Journals RSS : Gourt

Role of Epithelial Sodium Channels in the Renal Myogenic Response? [Letters to the Editor]

Response to Role of Epithelial Sodium Channels in the Renal Myogenic Response? [Letters to the Editor]

Human Nedd4L rs4149601 G Allele Generates Evolutionary New Isoform I With C2 Domain [Letters to the Editor]

Response to Human Nedd4L rs4149601 G Allele Generates Evolutionary New Isoform I With C2 Domain [Letters to the Editor]

Antihypertensive Treatment and Left Ventricular Mass Reduction: The Importance of Choosing the Comparator [Letters to the Editor]

Oral Carnitine Therapy and Insulin Resistance [Letters to the Editor]

Measurement of Blood Pressure in the Office: Recognizing the Problem and Proposing the Solution [Brief Reviews]

Plasticity of GABAergic Mechanisms Within the Nucleus of the Solitary Tract in Hypertension [Brief Reviews]

Therapeutic Implications of the Vasoprotective Axis of the Renin-Angiotensin System in Cardiovascular Diseases [Brief Reviews]

Angiotensin I-Converting Enzyme Inhibitors Are Allosteric Enhancers of Kinin B1 and B2 Receptor Function [Brief Reviews]

The beneficial effects of angiotensin I-converting enzyme (ACE) inhibitors go beyond the inhibition of ACE to decrease angiotensin (Ang) II or increase kinin levels. ACE inhibitors also affect kinin B1 and B2 receptor (B1R and B2R) signaling, which may underlie some of their therapeutic usefulness. They can indirectly potentiate the actions of bradykinin (BK) and ACE-resistant BK analogs on B2Rs to elevate arachidonic acid and NO release in laboratory experiments. Studies indicate that ACE inhibitors and some Ang metabolites increase B2R functions as allosteric enhancers by inducing a conformational change in ACE. This is transmitted to B2Rs via heterodimerization with ACE on the plasma membrane of cells. ACE inhibitors are also agonists of the B1R, at a Zn-binding sequence on the second extracellular loop that differs from the orthosteric binding site of the des-Arg-kinin peptide ligands. Thus, ACE inhibitors act as direct allosteric B1R agonists. When ACE inhibitors enhance B2R and B1R signaling, they augment NO production. Enhancement of B2R signaling activates endothelial NO synthase, yielding a short burst of NO; activation of B1Rs results in a prolonged high output of NO by inducible NO synthase. These actions, outside inhibiting peptide hydrolysis, may contribute to the pleiotropic therapeutic effects of ACE inhibitors in various cardiovascular disorders.

Aldosterone, Sodium, and Hypertension: Lessons From Torcetrapib? [Hypothesis]

Treatment of Isolated Left Ventricular Diastolic Dysfunction in Hypertension: Reaching Blood Pressure Target Matters [Editorial Commentaries]

Relative Plasma Volume Monitoring and Blood Pressure Control: An Overlooked Opportunity to Achieve Dry Weight in the Hemodialysis Patient [Editorial Commentaries]

Angiotensin II, Oxidant Signaling, and Hypertension: Down to a T? [Editorial Commentaries]

Going Out on a LIM and Cysteine-Rich Domains 1 Limb: A New Way to Block Calcineurin Activity [Editorial Commentaries]

Syndecan-1: A Critical Mediator in Cardiac Fibrosis [Editorial Commentaries]

Progress Toward Identifying Potential Markers for Preeclampsia: Role of Agonistic Autoantibody to the Angiotensin II Type I Receptor [Editorial Commentaries]

Inching Towards A Targeted Therapy for Preeclampsia [Editorial Commentaries]

Effect of Intensive Versus Standard Blood Pressure Lowering on Diastolic Function in Patients With Uncontrolled Hypertension and Diastolic Dysfunction [Original Articles]

Diastolic dysfunction may precede development of heart failure in hypertensive patients. We randomized 228 patients with uncontrolled hypertension, preserved ejection fraction, and diastolic dysfunction to 2 targeted treatment strategies: intensive, with a systolic blood pressure target of <130 mm Hg, or standard, with a systolic blood pressure target of <140 mm Hg, using a combination of valsartan, either 160 or 320 mg, plus amlodipine, either 5 or 10 mg, with other antihypertensive medications as needed. Echocardiographic assessment of diastolic function was performed at baseline and after 24 weeks in a prospective, open-label, blinded end point design. Blood pressure was reduced significantly in both groups, from 161.2±13.9/90.1±12.0 to 130.8±12.3/74.9±9.1 mm Hg (P<0.0001) in the intensive arm and from 162.1±13.2/93.7±12.2 to 137.0±12.9/79.6±11.0 mm Hg (P<0.0001) in the standard arm (P<0.003 for between-group comparisons). Myocardial relaxation velocity improved from 7.6±1.1 to 9.2±1.7 cm/s ( 1.54±1.4 cm/s; P<0.0001) in the intensive arm and from 7.5±1.3 to 9.0±1.9 cm/s ( 1.48±1.6 cm/s; P<0.0001) in the standard arm, with no difference between the 2 strategies in the achieved improvement (P=0.58). The degree of improvement in annular relaxation velocity was associated with the extent of systolic blood pressure reduction, and patients with the lowest achieved systolic blood pressure had the highest final diastolic relaxation velocities.

Syndecan-1 Amplifies Angiotensin II-Induced Cardiac Fibrosis [Original Articles]

Syndecan-1 (Synd1) is a transmembrane heparan sulfate proteoglycan that functions as a coreceptor for various growth factors and modulates signal transduction. The present study investigated whether Synd1, by affecting growth factor signaling, may play a role in hypertension-induced cardiac fibrosis and dysfunction. Expression of Synd1 was increased significantly in mouse hearts with angiotensin II–induced hypertension, which was spatially related to cardiac fibrosis. Angiotensin II significantly impaired fractional shortening and induced cardiac fibrosis in wild-type mice, whereas these effects were blunted in Synd1-null mice. Angiotensin II significantly increased cardiac expression of connective tissue growth factor and collagen type I and III in wild-type mice, which was blunted in Synd1-null mice. These findings were confirmed in vitro, where angiotensin II induced the expression of both connective tissue growth factor and collagen I in fibroblasts. The absence of Synd1 in either Synd1-null fibroblasts, after knockdown of Synd1 by short hairpin RNA, or after inhibition of heparan sulfates by protamine attenuated this increase, which was associated with reduced phosphorylation of Smad2. In conclusion, loss of Synd1 reduces cardiac fibrosis and dysfunction during angiotensin II–induced hypertension.

LIM and Cysteine-Rich Domains 1 Regulates Cardiac Hypertrophy by Targeting Calcineurin/Nuclear Factor of Activated T Cells Signaling [Original Articles]

LIM domain proteins are important regulators in cell growth, cell fate determination, cell differentiation, and remodeling of the cell cytoskeleton. LIM and cysteine-rich domains 1 (Lmcd1) is a novel protein that contain 2 LIM domains with regular spacing in the carboxy-terminal region. However, its roles in cardiac growth remain unknown. Here, we investigated whether Lmcd1 regulates cardiac hypertrophy in vitro and in vivo and elucidated the underlying molecular mechanisms. We used primary cultured cardiac myocytes and cardiac-specific Lmcd1 transgenic mice. In wild-type mice subjected to the aortic banding, cardiac hypertrophy was evident at 8 weeks. In transgenic mice, however, cardiac hypertrophy was significantly greater than that in wild-type mice, as estimated by heart weight:body weight ratio, cardiomyocyte area, and echocardiographic measurements, as well as cardiac atrial natriuretic peptide and B-type natriuretic peptide mRNA and protein levels. Our results further showed that cardiac fibrosis observed in wild-type aortic banding mice was augmented in transgenic aortic banding mice. Importantly, calcineurin activity and nuclear factor of activated T cells activation level were increased more in transgenic mice than those in wild-type mice after 8-week aortic banding. In vitro experiments in cardiac myocytes further revealed that angiotensin II-induced calcineurin activity and nuclear factor of activated T cells activation were enhanced by overexpression but blunted by downregulation of Lmcd1. In conclusion, our results suggest that Lmcd1 plays a critical role in the development of cardiac hypertrophy via activation of calcineurin/nuclear factor of activated T cells signaling pathway.

Blood Pressure Regulation in Humans: Calculation of an "Error Signal" in Control of Sympathetic Nerve Activity [Original Articles]

Within an individual, diastolic blood pressure (DBP) is negatively related to sympathetic burst incidence, such that lower pressure is associated with high burst incidence. Our goal was to explore the use of a calculation of a DBP "error signal" in the control of muscle sympathetic nerve activity in men and women. Baseline muscle sympathetic nerve activity was measured in healthy young men (n=22) and women (n=28). Women had significantly lower muscle sympathetic nerve activity than men (29±3 versus 43±2 bursts per 100 heartbeats; P<0.05). For each individual, the DBP at which there is a 50% likelihood of a muscle sympathetic nerve activity burst, the "T50" value, was calculated. Mean DBP was subtracted from the T50 blood pressure as an approximate error signal for burst activation. Error signal was negative in both sexes, indicating that DBP in both sexes was higher than the DBP value associated with a 50% burst likelihood. However, average error signal was significantly larger in women (–4±2 mm Hg) than in men (–1±0 mm Hg; P<0.05 versus women). We conclude that women operate at a mean DBP greater than their T50 compared with men, and this may be a contributing factor to low basal muscle sympathetic nerve activity in women. The relationship between error signal and burst incidence may provide important insight into the control of muscle sympathetic nerve activity across sexes and in various populations.

Sympathetic Nervous System Modulation of Inflammation and Remodeling in the Hypertensive Heart [Original Articles]

Chronic activation of the sympathetic nervous system is a key component of cardiac hypertrophy and fibrosis. However, previous studies have provided evidence that also implicate inflammatory cells, including mast cells (MCs), in the development of cardiac fibrosis. The current study investigated the potential interaction of cardiac MCs with the sympathetic nervous system. Eight-week–old male spontaneously hypertensive rats were sympathectomized to establish the effect of the sympathetic nervous system on cardiac MC density, myocardial remodeling, and cytokine production in the hypertensive heart. Age-matched Wistar Kyoto rats served as controls. Cardiac fibrosis and hypertension were significantly attenuated and left ventricular mass normalized, whereas cardiac MC density was markedly increased in sympathectomized spontaneously hypertensive rats. Sympathectomy normalized myocardial levels of interferon-, interleukin 6, and interleukin 10, but had no effect on interleukin 4. The effects of norepinephrine and substance P on isolated cardiac MC activation were investigated as potential mechanisms of interaction between the two. Only substance P elicited MC degranulation. Substance P was also shown to induce the production of angiotensin II by a mixed population of isolated cardiac inflammatory cells, including MCs, lymphocytes, and macrophages. These results demonstrate the ability of neuropeptides to regulate inflammatory cell function, providing a potential mechanism by which the sympathetic nervous system and afferent nerves may interact with inflammatory cells in the hypertensive heart.

Induction of Hypertension and Peripheral Inflammation by Reduction of Extracellular Superoxide Dismutase in the Central Nervous System [Original Articles]

The circumventricular organs (CVOs) lack a well-formed blood-brain barrier and produce superoxide in response to angiotensin II and other hypertensive stimuli. This increase in central superoxide has been implicated in the regulation of blood pressure. The extracellular superoxide dismutase (SOD3) is highly expressed in cells associated with CVOs and particularly with tanycytes lining this region. To understand the role of SOD3 in the CVOs in blood pressure regulation, we performed intracerebroventricular injection an adenovirus encoding Cre-recombinase (5x108 particles per milliliter) in mice with loxP sites flanking the SOD3 coding region (SOD3loxp/loxp mice). An adenovirus encoding red-fluorescent protein was injected as a control. Deletion of CVO SOD3 increased baseline blood pressure modestly and markedly augmented the hypertensive response to low-dose angiotensin II (140 ng/kg per day), whereas intracerebroventricular injection of adenovirus encoding red-fluorescent protein had minimal effects on these parameters. Adenovirus encoding Cre-recombinase–treated mice exhibited increased sympathetic modulation of heart rate and blood pressure variability, increased vascular superoxide production, and T-cell activation as characterized by increased circulating CD69+/CD3+ cells. Deletion of CVO SOD3 also markedly increased vascular T-cell and leukocyte infiltration caused by angiotensin II. We conclude that SOD3 in the CVO plays a critical role in the regulation of blood pressure, and its loss promotes T-cell activation and vascular inflammation, in part by modulating sympathetic outflow. These findings provide insight into how central signals produce vascular inflammation in response to hypertensive stimuli, such as angiotensin II.

Glutamatergic Receptor Activation in the Rostral Ventrolateral Medulla Mediates the Sympathoexcitatory Response to Hyperinsulinemia [Original Articles]

Hyperinsulinemia increases sympathetic nerve activity (SNA) and has been linked to cardiovascular morbidity in obesity. The rostral ventrolateral medulla (RVLM) plays a key role in the regulation of SNA and arterial blood pressure (ABP). Many sympathoexcitatory responses are mediated by glutamatergic receptor activation within the RVLM, and both the central renin–angiotensin and melanocortin systems are implicated in the sympathoexcitatory response to hyperinsulinemia. Therefore, we hypothesized that one or more of these neurotransmitters in the RVLM mediate the sympathoexcitatory response to insulin. Hyperinsulinemic–euglycemic clamps were performed in -chloralose anesthetized, male Sprague–Dawley rats by infusion of insulin (3.75 mU/kg per minute, IV) and 50% dextrose solution for 120 minutes. Physiological increases in plasma insulin elevated lumbar SNA, with no change in renal SNA, ABP, or blood glucose. Microinjection of the ionotropic glutamate receptor antagonist kynurenic acid into the RVLM significantly reduced lumbar SNA and ABP. Selective blockade of NMDA but not non-NMDA glutamate receptors resulted in similar reductions of lumbar SNA. In marked contrast, microinjection of the angiotensin II type 1 receptor antagonist losartan or the melanocortin 3/4 antagonist SHU9119 had no effect on lumbar SNA or ABP. Western blot analysis showed that insulin receptor expression is significantly lower in the RVLM than the hypothalamus, and direct microinjection of insulin into the RVLM did not significantly increase lumbar SNA. These findings suggest that hyperinsulinemia increases lumbar SNA by activation of a glutamatergic NMDA-dependent projection to the RVLM.

Angiotensin II Type 1 Receptor-Activated Caspase-3 Through Ras/Mitogen-Activated Protein Kinase/Extracellular Signal-Regulated Kinase in the Rostral Ventrolateral Medulla Is Involved in Sympathoexcitation in Stroke-Prone Spontaneously Hypertensive Rats [Original Articles]

In the rostral ventrolateral medulla (RVLM), angiotensin II-derived superoxide anions, which increase sympathetic nerve activity, induce a pressor response by activating the p38 mitogen-activated protein kinase (p38 MAPK) and extracellular signal-regulated kinase (ERK) pathway. The small G protein Ras mediates a caspase-3–dependent apoptotic pathway through p38 MAPK, ERK, and c-Jun N-terminal kinase. We hypothesized that angiotensin II type 1 receptors activate caspase-3 through the Ras/p38 MAPK/ERK/c-Jun N-terminal kinase pathway in the RVLM and that this pathway is involved in sympathoexcitation in stroke-prone spontaneously hypertensive rats (SHRSP), a model of human hypertension. The activities of Ras, p38 MAPK, ERK, and caspase-3 in the RVLM were significantly higher in SHRSP (14 to 16 weeks old) than in age-matched Wistar-Kyoto rats (WKY). The mitochondrial apoptotic proteins Bax and Bad in the RVLM were significantly increased in SHRSP compared with WKY. c-Jun N-terminal kinase activity did not differ between SHRSP and WKY. In SHRSP, intracerebroventricular infusion of a Ras inhibitor significantly reduced sympathetic nerve activity and improved baroreflex sensitivity, partially because of inhibition of the Ras/p38 MAPK/ERK, Bax, Bad, and caspase-3 pathway in the RVLM. Intracerebroventricular infusion of a caspase-3 inhibitor also inhibited sympathetic nerve activity and improved baroreflex sensitivity in SHRSP. Intracerebroventricular infusion of an angiotensin II type 1 receptor blocker in SHRSP partially inhibited the Ras/p38 MAPK/ERK, Bax, Bad, and caspase-3 pathway in the RVLM. These findings suggest that in SHRSP, angiotensin II type 1 receptor-activated caspase-3 acting through the Ras/p38 MAPK/ERK pathway in the RVLM might be involved in sympathoexcitation, which in turn plays a crucial role in the pathogenesis of hypertension.

Blood Pressure, Blood Flow, and Oxygenation in the Clipped Kidney of Chronic 2-Kidney, 1-Clip Rats: Effects of Tempol and Angiotensin Blockade [Original Articles]

Angiotensin II maintains renal cortical blood flow and renal oxygenation in the clipped kidney of early 2-kidney, 1-clip Goldblatt hypertensive (2K,1C) rats. The involvement of Ang II is believed to decline, whereas oxidative stress increases during the progression of 2K,1C hypertension. We investigated the hypothesis that the acute administration of drugs to inhibit reactive oxygen species (Tempol), angiotensin II type 1 receptors (candesartan), or angiotensin-converting enzyme (enalaprilat) lowers mean arterial pressure and increases kidney blood flow and oxygenation in the clipped kidney of chronic 2K,1C rats in contrast to sham controls. Twelve months after left renal artery clipping or sham, mean arterial pressure, renal cortical blood flow, and renal cortical and medullary oxygen tension were measured after acute administration of Tempol followed by enalaprilat or candesartan followed by enalaprilat. The mean arterial pressure of the 2K,1C rat was reduced by candesartan (–9%) and, more effectively, by Tempol (–35%). All of the applied treatments had similar blood pressure-lowering effects in sham rats (average: –21%). Only Tempol increased cortical blood flow (+35%) and cortical and medullary oxygen tensions (+17% and +94%, respectively) in clipped kidneys of 2K,1C rats. Administration of enalaprilat had no additional effect, except for a modest reduction in cortical blood flow in the clipped kidney of 2K,1C rats when coadministered with candesartan (–10%). In conclusion, acute administration of Tempol is more effective than candesartan in reducing the mean arterial blood pressure and improving renal blood perfusion and oxygenation in the clipped kidney of chronic 2K,1C rats.

Relative Plasma Volume Monitoring During Hemodialysis Aids the Assessment of Dry Weight [Original Articles]

Among hemodialysis patients, the assessment of dry weight remains a matter of clinical judgment because tests to assess dry weight have not been validated. The objective of this study was to evaluate and validate relative plasma volume (RPV) monitoring as a marker of dry weight. We performed RPV monitoring using the Crit-Line monitor at baseline and at 8 weeks in 150 patients participating in the Dry-Weight Reduction in Hypertensive Hemodialysis Patients Trial. The intervention group of 100 patients had dry weight probed, whereas 50 patients served as time controls. RPV slopes were defined as flat when they were less than the median (1.33% per hour) at the baseline visit. Among predominantly (87%) black hemodialysis patients, we found that flat RPV slopes suggest a volume-overloaded state for the following reasons: (1) probing dry weight in these patients led to steeper slopes; (2) those with flatter slopes at baseline had greater weight loss; (3) both baseline RPV slopes and the intensity of weight loss were found to be important for subsequent change in RPV slopes; and, most importantly, (4) RPV slopes predicted the subsequent reduction in interdialytic ambulatory systolic blood pressure. Those with the flattest slopes had the greatest decline in blood pressure on probing dry weight. Both baseline RPV slopes and the change in RPV slopes were important for subsequent changes in ambulatory systolic blood pressure. We conclude that RPV slope monitoring is a valid method to assess dry weight among hypertensive hemodialysis patients.

Shear Stress Mediates Endothelial Adaptations to Exercise Training in Humans [Original Articles]

Although episodic changes in shear stress have been proposed as the mechanism responsible for the effects of exercise training on the vasculature, this hypothesis has not been directly addressed in humans. We examined brachial artery flow-mediated dilation, an index of NO-mediated endothelial function, in healthy men in response to an acute bout of handgrip exercise and across an 8-week period of bilateral handgrip training. Shear stress responses were attenuated in one arm by cuff inflation to 60 mm Hg. Similar increases were observed in grip strength and forearm volume and girth in both limbs. Acute bouts of handgrip exercise increased shear rate (P<0.005) and flow-mediated dilation percentage (P<0.05) in the uncuffed limb, whereas no changes were evident in the cuffed arm. Handgrip training increased flow-mediated dilation percentage in the noncuffed limb at weeks 2, 4, and 6 (P<0.001), whereas no changes were observed in the cuffed arm. Brachial artery peak reactive hyperemia, an index of resistance artery remodeling, progressively increased with training in the noncuffed limb (P<0.001 and 0.004); no changes were evident in the cuffed arm. Neither acute nor chronic shear manipulation during exercise influenced endothelium-independent glyceryl trinitrate responses. These results demonstrate that exercise-induced changes in shear provide the principal physiological stimulus to adaptation in flow-mediated endothelial function and vascular remodeling in response to exercise training in healthy humans.

Reduced Ascending Aortic Strain and Distensibility: Earliest Manifestations of Vascular Aging in Humans [Original Articles]

Arterial stiffness predicts cardiovascular events beyond traditional risk factors. However, the relationship with aging of novel noninvasive measures of aortic function by MRI and their interrelationship with established markers of vascular stiffness remain unclear and currently limit their potential impact. Our aim was to compare age-related changes of central measures of aortic function with carotid distensibility, global carotid–femoral pulse wave velocity, and wave reflections. We determined aortic strain, distensibility, and aortic arch pulse wave velocity by MRI, carotid distensibility by ultrasound, and carotid–femoral pulse wave velocity by tonometry in 111 asymptomatic subjects (54 men, age range: 20 to 84 years). Central pressures were used to calculate aortic distensibility. Peripheral and central pulse pressure, augmentation index, and carotid–femoral pulse wave velocity increased with age, but aortic strain and aortic arch PWV were most closely and specifically related to aging. Ascending aortic (AA) strain and distensibility decreased, respectively, by 5.3±0.5% (R2=0.54, P<0.0001) and 13.6±1 kPa–1x10–3 (R2=0.62, P<0.0001), and aortic arch pulse wave velocity increased by 1.6±0.13 m/sec (R2=0.60, P<0.0001) for each decade of age after adjustment for gender, body size, and heart rate. We demonstrate in this study a dramatic decrease in AA distensibility before the fifth decade of life in individuals with diverse prevalence of risk factors free of overt cardiovascular disease. In particular, compared with other measures of aortic function, the best markers of subclinical large artery stiffening, were AA distensibility in younger and aortic arch pulse wave velocity in older individuals.

Intraaortic Pulse Pressure Amplification in Subjects at High Coronary Risk [Original Articles]

Peripheral (brachial) pulse pressure normally exceeds central (aortic) pulse pressure but is a less powerful predictor of cardiovascular risk. The difference between the 2 variables, called pulse pressure amplification, has never been specifically studied between the proximal and distal aorta in coronary patients. Our goal was to determine aortic pulse pressure amplification in subjects at high coronary risk, with emphasis on associated renal and inflammatory factors. Blood pressure was measured invasively in the ascending aorta, abdominal aorta (at the level of kidneys), and iliac artery in 101 subjects (mean age, 63±11 years; 61 men) undergoing coronary angiography. Independently of age, sex, and the presence of coronary stenosis, the increase of pulse pressure between the ascending and terminal aorta was over 10 mm Hg (P<0.001), whereas mean blood pressure remained unchanged. Pulse pressure amplification did not differ significantly between patients with and without coronary artery stenosis. Irrespective of confounding variables, high pulse pressure measured in the ascending aorta and at the level of renal arteries (but not in the iliac artery) was independently related to proteinuria. The increase in pulse pressure from the ascending aorta to the renal level was negatively associated with leukocyte count, even after multivariate adjustment (β coefficient, –0.19; 95% CI, –0.39 to 0.0; P<0.05). Increased plasma creatinine and aortic pulse wave velocity were independently and positively correlated (β coefficient, 0.36; CI, 0.18 to 0.54; P<0.001). Independently of coronary atherosclerosis, aortic pulse pressure integrates the predictive value of aortic, inflammatory, and renal factors.

Tumor Necrosis Factor-{alpha} Antagonists Improve Aortic Stiffness in Patients With Inflammatory Arthropathies: A Controlled Study [Original Articles]

The chronic inflammatory state of rheumatoid arthritis and other inflammatory arthropathies, such as ankylosing spondylitis and psoriatic arthritis, contributes to the accelerated atherosclerosis associated with these conditions. This study evaluates the effect of treatment with tumor necrosis factor (TNF)- antagonists on arterial stiffness in patients with inflammatory arthropathies. A total of 60 patients with rheumatoid arthritis, ankylosing spondylitis, or psoriatic arthritis and clinical indication for anti–TNF- therapy were included. Thirty-five patients started with anti–TNF- therapy and were compared with a nontreatment group of 25 patients. Aortic stiffness (aortic pulse wave velocity), augmentation index, and disease activity were assessed at baseline and after 3 months. Aortic pulse wave velocity (mean±SD) was reduced in the treatment group but not in the control group (–0.50±0.78 m/s versus 0.05±0.54 m/s, respectively; P=0.002). Concomitantly, C-reactive protein and the disease activity score were reduced in the treatment group (–9.3±20.2 mg/L [P<0.001] and –0.74±0.91 [P=0.004]). Augmentation index remained unchanged in both groups (0.1±7.1% versus –1.0±5.8%, respectively; P=0.53). In a multivariate linear regression model, only treatment with TNF- antagonist and change in mean arterial pressure predicted alterations in aortic pulse wave velocity. In summary, anti–TNF- therapy improved aortic stiffness in patients with inflammatory arthropathies. These findings support the idea that anti-inflammatory treatment has a favorable effect on cardiovascular risk in patients with inflammatory arthropathies.

Reactive Oxygen Species and Cyclooxygenase 2-Derived Thromboxane A2 Reduce Angiotensin II Type 2 Receptor Vasorelaxation in Diabetic Rat Resistance Arteries [Original Articles]

Angiotensin II has a key role in the control of resistance artery tone and local blood flow. Angiotensin II possesses 2 main receptors. Although angiotensin II type 1 receptor is well known and is involved in the vasoconstrictor and growth properties of angiotensin II, the role of the angiotensin II type 2 receptor (AT2R) remains much less understood. Although AT2R stimulation induces vasodilatation in normotensive rats, it induces vasoconstriction in pathological conditions involving oxidative stress and cyclooxygenase 2 expression. Thus, we studied the influence of cyclooxygenase 2 on AT2R-dependent tone in diabetes mellitus. Mesenteric resistance arteries were isolated from Zucker diabetic fatty (ZDF) and lean Zucker rats and studied using in vitro using wire myography. In ZDF rats, AT2R-induced dilation was lower than in lean rats (11% versus 21% dilation). Dilation in ZDF rats returned to the control (lean rats) level after acute superoxide reduction (Tempol and apocynin), cyclooxygenase 2 inhibition (NS398), or thromboxane A2 synthesis inhibition (furegrelate). Cyclooxygenase 2 expression and superoxide production were significantly increased in ZDF rat arteries compared with arteries of lean rats. After chronic treatment with Tempol, AT2R-dependent dilation was equivalent in ZDF and lean rats. Chronic treatment of ZDF rats with NS398 also restored AT2R-dependent dilation to the control (lean rats) level. Plasma thromboxane B2 (thromboxane A2 metabolite), initially high in ZDF rats, was decreased by chronic Tempol and by chronic NS398 to the level found in lean Zucker rats. Thus, in type 2 diabetic rats, superoxide and thromboxane A2 reduced AT2R-induced dilation. These findings are important to take into consideration when choosing vasoactive drugs for diabetic patients.

Cyclin-Dependent Kinase 5 Phosphorylates Endothelial Nitric Oxide Synthase at Serine 116 [Original Articles]

Nitric oxide (NO) production in endothelial cells (EC) is regulated by multisite phosphorylation of specific serine and threonine residues in endothelial NO synthase (eNOS). Among these, eNOS-Ser116 is phosphorylated in the basal state, and its phosphorylation contributes to basal NO production. Here, we investigated the mechanism by which eNOS-Ser116 is phosphorylated during the basal state using bovine aortic EC. Although a previous study suggested that protein kinase C was involved in eNOS-Ser116 phosphorylation, overexpression of various protein kinase C isoforms did not affect eNOS-Ser116 phosphorylation. An in silico analysis using a motif scan revealed that the eNOS-Ser116 residue might be a substrate for proline-directed protein kinases. Roscovitine, a specific inhibitor of cyclin-dependent kinase (CDK), 1, 2, and 5, but not an inhibitor of mitogen-activated protein kinase kinase or glycogen synthase kinase 3β, inhibited eNOS-Ser116 phosphorylation dose dependently. Furthermore, purified CDK1, 2, or 5 directly phosphorylated eNOS-Ser116 in vitro. Ectopic expression of the dominant-negative CDK5 but not dominant-negative CDK1 or dominant-negative CDK2 repressed eNOS-Ser116 phosphorylation and increased NO production. In addition, CDK5 activity was detected in bovine aortic EC, and coimmunoprecipitation and confocal microscopy studies revealed a colocalization of eNOS and CDK5. Cotransfection of CDK5 and p25, the specific CDK5 activator, increased eNOS-Ser116 phosphorylation and decreased NO production, but its parent molecule, p35, and p39, another activator, were not detected in bovine aortic EC, which suggests the existence of a novel CDK5 activator. Overall, this is the first study to find that CDK5 is a physiological kinase responsible for eNOS-Ser116 phosphorylation and regulation of NO production.

Tumor Necrosis Factor-{alpha}: A Possible Priming Agent for the Polymorphonuclear Leukocyte-Reduced Nicotinamide-Adenine Dinucleotide Phosphate Oxidase in Hypertension [Original Articles]

In the Sabra rat, oxidative stress (OS) and inflammation precede the development of hypertension. Inhibition of the phagocytic NADPH oxidase attenuates the rise in blood pressure. The present study was set to identify possible priming agents for this enzyme and to test the hypothesis that the phagocytic NADPH oxidase contributes to OS and inflammation. Sabra salt-sensitive and Sabra salt-resistant rats were salt loaded or provided regular chow for 60 days with or without apocynin to inhibit NADPH oxidase. Levels of interleukin 6, tumor necrosis factor-, and interferon- served as indices of inflammation. Extracellular and intracellular levels of the polymorphonuclear leukocyte tumor necrosis factor- receptors (p55 and p75) were assessed by flow cytometry in young and adult rats. NADPH oxidase activity and expression of p47phox were measured in polymorphonuclear leukocytes and aortic rings. Malondialdehyde and carbonylated fibrinogen served as indices of OS. Inflammatory and OS indices excluding interferon- were higher in the hypertensive state and reduced by apocynin. Levels of malondialdehyde and tumor necrosis factor- were elevated already in the prehypertensive state. No differences were found in the levels of p75. The extracellular expression of p55 was higher in adult Sabra salt-resistant compared with Sabra salt-sensitive rats (7.46±2.2% versus 2.1±0.5%; P<0.05), whereas levels of the intracellular p55 were higher in adult Sabra salt-sensitive rats (3.2±2% versus 1.1±0.5%; P<0.05). In young normotensive rats, the extracellular levels of p55 were higher in Sabra salt-sensitive compared with Sabra salt-resistant rats (10.6±5.2% versus 2.9±1.5%; P<0.01). Tumor necrosis factor- plays a role in activation of the polymorphonuclear leukocyte NADPH oxidase, thereby contributing to systemic OS, inflammation, and the development of hypertension in this model.

Vascular Endothelial Function Is Related to White Blood Cell Count and Myeloperoxidase Among Healthy Middle-Aged and Older Adults [Original Articles]

Endothelium-dependent dilation (EDD) is impaired with aging, but there is significant variability among healthy middle-aged and older adults. We tested the hypothesis that EDD is related to white blood cell (WBC) count in healthy men and women aged 55 to 75 years (n=48) who have a WBC count within the clinically normal range. The peak forearm blood flow response to intrabrachial artery infusion of acetylcholine was inversely related to WBC count (r=–0.38; P=0.004) and was 34% smaller in subjects with higher versus lower WBC count (more versus less than the median of 5.0x109 cells per liter; P=0.001). Vascular smooth muscle responsiveness to NO (peak forearm blood flow response to sodium nitroprusside) was inversely related to WBC count (r=–0.30; P=0.02) but did not fully explain the associations with EDD. Inhibition of NO with NG-monomethyl-l-arginine reduced EDD in subjects with lower (–56%; P=0.01) but not higher WBC count. Tetrahydrobiopterin selectively improved EDD in subjects with higher WBC count (+35%; P=0.01) by increasing NO bioavailability. EDD was related (P<0.05) to neutrophil, eosinophil, and monocyte but not lymphocyte or basophil counts. Myeloperoxidase, which is secreted by neutrophils and monocytes, consumes NO and produces molecules that oxidize tetrahydrobiopterin, was inversely related to EDD (r=–0.35; P=0.02), and was 42% higher in subjects with a higher WBC count (P=0.02). No other factors contributed to the relation between EDD and WBC count. Among healthy middle-aged and older adults, impaired EDD is related to higher neutrophil, eosinophil, and monocyte-based WBC count mediated by reduced responsiveness to NO and increased myeloperoxidase-associated reductions in tetrahydrobiopterin and NO bioavailability.

Aldosterone Abrogates Nuclear Factor {kappa}B-Mediated Tumor Necrosis Factor {alpha} Production in Human Neutrophils via the Mineralocorticoid Receptor [Original Articles]

Mineralocorticoid receptor (MR) activation by aldosterone controls salt homeostasis and inflammation in several tissues and cell types. Whether or not a functional MR exists in polymorphonuclear neutrophils is unknown. We investigated the hypothesis that aldosterone modulates inflammatory neutrophil responses via the MR. By flow cytometry, Western blot analysis, and microscopy, we found that neutrophils possess MR. Preincubation with aldosterone (10–11 to 10–6 M) dose-dependently inhibited nuclear factor B activation in interleukin (IL)-8– and granulocyte/macrophage colony-stimulating factor–treated neutrophils on fibronectin by IB Western blotting, electrophoretic mobility shift assay, and RT-PCR for IB mRNA. Aldosterone had no effect on tumor necrosis factor – and lipopolysaccharide-mediated nuclear factor B activation or on IL-8– and granulocyte/macrophage colony-stimulating factor–induced extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, or phosphatidylinositol 3-kinase/Akt activation. Spironolactone prevented nuclear factor B inhibition, indicating an MR-specific aldosterone effect. By RT-PCR, we found that neutrophils have 11β-hydroxysteroid dehydrogenase. Tumor necrosis factor , which is controlled by nuclear factor B, increased in the cell supernatant with IL-8 treatment. Aldosterone completely prevented this effect. RT-PCR showed a strong tumor necrosis factor mRNA increase with IL-8 that was blocked by aldosterone, excluding the possibility that the tumor necrosis factor increase was merely a consequence of secretion. Finally, conditioned medium from IL-8–treated neutrophils increased intercellular adhesion molecule–1 expression on endothelial cells and subsequently the adhesion of IL-8–treated neutrophils to endothelial cells. These effects were reduced when conditioned medium from aldosterone-pretreated neutrophils was used, and spironolactone blocked the aldosterone effect. Our data indicate that a functional MR exists in neutrophils mediating antiinflammatory effects that are at work when neutrophils interact with endothelial cells. These data could be relevant to MR-blockade treatment protocols.

Recombinant Vascular Endothelial Growth Factor 121 Infusion Lowers Blood Pressure and Improves Renal Function in Rats With PlacentalIschemia-Induced Hypertension [Original Articles]

Antagonism of vascular endothelial growth factor (VEGF) signaling by soluble fms-like tyrosine kinase 1 occurs during preeclampsia and is proposed to play an important role in the pathogenesis of preeclampsia. We reported recently that hypertension associated with chronic reductions in uteroplacental perfusion pressure (RUPP) is associated with increased soluble fms-like tyrosine kinase 1 and decreased free VEGF. Whether restoration of circulating VEGF can restore renal function and chronically decrease arterial pressure associated with placental ischemia remains unknown. We hypothesized that chronic infusion of VEGF121 would attenuate hypertension, increase glomerular filtration rate, and reverse the endothelial dysfunction associated with chronic RUPP. VEGF121 (at either 90 or 180 µg/kg per day) was administered for 5 days via osmotic minipump placed IP. Mean arterial pressure, renal function, and tissues were obtained on day 19 of pregnancy from RUPP+VEGF, RUPP, and normal pregnant dams. Mean arterial pressure was increased in the RUPP (131±3 mm Hg) compared with the normal pregnant (102±1 mm Hg) rats, and infusion of VEGF121 resolved the hypertension (105±5 mm Hg). Glomerular filtration rate was decreased in the RUPP dams (1.5±0.3 mL/min) and restored to normal pregnant levels (3.1±0.5 mL/min) by VEGF121 treatment (3.1±0.4 mL/min). Effective renal plasma flow, decreased by RUPP, was also increased by VEGF121 infusion. Relaxation to acetylcholine was enhanced by the VEGF treatment (P<0.05). These data demonstrate that chronic infusion of VEGF121 during late gestation restores glomerular filtration rate and endothelial function and reduces high blood pressure associated with placental ischemia. The present results suggest that VEGF121 may be a candidate molecule for management of preeclampsia and its related complications.

Angiotensin Receptor Agonistic Autoantibody Is Highly Prevalent in Preeclampsia: Correlation With Disease Severity [Original Articles]

Preeclampsia (PE), a syndrome affecting 5% of pregnancies, characterized by hypertension and proteinuria, is a leading cause of maternal and fetal morbidity and mortality. The condition is often accompanied by the presence of a circulating maternal autoantibody, the angiotensin II type I receptor agonistic autoantibody (AT1-AA). However, the prevalence of AT1-AA in PE remains unknown, and the correlation of AT1-AA titers with the severity of the disease remains undetermined. We used a sensitive and high-throughput luciferase bioassay to detect AT1-AA levels in the serum of 30 normal, 37 preeclamptic (10 mild and 27 severe), and 23 gestational hypertensive individuals. Here we report that AT1-AA is highly prevalent in PE (95%). Next, by comparing the levels of AT1-AA among women with mild and severe PE, we found that the titer of AT1-AA is proportional to the severity of the disease. Intriguingly, among severe preeclamptic patients, we discovered that the titer of AT1-AA is significantly correlated with the clinical features of PE: systolic blood pressure (r=0.56), proteinuria (r=0.70), and soluble fms-like tyrosine kinase-1 level (r=0.71), respectively. Notably, only AT1-AA, and not soluble fms-like tyrosine kinase-1, levels are elevated in gestational hypertensive patients. These data serve as compelling clinical evidence that AT1-AA is highly prevalent in PE, and its titer is strongly correlated to the severity of the disease.

Role of Endothelin in Mediating Soluble fms-Like Tyrosine Kinase 1-Induced Hypertension in Pregnant Rats [Original Articles]

Although soluble fms-like tyrosine kinase 1 (sFlt-1), an antagonist of vascular endothelial growth factor and placental growth factor, has been implicated in the pathogenesis of hypertension during preeclampsia, the mechanisms whereby enhanced sFlt-1 production leads to hypertension remain unclear. Both sFlt-1 and endothelin 1 productions are elevated in women with preeclampsia and in placental ischemic animal models of preeclampsia; however, the importance of endothelin 1 and sFlt-1 interactions in the control of blood pressure during pregnancy is unknown. The purpose of this study was to determine the role of endothelin 1 in mediating sFlt-1–induced hypertension in pregnant rats. To achieve this goal, sFlt-1 (3.7 µg/kg per day for 6 days) was infused into normal pregnant rats and pregnant rats treated with a selective endothelin type A receptor antagonist, ABT 627 (5 mg/kg per day for 6 days). Plasma concentration of sFlt-1 increased from 735±34 pg/mL in normal pregnant rats to 2498±645 pg/mL (P<0.05) with infusion of sFlt-1. Arterial pressure increased from 100±1 mm Hg in normal pregnant rats to 122±3 mm Hg (P<0.05) in sFlt-1–infused rats. Chronic increases in plasma sFlt-1 in normal pregnant rats increased preproendothelin mRNA expression in the renal cortices by 3-fold. In addition, chronic endothelin type A receptor blockade completely abolished the blood pressure response to sFlt-1 in pregnant rats (104±3 versus 100±1 mm Hg; P<0.05), whereas the endothelin A receptor antagonist had no effect on arterial pressure in NP rats (105±2 versus 100±1 mm Hg). In conclusion, this study demonstrates that endothelin 1, via endothelin type A receptor activation, plays an important role in mediating the hypertension in response to excess sFlt-1 during pregnancy.

Compliance, Safety, and Effectiveness of Fixed-Dose Combinations of Antihypertensive Agents: A Meta-Analysis [Original Articles]

Two or more antihypertensive agents are increasingly used to control blood pressure (BP) in hypertensive patients. However, it is unclear whether fixed-dose combinations (FDCs) of 2 antihypertensive agents in a single tablet provide greater benefits than the corresponding free-drug components given separately. A meta-analysis was performed to assess compliance, persistence, BP control, and safety associated with FDCs in comparison with their free-drug components. Fifteen included studies (n=32331) reported on ≥1 of the evaluated outcomes. In 3 cohort studies and 2 trials reporting on drug compliance (n=17 999), the use of FDCs was associated with significantly better compliance (odds ratio: 1.21 [95% CI: 1.03 to 1.43]; P=0.02) compared with its corresponding free-drug combinations. In 3 cohort studies (n=12 653), there was a nonsignificant improvement in persistence with therapy (odds ratio: 1.54 [95% CI: 0.95 to 2.49]; P=0.08), and in 5 trials (n=1775) the odds ratio for adverse effects for FDC use compared with free-drug combination use was 0.80 (95% CI: 0.58 to 1.11; P=0.19). In 9 trials (n=1671) with BP data, use of an FDC was associated with nonsignificant changes in systolic and diastolic BPs of 4.1 mm Hg (95% CI: –9.8 to 1.5; P=0.15) and 3.1 mm Hg (95% CI: –7.1 to 0.9; P=0.13), respectively. In these BP-lowering comparisons, there was heterogeneity associated with differences in study design but no publication bias. In conclusion, compared with free-drug combinations, FDCs of antihypertensive agents are associated with a significant improvement in compliance and with nonsignificant beneficial trends in BP and adverse effects.

Baseline Depressive Symptoms Are Not Associated With Clinically Important Levels of Incident Hypertension During Two Years of Follow-Up: The Multi-Ethnic Study of Atherosclerosis [Original Articles]

Previous longitudinal cohort studies have suggested an association between baseline depressive symptoms and incident hypertension. We assessed this possible association using data from the Multi-ethnic Study of Atherosclerosis, a population-based prospective cohort study of 6814 US adults from 4 different racial/ethnic groups. Baseline users of antihypertensive medications and participants lost to follow-up were excluded leaving 3914 participants. Patients with baseline depressive symptoms (n=622) were defined using a high score on the Center for Epidemiological Studies Depression Scale (≥16) or the use of an antidepressant medication. Hypertension was defined as systolic blood pressure of ≥140 mm Hg, diastolic blood pressure of ≥90 mm Hg or new use of antihypertensive medications plus physician diagnosis. Estimates were adjusted for known risk factors, including age, sex, baseline blood pressure, diabetes, and body mass index. Untreated blood pressure was estimated using an imputation approach. A total of 477 participants developed hypertension. Using relative risk regression, patients with baseline depressive symptoms did not have an increased risk of incident hypertension (relative risk: 1.02; 95% confidence interval [CI]: 0.99 to 1.05), although an association between tricyclic antidepressants and hypertension (relative risk: 1.20; 95% CI: 1.05 to 1.37) was observed in subgroup analysis. Depression, even after adjustment for covariates, was associated with small changes in systolic (+2.4 mm Hg; 95% CI: 0.2 to 4.7) and diastolic (+0.8 mm Hg; 95% CI: –0.6 to 2.3) blood pressures. Depressive symptoms may be associated with slight increases in blood pressure in this multiethnic cohort, but it is premature to conclude much without longer studies in other populations.

Is Longitudinal Pulse Pressure a Better Predictor of 24-Hour Urinary Albumin Excretion Than Other Indices of Blood Pressure? [Original Articles]

The strong relationship between urinary albumin excretion (UAE) and pulse pressure (PP) in cross-sectional studies suggests that pressure pulsatility may contribute to renal microvascular injury. The longitudinal relationships between UAE and the various indices of blood pressure (BP) are not well studied. We compared the associations of UAE with the longitudinal exposure to PP and systolic, diastolic, and mean BPs. UAE was measured from 24-hour urine collections in 450 community-dwelling subjects (age: 57±15 years, 53% women, all with UAE <200 µg/min). For each subject, longitudinal indices of BP were estimated by dividing the area under the curve of serial measurements of BP (median: 5) during 1 to 22 years preceding UAE measurement by the number of follow-up years. Median (interquartile range) UAE was 4.7 µg/min (3.3 to 7.8 µg/min) in women and 5.2 µg/min (3.7 to 9.8 µg/min) in men. In women, UAE was not related to longitudinal indices of BP. In men, in multivariable-adjusted models that included either longitudinal systolic and diastolic BPs or longitudinal PP and mean BP, UAE was independently associated with systolic (standardized regression coefficient [β]=0.227; P=0.03) but not with diastolic (β=–0.049; P=0.59) BP and with PP (β=0.216; P=0.01) but not with mean BP (β=0.032; P=0.72). Comparisons of these 2 models and stepwise regression analyses both indicated that, of the 4 longitudinal indices of BP, PP was the strongest predictor of UAE in men. The pulsatile component of BP confers the highest risk for BP-induced renal microvascular injury. Future studies should examine whether PP reduction provides additional renoprotection beyond that attained by conventional BP goals alone.

Peak C-Reactive Protein Level Predicts Long-Term Outcomes in Type B Acute Aortic Dissection [Original Articles]

Acute aortic dissection (AAD) is associated with an inflammatory reaction, as evidenced by elevated inflammatory markers, including C-reactive protein (CRP). The association between the peak CRP level and long-term outcomes in type B AAD has not been systematically investigated. The purpose of this study was to investigate whether the peak CRP level during admission predicts long-term outcomes in type B AAD. We conducted a clinical follow-up study of type B AAD. We divided the study population into 4 groups according to the tertiles of peak CRP levels (T1: 0.60 to 9.37 mg/dL; T2: 9.61 to 14.87 mg/dL; T3: 14.90 to 32.60 mg/dL; and unavailable peak CRP group). Multivariate Cox regression analysis was applied to investigate whether the tertiles of peak CRP predict adverse events even after adjusting for other variables. A total of 232 type B AAD patients were included in this analysis. The median follow-up period was 50 months. CRP reached its peak on day 4.5±1.7. Mean peak CRP values in T1, T2, and T3 were 6.4±2.4, 12.0±1.5, and 19.5±4.0 mg/dL, respectively. There were 65 events (39 deaths and 26 aortic events) during the follow-up. T3 and T2 (versus T1) were strong predictors of adverse events (T3: hazard ratio: 6.02 [95% CI: 2.44 to 14.87], P=0.0001; T2: hazard ratio: 3.25 [95% CI: 1.37 to 7.71], P=0.01) after controlling for all of the confounding factors. In conclusion, peak CRP is a strong predictor for adverse long-term events in patients with type B AAD.

63rd Annual Fall Conference and Scientific Session of the American Heart Association Council for High Blood Pressure Research in Association With the Council on the Kidney in Cardiovascular Disease [Preface]

Awards [Awards]

Endothelial and Vascular Muscle PPAR{gamma} in Arterial Pressure Regulation: Lessons From Genetic Interference and Deficiency [Novartis Award for Hypertension Research]

New Physiological Concepts of the Renin-Angiotensin System From the Investigation of Precursors and Products of Angiotensin I Metabolism [Novartis Award for Hypertension Research]

Cardioprotective Actions of Cyclic GMP: Lessons From Genetic Animal Models [Lewis K. Dahl Memorial Lecture]

N-Acetyl-Seryl-Aspartyl-Lysyl-Proline Attenuates Renal Injury and Dysfunction in Hypertensive Rats With Reduced Renal Mass: Council for High Blood Pressure Research [Original Articles Part 2]

N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is a naturally occurring peptide of which the plasma concentration is increased 4- to 5-fold by angiotensin-converting enzyme inhibitors. We reported previously that, in models of both hypertension and postmyocardial infarction, Ac-SDKP reduces cardiac inflammation and fibrosis. However, it is unknown whether Ac-SDKP can prevent or reverse renal injury and dysfunction in hypertension. In the present study, we tested the hypothesis that, in rats with 5/6 nephrectomy (5/6Nx)-induced hypertension, Ac-SDKP reduces renal damage, albuminuria, and dysfunction by decreasing inflammatory cell infiltration and renal fibrosis and by increasing nephrin protein. Ac-SDKP (800 µg/kg per day, SC via osmotic minipump) or vehicle was either started 7 days before 5/6Nx (prevention) and continued for 3 weeks or started 3 weeks after 5/6Nx (reversal) and continued for another 3 weeks. Rats with 5/6Nx developed high blood pressure, left ventricular hypertrophy, albuminuria, decreased glomerular filtration rate, and increased macrophage infiltration (inflammation) and renal collagen content (fibrosis). Ac-SDKP did not affect blood pressure or left ventricular hypertrophy in either group; however, it significantly reduced albuminuria, renal inflammation, and fibrosis and improved glomerular filtration rate in both prevention and reversal groups. Moreover, slit diaphragm nephrin protein expression in the glomerular filtration barrier was significantly decreased in hypertensive rats. This effect was partially prevented or reversed by Ac-SDKP. We concluded that Ac-SDKP greatly attenuates albuminuria and renal fibrosis and improves renal function in rats with 5/6Nx. These effects may be related to decreased inflammation (macrophages) and increased nephrin protein.

Protein Kinase C-Dependent NAD(P)H Oxidase Activation Induced by Type 1 Diabetes in Renal Medullary Thick Ascending Limb [Original Articles Part 2]

Type 1 diabetes provokes a protein kinase C (PKC)-dependent accumulation of superoxide anion in the renal medullary thick ascending limb (mTAL). We hypothesized that this phenomenon involves PKC-dependent NAD(P)H oxidase activation. The validity of this hypothesis was explored using mTAL suspensions prepared from rats with streptozotocin-induced diabetes and from sham (vehicle-treated) rats. Superoxide production was 5-fold higher in mTAL suspensions from diabetic rats compared with suspensions from sham rats. The NAD(P)H oxidase inhibitor apocynin caused an 80% decrease in superoxide production by mTAL from diabetic rats (P<0.05 vs untreated) without altering superoxide production by sham mTAL. NAD(P)H oxidase activity was >2-fold higher in mTAL from diabetic rats than in sham mTAL (P<0.05). Pretreatment with calphostin C (broad-spectrum PKC inhibitor) or rottlerin (PKC inhibitor) reduced NAD(P)H oxidase activity by 80% in both groups; however, PKC/β or PKCβ inhibition did not alter NAD(P)H oxidase activity in either group. Protein levels of Nox2, Nox4, and p47phox were significantly higher in diabetic mTAL than in mTAL from sham rats. In summary, elevated superoxide production by mTAL from diabetic rats was normalized by NAD(P)H oxidase inhibition. PKC-dependent, PKC-dependent, and total NAD(P)H oxidase activity was greater in mTAL from diabetic rats compared with sham. Protein levels of Nox2, Nox4, and p47phox were increased in mTAL from diabetic rats. We conclude that increased superoxide production by the mTAL during diabetes involves a PKC-dependent increase in NAD(P)H oxidase activity in concert with increased protein levels of catalytic and regulatory subunits of the enzyme.

Intrarenal Aminopeptidase N Inhibition Restores Defective Angiontesin II Type 2-Mediated Natriuresis in Spontaneously Hypertensive Rats [Original Articles Part 2]

The preferred ligand of angiotensin (Ang) II type 2 (AT2R)-mediated natriuresis is Ang III. The major enzyme responsible for the metabolism of Ang III is aminopeptidase N, which is selectively inhibited by compound PC-18. In this study, urine sodium excretion rates (UNaV), fractional excretion of sodium, fractional excretion of lithium, glomerular filtration rate, and mean arterial pressures were studied in prehypertensive and hypertensive spontaneously hypertensive rats (SHRs) and compared with age-matched Wistar-Kyoto rats (WKYs). Although renal interstitial infusion of Ang II type 1 receptor blocker candesartan increased UNaV in WKYs from a baseline of 0.05±0.01 to 0.17±0.04 µmol/min (P<0.01), identical infusions failed to increase UNaV in hypertensive SHRs. Coinfusion of AT2R antagonist PD-123319 abolished the natriuretic responses to candesartan in WKYs, indicating an AT2R-mediated effect. AT2R-mediated natriuresis was enabled in hypertensive SHRs by inhibiting the metabolism of Ang III with PC-18 (0.05±0.01 to 0.11±0.03 µmol/min; P<0.05). The defects in sodium excretion were present before the onset of hypertension in SHRs, because young WKYs demonstrated double the UNaV of SHRs (0.04±0.006 versus 0.02±0.003 µmol/min; P<0.01) at baseline. The increased UNaV of young WKYs was attributed to reduced renal proximal tubule sodium reabsorption, because increases in fractional excretion of sodium were paralleled by increases in fractional excretion of lithium. Renal interstitial PC-18 infusion ameliorated defective AT2R-mediated natriuresis in young SHRs by increasing fractional excretion of sodium and fractional excretion of lithium without changing the glomerular filtration rate. Thus, increased renal proximal tubule sodium retention is observed before the onset of hypertension in SHRs, and inhibition of the metabolism of Ang III ameliorates this pathophysiologic defect in sodium excretion.

Chronic Hypertension Enhances Presynaptic Inhibition by Baclofen in the Nucleus of the Solitary Tract [Original Articles Part 2]

The selective -aminobutyric acid B-subtype receptor agonist baclofen activates both presynaptic and postsynaptic receptors in the brain. Microinjection of baclofen into the nucleus of the solitary tract increases arterial pressure, heart rate, and sympathetic nerve discharge consistent with inhibition of the arterial baroreflex. The magnitude of these responses is enhanced in hypertension and is associated with increased postsynaptic GABAB receptor function. We tested whether a presynaptic mechanism contributes to the enhanced baclofen inhibition in hypertension. Whole-cell recordings of second-order baroreceptor neurons, identified by 4-(4-(dihexadecylamino)styryl)-N-methylpyridinium iodide labeling of aortic nerve, were obtained in brainstem slices from normotensive control and renal-wrap hypertensive rats. After 4 weeks, arterial blood pressure was 162±9 mm Hg in hypertensive (n=6) and 107±3 mm Hg in control rats (n=6/11; P<0.001). Baclofen reduced the amplitude of excitatory postsynaptic currents evoked by solitary tract stimulation and the EC50 of this inhibition was greater in control (1.5±0.5 µmol/L; n=6) than in hypertensive cells (0.6±0.1 µmol/L; n=9; P<0.05). Baclofen (1 µmol/L) elicited greater inhibition on evoked response in hypertensive (58±6%; n=9) than in control cells (40±6%; n=8; P<0.05). Another index of presynaptic inhibition, the paired-pulse ratio (ratio of second to first evoked response amplitudes at stimulus intervals of 40 ms), was greater in hypertensive (0.60±0.08; n=8) than in control cells (0.48±0.06; n=5; P<0.05). The results suggest that in renal-wrap hypertensive rats, baclofen causes an enhanced presynaptic inhibition of glutamate release from baroreceptor afferent terminals to second-order neurons in the nucleus of the solitary tract. This enhanced presynaptic inhibition could contribute to altered baroreflex function in hypertension.

Proton Magnetic Resonance Spectroscopy Detection of Neurotransmitters in Dorsomedial Medulla Correlate With Spontaneous Baroreceptor Reflex Function [Original Articles Part 2]

Control of heart rate variability via modulation of sympathovagal balance is a key function of nucleus tractus solitarii and the dorsal motor nucleus of the vagus localized in the dorsomedial medulla oblongata. Normal blood pressure regulation involves precise balance of glutamate (Glu)-glutamine--aminobutyric acid transmitter systems, and angiotensin II modulates these transmitters to produce tonic suppression of reflex function. It is not known, however, whether other brain transmitters/metabolites are indicators of baroreflex function. This study establishes the concept that comprehensive baseline transmitter/metabolite profiles obtained using in vivo 1H magnetic resonance spectroscopy in rats with well-characterized differences in resting blood pressure and baroreflex function can be used as indices of autonomic balance or baroreflex sensitivity. Transgenic rats with over-expression of renin [m(Ren2)27] or under-expression of glial-angiotensinogen (ASrAogen) were compared with Sprague-Dawley rats. Glu concentration in the dorsal medulla is significantly higher in ASrAogen rats compared with either Sprague-Dawley or (mRen2)27 rats. Glu levels and the ratio of Glu:glutamine correlated positively with indices of higher vagal tone consistent with the importance of these neurotransmitters in baroreflex function. Interestingly, the levels of choline-containing metabolites showed a significant positive correlation with spontaneous baroreflex sensitivity and a negative correlation with sympathetic tone. Thus, we demonstrate the concept that noninvasive assessment of neurochemical biomarkers may be used as an index of baroreflex sensitivity.

Early Life Stress Sensitizes Rats to Angiotensin II-Induced Hypertension and Vascular Inflammation in Adult Life [Original Articles Part 2]

Maternal separation during early life is an established chronic behavioral model of early life stress in rats. It is known that perinatal adverse environments increase activity of the renin-angiotensin (Ang) system, specifically Ang II, in adulthood. The aim of this study was to investigate whether the effects of early life stress augment the sensitivity of the Ang II pathway. Using Wistar Kyoto rats, the maternal separation (MS) protocol was performed by separating approximately half of the male pups from their mother 3 h/d from days 2 to 14 of life. Pups remaining with the mother at all times were used as controls. Maternal separation did not influence the plasma basal parameters, such as blood glucose, insulin, Ang II, Ang 1-7 and plasma renin activity. Furthermore, body weight, blood pressure, and heart rate were similar in MS and control rats. The acute pressor response to Ang II was not different in anesthetized MS and control rats. However, the chronic infusion of Ang II (65 ng/min SC) elicited an exaggerated hypertensive response in MS compared with control rats (P<0.05). Surprisingly, HR was dramatically increased during the second week of Ang II infusion in MS compared with control rats (P<0.05). This enhanced Ang II sensitivity was accompanied by a greater vascular inflammatory response in MS versus control rats. Chronic Ang II infusion increased vascular wall structure in both groups similarly. These data indicate that early life stress sensitizes rats to an increased hemodynamic and inflammatory response during Ang II–induced hypertension.

Interleukin 17 Promotes Angiotensin II-Induced Hypertension and Vascular Dysfunction [Original Articles Part 2]

We have shown previously that T cells are required for the full development of angiotensin II–induced hypertension. However, the specific subsets of T cells that are important in this process are unknown. T helper 17 cells represent a novel subset that produces the proinflammatory cytokine interleukin 17 (IL-17). We found that angiotensin II infusion increased IL-17 production from T cells and IL-17 protein in the aortic media. To determine the effect of IL-17 on blood pressure and vascular function, we studied IL-17–/– mice. The initial hypertensive response to angiotensin II infusion was similar in IL-17–/– and C57BL/6J mice. However, hypertension was not sustained in IL-17–/– mice, reaching levels 30-mm Hg lower than in wild-type mice by 4 weeks of angiotensin II infusion. Vessels from IL-17–/– mice displayed preserved vascular function, decreased superoxide production, and reduced T-cell infiltration in response to angiotensin II. Gene array analysis of cultured human aortic smooth muscle cells revealed that IL-17, in conjunction with tumor necrosis factor-, modulated expression of >30 genes, including a number of inflammatory cytokines/chemokines. Examination of IL-17 in diabetic humans showed that serum levels of this cytokine were significantly increased in those with hypertension compared with normotensive subjects. We conclude that IL-17 is critical for the maintenance of angiotensin II–induced hypertension and vascular dysfunction and might be a therapeutic target for this widespread disease.

Metabolomics in Angiotensin II-Induced Cardiac Hypertrophy [Original Articles Part 2]

Angiotensin II (Ang II) induces mitochondrial dysfunction. We tested whether Ang II alters the "metabolomic" profile. We harvested hearts from 8-week-old double transgenic rats harboring human renin and angiotensinogen genes (dTGRs) and controls (Sprague-Dawley), all with or without Ang II type 1 receptor (valsartan) blockade. We used gas chromatography coupled with time-of-flight mass spectrometry to detect 247 intermediary metabolites. We used a partial least-squares discriminate analysis and identified 112 metabolites that differed significantly after corrections (false discovery rate q <0.05). We found great differences in the use of fatty acids as an energy source, namely, decreased levels of octanoic, oleic, and linoleic acids in dTGR (all P<0.01). The increase in cardiac hypoxanthine levels in dTGRs suggested an increase in purine degradation, whereas other changes supported an increased ketogenic amino acid tyrosine level, causing energy production failure. The metabolomic profile of valsartan-treated dTGRs more closely resembled Sprague-Dawley rats than untreated dTGRs. Mitochondrial respiratory chain activity of cytochrome C oxidase was decreased in dTGRs, whereas complex I and complex II were unaltered. Mitochondria from dTGR hearts showed morphological alterations suggesting increased mitochondrial fusion. Cardiac expression of the redox-sensitive and the cardioprotective metabolic sensor sirtuin 1 was increased in dTGRs. Interestingly, valsartan changed the level of 33 metabolites and induced mitochondrial biogenesis in Sprague-Dawley rats. Thus, distinct patterns of cardiac substrate use in Ang II-induced cardiac hypertrophy are associated with mitochondrial dysfunction. The finding underscores the importance of Ang II in the regulation of mitochondrial biogenesis and cardiac metabolomics, even in healthy hearts.

Effects of Angiotensin Metabolites in the Coronary Vascular Bed of the Spontaneously Hypertensive Rat: Loss of Angiotensin II Type 2 Receptor-Mediated Vasodilation [Original Articles Part 2]

Because angiotensin (Ang) metabolites mediate functions independent of Ang II, we investigated their effects on coronary flow in spontaneously hypertensive rats (SHRs). Results were compared with those in the iliac artery and abdominal aorta and the coronary circulation of the Wistar rat. Ang II, III, and IV decreased coronary flow in SHRs and Wistar rats, with Ang III and IV being 10 and 1000 times less potent than Ang II. Ang-(1-7) decreased coronary flow at concentrations >1 µmol/L in SHRs. The Ang II type 1 receptor antagonist irbesartan blocked the effects of Ang II, III, and IV, whereas the Ang II type 2 receptor antagonist PD123319 blocked the effects of Ang-(1-7). The maximal Ang II- and III-induced decreases in coronary flow in SHRs were twice as large as those in Wistar rats. PD123319 enhanced the constrictor effects of Ang II and III in Wistar rats so that, in the presence of this drug, their effects were comparable to those in SHRs. In contrast, PD123319 did not alter the Ang II- and III-induced responses in SHRs and blocked the constrictor effect of Ang II in iliac arteries. Ang II type 2 receptor–mediated relaxation did not occur in iliac arteries and abdominal aortas, and the constrictor effects of Ang metabolites in these vessels were identical in Wistar rats and SHRs. In conclusion, coronary constriction induced by Ang II, Ang III, and Ang-(1-7) is enhanced in SHRs as compared with Wistar rats. This is attributable to the absence of counterregulatory Ang II type 2 receptor–mediated relaxation and/or a change of the Ang II type 2 receptor phenotype from relaxant to constrictor.

Angiotensin II Type 1 and Type 2 Receptors Regulate Basal Skeletal Muscle Microvascular Volume and Glucose Use [Original Articles Part 2]

Angiotensin II causes vasoconstriction via the type 1 receptor (AT1R) and vasodilatation through the type 2 receptor (AT2R). Both are expressed in muscle microvasculature, where substrate exchanges occur. Whether they modulate basal muscle microvascular perfusion and substrate metabolism is not known. We measured microvascular blood volume (MBV), a measure of microvascular surface area and perfusion, in rats during systemic infusion of angiotensin II at either 1 or 100 ng/kg per minute. Each caused a significant increase in muscle MBV. Likewise, administration of the AT1R blocker losartan increased muscle MBV by >3-fold (P<0.001). Hindleg glucose extraction and muscle interstitial oxygen saturation simultaneously increased by 2- to 3-fold. By contrast, infusing AT2R antagonist PD123319 significantly decreased muscle MBV by ≥80% (P<0.001). This was associated with a significant decrease in hindleg glucose extraction and muscle oxygen saturation. AT2R antagonism and inhibition of NO synthase each blocked the losartan-induced increase in muscle MBV and glucose uptake. In conclusion, angiotensin II acts on both AT1R and AT2R to regulate basal muscle microvascular perfusion. Basal AT1R tone restricts muscle MBV and glucose extraction, whereas basal AT2R activity increases muscle MBV and glucose uptake. Pharmacological manipulation of the balance of AT1R and AT2R activity affords the potential to improve glucose metabolism.

The Impact of Microsomal Prostaglandin E Synthase 1 on Blood Pressure Is Determined by Genetic Background [Original Articles Part 2]

Prostaglandin (PG)E2 has multiple actions that may affect blood pressure. It is synthesized from arachidonic acid by the sequential actions of phospholipases, cyclooxygenases, and PGE synthases. Although microsomal PGE synthase (mPGES)1 is the only genetically verified PGE synthase, results of previous studies examining the consequences of mPGES1 deficiency on blood pressure (BP) are conflicting. To determine whether genetic background modifies the impact of mPGES1 on BP, we generated mPGES1–/– mice on 2 distinct inbred backgrounds, DBA/1lacJ and 129/SvEv. On the DBA/1 background, baseline BP was similar between wild-type (WT) and mPGES1–/– mice. By contrast, on the 129 background, baseline BPs were significantly higher in mPGES1–/– animals than WT controls. During angiotensin II infusion, the DBA/1 mPGES1–/– and WT mice developed mild hypertension of similar magnitude, whereas 129-mPGES1–/– mice developed more severe hypertension than WT controls. DBA/1 animals developed only minimal albuminuria in response to angiotensin II infusion. By contrast, WT 129 mice had significantly higher levels of albumin excretion than WT DBA/1 and the extent of albuminuria was further augmented in 129 mPGES1–/– animals. In WT mice of both strains, the increase in urinary excretion of PGE2 with angiotensin II was attenuated in mPGES1–/– animals. Urinary excretion of thromboxane was unaffected by angiotensin II in the DBA/1 lines but increased more than 4-fold in 129 mPGES1–/– mice. These data indicate that genetic background significantly modifies the BP response to mPGES1 deficiency. Exaggerated production of thromboxane may contribute to the robust hypertension and albuminuria in 129 mPGES1-deficient mice.

mPGES-1 Protects Against DOCA-Salt Hypertension via Inhibition of Oxidative Stress or Stimulation of NO/cGMP [Original Articles Part 2]

Microsomal prostaglandin E synthase-1 (mPGES-1) is a recently characterized cytokine-inducible enzyme critically involved in pain and inflammatory response. However, its role in blood pressure regulation is still debatable. The present study was undertaken to examine the effect of mPGES-1 deletion on DOCA-salt hypertension. After 2 weeks of DOCA plus 1% NaCl as drinking fluid, hypertension and sodium retention were more severe in mPGES-1 knockout (KO) mice than in wild-type (WT) controls. The indices of oxidative stress including urinary 8-isprostane and renal thiobarbituric acid-reactive substances were only modestly increased or unchanged in the WT mice but more significantly increased in the KO mice after DOCA-salt. Conversely, in response to DOCA-salt, the indices of antioxidant systems including renal expression of superoxide dismutase-3 and urinary nitrate/nitrite excretion were all significantly elevated in the WT mice but remarkably suppressed in the KO mice. Tempol treatment (50 mg/kg per day) in DOCA-salt KO mice produced a marked attenuation of hypertension, sodium retention, and kidney injury. Immunoblotting demonstrated increased renal expression of mPGES-1 in DOCA-salt WT mice. DOCA-salt induced a nearly 5-fold increase in urinary PGE2 excretion in the WT mice, and this increase was completely abolished in the KO mice. Together, these results suggest that mPGES-1–derived PGE2 confers protection against DOCA-salt hypertension likely via inhibition of oxidative stress or stimulation of superoxide dismutase-3 and urinary nitrate/nitrite system.

Adrenic Acid Metabolites as Endogenous Endothelium-Derived and Zona Glomerulosa-Derived Hyperpolarizing Factors [Original Articles Part 2]

Adrenic acid (docosatetraenoic acid), an abundant fatty acid in the adrenal gland, is identical to arachidonic acid except for 2 additional carbons on the carboxyl end. Adrenic acid is metabolized by cyclooxygenases, cytochrome P450s, and lipoxygenases; however, little is known regarding the role of adrenic acid and its metabolites in vascular tone. Because of its abundance in the adrenal gland, we investigated the role of adrenic acid in vascular tone of bovine adrenal cortical arteries and its metabolism by bovine adrenal zona glomerulosa cells. In adrenal cortical arteries, adrenic acid caused concentration-dependent relaxations, which were inhibited by the epoxyeicosatrienoic acid antagonist 14,15-epoxyeicosa-5(Z)-enoic acid and the cytochrome P450 inhibitor SKF-525A. The large-conductance calcium-activated potassium channel blocker iberiotoxin or removal of the endothelium abolished these relaxations. Reverse-phase high-pressure liquid chromatography and liquid chromatography/mass spectrometry isolated and identified numerous adrenic acid metabolites from zona glomerulosa cells, including dihomo-epoxyeicosatrienoic acids and dihomo-prostaglandins. In denuded adrenal cortical arteries, adrenic acid caused concentration-dependent relaxations in the presence of zona glomerulosa cells but not in their absence. These relaxations were inhibited by SKF-525A, 14,15-epoxyeicosa-5(Z)-enoic acid, and iberiotoxin. Dihomo-16,17-epoxyeicosatrienoic acid caused concentration-dependent relaxations of adrenal cortical arteries, which were inhibited by 14,15-epoxyeicosa-5(Z)-enoic acid and high potassium. Our results suggest that adrenic acid relaxations of bovine adrenal cortical arteries are mediated by endothelial and zona glomerulosa cell cytochrome P450 metabolites. Thus, adrenic acid metabolites could function as endogenous endothelium-derived and zona glomerulosa-derived hyperpolarizing factors in the adrenal cortex and contribute to the regulation of adrenal blood flow.

Neointimal Hyperplasia and Vasoreactivity Are Controlled by Genetic Elements on Rat Chromosome 3 [Original Articles Part 2]

Neointimal hyperplasia (NIH) can lead to restenosis after clinical vascular interventions. NIH results from complex and poorly understood interactions between signaling cascades in the extracellular matrix and the disrupted endothelium, which lead to vessel occlusion. Quantitative trait loci (QTLs) were reported previously on rat chromosomes 3 and 6 through linkage analysis of postinjury NIH in midiliac arterial sections. In the current study, substitution mapping validated the RNO3 NIH QTL but not the RNO6 NIH QTL. The SHR.BN3 congenic strain had a 3-fold increase in the percentage of NIH compared with the parental spontaneously hypertensive rat strain. A double congenic study of RNO3+RNO6 NIH QTL segments suggested less than additive effects of these 2 genomic regions. To test the hypothesis that changes in vessel dynamics account for the differences in NIH formation, we performed vascular reactivity studies in the Brown Norway (BN), spontaneously hypertensive rat (SHR), SHR.BN3, and SHR.BN6 strains. De-endothelialized left common carotid artery rings of the SHR.BN3 showed an increased vascular responsiveness when treated with serotonin or prostaglandin F2, with significant differences in EC50 and maximum effect (P<0.01) values compared with the spontaneously hypertensive rat parental strain. Because both vascular reactivity and percentage of NIH formation in the SHR.BN3 strain are significantly higher than the SHR strain, we postulate that these traits may be associated and are controlled by genetic elements on RNO3. In summary, these results confirm that the RNO3 NIH QTL carries the gene(s) contributing to postinjury NIH formation.

Increased Angiotensin II in the Mesometrial Triangle of a Transgenic Rat Model of Preeclampsia [Original Articles Part 2]

The pregnant female human angiotensinogen (hAGN) transgenic rat mated with the male hrenin (hREN) transgenic rat is a model of preeclampsia with increased blood pressure, proteinuria, and placenta alterations of edema and necrosis. The reverse mating of female hRENxmale hAGN does not show preeclamptic features. Because the placenta is well-recognized to be a key contributor to the preeclamptic syndrome, our hypothesis is that local angiotensin peptide concentrations found in the placenta and its associated mesometrial triangle of the preeclamptic transgenic rat differ from the reverse mating. We characterized the angiotensin peptide content and the mRNA expression of hREN and hAGN of the mesometrial triangle and the placenta. Three groups of pregnant rats from the matings (Sprague-DawleyxSprague-Dawley, reverse mating, and female hAGNxmale hREN) were studied on day 21 of gestation. In the hAGNxhREN transgenic rat, angiotensin II is significantly increased in the placenta and mesometrial triangle vs Sprague-Dawley (24.2±3.9 vs 8.6±1.5 pg/mg protein; 27.8±5.5 vs 5.6±1.3 pg/mg protein; P<0.05), whereas in the reverse mating angiotensin II is increased in the placenta (19.1±1.7 vs 5.6±1.3 pg/mg protein; P<0.05) but unchanged in the mesometrial triangle (4.2±0.2 vs 8.6±1.5 pg/mg protein). The marked contrast in the expression of angiotensin II in the mesometrial triangle of the preeclamptic model vs the reverse mating suggests that local angiotensin II generated from the maternal parts of the uteroplacental unit may play a critical role in preeclampsia.

Resting Heart Rate Pattern During Follow-Up and Mortality in Hypertensive Patients [Original Articles Part 2]

There is a linear relationship between resting heart rate (HR) and mortality in normotensive and untreated hypertensive individuals. However, it is not clear whether HR is a marker of increased risk in hypertensive patients on treatment. We investigated the relationship between HR and mortality in patients with hypertension. We analyzed baseline HR, final HR, and HR change during follow-up in patients attending the Glasgow Blood Pressure Clinic. Using a threshold of 80 bpm, we classified patients into those who had a consistently high (high-high) or low (low-low) HR or patients whose HR increased (low-high) or decreased (high-low) over time. Survival analysis was carried out using Cox proportional hazards models adjusted for age, sex, body mass index, smoking, rate-limiting therapy, systolic blood pressure, and serum cholesterol. For each beat of HR change there was a 1% change in mortality risk. The highest risk of an all-cause event was associated with patients who had increased their HR by ≥5 bpm at the end of follow-up (1.51 [95% CI: 1.03 to 2.20]; P=0.035). Compared with low-low patients, high-high patients had a 78% increase in the risk of all-cause mortality (HR: 1.78 [95% CI: 1.31 to 2.41]; P<0.001). Cardiovascular mortality showed a similar pattern of results. Rate-limiting therapy did not have an independent effect on outcomes in this analysis. Change in HR achieved during follow-up of hypertensive patients is a better predictor of risk than baseline or final HR. After correction for rate-limiting therapy, HR remained a significant independent risk factor.

Focus on Obesity [Editorials]

Patch Instead of Pill: A Safer Menopausal Estrogen? [Editorials]

HDL Structure, Function, Therapeutics, and Imaging [ATVB In Focus]

Role of HDL, ABCA1, and ABCG1 Transporters in Cholesterol Efflux and Immune Responses [ATVB In Focus]

Atherosclerosis has been characterized as a chronic inflammatory response to cholesterol deposition in arteries, but the mechanisms linking cholesterol accumulation in macrophage foam cells to inflammation are poorly understood. Macrophage cholesterol efflux occurs at all stages of atherosclerosis and protects cells from free cholesterol and oxysterol-induced toxicity. The ATP-binding cassette transporters ABCA1 and ABCG1 are responsible for the major part of macrophage cholesterol efflux to serum or HDL in macrophage foam cells, but other less efficient pathways such as passive efflux are also involved. Recent studies have shown that the sterol efflux activities of ABCA1 and ABCG1 modulate macrophage expression of inflammatory cytokines and chemokines as well as lymphocyte proliferative responses. In macrophages, transporter deficiency causes increased signaling via various Toll-like receptors including TLR4. These studies have shown that the traditional roles of HDL and ABC transporters in cholesterol efflux and reverse cholesterol transport are mechanistically linked to antiinflammatory and immunosuppressive functions of HDL. The underlying mechanisms may involve modulation of sterol levels and lipid organization in cell membranes.

Signaling by the High-Affinity HDL Receptor Scavenger Receptor B Type I [ATVB In Focus]

Scavenger receptor B type I (SR-BI) plays an important role in mediating cholesterol exchange between cells, high-density lipoprotein (HDL) cholesterol, and other lipoproteins. SR-BI in hepatocytes is essential for reverse cholesterol transport and biliary secretion of HDL cholesterol; thus, it is atheroprotective. More recently, it has been discovered that the HDL–SR-BI tandem serves other functions that also likely contribute to HDL-related cardiovascular protection. A number of the latter mechanisms, particularly in endothelial cells, involve unique direct signal initiation by SR-BI that leads to the activation of diverse kinase cascades. SR-BI signaling occurs in response to plasma membrane cholesterol flux. It requires the C-terminal PDZ-interacting domain of the receptor, which mediates direct interaction with the adaptor molecule PDZK1; and the C-terminal transmembrane domain, which directly binds membrane cholesterol. In endothelium, direct SR-BI signaling in response to HDL results in enhanced production of the antiatherogenic molecule nitric oxide; in a nitric oxide–independent manner, it serves to maintain endothelial monolayer integrity. The role of SR-BI signaling in the numerous other cellular targets of HDL, including hepatocytes, macrophages, and platelets, and the basis by which SR-BI senses plasma membrane cholesterol movement to modify cell behavior are unknown. Further understanding of signaling by SR-BI will optimize the capacity to harness the mechanisms of action of HDL–SR-BI for cardiovascular benefit.

Dysfunctional HDL as a Diagnostic and Therapeutic Target [ATVB In Focus]

The atheroprotective effects of HDL are mediated by several mechanisms, including its role in reverse cholesterol transport and via its antiinflammatory properties. However, not all HDL is functionally similar. HDL and apolipoprotein A-I may become dysfunctional or even proinflammatory and thus promote atherosclerosis. ApoAI posttranslational modification can have a large impact on its function. Myeloperoxidase modification of apoAI impairs its function as a cholesterol acceptor, and the molecular changes induced by myeloperoxidase have been studied in detail. These studies provide the basis for the development of an oxidant-resistant form of apoAI and clinical measures of HDL modification and dysfunction, which may be useful as a treatment criterion.

Proteomics of Apolipoproteins and Associated Proteins From Plasma High-Density Lipoproteins [ATVB In Focus]

Proteomics studies have extended the list of identified apolipoproteins and associated proteins present in HDL and its subclasses. These proteins appear to cluster around specific functions related to lipid metabolism, inflammation, the immune system, hormone-binding, hemostasis, and antioxidant properties. Small studies suggest that there are substantial differences between the HDL proteome from cardiovascular disease patients and that from controls. Furthermore, dyslipidemia therapy shifts the HDL proteome from patients toward the profile observed in healthy controls. In addition, the proteome of HDL and LDL from patients with insulin resistance and peripheral atherosclerosis show significant differences with that of matched healthy controls. The proteome of HDL and LDL density subclasses have apolipoproteins and associated proteins profiles that suggest subclass-specific functions. However, proteomics studies of lipoproteins are few and small and should be interpreted with caution. Nonetheless rapid technical progress in proteomic platforms suggest that soon analysis time will be reduced and precise measurement of identified proteins will be possible. This, combined with controlled purification steps of HDL and its subclasses should provide further information about proteins involved in the particles postulated spectrum of functions, including those believed to be atheroprotective.

Structure and Function of HDL Mimetics [ATVB In Focus]

HDL mimetics have been constructed from a number of peptides and proteins with varying structures, all of which bind lipids found in HDL. HDL mimetics containing a peptide or protein have been constructed with as few as 4 and as many as 243 amino acid residues. Some HDL mimetics have been constructed with lipid but without a peptide or protein component. Some HDL mimetics promote cholesterol efflux, some have been shown to have a remarkable ability to bind oxidized lipids compared to human apolipoprotein A-I (apoA-I). Many of these peptides have been shown to have antiinflammatory properties. Based on studies in a number of animal models and in early human clinical trials, HDL mimetics appear to have promise as diagnostic and therapeutic agents.

High-Density Lipoprotein-Based Contrast Agents for Multimodal Imaging of Atherosclerosis [ATVB In Focus]

Lipoproteins, natural nanoparticles, have a well-recognized biological role and are highly suitable as a platform for delivering imaging agents. The ease with which both the exterior and interior of the particles can be modified permits the creation of multifunctional nanoparticles for imaging as well as the delivery of therapeutics. Importantly, their endogenous nature may make them biocompatible and biodegradable and allows them to avoid the recognition of the reticuloendothelial system. In particular, high-density lipoproteins (HDL) are of interest, because of their small size they can easily cross the endothelium and penetrate the underlying tissue. We summarize here the progress in establishing HDL as a vector for delivering a variety of diagnostically active materials to vulnerable atherosclerotic plaques in mouse models of atherosclerosis. By loading various types of image-enhancing compounds into either the core or surface of HDL, they can be visualized by different imaging modalities (MRI, CT, optical). By rerouting of HDL away from plaque macrophages, imaging of biological processes in diseases besides atherosclerosis may also be achieved.

Correlation Between Carotid Intimal/Medial Thickness and Atherosclerosis: A Point of View From Pathology [Brief Reviews]

A widely adopted surrogate for predicting rates of cardiovascular events involves measure of carotid intimal-medial thickness (CIMT) by B mode ultrasound, a technique available since the mid 1980s. The value of this modality remains in its ability to noninvasively assess cardiovascular risk beyond traditional factors identified by the Framingham risk score, and it is among the few available techniques for monitoring the effectiveness of pharmacotherapy on plaques. There are, however, existing limitations to this methodology. Perhaps the most important distinction is that IM thickness measurements are generally acquired in the common carotid artery, whereas advanced atherosclerotic disease occurs predominantly downstream in the internal carotid. Moreover, primary contributors to IM thickening are age and hypertension, which do not necessarily reflect the atherosclerotic process. Initiation of disease-related plaques begins as what is referred to as pathological intimal thickening; lesions characterized by the formation of lipid pools in the absence of a necrotic core. The eventual development of a necrotic core, however, is considered a key indicator of significant plaque advancement and recognized feature of lesion vulnerability. Necrotic cores are thought to arise from macrophage infiltration of lipid pools followed by secondary necrosis where defective clearance of debris, tissue disruption proteases, and intraplaque hemorrhage, likely contribute to its enlargement. Therefore, one of the primary limitations to CIMT is its inability to distinguish lesions with a necrotic core. Moreover, in most cases measures of plaque area or volume are generally considered better predictors of an inflammatory process consistent with atherosclerotic disease rather than intimal medial thickness.

The Value of Carotid Intima-Media Thickness for Predicting Cardiovascular Risk [Brief Reviews]

We reviewed prospective epidemiological data in the general population, mostly middle-aged to older persons, to determine the association of carotid intima-media thickness (CIMT) (assessed by B-mode ultrasonography) with cardiovascular risk. Reported risks were expressed as absolute (event risk per persons-years in subjects with a high CIMT) and relative (hazard ratio of high vs low CIMT). They were hardly comparable as the result of differences between the analyzed studies, including the site and procedure of CIMT measurement, the report of adjusted or unadjusted models, and the arbitrary cutoff point to evaluate the CIMTÆs ability to predict risk. Despite these heterogeneities, the following four main conclusions emerged: (1) CIMT was an independent but relatively modest (as judged by absolute risk) predictor of coronary heart disease (CHD); (2) CIMT was an independent predictor for stroke, slightly better than for CHD as judged by the relative risks of both events; (3) CIMT added little to the CHD prediction by risk factors, as judged by c statistic and receiver operating characteristic curve analysis (however, appropriate data for stroke on this important issue were lacking); and (4) the CHD prediction by CIMT was inferior to that by ultrasonography-assessed carotid plaque because plaque may be more representative of atherosclerosis than CIMT.

CD11c Expression in Adipose Tissue and Blood and Its Role in Diet-Induced Obesity [Integrative Physiology/Experimental Medicine]

Objective— To examine CD11c, a β2-integrin, on adipose tissue (AT) leukocytes and blood monocytes and its role in diet-induced obesity. Methods and Results— High-fat diet–induced obese C57BL/6 mice, CD11c-deficient mice, and obese humans were studied. CD11c, leukocytes, and chemokines/cytokines were examined in AT and/or blood by flow cytometry, RNase protection assay, quantitative polymerase chain reaction, or enzyme-linked immunosorbent assay. Obese C57BL/6 mice had increased CD11c in AT and blood compared with lean controls. CD11c messenger RNA positively correlated with monocyte chemoattractant protein 1 in human visceral AT. Obese humans with metabolic syndrome had a higher CD11c level on blood monocytes compared with lean humans. Low-fat diet–induced weight loss reduced blood monocyte CD11c in obese mice and humans. Mouse and human monocyte CD11c levels and mouse AT CD11c messenger RNA correlated with insulin resistance. CD11c deficiency in mice did not alter weight gain but decreased inflammation, evidenced by a lower T-cell number and reduced levels of major histocompatibility complex class II, C-C chemokine ligand 2 (CCL5), CCL4, and interferon in AT, and ameliorated insulin resistance and glucose intolerance associated with diet-induced obesity. Conclusions— Diet-induced obesity increased CD11c in both AT and blood in mice and humans. CD11c plays an important role in T-cell accumulation and activation in AT, and contributes to insulin resistance associated with obesity.

Natural Killer T Cells Are Involved in Adipose Tissues Inflammation and Glucose Intolerance in Diet-Induced Obese Mice [Integrative Physiology/Experimental Medicine]

Background— Macrophage and lymphocyte infiltration in adipose tissue may contribute to the pathogenesis of obesity-mediated metabolic disorders. Natural killer T (NKT) cells, which integrate proinflammatory cytokines, have been demonstrated in the atherosclerotic lesions and in visceral adipose tissue. Objective— To determine whether NKT cells are involved in glucose intolerance and adipose tissue inflammation in diet-induced obese mice. Methods and Results— To determine whether NKT cells are involved in the development of glucose intolerance, male β2-microglobulin knockout (KO) mice lacking NKT cells and C57BL/6J (wild-type) mice were fed with a high-fat diet (HFD) for 13 weeks. Body weight and visceral obesity were comparable between wild-type and KO mice. However, macrophage infiltration was reduced in adipose tissue and glucose intolerance was significantly ameliorated in KO mice. To further confirm that NKT cells are involved in these abnormalities, -galactosylceramide, 0.1 µg/g body weight, which specifically activates NKT cells, was administered after 13 weeks of HFD feeding. -Galactosylceramide significantly exacerbated glucose intolerance and macrophage infiltration as well as cytokine gene expression in adipose tissue. Conclusion— NKT cells play a crucial role in the development of adipose tissue inflammation and glucose intolerance in diet-induced obesity.

Leptin Enhances the Potency of Circulating Angiogenic Cells Via Src Kinase and Integrin {alpha}v{beta}5: Implications for Angiogenesis in Human Obesity [Integrative Physiology/Experimental Medicine]

Objective— To investigate the capacity of the adipokine leptin to promote angiogenesis by modulating the function of circulating angiogenic cells (CACs). Methods and Results— In vitro, leptin specifically promoted CAC adhesion to tubular endothelial structures and migration along outgrowing sprouts of endothelial cells. In vivo, stimulation of CACs with leptin increased their capacity to promote new vessel formation in the chorioallantoic membrane of chicken embryos and to improve neovascularization of ischemic murine hind limbs. These effects required the phosphorylation of vβ5 integrins, which depended on the interaction of leptin with its receptor ObR, and on Janus kinase (JAK) 2– and phospholipase C (PLC) -mediated activation of Src kinase. Protein tyrosine phosphatase 1B, a negative regulator of leptin signaling, was overexpressed in CACs from obese, hyperleptinemic individuals, and this was associated with insensitivity of CACs to the angiogenic effects of leptin. Weight loss (by 30±15 kg) normalized protein tyrosine phosphatase 1B expression in CACs and restored their responsiveness to leptin. A similar dose-dependent response was found after incubation of CACs from obese subjects with a protein tyrosine phosphatase 1B inhibitor ex vivo. Conclusion— Our results point to the ObR–Src kinase–vβ5 cross talk as a distinct novel component of the network of specific interactions between integrins and cytokine receptors in angiogenesis.

Loss of Endothelial Tie1 Receptor Impairs Lymphatic Vessel Development-Brief Report [Integrative Physiology/Experimental Medicine]

Objective— Studies of Tie1 gene-targeted embryos have demonstrated loss of blood vessel integrity, but the relevance of Tie1 in lymphatic vasculature development is unknown. We tested the hypothesis that the swelling observed in Tie1 mutant embryos is associated with lymphatic vascular defects. Methods and Results— We could extend the survival of the Tie1-deficient embryos in the ICR background, which allowed us to study their lymphatic vessel development. At embryonic day (E) 14.5, the Tie1–/– embryos had edema and hemorrhages and began to die. Immunohistochemical analysis revealed that they have abnormal lymph sacs. Tie1–/– mutants were swollen already at E12.5 without signs of hemorrhage. Their lymph sacs were abnormally patterned, suggesting that lymphatic malformations precede the blood vascular defects. We generated mice with a conditional Cre/loxP Tie1neo locus and found that the homozygous Tie1neo/neo hypomorphic embryos survived until E15.5 with lymphatic malformations resembling those seen in the Tie1–/– mutants. Conclusion— Our data show that loss of Tie1 results in lymphatic vascular abnormalities that precede the blood vessel phenotype. These findings indicate that Tie1 is involved in lymphangiogenesis and suggest differential requirements for Tie1 signaling in the two vascular compartments.

Role of Hypoxia-Inducible Factor 1{alpha} in T Cells as a Negative Regulator in Development of Vascular Remodeling [Integrative Physiology/Experimental Medicine]

Background and Purpose— Recent studies have shown that the cellular immune response in the development of vascular remodeling modulates the resulting pathological alterations. We show that hypoxia-inducible factor 1 (Hif-1) (specifically expressed in T cells) is involved in the immune response to vascular remodeling that accompanies arteriosclerosis. Methods and Results— To study the role of T cells in the development of vascular remodeling, femoral arterial injury induced by an external vascular polyethylene cuff was examined in mice lacking Hif-1 (specifically in T cells). We found that cuff placement caused prominent neointimal hyperplasia of the femoral artery in Hif-1– (T-cell)–deficient mice compared with that in control mice and that infiltration of inflammatory cells at the adventitia was markedly increased in the mutant mice. Studies to clarify the mechanism of augmented vascular remodeling in the mutant mice showed enhanced production of cytokines by activated T cells and augmented antibody production in response to a T-dependent antigen in the mutant mice. Conclusions— The results of this study revealed that Hif-1 in T cells plays a crucial role in vascular inflammation and remodeling in response to cuff injury as a negative regulator of T cell–mediated immune response. Potential new therapeutic strategies that target Hif-1 are described.

Nuclear Factor of Activated T Cells Regulates Osteopontin Expression in Arterial Smooth Muscle in Response to Diabetes-Induced Hyperglycemia [Integrative Physiology/Experimental Medicine]

Objective— Hyperglycemia is a recognized risk factor for cardiovascular disease in diabetes. Recently, we reported that high glucose activates the Ca2+/calcineurin-dependent transcription factor nuclear factor of activated T cells (NFAT) in arteries ex vivo. Here, we sought to determine whether hyperglycemia activates NFAT in vivo and whether this leads to vascular complications. Methods and Results— An intraperitoneal glucose-tolerance test in mice increased NFATc3 nuclear accumulation in vascular smooth muscle. Streptozotocin-induced diabetes resulted in increased NFATc3 transcriptional activity in arteries of NFAT-luciferase transgenic mice. Two NFAT-responsive sequences in the osteopontin (OPN) promoter were identified. This proinflammatory cytokine has been shown to exacerbate atherosclerosis and restenosis. Activation of NFAT resulted in increased OPN mRNA and protein in native arteries. Glucose-induced OPN expression was prevented by the ectonucleotidase apyrase, suggesting a mechanism involving the release of extracellular nucleotides. The calcineurin inhibitor cyclosporin A or the novel NFAT blocker A-285222 prevented glucose-induced OPN expression. Furthermore, diabetes resulted in higher OPN expression, which was significantly decreased by in vivo treatment with A-285222 for 4 weeks or prevented in arteries from NFATc3–/– mice. Conclusions— These results identify a glucose-sensitive transcription pathway in vivo, revealing a novel molecular mechanism that may underlie vascular complications of diabetes.

Hypercholesterolemia Induces Side-Specific Phenotypic Changes and Peroxisome Proliferator-Activated Receptor-{gamma} Pathway Activation in Swine Aortic Valve Endothelium [Integrative Physiology/Experimental Medicine]

Background— The endothelium of healthy aortic valves expresses different phenotypes on the aortic and ventricular sides. On the aortic side, which is susceptible to aortic valve sclerosis, there is a balanced coexpression of both propathological and protective pathways. Side-specific global gene expression can address endothelial phenotype balance in early aortic valve sclerosis. Methods and Results— Adult male swine were fed a hypercholesterolemic or an isocaloric normal diet for 2-week and 6-month periods. Hypercholesterolemia induced localized lipid insudation confined to the aortic side of the leaflet. Transcript profiling of valve endothelial populations showed that the susceptible aortic side was more sensitive to 2-week hypercholesterolemia than the ventricular side (1,325 vs 87 genes were differentially expressed). However, greater sensitivity was not evidence of a dysfunctional phenotype. Instead, pathway analyses identified differential expression of caspase 3–, peroxisome proliferator–activated receptor –, TNF-–, and nuclear factor-B–related pathways that were consistent with a protective endothelial phenotype. This was confirmed at the protein level at 2 weeks and persisted at 6 months. Conclusions— In a large animal model at high spatial resolution, endothelium on the pathosusceptible side of the aortic valve leaflet is responsive to hypercholesterolemia. Transcript profiles indicative of a protective phenotype were induced and persisted on the side prone to aortic valve sclerosis.

Human Apolipoprotein A-II Determines Plasma Triglycerides by Regulating Lipoprotein Lipase Activity and High-Density Lipoprotein Proteome [Integrative Physiology/Experimental Medicine]

Introduction— Apolipoprotein (apo) A-II is the second most abundant high-density lipoprotein (HDL) apolipoprotein. We assessed the mechanism involved in the altered postprandial triglyceride-rich lipoprotein metabolism of female human apoA-II-transgenic mice (hapoA-II-Tg mice), which results in up to an 11-fold increase in plasma triglyceride concentration. The relationships between apoA-II, HDL composition, and lipoprotein lipase (LPL) activity were also analyzed in a group of normolipidemic women. Methods and Results— Triglyceride-rich lipoprotein catabolism was decreased in hapoA-II-Tg mice compared to control mice. This suggests that hapoA-II, which was mainly associated with HDL during fasting and postprandially, impairs triglyceride-rich lipoprotein lipolysis. HDL isolated from hapoA-II-Tg mice impaired bovine LPL activity. Two-dimensional gel electrophoresis, mass spectrometry, and immunonephelometry identified a marked deficiency in the HDL content of apoA-I, apoC-III, and apoE in these mice. In normolipidemic women, apoA-II concentration was directly correlated with plasma triglyceride and inversely correlated with HDL-to-apoC-II+apoE/apoC-III ratios. HDL-mediated induction of LPL activity was inversely correlated with apoA-II and directly correlated with HDL-to-apoC-II+apoE/apoC-III ratios. Purified hapoA-II displaced apoC-II, apoC-III, and apoE from human HDL2. Human HDL3 was, compared to HDL2, enriched in apoA-II but poorer in apoC-II, apoC-III, and apoE. Conclusion— ApoA-II plays a crucial role in triglyceride catabolism by regulating LPL activity, at least in part, through HDL proteome modulation.

Metabolism of Very-Low-Density Lipoprotein and Low-Density Lipoprotein Containing Apolipoprotein C-III and Not Other Small Apolipoproteins [Integrative Physiology/Experimental Medicine]

Objective— We aimed to clarify the influence of apolipoprotein C-III (apoCIII) on human apolipoprotein B metabolism. Methods and Results— We studied the kinetics of 4 very-low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), and low-density lipoprotein (LDL) types containing: (1) otherApos–CIII–: none of apoCIII, apoAII, apoCI, apoCII, or apoE; (2) otherApos+CIII–: no apoCIII but at least one of the others; (3) otherApos–CIII+: apoCIII, but not any others; and (4) otherApos+CIII+: apoCIII and at least one other. VLDL and IDL otherApos–CIII+ and otherApos–CIII– had similar rates of lipolytic conversion to smaller particles. However, light LDL otherApos–CIII+ compared with otherApos–CIII– had much faster conversion to dense LDL as did light LDL otherApos+CIII+ compared with otherApos+CIII–. VLDL and IDL otherApos–CIII+ had minimal direct removal from circulation, whereas VLDL and IDL otherApos+CIII–, rich in apoE, showed fast clearance. Lipoproteins in fraction otherApos+CIII+ also rich in apoE had very low clearance. Conclusions— The results suggest that apoCIII strongly inhibits hepatic uptake of VLDL and IDL overriding the opposite influence of apoE when both are present. The presence of apoCIII on dense VLDL is not associated with slow conversion to IDL, a lipoprotein lipase-dependent process; but when on light LDL, apoCIII is associated with enhanced conversion to dense LDL, a process involving hepatic lipase.

The 5A Apolipoprotein A-I Mimetic Peptide Displays Antiinflammatory and Antioxidant Properties In Vivo and In Vitro [Integrative Physiology/Experimental Medicine]

Objectives— The apolipoprotein (apo)A-I mimetic peptide 5A is highly specific for ATP-binding cassette transporter (ABC)A1-mediated cholesterol efflux. We investigated whether the 5A peptide shares other beneficial features of apoA-I, such as protection against inflammation and oxidation. Methods— New Zealand white rabbits received an infusion of apoA-I, reconstituted high-density lipoprotein (HDL) containing apoA-I ([A-I]rHDL), or the 5A peptide complexed with phospholipids (1-palmitoyl-2-linoleoyl phosphatidylcholine [PLPC]), before inserting a collar around the carotid artery. Human coronary artery endothelial cells (HCAECs) were incubated with (A-I)rHDL or 5A/PLPC before stimulation with tumor necrosis factor . Results— ApoA-I, (A-I)rHDL, and 5A/PLPC reduced the collar-mediated increase in (1) endothelial expression of cell adhesion molecules vascular cell adhesion molecule-1 and intercellular adhesion molecule-1; (2) production, as well as the expression of the Nox4 catalytic subunits of the NADPH oxidase; and (3) infiltration of circulating neutrophils into the carotid intima–media. In HCAECs, both 5A/PLPC and (A-I)rHDL inhibited tumor necrosis factor-–induced intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 expression, as well as the nuclear factor B signaling cascade and production. The effects of the 5A/PLPC complex were no longer apparent in HCAECs knocked down for ABCA1. Conclusion— Like apoA-I, the 5A peptide inhibits acute inflammation and oxidative stress in rabbit carotids and HCAECs. In vitro, the 5A peptide exerts these beneficial effects through interaction with ABCA1.

Pharmacological Inhibition of C-C Chemokine Receptor 2 Decreases Macrophage Infiltration in the Aortic Root of the Human C-C Chemokine Receptor 2/Apolipoprotein E-/- Mouse: Magnetic Resonance Imaging Assessment [Integrative Physiology/Experimental Medicine]

Purpose— This study assessed the pharmacological effect of a novel selective C-C chemokine receptor (CCR) 2 antagonist (GSK1344386B) on monocyte/macrophage infiltration into atherosclerotic plaque using magnetic resonance imaging (MRI) in an atherosclerotic mouse model. Methods and Results— Apolipoprotein E–/– mice expressing human CCR2 were fed a Western diet (vehicle group) or a Western diet plus10 mg/kg per day of GSK1344386B (GSK1344386B group). After the baseline MRI, mice were implanted with osmotic pumps containing angiotensin II, 1000 ng/kg per minute, to accelerate lesion formation. After five weeks of angiotensin II administration, mice received ultrasmall superparamagnetic iron oxide, an MRI contrast agent for the assessment of monocyte/macrophage infiltration to the plaque, and underwent imaging. After imaging, mice were euthanized, and the heart and aorta were harvested for ex vivo MRI and histopathological examination. After 5 weeks of dietary dosing, there were no significant differences between groups in body or liver weight or plasma cholesterol concentrations. An in vivo MRI reflected a decrease in ultrasmall superparamagnetic iron oxide contrast agent uptake in the aortic arch of the GSK1344386B group (P<0.05). An ex vivo MRI of the aortic root also reflected decreased ultrasmall superparamagnetic iron oxide uptake in the GSK1344386B group and was verified by absolute iron analysis (P<0.05). Although there was no difference in aortic root lesion area between groups, there was a 30% reduction in macrophage area observed in the GSK1344386B group (P<0.05). Conclusion— An MRI was used to noninvasively assess the decreased macrophage content in the atherosclerotic plaque after selective CCR2 inhibition.

Absence of Myeloid COX-2 Attenuates Acute Inflammation but Does Not Influence Development of Atherosclerosis in Apolipoprotein E Null Mice [Integrative Physiology/Experimental Medicine]

Objective— The role of myeloid cell cyclooxygenase-2 (COX-2) in the progression of atherosclerosis has not been clearly defined. Methods and Results— We investigated the role of COX-2 expressed in the myeloid lineage in the development of atherosclerosis using a myeloid-specific COX-2–/– (COX-2–M/–M) mouse on a hyperlipidemic apolipoprotein (apo) E–/– background (COX-2–M/–M/apoE–/–). Myeloid COX-2 depletion resulted in significant attenuation of acute inflammation corresponding with decreased PGE2 levels in an air pouch model. COX-2 depletion in myeloid cells did not influence development of atherosclerosis in COX-2–M/–M/apoE–/– when compared to apoE–/– littermates fed either chow or western diets. The unanticipated lack of contribution of myeloid COX-2 to the development atherosclerosis is not attributable to altered maintenance, differentiation, or mobilization of myeloid and lymphoid populations. Moreover, myeloid COX-2 depletion resulted in unaltered serum prostanoid levels and cellular composition of atherosclerotic lesions of COX-2–M/–M/apoE–/– mice. Conclusions— Our results suggest that COX-2 expression in myeloid cells, including macrophages, does not influence the development of atherosclerosis in mice.

Hemorphin 7 Reflects Hemoglobin Proteolysis in Abdominal Aortic Aneurysm [Integrative Physiology/Experimental Medicine]

Objective— In human abdominal aortic aneurysm, the accumulation of blood-derived cells and proteases within the mural thrombus plays a pivotal role in the evolution toward vessel wall rupture. We sought to identify peptides released from abdominal aortic aneurysm specimens, characterized by an intraluminal thrombus. Methods and Results— Intraluminal thrombus samples were analyzed by differential proteomics, using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry. A 1309-Da peptide was detected in larger amounts in the newly formed luminal thrombus layer relative to older layers. It was identified as being LVVYPWTQRF (known as LVV-Hemorphin 7), a peptide generated from hemoglobin by cathepsin D. By immunohistochemical analysis, we showed that Hemorphin 7 (H7) colocalizes with cathepsin D and cathepsin G in the luminal layer of the intraluminal thrombus. In vitro, cathepsin G was able to generate H7 peptides at pH 7.4, whereas cathepsin D was only active in acidic conditions. Finally, H7 peptides were shown to be increased 3- to 4-fold in sera of abdominal aortic aneurysm patients relative to controls, and their levels were positively correlated with the volume of the thrombus. Conclusion— Our results suggest that circulating H7 peptides may reflect proteolysis of hemoglobin in the aneurysmal intraluminal thrombus and may be used as a biological marker of pathological vascular remodeling.

Altered Plasma Fibrin Clot Properties Are Associated With In-Stent Thrombosis [Integrative Physiology/Experimental Medicine]

Objectives— We sought to investigate whether patients with in-stent thrombosis (IST) display altered plasma fibrin clot properties. Methods and Results— We studied 47 definite IST patients, including 15 with acute, 26 subacute and 6 late IST, and 48 controls matched for demographics, cardiovascular risk factors, concomitant treatment and angiographic/stent parameters. Plasma clot permeability (Ks), which indicates a pore size, turbidity (lag phase, indicating the rate of fibrin clot formation, Absmax, maximum absorbance of a fibrin gel, reflecting the fiber thickness), lysis time (t50%) and maximum rate of d-dimer release from clots (D-Drate) were determined 2 to 73 (median 14.7) months after IST. Patients with IST had 21% lower Ks, 14% higher Absmax, 11% lower D-Drate, 30% longer t50% (all P<0.0001) and 5% shorter lag phase compared to controls (P=0.042). There were no correlations between clot variables and the time of IST or that from IST to blood sampling. Multiple regression analysis showed that Ks (odds ratio=0.36 per 0.1 µm2, P<0.001), D-Drate (odds ratio=0.16 per 0.01 mg/L/min, P<0.001) and stent length (odds ratio=1.1 per 1 mm, P=0.043) were independent predictors of IST (R2=0.58, P<0.001). Conclusions— IST patients tend to form dense fibrin clots resistant to lysis, and altered plasma fibrin clot features might contribute to the occurrence of IST.

L-4F Alters Hyperlipidemic (But Not Healthy) Mouse Plasma to Reduce Platelet Aggregation [Integrative Physiology/Experimental Medicine]

Background and Purpose— Hyperlipidemia is associated with platelet hyperreactivity. We hypothesized that L-4F, an apolipoprotein A-I mimetic peptide, would inhibit platelet aggregation in hyperlipidemic mice. Methods and Results— Injecting L-4F into apolipoprotein E (apoE)–null and low-density lipoprotein receptor–null mice resulted in a significant reduction in platelet aggregation in response to agonists; however, there was no reduction in platelet aggregation after injection of L-4F into wild-type (WT) mice. Consistent with these results, injection of L-4F into apoE-null mice prolonged bleeding time; the same result was not found in WT mice. Incubating L-4F in vitro with apoE-null platelet-rich plasma also resulted in decreased platelet aggregation. However, incubating washed platelets from either apoE-null or WT mice with L-4F did not alter aggregation. Compared with WT mice, unstimulated platelets from apoE-null mice contained significantly more 12-hydroxy 5,8,10,14-eicosatetraenoic acid, thromboxane A2, and prostaglandins D2 and E2. In response to agonists, platelets from L-4F–treated apoE-null mice formed significantly less thromboxane A2, prostaglandins D2 and E2, and 12-hydroxy 5,8,10,14-eicosatetraenoic acid. Conclusion— By binding plasma-oxidized lipids that cause platelet hyperreactivity in hyperlipidemic mice, L-4F decreases platelet aggregation.

Peroxisome Proliferator-Activated Receptor-{gamma} Coactivator 1-{alpha} Overexpression Prevents Endothelial Apoptosis by Increasing ATP/ADP Translocase Activity [Cell Biology/Signaling]

Objective— Fatty acids increase reactive oxygen species generation and cell apoptosis in endothelial cells. The peroxisome proliferator-activated receptor- coactivator 1- (PGC-1) is a transcriptional coactivator that increases mitochondrial biogenesis and fatty acid oxidation in various cells. This study was undertaken to investigate the possible preventive effect of PGC-1 on endothelial apoptosis and its molecular mechanism. Methods and Results— Treatment with linoleic acid in cultured human aortic endothelial cells increased reactive oxygen species generation and cell apoptosis. These effects appeared to be mediated by increases in cytosolic fat metabolites, ie, fatty acyl CoA, diacylglycerol, and ceramide, and consequent decreases in ATP/ADP translocase activity of adenine nucleotide translocator. Adenoviral overexpression of PGC-1 prevented linoleic acid-induced increases in reactive oxygen species generation and cell apoptosis in human aortic endothelial cells by increasing fatty acid oxidation, decreasing diacylglycerol and ceramide, and increasing ATP/ADP translocase activity. In isolated aorta, PGC-1 overexpression prevented linoleic acid-induced decrease in endothelium-dependent vasorelaxation, and this effect was abolished by adenine nucleotide translocator1 shRNA. Conclusions— PGC-1 regulates reactive oxygen species generation and apoptosis in endothelial cells by increasing fatty acid oxidation and enhancing ATP/ADP translocase activity. Measures to increase PGC-1 expression or ATP/ADP translocase activity in vascular cells may aid in the prevention or treatment of atherosclerosis.

Claudin-5 as a Novel Estrogen Target in Vascular Endothelium [Cell Biology/Signaling]

Objective— Estrogens have multiple effects on vascular physiology and function. In the present study, we look for direct estrogen target genes within junctional proteins. Methods and Results— We use murine endothelial cell lines of brain and heart origin, which express both subtypes of estrogen receptor, ER and ERβ. Treatment of these cells with 17β-estradiol (E2) led to an increase in transendothelial electric resistance and a most prominent upregulation of the tight junction protein claudin-5 expression. A significant increase of claudin-5 promoter activity, mRNA, and protein levels was detected in cells from both vascular beds. In protein lysates and in immunoreactions on brain sections from ovariectomized E2-treated mice, we noticed an increase in claudin-5 protein and mRNA content. Treatment of cells with a specific ERβ agonist, diarylpropionitrile, revealed the same effect as E2 stimulation. Moreover, we detected significantly lower claudin-5 mRNA and protein content in ERβ knockout mice. Conclusions— We describe claudin-5 as a novel estrogen target in vascular endothelium and show in vivo (brain endothelium) and in vitro (brain and heart endothelium) effects of estrogen on claudin-5 levels. The estrogen-induced increase in junctional protein levels may lead to an improvement in vascular structural integrity and barrier function of vascular endothelium.

Human Sickle Cell Blood Modulates Endothelial Heme Oxygenase Activity: Effects on Vascular Adhesion and Reactivity [Cell Biology/Signaling]

Objective— Sickle cell disease (SCD) is characterized by extensive hemolysis, increased cellular adhesion, and vaso-occlusion. Tissues from sickle patients express heme oxygenase-1 (HO-1), the enzyme that degrades free heme/hemoglobin to the signaling molecule carbon monoxide, and the antioxidants biliverdin/bilirubin. Here, we examined the HO response in endothelial cells exposed to human sickle blood and determined whether this response is beneficial for SCD. Methods and Results— We measured HO activity in human and bovine aortic endothelial cells incubated with human sickle or normal blood. Sickle blood increased HO activity, which was enhanced by hypoxia and was caused mainly by the red cell components of sickle blood. Oxidized hemoglobin was higher in sickle blood and increased markedly over time. Interestingly, HO activity correlated inversely with patients’ hemoglobin levels and positively with bilirubin and lactate dehydrogenase. HO-1 induction, exogenous biliverdin, or carbon monoxide markedly decreased adhesion of sickle blood to the endothelium, and sickle red cells partially inhibited relaxation mediated by carbon monoxide in isolated aortas. Conclusions— Our results highlight important associations between SCD and HO byproducts, which may counteract vascular complications of SCD.

Phospholipase A2-Modified Low-Density Lipoprotein Activates Macrophage Peroxisome Proliferator-Activated Receptors [Cell Biology/Signaling]

Background and Purpose— Peroxisome proliferator–activated receptors (PPARs) are ligand-activated transcription factors modulating metabolic and inflammatory responses of phagocytes to stimuli such as fatty acids and their metabolites. We studied the role of PPARs in macrophages exposed to low-density lipoprotein (LDL) modified by secretory phospholipase A2 (PLA). Methods and Results— By analyzing PPAR ligand–binding domain luciferase reporter activation, we observed that PLA-LDL transactivates PPAR and PPAR, but not PPAR. We confirmed that PLA-LDL induced PPAR response element reporter activation by endogenous PPAR and PPAR in human THP-1 macrophages. By using THP-1 cells with a stable knockdown of PPAR and PPAR, we showed that PLA-LDL–activated PPAR altered macrophage gene expression related to lipid metabolism and lipid droplet formation. Although PPAR/ silencing did not affect cholesterol and triglyceride accumulation in PLA-LDL–treated macrophages, PPAR activation by PLA-LDL attenuated macrophage inflammatory gene expression induced by interferon and lipopolysaccharide. Conclusions— PPAR activation by PLA-LDL does not influence lipid accumulation in PLA-LDL–treated macrophages. However, it attenuates macrophage inflammatory responses, thus contributing to an anti-inflammatory cell phenotype.

Vitamin D Receptor Activators Induce an Anticalcific Paracrine Program in Macrophages: Requirement of Osteopontin [Cell Biology/Signaling]

Objectives— Vascular calcification is highly correlated with morbidity and mortality, and it is often associated with inflammation. Vitamin D may regulate vascular calcification and has been associated with cardiovascular survival benefits. Methods and Results— We developed a macrophage/smooth muscle cell (SMC) coculture system and examined the effects of vitamin D receptor activators (VDRA), calcitriol and paricalcitol, on SMC matrix calcification. We found that treatment of SMC alone with VDRA had little effect on phosphate-induced SMC calcification in vitro. However, coculture with macrophages promoted SMC calcification, and this was strikingly inhibited by VDRA treatment. Several VDRA-induced genes, including bone morphogenetic protein-2 (BMP2), tumor necrosis factor-, and osteopontin, were identified as candidate paracrine factors for the protective effect of VDRA. Of these, osteopontin was further investigated and found to contribute significantly to the inhibitory actions of VDRA on calcification in macrophage/SMC cocultures. Conclusion— The ability of VDRA to direct a switch in the paracrine phenotype of macrophages from procalcific to anticalcific may contribute to their observed cardiovascular survival benefits.

Obesity Genotype Score and Cardiovascular Risk in Women With Type 2 Diabetes Mellitus [Clinical and Population Studies]

Objective— To investigate the associations between obesity-predisposing genetic variants, cardiovascular biomarkers, and cardiovascular disease (CVD) risk in women with preexisting type 2 diabetes mellitus. Methods and Results— We genotyped polymorphisms at nine established obesity loci in 1,395 women with diabetes from the Nurses’ Health Study: 449 women developed CVD, and 946 women did not develop CVD. A genetic risk score (GRS) was derived by summing risk alleles for each individual. Four polymorphisms (rs9939609 [FTO], rs11084753 [KCTD15], rs10838738 [MTCH2], and rs10938397 [GNPDA2]) showed nominally significant associations with CVD. The GRS combining all obesity loci was linearly related to CVD risk (P = 0.013 for trend). The odds ratio was 1.08 per risk allele (95% confidence interval, 1.02–1.15; P = 0.01) after adjustment for body mass index and other conventional risk factors. Women with the highest quartile of GRS had 53% (95% confidence interval, 6%–122%) increased CVD risk, compared with those in the lowest quartile of GRS (P = 0.024). In addition, a higher GRS was associated with lower adiponectin levels (P = 0.02). Further adjustment for body mass index and other covariates did not change the association (P = 0.006). A higher GRS was also correlated with lower levels of high-density lipoprotein (P = 0.01). Conclusion— Obesity-predisposing variants may jointly affect CVD risk among women with diabetes.

Conjoint Effects of Serum Calcium and Phosphate on Risk of Total, Cardiovascular, and Noncardiovascular Mortality in the Community [Clinical and Population Studies]

Objective— Hyperphosphatemia is a cardiovascular risk factor in patients with chronic kidney disease. Relations of circulating calcium (Ca) and phosphorus (Pi) to long-term mortality risk in the community require further investigation. Methods and Results— Associations of serum Ca and Pi to mortality were evaluated in a community-based cohort of 2176 men (mean age, 50.1 years). During follow-up (median, 29.8 years), 1009 men died, and 466 of these deaths resulted from cardiovascular causes. In Cox proportional hazards models, serum Pi and [CaxPi] were independent predictors of total mortality (hazard ratio per SD, 1.06; 95% CI, 1.01–1.12; P=0.03; 1.07; 95% CI, 1.01–1.12; P=0.01) and cardiovascular mortality (1.10; 95% CI, 1.02–1.18; P=0.01; 1.10; 95% CI, 1.03–1.19; P=0.008). Serum Ca was associated with risk of total mortality (1.08; 95% CI, 1.01–1.16; P=0.02) and noncardiovascular mortality (1.10; 95% CI, 1.01–1.21; P=0.04). Results were consistent after multivariate adjustments in subsamples of individuals with estimated glomerular filtration rate >90 mL/min and low-to-normal serum Ca and Pi. Conclusion— Circulating Ca and Pi levels are associated with risks of total, cardiovascular, and noncardiovascular mortality in the community, and their conjoint effects are additive. Additional studies are warranted to evaluate whether Ca and Pi are modifiable risk factors in the general population.

Postmenopausal Hormone Therapy and Risk of Idiopathic Venous Thromboembolism: Results From the E3N Cohort Study [Clinical and Population Studies]

Objective— Oral estrogen therapy increases venous thromboembolism risk among postmenopausal women. Although recent data showed transdermal estrogens may be safe with respect to thrombotic risk, the impact of the route of estrogen administration and concomitant progestogens is not fully established. Methods and Results— We used data from the E3N French prospective cohort of women born between 1925 and 1950 and biennially followed by questionnaires from 1990. Study population consisted of 80 308 postmenopausal women (average follow-up: 10.1 years) including 549 documented idiopathic first venous thromboembolism. Hazard ratios (HR) and 95% confidence intervals (CI) were estimated using Cox proportional models. Compared to never-users, past-users of hormone therapy had no increased thrombotic risk (HR=1.1; 95% CI: 0.8 to 1.5). Oral not transdermal estrogens were associated with increased thrombotic risk (HR=1.7; 95% CI: 1.1 to 2.8 and HR=1.1; 95% CI: 0.8 to 1.8; homogeneity: P=0.01). The thrombotic risk significantly differed by concomitant progestogens type (homogeneity: P<0.01): there was no significant association with progesterone, pregnanes, and nortestosterones (HR=0.9; 95% CI: 0.6 to 1.5, HR=1.3; 95% CI: 0.9 to 2.0 and HR=1.4; 95% CI: 0.7 to 2.4). However, norpregnanes were associated with increased thrombotic risk (HR=1.8; 95% CI: 1.2 to 2.7). Conclusions— In this large study, we found that route of estrogen administration and concomitant progestogens type are 2 important determinants of thrombotic risk among postmenopausal women using hormone therapy. Transdermal estrogens alone or combined with progesterone might be safe with respect to thrombotic risk.

Genetic Association and Interaction Analysis of USF1 and APOA5 on Lipid Levels and Atherosclerosis [Clinical and Population Studies]

Objective— USF1 is a ubiquitous transcription factor governing the expression of numerous genes of lipid and glucose metabolism. APOA5 is a well-established candidate gene regulating triglyceride (TG) levels and has been identified as a downstream target of upstream stimulatory factor. No detailed studies about the effect of APOA5 on atherosclerotic lesion formation have been conducted, nor has its potential interaction with USF1 been examined. Methods and Results— We analyzed allelic variants of USF1 and APOA5 in families (n=516) ascertained for atherogenic dyslipidemia and in an autopsy series of middle-aged men (n=300) with precise quantitative measurements of atherosclerotic lesions. The impact of previously associated APOA5 variants on TGs was observed in the dyslipidemic families, and variant rs3135506 was associated with size of fibrotic aortic lesions in the autopsy series. The USF1 variant rs2516839, associated previously with atherosclerotic lesions, showed an effect on TGs in members of the dyslipidemic families with documented coronary artery disease. We provide preliminary evidence of gene-gene interaction between these variants in an autopsy series with a fibrotic lesion area in the abdominal aorta (P=0.0028), with TGs in dyslipidemic coronary artery disease subjects (P=0.03), and with high-density lipoprotein cholesterol (P=0.008) in a large population cohort of coronary artery disease patients (n=1065) in which the interaction for TGs was not replicated. Conclusion— Our findings in these unique samples reinforce the roles of APOA5 and USF1 variants on cardiovascular phenotypes and suggest that both genes contribute to lipid levels and aortic atherosclerosis individually and possibly through epistatic effects.

Identification of Two Common Variants Contributing to Serum Apolipoprotein B Levels in Mexicans [Clinical and Population Studies]

Background and Purpose— Although the Mexican population has a high predisposition to dyslipidemias and premature coronary artery disease, this population is underinvestigated for the genetic factors conferring the high susceptibility. This study attempted to determine these genetic factors. Methods and Results— First, we investigated apolipoprotein B (apoB) levels in Mexican extended families with familial combined hyperlipidemia using a two-step testing strategy. In the screening step, we screened 5721 single-nucleotide polymorphisms (SNPs) for linkage signals with apoB. In the test step, we analyzed the 130 SNPs residing in regions of suggestive linkage signals for association with apoB. We identified significant associations with two SNPs (ie, rs1424032 [P=6.07x10–6] and rs1349411 [P=2.72x10–4]) that surpassed the significance level for the number of tests performed in the test step (P<3.84x10–4). Second, these SNPs were tested for replication in Mexican hyperlipidemic case-control samples. The same risk alleles as in the families with familial combined hyperlipidemia were significantly associated (P<0.05) with apoB in the case-control samples. The rs1349411 resides near the apoB messenger RNA editing enzyme (APOBEC1) involved in the processing of APOB messenger RNA in the small intestine. The rs1424032 resides in a highly conserved noncoding region predicted to function as a regulatory element. Conclusion— We identified two novel variants, rs1349411 and rs1424032, for serum apoB levels in Mexicans.

Atorvastatin Inhibits gp91phox Circulating Levels in Patients With Hypercholesterolemia [Clinical and Population Studies]

Objective— The inhibition of oxidative stress is among the most relevant pleiotropic effects of statins. The mechanism by which statins exert their antioxidant effect in vivo is still undefined. NADPH oxidase is among the most important sources of reactive oxygen species involved in atherosclerotic disease. Methods/Results— We developed an ELISA to evaluate serum levels of soluble-gp91phox, the catalytic core of phagocyte NADPH oxidase. In a cross-sectional study performed in 30 hypercholesterolemic patients and in 20 controls, serum soluble-gp91phox and urinary isoprostane, a marker of oxidative stress, were measured. The 2 variables were also measured in hypercholesterolemic patients, randomized to diet (n=15), or diet plus atorvastatin (10 mg daily, n=15) and followed for 30 days. Compared to controls, hypercholesterolemic patients had higher and significantly correlated (R=0.71; P<0.001) serum soluble-gp91phox (P<0.001) and urinary isoprostanes (P<0.001). After follow-up, the statin-allocated group showed a significant reduction of soluble-gp91phox (–33%, P<0.01), that paralleled that of isoprostanes (–37%, P<0.01) and cholesterol (–25%, P<0.01). The diet-allocated group showed only a weak reduction of cholesterol. Conclusion— Our study demonstrates that statins exert an antioxidant effect via inhibition of soluble gp91phox expression.

The Year's Successes in Failure: Circulation Research Takes a Look at the Key Research Developments of 2009 That Are Providing Hope in the Field of Heart Failure [News and Views]

The New Circulation Research: A Manifesto [Inaugural Editorials]

Vascular Dendritic Cells as Gatekeepers of Lipid Accumulation Within Nascent Atherosclerotic Plaques [Editorials]

Targeting Sarcoplasmic Reticulum Ca2+ Uptake to Improve Heart Failure: Hit or Miss [Editorials]

Another Broken Heart: Loss of Lamina-Associated Polypeptide 2{alpha} Causes Systolic Dysfunction [Editorials]

Be Still, My Beating Heart: Never! [Reviews]

Development of the Pacemaker Tissues of the Heart [Reviews]

Pacemaker and conduction system myocytes play crucial roles in initiating and regulating the contraction of the cardiac chambers. Genetic defects, acquired diseases, and aging cause dysfunction of the pacemaker and conduction tissues, emphasizing the clinical necessity to understand the molecular and cellular mechanisms of their development and homeostasis. Although all cardiac myocytes of the developing heart initially possess pacemaker properties, the majority differentiates into working myocardium. Only small populations of embryonic myocytes will form the sinus node and the atrioventricular node and bundle. Recent efforts have revealed that the development of these nodal regions is achieved by highly localized suppression of working muscle differentiation, and have identified transcriptional repressors that mediate this process. This review will summarize and reflect new experimental findings on the cellular origin and the molecular control of differentiation and morphogenesis of the pacemaker tissues of the heart. It will also shed light on the etiology of inborn and acquired errors of nodal tissues.

Mapping Cardiac Pacemaker Circuits: Methodological Puzzles of the Sinoatrial Node Optical Mapping [Reviews]

Abstract: Historically, milestones in science are usually associated with methodological breakthroughs. Likewise, the advent of electrocardiography, microelectrode recordings and more recently optical mapping have ushered in new periods of significance of advancement in elucidating basic mechanisms in cardiac electrophysiology. As with any novel technique, however, data interpretation is challenging and should be approached with caution, as it cannot be simply extrapolated from previously used methodologies and with experience and time eventually becomes validated. A good example of this is the use of optical mapping in the sinoatrial node (SAN): when microelectrode and optical recordings are obtained from the same site in myocardium, significantly different results may be noted with respect to signal morphology and as a result have to be interpreted by a different set of principles. Given the rapid spread of the use of optical mapping, careful evaluation must be made in terms of methodology with respect to interpretation of data gathered by optical sensors from fluorescent potential-sensitive dyes. Different interpretations of experimental data may lead to different mechanistic conclusions. This review attempts to address the origin and interpretation of the "double component" morphology in the optical action potentials obtained from the SAN region. One view is that these 2 components represent distinctive signals from the SAN and atrial cells and can be fully separated with signal processing. A second view is that the first component preceding the phase 0 activation represents the membrane currents and intracellular calcium transients induced diastolic depolarization from the SAN. Although the consensus from both groups is that ionic mechanisms, namely the joint action of the membrane and calcium automaticity, are important in the SAN function, it is unresolved whether the double-component originates from the recording methodology or represents the underlying physiology. This overview aims to advance a common understanding of the basic principles of optical mapping in complex 3D anatomic structures.

Protein Acetylation in the Cardiorenal Axis: The Promise of Histone Deacetylase Inhibitors [Reviews]

Abstract: Acetylation of histone and nonhistone proteins provides a key mechanism for controlling signaling and gene expression in heart and kidney. Pharmacological inhibition of protein deacetylation with histone deacetylase (HDAC) inhibitors has shown promise in preclinical models of cardiovascular and renal disease. Efficacy of HDAC inhibitors appears to be governed by pleiotropic salutary actions on a variety of cell types and pathophysiological processes, including myocyte hypertrophy, fibrosis, inflammation and epithelial-to-mesenchymal transition, and occurs at compound concentrations below the threshold required to elicit toxic side effects. We review the roles of acetylation/deacetylation in the heart and kidney and provide rationale for extending HDAC inhibitors into clinical testing for indications involving these organs.

Protein S-Nitrosylation and Cardioprotection [Reviews]

Abstract: Nitric oxide (NO) plays an important role in the regulation of cardiovascular function. In addition to the classic NO activation of the cGMP-dependent pathway, NO can also regulate cell function through protein S-nitrosylation, a redox dependent, thiol-based, reversible posttranslational protein modification that involves attachment of an NO moiety to a nucleophilic protein sulfhydryl group. There are emerging data suggesting that S-nitrosylation of proteins plays an important role in cardioprotection. Protein S-nitrosylation not only leads to changes in protein structure and function but also prevents these thiol(s) from further irreversible oxidative/nitrosative modification. A better understanding of the mechanism regulating protein S-nitrosylation and its role in cardioprotection will provide us new therapeutic opportunities and targets for interventions in cardiovascular diseases.

Human Skeletal Muscle Drug Transporters Determine Local Exposure and Toxicity of Statins [Molecular Medicine]

Rationale: The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, or statins, are important drugs used in the treatment and prevention of cardiovascular disease. Although statins are well tolerated, many patients develop myopathy manifesting as muscle aches and pain. Rhabdomyolysis is a rare but severe toxicity of statins. Interindividual differences in the activities of hepatic membrane drug transporters and metabolic enzymes are known to influence statin plasma pharmacokinetics and risk for myopathy. Interestingly, little is known regarding the molecular determinants of statin distribution into skeletal muscle and its relevance to toxicity. Objective: We sought to identify statin transporters in human skeletal muscle and determine their impact on statin toxicity in vitro. Methods and Results: We demonstrate that the uptake transporter OATP2B1 (human organic anion transporting polypeptide 2B1) and the efflux transporters, multidrug resistance–associated protein (MRP)1, MRP4, and MRP5 are expressed on the sarcolemmal membrane of human skeletal muscle fibers and that atorvastatin and rosuvastatin are substrates of these transporters when assessed using a heterologous expression system. In an in vitro model of differentiated, primary human skeletal muscle myoblast cells, we demonstrate basal membrane expression and drug efflux activity of MRP1, which contributes to reducing intracellular statin accumulation. Furthermore, we show that expression of human OATP2B1 in human skeletal muscle myoblast cells by adenoviral vectors increases intracellular accumulation and toxicity of statins and such effects were abrogated when cells overexpressed MRP1. Conclusions: These results identify key membrane transporters as modulators of skeletal muscle statin exposure and toxicity.

Roles for Endoplasmic Reticulum-Associated Degradation and the Novel Endoplasmic Reticulum Stress Response Gene Derlin-3 in the Ischemic Heart [Cellular Biology]

Rationale: Stresses, such as ischemia, impair folding of nascent proteins in the rough endoplasmic reticulum (ER), activating the unfolded protein response, which restores efficient ER protein folding, thus leading to protection from stress. In part, the unfolded protein response alleviates ER stress and cell death by increasing the degradation of terminally misfolded ER proteins via ER-associated degradation (ERAD). ERAD is increased by the ER stress modulator, activating transcription factor (ATF)6, which can induce genes that encode components of the ERAD machinery. Objective: Recently, it was shown that the mouse heart is protected from ischemic damage by ATF6; however, ERAD has not been studied in the cardiac context. A recent microarray study showed that the Derlin-3 (Derl3) gene, which encodes an important component of the ERAD machinery, is robustly induced by ATF6 in the mouse heart. Methods and Results: In the present study, activated ATF6 induced Derl3 in cultured cardiomyocytes, and in the heart, in vivo. Simulated ischemia (sI), which activates ER stress, induced Derl3 in cultured myocytes, and in an in vivo mouse model of myocardial infarction, Derl3 was also induced. Derl3 overexpression enhanced ERAD and protected cardiomyocytes from simulated ischemia–induced cell death, whereas dominant-negative Derl3 decreased ERAD and increased simulated ischemia–induced cardiomyocyte death. Conclusions: This study describes a potentially protective role for Derl3 in the heart, and is the first to investigate the functional consequences of enhancing ERAD in the cardiac context.

Rad As a Novel Regulator of Excitation-Contraction Coupling and {beta}-Adrenergic Signaling in Heart [Cellular Biology]

Rationale: Rad (Ras associated with diabetes) GTPase, a monomeric small G protein, binds to Cavβ subunit of the L-type Ca2+ channel (LCC) and thereby regulates LCC trafficking and activity. Emerging evidence suggests that Rad is an important player in cardiac arrhythmogenesis and hypertrophic remodeling. However, whether and how Rad involves in the regulation of excitation–contraction (EC) coupling is unknown. Objective: This study aimed to investigate possible role of Rad in cardiac EC coupling and β-adrenergic receptor (βAR) inotropic mechanism. Methods and Results: Adenoviral overexpression of Rad by 3-fold in rat cardiomyocytes suppressed LCC current (ICa), [Ca2+]i transients, and contractility by 60%, 42%, and 38%, respectively, whereas the "gain" function of EC coupling was significantly increased, due perhaps to reduced "redundancy" of LCC in triggering sarcoplasmic reticulum release. Conversely, 70% Rad knockdown by RNA interference increased ICa (50%), [Ca2+]i transients (52%) and contractility (58%) without altering EC coupling efficiency; and the dominant negative mutant RadS105N exerted a similar effect on ICa. Rad upregulation caused depolarizing shift of LCC activation and hastened time-dependent LCC inactivation; Rad downregulation, however, failed to alter these attributes. The Na+/Ca2+ exchange activity, sarcoplasmic reticulum Ca2+ content, properties of Ca2+ sparks and propensity for Ca2+ waves all remained unperturbed regardless of Rad manipulation. Rad overexpression, but not knockdown, negated βAR effects on ICa and Ca2+ transients. Conclusion: These results establish Rad as a novel endogenous regulator of cardiac EC coupling and βAR signaling and support a parsimonious model in which Rad buffers Cavβ to modulate LCC activity, EC coupling, and βAR responsiveness.

GTP Cyclohydrolase I Phosphorylation and Interaction With GTP Cyclohydrolase Feedback Regulatory Protein Provide Novel Regulation of Endothelial Tetrahydrobiopterin and Nitric Oxide [Cellular Biology]

Rationale: GTP cyclohydrolase I (GTPCH-1) is the rate-limiting enzyme involved in de novo biosynthesis of tetrahydrobiopterin (BH4), an essential cofactor for NO synthases and aromatic amino acid hydroxylases. GTPCH-1 undergoes negative feedback regulation by its end-product BH4 via interaction with the GTP cyclohydrolase feedback regulatory protein (GFRP). Such a negative feedback mechanism should maintain cellular BH4 levels within a very narrow range; however, we recently identified a phosphorylation site (S81) on human GTPCH-1 that markedly increases BH4 production in response to laminar shear. Objective: We sought to define how S81 phosphorylation alters GTPCH-1 enzyme activity and how this is modulated by GFRP. Methods and Results: Using prokaryotically expressed proteins, we found that the GTPCH-1 phospho-mimetic mutant (S81D) has increased enzyme activity, reduced binding to GFRP and resistance to inhibition by GFRP compared to wild-type GTPCH-1. Using small interfering RNA or overexpressing plasmids, GFRP was shown to modulate phosphorylation of GTPCH-1, BH4 levels, and NO production in human endothelial cells. Laminar, but not oscillatory shear stress, caused dissociation of GTPCH-1 and GFRP, promoting GTPCH-1 phosphorylation. We also found that both GTPCH-1 phosphorylation and GFRP downregulation prevents endothelial NO synthase uncoupling in response to oscillatory shear. Finally oscillatory shear was associated with impaired GTPCH-1 phosphorylation and reduced BH4 levels in vivo. Conclusions: These studies provide a new mechanism for regulation of endothelial GTPCH-1 by its phosphorylation and interplay with GFRP. This mechanism allows for escape from GFRP negative feedback and permits large amounts of BH4 to be produced in response to laminar shear stress.

Cyclooxygenase-2-Dependent Prostacyclin Formation and Blood Pressure Homeostasis: Targeted Exchange of Cyclooxygenase Isoforms in Mice [Integrative Physiology]

Rationale: Cyclooxygenase (COX)-derived prostanoids (PGs) are involved in blood pressure homeostasis. Both traditional nonsteroidal antiinflammatory drugs (NSAIDs) that inhibit COX-1 and COX-2 and NSAIDs designed to be selective for inhibition of COX-2 cause sodium retention and elevate blood pressure. Objective: To elucidate the role of COX-2 in blood pressure homeostasis using COX-1>COX-2 mice, in which the COX-1 expression is controlled by COX-2 regulatory elements. Methods and Results: COX-1>COX-2 mice developed systolic hypertension relative to wild types (WTs) on a high-salt diet (HSD); this was attenuated by a PGI2 receptor agonist. HSD increased expression of COX-2 in WT mice and of COX-1 in COX-1>COX-2 mice in the inner renal medulla. The HSD augmented in all strains urinary prostanoid metabolite excretion, with the exception of the major PGI2 metabolite that was suppressed on regular chow and unaltered by the HSD in both mutants. Furthermore, inner renal medullary expression of the receptor for PGI2, but not for other prostanoids, was depressed by HSD in WT and even more so in both mutant strains. Increasing osmolarity augmented expression of COX-2 in WT renal medullary interstitial cells and again the increase in formation of PGI2 observed in WTs was suppressed in cells derived from both mutants. Intramedullary infusion of the PGI2 receptor agonist increased urine volume and sodium excretion in mice. Conclusions: These studies suggest that dysregulated expression of the COX-2 dependent, PGI2 biosynthesis/response pathway in the renal inner renal medulla undermines the homeostatic response to a HSD. Inhibition of this pathway may contribute directly to the hypertensive response to NSAIDs.

Lamina-Associated Polypeptide 2{alpha} Loss Impairs Heart Function and Stress Response in Mice [Integrative Physiology]

Rationale: Lamina-associated polypeptide (LAP)2 is a mammalian chromatin-binding protein that interacts with a fraction of A-type lamins in the nuclear interior. Because mutations in lamins and LAP2 lead to cardiac disorders in humans, we hypothesized that these factors may play important roles in heart development and adult tissue homeostasis. Objective: We asked whether the presence of LAP2 was required for normal cardiac function. Methods and Results: To study the molecular mechanisms of the disease, we analyzed heart structure and function in complete and conditional Lap2–/– mice as well as Lap2–/–/Mdx mutants. Unlike conditional deletion of LAP2 in late embryonic striated muscle, its complete knockout caused systolic dysfunction in young mice, accompanied by sporadic fibrosis in old animals, as well as deregulation of major cardiac transcription factors GATA4 and myocyte enhancer factor 2c. Activation of compensatory pathways, including downregulation of β-adrenergic receptor signaling, resulted in reduced responsiveness of the myocardium to chronic β-adrenergic stimulation and stalled the progression of LAP2-deficient hearts from hypertrophy toward cardiac failure. Dystrophin deficiency in an Mdx background resulted in a transient rescue of the Lap2–/– phenotype. Conclusions: Our data suggest a novel role of LAP2 in the maintenance of cardiac function under normal and stress conditions.

Phospholamban Ablation Rescues Sarcoplasmic Reticulum Ca2+ Handling but Exacerbates Cardiac Dysfunction in CaMKII{delta}C Transgenic Mice [Integrative Physiology]

Rationale: We previously showed that transgenic mice expressing Ca2+/calmodulin-dependent protein kinase II C (CaMKII-TG) develop dilated cardiomyopathy associated with increased ryanodine receptors (RyR2) phosphorylation, enhanced sarcoplasmic reticulum (SR) Ca2+ leak and lowering of SR Ca2+ load. We hypothesized that phospholamban (PLN) ablation would restore SR Ca2+ load and prevent the decreased ventricular contractility, dilation and mortality seen in CaMKII-TG. Objective: Our objectives were to generate CaMKII-TG mice lacking PLN, determine whether the maladaptive effects of cardiac CaMKIIC expression were corrected, and establish the mechanistic basis for these changes. Methods and Results: CaMKII-TG were crossed with PLN knockout (PLN-KO) mice to generate KO/TG mice. Myocytes from wild type (WT), CaMKII-TG, PLN-KO and KO/TG were compared. The decreased SR Ca2+ load and twitch Ca2+ transients seen in CaMKII-TG were normalized in KO/TG. Surprisingly the heart failure phenotype was exacerbated, as indicated by increased left ventricular dilation, decreased ventricular function, increased apoptosis and greater mortality. In KO/TG myocytes SR Ca2+ sparks and leak were significantly increased, presumably because of the combined effects of restored SR Ca2+ load and RyR2 phosphorylation. Mitochondrial Ca2+ loading was increased in cardiomyocytes from KO/TG versus WT or CaMKII-TG mice and this was dependent on elevated SR Ca2+ sparks. Cardiomyocytes from KO/TG showed poor viability, improved by inhibiting SR Ca2+ release and mitochondrial Ca2+ loading. Conclusions: Normalizing cardiomyocyte SR Ca2+ loading in the face of elevated CaMKII and RyR2 phosphorylation leads to enhanced SR Ca2+ leak and mitochondrial Ca2+ elevation, associated with exacerbated cell death, heart failure and mortality.

Vascular Smooth Muscle Cell Apoptosis Induces Interleukin-1-Directed Inflammation: Effects of Hyperlipidemia-Mediated Inhibition of Phagocytosis [Integrative Physiology]

Rationale: Atherosclerosis is characterized by lipid accumulation in the vessel wall, inflammation, and both macrophage and vascular smooth muscle cell (VSMC) apoptosis. However, whereas VSMC apoptosis in mice with established atherosclerotic plaques or hyperlipidemia increases serum levels of the proatherogenic cytokines monocyte chemotactic protein (MCP)-1, tumor necrosis factor , and interleukin (IL)-6, the link between hyperlipidemia, apoptosis and inflammation, and the mechanisms by which apoptotic cells promote inflammation in atherosclerosis are unknown. Objective: To determine whether hyperlipidemia affects apoptotic cell clearance, and identify the molecular pathways downstream of VSMC apoptosis that may promote inflammation. Methods and Results: We find that human VSMCs are potent and efficient phagocytes of apoptotic human VSMCs, but phagocytosis is significantly reduced by oxidized low-density lipoprotein in vitro or hyperlipidemia in vivo. Necrotic human aortic VSMCs release IL-1, which induces IL-6 and MCP-1 production from viable human VSMCs in vitro. In contrast, secondary necrotic VSMCs release both IL-1 and caspase-activated IL-1β, augmenting IL-6 and MCP-1 production. Conditionally inducing VSMC apoptosis in situ in hyperlipidemic SM22-hDTR/ApoE–/– mice to levels seen in human plaques increases serum MCP-1, tumor necrosis factor , and IL-6, which is prevented by blocking IL-1. Conclusions: We conclude that VSMC necrosis releases IL-1, whereas secondary necrosis of apoptotic VSMCs releases both IL-1 and β. IL-1 from necrotic VSMCs induces the surrounding viable VSMCs to produce proinflammatory cytokines. Thus, failed clearance of apoptotic VSMCs caused by hyperlipidemia in vivo may promote the increased serum cytokines and chronic inflammation associated with atherosclerosis.

Brain-Selective Overexpression of Human Angiotensin-Converting Enzyme Type 2 Attenuates Neurogenic Hypertension [Integrative Physiology]

Rationale: Angiotensin converting enzyme type 2 (ACE2) is a new member of the brain renin-angiotensin system, that might be activated by an overactive renin-angiotensin system. Objective: To clarify the role of central ACE2 using a new transgenic mouse model with human (h)ACE2 under the control of a synapsin promoter, allowing neuron-targeted expression in the central nervous system. Methods and Results: Syn-hACE2 (SA) transgenic mice exhibit high hACE2 protein expression and activity throughout the brain. Baseline hemodynamic parameters (telemetry), autonomic function, and spontaneous baroreflex sensitivity (SBRS) were not significantly different between SA mice and nontransgenic littermates. Brain-targeted ACE2 overexpression attenuated the development of neurogenic hypertension (Ang II infusion: 600 ng/kg per minute for 14 days) and the associated reduction of both SBRS and parasympathetic tone. This prevention of hypertension by ACE2 overexpression was reversed by blockade of the Ang-(1-7) receptor (d-Ala7-Ang-[1-7]; 600 ng/kg per minute). Brain angiotensin II type 2 (AT2)/AT1 and Mas/AT1 receptor ratios were significantly increased in SA mice. They remained higher following Ang II infusion but were dramatically reduced after Ang-(1-7) receptor blockade. ACE2 overexpression resulted in increased NOS and NO levels in the brain, and prevented the Ang II–mediated decrease in NOS expression in regions modulating blood pressure regulation. Conclusions: ACE2 overexpression attenuates the development of neurogenic hypertension partially by preventing the decrease in both SBRS and parasympathetic tone. These protective effects might be mediated by enhanced NO release in the brain resulting from Mas and AT2 receptor upregulation. Taken together, our data highlight the compensatory role of central ACE2 and its potential benefits as a therapeutic target for neurogenic hypertension.

Resident Intimal Dendritic Cells Accumulate Lipid and Contribute to the Initiation of Atherosclerosis [Integrative Physiology]

Rationale: Atherosclerosis is an inflammatory disease in which leukocytes and oxidatively modified lipids accumulate in the arterial intima. Previously, we showed that dendritic cells (DCs) accumulate preferentially in regions predisposed to atherosclerosis in the normal murine aortic intima. The function of these cells in atherogenesis is unknown. Objective: Our goal was to determine the role of resident intimal DCs in the initiation of atherosclerosis. Methods and Results: En face immunostaining of nascent atherosclerotic lesions in low-density lipoprotein receptor–deficient (Ldlr–/–) mice fed a cholesterol-rich diet for 5 or 10 days demonstrated that foam cells expressed DC markers CD11c, 33D1, and major histocompatibility complex class II. Transmission electron microscopy revealed that the majority of intimal lipid was intracellular. The role of resident intimal DCs in lesion formation was verified by their conditional depletion using transgenic mice expressing the simian diphtheria toxin receptor in CD11c+ cells. A single injection of diphtheria toxin depleted intimal CD11c+ DCs by >98% within 24 hours, with 25% and 75% recovery at 1 and 3 weeks, respectively. When bred onto the Ldlr–/– background, intimal DC depletion with diphtheria toxin during 5 days of lesion formation reduced the intimal lipid area by 55% relative to undepleted controls. Transmission electron microscopy revealed few foam cells in DC-depleted mice and abundant accumulation of subendothelial extracellular lipid. Conclusions: Induction of hypercholesterolemia in mice triggers rapid ingestion of lipid by resident intimal DCs, which initiate nascent foam cell lesion formation.