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A 63-year old Caucasian female with history of aortic valve replacement and CAD with prior drug-eluting stent placement (DES) to proximal LAD and LCX, presented as a hospital transfer with recurrent episodes of chest pain. Chest discomfort was described as severe chest pressure, radiating to bilateral upper extremities. Episodes were not associated with significant exertion and frequently occurred at night/early morning. Of note, the patient had undergone coronary angiography at the outside hospital, revealing no in-stent restenosis or new coronary lesions. Soon after the patient’s arrival in the CCU, she developed profound chest pressure, accompanied by hypotension, diaphoresis and nausea. Her 12-lead ECG revealed 2-mm ST-elevation in aVR, accompanied by ST-depression in inferior, anterior, and apical leads. Initial high-sensitivity (hs)-troponin T was 26 ng/L. She was given sublingual nitroglycerin and was started on a nitroglycerin and nicardipine infusion, with resolution of symptoms after ~30 minutes, followed by normalization of hs-troponin T. A transthoracic echocardiogram did not reveal focal left ventricular (LV) wall motion abnormalities, mild LV hypertrophy was present. Given the patients’ presentation with no in-stent restenosis or new lesions, frequent episodes of chest pain-not related to exertion with occurrence at nighttime and early morning, her presentation was deemed to be consistent with epicardial coronary vasospasm. Subsequently, the patients’ anti-anginal regimen was uptitrated to include extended-release nitrates, calcium-channel blockade, and I Na blockade. Unfortunately, she continued to have frequent episodes of profound angina, accompanied by hypotension, and significant ischemic ECG changes. This prompted us to pursue bilateral sympathectomy. Subsequently, the patient had resolution of prior symptoms. She was continued on an anti-vasospastic regimen, consisting of extended release nitrates and calcium-channel blockade. This case represents the challenging management of medication-resistant epicardial coronary artery vasospasm. As previously described, sympathectomy remains an effective therapeutic option for management in these difficult and life-threatening situations.

Introduction: Vasoplegia is a severe complication after cardiac surgery and is associated with impaired clinical outcome. Pre-operative heart failure (HF) is considered an independent predictor of post-operative vasoplegia. We hypothesize that HF patients are more susceptible to vasoplegia due to altered vascular responsiveness. In this study, vasoresponsiveness in patients undergoing cardiac surgery for HF is investigated. Methods: A prospective, observational study was conducted at Leiden University Medical Center. We included patients with HF (N=18) and without HF (N=18) who underwent cardiac surgery on cardiopulmonary bypass. Vasoresponsiveness was assessed at 4 different timepoints: 1) before induction, 2) after induction, 3) after cessation of cardiopulmonary bypass and 4) on the first postoperative day. The vascular response was recorded as change in systemic vascular resistance (SVR) after the administration of phenylephrine (bolus 2 μg/kg). Results: Thirty-six patients were included [67 (61-71) years, 78% male]. Vascular responsiveness was significantly attenuated in patients with HF compared to controls. The response to phenylephrine was already diminished at baseline in HF patients and was almost abolished after cessation of cardiopulmonary bypass (Figure). Roughly the same pattern of vasoresponsiveness was found when excluding patients that received norepinephrine. Moreover, HF patients required a significantly higher amount of noradrenaline [169.80 (IQR 14.77-318.97) ug/kg] compared to controls [3.61 (IQR 0-41.60) ug/kg] to maintain similar SVR during the first 24h postoperatively. Conclusions: The vascular responsiveness is altered in patients with HF and this might explain the higher prevalence of vasoplegia in this patient population.

Introduction: Coronary vasospasm is a side effect of 5-FU (fluorouracil). Beyond switching chemotherapy regimens, there are no established treatments for vasospasm. Limited data exist regarding the efficacy of calcium channel blockers (CCBs) and/or nitrates with 5-FU vasospasm. Methods: We conducted a retrospective analysis of all patients at Massachusetts General Hospital who were diagnosed with 5-FU coronary vasospasm. As part of institutional practice, those referred to cardio-oncology receive pre-treatment with vasospasm therapies (either combination [nitrates and CCBs] or single agent therapy [nitrates or CCBs]) prior to re-challenge with 5-FU. Our primary outcomes were the safety and effectiveness of this cardiac pre-treatment approach. Results: Among 7,711 patients who received 5-FU from Jan 2001 to Jul 2017, 86 (1.11%) developed coronary vasospasm. Overall 64 patients continued to receive subsequent 5-FU, while 22 stopped. Of the 64 who continued, 60 were referred to cardio-oncology and prescribed CCBs and/or nitrates prior to subsequent 5-FU. Of these 60, 52% (31/60) received both nitrates and CCBs, 20% (12/60) received CCBs alone, and 28% (17/60) received nitrates alone, while 4 continued 5-FU without cardiac pre-treatment. Chest pain (without a myocardial infarct) recurred in 11.7% (7/60) among those who received cardiac pre-treatment vs. 50% (2/4) among those who did not (P=0.033). When those who stopped were compared to those who continued 5-FU (with cardiac pre-treatment), the cardiovascular profile, stage and type of cancer were similar. As compared to patients who stopped 5-FU, those who continued after nitrates/CCBs had improved progression free (HR 0.56, P=0.076 [95% CI 0.30-1.06]) and overall (HR 0.29, P Conclusion: In the largest report of 5-FU associated vasospasm, re-challenge after pre-treatment with CCBs and nitrates was safe and effective, thus allowing patients to receive additional 5-FU.

Introduction: Recent evidences suggest that sex and estrogen can influence cerebrovascular reactivity. We investigated the role of prostanoids and the estrogen receptor (ER) ex vivo in isolated middle cerebral arteries from male (M-MCA) and female (F-MCA) rats. Methods: MCA segments isolated from 20-week-old male and female Sprague-Dawley rats were mounted on a wire Multi Myograph (DMT) to measure isometric tone. Acetylcholine (ACh) was used to assess endothelium integrity. Concentration response curves to the thromboxane analog U46619 (U4, 10 -11 -10 -5 M) were performed in arteries intact or pre-treated with indomethacin (I,10 -5 M) or the ER antagonist G15 (G, 10 -6 M). Data were acquired with Powerlab 8 (ADInstruments) and recorded with LabChart v8 (ADInstruments). Response to K + was expressed in mN/mm whereas U4 maximal contraction as percent of maximal response to 75mM K + (%K MAX ) and sensitivity as pD 2 (-LogEC 50 ). Results: M-MCA (n=10) and F-MCA (n=13) displayed similar optimal diameters (M vs. F; 214±12 vs. 218±5 μm, p>0.05) and ACh-dependent relaxation (M vs. F; 71±11 vs. 78±10 % pre-constricted tone; 7.3±0.4 vs. 7.2±0.2, p>0.05). A greater contraction to K + was observed in M-MCA (M vs. F; 1±0.2 vs. 0.6±0.1 mN/mm, pvs. F; 116±6 vs. 109±10 %K MAX ) and sensitivity (M vs. F; 7.2±0.2 vs. 7.3±0.2) to U4. Pre-incubation with Indomethacin lowered maximal response and sensitivity (M vs. M-I; 116±6 vs. 97±5%K MAX, pvs. 6.8±0.1, pvs. F-I; 109±9 vs. 113±7%K MAX ; 7.3±0.2 vs. 7.3±0.2). Pre-treatment with G15 increased U4 sensitivity in M-MCA (M vs. M-G; 7.3±0.2 vs. 7.8±0.2, pvs. 7.4±0.3). Conclusions: Our data demonstrate sex differences in thromboxane-dependent contraction of MCA in middle-aged rats. Hormonal changes during the estrous cycle may contribute to a greater variability of F-MCA responses.

Insufficient sleep, defined as chronic short sleep duration (A receptor antagonist) and ACh + BQ-123 in both groups and after the exercise intervention in the short sleepers. As expected, forearm vasodilator responses to ACh were lower (20%; PA receptor blockade. These results indicate that regular aerobic exercise can reverse the negative influence of insufficient sleep on endothelial vasomotor function, independent of changes in nightly sleep duration.

Background: The perivascular adipose tissue (PVAT) surrounding vessels constitutes a distinct functional integral layer of the vasculature required to preserve vascular tone under physiological conditions. However, there is little information on the relationship between PVAT and blood pressure regulation, including its potential contributions to circadian blood pressure variation. Methods: Using unique brown adipocyte–specific aryl hydrocarbon receptor nuclear translocator-like protein 1 (Bmal1) and angiotensinogen knockout mice, we determined the vasoactivity of homogenized PVAT in aortic rings and how brown adipocyte peripheral expression of Bmal1 and angiotensinogen in PVAT regulates the amplitude of diurnal change in blood pressure in mice. Results: We uncovered a peripheral clock in PVAT and demonstrated that loss of Bmal1 in PVAT reduces blood pressure in mice during the resting phase, leading to a superdipper phenotype. PVAT extracts from wild-type mice significantly induced contractility of isolated aortic rings in vitro in an endothelium-independent manner. This property was impaired in PVAT from brown adipocyte–selective Bmal1-deficient (BA-Bmal1-KO) mice. The PVAT contractile properties were mediated by local angiotensin II, operating through angiotensin II type 1 receptor–dependent signaling in the isolated vessels and linked to PVAT circadian regulation of angiotensinogen. Indeed, angiotensinogen mRNA and angiotensin II levels in PVAT of BA-Bmal1-KO mice were significantly reduced. Systemic infusion of angiotensin II, in turn, reduced Bmal1 expression in PVAT while eliminating the hypotensive phenotype during the resting phase in BA-Bmal1-KO mice. Angiotensinogen, highly expressed in PVAT, shows circadian expression in PVAT, and selective deletion of angiotensinogen in brown adipocytes recapitulates the phenotype of selective deletion of Bmal1 in brown adipocytes. Furthermore, angiotensinogen is a transcriptional target of Bmal1 in PVAT. Conclusions: These data indicate that local Bmal1 in PVAT regulates angiotensinogen expression and the ensuing increase in angiotensin II, which acts on smooth muscle cells in the vessel walls to regulate vasoactivity and blood pressure in a circadian fashion during the resting phase. These findings will contribute to a better understanding of the cardiovascular complications of circadian disorders, alterations in the circadian dipping phenotype, and cross-talk between systemic and peripheral regulation of blood pressure.

Articles, see p 2451 and p 2468 Pulmonary arterial hypertension (PAH) is an obstructive vasculopathy in which vascular cells proliferate too rapidly or are resistant to apoptosis. PAH is also characterized by increases in inflammation, vasoconstriction, and fibrosis. Ultimately, these vascular changes increase right ventricular afterload, leading to heart failure and death. Pulmonary vascular cells from patients with PAH have adaptations that ensure that their high rates of proliferation are not thwarted by energy limitations, mitochondria-mediated apoptosis, or inadequate rates of mitotic fission.1 Histological examination reveals the consequences of this proliferative diathesis, including intimal hyperplasia and plexiform lesions, medial hypertrophy, and adventitial fibrosis. The resulting endothelial dysfunction favors thrombosis and vasoconstriction; the hyperplasia of pulmonary artery smooth muscle cells (PASMCs) favors vasoconstriction; and the adventitial fibrosis results in stiff, noncompliant arteries. Two articles in this issue of Circulation describe a metabolic adaptation that is good for the abnormal cell but bad for the patient, namely a shift in glucose metabolism called the Warburg phenomenon. Otto Warburg, a German Nobel Laureate, described the predilection of cancer to support its rapid growth by a reliance on glycolysis despite an abundance of available oxygen. Warburg metabolism is a failure of 2 fundamental pathways: glucose metabolism and mitochondrial oxygen sensing. Glycolysis and glucose oxidation are normally coupled, meaning they occur in proportion to each other. The final products of glycolysis are ATP and pyruvate. Pyruvate enters the mitochondria via the mitochondrial pyruvate transporter and acts as the substrate for pyruvate dehydrogenase (PDH), which regulates glucose oxidation and supplies acetyl CoA to the Krebs cycle (Figure). Warburg metabolism represents a state of uncoupled glycolysis in that glycolysis is disproportionately elevated compared with mitochondrial pyruvate utilization and glucose oxidation. Warburg metabolism allows the cell a source of energy (glycolysis) while suppressing mitochondrial apoptosis and stimulating …

Article, see p 1509 Heart failure (HF) remains one of the largest health problems in the Western world that caused an estimated 37.6 million HF or HF-related hospitalizations in the United States between 2001 and 2009.1 After an insult to the myocardium, cardiac output drops, which will lead to impairment of the systemic circulation and ultimately signs and symptoms of HF. Indeed, tissue and organ perfusion generally decreases in HF, despite the fact that local compensatory mechanisms are activated. To maintain sufficient perfusion of vital organs and peripheral tissues, vasoconstrictor mechanisms in the systemic arterial bed become active in response to stimulation of the sympathetic nervous and renin-angiotensin system.2 This has paved the way for the use of arterial vasodilators in HF because long-term systemic vasoconstriction leads to left ventricular hypertrophy and failure and ultimately refractory HF. As a consequence of left-sided HF, pulmonary congestion may occur. Previously, pulmonary congestion was considered mainly a marker of severity of left-sided HF, but there is increasing evidence that pulmonary congestion may also be actively contributing to the HF syndrome and impair prognosis.3 Along with previous attempts to indirectly monitor signs related to pulmonary congestion in HF,4 there is a (renewed) interest in more directly measuring pulmonary artery (PA) pressures in HF. Moreover, elevated PA pressures have been shown to strongly associate with an adverse prognosis and increased risk for hospitalization in patients with HF (Figure).5 For this reason, the pulmonary circulation as a therapeutic target in HF has gained increasing interest in recent years. Figure. Association between pulmonary pressures and outcomes in outpatients with heart failure …

Introduction: The efficacy of epinephrine (Ep) administration to cardiopulmonary arrest (CPA) patients is still controversial, and correlation between plasma Ep levels of and prognosis in CPA patients is unclear. Hypothesis: We hypothesized that the more EP is administered, the higher the rate of ROSC becomes in CPA patients. The purpose of this study is to evaluate whether a dose of Ep influenced the prognosis in CPA patients. Methods: This was a prospective, observational clinical study, approved by the ethics committee of Gunma University Hospital (IRB #14-13). CPA patients transferred to our hospital were enrolled prospectively between July 2014 and July 2017. The levels of Ep in the plasma were measured using blood samples immediately obtained at the time of arrival to our hospital. Patients were divided into the four groups based on the prehospital administered dose of EP; patients without prehospital Ep administration (group Z), prehospital administration of 1mg Ep (group O), 2mg Ep (group T) and 4mg Ep (group F). The plasma Ep levels and the conditions of resuscitation were compared among those groups. The IBM SPSS Statistics 25 software was used for statistical analysis. Data are shown as median (Q1, Q3). The Kruskal-Wallis or chi-square tests were used to conduct the comparisons among four groups. P Results: We analyzed 150 patients. There were 96 patients in group Z, 38 in group O, 11 in group T and 5 in group F. There was no significant difference in prehospital resuscitation time among 4 groups, and the level of Ep in the plasma obtained immediately after arrival at hospital was the highest in the group F with a significant difference {286.0 (247.2, 424.9) ng/ml in group F, 244.0 (22.1, 620.3) ng/ml in group T, 1.6 (0.5, 4.5) ng/ml in group O and 2.0 (0.4, 4.5) ng/ml in group Z, respectively, p Conclusions: Our results suggest that the prehospital administered dose of Ep is not relates with the acquisition of ROSC in patients with CPA.

Introduction: Acute myocardial infarction (AMI) is a major cause of cardiac arrest. Coronary vasospasm has been suggested to play a significant role in the acute phase of MI. Vasospastic angina (VSA) is characterized by spontaneous attacks of chest pain, caused by transient coronary vasospasm. Severe coronary vasospasm might cause AMI, fatal arrhythmia, and subsequent cardiac arrest, however, there have been limited studies about the clinical characteristics and prognosis of patients with VSA regarding cardiac arrest. This study investigated the clinical features and outcomes of VSA compared to other MI in patients survived out-of-hospital cardiac arrest (OHCA). Method and Results: We analyzed the data of adult OHCA patients from the prospectively collected post cardiac arrest syndrome (PCAS) registry of 3 urban academic hospitals from December 2013 to December 2017. During the study period, 721 OHCAs were collected, including 18 diagnosed with VSA and 133 with other MI as primary cause of cardiac arrest. Compared to other MI, more patients achieved pre-hospital return of spontaneous circulation (ROSC) in VSA group (72.2% vs. 47.4%, p =0.041). The advanced cardiovascular life support (ACLS) time (median, 4.0 min vs. 10.0 min; p =0.008) was shorter in VSA group. The SOFA (5.0 vs. 9.0, p =0.007) and APACHE II score (20.5 vs. 25.0, p =0.007) at admission were lower in VSA group. The proportion of the patients who underwent coronary angiography (CAG) did not differ between the two groups and there were no patients with a newly diagnosed coronary obstructive lesion in CAG performed after ROSC. More patients in VSA group had good neurologic outcome at hospital discharge (77.8% vs. 47.3%, p =0.022), 28 days (76.5% vs. 48.4%, p =0.038), and 6 months (76.5% vs. 47.7%, p =0.037). All-cause mortality rate at in-hospital (5.6% vs. 42.1%, p =0.002) and 28 days (11.2% vs. 44.5%, p =0.009) were lower in VSA group. In the subgroup analysis of patients who underwent CAG, more patients with VSA showed lower mortality rate at in-hospital (0.0% vs. 35.4%, p =0.003) and 28 days (6.7% vs. 38.0%, p =0.019). Conclusion: In patients survived OHCA, patients with VSA more achieved pre-hospital ROSC and showed better neurologic outcome and lower mortality rate compared to those with other MI.

Introduction: Cerebral vasoconstriction in the post-resuscitation phase worsens neurological outcome. Nitric oxide (NO) plays important roles mediating vasodilatation and anti-apoptotic protection. We therefore designed an Au-polymersomes/S-nitrosoglutathione (Au-PLGA/GSNO) nanoparticle that can be triggered by ultrasound (US) to release NO, and investigated its roles in mitigating cerebral vasoconstriction and neuronal apoptosis post-CPR. Hypothesis: Spatiotemporally controlled, US-triggered NO release by Au-PLGA/GSNO improves post-CPR cerebral perfusion and confers anti-apoptotic neuroprotection. Methods: Using an established rat model of asphyxia cardiac arrest and CPR, Au-PLGA/GSNO (7500 PPM, 0.4 ml) was infused with simultaneous US (1 MHz) stimulation at the brain 10 min after ROSC. Brain tissue perfusion was continuously recorded by OxyFLO probe and cerebral vasculature videoed by CytoCam. The blood was sampled 2 h post-CPR for measurement of nitrate/nitrite, and the brain harvested for measurement of casepase-3, endothelial NO synthase (eNOS) and protein kinase B (Akt). In a subgroup the brain was harvested at 24 h for TUNEL stain. Results: After CPR, marked cerebral vasoconstriction was noted on CytoCam while brain perfusion significantly reduced to ~0.5 folds that of baseline. After Au-PLGA/GSNO infusion and US stimulation, cerebral vasoconstriction was ameliorated and the brain perfusion significantly enhanced ( P < 0.05 vs. CPR control). The plasma NO indicated by nitrate/nitrite 2 h post-CPR was significantly increased ( P < 0.01) while cleaved caspase-3/caspase-3 of the brain markedly reduced ( P < 0.001). TUNEL stain of the hippocampus CA1 and CA3 regions were also remarkably abrogated, suggesting anti-apoptotic neuroprotection. Specifically, the phosphorylated (p)-eNOS/eNOS and p-Akt/Akt were also increased ( P < 0.01 and 0.001, respectively), indicating reciprocating activation of Akt-eNOS signaling upstream of NO. Conclusion: Spatiotemporally controlled US-triggered NO release by Au-PLGA/GSNO mitigates cerebral vasoconstriction, improves brain perfusion and confers anti-apoptotic neuroprotection post-CPR via reciprocating Akt-eNOS-NO signaling.

It is estimated that >140 million people live above 2500 m in various regions of the world.1 There are many challenges to living at high altitude, but chronic exposure to alveolar hypoxia is prominent among them. Inspired Po2 falls from ≈150 mm Hg at sea level to ≈100 mm Hg at 3000 m and 43 mm Hg on the summit of Everest (8400 m).2,3 The body responds by hyperventilating, increasing resting heart rate, and stimulating red cell production in an attempt to maintain the oxygen content of arterial blood at or above sea level values.2 However, hypoxic pulmonary vasoconstriction (HPV) and vascular remodeling, together with increased erythropoiesis, place an increased pressure load on the right ventricle (RV). How well healthy humans adapt to hypoxia depends on their rate of ascent to altitude, the severity and duration of their exposure, and their genetic background. ### Pulmonary Vascular Response to Hypoxia For most mammals, including humans, a rise in pulmonary artery pressure (PAP) is an early and inevitable consequence of ascent to high altitude. Resting mean PAP increases along a parabolic curve from 15 mm Hg at 2000 m to ≈30 mm Hg at 4500 m.4 The exceptions and interindividual variation in the magnitude of response offer a natural experiment that might provide insight into fundamental underlying mechanisms ( vide infra ). The initial rise in PAP on exposure to hypoxia is attributed to HPV. With chronic hypoxia, other mechanisms that likely drive vascular remodeling soon contribute to the elevated pressure (Figure 1A). After 2 or 3 weeks of hypoxia, there is little response to rebreathing 100% oxygen, indicating a structural resistance to pulmonary blood flow rather than one based solely on increased vasomotor tone.6 A fall in PAP on re-exposure to a normal oxygen environment is evident in rats monitored by telemetry over days …

Background— Phenotypic modulation or switching of vascular smooth muscle cells from a contractile/quiescent to a proliferative/synthetic phenotype plays a key role in vascular proliferative disorders such as atherosclerosis and restenosis. Although several calcium handling proteins that control differentiation of smooth muscle cells have been identified, the role of protein phosphatase inhibitor 1 (I-1) in the acquisition or maintenance of the contractile phenotype modulation remains unknown. Methods and Results— In human coronary arteries, I-1 and sarco/endoplasmic reticulum Ca 2+ -ATPase expression is specific to contractile vascular smooth muscle cells. In synthetic cultured human coronary artery smooth muscle cells, protein phosphatase inhibitor 1 (I-1 target) is highly expressed, leading to a decrease in phospholamban phosphorylation, sarco/endoplasmic reticulum Ca 2+ -ATPase, and cAMP-responsive element binding activity. I-1 knockout mice lack phospholamban phosphorylation and exhibit vascular smooth muscle cell arrest in the synthetic state with excessive neointimal proliferation after carotid injury, as well as significant modifications of contractile properties and relaxant response to acetylcholine of femoral artery in vivo. Constitutively active I-1 gene transfer decreased neointimal formation in an angioplasty rat model by preventing vascular smooth muscle cell contractile to synthetic phenotype change. Conclusions— I-1 and sarco/endoplasmic reticulum Ca 2+ -ATPase synergistically induce the vascular smooth muscle cell contractile phenotype. Gene transfer of constitutively active I-1 is a promising therapeutic strategy for preventing vascular proliferative disorders.

Background— This investigation examined the mechanisms by which coronary perivascular adipose tissue (PVAT)–derived factors influence vasomotor tone and the PVAT proteome in lean versus obese swine. Methods and Results— Coronary arteries from Ossabaw swine were isolated for isometric tension studies. We found that coronary ( P =0.03) and mesenteric ( P =0.04) but not subcutaneous adipose tissue augmented coronary contractions to KCl (20 mmol/L). Inhibition of Ca V 1.2 channels with nifedipine (0.1 µmol/L) or diltiazem (10 µmol/L) abolished this effect. Coronary PVAT increased baseline tension and potentiated constriction of isolated arteries to prostaglandin F2α in proportion to the amount of PVAT present (0.1–1.0 g). These effects were elevated in tissues obtained from obese swine and were observed in intact and endothelium denuded arteries. Coronary PVAT also diminished H 2 O 2 -mediated vasodilation in lean and, to a lesser extent, in obese arteries. These effects were associated with alterations in the obese coronary PVAT proteome (detected 186 alterations) and elevated voltage-dependent increases in intracellular [Ca 2+ ] in obese smooth muscle cells. Further studies revealed that the Rho-kinase inhibitor fasudil (1 µmol/L) significantly blunted artery contractions to KCl and PVAT in lean but not obese swine. Calpastatin (10 μmol/L) also augmented contractions to levels similar to that observed in the presence of PVAT. Conclusions— Vascular effects of PVAT vary according to anatomic location and are influenced by an obese phenotype. Augmented contractile effects of obese coronary PVAT are related to alterations in the PVAT proteome (eg, calpastatin), Rho-dependent signaling, and the functional contribution of K + and Ca V 1.2 channels to smooth muscle tone.

Background— The circadian variation in the incidence of cardiovascular events may be attributable to the circadian changes in vascular contractility. The circadian rhythm of vascular contractility is determined by the interplay between the central and peripheral clocks. However, the molecular mechanism of the vascular intrinsic clock that generates the circadian rhythm of vascular contractility still remains largely unknown. Methods and Results— The agonist-induced phosphorylation of myosin light chain in cultured smooth muscle cells synchronized by dexamethasone pulse treatment exhibited an apparent circadian oscillation, with a 25.4-hour cycle length. The pharmacological inhibition and knockdown of Rho-associated kinase 2 (ROCK2) abolished the circadian rhythm of myosin light chain phosphorylation. The expression and activity of ROCK2 exhibited a circadian rhythm in phase with that of myosin light chain phosphorylation. A clock gene, RORα, activated the promoter of the ROCK2 gene, whereas its knockdown abolished the rhythmic expression of ROCK2. In the mouse aorta, ROCK2 expression exhibited the circadian oscillation, with a peak at Zeitgeber time 0/24 and a nadir at Zeitgeber time 12. The myofilament Ca 2+ sensitization induced by GTPγS and U46619, a thromboxane A2 analog, at Zeitgeber time 0/24 was greater than that seen at Zeitgeber time 12. The circadian rhythm of ROCK2 expression and myofilament Ca 2+ sensitivity was abolished in staggerer mutant mice, which lack a functional RORα. Conclusions— ROCK2 plays a pivotal role in generating the intrinsic circadian rhythm of vascular contractility by receiving a cue from RORα. The ROCK2-mediated intrinsic rhythm of vascular contractility may underlie the diurnal variation of the incidence of cardiovascular diseases.

Background— Puma (p53-upregulated modulator of apoptosis) is a proapoptotic Bcl-2 family protein that serves as a general sensor in response to pathological apoptotic stimuli. In previous work, we demonstrated that puma ablation protects the heart from reperfusion injury in a Langendorff setting. Consistent with this, downregulation of Puma in isolated cardiac myocytes prevented apoptosis induced by different proapoptotic agents. Here, we extended our research to investigate the role of Puma, a downstream mediator of p53, in the development of heart failure using Puma −/− mice. Methods and Results— Mice underwent transverse aortic constriction, and the characteristics of cardiac remodeling were analyzed by echocardiography, histology, and gene expression at multiple time points after surgery. Four weeks after the operation, puma deletion attenuated pressure overload–induced apoptosis and fibrosis; however, it did not affect hypertrophy and angiogenesis and maintained functional performance (fractional shortening, 39% versus 25.2% in Puma −/− versus WT mice, respectively). Even at 12 weeks after transverse aortic constriction, Puma −/− mice displayed only slightly reduced contractility. In addition, transverse aortic constriction induced puma expression in a partially p53-dependent manner. To corroborate these findings, we studied another heart failure model in which heart-specific mdm4 deletion leads to p53 activation and dilated cardiomyopathy. In these mice, Puma was upregulated and its deletion rescued the cardiomyopathy phenotype. Conclusions— Our data indicate that Puma might be a critical component of the apoptotic signaling pathways that contribute to ventricular remodeling and heart failure. Therefore, Puma inactivation may serve as a preferential target to prevent heart failure induced by cellular stress.

Background— Nitrite can be converted to nitric oxide (NO) by a number of different biochemical pathways. In newborn lambs, an aerosol of inhaled nitrite has been found to reduce pulmonary blood pressure, possibly acting via conversion to NO by reaction with intraerythrocytic deoxyhemoglobin. If so, the vasodilating effects of nitrite would be attenuated by free hemoglobin in plasma that would rapidly scavenge NO. Methods and Results— Pulmonary vascular pressures and resistances to flow were measured in anesthetized newborn lambs. Plasma hemoglobin concentrations were then elevated, resulting in marked pulmonary hypertension. This effect was attenuated if infused hemoglobin was first oxidized to methemoglobin, which does not scavenge NO. These results further implicate NO as a tonic pulmonary vasodilator. Next, while free hemoglobin continued to be infused, the lambs were given inhaled NO gas (20 ppm), inhaled sodium nitrite aerosol (0.87 mol/L), or an intravascular nitrite infusion (3 mg/h bolus, 5 mg · kg −1 · h −1 infusion). Inhaled NO and inhaled nitrite aerosol both resulted in pulmonary vasodilation. Intravascular infusion of nitrite, however, did not. Increases in exhaled NO gas were observed in lambs while breathing the nitrite aerosol (≈20 ppb NO) but not during intravascular infusion of nitrite. Conclusions— We conclude that the pulmonary vasodilating effect of inhaled nitrite results from its conversion to NO in airway and parenchymal lung tissue and is not dependent on reactions with deoxyhemoglobin in the pulmonary circulation. Inhaled nitrite aerosol remains a promising candidate to reduce pulmonary hypertension in clinical application.

Pulmonary arterial hypertension (PAH) is a syndrome in which pulmonary arterial obstruction increases pulmonary vascular resistance, which leads to right ventricular (RV) failure and a 15% annual mortality rate. The present review highlights recent advances in the basic science of PAH. New concepts clarify the nature of PAH and provide molecular blueprints that explain how PAH is initiated and maintained. Five basic science concepts provide a framework to understand and treat PAH: (1) Endothelial dysfunction creates an imbalance that favors vasoconstriction, thrombosis, and mitogenesis. Restoration of this balance by inhibition of endothelin and thromboxane or augmentation of nitric oxide (NO) and prostacyclin is the paradigm on which most current therapy is based. (2) PAH has a genetic component. Mutations (bone morphogenetic protein receptor-2 [BMPR2]) and single-nucleotide polymorphisms (SNPs; ion channels and transporter genes) predispose to PAH. (3) Excess proliferation, impaired apoptosis, and glycolytic metabolism in pulmonary artery smooth muscle, fibroblasts, and endothelial cells suggest analogies to cancer. Many experimental therapies reduce PAH by decreasing the proliferation/apoptosis ratio; these include inhibitors of pyruvate dehydrogenase kinase (PDK), serotonin transporters (SERT), survivin, 3-hydroxy-3-methylglutaryl coenzyme A reductase, transcription factors (hypoxia-inducible factor [HIF]-1α and nuclear factor of activated T lymphocytes [NFAT]), and tyrosine kinases. Augmentation of voltage-gated K+ channels (Kv1.5) and BMPR2 signaling also addresses this imbalance. Tyrosine kinase inhibitors used to treat cancer are currently in phase 1 PAH trials. (4) Refractory vasoconstriction may occur due to rho kinase activation. Fewer than 20% of PAH patients respond to conventional vasodilators; however, refractory vasoconstriction may respond to rho kinase inhibitors. (5) The RV can be targeted therapeutically. Although increased afterload initiates RV failure, which is the major cause of death/dysfunction in PAH, the RV may be amenable to cardiac-targeted therapies. The RV in PAH has features of ischemic, hibernating myocardium. Guided by these new …

Background— Obesity causes hypertension and sympathoactivation, a process proposed to be mediated by leptin. Protein tyrosine phosphatase 1B (PTP1B), a major new pharmaceutical target in the treatment of obesity and type II diabetes mellitus, constrains the metabolic actions of leptin, but the extent to which PTP1B regulates its cardiovascular effects is unclear. This study examined the hypothesis that PTP1B is a negative regulator of the cardiovascular effects of leptin. Methods and Results— PTP1B knockout mice had lower body fat but higher mean arterial pressure (116±5 versus 105±5 mm Hg, P P P P 1 -adrenergic receptor subtypes, consistent with blunted constriction to phenylephrine. Conclusions— These data indicate that PTP1B is a key regulator of the cardiovascular effects of leptin and that reduced vascular adrenergic reactivity provides a compensatory limit to the effects of leptin on mean arterial pressure.

The microcirculation is generally taken to include the smallest arteries, the arterioles, capillaries, and venules.1,2 Exchange of gases, nutrients, and metabolites between the blood and tissues occurs almost exclusively in the microcirculation, and adequate perfusion via the microcirculatory network is essential for the integrity of tissue and organ function. Our aim in the present article is to bring together recent clinical and experimental research indicating that inadequate perfusion may underlie much of the tissue and organ dysfunction associated with chronic conditions including hypertension, obesity, and diabetes mellitus. Given the high level of research activity in this field, our review can be neither systematic nor comprehensive. We apologize to the many authors whose important contributions are not acknowledged here; additional references are given in a Data Supplement on the journal’s Web site. In addition to providing the large surface area needed for blood-tissue exchange, the microcirculation largely controls the perfusion of tissues in response to varying metabolic requirements. Large and medium-sized arteries and veins offer relatively little resistance to the flow of blood, and work pioneered by DeLano and colleagues3 showed that some 70% to 90% of the systemic arterial pressure is delivered to the microcirculation, where the main resistance to flow is offered. The microcirculation, therefore, largely determines local and overall peripheral resistance. The precapillary elements of the microcirculation also protect the fragile capillaries from the potentially damaging pressures that occur in the larger arteries. An abrupt increase in pressure brings about a rapid and reversible vasoconstriction of small resistance vessels due to their inherent myogenic tone.4 Prolonged elevations of pressure can cause a range of more lasting changes in the microcirculation, 2 of which, remodeling of small arteries and arterioles and rarefaction of arterioles and capillaries, will be considered briefly below. ### Small Artery and Arteriolar Remodeling Small arteries and arterioles, with …

Background— Angiotensin (Ang) II–induced target-organ damage involves innate and acquired immunity. Mice deficient for the helix-loop-helix transcription factor inhibitor of differentiation (Id2 −/− ) lack Langerhans and splenic CD8a+ dendritic cells, have reduced natural killer cells, and have altered CD8 T-cell memory. We tested the hypothesis that an alteration in the number and quality of circulating blood cells caused by Id2 deletion would ameliorate Ang II–induced target-organ damage. Methods and Results— We used gene-deleted and transgenic mice. We conducted kidney and bone marrow transplants. In contrast to Ang II–infused Id2 +/− , Id2 −/− mice infused with Ang II remained normotensive and failed to develop albuminuria or renal damage. Bone marrow transplant of Id2 +/− bone marrow to Id2 −/− mice did not restore the blunted blood pressure response to Ang II. Transplantation of Id2 −/− kidneys to Id2 +/− mice also could not prevent Ang II–induced hypertension and renal damage. We verified the Ang II resistance in Id2 −/− mice in a model of local tissue Ang II production by crossing hypertensive mice transgenic for rat angiotensinogen with Id2 −/− or Id2 +/− mice. Angiotensinogen-transgenic Id2 +/− mice developed hypertension, albuminuria, and renal injury, whereas angiotensinogen-transgenic Id2 −/− mice did not. We also found that vascular smooth muscle cells from Id2 −/− mice showed an antisenescence phenotype. Conclusions— Our bone marrow and kidney transplant experiments suggest that alterations in circulating immune cells or Id2 in the kidney are not responsible for Ang II resistance. The present studies identify a previously undefined role for Id2 in the pathogenesis of Ang II–induced hypertension.

Background— One of the major obstacles hindering the clinical development of a cell-free, hemoglobin-based oxygen carrier (HBOC) is systemic vasoconstriction. Methods and Results— Experiments were performed in healthy mice and lambs by infusion of either murine tetrameric hemoglobin (0.48 g/kg) or glutaraldehyde-polymerized bovine hemoglobin (HBOC-201, 1.44 g/kg). We observed that intravenous infusion of either murine tetrameric hemoglobin or HBOC-201 induced prolonged systemic vasoconstriction in wild-type mice but not in mice congenitally deficient in endothelial nitric oxide (NO) synthase (NOS3). Treatment of wild-type mice by breathing NO at 80 ppm in air for 15 or 60 minutes or with 200 ppm NO for 7 minutes prevented the systemic hypertension induced by subsequent intravenous administration of murine tetrameric hemoglobin or HBOC-201 and did not result in conversion of plasma hemoglobin to methemoglobin. Intravenous administration of sodium nitrite (48 nmol) 5 minutes before infusion of murine tetrameric hemoglobin also prevented the development of systemic hypertension. In awake lambs, breathing NO at 80 ppm for 1 hour prevented the systemic hypertension caused by subsequent infusion of HBOC-201. Conclusions— These findings demonstrate that HBOC can cause systemic vasoconstriction by scavenging NO produced by NOS3. Moreover, in 2 species, inhaled NO administered before the intravenous infusion of HBOC can prevent systemic vasoconstriction without causing methemoglobinemia.

Background— Constriction of the ductus arteriosus (DA) is initiated at birth by inhibition of O 2 -sensitive K + channels in DA smooth muscle cells. Subsequent membrane depolarization and calcium influx through L-type calcium channels initiates functional closure. We hypothesize that Rho-kinase activation is an additional mechanism that sustains DA constriction. Methods and Results— The effect of increased P o 2 on the activity and expression of Rho-kinase was assessed in DAs from neonates with hypoplastic left-heart syndrome (n=15) and rabbits (339 term and 99 preterm rabbits). Rho-kinase inhibitors (Y-27632 and fasudil) prevent and reverse O 2 constriction. Heterogeneity exists in the sensitivity of constrictors (P o 2 =endothelin=phenylephrine>KCl) and of fetal vessels (DA=pulmonary artery>aorta) to Rho-kinase inhibition. Inhibition of L-type calcium channels (nifedipine) or removal of extracellular calcium inhibits approximately two thirds of O 2 constriction. Residual DA constriction reflects calcium sensitization, which persists after removal of extracellular calcium and blocking of sarcoplasmic reticulum Ca 2+ -ATPase. In term DA, an increase in P o 2 activates Rho-kinase and thereby increases RhoB and ROCK-1 expression. Activation of Rho-kinase in DA smooth muscle cells is initiated by a P o 2 -dependent, rotenone-sensitive increase in mitochondrion-derived reactive O 2 species. O 2 effects on Rho-kinase are mimicked by exogenous H 2 O 2 . In preterm DAs, immaturity of mitochondrial reactive oxygen species generation is associated with reduced and delayed O 2 constriction and lack of P o 2 -dependent upregulation of Rho-kinase expression. Conclusions— O 2 activates Rho-kinase and increases Rho-kinase expression in term DA smooth muscle cells by a redox-regulated, positive-feedback mechanism that promotes sustained vasoconstriction. Conversely, Rho-kinase inhibitors may be useful in maintaining DA patency, as a bridge to congenital heart surgery.

Background: PAH (Group I) is a disease of the small pulmonary arteries, characterized by vasoconstriction, vascular proliferation and remodeling. PH-HFpEF (Group II) is known to occur secondary to the LV diastolic dysfunction and is recognized as a clinical complication of metabolic syndrome, with no currently approved therapies. Since the use of conventional therapies for PAH in PH-HFpEF patients is often ineffective, targeting both pulmonary vascular disease and metabolic disorders appears to be a key strategy for the treatment of PH-HFpEF. We, as well as others, have reported that nitrite exhibits therapeutic efficacy in moderate PAH models and improves glucose metabolism. In addition, metformin, the canonical AMPK activator and the first-line anti-diabetic drug, has been found to prevent the development of moderate PAH. In this work, we examined the effects of nitrite and metformin in PH-HFpEF, and compared their effects in severe PAH. Methods and Results: Nitrite (100mg/L) and metformin (300mg/kg) were given in drinking water to rats with Su5416/hypoxia-induced severe PAH or to rats with Su5416/metabolic syndrome-induced PH-HFpEF for 6 and 14 weeks, respectively. In the PH-HFpEF rats, our data showed that both nitrite and metformin treatments reduced pulmonary pressures and pathological vascular remodeling and limited hyperglycemia, which correlated with increased SIRT3-AMPK activation in skeletal muscles and AMPK activation in pulmonary vessels. Similar protective effect of nitrite was also observed in Su5416/hypoxia rats, together with increased activation of SIRT3 and AMPK in skeletal muscles. However, metformin failed to reduce pulmonary pressures and vascular remodeling in Su5416/hypoxia rats, with unchanged activation levels of SIRT3 and AMPK, suggesting metformin may favorably affect PH only in the presence of metabolic disorders. In addition, the AMPK agonist AICAR was found incapable of limiting PAH in Su5416/hypoxia rats. Conclusions: Our findings indicate that oral nitrite treatment limits PH-HFpEF, as well as PAH, while metformin treatment more preferentially improves PH-HFpEF. Our findings also indicate that activation of SIRT3-AMPK may represent a potential therapeutic route in the treatment of both PAH and PH-HFpEF.

Introduction: Up4A, a novel endothelium-derived vasoactive agent, is proposed to play a role in cardiovascular disorder via activation of P2 receptors (P2R). Here, we evaluated the vasoactive effect of Up4A on coronary flow (CF) in ApoE knockout mice (KO) with and without high fat diet (HFD). Methods: Functional studies were conducted in isolated mouse hearts using Langendorff and isolated coronary arteries (diameter≈100 μm) using wire Myograph. Immunofluorescence was performed for P2X1R expression in isolated coronary arteries. Results: There was no significant difference in baseline CF (ml/min/g) among wild type (WT, 18.4±0.5), ApoE KO (17.6±1.1) and ApoE KO+HFD (15.9±1.0). Infusion of Up4A (10-9-10-5 M) resulted in a dose-dependent reduction in CF in WT (by ~38%), which was similar to ApoE KO (by ~40%). In isolated coronary arteries of WT, Up4A produced a mild contraction up to ~20%, indicating a vasoconstrictor effect of Up4A. Notably, Up4A further decreased CF in ApoE+HFD as compared to ApoE. Moreover, selective P2X1R antagonist MRS2159 (10 μM) restored Up4A-mediated decrease in CF more in ApoE+HFD than ApoE (Figure). Finally, the percentage expression of vasoconstrictor P2X1R on smooth muscle cells (SMC) of coronary arteries was higher in ApoE+HFD than ApoE, which was due to a markedly reduced vasodilator P2X1R expression on EC in ApoE+HFD (8.7±0.6 fluorescence intensity in A.U.) than ApoE (22.1±0.5), while P2X1R expression on SMC was similar between ApoE+HFD (6.3±0.4) and ApoE (8.4±0.3). Conclusion: Up4A exerts a vasoconstrictor influence in mouse coronary microcirculation. Up4A decreases CF in WT to a similar extent in ApoE, whereas HFD exacerbates Up4A-decreased CF in ApoE possibly through downregulation of vasodilator P2X1R on EC. These findings indicate that Up4A alters CF in atherosclerosis and P2X1R may represent a potential therapeutic target.

Introduction: Advanced heart failure (HF) patients may stay for a long period of time in the hospital for the treatment of HF and prolonged inactivity causes complications that result in worsening of exercise tolerance. On the other hand, there is evidence that treatment with muscle low-frequency electrical stimulation (FES) provides important benefits for HF patients. This study investigated the effects of FES on neurovascular control and exercise tolerance in HF hospitalized patients. Methods and results: Thirty hospitalized patients for treatment of decompensated HF, functional class IV NYHA and ejection fraction ≤ 30% were consecutively randomised into two groups: 1) FES(n= 15; 54±2 years) and control(n= 15; 49±2 years). Muscle sympathetic nerve activity (MSNA) was directly recorded via microneurography and blood flow by venous occlusion plethysmography. Heart rate and blood pressure were evaluated on a beat-to-beat basis (Finometer). Exercise tolerance by six-minute walk test, quadriceps muscle strength by a dynamometer and quality of life by Minnesota Questionnaire. FES consisted of stimulating the lower limbs at 10 Hz frequency, 150 ms pulse width and 70 mA intensity for 60 min/day for 7 days/week for 10 consecutive days. The control group underwent electrical stimulation at intensity of 20 mA. Baseline characteristics were similar between groups, except age that was higher in FES group. FES significantly decreased MSNA burst frequency (49 ±5 vs. 35±5, P= 0.002) and burst incidence (61 ±6 vs. 49±7, P= 0.04). In addition, FES significantly increased leg blood flow (0.88 ±0.09 vs. 1.47 ±0.14, P Conclusion: FES improves MSNA and vasoconstriction and increases exercise tolerance, muscle strength and quality of life in hospitalised HF patients. These findings support the use of FES as an adjuvant therapy for patients hospitalised to achieve the stabilisation of HF.

Lankadeva et al1 recently published a very interesting article investigating the mechanisms underlying hypertension and chronic kidney disease in children born with reduced congenital renal mass, performing a study in sheep with fetal uninephrectomy. The authors described that renal dysfunction after congenital renal mass reduction is associated with impaired regulation of renal hemodynamics by nitric oxide (NO). We think that this is a very interesting and well-designed study and supports the assumptions described in other diseases such as diabetic nephropathy.2 Conflicting findings have been reported with regard to NO synthesis and release in type 1 and type …

I read with great interest the article by Lankadeva and colleagues1 dealing with the relationship between renal dysfunction and nitric oxide bioavailability in the sheep with a congenital renal mass reduction. The results of their study demonstrated that basal mean arterial pressure was higher, whereas glomerular filtration rate and renal blood flow were lower in the sheep with fetal uninephrectomy (uni-x sheep) than in the sham controls. N ω-Nitro-l-arginine methyl ester, a nitric oxide synthase (NOS) inhibitor, increased blood pressure to the same extent in both groups, whereas glomerular filtration rate and renal blood flow decreased …

Background— Protease-activated receptor type 1 (PAR-1) has been proposed as the principal thrombin receptor in humans, although its actions in vivo have not been defined. The aim of the present study was to determine the direct vascular actions of PAR-1 agonism in humans. Methods and Results— Dorsal hand vein diameter was measured by the Aellig technique in 14 healthy volunteers during local intravenous SFLLRN (PAR-1 agonist; 0.05 to 15 nmol/min) and SLIGKV (PAR-2 agonist; 1.6 to 160 nmol/min) infusions. The venous effects of SFLLRN were further assessed in the presence or absence of norepinephrine or the glycoprotein IIb/IIIa antagonist tirofiban. Forearm blood flow was measured by venous occlusion plethysmography in 16 volunteers during infusion of SFLLRN (1 to 50 nmol/min), SLIGKV (160 to 800 nmol/min), and the endothelium-dependent vasodilator bradykinin (100 to 1000 pmol/min). Platelet-monocyte binding (a sensitive measure of platelet activation) and plasma tissue plasminogen activator (tPA), plasminogen-activator inhibitor 1, and von Willebrand factor concentrations were measured at intervals throughout the study. SFLLRN caused dose-dependent venoconstriction ( P P P P P P Conclusions— We have demonstrated that PAR-1 agonism causes platelet activation, venous constriction, arterial dilatation, and tPA release in vivo in humans. These unique and contrasting effects provide important insights into the physiological and pathophysiological role of thrombin in the human venous and arterial circulations.

Background— The degree of pulmonary hypertension in healthy subjects exposed to acute hypobaric hypoxia at high altitude was found to be related to increased plasma endothelin (ET)-1. The aim of the present study was to investigate the effects of ET-1 antagonism on pulmonary hypertension, renal water, and sodium balance under acute and prolonged exposure to high-altitude–associated hypoxia. Methods and Results— In a double-blind fashion, healthy volunteers were randomly assigned to receive bosentan (62.5 mg for 1 day and 125 mg for the following 2 days; n=10) or placebo (n=10) at sea level and after rapid ascent to high altitude (4559 m). At sea level, bosentan did not induce any significant changes in hemodynamic or renal parameters. At altitude, bosentan induced a significant reduction of systolic pulmonary artery pressure (21±7 versus 31±7 mm Hg, P 2 OCl/glomerular filtration rate) were significantly reduced versus placebo after 2 days of ET-1 antagonism (1100±200 versus 1610±590 mL; −6.7±3.5 versus −1.8±4.8 mL/min, P Conclusions— The present results indicate that the early beneficial effect of ET-1 antagonism on pulmonary blood pressure is followed by an impairment in volume adaptation. These findings must be considered for the prevention and treatment of acute mountain sickness.

Background— During septic shock, the vasoconstrictor response to norepinephrine is seriously blunted. Animal experiments suggest that hyperpolarization of smooth muscle cells by opening of potassium (K) channels underlies this phenomenon. In the present study, we examined whether K-channel blockers and/or nitric oxide (NO) synthase inhibition could restore norepinephrine sensitivity during experimental human endotoxemia. Methods and Results— Volunteers received 2 ng/kg Escherichia coli endotoxin intravenously. Forearm blood flow (FBF) was measured with venous occlusion plethysmography. Infusion of 4 dose steps of norepinephrine into the brachial artery decreased the FBF ratio (ratio of FBF in the experimental arm to FBF in the control arm) to 84±4%, 70±4%, 55±4%, and 38±4% (mean±SEM) of its baseline value. After endotoxin administration, norepinephrine-induced vasoconstriction was attenuated (FBF ratio, 101±4%, 92±4%, 83±6%, and 56±7%; n=30; P= 0.0018; pooled data). Intrabrachial infusion of the K-channel blocker tetraethylammonium (TEA) completely restored the vasoconstrictor response to norepinephrine from 104±5%, 93±7%, 93±12%, and 69±12% to 89±9%, 73±4%, 59±5%, and 46±8% (n=6; P =0.045). Other K-channel blockers did not affect the response to norepinephrine. The NO synthase inhibitor N G -monomethyl- l -arginine (L-NMMA; 0.2 mg · min −1 · dL −1 intra-arterially) also restored the norepinephrine sensitivity. In the presence of L-NMMA, TEA did not have an additional effect on the norepinephrine-induced vasoconstriction (n=6; P =0.9). Conclusions— The K-channel blocker TEA restores the attenuated vasoconstrictor response to norepinephrine during experimental human endotoxemia. Coadministration of L-NMMA abolishes this potentiating effect of TEA, suggesting that NO mediates the endotoxin-induced effect on vascular K channels. In the absence of an effect of the selective adenosine triphosphate–dependent K-channel blocker tolbutamide, we conclude that the blunting effect of endotoxin on norepinephrine-induced vasoconstriction is caused by NO-mediated activation of calcium-activated K channels in the vascular wall.

Background— The cause of pulmonary arterial hypertension (PAH) was investigated in humans and fawn hooded rats (FHR), a spontaneously pulmonary hypertensive strain. Methods and Results— Serial Doppler echocardiograms and cardiac catheterizations were performed in FHR and FHR/BN1, a consomic control that is genetically identical except for introgression of chromosome 1. PAH began after 20 weeks of age, causing death by &60 weeks. FHR/BN1 did not develop PAH. FHR pulmonary arterial smooth muscle cells (PASMCs) had a rarified reticulum of hyperpolarized mitochondria with reduced expression of electron transport chain components and superoxide dismutase-2. These mitochondrial abnormalities preceded PAH and persisted in culture. Depressed mitochondrial reactive oxygen species (ROS) production caused normoxic activation of hypoxia inducible factor (HIF-1α), which then inhibited expression of oxygen-sensitive, voltage-gated K + channels (eg, Kv1.5). Disruption of this mitochondrial-HIF-Kv pathway impaired oxygen sensing (reducing hypoxic pulmonary vasoconstriction, causing polycythemia), analogous to the pathophysiology of chronically hypoxic Sprague-Dawley rats. Restoring ROS (exogenous H 2 O 2 ) or blocking HIF-1α activation (dominant-negative HIF-1α) restored Kv1.5 expression/function. Dichloroacetate, a mitochondrial pyruvate dehydrogenase kinase inhibitor, corrected the mitochondrial-HIF-Kv pathway in FHR-PAH and human PAH PASMCs. Oral dichloroacetate regressed FHR-PAH and polycythemia, increasing survival. Chromosome 1 genes that were dysregulated in FHRs and relevant to the mitochondria-HIF-Kv pathway included HIF-3α (an HIF-1α repressor), mitochondrial cytochrome c oxidase, and superoxide dismutase-2. Like FHRs, human PAH-PASMCs had dysmorphic, hyperpolarized mitochondria; normoxic HIF-1α activation; and reduced expression/activity of HIF-3α, cytochrome c oxidase, and superoxide dismutase-2. Conclusions— FHRs have a chromosome 1 abnormality that disrupts a mitochondria-ROS-HIF-Kv pathway, leading to PAH. Similar abnormalities occur in idiopathic human PAH. This study reveals an intersection between oxygen-sensing mechanisms and PAH. The mitochondria-ROS-HIF-Kv pathway offers new targets for PAH therapy.

Background— Vasoreactivity tests are fundamental in evaluating pulmonary arterial hypertension (PAH). Mutations of the transforming growth factor-β type II receptor gene, BMPR2 , predispose to the development of pulmonary hypertension and may alter the response to vasodilators. Previous investigations have not examined the relationship of BMPR2 mutations to vasoreactivity. Methods and Results— We identified 133 consecutive unrelated patients with either idiopathic or familial PAH. Sixty-six patients were excluded because we lacked either DNA samples (n=18) or complete data from a vasoreactivity test (n=48). The remaining 67 patients were screened for BMPR2 DNA sequence variations, and specific variations were confirmed by gene sequencing. The vasoreactivity of patients with nonsynonymous BMPR2 variations was compared with that of patients without nonsynonymous BMPR2 variations. We found nonsynonymous BMPR2 variations in 27 of 67 patients with idiopathic (n=16 of 52) or familial (n=11 of 15) PAH. Vasoreactivity was identified in 3.7% of 27 patients with nonsynonymous BMPR2 variations and in 35% of 40 patients without nonsynonymous BMPR2 variations ( P =0.003). Five of the 27 nonsynonymous variations occur commonly in healthy individuals. None of the remaining 22 patients with BMPR2 variations demonstrated vasoreactivity, and the analysis remained unchanged when we assumed that nonsynonymous BMPR2 variations were present in all 15 patients with familial PAH. Conclusions— Patients with familial or idiopathic PAH and nonsynonymous BMPR2 variations are unlikely to demonstrate vasoreactivity. Further trials are required to determine whether long-term therapy can be directed by tests for BMPR2 variations.

Background— The vasoconstricting peptide endothelin-1 (ET-1) has been associated with atherosclerotic cardiovascular disease, vascular smooth muscle cell (VSMC) growth stimulation, and intimal thickening. ET-1 binds 2 receptor subtypes, endothelin A and B, and the ET A receptor mediates vasoconstriction and VSMC growth. This study aims to quantitatively assess arterial remodeling variables and compare them with changes in ET-1, ET A , and ET B expression in the internal mammary artery (IMA). Methods and Results— Specimens from 55 coronary artery disease (CAD) patients (45 men, 10 women; mean age 65 years) and 14 control IMA specimens (from 7 men and 7 women; mean age 45 years) were collected. IMA cross sections were assessed by histochemical and immunohistochemical staining methods to quantify the levels of medionecrosis, fibrosis, VSMC growth, ET-1, ET A , ET B , and macrophage infiltration. The percentage area of medionecrosis in the patients was almost double that in the controls (31.85±14.52% versus 17.10±9.96%, P =0.0006). Total and type 1 collagen was significantly increased compared with controls (65.8±18.3% versus 33.7±13.7%, P =0.07, and 14.2±10.0% versus 4.8±2.8%, P =0.01, respectively). Despite ACE and/or statin therapy, ET-1 expression and cell cycling were significantly elevated in the patient IMAs relative to the controls (46.27±18.46 versus 8.56±8.42, P =0.0001, and 37.29±12.88 versus 11.06±8.18, P =0.0001, respectively). ET A and ET B staining was elevated in the patient vessels (46.88±11.52% versus 18.58±7.65%, P =0.0001, and 42.98±7.08% versus 34.73±5.20%, P =0.0067, respectively). A mild presence of macrophages was noted in all sections. Conclusions— Elevated distribution of collagen indicative of fibrosis coupled with increased cell cycling and high levels of ET-1 and ET A expression in the absence of chronic inflammation suggests altered IMA VSMC regulation is fundamental to the remodeling process.

Background— Hypertension, a major cause of cardiovascular morbidity and mortality, can result from chronic hypoxia; however, the pathogenesis of this disorder is unknown. We hypothesized that downregulation of the maxi-K + channel β 1 -subunit by hypoxia decreases the ability of these channels to hyperpolarize arterial smooth muscle cells, thus favoring vasoconstriction and hypertension. Methods and Results— Lowering O 2 tension produced a decrease of maxi-K + β 1 -subunit mRNA levels in rat (aortic and basilar) and human (mammary) arterial myocytes. This was paralleled by a reduction of the β 1 -subunit protein level as determined by immunocytochemistry and flow cytometry. Exposure to hypoxia also produced a decrease of open probability, mean open time, and sensitivity to the xenoestrogen tamoxifen of single maxi-K + channels recorded from patch-clamped dispersed myocytes. The number of channels per patch and the single-channel conductance were not altered. The vasorelaxing force of maxi-K + channels was diminished in rat and human arterial rings exposed to low oxygen tension. Conclusions— These results indicate that a decrease of the maxi-K + channel β 1 -subunit expression in arterial myocytes is a key factor in the vasomotor alterations induced by hypoxia.

Background— Secondary erythrocytosis results in increased shear stress in cyanotic congenital heart disease (CCHD), which may modify the balance between vasodilators and vasoconstrictors and affect systemic endothelial function. Because no data are available on systemic vasomotion, systemic endothelial function and nitric oxide (NO) availability were investigated in CCHD patients. Methods and Results— Responses to arterial endothelium-dependent (acetylcholine [Ach]) and -independent (sodium nitroprusside [SNP]) vasodilation, NO synthase blockade ( N G -monomethyl- l -arginine [ l -NMMA]), endothelin-1 (ET-1), and ET-1 receptor blockade by BQ-123 in 11 CCHD patients (O 2 saturation −1 · 100 mL −1 of forearm volume [FAV], P −1 · 100 mL −1 of FAV; P >0.1), the response to Ach was markedly reduced in CCHD (maximal increase in FBF, 2.8±0.8 versus 37.5±4.4 mL · min −1 · 100 mL −1 of FAV; P l -NMMA was less effective in CCHD (decrease in FBF, 25±6% versus 40±4%; P P Conclusions— Systemic endothelial dysfunction is evident in CCHD patients as shown by strikingly reduced endothelial vasodilation to Ach. The response to exogenous ET-1 is reduced, possibly because of elevated endogenous ET-1 levels, but the effects of endogenous ET-1 on arterial tone are not enhanced, as indicated by the similar response to ET-1 blockade.

Background— Bone morphogenetic protein receptor-2 (BMPR2)–heterozygous, mutant ( BMPR2 +/− ) mice have a genetic trait similar to that of certain patients with idiopathic pulmonary arterial hypertension (IPAH). To understand the role of BMPR2 in the development of IPAH, we examined the phenotype of BMPR2 +/− mice and their response to inflammatory stress. Methods and Results— BMPR2 +/− mice were found to have the same life span, right ventricular systolic pressure (RVSP), and lung histology as those of wild-type mice under unstressed conditions. However, when treated with recombinant adenovirus expressing 5-lipoxygenase (Ad5LO), BMPR2 +/− mice exhibited significantly higher RVSP than wild-type mice. The increase of RVSP occurred in the first 2 weeks after Ad5LO delivery. Modest but significant muscularization of distal pulmonary arterioles appeared in BMPR2 +/− mice 4 weeks after Ad5LO treatment. Measurement of urinary metabolites of vasoactive molecules showed that cysteinyl leukotrienes, prostacyclin metabolites, and PGE 2 were all increased to a similar degree in both BMPR2 +/− and wild-type mice during 5LO transgene expression, whereas urinary endothelin-1 remained undetectable. Urinary thromboxane A 2 metabolites, in contrast, were significantly higher in BMPR2 +/− than in wild-type mice and paralleled the increase in RVSP. Platelet activation markers, serotonin, and soluble P-selectin showed a trend toward higher concentrations in BMPR2 +/− than wild-type mice. Cell culture studies found that BMP treatment reduced interleukin-1β–stimulated thromboxane A 2 production in the pulmonary epithelial cell line A549. Conclusions— BMPR2 +/− mice do not develop pulmonary hypertension spontaneously; however, under inflammatory stress, they are more susceptible to an increase in RVSP, thromboxane A 2 production, and vascular remodeling than wild-type mice.

Background— We previously identified the G-protein–coupled receptor Mas, encoded by the Mas proto-oncogene, as an endogenous receptor for the heptapeptide angiotensin-(1-7); however, the receptor is also suggested to be involved in actions of angiotensin II. We therefore tested whether this could be mediated indirectly through an interaction with the angiotensin II type 1 receptor, AT 1 . Methods and Results— In transfected mammalian cells, Mas was not activated by angiotensin II; however, AT 1 receptor–mediated, angiotensin II–induced production of inositol phosphates and mobilization of intracellular Ca 2+ was diminished by 50% after coexpression of Mas , despite a concomitant increase in angiotensin II binding capacity. Mas and the AT 1 receptor formed a constitutive hetero-oligomeric complex that was unaffected by the presence of agonists or antagonists of the 2 receptors. In vivo, Mas acts as an antagonist of the AT 1 receptor; mice lacking the Mas gene show enhanced angiotensin II–mediated vasoconstriction in mesenteric microvessels. Conclusions— These results demonstrate that Mas can hetero-oligomerize with the AT 1 receptor and by so doing inhibit the actions of angiotensin II. This is a novel demonstration that a G-protein–coupled receptor acts as a physiological antagonist of a previously characterized receptor. Consequently, the AT 1 -Mas complex could be of great importance as a target for pharmacological intervention in cardiovascular diseases.

Background— Chronic orthostatic intolerance (COI) occurs in postural tachycardia syndrome (POTS) and in some individuals with repeated neurocardiogenic syncope/presyncope (NCS), without POTS. This study addressed whether patients with COI and POTS or NCS have neurocirculatory abnormalities during supine rest. Methods and Results— Adult patients referred for COI who had POTS (n=90, mean±SEM age 40±1 years, 86% women) or NCS (n=36, 41±2 years old, 78% women) underwent measurements of plasma levels of catecholamines and forearm hemodynamics. Comparison data were obtained from 32 age- and gender-matched normal volunteers (39±2 years old, 81% women). The POTS group had a relatively fast mean heart rate (79±2 bpm) during supine rest compared with the NCS group (69±1.6 bpm, P =0.03) and normal volunteers (66±3 bpm, P =0.0004). The POTS group also had higher mean arterial norepinephrine (1.61±0.11 nmol/L, n=37) and epinephrine (0.39±0.03 nmol/L, n=37) concentrations than the NCS group (1.03±0.12 nmol/L, n=20, P =0.0012; 0.21±0.03 nmol/L, n=20, P =0.0005) and normal volunteers (1.13±0.11 nmol/L, n=20, P =0.006; 0.17±0.03 nmol/L, n=15, P =0.0001). The NCS group had higher mean forearm vascular resistance (52±6 U) than the POTS group (36±2 U, P =0.003). Conclusions— Overall, POTS features increased heart rate and sympathetic nervous and adrenomedullary hormonal system outflows during supine rest. Increased sympathetic outflow may contribute to the relative tachycardia in POTS. NCS features forearm vasoconstriction during supine rest but not sympathoneural or adrenomedullary activation.