Myocardial infarction with non-obstructive coronary arteries (MINOCA) is a heterogeneous and diverse disease entity, which accounts for about 6 % of all acute myocardial infarction (AMI) cases. In patients with chest pain and acute myocardial injury detected by a highly sensitive troponin assay, the absence of epicardial coronary stenosis of 50 % or greater on angiography leads to the working diagnosis of MINOCA. The updated JCS/CVIT/JCC 2023 Guideline described MINOCA as a new disease concept and recommended a multimodality approach to uncovering the underlying causes of MINOCA. Cardiac magnetic resonance (CMR) is useful in not only making a definite diagnosis of MINOCA, but also excluding non-ischemic causes that mimic AMI such as takotsubo cardiomyopathy and myocarditis. Meanwhile, intracoronary imaging, particularly optical coherence tomography (OCT), enables us to evaluate precisely intracoronary morphological alterations including plaque disruption and spontaneous coronary artery dissection which are not revealed by angiographic findings alone. Recent studies have shown that an initial workup with the combination of CMR and OCT could provide a definite diagnosis in a significant percentage of patients suspected of MINOCA. Consecutively, patients with inconclusive results of a series of CMR and OCT implementation are eligible for assessing the potential for coronary functional abnormalities or blood coagulopathy as another factor involved in the development of MINOCA. Although uncovering the pathogenesis of MINOCA might be essential for establishing an individualized treatment approach, significant knowledge gaps in terms of secondary prevention strategies for MINOCA focusing on the improvement of long-term prognosis remain to be overcome. In this review, we summarize our current understanding of MINOCA and highlight contemporary diagnostic approaches for patients with suspected MINOCA.

UNLABELLED: A 17-year-old male was diagnosed with acute myocarditis based on the presence of CD3-positive T-lymphocytes in myocardial biopsy, normal coronary angiography, and focal increase in late gadolinium enhancement, T2 intensity and native T1 value. On day 2, the patient suffered from recurrence of chest pain with new ST segment elevations on electrocardiogram. A transient metabolic alteration (inversed lactate level of the coronary sinus relative to that of the coronary artery) accompanied by chest pain and electrocardiographic changes without epicardial coronary spasm in acetylcholine provocation test led to the diagnosis of microvascular angina, which is characterized by a transient myocardial ischemia secondary to a dysfunction of the resistance coronary vessels (<500 μm) that, because of their small size, are not visualized at coronary angiography. Benidipine, a dihydropyridine calcium channel antagonist, was started for chest pain due to microvascular angina. On 6 months after admission, when the findings of cardiac magnetic resonance were recovered, intracoronary infusion of acetylcholine did not induce chest pain, electrocardiographic changes, epicardial coronary spasm, and adverse changes of lactate levels of the coronary artery and sinus. The patient had no chest symptoms 2 years after discontinuation of benidipine. LEARNING OBJECTIVE: The present case of microvascular angina, which was complicated with acute myocarditis on acute phase and recovered in chronic phase, indicates an association of myocardial inflammation with reversible coronary microvascular dysfunction.

Professor Maseri pioneered the research and treatment of coronary vasomotion abnormalities represented by coronary vasospasm and coronary microvascular dysfunction (CMD). These mechanisms can cause myocardial ischaemia even in the absence of obstructive coronary artery disease, and have been appreciated as an important aetiology and therapeutic target with major clinical implications in patients with ischaemia with non-obstructive coronary artery disease (INOCA). Coronary microvascular spasm is one of the key mechanisms responsible for myocardial ischaemia in patients with INOCA. Comprehensive assessment of coronary vasomotor reactivity by invasive functional coronary angiography or interventional diagnostic procedure is recommended to identify the underlying mechanisms of myocardial ischaemia and to tailor the best treatment and management based on the endotype of INOCA. This review highlights the pioneering works of Professor Maseri and contemporary research on coronary vasospasm and CMD with reference to endothelial dysfunction, Rho-kinase activation and inflammation.

Due to the coronavirus disease 2019 (COVID-19) pandemic, the first state of emergency had been declared from April 7 to May 25, 2020, in Japan. This pandemic might affect the management for patients with acute myocardial infarction (AMI). Method and Results: To evaluate the critical care and outcomes of AMI patients during the COVID-19 outbreak, we examined the patients with AMI hospitalized in 2020 (n = 1186) and those in 2017-2019 (n = 4877) using a database of the Miyagi AMI Registry Study. The door-to-device time under the emergency declaration became longer as compared with that of the same period in 2017-2019 [83(65-111) vs 74(54-108) min, p = 0.04]. Importantly, the time delay was noted in only patients with Killip class I on arrival, but not in those with Killip class II-IV. Meanwhile, there were no significant changes in the duration from the symptom onset to hospital arrival, the use rate of ambulance and the performance rate of primary percutaneous coronary intervention before and after the COVID-19 outbreak. Eventually, in-hospital mortality had not deteriorated under the state of emergency (6.7 vs 7.8 %, P = 0.69). Conclusion: The emergence of the COVID-19 outbreak seemed to affect AMI management and highlight understanding the barriers to cardiovascular critical care.

BACKGROUND : Cardiac sarcoidosis is a chronic, inflammatory disease that can affect the heart and often results in heart failure and lethal arrhythmias. A multimodality imaging approach without endomyocardial biopsy allows for the diagnosis of isolated cardiac sarcoidosis. Coronary vasomotion abnormalities are highly prevalent in various cardiovascular and inflammatory diseases. It remains unknown whether active myocardial inflammation due to cardiac sarcoidosis is associated with coronary vasomotion abnormalities. CASE SUMMARY : A 68-year-old man without a past medical history experienced an out-of-hospital cardiac arrest due to ventricular fibrillation and was successfully resuscitated without neurologic sequelae. Coronary angiography showed normal coronary arteries; however, intracoronary acetylcholine provocation testing demonstrated both epicardial coronary and coronary microvascular spasm. He was diagnosed with isolated cardiac sarcoidosis by fulfilling the diagnostic criteria proposed by the Japanese Circulation Society 2016 diagnostic guidelines, including fatal ventricular arrhythmia, focal left ventricular wall asynergy, increased myocardial fluorodeoxyglucose uptake by positron emission tomography, and late gadolinium enhancement by cardiac magnetic resonance in the heart. He was treated with calcium-channel blocker for coronary artery spasm and prednisolone for cardiac sarcoidosis and underwent implantation of an implantable cardioverter-defibrillator for secondary prevention. Following the treatment, the severity of coronary artery spasm was reduced along with regression of the myocardial inflammation. DISCUSSION : Epicardial coronary artery and coronary microvascular spasm can be accompanied by active myocardial inflammation of isolated cardiac sarcoidosis, and the treatment with corticosteroid and calcium-channel blocker may be effective for relieving the severity of coronary artery spasm in association with regression of myocardial inflammation of the disease.

OBJECTIVES: Delirium is an important prognostic factor in postoperative patients undergoing cardiovascular surgery and intervention, including transcatheter aortic valve implantation (TAVI). However, delirium after transcatheter aortic valve implantation (DAT) is difficult to predict and its pathophysiology is still unclear. We aimed to investigate whether preoperative cerebral blood flow (CBF) is associated with DAT and, if so, whether CBF measurement is useful for predicting DAT. METHODS: We evaluated CBF in 50 consecutive patients before TAVI (84.7±4.5 yrs., 36 females) using 99mTc ethyl cysteinate dimer single-photon emission computed tomography. Preoperative CBF of the DAT group (N = 12) was compared with that of the non-DAT group (N = 38) using whole brain voxel-wise analysis with SPM12 and region of interest-based analysis with the easy-Z score imaging system. Multivariable logistic regression analysis with the presence of DAT was used to create its prediction model. RESULTS: The whole brain analysis showed that preoperative CBF in the insula was lower in the DAT than in the non-DAT group (P<0.05, family-wise error correction). Decrease extent ratio in the insula of the DAT group (17.6±11.5%) was also greater relative to that of the non-DAT group (7.0±11.3%) in the region of interest-based analysis (P = 0.007). A model that included preoperative CBF in the insula and conventional indicators (frailty index, short physical performance battery and mini-mental state examination) showed the best predictive power for DAT (AUC 0.882). CONCLUSIONS: These results suggest that preoperative CBF in the insula is associated with DAT and may be useful for its prediction.

ABSTRACT: The endothelium plays a pivotal role in the regulation of vascular tone by synthesizing and liberating endothelium-derived relaxing factors inclusive of vasodilator prostaglandins (e.g. prostacyclin), nitric oxide (NO), and endothelium-dependent hyperpolarization (EDH) factors in a distinct blood vessel-size dependent manner. Large conduit arteries are predominantly regulated by NO and small resistance arteries by EDH factors. Accumulating evidence over the past few decades has demonstrated that endothelial dysfunction and coronary vasomotion abnormalities play crucial roles in the pathogenesis of various cardiovascular diseases. Structural and functional alterations of the coronary microvasculature have been coined as coronary microvascular dysfunction (CMD), which is highly prevalent and associated with adverse clinical outcomes in many clinical settings. The major mechanisms of coronary vasomotion abnormalities include enhanced coronary vasoconstrictive reactivity at epicardial and microvascular levels, impaired endothelium-dependent and -independent coronary vasodilator capacities, and elevated coronary microvascular resistance caused by structural factors. Recent experimental and clinical research has highlighted CMD as the systemic small artery disease beyond the heart, emerging modulators of vascular functions, novel insight into the pathogenesis of cardiovascular diseases associated with CMD, and potential therapeutic interventions to CMD with major clinical implications. Herein, we will summarize the current knowledge on the endothelial modulation of vascular tone as well as the pathogenesis of coronary macro- and microvascular diseases from bench to bedside, with a special emphasis placed on the mechanisms and clinical implications of CMD.

[Figure: see text].

OBJECTIVES: Whether there are prognostic links between coronary morphologies and coronary functional abnormalities was examined in ischemia and nonobstructive coronary artery disease (INOCA) patients. BACKGROUND: Although INOCA has attracted much attention, little is known about the prognostic impact of coronary morphologies in this disorder. METHODS: A total of 329 consecutive INOCA patients were enrolled and underwent spasm provocation testing combined with lactate sampling for diagnosis of epicardial and microvascular spasm (MVS). On the basis of the functional tests, the patients were classified into 4 groups: a control group without epicardial spasm or MVS (n = 32), MVS alone (n = 51), diffuse spasm in ≥2 coronary segments (n = 204), and focal spasm in 1 segment (n = 42). In this population, optical coherence tomography imaging of the left anterior descending coronary artery was performed for evaluation of adventitial vasa vasorum (AVV) and intraplaque neovessels (IPN). Index of microcirculatory resistance was also measured. RESULTS: MVS frequently coexisted with diffuse (70%) and focal spasm (68%) with a good correlation between AVV and index of microcirculatory resistance (R = 0.353; p = 0.022). For a median follow-up of 1,043 days, focal spasm showed the worst prognosis (log rank p = 0.005), for which IPN was a significant prognostic factor. By contrast, diffuse spasm showed the greatest AVV with an intermediate prognosis. The prognostic value of INOCA was significantly enhanced by adding AVV and IPN to the physiological indices (area under the curve = 0.88 vs. 0.76; p = 0.048). CONCLUSIONS: These results provide the first evidence that there are important prognostic links between coronary morphologies (evaluated by optical coherence tomography) and coronary functional abnormalities in patients with INOCA, indicating the importance of both evaluations in this population.

Approximately one-half of patients undergoing diagnostic coronary angiography for angina have no significant coronary atherosclerotic stenosis. This clinical condition has recently been described as ischaemia with non-obstructive coronary arteries (INOCA). Coronary functional abnormalities are central to the pathogenesis of INOCA, including epicardial coronary spasm and coronary microvascular dysfunction composed of a variable combination of increased vasoconstrictive reactivity and/or reduced vasodilator function. During the last decade - in INOCA patients in particular - evidence for the prognostic impact of coronary functional abnormalities has accumulated and various non-invasive and invasive diagnostic techniques have enabled the evaluation of coronary vasomotor function in a comprehensive manner. In this review, the authors briefly summarise the recent advances in the understanding of pathophysiology and diagnosis of epicardial coronary artery spasm and coronary microvascular dysfunction.

There is accumulating evidence highlighting a close relationship between inflammation and coronary microvascular dysfunction (CMD) in various experimental and clinical settings, with major clinical implications. Chronic low-grade vascular inflammation plays important roles in the underlying mechanisms behind CMD, especially in patients with coronary artery disease, obesity, heart failure with preserved ejection fraction and chronic inflammatory rheumatoid diseases. The central mechanisms of coronary vasomotion abnormalities comprise enhanced coronary vasoconstrictor reactivity, reduced endothelium-dependent and -independent coronary vasodilator capacity and increased coronary microvascular resistance, where inflammatory mediators and responses are substantially involved. How to modulate CMD to improve clinical outcomes of patients with the disorder and whether CMD management by targeting inflammatory responses can benefit patients remain challenging questions in need of further research. This review provides a concise overview of the current knowledge of the involvement of inflammation in the pathophysiology and molecular mechanisms of CMD from bench to bedside.

<sec><title>Introduction:</title> Structural and functional abnormalities of coronary microvasculature, referred to as coronary microvascular dysfunction (CMD), have been implicated in a wide range of cardiovascular diseases and have gained growing attention in patients with chest pain with no obstructive coronary artery disease, especially in females. The central mechanisms of coronary vasomotion abnormalities encompass enhanced coronary vasoconstrictive reactivity (ie, coronary spasm), reduced endothelium-dependent and -independent coronary vasodilator capacities, and increased coronary microvascular resistance. The 2 major endothelium-derived relaxing factors, nitric oxide (NO) and endothelium-dependent hyperpolarization (EDH) factors, modulate vascular tone in a distinct vessel size–dependent manner; NO mainly mediates vasodilatation of relatively large, conduit vessels, while EDH factors in small resistance vessels. Endothelium-dependent hyperpolarization–mediated vasodilatation is more prominent in female resistance arteries, where estrogens exert beneficial effects on endothelium-dependent vasodilatation via multiple mechanisms. In the clinical settings, therapeutic approaches targeting NO are disappointing for the treatment of various cardiovascular diseases, where endothelial dysfunction and CMD are substantially involved. </sec><sec><title>Significance:</title> In this review, we will discuss the current knowledge on the pathophysiology and molecular mechanisms of endothelial function and coronary vasomotion abnormalities from bench to bedside, with a special reference to gender differences. </sec><sec><title>Results:</title> Recent experimental and clinical studies have demonstrated distinct gender differences in endothelial function and coronary vasomotion abnormalities with major clinical implications. Moreover, recent landmark clinical trials regarding the management of stable coronary artery disease have questioned the benefit of percutaneous coronary intervention, supporting the importance of the coronary microvascular physiology. </sec><sec><title>Conclusion:</title> Further characterization and a better understanding of the gender differences in basic vascular biology as well as those in cardiovascular diseases are indispensable to improve health care and patient outcomes in cardiovascular medicine. </sec>

AIMS: The aim of this study was to test the hypothesis that coronary microvascular endothelial dysfunction (CMED) is associated with epicardial coronary atherosclerosis. METHODS AND RESULTS: We performed a cross-sectional analysis of a comprehensive invasive assessment of coronary physiology with a focus on endothelium-dependent coronary microvascular function and virtual-histology intravascular ultrasound (VH-IVUS) in a total of 148 consecutive patients with chest pain and angiographically normal coronary arteries or non-obstructive coronary artery disease (CAD). Endothelium-dependent coronary vascular reactivity was evaluated by graded doses of intracoronary acetylcholine (ACh). CMED was defined as a percent increase in coronary blood flow of ≤50% in response to ACh. Patients with CMED (n=87) showed more vulnerable plaque characteristics as compared to those without (n=61); they showed higher plaque burden in association with larger necrotic core volume and higher frequency of imaged arteries containing at least one VH-IVUS-derived thin-capped fibroatheroma (TCFA) (n=22 [25.3%] vs 5 [8.2%], p=0.008). Multivariate logistic regression analysis revealed that CMED was an independent predictor of VH-IVUS-derived TCFA (adjusted odds ratio 2.28 [95% confidence interval: 1.30-4.02], p=0.004). CONCLUSIONS: Independently of conventional coronary risk factors, CMED was associated with vulnerable plaque characteristics in patients with non-obstructive CAD.

The endothelium plays crucial roles in modulating vascular tone by synthesizing and releasing endothelium-derived relaxing factors (EDRFs), including vasodilator prostaglandins, nitric oxide (NO) and endothelium-dependent hyperpolarization (EDH) factors. Thus, endothelial dysfunction is the hallmark of atherosclerotic cardiovascular diseases. Importantly, the contribution of EDRFs to endothelium-dependent vasodilatation varies in a distinct vessel size-dependent manner; NO mainly mediates vasodilatation of relatively large, conduit vessels (eg epicardial coronary arteries), while EDH factors in small resistance vessels (eg coronary microvessels). Endothelium-derived hydrogen peroxide (H2 O2 ) is a physiological signalling molecule serving as one of the major EDH factors especially in microcirculations and has gained increasing attention in view of its emerging relevance for cardiovascular diseases. In the clinical settings, therapeutic approaches targeting NO (eg NO donors) or non-specific elimination of reactive oxygen species (eg antioxidant supplements) are disappointingly ineffective for the treatment of various cardiovascular diseases, in which endothelial dysfunction and coronary microvascular dysfunction are substantially involved. These lines of evidence indicate the potential importance of the physiological balance between NO and H2 O2 /EDH factor. Further characterization and better understanding of endothelium-dependent vasodilatations are important to develop novel therapeutic strategies in cardiovascular medicine. In this MiniReview, we will briefly summarize the current knowledge on the emerging regulatory roles of endothelium-dependent vasodilatations in the cardiovascular system, with a special reference to the two major EDRFs, NO and H2 O2 /EDH factor, in health and disease.

Background: Hyperhomocysteinemia (HHcy) has been proposed as an important cardiovascular risk factor (cRF). However, little is known about the association between plasma homocysteine levels and peripheral microvascular endothelial dysfunction (PMED), which is an integrated index of vascular health. Methods: This cross-sectional and retrospective cohort study included patients who underwent non-invasive PMED assessment using reactive hyperemia peripheral arterial tonometry (RH-PAT). The association between HHcy and PMED, and its impact on MACE (all-cause mortality and atherosclerotic cardiovascular events) was investigated. Results: A total of 257 patients were enrolled (HHcy > 10.0 µmol/L, N = 51; lower levels of homocysteine [LHcy] ≤ 10 µmol/L, N = 206). Patients with HHcy were older, predominantly males, and with more comorbidities than patients with LHcy (p < 0.05 for all). RH-PAT index was lower in patients with HHcy versus LHcy (p = 0.01). A significant association between HHcy and PMED was observed in older (≥60 years), obese (≥30 kg/m2), present/past smokers and hypertensive patients. HHcy was significantly associated with PMED even after adjusting for other cRF and B-vitamins supplementation. HHcy was associated with an increased risk of MACE with a hazard ratio of 3.65 (95% CI 1.41-9.48, p = 0.01) and an adjusted hazard ratio of 2.44 (95% CI 0.91-6.51, p = 0.08) after adjustment for age (≥60 years). Conclusion: HHcy was independently associated with PMED after adjusting for cRF and B-vitamins supplementation. Thus, the link between homocysteine and MACE could be mediated by endothelial dysfunction, and will require further clarification with future studies.

OBJECTIVE: It remains to be elucidated whether and how endothelial functions are impaired in peripheral circulation of patients with coronary functional disorders, such as vasospastic angina (VSA) and microvascular angina (MVA). We simultaneously examined endothelial functions of peripheral conduit and resistance arteries in patients with coronary functional disorders, with a special reference to NO and endothelium-dependent hyperpolarization factors. Approach and Results: Based on the results of invasive coronary acetylcholine testing and coronary physiological measurements, we divided 43 patients into 3 groups; VSA, MVA, and VSA+MVA. Endothelium-dependent vasodilatations of the brachial artery and fingertip arterioles to intra-arterial infusion of bradykinin were simultaneously evaluated by ultrasonography and peripheral arterial tonometry, respectively. To assess NO and endothelium-dependent hyperpolarization factors, measurements were repeated after oral aspirin and intra-arterial infusion of NG-monomethyl-L-arginine. Additionally, endothelium-independent vasodilatations to sublingual nitroglycerin and plasma levels of biomarkers for endothelial functions were measured. Surprisingly, digital vasodilatations to bradykinin were almost absent in patients with MVA alone and those with VSA+MVA compared with those with VSA alone. Mechanistically, both NO- and endothelium-dependent hyperpolarization-mediated digital vasodilatations were markedly impaired in patients with MVA alone. In contrast, endothelium-independent vasodilatations to nitroglycerin were comparable among the 3 groups. Plasma levels of soluble VCAM (vascular cell adhesion molecule)-1 were significantly higher in patients with MVA alone compared with those with VSA alone. CONCLUSIONS: These results provide the first evidence that both NO- and endothelium-dependent hyperpolarization-mediated digital vasodilatations are markedly impaired in MVA patients, suggesting that MVA is a cardiac manifestation of the systemic small artery disease.

Background Coronary artery disease risk factors are associated with atrial fibrillation (AF) and coronary endothelial dysfunction (CED). We hypothesized that CED is associated with increased risk of incident AF among patients with chest pain and nonobstructive coronary artery disease. Methods and Results Three hundred patients with chest pain, nonobstructive coronary artery disease, and no history of AF underwent intracoronary acetylcholine infusion for evaluation of baseline epicardial (decrease in mid-left anterior descending coronary artery diameter in response to acetylcholine) and microvascular (<50% increase in coronary blood flow in response to acetylcholine) CED. Primary outcome was incident AF over a mean follow-up period of 10.5±5.5 years. Mean age was 53.3±10.8 years, and 70% were women. Baseline clinical and echocardiographic characteristics were similar between patients with CED (n=256) and those with normal endothelial function (n=44). Overall, 35 of 300 (12%) patients developed AF, among whom 34 of 35 (97%) had CED at baseline. Compared with normal endothelial function, the presence of CED was associated with 11% increased absolute risk and 5.8-fold increased relative risk of incident AF. Moreover, CED (odds ratio, 3.87; 95% CI, 1.27-47.0) and increased (>34 mL/m2) left atrial volume index (odds ratio, 3.87; 95% CI, 1.60-9.11) were independent predictors of incident AF. Conclusions Patients with normal coronary endothelial function, as compared with those with CED and similar AF risk factors, have significantly lower incidence of AF on long-term follow-up. The potential mechanistic link between vascular dysfunction and AF development warrants further investigation.

AIMS: Cardiovascular health metrics predict the risk not only of cardiovascular diseases but also of several types of cancers. Microvascular endothelial dysfunction can predict future cardiovascular adverse events, but the predictive value of microvascular endothelial dysfunction for future risk of solid-tumor cancer has not been characterized. METHODS: A total of 488 patients who underwent microvascular endothelial function assessment using reactive hyperemia peripheral arterial tonometry were included in this study. Microvascular endothelial dysfunction was defined as a reactive hyperemia peripheral arterial tonometry index ≤2.0. RESULTS: Of 221 patients with a baseline reactive hyperemia peripheral arterial tonometry index ≤2.0, 21 patients (9.5%) were diagnosed with incident solid-tumor cancer during follow-up, whereas of 267 patients with a baseline reactive hyperemia peripheral arterial tonometry index >2.0, 10 patients (3.7%) were diagnosed with incident solid-tumor cancer during follow-up (p = 0.009). Patients with a reactive hyperemia peripheral arterial tonometry index ≤2.0 had lower solid-tumor cancer-free survival compared to patients with a reactive hyperemia peripheral arterial tonometry index >2.0 (log-rank p = 0.017) (median follow-up 6.0 (3.0-9.1) years). Cox proportional hazard analyses showed that a reactive hyperemia peripheral arterial tonometry index ≤2.0 predicted the incidence of solid-tumor cancer, with a hazard ratio of 2.52 (95% confidence interval 1.17-5.45; p = 0.019) after adjusting for age, sex, and coronary artery disease, 2.83 (95% confidence interval 1.30-6.17; p = 0.009) after adjusting for diabetes mellitus, hypertension, smoking status, and body mass index >30 kg/m2, 2.79 (95% confidence interval 1.21-6.41; p = 0.016) after adjusting for fasting plasma glucose, systolic blood pressure, smoking status (current or former), and body mass index, and 2.43 (95% confidence interval 1.10-5.34; p = 0.028) after adjusting for Framingham risk score. CONCLUSION: Microvascular endothelial dysfunction, as defined by a reactive hyperemia peripheral arterial tonometry index ≤2.0, was associated with a greater than two-fold increased risk of solid-tumor cancer. Microvascular endothelial dysfunction may be a useful marker to predict the future risk of solid-tumor cancer, in addition to its known ability to predict cardiovascular disease. Further research is necessary to develop adequate cancer screening strategies for patients with microvascular endothelial dysfunction.

Diffuse and focal epicardial coronary disease and coronary microvascular abnormalities may exist side-by-side. Identifying the contributions of each of these three players in the coronary circulation is a difficult task. Yet identifying coronary microvascular dysfunction (CMD) as an additional player in patients with coronary artery disease (CAD) may provide explanations of why symptoms may persist frequently following and why global coronary flow reserve may be more prognostically important than fractional flow reserve measured in a single vessel before percutaneous coronary intervention. This review focuses on the challenges of identifying the presence of CMD in the context of diffuse non-obstructive CAD and obstructive CAD. Furthermore, it is going to discuss the pathophysiology in this complex situation, examine the clinical context in which the interaction of the three components of disease takes place and finally look at non-invasive diagnostic methods relevant for addressing this question.

Endothelium-dependent hyperpolarization (EDH) factor is one of endothelium-derived relaxing factors and plays important roles especially in microvessels. We have previously demonstrated that endothelium-derived hydrogen peroxide (H2O2) is an EDH factor produced by all types of nitric oxide synthases (NOSs), including endothelial NOS (eNOS), neuronal NOS (nNOS), and inducible NOS. Recent studies have suggested the association between coronary microvascular dysfunction and cardiac diastolic dysfunction. However, the role of EDH in this issue remains to be fully elucidated. We thus examined whether EDH plays an important role in coronary microcirculation and if so, whether endothelial dysfunction, especially impaired EDH, is involved in the pathogenesis of cardiac diastolic dysfunction in mice. Using a Langendorff-perfused heart experiment, we examined the increase in coronary flow in response to bradykinin in the presence of indomethacin and N-nitro-L-arginine (EDH condition) in wild-type, eNOS-knockout (KO), and nNOS/eNOS-double-KO mice. Compared with wild-type mice, EDH-mediated relaxations were increased in eNOS-KO mice but were significantly reduced in n/eNOS-KO mice. Catalase, a specific H2O2 scavenger, markedly inhibited EDH-mediated relaxations in all 3 genotypes, indicating compensatory roles of nNOS-derived H2O2 as an EDH factor in coronary microcirculation. Although both eNOS-KO and n/eNOS-KO mice exhibited similar extents of cardiac morphological changes, only n/eNOS-KO mice exhibited cardiac diastolic dysfunction. The expression of oxidized protein kinase G I-α (PKGIα) in the heart was significantly increased in eNOS-KO mice compared with n/eNOS-KO mice. These results indicate that EDH/H2O2 plays important roles in maintaining coronary microcirculation and cardiac diastolic function through oxidative PKGIα activation.

Although increased levels of reactive oxygen species (ROS) are involved in the pathogenesis of cardiovascular diseases, the importance of physiological ROS has also been emerging. We have previously demonstrated that endothelium-derived H2O2 is an endothelium-dependent hyperpolarization (EDH) factor and that loss of endothelial caveolin-1 reduces EDH/H2O2 in the microcirculation. Caveolin-1 (Cav-1) is a scaffolding/regulatory protein that interacts with diverse signaling pathways, including angiogenesis. However, it remains unclear whether endothelial Cav-1 plays a role in ischemic angiogenesis by modulating EDH/H2O2. In the present study, we thus addressed this issue in a mouse model of hindlimb ischemia using male endothelium-specific Cav-1 (eCav-1) knockout (KO) mice. In isometric tension experiments with femoral arteries from eCav-1-KO mice, reduced EDH-mediated relaxations to acetylcholine and desensitization of sodium nitroprusside-mediated endothelium-independent relaxations were noted ( n = 4~6). An ex vivo aortic ring assay also showed that the extent of microvessel sprouting was significantly reduced in eCav-1-KO mice compared with wild-type (WT) littermates ( n = 12 each). Blood flow recovery at 4 wk assessed with a laser speckle flowmeter after femoral artery ligation was significantly impaired in eCav-1-KO mice compared with WT littermates ( n = 10 each) and was associated with reduced capillary density and muscle fibrosis in the legs ( n = 6 each). Importantly, posttranslational protein modifications by reactive nitrogen species and ROS, as evaluated by thiol glutathione adducts and nitrotyrosine, respectively, were both increased in eCav-1-KO mice ( n = 6~7 each). These results indicate that endothelial Cav-1 plays an important role in EDH-mediated vasodilatation and ischemic angiogenesis through posttranslational protein modifications by nitrooxidative stress in mice in vivo. NEW & NOTEWORTHY Although increased levels of reactive oxygen species (ROS) are involved in the pathogenesis of cardiovascular diseases, the importance of physiological ROS has also been emerging. The present study provides a line of novel evidence that endothelial caveolin-1 plays important roles in endothelium-dependent hyperpolarization and ischemic angiogenesis in hindlimb ischemia in mice through posttranslational protein modifications by reactive nitrogen species and ROS in mice in vivo.

Axitinib has emerged as a promising antineoplastic agent for the treatment of advanced renal cell carcinoma. Although the administration of axitinib was well-tolerated in clinical trials, the real-world safety and tolerability remain unverified. We herein report a patient with metastatic renal cell carcinoma who suddenly developed life-threatening hyperkalemia following the initiation of axitinib treatment. Although hyperkalemia has been reported with an incidence of <10%, acute severe hyperkalemia may be a considerably critical adverse event of axitinib therapy, especially in patients with risk factors for hyperkalemia. An abundance of caution for unusual and unpredictable toxicities is warranted when using axitinib.

Endothelium-dependent hyperpolarization (EDH) plays important roles in the systemic circulation, whereas its role in the pulmonary circulation remains largely unknown. Furthermore, the underlying mechanisms of pulmonary hypertension (PH) also remain to be elucidated. We thus aimed to elucidate the role of EDH in pulmonary circulation in general and in PH in particular. In isolated perfused lung and using male wild-type mice, endothelium-dependent relaxation to bradykinin (BK) was significantly reduced in the presence of Nω-nitro-l-arginine by ~50% compared with those in the presence of indomethacin, and the combination of apamin plus charybdotoxin abolished the residual relaxation, showing the comparable contributions of nitric oxide (NO) and EDH in the pulmonary microcirculation under physiological conditions. Catalase markedly inhibited EDH-mediated relaxation, indicating the predominant contribution of endothelium-derived H2O2. BK-mediated relaxation was significantly reduced at day 1 of hypoxia, whereas it thereafter remained unchanged until day 28. EDH-mediated relaxation was diminished at day 2 of hypoxia, indicating a transition from EDH to NO in BK-mediated relaxation before the development of hypoxia-induced PH. Mechanistically, chronic hypoxia enhanced endothelial NO synthase expression and activity associated with downregulation of caveolin-1. Nitrotyrosine levels were significantly higher in vascular smooth muscle of pulmonary microvessels under chronic hypoxia than under normoxia. A similar transition of the mediators in BK-mediated relaxation was also noted in the Sugen hypoxia mouse model. These results indicate that EDH plays important roles in the pulmonary microcirculation in addition to NO under normoxic conditions and that impaired EDH-mediated relaxation and subsequent nitrosative stress may be potential triggers of the onset of PH. NEW & NOTEWORTHY This study provides novel evidence that both endothelium-dependent hyperpolarization and nitric oxide play important roles in endothelium-dependent relaxation in the pulmonary microcirculation under physiological conditions in mice and that hypoxia first impairs endothelium-dependent hyperpolarization-mediated relaxation, with compensatory upregulation of nitric oxide, before the development of hypoxia-induced pulmonary hypertension.

AIMS: Nitric oxide (NO) and endothelium-dependent hyperpolarization (EDH) play important roles in maintaining cardiovascular homeostasis. We have previously demonstrated that endothelial NO synthase (eNOS) plays diverse roles depending on vessel size, as a NO generating system in conduit arteries and an EDH-mediated system in resistance arteries, for which caveolin-1 (Cav-1) is involved. However, the physiological role of endothelial Cav-1 in microvessels remains to be elucidated. METHODS AND RESULTS: We newly generated endothelium-specific Cav-1-knockout (eCav-1-KO) mice. eCav-1-KO mice showed loss of endothelial Cav-1/eNOS complex and had cardiac hypertrophy despite normal blood pressure. In eCav-1-KO mice, as compared to wild-type controls, the extent of eNOS phosphorylation at inhibitory Thr495 was significantly reduced in mesenteric arteries and the heart. Isometric tension and Langendorff-perfused heart experiments showed that NO-mediated responses were enhanced, whereas EDH-mediated responses were reduced in coronary microcirculation in eCav-1-KO mice. Immunohistochemistry showed increased level of 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP), a marker of nitrative stress, in the heart from eCav-1-KO mice. S-guanylation of cardiac H-Ras in eCav-1-KO mice was also significantly increased compared with wild-type controls. CONCLUSIONS: These results suggest that eCav-1 is involved in the protective role of EDH against nitrative stress caused by excessive NO to maintain cardiac microvascular homeostasis.

Thyroid storm is a life-threatening disorder that remains a therapeutic challenge. Although β-blockers are the mainstay for treatment, their use can be challenging in cases complicated by rapid atrial fibrillation and decompensated heart failure. We present a case of thyroid storm-associated atrial fibrillation and decompensated heart failure complicated by gastrointestinal dysfunction secondary to diffuse peritonitis that was successfully managed by a switching therapy, in which the continuous intravenous administration of landiolol was changed to bisoprolol via transdermal patch, in the acute phase treatment. This switching therapy may offer a promising therapeutic option for this potentially lethal disorder.

The endothelium plays important roles in modulating vascular tone by synthesizing and releasing a variety of endothelium-derived relaxing factors, including vasodilator prostaglandins, NO, and endothelium-dependent hyperpolarization factors, as well as endothelium-derived contracting factors. Endothelial dysfunction is mainly caused by reduced production or action of these relaxing mediators. Accumulating evidence has demonstrated that endothelial functions are essential to ensure proper maintenance of vascular homeostasis and that endothelial dysfunction is the hallmark of a wide range of cardiovascular diseases associated with pathological conditions toward vasoconstriction, thrombosis, and inflammatory state. In the clinical settings, evaluation of endothelial functions has gained increasing attention in view of its emerging relevance for cardiovascular disease. Recent experimental and clinical studies in the vascular biology field have demonstrated a close relationship between endothelial functions and cardiovascular disease and the highlighted emerging modulators of endothelial functions, new insight into cardiovascular disease associated with endothelial dysfunction, and potential therapeutic and diagnostic targets with major clinical implications. We herein will summarize the current knowledge on endothelial functions from bench to bedside with particular focus on recent publications in Arteriosclerosis, Thrombosis, and Vascular Biology.

Paroxysmal sympathetic hyperactivity (PSH) is a distinct syndrome of episodic sympathetic hyperactivities following severe acquired brain injury, characterized by paroxysmal transient fever, tachycardia, hypertension, tachypnea, excessive diaphoresis and specific posturing. PSH remains to be an under-recognized condition with a diagnostic pitfall especially in the intensive care unit (ICU) settings due to the high prevalence of concomitant diseases that mimic PSH. A consensus set of diagnostic criteria named PSH-Assessment Measure (PSH-AM) has been developed recently, which is consisted of two components: a diagnosis likelihood tool derived from clinical characteristics of PSH, and a clinical feature scale assigned to the severity of each sympathetic hyperactivity. We herein present a case series of patients with PSH who were diagnosed and followed by using PSH-AM in our tertiary institutional medical and surgical ICU between April 2015 and March 2017 in order to evaluate the clinical efficacy of PSH-AM. Among 394 survivors of 521 patients admitted with acquired brain injury defined as acute brain injury at all levels of severity regardless of the presence of altered consciousness, including traumatic brain injury, stroke, infectious disease, and encephalopathy, 6 patients (1.5%) were diagnosed as PSH by using PSH-AM. PSH-AM served as a useful scoring system for early objective diagnosis, assessment of severity, and serial evaluation of treatment efficacy in the management of PSH in the ICU settings. In conclusion, critical care clinicians should consider the possibility of PSH and can use PSH-AM as a useful diagnostic and guiding tool in the management of PSH.

Accumulating evidence has demonstrated the importance of reactive oxygen species (ROS) as an essential second messenger in health and disease. Endothelial dysfunction is the hallmark of atherosclerotic cardiovascular diseases, in which pathological levels of ROS are substantially involved. The endothelium plays a crucial role in modulating tone of underlying vascular smooth muscle by synthesizing and releasing nitric oxide (NO) and endothelium-dependent hyperpolarization (EDH) factors in a distinct vessel size-dependent manner through the diverse roles of the endothelial NO synthases (NOSs) system. Endothelium-derived hydrogen peroxide (H2O2) is a physiological signaling molecule serving as one of the major EDH factors especially in microcirculations and has gained increasing attention in view of its emerging relevance for cardiovascular homeostasis. In the clinical settings, it has been reported that antioxidant supplements are unexpectedly ineffective to prevent cardiovascular events. These lines of evidence indicate the potential importance of the physiological balance between NO and H2O2/EDH through the diverse functions of endothelial NOSs system in maintaining cardiovascular homeostasis. A better understanding of cardiovascular redox signaling is certainly needed to develop novel therapeutic strategies in cardiovascular medicine. In this review, we will briefly summarize the current knowledge on the emerging regulatory roles of redox signaling pathways in cardiovascular homeostasis, with particular focus on the two endothelial NOSs-derived mediators, NO and H2O2/EDH.

The specific roles of nitric oxide (NO) synthases (NOSs) in bladder smooth muscle remain to be elucidated. We examined the roles of NOSs in β-adrenoceptor (AR)-mediated bladder relaxation. Male mice (C57BL6) deficient of neuronal NOS [nNOS-knockout (KO)], endothelial NOS (eNOS-KO), neuronal/endothelial NOS (n/eNOS-KO), neuronal/endothelial/inducible NOS (n/e/iNOS-KO), and their controls [wild-type (WT)] were used. Immunohistochemical analysis was performed in the bladder. Then the responses to relaxing agents and the effects of several inhibitors on the relaxing responses were examined in bladder strips precontracted with carbachol. Immunofluorescence staining showed expressions of nNOS and eNOS in the urothelium and smooth muscle of the bladder. Isoproterenol-induced relaxations were significantly reduced in nNOS-KO mice and were further reduced in n/eNOS-KO and n/e/iNOS-KO mice compared with WT mice. The relaxation in n/e/iNOS-KO mice was almost the same as in n/eNOS-KO mice. Inhibition of Ca2+-activated K+ (KCa) channel with charybdotoxin and apamin abolished isoproterenol-induced bladder relaxation in WT mice. Moreover, direct activation of KCa channel with NS1619 caused comparable extent of relaxations among WT, nNOS-KO, and n/eNOS-KO mice. In contrast, NONOate (a NO donor) or hydrogen peroxide (H2O2) (another possible relaxing factor from eNOS) caused minimal relaxations, and catalase (H2O2 scavenger) had no inhibitory effects on isoproterenol-induced relaxations. These results indicate that both nNOS and eNOS are substantially involved in β-AR-mediated bladder relaxations in a NO- or H2O2-independent manner through activation of KCa channels.

Biguanides are a drug of choice for the treatment of type 2 diabetes mellitus. Although they can cause lactic acidosis in susceptible patients with predisposing risk factors, the incidence of lactic acidosis is reported to be very low when they are used properly. We herein present a case of biguanide-associated severe lactic acidosis complicated with thiamine deficiency that was provoked without predisposing factors for thiamine deficiency. Diabetic patients taking biguanide may be predisposed to thiamine deficiency, even when there is no evidence of risk factors, and the high-dose administration of thiamine may be essential in the treatment of this otherwise under-recognized disorder.

Endothelium-dependent relaxations are predominantly regulated by nitric oxide (NO) in large conduit arteries and by endothelium-dependent hyperpolarization (EDH) in small resistance vessels. Although the nature of EDH factors varies depending on species and vascular beds, we have previously demonstrated that endothelial NO synthases (eNOS)-derived hydrogen peroxide (H2O2) is an EDH factor in animals and humans. This vessel size-dependent contribution of NO and EDH is, at least in part, attributable to the diverse roles of endothelial NOSs system; in large conduit arteries, eNOS mainly serves as a NO-generating system to elicit soluble guanylate cyclase-cyclic guanosine monophosphate-mediated relaxations, whereas in small resistance vessels, it serves as a superoxide-generating system to cause EDH/H2O2-mediated relaxations. Endothelial caveolin-1 may play an important role for the diverse roles of NOSs. Although reactive oxygen species are generally regarded harmful, the physiological roles of H2O2 have attracted much attention as accumulating evidence has shown that endothelium-derived H2O2 contributes to cardiovascular homeostasis. The diverse functions of endothelial NOSs system with NO and EDH/H2O2 could account for a compensatory mechanism in the setting of endothelial dysfunction. In this review, we will briefly summarize the current knowledge on the diverse functions of endothelial NOSs system: NO and EDH/H2O2.

OBJECTIVE: Endothelium-derived nitric oxide (NO) and endothelium-dependent hyperpolarization (EDH) play important roles in modulating vascular tone in a distinct vessel size-dependent manner; NO plays a dominant role in conduit arteries and EDH in resistance vessels. We have recently demonstrated that endothelial NO synthase (eNOS) is functionally suppressed in resistance vessels through caveolin-1 (Cav-1)-dependent mechanism, switching its function from NO to EDH/hydrogen peroxide generation in mice. Here, we examined the possible importance of the physiological balance between NO and EDH in cardiovascular homeostasis. APPROACH AND RESULTS: We used 2 genotypes of mice in which eNOS activity is genetically upregulated; Cav-1-knockout (Cav-1-KO) and endothelium-specific eNOS transgenic (eNOS-Tg) mice. Isometric tension recordings and Langendorff experiments with isolated perfused hearts showed that NO-mediated relaxations were significantly enhanced, whereas EDH-mediated relaxations were markedly reduced in microcirculations. Importantly, impaired EDH-mediated relaxations of small mesenteric arteries from Cav-1-KO mice were completely rescued by crossing the mice with those with endothelium-specific overexpression of Cav-1. Furthermore, both genotypes showed altered cardiovascular phenotypes, including cardiac hypertrophy in Cav-1-KO mice and hypotension in eNOS-Tg mice. Finally, we examined cardiac responses to chronic pressure overload by transverse aortic constriction in vivo. When compared with wild-type mice, both Cav-1-KO and eNOS-Tg mice exhibited reduced survival after transverse aortic constriction associated with accelerated left ventricular systolic dysfunction, reduced coronary flow reserve, and enhanced myocardial hypoxia. CONCLUSIONS: These results indicate that excessive endothelium-derived NO with reduced EDH impairs cardiovascular homeostasis in mice in vivo.

Angiotensin II (Ang II), a potent precursor of hypertrophy and heart failure, upregulates neuronal nitric oxide synthase (nNOS or NOS1) in the myocardium. Here, we investigate the involvement of type 1 and 2 angiotensin receptors (AT1R and AT2R) and molecular mechanisms mediating Ang II-upregulation of nNOS. Our results showed that pre-treatment of left ventricular (LV) myocytes with antagonists of AT1R or AT2R (losartan, PD123319) and ROS scavengers (apocynin, tiron or PEG-catalase) blocked Ang II-upregulation of nNOS. Surface biotinylation or immunocytochemistry experiments demonstrated that AT1R expression in plasma membrane was progressively decreased (internalization), whereas AT2R was increased (membrane trafficking) by Ang II. Inhibition of AT1R or ROS scavengers prevented Ang II-induced translocation of AT2R to plasma membrane, suggesting an alignment of AT1R-ROS-AT2R. Furthermore, Ang II increased eNOS-Ser(1177) but decreased eNOS-Thr(495), indicating concomitant activation of eNOS. Intriguingly, ROS scavengers but not AT2R antagonist prevented Ang II-activation of eNOS. NOS inhibitor (L-NG-Nitroarginine Methyl Ester, L-NAME) or eNOS gene deletion (eNOS(-/-)) abolished Ang II-induced membrane trafficking of AT2R, nNOS protein expression and activity. Mechanistically, S-nitrosation of AT2R was increased by sodium nitroprusside (SNP), a NO donor. Site-specific mutagenesis analysis reveals that C-terminal cysteine 349 in AT2R is essential in AT2R translocation to plasma membrane. Taken together, we demonstrate, for the first time, that Ang II upregulates nNOS protein expression and activity via AT1R/ROS/eNOS-dependent S-nitrosation and membrane translocation of AT2R. Our results suggest a novel crosstalk between AT1R and AT2R in regulating nNOS via eNOS in the myocardium under pathogenic stimuli.

Dyslipidemia is a life-style disorder and is one of the important risk factors of cardiovascular diseases. Nitric oxide (NO) exerts beneficial effects on lipid metabolism through activation of hepatic sterol regulatory element-binding protein (SREBP)-2, a transcriptional factor for cholesterol metabolism and expression of LDL receptor, while Rho-kinase, an effecter protein of small G protein, RhoA, contributes to the pathogenesis of metabolic syndrome through suppressing the whole body energy consumption. However, the crosstalk between NO and Rho-kinase in regulation of lipid metabolism remains to be elucidated. In the present study, we used male wild-type (WT) mice and mice lacking three isoforms of NO synthase (NOSs(-/-)). WT mice were fed either normal diet (ND) or high-fat diet (HFD), while NOSs(-/-) mice were fed ND with or without a selective Rho-kinase inhibitor, fasudil (100 mg/kg/day), for 6 weeks. At 6 weeks, plasma NOx concentration was significantly decreased and Rho-kinase activity and lipid levels were significantly elevated in HFD-fed WT mice and NOSs(-/-) mice compared with ND-fed WT mice. In the liver, SREBP-2 activity was reduced in NOSs(-/-) mice. Fasudil ameliorated lipid levels in HFD-fed WT mice and NOSs(-/-) mice without affecting SREBP-2 activity or LDL receptor expression, whereas it significantly enhanced phosphorylation of AMP-activated kinase (AMPK) in the liver and skeletal muscle. Importantly, the beneficial metabolic effects of fasudil were absent in HFD-fed AMPK(-/-) mice. These results provide the first evidence that NO and Rho-kinase play opposing roles for the lipid metabolism, suggesting that Rho-kinase inhibitors could be novel therapeutic agents of dyslipidemia.

Reactive oxygen species (ROS) have been considered to play a major role in the pathogenesis of cardiovascular diseases. However, this notion needs to be revised since recent evidence indicates that vascular-derived hydrogen peroxide (H2O2) serves as an important signaling molecule in the cardiovascular system at its low physiological concentrations. At low concentrations, H2O2 can act as a second messenger, transducing the oxidative signal into biological responses through post-translational protein modification. These structural changes ultimately lead to altered cellular function. Intracellular redox status is closely regulated by the balance between oxidant and antioxidant systems and their imbalance can cause oxidative or reductive stress, leading to cellular damage and dysregulation. For example, excessive H2O2 deteriorates vascular functions and promotes vascular disease through multiple pathways. Furthermore, cyclophilin A (CyPA) has been shown to be secreted from vascular smooth muscle cells and to augment the destructive effects of ROS, linking it to the development of many cardiovascular diseases. Thus, it is important to understand the H2O2 signaling and the roles of downstream effectors such as CyPA in the vascular system in order to develop new therapeutic strategies for cardiovascular diseases. In this review, we will discuss the dual roles of vascular-derived H2O2 in mediating vascular functions (physiological roles) and promoting vascular diseases (pathological roles), with particular emphasis on the function of CyPA. This article is part of a Special Issue entitled "Redox Signalling in the Cardiovascular System".

OBJECTIVE: Vascular endothelium plays an important role to maintain cardiovascular homeostasis through several mechanisms, including endothelium-dependent hyperpolarization (EDH). We have recently demonstrated that EDH is involved in endothelial metabolic regulation in mice. However, it remains to be examined whether AMP-activated protein kinase (AMPK), an important metabolic regulator, is involved in EDH and if so, whether endothelial AMPK (eAMPK) plays a role for circulatory regulation. APPROACH AND RESULTS: We examined the role of eAMPK in EDH, using mice with endothelium-specific deficiency of α-catalytic subunit of AMPK, either α1 (eAMPKα1 (-/-)α2 (+/+)) or α2 (eAMPKα1 (+/+)α2 (-/-)) alone or both of them (eAMPKα1 (-/-)α2 (-/-)). We performed telemetry, organ chamber, electrophysiological, and Langendorff experiments to examine blood pressure, vascular responses, hyperpolarization of membrane potential, and coronary flow responses, respectively. Hypertension was noted throughout the day in eAMPKα1 (-/-)α2 (-/-) and eAMPKα1 (-/-)α2 (+/+) but not in eAMPKα1 (+/+)α2 (-/-) mice when compared with respective control. Importantly, endothelium-dependent relaxations, EDH, and coronary flow increase were all significantly reduced in eAMPKα1 (-/-)α2 (-/-) and eAMPKα1 (-/-)α2 (+/+) but not in eAMPKα1 (+/+)α2 (-/-) mice. In contrast, endothelium-independent relaxations to sodium nitroprusside (a NO donor), NS-1619 (a Ca(2+)-activated K(+) channel opener), and exogenous H2O2 were almost comparable among the groups. In eAMPKα1 (-/-)α2 (-/-) mice, antihypertensive treatment with hydralazine or long-term treatment with metformin (a stimulator of AMPK) failed to restore EDH-mediated responses. CONCLUSIONS: These results provide the first direct evidence that α1 subunit of eAMPK substantially mediates EDH responses of microvessels and regulates blood pressure and coronary flow responses in mice in vivo, demonstrating the novel role of eAMPK in cardiovascular homeostasis.

BACKGROUND: Metabolic disorders, caused by excessive calorie intake and low physical activity, are important cardiovascular risk factors. Rho-kinase, an effector protein of the small GTP-binding protein RhoA, is an important cardiovascular therapeutic target and its activity is increased in patients with metabolic syndrome. We aimed to examine whether Rho-kinase inhibition improves high-fat diet (HFD)-induced metabolic disorders, and if so, to elucidate the involvement of AMP-activated kinase (AMPK), a key molecule of metabolic conditions. METHODS AND RESULTS: Mice were fed a high-fat diet, which induced metabolic phenotypes, such as obesity, hypercholesterolemia and glucose intolerance. These phenotypes are suppressed by treatment with selective Rho-kinase inhibitor, associated with increased whole body O2 consumption and AMPK activation in the skeletal muscle and liver. Moreover, Rho-kinase inhibition increased mRNA expression of the molecules linked to fatty acid oxidation, mitochondrial energy production and glucose metabolism, all of which are known as targets of AMPK in those tissues. In systemic overexpression of dominant-negative Rho-kinase mice, body weight, serum lipid levels and glucose metabolism were improved compared with littermate control mice. Furthermore, in AMPKα2-deficient mice, the beneficial effects of fasudil, a Rho-kinase inhibitor, on body weight, hypercholesterolemia, mRNA expression of the AMPK targets and increase of whole body O2 consumption were absent, whereas glucose metabolism was restored by fasudil to the level in wild-type mice. In cultured mouse myocytes, pharmacological and genetic inhibition of Rho-kinase increased AMPK activity through liver kinase b1 (LKB1), with up-regulation of its targets, which effects were abolished by an AMPK inhibitor, compound C. CONCLUSIONS: These results indicate that Rho-kinase inhibition ameliorates metabolic disorders through activation of the LKB1/AMPK pathway, suggesting that Rho-kinase is also a novel therapeutic target of metabolic disorders.