Identification of Celastramycin as a Novel Therapeutic Agent for Pulmonary Arterial Hypertension.

RATIONALE: Pulmonary arterial hypertension (PAH) is characterized by enhanced proliferation of pulmonary artery smooth muscle cells (PASMCs) accompanying increased production of inflammatory factors and adaptation of the mitochondrial metabolism to a hyperproliferative state. However, all the drugs in clinical use target pulmonary vascular dilatation, which may not be effective for patients with advanced PAH. OBJECTIVE: We aimed to discover a novel drug for PAH that inhibits PASMC proliferation. METHODS AND RESULTS: We screened 5562 compounds from original library using high-throughput screening system to discover compounds which inhibit proliferation of PASMCs from patients with PAH (PAH-PASMCs). We found that celastramycin, a benzoyl pyrrole-type compound originally found in a bacteria extract, inhibited the proliferation of PAH-PASMCs in a dose-dependent manner with relatively small effects on PASMCs from healthy donors. Then, we made 25 analogs of celastramycin and selected the lead compound, which significantly inhibited cell proliferation of PAH-PASMCs and reduced cytosolic reactive oxygen species levels. Mechanistic analysis demonstrated that celastramycin reduced the protein levels of HIF-1α (hypoxia-inducible factor 1α), which impairs aerobic metabolism, and κB (nuclear factor-κB), which induces proinflammatory signals, in PAH-PASMCs, leading to reduced secretion of inflammatory cytokine. Importantly, celastramycin treatment reduced reactive oxygen species levels in PAH-PASMCs with increased protein levels of Nrf2 (nuclear factor erythroid 2-related factor 2), a master regulator of cellular response against oxidative stress. Furthermore, celastramycin treatment improved mitochondrial energy metabolism with recovered mitochondrial network formation in PAH-PASMCs. Moreover, these celastramycin-mediated effects were regulated by ZFC3H1 (zinc finger C3H1 domain-containing protein), a binding partner of celastramycin. Finally, celastramycin treatment ameliorated pulmonary hypertension in 3 experimental animal models, accompanied by reduced inflammatory changes in the lungs. CONCLUSIONS: These results indicate that celastramycin ameliorates pulmonary hypertension, reducing excessive proliferation of PAH-PASMCs with less inflammation and reactive oxygen species levels, and recovered mitochondrial energy metabolism. Thus, celastramycin is a novel drug for PAH that targets antiproliferative effects on PAH-PASMCs.

Clinical risk factors of stroke and major bleeding in patients with non-valvular atrial fibrillation under rivaroxaban: the EXPAND Study sub-analysis.

For Japanese patients with non-valvular atrial fibrillation (NVAF), the risk of stroke and major bleeding events was assessed by using the CHADS2, CHA2DS2-VASc, and HAS-BLED scores. The risk factors for embolism and major bleeding under DOAC may be different from current reports. We analyzed the data set of the EXPAND Study to determine the risk factors for events among Japanese NVAF patients in the era of direct oral anticoagulant. Using the data of EXPAND Study, the validity for predictability of the CHADS2, CHA2DS2-VASc, and HAS-BLED scores was identified using the receiver operating characteristic curve analysis. Multivariate analysis was performed with the Cox proportional hazard model to determine the independent risk factors for stroke/systemic embolism and major bleeding among NVAF patients receiving rivaroxaban. Explanatory variables were selected based on the univariate analysis. A total of 7141 patients (mean age 71.6 ± 9.4 years, women 32.3%, and rivaroxaban 15 mg per day 56.5%) were included. Incidence rates of stroke/systemic embolism and major bleeding were 1.0%/year and 1.2%/year, respectively. The multivariate analysis revealed that only history of stroke was associated with stroke/systemic embolism (hazard ratio 3.4, 95% confidence interval 2.5-4.7, p < 0.0001). By contrast, age (1.7, 1.1-2.6, p = 0.0263), creatinine clearance (CrCl) 30-49 mL/min (1.6, 1.2-2.2, p = 0.0011), liver dysfunction (1.7, 1.1-2.8, p = 0.0320), history/disposition of bleeding (1.8, 1.0-3.0, p = 0.0348), and concomitant use of antiplatelet agents (1.6, 1.2-2.3, p = 0.0030) were associated with major bleeding. This sub-analysis showed that some components of the HAS-BLED score were independently associated with major bleeding in Japanese NVAF patients receiving anticoagulation therapy by rivaroxaban. Additionally, CrCl value of 30-49 mL/min was an independent predictor of major bleeding in patients receiving rivaroxaban.

The EXPAND study: Efficacy and safety of rivaroxaban in Japanese patients with non-valvular atrial fibrillation.

AIMS: The EXPAND study examined the real-world efficacy and safety of rivaroxaban for the prevention of stroke and systemic embolism (SE) in Japanese patients with non-valvular atrial fibrillation (NVAF). METHODS AND RESULTS: This multicenter, prospective, non-interventional, observational, cohort study was conducted at 684 medical centers in Japan. A total of 7141 NVAF patients ≥20 years of age (mean, 71.6 ±â€¯9.4 years) who were being or about to be treated with rivaroxaban (10 mg/day, 43.5%; 15 mg/day, 56.5%) were followed for an average of 897.1 (±206.8) days with a high follow-up rate (99.65%). The mean CHADS2 score at baseline was 2.1 (1.3) (0-1, 37%; 2, 29%; ≥3, 34%). The total incidence rate of symptomatic stroke and SE (primary efficacy endpoint) was 1.0%/year, and 0.5%, 0.9%, and 1.7%/year for those with CHADS2 scores of 0-1, 2, and ≥3, respectively. Cumulative incidence rates for major bleeding (primary safety endpoint) and non-major bleeding (secondary safety endpoint) were 1.2%/year and 4.9%/year, respectively. Differences were noted between new and current users only for major bleeding event rate (1.7% vs. 1.1%/year, P = 0.0024). Comparisons with previous studies suggested that rivaroxaban is effective and safe for low-risk patients (0-1 CHADS2), as shown for warfarin in the XANTUS international prospective post-marketing study. CONCLUSIONS: The EXPAND study demonstrated that low dosages of rivaroxaban for Japanese NVAF patients in real-world clinical practice, including those with CHADS2 scores 0-1, resulted in low rates of stroke and SE, and major and non-major bleeding.

The Dramatic Recovery of a Patient with Biguanide-associated Severe Lactic Acidosis Following Thiamine Supplementation.

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.

Crucial roles of nitric oxide synthases in ß-adrenoceptor-mediated bladder relaxation in mice.

The specific roles of nitric oxide (NO) synthases (NOSs) in bladder smooth muscle remain to be elucidated. We examined the roles of NOSs in ß-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 ß-AR-mediated bladder relaxations in a NO- or H2O2-independent manner through activation of KCa channels.

Study Design and Baseline Characteristics of the EXPAND Study: Evaluation of Effectiveness and Safety of Xa Inhibitor, Rivaroxaban for the Prevention of Stroke and Systemic Embolism in a Nationwide Cohort of Japanese Patients Diagnosed as Non-Valvular Atrial Fibrillation.

The use of rivaroxaban, a factor Xa inhibitor, has been increasing for prevention of ischemic stroke and systemic embolism in patients with non-valvular atrial fibrillation (AF) in Japan. We conducted the nationwide multicenter study, termed as the EXPAND Study, to address its effectiveness and safety in the real-world practice of patients with non-valvular AF in Japan. The EXPAND Study is a prospective, non-interventional, observational cohort study to evaluate the effectiveness and safety of rivaroxaban in non-valvular AF patients in a real-world clinical practice. A total of 7,178 patients with non-valvular AF were enrolled in 684 medical institutes between November 20, 2012 and June 30, 2014. As for the baseline demographic and clinical characteristics of 7,164 patients, the proportion of female patients was 32.2%, and those of patients with creatinine clearance < 50 mL/min and non-paroxysmal (persistent or permanent) AF were 21.8% and 55.1%, respectively. The proportions of patients complicated with hypertension, congestive heart failure, diabetes mellitus, and a history of ischemic stroke were 70.9%, 25.9%, 24.3%, and 20.2%, respectively. The proportions of patients with a CHADS2 score ≤ 1 and a CHA2DS2-VASc score ≤ 1 were 37.3% and 13.6%, respectively. They were followed up until March 31, 2016 for a mean follow-up period of approximately 2.5 years. The findings of the EXPAND Study will help to establish an appropriate treatment with rivaroxaban for Japanese patients with non-valvular AF.

Prognostic Impact of Loop Diuretics in Patients With Chronic Heart Failure　- Effects of Addition of Renin-Angiotensin-Aldosterone System Inhibitors and ß-Blockers.

BACKGROUND: It remains to be elucidated whether addition of renin-angiotensin-aldosterone system (RAAS) inhibitors and/or ß-blockers to loop diuretics has a beneficial prognostic impact on chronic heart failure (CHF) patients. METHODS AND RESULTS: From the Chronic Heart failure Analysis and Registry in the Tohoku district 2 (CHART-2) Study (n=10,219), we enrolled 4,134 consecutive patients with symptomatic stage C/D CHF (mean age, 69.3 years, 67.7% male). We constructed Cox models for composite of death, myocardial infarction, stroke and HF admission. On multivariate inverse probability of treatment weighted (IPTW) Cox modeling, loop diuretics use was associated with worse prognosis with hazard ratio (HR) 1.28 (P<0001). Furthermore, on IPTW multivariate Cox modeling for multiple treatments, both low-dose (<40 mg/day) and high-dose (≥40 mg/day) loop diuretics were associated with worse prognosis with HR 1.32 and 1.56, respectively (both P<0.001). Triple blockade with RAS inhibitor(s), mineral corticoid (aldosterone) receptor antagonist(s) (MRA), and ß-blocker(s) was significantly associated with better prognosis in those on low-dose but not on high-dose loop diuretics. CONCLUSIONS: Chronic use of loop diuretics is significantly associated with worse prognosis in CHF patients in a dose-dependent manner, whereas the triple combination of RAAS inhibitor(s), MRA, and ß-blocker(s) is associated with better prognosis when combined with low-dose loop diuretics. (Circ J 2016; 80: 1396-1403).

Nitric oxide mediates selective degeneration of hypothalamic orexin neurons through dysfunction of protein disulfide isomerase.

We addressed the role of nitric oxide (NO) in orexin neuron degeneration that has been observed under various pathological conditions. Administration of an NO donor NOC18 (50 nmol) into the third ventricle of mice resulted in a significant decrease of orexin-immunoreactive (-IR) neurons, in contrast to a modest change in melanin-concentrating hormone-IR neurons. In addition, NOC18 promoted formation of orexin-A-IR aggregates within orexin neurons. An endoplasmic reticulum stress inducer tunicamycin replicated the effect of NOC18 with regard to decrease of orexin-IR neurons and formation of aggregates. We also found that NOC18 caused an increase in S-nitrosation of protein disulfide isomerase (PDI) and a decrease in PDI activity in hypothalamic tissues. Moreover, PDI inhibitors, such as cystamine and securinine, caused a selective decrease of orexin neurons and promoted formation of orexin-A-IR aggregates. Aggregate formation in orexin-IR neurons was also induced by local injection of small interfering RNA targeting PDI. Interestingly, sleep deprivation for 7 consecutive days induced a selective decrease of orexin-IR neurons, which was preceded by aggregate formation in orexin-IR neurons and an increase in S-nitrosated PDI in the hypothalamus. Activity of neuronal NO synthase (nNOS)-positive neurons in the lateral hypothalamus as assessed by c-Fos expression was elevated in response to sleep deprivation. Finally, sleep deprivation-induced decrease of orexin-IR neurons, formation of aggregates, and S-nitrosation of PDI were not observed in nNOS knock-out mice. These results indicate that nNOS-derived NO may mediate specific pathological events in orexin neurons, including neuropeptide misfolding via S-nitrosation and inactivation of PDI.

Usefulness of the noncontact mapping system to elucidate the conduction property for the treatment of common atrial flutter.

BACKGROUND: The functional role of the cavotricuspid isthmus (CTI) for common atrial flutter (cAFL) remains to be elucidated. In the present study, we examined whether the EnSite system (St. Jude Medical, St. Paul, MN, USA), a noncontact mapping system, is useful to evaluate the conduction properties of CTI to minimize radiofrequency (RF) ablation applications for cAFL. METHODS: We enrolled 22 consecutive patients with cAFL (64.1 ± 9.5 years old, M/F 21/1) treated with the EnSite system and examined the conduction properties during cAFL and during atrial pacing. In addition, the effectiveness of the system was evaluated in comparison with the conventional ablation group (67 ± 8.9 years old, n = 15, M/F 13/2). RESULT: In 11 out of the 22 patients, CTI block line was achieved by fewer RF applications on a presumed single activation pathway which the EnSite system showed (point ablation [PA] group), and the remaining 11 patients needed additional linear ablation (additional ablation [AA] group). The number of RF applications in the PA group was significantly smaller than that in the conventional group. During the lower lateral right atrial pacing at a cycle length of 600 ms, the CV of the CTI in the PA group was smaller compared to that in the AA group (1.36 ± 0.61 vs 2.17 ± 0.66 m/s, P < 0.05), although the CV during cAFL (averaged cycle length 245 ± 34 ms) was not different in both groups. CONCLUSIONS: These results indicate that targeting the presumed single line identified by EnSite could be an optional therapy for cAFL RF ablation, and diverse conduction properties in CTI are related to the success rate of this procedure. (PACE 2012;35:1464-1471).

Long-term treatment with nifedipine suppresses coronary hyperconstricting responses and inflammatory changes induced by paclitaxel-eluting stent in pigs in vivo: possible involvement of Rho-kinase pathway.

AIMS: Accumulating evidence indicates that coronary vasoconstricting responses are enhanced at the edges of coronary segment implanted with a drug-eluting stent (DES) compared with a bare-metal stent (BMS) in humans. We have recently demonstrated that Rho-kinase pathway plays an important role in DES-induced coronary hyperconstricting responses associated with inflammatory changes in pigs in vivo. This study examined whether long-term treatment with calcium channel blocker suppresses DES-induced coronary hyperconstricting responses in pigs in vivo. METHODS AND RESULTS: Paclitaxel-eluting stent (PES) and a BMS were randomly implanted in the left coronary arteries in male domestic pigs with and without long-acting nifedipine (NIF, 4 mg/kg/day) for 4 weeks (n = 7 each). Coronary vasomotion was evaluated by quantitative coronary angiography at least 24 h after withdrawal of NIF to avoid its direct effects on coronary vasomotion. In the control group (without NIF), coronary vasoconstricting responses to serotonin (10 and 100 µg/kg, i.c.) were significantly enhanced at the PES site compared with the BMS site (P = 0.009), which were abolished by hydroxyfasudil (90 and 300 µg/kg, i.c.), a selective Rho-kinase inhibitor. The PES-induced vasoconstricting responses were significantly inhibited in the NIF group (P = 0.019). Histological examination showed that inflammatory cell accumulation and microthrombus formation were enhanced at the PES site compared with the BMS site (P < 0.05), both of which were significantly suppressed by NIF associated with reduced Rho-kinase expression and activity (P < 0.05). CONCLUSION: These results indicate that long-term treatment with NIF suppresses PES-induced coronary abnormalities partly through Rho-kinase pathway inhibition in vivo.

Fish oil and omega-3 fatty acids in cardiovascular disease: do they really work?

Omega-3 fatty acids, which are found abundantly in fish oil, exert pleiotropic cardiometabolic effects with a diverse range of actions. The results of previous studies raised a lot of interest in the role of fish oil and omega-3 fatty acids in primary and secondary prevention of cardiovascular diseases. The present review will focus on the current clinical uses of omega-3 fatty acids and provide an update on their effects. Since recently published trials in patients with coronary artery diseases or post-myocardial infarction did not show an effect of omega-3 fatty acids on major cardiovascular endpoints, this review will examine the limitations of those data and suggest recommendations for the use of omega-3 fatty acids.

Long-term treatment with eicosapentaenoic acid ameliorates myocardial ischemia-reperfusion injury in pigs in vivo. -Involvement of Rho-kinase pathway inhibition-.

BACKGROUND: Eicosapentaenoic acid (EPA), the major n-3 fatty acid in fish oil, exerts cardioprotective effects against ischemic heart disease; however, the detailed mechanisms remain to be elucidated. Rho-kinase plays an important role in the pathogenesis of cardiovascular diseases including ischemia-reperfusion (I/R) injury. Thus, the hypothesis that long-term EPA treatment ameliorates myocardial I/R injury through Rho-kinase pathway inhibition in pigs in vivo was investigated. METHODS AND RESULTS: Male pigs were treated with either a control chow or EPA (600·mg·kg⁻¹·day⁻¹) for 3 weeks (n=8 each) and were subjected to myocardial ischemia by 90-min occlusion of the left circumflex coronary artery and subsequent 60-min reperfusion. The EPA group had an increased EPA level in red blood cells (4.4 ± 0.3mol%). The EPA treatment significantly ameliorated myocardial I/R injury, including regional wall motion abnormality (EPA 5.3 ± 3.6 vs. control 35.1 ± 3.8 unit, P<0.0001), left ventricular ejection fraction (EPA 43 ± 9% vs. control 32 ± 7%, P<0.05), occurrence of ventricular arrhythmias (EPA 181 ± 73 vs. control 389 ± 51 events, P<0.0001) and histological accumulation of inflammatory cells (P<0.01). Importantly, the EPA treatment significantly inhibited myocardial Rho-kinase activity (assessed by the extent of the myosin-binding subunit phosphorylation) (EPA 0.47 ± 0.11 vs. control 0.77 ± 0.14, P<0.05) and preserved myocardial eNOS activity (EPA 0.56 ± 0.13 vs. control 0.23 ± 0.07, P<0.01) with a significant correlation noted between them. CONCLUSIONS: Long-term treatment with EPA ameliorates I/R injury partly through Rho-kinase pathway inhibition in vivo.

[ACTION study: efficacy of nifedipine in preventing cardiovascular disease in patients with coronary artery disease].

Recent reports show that nifedipine not only causes vasodilation but also exerts beneficial effects on the endothelium of blood vessels. Some clinical trials evaluated nifedipine GITS (gastrointestinal therapeutic system) in patients with coronary artery disease. The investigators found that the treatment with nifedipine improved acetylcholine reactivity in coronary arteries and inhibited increases in coronary plaque volume. Furthermore, the large randomised, double-blind, placebo-controlled ACTION (A Coronary Disease Trial Investigating Outcome with Nifedipine GITS) study in patients with stable angina pectoris revealed that the treatment with nifedipine GITS led to significant reductions in the onset of overt heart failure and in the need for coronary angiography or coronary artery bypass graft surgery. These data indicate that a direct action on blood vessels, rather than coronary vasodilating or antihypertensive effects, might be responsible for improved prognosis with nifedipine.

Combination therapy with cerivastatin and nifedipine improves endothelial dysfunction after balloon injury in porcine coronary arteries.

HMG-CoA reductase inhibitors and calcium channel blockers have antiatherogenic effects; however, their mechanisms remain to be elucidated. This study examined the effect of cerivastatin and/or nifedipine on the endothelial dysfunction in porcine balloon-injured coronary arteries. Normal male pigs were randomly divided into the following four groups: control, cerivastatin (1 mg/kg/d PO), nifedipine (4 mg/kg/d PO), and their combination (n = 10 each). We started the treatments 3 days before balloon injury in the proximal left coronary arteries and continued for 4 weeks after the procedure. Then, we examined endothelial vasodilator functions ex vivo in organ chambers and in vitro by Western blotting for eNOS expression. Endothelium-dependent relaxations to serotonin, but not those to bradykinin or the calcium ionophore A23187 or endothelium-independent relaxations to sodium nitroprusside, were significantly impaired by balloon injury. The monotherapy with cerivastatin or nifedipine partially improved, and their combination supernormalized the relaxations to serotonin without affecting those to bradykinin or A23187 or endothelium-independent relaxations to sodium nitroprusside. The expression of eNOS was significantly reduced by balloon injury and normalized by the combination therapy. These results indicate that the combination therapy improves endothelial dysfunction after balloon injury, in which the up-regulation of eNOS may be involved.

Effects of nitric oxide synthase isoform deletion on oxytocin and vasopressin messenger RNA in mouse hypothalamus.

The effects of neuronal, endothelial, or inducible nitric oxide synthase gene disruption on the expression of oxytocin and vasopressin gene were examined in the hypothalamus (paraventricular, supraoptic, suprachiasmatic, and anterior commissural nuclei) and extrahypothalamus (bed nucleus of the stria terminalis). The oxytocin messenger RNA levels in the anterior commissural nucleus of neuronal nitric oxide synthase knockout mice were significantly higher than in control mice, but not in endothelial or inducible nitric oxide synthase knockout mice. In contrast, no significant effects of neuronal, endothelial, or inducible nitric oxide synthase gene disruption on oxytocin and vasopressin messenger RNA levels in the other hypothalamic and extrahypothalamic nuclei were observed. These results suggest that neuronal nitric-oxide-synthase-derived nitric oxide may be involved in the regulation of oxytocin gene expression in the anterior commissural nucleus.

Long-term inhibition of Rho-kinase suppresses neointimal formation after stent implantation in porcine coronary arteries: involvement of multiple mechanisms.

OBJECTIVE: We recently demonstrated that Rho-kinase, an effector of the small GTPase Rho, is substantially involved in the pathogenesis of arteriosclerosis. In this study, we examined whether Rho-kinase is also involved in in-stent restenosis and if so, what mechanism is involved. METHODS AND RESULTS: Pigs underwent stent implantation in the left coronary artery with or without administration of fasudil (30 mg/kg per day orally), a specific Rho-kinase inhibitor, starting 2 days before the procedure for a duration of 4 weeks. On day 28, reductions in coronary diameter and neointimal formation associated with macrophage accumulation, collagen deposition, and transforming growth factor (TGF)-beta1 expression were noted at the stent site, and all were significantly suppressed by fasudil. On day 7, fasudil significantly increased the frequency of TUNEL-positive apoptotic cells, while it tended to reduce that of bromodeoxyuridine-positive proliferating cells in the neointima. Western blot analysis on day 7 demonstrated that phosphorylations of the ezrin/radixin/moesin family (a marker of Rho-kinase activity in vivo) and protein expression of monocyte chemoattractant protein-1and bcl-2 were upregulated at the stent site and were significantly suppressed by fasudil. CONCLUSIONS: These results indicate that long-term inhibition of Rho-kinase suppresses in-stent neointimal formation by multiple mechanisms, including reduced vascular inflammation, enhanced apoptosis, and decreased collagen deposition.