Serum zinc concentration in patients with myocardial infarction: a retrospective study.

BACKGROUND: Zinc regulates the oxidative stress and inflammatory signaling cascade and affects the development and deterioration of cardiovascular disease. We investigated the prognosis of developing heart failure in patients with myocardial infarction. METHODS: Patients with myocardial infarction (n = 243) were divided using the median value of zinc concentration on admission into low (< 66 µg/dL at admission, n = 111) and high zinc group (≥ 66 µg/dL at admission, n = 132). During follow-up (mean ± SD: 734 ± 597 days; median 691 days), admission due to heart failure was observed in 12 patients: 10 and 2 cases in the low and high zinc groups, respectively. RESULTS: The risk of admission due to heart failure was significantly higher in the low zinc than in the high zinc group (P = 0.0043). Relative to the high zinc group, the hazard ratio for admission due to heart failure was 15.7 (95% confidence interval 1.11-221, P = 0.042) via adjusted Cox proportional hazards analysis. Even after propensity score matching, the risk of admission due to heart failure was significantly higher in the low zinc than in the high zinc group (P = 0.048). CONCLUSION: Low serum zinc concentration may be a risk factor for admission due to heart failure after myocardial infarction.

A case of syncope after exertion due to sigmoid septum evaluated by exercise stress echocardiography.

A sigmoid septum is associated with sharp angulation and aging of the aortic root; however, it does not affect the pressure gradient in the left ventricular outflow tract and is generally asymptomatic. This report describes a 73-year-old woman who presented with syncope after exertion. Echocardiography revealed that the cause was left ventricular outflow tract stenosis associated with a sigmoid septum; her symptoms improved with beta-blocker therapy. Exercise stress echocardiography was performed to determine treatment efficacy. Sigmoid septum causes syncope on exertion; however, drug therapy is effective. Exercise stress echocardiography is effective in determining treatment efficacy. If syncope is present, a sigmoid septum should be considered as a cause. Learning objectives: 1.A sigmoid septum is part of or resembles hypertrophic cardiomyopathy, resulting in left ventricular outflow tract (LVOT) stenosis that is exacerbated by exertion and may cause syncope.2.A sigmoid septum is a differential diagnosis for the cause of syncope and is diagnosed using cardiac echocardiography.3.LVOT stenosis due to a sigmoid septum can be improved with drug therapy such as beta-blockers.4.The effects of beta-blocker therapy can be determined by exercise stress echocardiography.

Effect of constipation on readmission for heart failure in patients with acute heart failure.

AIMS: Constipation is a common gastrointestinal disorder that is associated with a high cardiovascular event rate in the general population. Although constipation is common in patients with cardiovascular diseases, only a few studies have examined the relationship between constipation and the prognosis of patients with heart failure. This study aimed to evaluate the effects of constipation on the prognosis of patients with acute heart failure. METHODS AND RESULTS: We investigated 397 patients admitted to our hospital from December 2020 to December 2022 with acute heart failure (mean age, 81 ± 13 years; 54% men). Patients with constipation were defined as those either taking laxatives regularly or diagnosed with constipation according to the International Statistical Classification of Diseases and Related Health Problems. During the follow-up periods (median, 173 days), 35 patients died, and 74 experienced readmission due to heart failure. Kaplan-Meier analysis before and after propensity score matching using 14 variables revealed that the risk of readmission due to heart failure was significantly higher in patients with constipation than in those without (before: log-rank P = 0.014, after: log-rank P = 0.0027). The adjusted Cox proportional hazards analysis revealed that the hazard ratio for readmission due to heart failure was 2.61 (95% confidence interval, 1.38-4.94, P = 0.0032). The risk of all-cause death was not significantly different between the two groups (hazard ratio, 1.76; 95% confidence interval, 0.61-5.06; P = 0.30). CONCLUSIONS: Constipation status was strongly associated with a higher risk of readmission for heart failure in patients with acute heart failure.

Higher Recurrence Rate of Acute Coronary Syndrome in Patients with Multiple-Time Myocardial Infarction.

The recurrence rate of acute coronary syndrome (ACS) in patients after first-time myocardial infarction (MI) is over ten times higher than in the general population. However, it is unclear whether patients with multiple-time MI have an even higher recurrence rate of MI. This study aimed to compare the recurrence rate in patients with multiple-time MI with the rate in patients after first-time MI. We retrospectively studied 794 consecutive MI patients who were discharged. Recurrent ACS was investigated in patients with previous MI (n = 46) and without previous MI (n = 748). During the follow-up periods (mean ± SD: 757 ± 733 days), recurrent ACS occurred in 47 cases without previous MI and in 7 cases with previous MI. Kaplan-Meier analysis revealed that the risk of recurrent ACS was significantly higher in patients with previous MI than in patients without previous MI. ACS recurrence rates one year from the onset were 4.2% in patients without previous MI and 11.9% in patients with previous MI. Landmark analysis revealed that the higher recurrence rate in patients with previous MI was as high as 14.1% from 1 year after the onset to 2 years. In conclusion, the risk of recurrent ACS may be significantly higher in patients with multiple-time MI than in patients after first-time MI.

Identification of Celastrol as a Novel Therapeutic Agent for Pulmonary Arterial Hypertension and Right Ventricular Failure Through Suppression of Bsg (Basigin)/CyPA (Cyclophilin A).

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.

Identification of Adipsin as a Novel Prognostic Biomarker in Patients With Coronary Artery Disease.

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<0.001]) and rehospitalization (HR, 2.4; 95% CI, 1.7-3.5 [P<0.001]). Interestingly, higher high-sensitivity C-reactive protein levels (≥1 mg/L) were significantly correlated with all-cause death (HR, 3.2; 95% CI, 1.7-5.9 [P<0.001]) and rehospitalization (HR, 1.5, 95% CI, 1.1-1.9 [P<0.01]). Importantly, the combination of adipsin (≥400 ng/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<0.001]). Finally, the receiver operating characteristic curve demonstrated that serum adipsin levels predict the death caused by acute myocardial infarction in patients with coronary artery disease (C-statistic, 0.847). 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.

Diagnostic and Prognostic Significance of Serum Levels of SeP (Selenoprotein P) in Patients With Pulmonary Hypertension.

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<0.01). Importantly, we found that the absolute change in SeP of patients with PAH (ΔSeP) in response to the initiation of PAH-specific therapy significantly correlated with the absolute change in mean pulmonary artery pressure, pulmonary vascular resistance (ΔPVR), and cardiac index (ΔCI; R=0.78, 0.76, and -0.71 respectively, all P<0.0001). Moreover, increase in ΔSeP during the follow-up predicted poor outcome of PAH. CONCLUSIONS: Serum SeP is a novel biomarker for diagnosis and assessment of treatment efficacy and long-term prognosis in patients with PAH.

Identification of Emetine as a Therapeutic Agent for Pulmonary Arterial Hypertension: Novel Effects of an Old Drug.

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.

ADAMTS8 Promotes the Development of Pulmonary Arterial Hypertension and Right Ventricular Failure: A Possible Novel Therapeutic Target.

RATIONALE: Pulmonary arterial hypertension (PAH) is characterized by pulmonary vascular remodeling with aberrant pulmonary artery smooth muscle cells (PASMCs) proliferation, endothelial dysfunction, and extracellular matrix remodeling. OBJECTIVE: Right ventricular (RV) failure is an important prognostic factor in PAH. Thus, we need to elucidate a novel therapeutic target in both PAH and RV failure. METHODS AND RESULTS: We performed microarray analysis in PASMCs from patients with PAH (PAH-PASMCs) and controls. We found a ADAMTS8 (disintegrin and metalloproteinase with thrombospondin motifs 8), a secreted protein specifically expressed in the lung and the heart, was upregulated in PAH-PASMCs and the lung in hypoxia-induced pulmonary hypertension (PH) in mice. To elucidate the role of ADAMTS8 in PH, we used vascular smooth muscle cell-specific ADAMTS8-knockout mice (ADAMTSΔSM22). Hypoxia-induced PH was attenuated in ADAMTSΔSM22 mice compared with controls. ADAMTS8 overexpression increased PASMC proliferation with downregulation of AMPK (AMP-activated protein kinase). In contrast, deletion of ADAMTS8 reduced PASMC proliferation with AMPK upregulation. Moreover, deletion of ADAMTS8 reduced mitochondrial fragmentation under hypoxia in vivo and in vitro. Indeed, PASMCs harvested from ADAMTSΔSM22 mice demonstrated that phosphorylated DRP-1 (dynamin-related protein 1) at Ser637 was significantly upregulated with higher expression of profusion genes (Mfn1 and Mfn2) and improved mitochondrial function. Moreover, recombinant ADAMTS8 induced endothelial dysfunction and matrix metalloproteinase activation in an autocrine/paracrine manner. Next, to elucidate the role of ADAMTS8 in RV function, we developed a cardiomyocyte-specific ADAMTS8 knockout mice (ADAMTS8ΔαMHC). ADAMTS8ΔαMHC mice showed ameliorated RV failure in response to chronic hypoxia. In addition, ADAMTS8ΔαMHC mice showed enhanced angiogenesis and reduced RV ischemia and fibrosis. Finally, high-throughput screening revealed that mebendazole, which is used for treatment of parasite infections, reduced ADAMTS8 expression and cell proliferation in PAH-PASMCs and ameliorated PH and RV failure in PH rodent models. CONCLUSIONS: These results indicate that ADAMTS8 is a novel therapeutic target in PAH.

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.

Identification of Novel Therapeutic Targets for Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) and chronic thromboembolic pulmonary hypertension (CTEPH) are fatal diseases; however, their pathogenesis still remains to be elucidated. We have recently screened novel pathogenic molecules and have performed drug discovery targeting those molecules. Pulmonary artery smooth muscle cells (PASMCs) in patients with PAH (PAH-PASMCs) have high proliferative properties like cancer cells, which leads to thickening and narrowing of distal pulmonary arteries. Thus, we conducted a comprehensive analysis of PAH-PASMCs and lung tissues to search for novel pathogenic proteins. We validated the pathogenic role of the selected proteins by using tissue-specific knockout mice. To confirm its clinical significance, we used patient-derived blood samples to evaluate the potential as a biomarker for diagnosis and prognosis. Finally, we conducted a high throughput screening and found inhibitors for the pathogenic proteins.

Different roles of myocardial ROCK1 and ROCK2 in cardiac dysfunction and postcapillary pulmonary hypertension in mice.

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.

Small GTP-Binding Protein GDP Dissociation Stimulator Prevents Thoracic Aortic Aneurysm Formation and Rupture by Phenotypic Preservation of Aortic Smooth Muscle Cells.

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.

Selenoprotein P Promotes the Development of Pulmonary Arterial Hypertension: Possible Novel Therapeutic Target.

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.

Activated TAFI Promotes the Development of Chronic Thromboembolic Pulmonary Hypertension: A Possible Novel Therapeutic Target.

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.

Prognostic Impacts of Plasma Levels of Cyclophilin A in Patients With Coronary Artery Disease.

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<0.001) than CyPA (≥12 ng/mL) or hsCRP (≥1 mg/L) alone. 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.

Protective Roles of Endothelial AMP-Activated Protein Kinase Against Hypoxia-Induced Pulmonary Hypertension in Mice.

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.

SmgGDS as a Crucial Mediator of the Inhibitory Effects of Statins on Cardiac Hypertrophy and Fibrosis: Novel Mechanism of the Pleiotropic Effects of Statins.

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.

Basigin Promotes Cardiac Fibrosis and Failure in Response to Chronic Pressure Overload in Mice.

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.

RhoA/Rho-Kinase in the Cardiovascular System.

Twenty years ago, Rho-kinase was identified as an important downstream effector of the small GTP-binding protein, RhoA. Thereafter, a series of studies demonstrated the important roles of Rho-kinase in the cardiovascular system. The RhoA/Rho-kinase pathway is now widely known to play important roles in many cellular functions, including contraction, motility, proliferation, and apoptosis, and its excessive activity induces oxidative stress and promotes the development of cardiovascular diseases. Furthermore, the important role of Rho-kinase has been demonstrated in the pathogenesis of vasospasm, arteriosclerosis, ischemia/reperfusion injury, hypertension, pulmonary hypertension, and heart failure. Cyclophilin A is secreted by vascular smooth muscle cells and inflammatory cells and activated platelets in a Rho-kinase-dependent manner, playing important roles in a wide range of cardiovascular diseases. Thus, the RhoA/Rho-kinase pathway plays crucial roles under both physiological and pathological conditions and is an important therapeutic target in cardiovascular medicine. Recently, functional differences between ROCK1 and ROCK2 have been reported in vitro. ROCK1 is specifically cleaved by caspase-3, whereas granzyme B cleaves ROCK2. However, limited information is available on the functional differences and interactions between ROCK1 and ROCK2 in the cardiovascular system in vivo. Herein, we will review the recent advances about the importance of RhoA/Rho-kinase in the cardiovascular system.