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Objective: Excessive proliferation and apoptosis resistance are special characteristics of pulmonary artery smooth muscle cells (PASMCs) in pulmonary arterial hypertension (PAH). However, the drugs in clinical use for PAH target vascular dilatation, which do not exert adequate effects in patients with advanced PAH. Here, we report a novel therapeutic effect of emetine, a principal alkaloid extracted from the root of ipecac clinically used as an emetic and antiprotozoal drug. Approach and Results: We performed stepwise screenings for 5562 compounds from original library. First, we performed high-throughput screening with PASMCs from patients with PAH (PAH-PASMCs) and found 80 compounds that effectively inhibited proliferation. Second, we performed the repeatability and counter assay. Finally, we performed a concentration-dependent assay and found that emetine inhibits PAH-PASMC proliferation. Interestingly, emetine significantly reduced protein levels of HIFs (hypoxia-inducible factors; HIF-1α and HIF-2α) and downstream PDK1 (pyruvate dehydrogenase kinase 1). Moreover, emetine significantly reduced the protein levels of RhoA (Ras homolog gene family, member A), Rho-kinases (ROCK1 and ROCK2 [rho-associated coiled-coil containing protein kinases 1 and 2]), and their downstream CyPA (cyclophilin A), and Bsg (basigin) in PAH-PASMCs. Consistently, emetine treatment significantly reduced the secretion of cytokines/chemokines and growth factors from PAH-PASMCs. Interestingly, emetine reduced protein levels of BRD4 (bromodomain-containing protein 4) and downstream survivin, both of which are involved in many cellular functions, such as cell cycle, apoptosis, and inflammation. Finally, emetine treatment ameliorated pulmonary hypertension in 2 experimental rat models, accompanied by reduced inflammatory changes in the lungs and recovered right ventricular functions. Conclusions: Emetine is an old but novel drug for PAH that reduces excessive proliferation of PAH-PASMCs and improves right ventricular functions.

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.

Objective:Pulmonary arterial hypertension is characterized by abnormal proliferation of pulmonary artery smooth muscle cells and vascular remodeling, which leads to right ventricular (RV) failure. Bsg (Basigin) is a transmembrane glycoprotein that promotes myofibroblast differentiation, cell proliferation, and matrix metalloproteinase activation. CyPA (cyclophilin A) binds to its receptor Bsg and promotes pulmonary artery smooth muscle cell proliferation and inflammatory cell recruitment. We previously reported that Bsg promotes cardiac fibrosis and failure in the left ventricle in response to pressure-overload in mice. However, the roles of Bsg and CyPA in RV failure remain to be elucidated.Approach and Results:First, we found that protein levels of Bsg and CyPA were upregulated in the heart of hypoxia-induced pulmonary hypertension (PH) in mice and monocrotaline-induced PH in rats. Furthermore, cardiomyocyte-specific Bsg-overexpressing mice showed exacerbated RV hypertrophy, fibrosis, and dysfunction compared with their littermates under chronic hypoxia and pulmonary artery banding. Treatment with celastrol, which we identified as a suppressor of Bsg and CyPA by drug screening, decreased proliferation, reactive oxygen species, and inflammatory cytokines in pulmonary artery smooth muscle cells. Furthermore, celastrol treatment ameliorated RV systolic pressure, hypertrophy, fibrosis, and dysfunction in hypoxia-induced PH in mice and SU5416/hypoxia-induced PH in rats with reduced Bsg, CyPA, and inflammatory cytokines in the hearts and lungs.Conclusions:These results indicate that elevated Bsg in pressure-overloaded RV exacerbates RV dysfunction and that celastrol ameliorates RV dysfunction in PH model animals by suppressing Bsg and its ligand CyPA. Thus, celastrol can be a novel drug for PH and RV failure that targets Bsg and CyPA.Graphic Abstract:A graphic abstract is available for this article.

Background: Excessive proliferation and apoptosis resistance of pulmonary artery smooth muscle cells (PASMCs) are key mechanisms of pulmonary arterial hypertension (PAH). Despite the multiple combination therapy, a considerable number of patients develop severe pulmonary hypertension (PH) because of the lack of diagnostic biomarker and antiproliferative therapies for PASMCs. Methods: Microarray analyses were used to identify a novel therapeutic target for PAH. In vitro experiments, including lung and serum samples from patients with PAH, cultured PAH-PASMCs, and high-throughput screening of 3336 low-molecular-weight compounds, were used for mechanistic study and exploring a novel therapeutic agent. Five genetically modified mouse strains, including PASMC-specific selenoprotein P (SeP) knockout mice and PH model rats, were used to study the role of SeP and therapeutic capacity of the compounds for the development of PH in vivo. Results: Microarray analysis revealed a 32-fold increase in SeP in PAH-PASMCs compared with control PASMCs. SeP is a widely expressed extracellular protein maintaining cellular metabolism. Immunoreactivity of SeP was enhanced in the thickened media of pulmonary arteries in PAH. Serum SeP levels were also elevated in patients with PH compared with controls, and high serum SeP predicted poor outcome. SeP-knockout mice ( SeP –/– ) exposed to chronic hypoxia showed significantly reduced right ventricular systolic pressure, right ventricular hypertrophy, and pulmonary artery remodeling compared with controls. In contrast, systemic SeP-overexpressing mice showed exacerbation of hypoxia-induced PH. Furthermore, PASMC-specific SeP –/– mice showed reduced hypoxia-induced PH compared with controls, whereas neither liver-specific SeP knockout nor liver-specific SeP-overexpressing mice showed significant differences with controls. Altogether, protein levels of SeP in the lungs were associated with the development of PH. Mechanistic experiments demonstrated that SeP promotes PASMC proliferation and resistance to apoptosis through increased oxidative stress and mitochondrial dysfunction, which were associated with activated hypoxia-inducible factor-1α and dysregulated glutathione metabolism. It is important to note that the high-throughput screening of 3336 compounds identified that sanguinarine, a plant alkaloid with antiproliferative effects, reduced SeP expression and proliferation in PASMCs and ameliorated PH in mice and rats. Conclusions: These results indicate that SeP promotes the development of PH, suggesting that it is a novel biomarker and therapeutic target of the disorder.

Objective: Despite the recent progress in upfront combination therapy for pulmonary arterial hypertension (PAH), useful biomarkers for the disorder still remain to be developed. SeP (Selenoprotein P) is a glycoprotein secreted from various kinds of cells including pulmonary artery smooth muscle cells to maintain cellular metabolism. We have recently demonstrated that SeP production from pulmonary artery smooth muscle cells is upregulated and plays crucial roles in the pathogenesis of PAH. However, it remains to be elucidated whether serum SeP levels could be a useful biomarker for PAH. Approach and Results: We measured serum SeP levels and evaluated their prognostic impacts in 65 consecutive patients with PAH and 20 controls during follow-up (mean, 1520 days; interquartile range, 1393–1804 days). Serum SeP levels were measured using a newly developed sol particle homogeneous immunoassay. The patients with PAH showed significantly higher serum SeP levels compared with controls. Higher SeP levels (cutoff point, 3.47 mg/L) were associated with the outcome (composite end point of all-cause death and lung transplantation) in patients with PAH (hazard ratio, 4.85 [1.42–16.6]; P R =0.78, 0.76, and −0.71 respectively, all P Conclusions: Serum SeP is a novel biomarker for diagnosis and assessment of treatment efficacy and long-term prognosis in patients with PAH.

Background Circulating proteins are exposed to vascular endothelial layer and influence their functions. Among them, adipsin is a member of the trypsin family of peptidases and is mainly secreted from adipocytes, monocytes, and macrophages, catalyzing the rate‐limiting step of the alternative complement pathway. However, its pathophysiological role in cardiovascular disease remains to be elucidated. Here, we examined whether serum adipsin levels have a prognostic impact in patients with coronary artery disease. Methods and Results In 370 consecutive patients undergoing diagnostic coronary angiography, we performed a cytokine array analysis for screening serum levels of 50 cytokines/chemokines and growth factors. Among them, classification and regression analysis identified adipsin as the best biomarker for prediction of their long‐term prognosis (median 71 months; interquartile range, 55–81 months). Kaplan–Meier curve showed that higher adipsin levels (≥400 ng/ mL ) were significantly associated with all‐cause death (hazard ratio [HR], 4.2; 95% CI, 1.7–10.6 [ P HR , 2.4; 95% CI, 1.7–3.5 [ P HR, 3.2; 95% CI, 1.7–5.9 [ P HR, 1.5, 95% CI, 1.1–1.9 [ P mL ) and high‐sensitivity C‐reactive protein (≥1 mg/L) was more significantly associated with all‐cause death ( HR, 21.0; 95% CI, 2.9–154.1 [ P Conclusions These results indicate that adipsin is a novel biomarker that predicts all‐cause death and rehospitalization in patients with coronary artery disease, demonstrating the novel aspects of the alternative complementary system in the pathogenesis of coronary artery disease.

Background 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 mitochondrial metabolism to a hyperproliferative state. However, at present, since all the drugs in clinical use target pulmonary vascular dilatation, they may not be so effective for patients with advanced PAH. Purposes We aimed to discover a novel drug for PAH that inhibits PASMC proliferation. Methods In the first screening, we examined 5,562 compounds from our original library using high-throughput screening system to discover a compound that inhibits proliferation of PASMCs from PAH patients (PAH-PASMCs). In the second screening, we performed concentration-dependent assays and counter assays with PAH-PASMCs and PASMCs from healthy donors. We also performed apoptosis assays and mechanistic analysis for inflammation, reactive oxygen species (ROS), and mitochondrial function. Results 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 minimal effects on PASMCs from healthy donors. Moreover, celastramycin inhibited proliferation with minimal increase in apoptosis and low rate of cell death. Then, we synthesized 25 analogues of celastramycin, and finally selected the lead compound that significantly inhibited proliferation of PAH-PASMCs and reduced cytosolic ROS levels. Mechanistic analysis demonstrated that celastramycin reduced the protein levels of hypoxia-inducible factor-1α, which was abnormally activated in PAH-PASMCs and impaired aerobic metabolism, and nuclear factor-κB, which induces pro-inflammatory signals, in PAH-PASMCs compared with vehicle controls, leading to reduced secretion of inflammatory cytokines. Importantly, celastramycin treatment reduced the ROS levels in PAH-PASMCs with increased protein levels of NF-E2-related factor 2 (Nrf2), a master regulator of cellular response against oxidative stress. Furthermore, celastramycin treatment improved mitochondrial energy metabolism with recovered mitochondrial network formation in PAH-PASMCs. We also discovered that celastramycin-mediated effects on these transcriptional modulators could be regulated by zinc finger C3H1 domain-containing protein, which is a binding partner of celastramycin. Finally, celastramycin treatment ameliorated pulmonary hypertension in three experimental animal models of PH in mice and rats, accompanied by reduced inflammatory changes in the lungs. Conclusions These results indicate that celastramycin ameliorates pulmonary hypertension through inhibition of excessive proliferation of PAH-PASMCs, for which its anti-inflammatory and beneficial effects on mitochondrial energy metabolism may be involved. Thus, celastramycin could be a novel drug for PAH as it exerts anti-proliferative effects on PAH-PASMCs.

Background Despite the recent progress in upfront combination therapy for pulmonary arterial hypertension (PAH), a useful biomarker for the disorder still remains to be developed. Selenoprotein P (SeP) is a glycoprotein secreted mainly from hepatocytes but also from other various kinds of cells, including pulmonary artery smooth muscle cells (PASMCs), to maintain selenium homeostasis and cellular energy metabolism. We have recently demonstrated that SeP expression in PASMCs is markedly up-regulated in PAH patients and plays crucial roles in the pathogenesis of the disorder. In this study, we thus examined whether serum levels of SeP could be a useful biomarker for the disorder. Methods In the experimental study, we performed gene expression microarray and in silico analyses to identify a novel therapeutic target for PAH. We also used the lung, serum, and cultured PAMSCs derived from patients with PAH for mechanistic experiments. In the clinical study, we enrolled a total of 65 consecutive patients with PAH who underwent right heart catheterization for hemodynamic assessment. We measured serum SeP levels and evaluated their prognostic impacts during follow-up (mean 1,520 days, IQR: 1,393–1,804 days). Serum SeP level was measured using a newly developed sol particle homogeneous immunoassay. As controls, we collected serum samples from 20 controls without any known cardiac disorders evaluated by hematological examination, echocardiography, and coronary angiography. In PAH patients, we examined the relationship between baseline SeP levels and composite endpoint of all-cause death and lung transplantation. The correlation between the absolute changes in SeP and those in hemodynamic parameters during follow-up were also examined. Results In the experimental study, SeP promoted PASMC proliferation through increased oxidative stress and mitochondrial metabolic dysfunction, which were associated with activated HIF-1α and dysregulated glutathione metabolism. In the clinical study, PAH patients showed significantly higher levels of serum SeP compared with controls (3.07±0.57 vs. 2.43±0.25 mg/L, P Figure 1 Conclusions These results indicate that SeP is a novel therapeutic target of PAH and that serum SeP levels are a novel biomarker for diagnosis and assessment of treatment efficacy and long-term prognosis in PAH patients. Acknowledgement/Funding Grants-in-aid for Scientific Research from the Japan Agency for Medical Research and Development, Tokyo, Japan (16ek0109176h0001, 17ek0109227h0001).

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.We aimed to discover a novel drug for PAH that inhibits PASMC proliferation.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.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.

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.

Objective— Basigin (Bsg) is a transmembrane glycoprotein that activates matrix metalloproteinases and promotes inflammation. However, the role of Bsg in the pathogenesis of cardiac hypertrophy and failure remains to be elucidated. We examined the role of Bsg in cardiac hypertrophy and failure in mice and humans. Approach and Results— We performed transverse aortic constriction in Bsg +/– and in wild-type mice. Bsg +/– mice showed significantly less heart and lung weight and cardiac interstitial fibrosis compared with littermate controls after transverse aortic constriction. Both matrix metalloproteinase activities and oxidative stress in loaded left ventricle were significantly less in Bsg +/– mice compared with controls. Echocardiography showed that Bsg +/– mice showed less hypertrophy, less left ventricular dilatation, and preserved left ventricular fractional shortening compared with littermate controls after transverse aortic constriction. Consistently, Bsg +/– mice showed a significantly improved long-term survival after transverse aortic constriction compared with Bsg +/+ mice, regardless of the source of bone marrow ( Bsg +/+ or Bsg +/– ). Conversely, cardiac-specific Bsg-overexpressing mice showed significantly poor survival compared with littermate controls. Next, we isolated cardiac fibroblasts and examined their responses to angiotensin II or mechanical stretch. Both stimuli significantly increased Bsg expression, cytokines/chemokines secretion, and extracellular signal–regulated kinase/Akt/JNK activities in Bsg +/+ cardiac fibroblasts, all of which were significantly less in Bsg +/– cardiac fibroblasts. Consistently, extracellular and intracellular Bsg significantly promoted cardiac fibroblast proliferation. Finally, serum levels of Bsg were significantly elevated in patients with heart failure and predicted poor prognosis. Conclusions— These results indicate the crucial roles of intracellular and extracellular Bsg in the pathogenesis of cardiac hypertrophy, fibrosis, and failure in mice and humans.

Although postcapillary pulmonary hypertension (PH) is an important prognostic factor for patients with heart failure (HF), its pathogenesis remains to be fully elucidated. To elucidate the different roles of Rho-kinase isoforms, ROCK1 and ROCK2, in cardiomyocytes in response to chronic pressure overload, we performed transverse aortic constriction (TAC) in cardiac-specific ROCK1-deficient (cROCK1−/−) and ROCK2-deficient (cROCK2−/−) mice. Cardiomyocyte-specific ROCK1 deficiency promoted pressure-overload-induced cardiac dysfunction and postcapillary PH, whereas cardiomyocyte-specific ROCK2 deficiency showed opposite results. Histological analysis showed that pressure-overload-induced cardiac hypertrophy and fibrosis were enhanced in cROCK1−/− mice compared with controls, whereas cardiac hypertrophy was attenuated in cROCK2−/− mice after TAC. Consistently, the levels of oxidative stress were up-regulated in cROCK1−/− hearts and down-regulated in cROCK2−/− hearts compared with controls after TAC. Furthermore, cyclophilin A (CyPA) and basigin (Bsg), both of which augment oxidative stress, enhanced cardiac dysfunction and postcapillary PH in cROCK1−/− mice, whereas their expressions were significantly lower in cROCK2−/− mice. In clinical studies, plasma levels of CyPA were significantly increased in HF patients and were higher in patients with postcapillary PH compared with those without it. Finally, high-throughput screening demonstrated that celastrol, an antioxidant and antiinflammatory agent, reduced the expressions of CyPA and Bsg in the heart and the lung, ameliorating cardiac dysfunction and postcapillary PH induced by TAC. Thus, by differentially affecting CyPA and Bsg expressions, ROCK1 protects and ROCK2 jeopardizes the heart from pressure-overload HF with postcapillary PH, for which celastrol may be a promising agent.

Background: Thoracic aortic aneurysm (TAA) and dissection are fatal diseases that cause aortic rupture and sudden death. The small GTP-binding protein GDP dissociation stimulator (SmgGDS) is a crucial mediator of the pleiotropic effects of statins. Previous studies revealed that reduced force generation in aortic smooth muscle cells (AoSMCs) causes TAA and thoracic aortic dissection. Methods: To examine the role of SmgGDS in TAA formation, we used an angiotensin II (1000 ng·min −1 ·kg −1 , 4 weeks)–induced TAA model. Results: We found that 33% of Apoe −/− SmgGDS +/− mice died suddenly as a result of TAA rupture, whereas there was no TAA rupture in Apoe −/− control mice. In contrast, there was no significant difference in the ratio of abdominal aortic aneurysm rupture between the 2 genotypes. We performed ultrasound imaging every week to follow up the serial changes in aortic diameters. The diameter of the ascending aorta progressively increased in Apoe −/− SmgGDS +/− mice compared with Apoe −/− mice, whereas that of the abdominal aorta remained comparable between the 2 genotypes. Histological analysis of Apoe −/− SmgGDS +/− mice showed dissections of major thoracic aorta in the early phase of angiotensin II infusion (day 3 to 5) and more severe elastin degradation compared with Apoe −/− mice. Mechanistically, Apoe −/− SmgGDS +/− mice showed significantly higher levels of oxidative stress, matrix metalloproteinases, and inflammatory cell migration in the ascending aorta compared with Apoe −/− mice. For mechanistic analyses, we primary cultured AoSMCs from the 2 genotypes. After angiotensin II (100 nmol/L) treatment for 24 hours, Apoe −/− SmgGDS +/− AoSMCs showed significantly increased matrix metalloproteinase activity and oxidative stress levels compared with Apoe −/− AoSMCs. In addition, SmgGDS deficiency increased cytokines/chemokines and growth factors in AoSMCs. Moreover, expressions of fibrillin-1 ( FBN1 ), α-smooth muscle actin ( ACTA2 ), myosin-11 ( MYH11 ), MYLLK , and PRKG1 , which are force generation genes, were significantly reduced in Apoe −/− SmgGDS +/− AoSMCs compared with Apoe −/− AoSMCs. A similar tendency was noted in AoSMCs from patients with TAA compared with those from control subjects. Finally, local delivery of the SmgGDS gene construct reversed the dilation of the ascending aorta in Apoe −/− SmgGDS +/− mice. Conclusions: These results suggest that SmgGDS is a novel therapeutic target for the prevention and treatment of TAA.

Background: Thoracic aortic aneurysm (TAA) and dissection (TAD) are fatal diseases, which cause aortic rupture and sudden death. The small GTP-binding protein GDP dissociation stimulator (SmgGDS) is a crucial mediator of the pleiotropic effects of statins. Previous studies revealed that reduced force generation in AoSMCs causes TAA and TAD. Methods and Results: To examine the role of SmgGDS in TAA formation, we employed an angiotensin II (AngII, 1,000 ng/min/kg, 4weeks)-induced TAA model in Apoe -/- SmgGDS +/- mice, in which 33% died suddenly due to TAA rupture, whereas there was no TAA rupture in Apoe -/- control mice. In contrast, there was no significant difference in the ratio of AAA rupture between the two genotypes. We performed ultrasound imaging every week to follow the serial changes in aortic diameters. The diameter of the ascending aorta progressively increased in Apoe -/- SmgGDS +/- mice compared with Apoe -/- mice, whereas that of the abdominal aorta remained comparable between the two genotypes. Histological analysis of Apoe -/- SmgGDS +/- mice showed dissections of major thoracic aorta in the early phase of AngII infusion (day 3~5) and more severe elastin degradation compared with Apoe -/- mice. Mechanistically, Apoe -/- SmgGDS +/- mice showed significantly higher levels of oxidative stress, matrix metalloproteinases, and inflammatory cell migration in the ascending aorta compared with Apoe -/- mice. For mechanistic analyses, we primary cultured AoSMCs from the 2 genotypes. After AngII (100 nM) treatment for 24 hours, Apoe -/- SmgGDS +/- AoSMCs showed significantly increased MMP activity and oxidative stress levels compared with Apoe -/- AoSMCs. In addition, SmgGDS deficiency increased cytokines/chemokines and growth factors in AoSMCs. Moreover, expressions of FBN1 , ACTA2 , MYH11 , MLK and PRKG1 , which are force generation genes, were significantly reduced in Apoe -/- SmgGDS +/- AoSMCs compared with Apoe -/- AoSMCs. Similar tendency was noted in AoSMCs from TAA patients compared with those from controls. Finally, local delivery of the SmgGDS gene construct reversed the dilation of the ascending aorta in Apoe -/- SmgGDS +/- mice. Conclusions: These results suggest that SmgGDS is a novel therapeutic target for the prevention and treatment of TAA.

From August 26th to 30th, the 2017 Annual Congress of the European Society of Cardiology (ESC 2017) was held in Barcelona, Spain. Despite the terrorism tradegy just before the ESC congress, the congress attracted many medical professionals from all over the world to discuss the recent topics in cardiovascular medicine in more than 500 sessions, including COMPASS (Cardiovascular OutcoMes for People using Anticoagulation StrategieS Trial), CANTOS (Canakinumab Anti-Inflammatory Thrombosis Outcomes Study), and ORION (which assessed the effect of a novel siRNA inhibitor to PCSK9 on reductions in low-density lipoprotein cholesterol). Japanese cardiologists and the Japanese Circulation Society greatly contributed to the congress. This report briefly introduces some late-breaking registry results, late-breaking clinical trials, and ESC Guidelines from the ESC 2017 Congress.

Background: Statins reduce the incidence and development of thoracic aortic aneurysm (TAA) and rupture. We have previously identified that small GTP-binding protein GDP dissociation stimulator (SmgGDS) is a crucial mediator of the pleiotropic effects of statins. Methods and Results: To examine the role of SmgGDS in TAA formation, Apoe -/- and Apoe -/- SmgGDS +/- (DKO) mice were infused with angiotensin II (AngII, 1,000 ng/min/kg) for 4 weeks. There was no significant difference in blood pressure between the 2 genotypes in response to the AngII treatment. However, during the follow-up, 36% of DKO mice died suddenly due to TAA rupture, whereas there was no TAA rupture in Apoe -/- mice ( P n =14 each). Histological analysis of DKO mice showed dissections of major thoracic aorta in the early phase of AngII infusion (day 3~5). We performed ultrasound imaging every week to follow the serial changes in aortic diameters. Diameter of the ascending aorta was progressively and significantly increased in DKO mice compared with Apoe -/- mice (1.64±0.06 vs. 1.43±0.05 mm at 4 weeks, P n =14 each), whereas that of the abdominal aorta was comparable between the 2 genotypes. Indeed, there was no significant difference in the incidence of AngII-induced abdominal aortic aneurysm (AAA) formation between the 2 genotypes (both 75%). Western blotting demonstrated that AngII-induced activations of JNK and ERK were significantly higher in the thoracic aorta of DKO mice compared with Apoe -/- mice ( P n =6 each). For mechanistic analyses, we primary cultured aortic smooth muscle cells (AoSMCs) from the 2 genotypes. After AngII (100 nM) treatment for 24 hours, DKO AoSMCs showed significantly increased JNK activity, cyclophilin A secretion, and oxidative stress levels compared with Apoe -/- AoSMCs ( P n =6 each). Interestingly, AngII-induced upregulation of Nrf2, a master regulator of cellular responses against environmental stresses, was significantly less in DKO AoSMCs compared with Apoe -/- AoSMCs ( P n =6). Finally, expressions of matrix metalloproteinase-2 and -9 were significantly increased in DKO AoSMCs compared with Apoe -/- AoSMCs. Conclusions: These results suggest that SmgGDS is a novel therapeutic target for the prevention and treatment of TAA.

Rationale: Pulmonary hypertension is a fatal disease; however, its pathogenesis still remains to be elucidated. Thrombin-activatable fibrinolysis inhibitor (TAFI) is synthesized by the liver and inhibits fibrinolysis. Plasma TAFI levels are significantly increased in chronic thromboembolic pulmonary hypertension (CTEPH) patients. Objective: To determine the role of activated TAFI (TAFIa) in the development of CTEPH. Methods and Results: Immunostaining showed that TAFI and its binding partner thrombomodulin (TM) were highly expressed in the pulmonary arteries (PAs) and thrombus in patients with CTEPH. Moreover, plasma levels of TAFIa were increased 10-fold in CTEPH patients compared with controls. In mice, chronic hypoxia caused a 25-fold increase in plasma levels of TAFIa with increased plasma levels of thrombin and TM, which led to thrombus formation in PA, vascular remodeling, and pulmonary hypertension. Consistently, plasma clot lysis time was positively correlated with plasma TAFIa levels in mice. Additionally, overexpression of TAFIa caused organized thrombus with multiple obstruction of PA flow and reduced survival rate under hypoxia in mice. Bone marrow transplantation showed that circulating plasma TAFI from the liver, not in the bone marrow, was activated locally in PA endothelial cells through interactions with thrombin and TM. Mechanistic experiments demonstrated that TAFIa increased PA endothelial permeability, smooth muscle cell proliferation, and monocyte/macrophage activation. Importantly, TAFIa inhibitor and peroxisome proliferator–activated receptor-α agonists significantly reduced TAFIa and ameliorated animal models of pulmonary hypertension in mice and rats. Conclusions: These results indicate that TAFIa could be a novel biomarker and realistic therapeutic target of CTEPH.

Objective— Cyclophilin A (CyPA) is secreted from vascular smooth muscle cells, inflammatory cells, and activated platelets in response to oxidative stress. We have recently demonstrated that plasma CyPA level is a novel biomarker for diagnosing coronary artery disease. However, it remains to be elucidated whether plasma CyPA levels also have a prognostic impact in such patients. Approach and Results— In 511 consecutive patients undergoing diagnostic coronary angiography, we measured the plasma levels of CyPA, high-sensitivity C-reactive protein (hsCRP), and brain natriuretic peptide and evaluated their prognostic impacts during the follow-up (42 months, interquartile range: 25–55 months). Higher CyPA levels (≥12 ng/mL) were significantly associated with all-cause death, rehospitalization, and coronary revascularization. Higher hsCRP levels (≥1 mg/L) were also significantly correlated with the primary end point and all-cause death, but not with rehospitalization or coronary revascularization. Similarly, higher brain natriuretic peptide levels (≥100 pg/mL) were significantly associated with all-cause death and rehospitalization, but not with coronary revascularization. Importantly, the combination of CyPA (≥12 ng/mL) and hsCRP (≥1 mg/L) was more significantly associated with all-cause death (hazard ratio, 21.2; 95% confidence interval, 4.9–92.3,; P Conclusions— The results indicate that plasma CyPA levels can be used to predict all-cause death, rehospitalization, and coronary revascularization in patients with coronary artery disease and that when combined with other biomarkers (hsCRP and brain natriuretic peptide levels), the CyPA levels have further enhanced prognostic impacts in those patients.

Background: Excessive proliferation of pulmonary artery smooth muscle cells (PASMCs) is the main characteristics in pulmonary arterial hypertension (PAH); however, its mechanism still remains unclear. Methods & Results: To explore a novel therapeutic target in PAH, we performed a gene expression microarray screening using PASMCs from patients with PAH (PAH-PASMCs) and those from controls and found up-regulation of selenoprotein P (SeP) in PAH-PASMCs (>5-fold, P2 10%) increased SeP expression in the lung. In PAH-PASMCs, hypoxia (O 2 2%, 24 hours) increased SeP expression compared with normoxic controls (O 2 21%) (1.6-fold, P SeP -/- ) were exposed to hypoxia (O 2 10%) for 4 weeks, which resulted in suppression of right ventricular systolic pressure (RVSP), RV hypertrophy and PA remodeling compared with controls (all P SeP -/- PASMCs compared with SeP +/+ PASMCs (P SeP -/- PASMCs showed ERK1/2 inactivation, AMPK activation, and reduced secretion of pro-inflammatory cytokines. SMC-targeted deletion of SeP suppressed the development of hypoxia-induced PH compared with controls (all P0.05, n=8-9). In the clinical study, serum SeP levels were significantly elevated in PAH patients (n=203) compared with controls (n=20) (P Conclusions: These results indicate that SeP, especially in PASMCs, promotes PA remodeling, suggesting that it is a novel therapeutic target for PAH.

The detailed molecular mechanisms of the pleiotropic effects of statins remain to be fully elucidated. Here, we hypothesized that cardioprotective effects of statins are mediated by small GTP-binding protein GDP dissociation stimulator (SmgGDS). SmgGDS +/– and wild-type (WT) mice were treated with continuous infusion of angiotensin II (Ang II) for 2 weeks with and without oral treatment with atorvastatin or pravastatin. At 2 weeks, the extents of Ang II–induced cardiac hypertrophy and fibrosis were comparable between the 2 genotypes. However, statins significantly attenuated cardiomyocyte hypertrophy and fibrosis in WT mice, but not in SmgGDS +/– mice. In SmgGDS +/– cardiac fibroblasts (CFs), Rac1 expression, extracellular signal–regulated kinases 1/2 activity, Rho-kinase activity, and inflammatory cytokines secretion in response to Ang II were significantly increased when compared with WT CFs. Atorvastatin significantly reduced Rac1 expression and oxidative stress in WT CFs, but not in SmgGDS +/– CFs. Furthermore, Bio-plex analysis revealed significant upregulations of inflammatory cytokines/chemokines and growth factors in SmgGDS +/– CFs when compared with WT CFs. Importantly, conditioned medium from SmgGDS +/– CFs increased B-type natriuretic peptide expression in rat cardiomyocytes to a greater extent than that from WT CFs. Furthermore, atorvastatin significantly increased SmgGDS secretion from mouse CFs. Finally, treatment with recombinant SmgGDS significantly reduced Rac1 expression in SmgGDS +/– CFs. These results indicate that both intracellular and extracellular SmgGDS play crucial roles in the inhibitory effects of statins on cardiac hypertrophy and fibrosis, partly through inhibition of Rac1, Rho kinase, and extracellular signal–regulated kinase 1/2 pathways, demonstrating the novel mechanism of the pleiotropic effects of statins.

Rationale: Endothelial AMP-activated protein kinase (AMPK) plays an important role for vascular homeostasis, and its role is impaired by vascular inflammation. However, the role of endothelial AMPK in the pathogenesis of pulmonary arterial hypertension (PAH) remains to be elucidated. Objective: To determine the role of endothelial AMPK in the development of PAH. Methods and Results: Immunostaining showed that endothelial AMPK is downregulated in the pulmonary arteries of patients with PAH and hypoxia mouse model of pulmonary hypertension (PH). To elucidate the role of endothelial AMPK in PH, we used endothelial-specific AMPK-knockout mice ( eAMPK –/– ), which were exposed to hypoxia. Under normoxic condition, eAMPK –/– mice showed the normal morphology of pulmonary arteries compared with littermate controls ( eAMPK flox/flox ). In contrast, development of hypoxia-induced PH was accelerated in eAMPK –/– mice compared with controls. Furthermore, the exacerbation of PH in eAMPK –/– mice was accompanied by reduced endothelial function, upregulation of growth factors, and increased proliferation of pulmonary artery smooth muscle cells. Importantly, conditioned medium from endothelial cells promoted pulmonary artery smooth muscle cell proliferation, which was further enhanced by the treatment with AMPK inhibitor. Serum levels of inflammatory cytokines, including tumor necrosis factor-α and interferon-γ were significantly increased in patients with PAH compared with healthy controls. Consistently, endothelial AMPK and cell proliferation were significantly reduced by the treatment with serum from patients with PAH compared with controls. Importantly, long-term treatment with metformin, an AMPK activator, significantly attenuated hypoxia-induced PH in mice. Conclusions: These results indicate that endothelial AMPK is a novel therapeutic target for the treatment of PAH.

Aims The pleiotropic effects of HMG-CoA reductase inhibitors (statins) independent of cholesterol-lowering effects have attracted much attention. We have recently demonstrated that the pleiotropic effects of statins are partly mediated through up-regulation of small GTP-binding protein dissociation stimulator (SmgGDS) with a resultant Rac1 degradation and reduced oxidative stress. However, it remains to be elucidated what molecular mechanisms are involved. Methods and results To first determine in what tissue statins up-regulate SmgGDS expression, we examined the effects of two statins (atorvastatin 10 mg/kg per day and pravastatin 50 mg/kg per day for 1 week) on SmgGDS expression in mice in vivo . The two statins increased SmgGDS expression especially in the aorta. Atorvastatin also increased SmgGDS expression in cultured human umbilical venous endothelial cells (HUVEC) and human aortic endothelial cells, but not in human aortic vascular smooth muscle cells. Furthermore, Akt phosphorylation was transiently enhanced only in HUVEC in response to atorvastatin. Then, to examine whether Akt is involved for up-regulation of SmgGDS by statins, we knocked out Akt1 by its siRNA in HUVEC, which abolished the effects by atorvastatin to up-regulate SmgGDS. Furthermore, when we knocked down β1-integrin to elucidate the upstream molecule of Akt1, the effect of atorvastatin to up-regulate SmgGDS was abolished. Finally, we confirmed that Akt activator, SC79, significantly up-regulate SmgGDS in HUVEC. Conclusion These results indicate that statins selectively up-regulate SmgGDS in endothelial cells, for which the β1-integrin/Akt1 pathway may be involved, demonstrating the novel aspects of the pleiotropic effects of statins.

Background: Statins are widely known to exert beneficial pleiotropic effects mediated by anti-oxidative and anti-inflammatory mechanisms, independent of their LDL-cholesterol lowering effect. However, the detailed molecular mechanisms of the pleiotropic effects of statins remain to be fully elucidated. Here, we demonstrate that small GTP-binding protein GDP dissociation stimulator (SmgGDS) is a crucial mediator of the pleiotropic effects of statins. Methods and Results: SmgGDS +/- and wild-type (WT) mice were treated with continuous infusion of angiotensin II (AngII) for 2 weeks with and without oral treatment with atorvastatin (10 mg/kg/day) or pravastatin (50 mg/kg/day). At 2 weeks, the extents of AngII-induced cardiac hypertrophy and fibrosis were comparable between the 2 genotypes. However, statins significantly attenuated cardiomyocyte hypertrophy, fibrosis and left ventricular diastolic dysfunction in WT mice, but not in SmgGDS +/- mice. Since SmgGDS was highly expressed in cardiac fibroblasts (CFs) in the heart, we then examined the role of SmgGDS in cultured CFs from WT and SmgGDS +/- mice. In SmgGDS +/- CFs, Rac1 expression, ERK1/2 activity, Rho-kinase activity and inflammatory cytokines secretion in response to AngII were significantly increased as compared with WT CFs. Atorvastatin significantly reduced Rac1 expression and oxidative stress in WT CFs, but not in SmgGDS +/- CFs. Furthermore, Bio-plex analysis revealed significant up-regulations of inflammatory cytokines/chemokines and growth factors in SmgGDS +/- CFs as compared with WT CFs. Importantly, conditioned medium from SmgGDS +/- CFs increased BNP expression in neonatal rat cardiomyocytes to a greater extent than that from WT CFs. Furthermore, atorvastatin significantly increased SmgGDS secretion from mouse and human CFs. Finally, treatment with recombinant SmgGDS significantly reduced Rac1 expression in SmgGDS +/- CFs. Conclusion: These results indicate that both intracellular and extracellular SmgGDS play a crucial role in the inhibitory effects of statins on cardiac hypertrophy and fibrosis, partly through inhibition of Rac1, Rho-kinase and ERK1/2 pathways, demonstrating the novel mechanism of the pleiotropic effects of statins.