensin II-Induced Abdominal Aortic Aneurysm Formation in Apolipoprotein E-Knockout Mice: Possible Role of ERK [Integrative Physiology/Experimental Medicine]

Objective— Abdominal aortic aneurysm (AAA) is a life-threatening disease affecting almost 10% of the population over age 65. Generation of AAAs by infusion of angiotensin (Ang) II in apolipoprotein E-knockout (ApoE–/–) mice is an animal model which supports an imbalance of the renin-angiotensin system in the pathogenesis of AAA. The effect of statins on AngII-mediated AAA formation and the associated neovascularization is not known. Here we determined the effect of simvastatin and the ERK inhibitor, CI1040, on AngII-stimulated AAA formation. Methods and Results— ApoE–/– mice infused for 28 days with AngII using osmotic minipumps were treated with placebo, 10 mg/kg/d simvastatin, or 100 mg/kg/d CI1040. 95% of AngII-treated mice developed AAA with neovascularization of the lesion, increased ERK phosphorylation, MCP-1 secretion, and MMP activity. These effects were markedly reversed by simvastatin and in part by CI1040. Furthermore, simvastatin and the ERK inhibitor U0126 reversed AngII-stimulated angiogenesis and MMP secretion by human umbilical vein endothelial cells. Conclusions— These data support the conclusion that simvastatin interferes with AAA formation induced by AngII in ApoE–/– mice at least in part via ERK inhibition.

Smooth Muscle LDL Receptor-Related Protein-1 Inactivation Reduces Vascular Reactivity and Promotes Injury-Induced Neointima Formation [Integrative Physiology/Experimental Medicine]

Objective— Defective smooth muscle expression of LDL receptor-related protein-1 (Lrp1) increases atherosclerosis in hypercholesterolemic mice. This study explored the importance of smooth muscle Lrp1 expression under normolipidemic conditions. Methods and Results— Smooth muscle cells isolated from control (smLrp1+/+) and smooth muscle-specific Lrp1 knockout (smLrp1–/–) mice were characterized based on morphology, smooth muscle marker protein expression levels, and growth rates in vitro. Vascular functions were assessed by aortic constrictive response to agonist stimulation in situ and neointimal hyperplasia to carotid arterial injury in vivo. The smLrp1–/– smooth muscle cells displayed reduced -actin and calponin expression and an accelerated growth rate attribtuable to sustained phosphorylation of platelet-derived growth factor receptor (PRGFR) and protein kinase B/Akt. Vasoconstrictive response to agonist stimulation was impaired in aortic rings isolated from smLrp1–/– mice. Injury-induced neointimal hyperplasia was significantly increased in smLrp1–/– mice. The increase in neointima was associated with corresponding elevated activation of PDGFR signaling pathway. Conclusions— Smooth muscle expression of Lrp1 is important in maintaining normal vascular functions under normolipidemic conditions. The absence of Lrp1 expression results in greater smooth muscle cell proliferation, deficient contractile protein expression, impairment of vascular contractility, and promotion of denudation-induced neointimal hyperplasia.

Thiol Oxidative Stress Induced by Metabolic Disorders Amplifies Macrophage Chemotactic Responses and Accelerates Atherogenesis and Kidney Injury in LDL Receptor-Deficient Mice [Integrative Physiology/Experimental Medicine]

Background— Strengthening the macrophage glutathione redox buffer reduces macrophage content and decreases the severity of atherosclerotic lesions in LDL receptor-deficient (LDLR–/–) mice, but the underlying mechanisms were not clear. This study examined the effect of metabolic stress on the thiol redox state, chemotactic activity in vivo, and the recruitment of macrophages into atherosclerotic lesions and kidneys of LDL-R–/– mice in response to mild, moderate, and severe metabolic stress. Methods and Results— Reduced glutathione (GSH) and glutathione disulfide (GSSG) levels in peritoneal macrophages isolated from mildly, moderately, and severe metabolically-stressed LDL-R–/– mice were measured by HPLC, and the glutathione reduction potential (Eh) was calculated. Macrophage Eh correlated with the macrophage content in both atherosclerotic (r2=0.346, P=0.004) and renal lesions (r2=0.480, P=0.001) in these mice as well as the extent of both atherosclerosis (r2=0.414, P=0.001) and kidney injury (r2=0.480, P=0.001). Compared to LDL-R–/– mice exposed to mild metabolic stress, macrophage recruitment into MCP-1-loaded Matrigel plugs injected into LDL-R–/– mice increased 2.6-fold in moderately metabolically-stressed mice and 9.8-fold in severely metabolically-stressed mice. The macrophage Eh was a strong predictor of macrophage chemotaxis (r2=0.554, P<0.001). Conclusion— Thiol oxidative stress enhances macrophage recruitment into vascular and renal lesions by increasing the responsiveness of macrophages to chemoattractants. This novel mechanism contributes at least in part to accelerated atherosclerosis and kidney injury associated with dyslipidemia and diabetes in mice.

Ccl2 and Ccl3 Mediate Neutrophil Recruitment via Induction of Protein Synthesis and Generation of Lipid Mediators [Integrative Physiology/Experimental Medicine]

Objective— Although the chemokines monocyte chemoattractant protein-1 (Ccl2/JE/MCP-1) and macrophage inflammatory protein-1 (Ccl3/MIP-1) have recently been implicated in neutrophil migration, the underlying mechanisms remain largely unclear. Methods and Results— Stimulation of the mouse cremaster muscle with Ccl2/JE/MCP-1 or Ccl3/MIP-1 induced a significant increase in numbers of firmly adherent and transmigrated leukocytes (>70% neutrophils) as observed by in vivo microscopy. This increase was significantly attenuated in mice receiving an inhibitor of RNA transcription (actinomycin D) or antagonists of platelet activating factor (PAF; BN 52021) and leukotrienes (MK-886; AA-861). In contrast, leukocyte responses elicited by PAF and leukotriene-B4 (LTB4) themselves were not affected by actinomycin D, BN 52021, MK-886, or AA-861. Conversely, PAF and LTB4, but not Ccl2/JE/MCP-1 and Ccl3/MIP-1, directly activated neutrophils as indicated by shedding of CD62L and marked upregulation of CD11b. Moreover, Ccl2/JE/MCP-1- and Ccl3/MIP-1-elicited leakage of fluorescein isothiocyanate dextran as well as collagen IV remodeling within the venular basement membrane were completely absent in neutrophil-depleted mice. Conclusions— Ccl2/JE/MCP-1 and Ccl3/MIP-1 mediate firm adherence and (subsequent) transmigration of neutrophils via protein synthesis and secondary generation of leukotrienes and PAF, which in turn directly activate neutrophils. Thereby, neutrophils facilitate basement membrane remodeling and promote microvascular leakage.

Bone Marrow-Derived Cell-Specific Chemokine (C-C Motif) Receptor-2 Expression is Required for Arteriolar Remodeling [Integrative Physiology/Experimental Medicine]

Objective— Bone marrow-derived cells (BMCs) and inflammatory chemokine receptors regulate arteriogenesis and angiogenesis. Here, we tested whether arteriolar remodeling in response to an inflammatory stimulus is dependent on BMC-specific chemokine (C-C motif) receptor 2 (CCR2) expression and whether this response involves BMC transdifferentiation into smooth muscle. Methods and Results— Dorsal skinfold window chambers were implanted into C57Bl/6 wild-type (WT) mice, as well as the following bone marrow chimeras (donor-host): WT-WT, CCR2–/–-WT, WT-CCR2–/–, and EGFP+-WT. One day after implantation, tissue MCP-1 levels rose from "undetectable" to 463pg/mg, and the number of EGFP+ cells increased more than 4-fold, indicating marked inflammation. A 66% (28 µm) increase in maximum arteriolar diameter was observed over 7 days in WT-WT mice. This arteriolar remodeling response was completely abolished in CCR2–/–-WT mice but largely rescued in WT-CCR2–/– mice. EGFP+ BMCs were numerous throughout the tissue, but we found no evidence that EGFP+ BMCs transdifferentiate into smooth muscle, based on examination of >800 arterioles and venules. Conclusions— BMC-specific CCR2 expression is required for injury/inflammation-associated arteriolar remodeling, but this response is not characterized by the differentiation of BMCs into smooth muscle.

CXCR4 Expression Determines Functional Activity of Bone Marrow-Derived Mononuclear Cells for Therapeutic Neovascularization in Acute Ischemia [Integrative Physiology/Experimental Medicine]

Objective— Bone marrow–derived mononuclear cells (BMCs) improve the functional recovery after ischemia. However, BMCs comprise a heterogeneous mixture of cells, and it is not known which cell types are responsible for the induction of neovascularization after cell therapy. Because cell recruitment is critically dependent on the expression of the SDF-1-receptor CXCR4, we examined whether the expression of CXCR4 may identify a therapeutically active population of BMCs. Methods and Results— Human CXCR4+ and CXCR4– BMCs were sorted by magnetic beads. CXCR4+ BMCs showed a significantly higher invasion capacity under basal conditions and after SDF-1 stimulation. Hematopoietic or mesenchymal colony-forming capacity did not differ between CXCR4+ and CXCR4– BMCs. Injection of CXCR4+ BMCs in mice after induction of hindlimb ischemia significantly improved the recovery of perfusion compared to injection of CXCR4– BMCs. Likewise, capillary density was significantly increased in CXCR4+ BMC-treated mice. Because part of the beneficial effects of cell therapy were attributed to the release of paracrine effectors, we analyzed BMC supernatants for secreted factors. Importantly, supernatants of CXCR4+ BMCs were enriched in the proangiogenic cytokines HGF and PDGF-BB. Conclusion— CXCR4+ BMCs exhibit an increased therapeutic potential for blood flow recovery after acute ischemia. Mechanistically, their higher migratory capacity and their increased release of paracrine factors may contribute to enhanced tissue repair.

Monocyte Chemoattractant Proteins Mediate Myocardial Microvascular Dysfunction in Swine Renovascular Hypertension [Integrative Physiology/Experimental Medicine]

Background— Monocyte chemoattractant proteins (MCPs) play an important role in mediating inflammatory processes. Hypertension (HTN) is associated with inflammation as well as impaired cardiac microcirculatory function and structure, but the contribution of MCPs to these alterations remained unclear. This study tested the hypothesis that MCPs regulate cardiac microvascular function and structure in experimental HTN. Methods and Results— Pigs (n=6 per group) were studied after 10 weeks of normal, renovascular HTN, or renovascular HTN+ bindarit (MCPs inhibitor, 50 mg/kg/d PO). Left ventricular (LV) function, myocardial microvascular permeability, and fractional vascular volume were assessed by fast computed tomography before and after adenosine infusion (400 µg/kg/min). Myocardial fibrosis, inflammation, and microvascular remodeling were determined ex vivo. Hypertension was not altered by bindarit, but LV hypertrophy and diastolic function were improved. In response to adenosine, myocardial microvascular permeability increased in HTN (from 0.0083±0.0009 to 0.0103±0.0011 AU, P=0.038 versus baseline) and fractional vascular volume decreased, whereas both remained unchanged in normal and HTN+bindarit pigs. HTN upregulated endothelin-1 expression, myocardial inflammation, and microvascular wall thickening, which were inhibited by bindarit. Conclusions— MCPs partly mediate myocardial inflammation, fibrosis, vascular remodeling, and impaired vascular integrity induced by hypertension. Inhibition of MCPs could potentially be a therapeutic target in hypertensive cardiomyopathy.

Stimulation of Coronary Collateral Growth by Granulocyte Stimulating Factor: Role of Reactive Oxygen Species [Integrative Physiology/Experimental Medicine]

Objective— The purpose of this study was to determine whether G-CSF promotes coronary collateral growth (CCG) and decipher the mechanism for this stimulation. Methods and Results— In a rat model of repetitive episodic myocardial ischemia (RI, 40 seconds LAD occlusion every 20 minutes for 2 hours and 20 minutes, 3 times/d for 5 days) CCG was deduced from collateral-dependent flow (flow to LAD region during occlusion). After RI, G-CSF (100 µg/kg/d) increased CCG (P<0.01) (0.47±0.15) versus vehicle (0.14±0.06). Surprisingly, G-CSF treatment without RI increased CCG (0.57±0.18) equal to G-CSF+RI. We evaluated ROS by dihydroethidine (DHE) fluorescence (LV injection, 60 µg/kg, during two episodes of ischemia). DHE fluorescence was double in G-CSF+RI versus vehicle+RI (P<0.01), and even higher in G-CSF without RI (P<0.01). Interestingly, the DHE signal did not colocalize with myeloperoxidase (immunostaining, neutrophil marker) but appeared in cardiac myocytes. The study of isolated cardiac myocytes revealed the cytokine stimulates ROS which elicit production of angiogenic factors. Apocynin inhibited G-CSF effects both in vivo and in vitro. Conclusions— G-CSF stimulates ROS production directly in cardiomyocytes, which plays a pivotal role in triggering adaptations of the heart to ischemia including growth of the coronary collaterals.

Somitovasculin, a Novel Endothelial-Specific Transcript Involved in the Vasculature Development [Integrative Physiology/Experimental Medicine]

Objective— We recently isolated and characterized endothelial-like CD31+ cells derived from mouse embryonic stem (mES) cells and identified their transcriptome. The main objective of this study was to determine the functional relevance of the transcripts of unknown function (TUF) for vasculature development. Methods and Results— We selected 2 TUFs of more than 27 to study their role for blood vessel development in zebrafish. Morpholino (MO) knockdown of the zebrafish orthologs of the first TUF (TUF1, mouse cDNA BC022623) showed disruption of the intersegmental vessels (ISV) at 2 days postfertilization as observed by live imaging of fli:EGFP-transgenic embryos. The morphants showed abnormal blood circulation, but no effect on hematopoiesis was observed as demonstrated by gata-1 in situ hybridizations. Because knockdown of TUF1 resulted in disruption of the ISV patterning we named the TUF1 somitovasculin. TUF2 has been identified as cDNA clone BC020535. The MO knockdown of TUF2 resulted in a phenotype with an enlarged heart and the embryos lacked circulation completely. Conclusion— We have shown the participation of a novel transcript (named somitovasculin) in circulatory vessel development. The combination of expression profiling in differentiating mES cells and the zebrafish model has the potential for rapid identification and functional characterization of TUFs.

Therapeutic Potential of Unrestricted Somatic Stem Cells Isolated from Placental Cord Blood for Cardiac Repair Post Myocardial Infarction [Integrative Physiology/Experimental Medicine]

Objective— Unrestricted somatic stem cells (USSCs) were successfully identified from human cord blood. However, the efficacy of USSC transplantation for improving left ventricular (LV) function post myocardial infarction (MI) is still controversial. Methods and Results— PBS, 1x106 human fibroblasts (Fbr), 1x105 USSCs (LD), or 1x106 USSCs (HD) were transplanted intramyocardially 20 minutes after ligating the LAD of nude rats. Echocardiography and a microtip conductance catheter at day 28 revealed a dose-dependent improvement of LV function after USSC transplantation. Necropsy examination revealed dose-dependent augmentation of capillary density and inhibition of LV fibrosis. Dual-label immunohistochemistry for cardiac troponin-I and human nuclear antigen (HNA) demonstrated that human cardiomyocytes (CMCs) were dose-dependently generated in ischemic myocardium 28 days after USSC transplantation. Similarly, dual-label immunostaining for smooth muscle actin and class I human leukocyte antigen or that for von Willebrand factor and HNA also revealed a dose-dependent vasculogenesis after USSC transplantation. RT-PCR indicated that expression of human-specific genes of CMCs, smooth muscle cells, and endothelial cell markers in infarcted myocardium were significantly augmented in USSC-treated animals compared with control groups. Conclusions— USSC transplantation leads to functional improvement and recovery from MI and exhibits a significant and dose-dependent potential for concurrent cardiomyogenesis and vasculogenesis.

c-Jun DNAzymes Inhibit Myocardial Inflammation, ROS Generation, Infarct Size, and Improve Cardiac Function After Ischemia-Reperfusion Injury [Integrative Physiology/Experimental Medicine]

Objectives— Coronary reperfusion has been the mainstay therapy for reduced infarct size after a heart attack. However, this intervention also results in myocardial injury by initiating a marked inflammatory reaction, and new treatments are keenly sought. Methods and Results— The basic-region leucine zipper protein, c-Jun is poorly expressed in the normal myocardium and is induced within 24 hours after myocardial ischemia-reperfusion injury. Synthetic catalytic DNA molecules (DNAzymes) targeting c-Jun (Dz13) reduce infarct size in the area-at-risk (AAR) regardless of whether it is delivered intramyocardially at the initiation of ischemia or at the time of reperfusion. Dz13 attenuates neutrophil infiltration, c-Jun and ICAM-1 expression in vascular endothelium, cardiomyocyte apoptosis, and the generation of reactive oxygen species in the reperfused myocardium. It inhibits infiltration into the AAR of complement 3 (C3), C3a receptor (C3aR), membrane attack complex-1 (Mac-1), or matrix metalloproteinase-2 (MMP-2) positive inflammatory cells. Dz13 also improves cardiac function without influencing myocardial vascularity or fibrosis. Conclusion— These findings demonstrate the regulatory role of c-Jun in the pathogenesis of myocardial inflammation and infarction following ischemia-reperfusion injury, and inhibition of this process using catalytic DNA.

Nrf2 Protects Against Maladaptive Cardiac Responses to Hemodynamic Stress [Integrative Physiology/Experimental Medicine]

Background— Reactive oxygen species (ROS) play an important role in the maintenance of cardiovascular homeostasis. The present study sought to determine whether nuclear factor erythroid-2 related factor 2 (Nrf2), a master gene of the endogenous antioxidant defense system, is a critical regulator of the cardiac hypertrophic response to pathological stress. Methods and Results— Cardiac hypertrophy and dysfunction were established in mice by transverse aortic constriction (TAC). Nrf2 expression was transiently increased and then declined to the basal level while impairment of cardiac function proceeded. The knockout of Nrf2 (Nrf2–/–) did not cause any apparent structural and functional abnormalities in the unstressed heart. However, Nrf2–/– mice after TAC developed pathological cardiac hypertrophy, significant myocardial fibrosis and apoptosis, overt heart failure, and increased mortality, which were associated with elevated myocardial levels of 4-hydroxy-2-nonenal and 8-hydroxydeoxyguanosine and a complete blockade of the myocardial expression of several antioxidant genes. Overexpression of Nrf2 dramatically inhibited hypertrophic factor–induced ROS production and growth in both cardiomyocytes and cardiac fibroblasts, whereas knockdown of Nrf2 exerted opposite effects in both cells. Conclusions— These findings demonstrate that activation of Nrf2 provides a novel mechanism to protect the murine heart against pathological cardiac hypertrophy and heart failure via suppressing oxidative stress.

Activation of Nrf2 in Endothelial Cells Protects Arteries From Exhibiting a Proinflammatory State [Integrative Physiology/Experimental Medicine]

Objective— Proinflammatory mediators influence atherosclerosis by inducing adhesion molecules (eg, VCAM-1) on endothelial cells (ECs) via signaling intermediaries including p38 MAP kinase. Regions of arteries exposed to high shear stress are protected from inflammation and atherosclerosis, whereas low-shear regions are susceptible. Here we investigated whether the transcription factor Nrf2 regulates EC activation in arteries. Methods and Results— En face staining revealed that Nrf2 was activated in ECs at an atheroprotected region of the murine aorta where it negatively regulated p38–VCAM-1 signaling, but was expressed in an inactive form in ECs at an atherosusceptible site. Treatment with sulforaphane, a dietary antioxidant, activated Nrf2 and suppressed p38–VCAM-1 signaling at the susceptible site in wild-type but not Nrf2–/– animals, indicating that it suppresses EC activation via Nrf2. Studies of cultured ECs revealed that Nrf2 inactivates p38 by suppressing an upstream activator MKK3/6 and by enhancing the activity of the negative regulator MKP-1. Conclusions— Nrf2 prevents ECs at the atheroprotected site from exhibiting a proinflammatory state via the suppression of p38–VCAM-1 signaling. Pharmacological activation of Nrf2 reduces EC activation at atherosusceptible sites and may provide a novel therapeutic strategy to prevent or reduce atherosclerosis.

Dynamic Observation of Mechanically-Injured Mouse Femoral Artery Reveals an Antiinflammatory Effect of Renin Inhibitor [Integrative Physiology/Experimental Medicine]

Objective— The renin-angiotensin-aldosterone system (RAS) plays a central role in atherosclerosis. To investigate the effects of a direct renin inhibitor aliskiren on vascular inflammation, we conducted leukocyte adhesion assays in vivo and in vitro using a novel real-time imaging system. Methods and Results— Aliskiren (10 mg/kg/d) or PBS was administered to C57BL/6 mice (6–7 weeks of age; Oriental Yeast, Tokyo, Japan) for 2 weeks via an osmotic pump. Blood pressure was not significantly changed in the 2 groups throughout the experimental period. A perivascular cuff injury was then introduced to the femoral artery and real-time intravital microscopic observation was conducted 24 hours after injury. The number of adherent leukocytes was elevated in the injured mice without aliskiren (43.8±9.3/10–2 mm2), whereas that was significantly reduced in the mice with aliskiren (18.4±4.4, P<0.05). Treatment of human umbilical vein endothelial cells (HUVECs) with aliskiren significantly reduced the adhesion of THP-1 cells to TNF-–activated HUVECs (P<0.05). Interestingly, TNF-–induced renin activity and angiotensin II production in HUVECs were also blunted by aliskiren. Furthermore, exogenous renin and angiotensin II abrogated the aliskiren-mediated reduction of THP-1 cell adhesion to HUVECs. Conclusions— Our in vivo and in vitro findings indicate a pivotal role for renin inhibition in vascular inflammation independent of blood pressure.

The {alpha}11{beta}1 Integrin Has a Mechanistic Role in Control of Interstitial Fluid Pressure and Edema Formation in Inflammation [Integrative Physiology/Experimental Medicine]

Objective— Collagen-binding integrins may be involved in controlling interstitial fluid pressure (Pif), transcapillary fluid flux, and tissue fluid volume. Our aim was to explore whether the newly discovered collagen binding 11β1 integrin has a mechanistic role in inflammatory edema formation. Methods and Results— In collagen matrices seeded with a mixture of mast cells and fibroblasts, fibroblasts lacking the 11 integrin subunit (11–/–) contracted collagen gels less efficiently than control fibroblasts, suggesting that the 11β1 integrin is able to mediate tensile force in connective tissues. In 11–/– mice, control Pif in skin did not differ from the pressure found in wild-type mice. Whereas a reduction in Pif was found in control mice after inducing inflammation, thereby contributing to fluid extravasation and edema formation, such a reduction was not seen in 11–/– mice. That this effect is mediated through the extracellular compartment is suggested by a similar plasma protein extravasation ratio in 11–/– and wild-type mice. Conclusions— Our data suggest that 11β1 integrins on dermal fibroblasts mediate collagen lattice remodeling and have a mechanistic role in controlling Pif in inflammation and thereby fluid extravasation and edema formation in vivo.

PPAR{beta}/{delta} Agonists Modulate Platelet Function via a Mechanism Involving PPAR Receptors and Specific Association/Repression of PKC{alpha}-Brief Report [Cell Biology/Signaling]

Objectives— Peroxisome proliferator-activated receptor β/ (PPARβ/) is a nuclear receptor found in platelets. PPARβ/ agonists acutely inhibit platelet function within a few minutes of addition. As platelets are anucleated, the effects of PPARβ/ agonists on platelets must be nongenomic. Currently, the particular role of PPARβ/ receptors and their intracellular signaling pathways in platelets are not known. Methods and Results— We have used mice lacking PPARβ/ (PPARβ/–/–) to show the effects of the PPARβ/ agonist GW501516 on platelet adhesion and cAMP levels are mediated specifically by PPARβ/, however GW501516 had no PPARβ/-specific effect on platelet aggregation. Studies in human platelets showed that PKC, which can mediate platelet activation, was bound and repressed by PPARβ/ after platelets were treated with GW501516. Conclusions— These data provide evidence of a novel mechanism by which PPAR receptors influence platelet activity and thereby thrombotic risk.

Foxp3 Regulates Megakaryopoiesis and Platelet Function [Cell Biology/Signaling]

Objective— Platelets are crucial for hemostasis and are vital regulators of inflammation. Foxp3 is a key transcription factor for T regulatory cell development. Humans with IPEX (immune dysregulation, polyendocrinopathy, enteropathy, x-linked) and the scurfy (Foxp3sf) mouse have mutations in the Foxp3 gene that lead to a host of pathologies including autoimmunity and skin diseases. Scurfy mice and some humans with IPEX are also thrombocytopenic. The purpose of this study was to determine whether the absence of functional Foxp3 leads to defects in megakaryocytes and platelets. Methods and Results— We discovered that human and mouse megakaryocytes express Foxp3 mRNA and protein. Using shRNA and Foxp3sf mice, we demonstrated that Foxp3-deficient mouse and human megakaryocyte progenitors exhibited proliferation defects. Striking platelet abnormalities were observed in both an IPEX patient and Foxp3sf mice. Impaired platelet spreading and release of TGF-β and CD40 ligand (CD40L), and abnormal levels of plasma CD40L were observed in a case of IPEX syndrome. Foxp3sf mice were thrombocytopenic and had increased platelet volume and altered serum levels of CD40L, TXB2, and TGF-β. Conclusion— These findings provide compelling new evidence that Foxp3 is needed for proper megakaryopoiesis and plays a role in regulating platelet function including spreading and release.

Rapid Procoagulant Phosphatidylserine Exposure Relies on High Cytosolic Calcium Rather Than on Mitochondrial Depolarization [Cell Biology/Signaling]

Objective— Relationships between intracellular Ca2+ concentration ([Ca2+]cyt) and apoptotic events, such as mitochondrial depolarization (m loss) and Bcl-2 and Bad phosphorylation, were analyzed in platelets and Jurkat cells in relation to rapid procoagulant phosphatidylserine (PS) exposure. Methods and Results— Platelets were stimulated with A23187, thapsigargin (TG) and thrombin plus convulxin (Thr/Cvx), and Jurkat cells with ionomycin, in the presence or absence of cyclosporin A (CsA), a mitochondrial permeability transition pore inhibitor. m loss occurred when platelets were stimulated in Ca2+ medium in conditions exposing PS, but also in EGTA medium. CsA inhibited PS exposure, [Ca2+]cyt increase, and m loss in platelets stimulated with TG and Thr/Cvx, but had no inhibitory effect on A23187 stimulation. CsA reduced TG-induced Ca2+ release from the endoplasmic reticulum and, consequently, external Ca2+ influx. In ionomycin-stimulated Jurkat cells, rapid PS exposure was evidenced but not m loss, and CsA did not inhibit the process. The status of phosphorylated Bad and Bcl-2 in both cell types remained unchanged on stimulation. Conclusions— Whether m loss occurs or not, PS exposure is triggered by a high [Ca2+]cyt increase. Data further demonstrate that CsA prevents membrane scrambling by inhibiting the high [Ca2+]cyt increase, independently of its effect on mitochondrial permeability transition pore.

Disinhibition of SOD-2 Expression to Compensate for a Genetically Determined NO Deficit in Endothelial Cells-Brief Report [Cell Biology/Signaling]

Objective— Homozygosity for the –786C-variant of the human nos-3 gene is a risk factor for coronary artery disease (CAD). Interestingly, affected individuals develop CAD more frequently but not earlier than the general population. Methods and Results— Genotyped primary human umbilical vein endothelial cells (ECs) were exposed to fluid shear stress (FSS) and analyzed for nitric oxide (NO) and superoxide anion (O2–) formation as well as mRNA and protein expression of different antioxidant enzymes. Dysfunctional CC-genotype ECs failed to upregulate NO synthase expression in response to FSS and exhibited a reduced NO synthesis capacity when compared to functionally intact TT-genotype ECs. However, only CC-genotype ECs responded to FSS with an Egr-1–mediated increase in manganese-containing superoxide dismutase (SOD-2) expression, shielding them from endothelin-1–induced oxidative stress in a NO-independent manner. Conclusions— This FSS-induced rise in SOD-2 expression in CC-genotype ECs effectively stabilizes their antiatherosclerotic phenotype and may explain not only the comparatively slow onset of CAD in homozygous carriers of the C-allele of the nos-3 gene but also define a general strategy for preventing endothelial dysfunction at the outset of atherosclerosis.

Upregulation of Glutathione Peroxidase Offsets Stretch-Induced Proatherogenic Gene Expression in Human Endothelial Cells [Cell Biology/Signaling]

Objective— Localization of atherosclerotic plaques typically correlates with areas of biomechanical strain where shear stress is decreased while stretch, thought to promote atherogenesis through enhanced oxidative stress, is increased. Methods and Results— In human cultured endothelial cells, nitric oxide synthase expression was exclusively shear stress–dependent whereas expression of glutathione peroxidase-1 (GPx-1), but not that of Cu2+/Zn2+-superoxide dismutase or Mn2+-superoxide dismutase, was upregulated solely in response to cyclic stretch. GPx-1 expression was also enhanced in isolated mouse arteries perfused at high pressure. Combined pharmacological and decoy oligodeoxynucleotide blockade revealed that activation of p38 MAP kinase followed by nuclear translocation of CCAAT/enhancer binding protein plays a pivotal role in stretch-induced GPx-1 expression in human endothelial cells. Antisense oligodeoxynucleotide knockdown of GPx-1 reinforced both their capacity to generate hydrogen peroxide and the transient stretch-induced expression of CD40, monocyte chemoatractant protein-1, and vascular cell adhesion molecule-1. Consequently, THP-1 monocyte adhesion to the GPx-1–depleted cells was augmented. Conclusions— Stretch-induced proatherosclerotic gene expression in human endothelial cells seems to be hydrogen peroxide-mediated. The concomitant rise in GPx-1 expression, but not that of other antioxidant enzymes, may comprise an adaptive mechanism through which the cells maintain their antiatherosclerotic properties in spite of a decreased bioavailability of nitric oxide.

Flow Activation of AMP-Activated Protein Kinase in Vascular Endothelium Leads to Kruppel-Like Factor 2 Expression [Cell Biology/Signaling]

Objective— Vascular endothelial cells (ECs) confer atheroprotection at locations of the arterial tree where pulsatile laminar flow (PS) exists with a high shear stress and a large net forward direction. We investigated whether the PS-induced expression of the transcription factor Krüppel-Like Factor 2 (KLF2) in cultured ECs and its expression in the mouse aorta is regulated by AMP-activated protein kinase (AMPK). Methods and Results— AMPK inhibition by Compound C or siRNA had a significant blocking effect on the PS-induced KLF2 expression. The induction of KLF2 by PS led to the increase in eNOS and the suppression of ET-1, which could be reversed by KLF2 siRNA. In addition, PS induced the phosphorylation of ERK5 and MEF2 which are necessary for the KLF2 expression. These mechanotransduction events were abrogated by the blockade of AMPK. Furthermore, the phosphorylation levels of ERK5 and MEF2, as well as the expression of KLF2, were significantly reduced in the aorta of AMPK2 knockout mice when compared with wild-type control mice. Conclusion— The flow-mediated AMPK activation is a newly defined KLF2 regulatory pathway in vascular endothelium that acts via ERK5/MEF2.

Alzheimer Disease-Associated Peptide, Amyloid {beta}40, Inhibits Vascular Regeneration With Induction of Endothelial Autophagy [Cell Biology/Signaling]

Objective— Although the majority of cases of Alzheimer disease (AD) are known to be attributable to the sporadic (nongenetic) form of the disease, the mechanism underlying its cause and progression still remains unclear. Methods and Results— We found that vascular β-amyloid (Aβ), Aβ40, inhibited the proliferative activity of human brain vascular endothelial cells (HBECs) without toxic effects on them. This peptide also inhibited tube formation and migration of HBECs. Moreover, Aβ40 inhibited ex vivo hippocampal revascularization, reendothelialization, and the differentiation of adult endothelial progenitor cells. Importantly, Aβ40 suppressed the proliferative activity of HBECs through the induction of "self-digesting" autophagy. This induction involved the intracellular regulation of class 3 phosphatidylinositol 3-kinase (PI3K) as well as Akt signaling in HBECs. Furthermore, tissue culture of murine brain sections from GFP-LC3 transgenic mice revealed that Aβ40 not only reduced the vessel density in hippocampal lesions, but also induced autophagy in neurovascular ECs. Conclusions— Our present findings indicate that the initial progression of AD might be in part driven by Aβ40-induced endothelial autophagy and impairment of neurovascular regeneration, suggesting important implications for therapeutic approaches to AD.

The Phosphorylation Motif at Serine 225 Governs the Localization and Function of Sphingosine Kinase 1 in Resistance Arteries [Cell Biology/Signaling]

Objective— The purpose of this study was to characterize a phosphorylation motif at serine 225 as a molecular switch that regulates the pressure-dependent activation of sphingosine kinase 1 (Sk1) in resistance artery smooth muscle cells. Methods and Results— In isolated hamster gracilis muscle resistance arteries, pressure-dependent activation/translocation of Sk1 by ERK1/2 was critically dependent on its serine 225 phosphorylation site. Specifically, expression of Sk1S225A reduced resting and myogenic tone, resting Ca2+, pressure-induced Ca2+ elevations, and Ca2+ sensitivity. The lack of function of the Sk1S225A mutant could not be entirely overcome by forced localization to the plasma membrane via a myristoylation/palmitylation motif; the membrane anchor also significantly inhibited the function of the wild-type Sk1 enzyme. In both cases, Ca2+ sensitivity and myogenic tone were attenuated, whereas Ca2+ handling was normalized/enhanced. These discrete effects are consistent with cell surface receptor-mediated effects (Ca2+ sensitivity) and intracellular effects of S1P (Ca2+ handling). Accordingly, S1P2 receptor inhibition (1µmol/L JTE013) attenuated myogenic tone without effect on Ca2+. Conclusions— Translocation and precise subcellular positioning of Sk1 is essential for full Sk1 function; and two distinct S1P pools, proposed to be intra- and extracellular, contribute to the maintenance of vascular tone.

Liver X Receptor-Mediated Induction of Cholesteryl Ester Transfer Protein Expression Is Selectively Impaired in Inflammatory Macrophages [Cell Biology/Signaling]

Objective— Cholesteryl ester transfer protein (CETP) is a target gene for the liver X receptor (LXR). The aim of this study was to further explore this regulation in the monocyte-macrophage lineage and its modulation by lipid loading and inflammation, which are key steps in the process of atherogenesis. Methods and Results— Exposure of bone marrow–derived macrophages from human CETP transgenic mice to the T0901317 LXR agonist increased CETP, PLTP, and ABCA1 mRNA levels. T0901317 also markedly increased CETP mRNA levels and CETP production in human differentiated macrophages, whereas it had no effect on CETP expression in human peripheral blood monocytes. In inflammatory mouse and human macrophages, LXR-mediated CETP gene upregulation was inhibited, even though ABCA1, ABCG1, and SREBP1c inductions were maintained. The inhibition of CETP gene response to LXR agonists in inflammatory cells was independent of lipid loading (ie, oxidized LDL increased CETP production in noninflammatory macrophages with a synergistic effect of synthetic LXR agonists). Conclusion— LXR-mediated induction of human CETP expression is switched on during monocyte-to-macrophage differentiation, is magnified by lipid loading, and is selectively lost in inflammatory macrophages, which suggests that inflammatory cells may not increase the circulating CETP pool on LXR agonist treatment.

Stimulation of Cholesterol Efflux by LXR Agonists in Cholesterol-Loaded Human Macrophages Is ABCA1-Dependent but ABCG1-Independent [Cell Biology/Signaling]

Objective— Maintenance of cholesterol homeostasis in human macrophages is essential to prevent foam cell formation. We evaluated the relative contribution of the ABCA1 and ABCG1 transporters to cholesterol efflux from human macrophages, and of the capacity of LXR agonists to reduce foam cell formation by stimulating export of cellular cholesterol. Methods and Results— ABCG1 mRNA levels were strongly increased in acLDL-loaded THP-1 macrophages and in HMDM on stimulation with LXR agonists. However, silencing of ABCG1 expression using ABCG1-specific siRNA indicated that ABCG1 was not essential for cholesterol efflux to HDL in cholesterol-loaded human macrophages stimulated with LXR agonists. Indeed, ABCA1 was solely responsible for the stimulation of cholesterol efflux to HDL on LXR activation, as this effect was abolished in HMDM from Tangier patients. Furthermore, depletion of cellular ATP indicated that the LXR-induced export of cholesterol was an ATP-dependent transport mechanism in human macrophages. Finally, use of an anti–Cla-1 blocking antibody identified the Cla-1 receptor as a key component in cholesterol efflux to HDL from cholesterol-loaded human macrophages. Conclusion— Our data indicate that stimulation of cholesterol efflux to HDL by LXR agonists in human foam cells involves an ATP-dependent transport mechanism mediated by ABCA1 that it appears to be independent of ABCG1 expression.

Inhibition of Long-Chain Acyl Coenzyme A Synthetases During Fatty Acid Loading Induces Lipotoxicity in Macrophages [Cell Biology/Signaling]

Objectives— Obesity is often associated with hypertriglyceridemia and elevated free fatty acids (FFAs), which are independent risk factors for cardiovascular disease and diabetes. Although impairment of cholesterol homeostasis is known to induce toxicity in macrophages, the consequence of altered fatty acid homeostasis is not clear. Methods and Results— Long-chain acyl CoA synthetases (ACSLs) play a critical role in fatty acid homeostasis by channeling fatty acids to diverse metabolic pools. We treated mouse peritoneal macrophages (MPMs) with VLDL or FFAs in the presence of triacsin C, an inhibitor of the 3 ACSL isoforms present in macrophages. Treatment of macrophages with VLDL and triacsin C resulted in reduced TG accumulation but increased intracellular FFA levels, which induced lipotoxicity characterized by apoptosis. Treatment of MPMs with the saturated fatty acid stearic acid in the presence of triacsin C increased intracellular stearic acid and induced apoptosis. Stromal vascular cells collected from high-fat diet–fed mice displayed foam cell morphology and exhibited increased mRNA levels of macrophage markers and ACSL1. Importantly, all of these changes were associated with increased FFA level in AT. Conclusions— Inhibition of ACSLs during fatty acid loading results in apoptosis via accumulation of FFAs. Our data have implications in understanding the consequences of dysregulated fatty acid metabolism in macrophages.

Saturated Fatty Acids Do Not Directly Stimulate Toll-Like Receptor Signaling [Cell Biology/Signaling]

Objective— Toll-like receptors (TLRs) initiate inflammatory signaling in response to conserved microbial molecules. It has been proposed that dietary saturated fatty acids (SFAs) may also serve as endogenous ligands of TLR2 or TLR4, thereby promoting diseases associated with inflammation and dyslipidemia, including atherosclerosis and insulin resistance. Methods and Results— We investigated the effects of SFAs on TLR-dependent signaling using a broad range of cell types and readouts. In HEK-293 cells transfected with TLR2, TLR4, or TLR5, SFAs complexed with fatty-acid-free bovine serum albumin (BSA)-stimulated TLR-dependent signaling. However, SFAs alone did not elicit a similar response. Further analysis showed that the effect seen with the complexed SFAs was attributable to LPS and lipopeptide contamination of fatty-acid-free BSA. Additional studies in macrophages, endothelial cells, smooth muscle cells, adipocytes, skeletal muscle cells, and human peripheral blood mononuclear cells confirmed the lack of stimulation of TLR-dependent signaling pathways or expression of TLR-target genes by SFAs. Conclusions— SFAs do not directly stimulate TLR-dependent signaling, suggesting that alternative mechanisms link dietary fat intake with TLR-associated pathologies. LPS and lipopeptide contamination of the widely used reagent fatty-acid-free BSA explains the previously reported stimulation of TLR2 and TLR4 by SFAs.

Statins Block Calcific Nodule Formation of Valvular Interstitial Cells by Inhibiting {alpha}-Smooth Muscle Actin Expression [Cell Biology/Signaling]

Objective— Calcific aortic stenosis, characterized by excessive fibrosis and deposition of bone-like calcified tissue, affects roughly 2% to 3% of the U.S. population over the age of 65. Recent studies have suggested that statins have a positive effect on the progression of aoritic stenosis, likely because of their ability to affect the resident cell population, known as valvular interstitial cells (VICs). VICs are fibroblastic cells that can differentiate to form activated myofibroblasts, displaying increased alpha smooth muscle actin (SMA) expression, contractility, and collagen production. Methods and Results— In culture, VICs spontaneously form multicellular aggregates that subsequently develop into calcified nodules, providing an in vitro model for aortic stenosis. Using real-time microscopic tracking, we observed that confluent VIC monolayers spontaneously contract into rounded nodules, suggesting that myofibroblastic contractility is a critical step in the process of nodule formation. Overexpression of SMA increased VIC calcific nodule formation and contractility, whereas knockdown of SMA with siRNAs reduced these phenotypes, suggesting that the expression and contractile properties of SMA are essential to the formation of nodules. Statin treatment of VICs reduced SMA expression, inhibited contractility, and decreased nodule formation. When statins were used to treat preformed nodules, no decrease in the number of calcified nodules was observed, suggesting that statins may play more of a preventative role in aortic stenosis than a cure. Conclusions— Our studies provide evidence of a causal relationship between VIC myofibroblastic activity and initial VIC calcific nodule formation. Furthermore, we demonstrate that pravastatin inhibition of calcific nodule formation is related to inhibition of myofibroblastic activity.

Genome-Wide Association Identifies the ABO Blood Group as a Major Locus Associated With Serum Levels of Soluble E-Selectin [Clinical and Population Studies]

Background— Elevated serum soluble E-selectin levels have been associated with a number of diseases. Although E-selectin levels are heritable, little is known about the specific genetic factors involved. E-selectin levels have been associated with the ABO blood group phenotype. Methods and Results— We performed a high-resolution genome-wide association study of serum soluble E-selectin levels in 685 white individuals with type 1 diabetes from the Diabetes Control and Complications Trial (DCCT)/Epidemiology of Diabetes Intervention and Complications (EDIC) study to identify major loci influencing levels. Highly significant evidence for association (P=10–29) was observed for rs579459 near the ABO blood group gene, accounting for 19% of the variance in E-selectin levels. Levels of E-selectin were higher in O/O than O/A heterozygotes, which were likewise higher than A/A genotypes. Analysis of subgroups of A alleles reveals heterogeneity in the association, and even after this was accounted for, an intron 1 SNP remained significantly associated. We replicate the ABO association in nondiabetic individuals. Conclusion— ABO is a major locus for serum soluble E-selectin levels. We excluded population stratification, fine-mapped the association to sub-A alleles, and also document association with additional variation in the ABO region.

FVII, FVIIa, and Downstream Markers of Extrinsic Pathway Activation Differ by EPCR Ser219Gly Variant in Healthy Men [Clinical and Population Studies]

Objective— The purpose of this study was to determine the effect of a variant in EPCR (Ser219Gly), previously shown to affect EPCR shedding, on plasma FVII, FVIIa, and downstream markers of activated coagulation. Methods and Results— Statistical analysis was undertaken in 2000 healthy middle aged men (NPHSII). Higher soluble EPCR levels were confirmed for Gly allele carriers (P<0.0001). Significantly higher levels of FVII, FVIIa, and downstream markers of activated coagulation in the extrinsic pathway (FIX activation pep [FIXpep]; FX activation pep [FXpep]), and prothrombin F1+2 (F1+2) were identified in baseline samples, in Gly carriers compared to Ser/Ser (P<=0.04 for trend). In repeat samples collected for up to 5 years, levels of FVII and F1+2 were higher in Gly allele carriers compared to Ser/Ser by (FVII: 6.9% CI 5.5 to 8.4 in Ser/Gly; and 23.4% CI 16.3 to 30.8 in Gly/Gly, P<0.0001), (F1+2: 8.1% CI 5.2 to 11.1 in Ser/Gly; 25.2% CI 11.8 to 40.3 in Gly/Gly, P<0.04), confirming reproducibility of findings at baseline. Molar ratios for FIXpep, FXpep, and F1+2 to FVIIa were constant in Ser/Ser and Ser/Gly but tended to be higher in Gly/Gly, reaching statistical significance for FIXpep:FVIIa (P=0.04). Conclusions— These data suggest that higher levels of FVII and FVIIa circulate when EPCR shedding is greatest. Furthermore, these results suggest consequences for activation of extrinsic coagulation.

Ion Mobility Analysis of Lipoprotein Subfractions Identifies Three Independent Axes of Cardiovascular Risk [Clinical and Population Studies]

Objective— Whereas epidemiological studies show that levels of low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) predict incident cardiovascular disease (CVD), there is limited evidence relating lipoprotein subfractions and composite measures of subfractions to risk for CVD in prospective cohort studies. Methods and Results— We tested whether combinations of lipoprotein subfractions independently predict CVD in a prospective cohort of 4594 initially healthy men and women (the Malmö Diet and Cancer Study, mean follow-up 12.2 years, 377 incident cardiovascular events). Plasma lipoproteins and lipoprotein subfractions were measured at baseline with a novel high-resolution ion mobility technique. Principal component analysis (PCA) of subfraction concentrations identified 3 major independent (ie, zero correlation) components of CVD risk, one representing LDL-associated risk, a second representing HDL-associated protection, and the third representing a pattern of decreased large HDL, increased small/medium LDL, and increased triglycerides. The last corresponds to the previously described "atherogenic lipoprotein phenotype." Several genes that may underlie this phenotype—CETP, LIPC, GALNT2, MLXIPL, APOA1/A5, LPL—are suggested by SNPs associated with the combination of small/medium LDL and large HDL. Conclusion— PCA on lipoprotein subfractions yielded three independent components of CVD risk. Genetic analyses suggest these components represent independent mechanistic pathways for development of CVD.

Controlling Myocyte cGMP: Phosphodiesterase 1 Joins the Fray [Editorials]

Origin of Cardiac Fibroblasts and the Role of Periostin [Reviews]

Abstract: Cardiac fibroblasts are the most populous nonmyocyte cell type within the mature heart and are required for extracellular matrix synthesis and deposition, generation of the cardiac skeleton, and to electrically insulate the atria from the ventricles. Significantly, cardiac fibroblasts have also been shown to play an important role in cardiomyocyte growth and expansion of the ventricular chambers during heart development. Although there are currently no cardiac fibroblast-restricted molecular markers, it is generally envisaged that the majority of the cardiac fibroblasts are derived from the proepicardium via epithelial-to-mesenchymal transformation. However, still relatively little is known about when and where the cardiac fibroblasts cells are generated, the lineage of each cell, and how cardiac fibroblasts move to reside in their final position throughout all four cardiac chambers. In this review, we summarize the present understanding regarding the function of Periostin, a useful marker of the noncardiomyocyte lineages, and its role during cardiac morphogenesis. Characterization of the cardiac fibroblast lineage and identification of the signals that maintain, expand and regulate their differentiation will be required to improve our understanding of cardiac function in both normal and pathophysiological states.

Smooth Muscle Cell {alpha}2{delta}-1 Subunits Are Essential for Vasoregulation by CaV1.2 Channels [Cellular Biology]

Rationale: Voltage-dependent L-type (CaV1.2) Ca2+ channels are a heteromeric complex formed from pore-forming 1 and auxiliary 2 and β subunits. CaV1.2 channels are the principal Ca2+ influx pathway in arterial myocytes and regulate multiple physiological functions, including contraction. The macromolecular composition of arterial myocyte CaV1.2 channels remains poorly understood, with no studies having examined the molecular identity or physiological functions of 2 subunits. Objective: We investigated the functional significance of 2 subunits in myocytes of resistance-size (100 to 200 µm diameter) cerebral arteries. Methods and Results: 2-1 was the only 2 isoform expressed in cerebral artery myocytes. Pregabalin, an 2-1/-2 ligand, and an 2-1 antibody, inhibited CaV1.2 currents in isolated myocytes. Acute pregabalin application reversibly dilated pressurized arteries. Using a novel application of surface biotinylation, data indicated that >95% of CaV1.2 1 and 2-1 subunits were present in the arterial myocyte plasma membrane. 2-1 knockdown using short hairpin RNA reduced plasma membrane-localized CaV1.2 1 subunits, caused a corresponding elevation in cytosolic CaV1.2 1 subunits, decreased intracellular Ca2+ concentration, inhibited pressure-induced vasoconstriction ("myogenic tone"), and attenuated pregabalin-induced vasodilation. Prolonged (24-hour) pregabalin exposure did not alter total 2-1 or CaV1.2 1 proteins but decreased plasma membrane expression of each subunit, which reduced myogenic tone. Conclusions: 2-1 is essential for plasma membrane expression of arterial myocyte CaV1.2 1 subunits. 2-1 targeting can block CaV1.2 channels directly and inhibit surface expression of CaV1.2 1 subunits, leading to vasodilation. These data identify 2-1 as a novel molecular target in arterial myocytes, the manipulation of which regulates contractility.

Role of Ca2+/Calmodulin-Stimulated Cyclic Nucleotide Phosphodiesterase 1 in Mediating Cardiomyocyte Hypertrophy [Integrative Physiology]

Rationale: Cyclic nucleotide phosphodiesterases (PDEs) through the degradation of cGMP play critical roles in maintaining cardiomyocyte homeostasis. Ca2+/calmodulin (CaM)-activated cGMP-hydrolyzing PDE1 family may play a pivotal role in balancing intracellular Ca2+/CaM and cGMP signaling; however, its function in cardiomyocytes is unknown. Objective: Herein, we investigate the role of Ca2+/CaM-stimulated PDE1 in regulating pathological cardiomyocyte hypertrophy in neonatal and adult rat ventricular myocytes and in the heart in vivo. Methods and Results: Inhibition of PDE1 activity using a PDE1-selective inhibitor, IC86340, or downregulation of PDE1A using siRNA prevented phenylephrine induced pathological myocyte hypertrophy and hypertrophic marker expression in neonatal and adult rat ventricular myocytes. Importantly, administration of the PDE1 inhibitor IC86340 attenuated cardiac hypertrophy induced by chronic isoproterenol infusion in vivo. Both PDE1A and PDE1C mRNA and protein were detected in human hearts; however, PDE1A expression was conserved in rodent hearts. Moreover, PDE1A expression was significantly upregulated in vivo in the heart and myocytes from various pathological hypertrophy animal models and in vitro in isolated neonatal and adult rat ventricular myocytes treated with neurohumoral stimuli such as angiotensin II (Ang II) and isoproterenol. Furthermore, PDE1A plays a critical role in phenylephrine-induced reduction of intracellular cGMP- and cGMP-dependent protein kinase (PKG) activity and thereby cardiomyocyte hypertrophy in vitro. Conclusions: These results elucidate a novel role for Ca2+/CaM-stimulated PDE1, particularly PDE1A, in regulating pathological cardiomyocyte hypertrophy via a cGMP/PKG-dependent mechanism, thereby demonstrating Ca2+ and cGMP signaling cross-talk during cardiac hypertrophy.

Nitro-Fatty Acid Inhibition of Neointima Formation After Endoluminal Vessel Injury [Integrative Physiology]

Rationale: Fatty acid nitroalkenes are endogenously generated electrophilic byproducts of nitric oxide and nitrite-dependent oxidative inflammatory reactions. Existing evidence indicates nitroalkenes support posttranslational protein modifications and transcriptional activation that promote the resolution of inflammation. Objective: The aim of this study was to assess whether in vivo administration of a synthetic nitroalkene could elicit antiinflammatory actions in vivo using a murine model of vascular injury. Methods and Results: The in vivo administration (21 days) of nitro-oleic acid (OA-NO2) inhibited neointimal hyperplasia after wire injury of the femoral artery in a murine model (OA-NO2 treatment resulted in reduced intimal area and intima to media ratio versus vehicle- or oleic acid (OA)-treated animals,P<0.0001). Increased heme oxygenase (HO)-1 expression accounted for much of the vascular protection induced by OA-NO2 in both cultured aortic smooth muscle cells and in vivo. Inhibition of HO by Sn(IV)-protoporphyrin or HO-1 small interfering RNA reversed OA-NO2–induced inhibition of platelet-derived growth factor-stimulated rat aortic smooth muscle cell migration. The upregulation of HO-1 expression also accounted for the antistenotic actions of OA-NO2 in vivo, because inhibition of neointimal hyperplasia following femoral artery injury was abolished in HO-1–/– mice (OA-NO2–treated wild-type versus HO-1–/– mice, P=0.016). Conclusions: In summary, electrophilic nitro-fatty acids induce salutary gene expression and cell functional responses that are manifested by a clinically significant outcome, inhibition of neointimal hyperplasia induced by arterial injury.

Induction of the CXC Chemokine Interferon-{gamma}-Inducible Protein 10 Regulates the Reparative Response Following Myocardial Infarction [Integrative Physiology]

Rationale: Interferon--inducible protein (IP)-10/CXCL10, an angiostatic and antifibrotic chemokine with an important role in T-cell trafficking, is markedly induced in myocardial infarcts, and may regulate the reparative response. Objective: To study the role of IP-10 in cardiac repair and remodeling. Methods and Results: We studied cardiac repair in IP-10-null and wild-type (WT) mice undergoing reperfused infarction protocols and examined the effects of IP-10 on cardiac fibroblast function. IP-10-deficient and WT animals had comparable acute infarct size. However, the absence of IP-10 resulted in a hypercellular early reparative response and delayed contraction of the scar. Infarcted IP-10–/– hearts exhibited accentuated early dilation, followed by rapid wall thinning during infarct maturation associated with systolic dysfunction. Although IP-10-null and WT mice had comparable cytokine expression, the absence of IP-10 was associated with marked alterations in the cellular content of the infarct. IP-10–/– infarcts had more intense infiltration with CD45+ leukocytes, Mac-2+ macrophages, and -smooth muscle actin (-SMA)+ myofibroblasts than WT infarcts but exhibited reduced recruitment of the subpopulations of leukocytes, T lymphocytes and -SMA+ cells that expressed CXCR3, the IP-10 receptor. IP-10 did not modulate cardiac fibroblast proliferation and apoptosis but significantly inhibited basic fibroblast growth factor-induced fibroblast migration. In addition, IP-10 enhanced growth factor-mediated wound contraction in fibroblast-populated collagen lattices. Conclusions: Endogenous IP-10 is an essential inhibitory signal that regulates the cellular composition of the healing infarct and promotes wound contraction, attenuating adverse remodeling. IP-10-mediated actions may be due, at least in part, to direct effects on fibroblast migration and function.

Dystroglycan Matrix Receptor Function in Cardiac Myocytes Is Important for Limiting Activity-Induced Myocardial Damage [Integrative Physiology]

Rationale: Genetic mutations in a number of putative glycosyltransferases lead to the loss of glycosylation of dystroglycan and loss of its laminin-binding activity in genetic forms of human muscular dystrophy. Human patients and glycosylation defective myd mice develop cardiomyopathy with loss of dystroglycan matrix receptor function in both striated and smooth muscle. Objective: To determine the functional role of dystroglycan in cardiac muscle and smooth muscle in the development of cardiomyopathy in muscular dystrophies. Methods and Results: Using cre/lox–mediated gene targeting, we show here that loss of dystroglycan function in ventricular cardiac myocytes is sufficient to induce a progressive cardiomyopathy in mice characterized by focal cardiac fibrosis, increase in cardiac mass, and dilatation ultimately leading to heart failure. In contrast, disruption of dystroglycan in smooth muscle is not sufficient to induce cardiomyopathy. The specific loss of dystroglycan function in cardiac myocytes causes the accumulation of large, clustered patches of myocytes with membrane damage, which increase in number in response to exercise-induced cardiac stress, whereas exercised mice with normal dystroglycan expression accumulate membrane damage limited to individual myocytes. Conclusions: Our findings suggest dystroglycan function as an extracellular matrix receptor in cardiac myocytes plays a primary role in limiting myocardial damage from spreading to neighboring cardiac myocytes, and loss of dystroglycan matrix receptor function in cardiac muscle cells is likely important in the development of cardiomyopathy in glycosylation-deficient muscular dystrophies.

Gene Therapy With the Angiogenic Cytokine Secretoneurin Induces Therapeutic Angiogenesis by a Nitric Oxide-Dependent Mechanism [Integrative Physiology]

Rationale: The neuropeptide secretoneurin induces angiogenesis and postnatal vasculogenesis and is upregulated by hypoxia in skeletal muscle cells. Objective: We sought to investigate the effects of secretoneurin on therapeutic angiogenesis. Methods and Results: We generated a secretoneurin gene therapy vector. In the mouse hindlimb ischemia model secretoneurin gene therapy by intramuscular plasmid injection significantly increased secretoneurin content of injected muscles, improved functional parameters, reduced tissue necrosis, and restored blood perfusion. Increased muscular density of capillaries and arterioles/arteries demonstrates the capability of secretoneurin gene therapy to induce therapeutic angiogenesis and arteriogenesis. Furthermore, recruitment of endothelial progenitor cells was enhanced by secretoneurin gene therapy consistent with induction of postnatal vasculogenesis. Additionally, secretoneurin was able to activate nitric oxide synthase in endothelial cells and inhibition of nitric oxide inhibited secretoneurin-induced effects on chemotaxis and capillary tube formation in vitro. In vivo, secretoneurin induced nitric oxide production and inhibition of nitric oxide attenuated secretoneurin-induced effects on blood perfusion, angiogenesis, arteriogenesis, and vasculogenesis. Secretoneurin also induced upregulation of basic fibroblast growth factor and platelet-derived growth factor-B in endothelial cells. Conclusions: In summary, our data indicate that gene therapy with secretoneurin induces therapeutic angiogenesis, arteriogenesis, and vasculogenesis in the hindlimb ischemia model by a nitric oxide–dependent mechanism.

Differential Healing After Sirolimus, Paclitaxel, and Bare Metal Stent Placement in Combination With Peroxisome Proliferator-Activator Receptor {gamma} Agonists: Requirement for mTOR/Akt2 in PPAR{gamma} Activation [Integrative Physiology]

Rationale: Sirolimus-eluting coronary stents (SESs) and paclitaxel-eluting coronary stents (PESs) are used to reduce restenosis but have different sites of action. The molecular targets of sirolimus overlap with those of the peroxisome proliferator-activated receptor (PPAR) agonist rosiglitazone (RSG) but the consequence of this interaction on endothelialization is unknown. Objective: Using the New Zealand white rabbit iliac model of stenting, we examined the effects of RSG on SESs, PESs, and bare metal stents endothelialization. Methods and Results: Animals receiving SESs, PESs, or bare metal stents and either RSG (3 mg/kg per day) or placebo were euthanized at 28 days, and arteries were evaluated by scanning electron microscopy. Fourteen-day organ culture and Western blotting of iliac arteries and tissue culture experiments were conducted. Endothelialization was significantly reduced by RSG in SESs but not in PESs or bare metal stents. Organ culture revealed reduced vascular endothelial growth factor in SESs receiving RSG compared to RSG animals receiving bare metal stent or PESs. Quantitative polymerase chain reaction in human aortic endothelial cells (HAECs) revealed that sirolimus (but not paclitaxel) inhibited RSG-induced vascular endothelial growth factor transcription. Western blotting demonstrated that inhibition of molecular signaling in SES+RSG–treated arteries was similar to findings in HAECs treated with RSG and small interfering RNA to PPAR, suggesting that sirolimus inhibits PPAR. Transfection of HAECs with mTOR (mammalian target of rapamycin) short hairpin RNA and with Akt2 small interfering RNA significantly inhibited RSG-mediated transcriptional upregulation of heme oxygenase-1, a PPAR target gene. Chromatin immunoprecipitation assay demonstrated sirolimus interferes with binding of PPAR to its response elements in heme oxygenase-1 promoter. Conclusions: mTOR/Akt2 is required for optimal PPAR activation. Patients who receive SESs during concomitant RSG treatment may be at risk for delayed stent healing.

Deletion of Protein Tyrosine Phosphatase 1b Improves Peripheral Insulin Resistance and Vascular Function in Obese, Leptin-Resistant Mice via Reduced Oxidant Tone [Integrative Physiology]

Rationale: Obesity is a risk factor for cardiovascular dysfunction, yet the underlying factors driving this impaired function remain poorly understood. Insulin resistance is a common pathology in obese patients and has been shown to impair vascular function. Whether insulin resistance or obesity, itself, is causal remains unclear. Objective: The present study tested the hypothesis that insulin resistance is the underlying mediator for impaired NO-mediated dilation in obesity by genetic deletion of the insulin-desensitizing enzyme protein tyrosine phosphatase (PTP)1B in db/db mice. Methods and Results: The db/db mouse is morbidly obese, insulin-resistant, and has tissue-specific elevation in PTP1B expression compared to lean controls. In db/db mice, PTP1B deletion improved glucose clearance, dyslipidemia, and insulin receptor signaling in muscle and fat. Hepatic insulin signaling in db/db mice was not improved by deletion of PTP1B, indicating specific amelioration of peripheral insulin resistance. Additionally, obese mice demonstrate an impaired endothelium dependent and independent vasodilation to acetylcholine and sodium nitroprusside, respectively. This impairment, which correlated with increased superoxide in the db/db mice, was corrected by superoxide scavenging. Increased superoxide production was associated with increased expression of NAD(P)H oxidase 1 and its molecular regulators, Noxo1 and Noxa1. Conclusions: Deletion of PTP1B improved both endothelium dependent and independent NO-mediated dilation and reduced superoxide generation in db/db mice. PTP1B deletion did not affect any vascular function in lean mice. Taken together, these data reveal a role for peripheral insulin resistance as the mediator of vascular dysfunction in obesity.

TRPC1 Channels Are Critical for Hypertrophic Signaling in the Heart [Integrative Physiology]

Rationale: Cardiac muscle adapts to increase workload by altering cardiomyocyte size and function resulting in cardiac hypertrophy. G protein–coupled receptor signaling is known to govern the hypertrophic response through the regulation of ion channel activity and downstream signaling in failing cardiomyocytes. Objective: Transient receptor potential canonical (TRPC) channels are G protein–coupled receptor operated channels previously implicated in cardiac hypertrophy. Our objective of this study is to better understand how TRPC channels influence cardiomyocyte calcium signaling. Methods and Results: Here, we used whole cell patch clamp of adult cardiomyocytes to show upregulation of a nonselective cation current reminiscent of TRPC channels subjected to pressure overload. This TRPC current corresponds to the increased TRPC channel expression noted in hearts of mice subjected to pressure overload. Importantly, we show that mice lacking TRPC1 channels are missing this putative TRPC current. Moreover, Trpc1–/– mice fail to manifest evidence of maladaptive cardiac hypertrophy and maintain preserved cardiac function when subjected to hemodynamic stress and neurohormonal excess. In addition, we provide a mechanistic basis for the protection conferred to Trpc1–/– mice as mechanosensitive signaling through calcineurin/NFAT, mTOR and Akt is altered in Trpc1–/– mice. Conclusions: From these studies, we suggest that TRPC1 channels are critical for the adaptation to biomechanical stress and TRPC dysregulation leads to maladaptive cardiac hypertrophy and failure.

Whole Body UVA Irradiation Lowers Systemic Blood Pressure by Release of Nitric Oxide From Intracutaneous Photolabile Nitric Oxide Derivates [Clinical/Translational Research]

Rationale: Human skin contains photolabile nitric oxide derivates like nitrite and S-nitroso thiols, which after UVA irradiation, decompose and lead to the formation of vasoactive NO. Objective: Here, we investigated whether whole body UVA irradiation influences the blood pressure of healthy volunteers because of cutaneous nonenzymatic NO formation. Methods and Results: As detected by chemoluminescence detection or by electron paramagnetic resonance spectroscopy in vitro with human skin specimens, UVA illumination (25 J/cm2) significantly increased the intradermal levels of free NO. In addition, UVA enhanced dermal S-nitrosothiols 2.3-fold, and the subfraction of dermal S-nitrosoalbumin 2.9-fold. In vivo, in healthy volunteers creamed with a skin cream containing isotopically labeled 15N-nitrite, whole body UVA irradiation (20 J/cm2) induced significant levels of 15N-labeled S-nitrosothiols in the blood plasma of light exposed subjects, as detected by cavity leak out spectroscopy. Furthermore, whole body UVA irradiation caused a rapid, significant decrease, lasting up to 60 minutes, in systolic and diastolic blood pressure of healthy volunteers by 11±2% at 30 minutes after UVA exposure. The decrease in blood pressure strongly correlated (R2=0.74) with enhanced plasma concentration of nitrosated species, as detected by a chemiluminescence assay, with increased forearm blood flow (+26±7%), with increased flow mediated vasodilation of the brachial artery (+68±22%), and with decreased forearm vascular resistance (–28±7%). Conclusions: UVA irradiation of human skin caused a significant drop in blood pressure even at moderate UVA doses. The effects were attributed to UVA induced release of NO from cutaneous photolabile NO derivates.