澤城 大悟

Several genetic defects, including a mutation in myosin heavy chain 11 (Myh11), are reported to cause familial thoracic aortic aneurysm and dissection (FTAAD). We recently showed that mice lacking K1256 of Myh11 developed aortic dissection when stimulated with angiotensin II, despite the absence of major pathological phenotypic abnormalities prior to stimulation. In this study, we used a comprehensive, data-driven, unbiased, multi-omics approach to find underlying changes in transcription and metabolism that predispose the aorta to dissection in mice harboring the Myh11 K1256del mutation. Pathway analysis of transcriptomes showed that genes involved in membrane transport were downregulated in homozygous mutant (Myh11ΔK/ΔK) aortas. Furthermore, expanding the analysis with metabolomics showed that two mechanisms that raise the cytosolic Ca2+ concentration-multiple calcium channel expression and ADP-ribose synthesis-were attenuated in Myh11ΔK/ΔK aortas. We suggest that the impairment of the Ca2+ influx attenuates aortic contraction and that suboptimal contraction predisposes the aorta to dissection.

Adipose tissue macrophages (ATMs) play an important role in obesity and inflammation, and they accumulate in adipose tissue (AT) with aging. Furthermore, increased ATM senescence has been shown in obesity-related AT remodeling and dysfunction. However, ATM senescence and its role are unclear in age-related AT dysfunction. Here, we show that ATMs (a) acquire a senescence-like phenotype during chronological aging; (b) display a global decline of basic macrophage functions such as efferocytosis, an essential process to preserve AT homeostasis by clearing dysfunctional or apoptotic cells; and (c) promote AT remodeling and dysfunction. Importantly, we uncover a major role for the age-associated accumulation of osteopontin (OPN) in these processes in visceral AT. Consistently, loss or pharmacologic inhibition of OPN and bone marrow transplantation of OPN-/- mice attenuate the ATM senescence-like phenotype, preserve efferocytosis, and finally restore healthy AT homeostasis in the context of aging. Collectively, our findings implicate pharmacologic OPN inhibition as a viable treatment modality to counter ATM senescence-mediated AT remodeling and dysfunction during aging.

BACKGROUND: Aging myocardium undergoes progressive cardiac hypertrophy and interstitial fibrosis with diastolic and systolic dysfunction. Recent metabolomics studies shed light on amino acids in aging. The present study aimed to dissect how aging leads to elevated plasma levels of the essential amino acid phenylalanine and how it may promote age-related cardiac dysfunction. METHODS: We studied cardiac structure and function, together with phenylalanine catabolism in wild-type (WT) and p21-/- mice (male; 2-24 months), with the latter known to be protected from cellular senescence. To explore phenylalanine's effects on cellular senescence and ectopic phenylalanine catabolism, we treated cardiomyocytes (primary adult rat or human AC-16) with phenylalanine. To establish a role for phenylalanine in driving cardiac aging, WT male mice were treated twice a day with phenylalanine (200 mg/kg) for a month. We also treated aged WT mice with tetrahydrobiopterin (10 mg/kg), the essential cofactor for the phenylalanine-degrading enzyme PAH (phenylalanine hydroxylase), or restricted dietary phenylalanine intake. The impact of senescence on hepatic phenylalanine catabolism was explored in vitro in AML12 hepatocytes treated with Nutlin3a (a p53 activator), with or without p21-targeting small interfering RNA or tetrahydrobiopterin, with quantification of PAH and tyrosine levels. RESULTS: Natural aging is associated with a progressive increase in plasma phenylalanine levels concomitant with cardiac dysfunction, whereas p21 deletion delayed these changes. Phenylalanine treatment induced premature cardiac deterioration in young WT mice, strikingly akin to that occurring with aging, while triggering cellular senescence, redox, and epigenetic changes. Pharmacological restoration of phenylalanine catabolism with tetrahydrobiopterin administration or dietary phenylalanine restriction abrogated the rise in plasma phenylalanine and reversed cardiac senescent alterations in aged WT mice. Observations from aged mice and human samples implicated age-related decline in hepatic phenylalanine catabolism as a key driver of elevated plasma phenylalanine levels and showed increased myocardial PAH-mediated phenylalanine catabolism, a novel signature of cardiac aging. CONCLUSIONS: Our findings establish a pathogenic role for increased phenylalanine levels in cardiac aging, linking plasma phenylalanine levels to cardiac senescence via dysregulated phenylalanine catabolism along a hepatic-cardiac axis. They highlight phenylalanine/PAH modulation as a potential therapeutic strategy for age-associated cardiac impairment.

In the context of obesity, senescent cells accumulate in white adipose tissue (WAT). The cellular underpinnings of WAT senescence leading to insulin resistance are not fully elucidated. The objective of the current study was to evaluate the presence of WAT senescence early after initiation of high-fat diet (HFD, 1-10 weeks) in 5-month-old male C57BL/6J mice and the potential role of energy metabolism. We first showed that WAT senescence occurred 2 weeks after HFD as evidenced in whole WAT by increased senescence-associated ß-galactosidase activity and cyclin-dependent kinase inhibitor 1A and 2A expression. WAT senescence affected various WAT cell populations, including preadipocytes, adipose tissue progenitors, and immune cells, together with adipocytes. WAT senescence was associated with higher glycolytic and mitochondrial activity leading to enhanced ATP content in HFD-derived preadipocytes, as compared with chow diet-derived preadipocytes. One-month daily exercise, introduced 5 weeks after HFD, was an effective senostatic strategy, since it reversed WAT cellular senescence, while reducing glycolysis and production of ATP. Interestingly, the beneficial effect of exercise was independent of body weight and fat mass loss. We demonstrated that WAT cellular senescence is one of the earliest events occurring after HFD initiation and is intimately linked to the metabolic state of the cells. Our data uncover a critical role for HFD-induced elevated ATP as a local danger signal inducing WAT senescence. Exercise exerts beneficial effects on adipose tissue bioenergetics in obesity, reversing cellular senescence, and metabolic abnormalities.

AIMS: Increase of cardiac cAMP bioavailability and PKA activity through adenylyl-cyclase 8 (AC8) overexpression enhances contractile function in young transgenic mice (AC8TG). Ageing is associated with decline of cardiac contraction partly by the desensitization of β-adrenergic/cAMP signalling. Our objective was to evaluate cardiac cAMP signalling as age increases between 2 months and 12 months and to explore whether increasing the bioavailability of cAMP by overexpression of AC8 could prevent cardiac dysfunction related to age. METHODS AND RESULTS: Cardiac cAMP pathway and contractile function were evaluated in AC8TG and their non-transgenic littermates (NTG) at 2- and 12 months old. AC8TG demonstrated increased AC8, PDE1, 3B and 4D expression at both ages, resulting in increased phosphodiesterase and PKA activity, and increased phosphorylation of several PKA targets including sarco(endo)plasmic-reticulum-calcium-ATPase (SERCA2a) cofactor phospholamban (PLN) and GSK3α/β a main regulator of hypertrophic growth and ageing. Confocal immunofluorescence revealed that the major phospho-PKA substrates were co-localized with Z-line in 2-month-old NTG but with Z-line interspace in AC8TG, confirming the increase of PKA activity in the compartment of PLN/SERCA2a. In both 12-month-old NTG and AC8TG, PLN and GSK3α/β phosphorylation was increased together with main localization of phospho-PKA substrates in Z-line interspaces. Haemodynamics demonstrated an increased contractile function in 2- and 12-month-old AC8TG, but not in NTG. In contrast, echocardiography and tissue Doppler imaging (TDI) performed in conscious mice unmasked myocardial dysfunction with a decrease of systolic strain rate in both old AC8TG and NTG. In AC8TG TDI showed a reduced strain rate even in 2-month-old animals. Development of age-related cardiac dysfunction was accelerated in AC8TG, leading to heart failure (HF) and premature death. Histological analysis confirmed early cardiomyocyte hypertrophy and interstitial fibrosis in AC8TG when compared with NTG. CONCLUSION: Our data demonstrated an early and accelerated cardiac remodelling in AC8TG mice, leading to the development of HF and reduced lifespan. Age-related reorganization of cAMP/PKA signalling can accelerate cardiac ageing, partly through GSK3α/β phosphorylation.

Obesity is characterized by accumulation of adipose tissue and is one the most important risk factors in the development of insulin resistance. Carbon monoxide-releasing (CO-releasing) molecules (CO-RMs) have been reported to improve the metabolic profile of obese mice, but the underlying mechanism remains poorly defined. Here, we show that oral administration of CORM-401 to obese mice fed a high-fat diet (HFD) resulted in a significant reduction in body weight gain, accompanied by a marked improvement in glucose homeostasis. We further unmasked an action we believe to be novel, by which CO accumulates in visceral adipose tissue and uncouples mitochondrial respiration in adipocytes, ultimately leading to a concomitant switch toward glycolysis. This was accompanied by enhanced systemic and adipose tissue insulin sensitivity, as indicated by a lower blood glucose and increased Akt phosphorylation. Our findings indicate that the transient uncoupling activity of CO elicited by repetitive administration of CORM-401 is associated with lower weight gain and increased insulin sensitivity during HFD. Thus, prototypic compounds that release CO could be investigated for developing promising insulin-sensitizing agents.

BACKGROUND: Aging induces cardiac structural and functional changes linked to the increased deposition of extracellular matrix proteins, including OPN (osteopontin), conducing to progressive interstitial fibrosis. Although OPN is involved in various pathological conditions, its role in myocardial aging remains unknown. METHODS: OPN deficient mice (OPN-/-) with their wild-type (WT) littermates were evaluated at 2 and 14 months of age in terms of cardiac structure, function, histology and key molecular markers. OPN expression was determined by reverse-transcription polymerase chain reaction, immunoblot and immunofluorescence. Luminex assays were performed to screen plasma samples for various cytokines/adipokines in addition to OPN. Similar explorations were conducted in aged WT mice after surgical removal of visceral adipose tissue (VAT) or treatment with a small-molecule OPN inhibitor agelastatin A. Primary WT fibroblasts were incubated with plasma from aged WT and OPN-/- mice, and evaluated for senescence (senescence-associated β-galactosidase and p16), as well as fibroblast activation markers (Acta2 and Fn1). RESULTS: Plasma OPN levels increased in WT mice during aging, with VAT showing the strongest OPN induction contrasting with myocardium that did not express OPN. VAT removal in aged WT mice restored cardiac function and decreased myocardial fibrosis in addition to a substantial reduction of circulating OPN and transforming growth factor β levels. OPN deficiency provided a comparable protection against age-related cardiac fibrosis and dysfunction. Intriguingly, a strong induction of senescence in cardiac fibroblasts was observed in both VAT removal and OPN-/- mice. The addition of plasma from aged OPN-/- mice to cultures of primary cardiac fibroblasts induced senescence and reduced their activation (compared to aged WT plasma). Finally, Agelastatin A treatment of aged WT mice fully reversed age-related myocardial fibrosis and dysfunction. CONCLUSIONS: During aging, VAT represents the main source of OPN and alters heart structure and function via its profibrotic secretome. As a proof-of-concept, interventions targeting OPN, such as VAT removal and OPN deficiency, rescued the heart and induced a selective modulation of fibroblast senescence. Our work uncovers OPN's role in the context of myocardial aging and proposes OPN as a potential new therapeutic target for a healthy cardiac aging.

AIM: To clarify whether carotid atherosclerosis and its risk factors are associated with cognitive decline. METHODS: We evaluated 206 individuals who visited our center for health screening. We carried out physical examinations, blood tests, intima-media thickness (IMT) measurement by carotid ultrasonography, brain magnetic resonance imaging scanning and cognitive function assessments. A total of 30 individuals, who had significant cerebrovascular lesions detected in magnetic resonance imaging scans, were excluded. To detect early cognitive decline, we defined "cognitive impairment (CI)" when a patient satisfied at least one of three criteria. These were Mini-Mental State Examination score <24, clock-drawing test score <4 coexisting with forgetfulness and Wechsler Memory Scale-revised delayed recall score below the normal range for the duration of education (>16 years of education: ≥9, 10-15 years: ≥5, 0-9 years: ≥3). RESULTS: Among 176 individuals, 27 were placed in the CI group. IMT was significantly higher in the CI group as compared with the non-CI group (mean ± SD: 2.0 ± 1.0 vs 1.7 ± 0.7, P = 0018 by Student's t-test). Other atherosclerotic risk factors, such as blood pressure, low-density lipoprotein cholesterol, and hemoglobin A1c, were not significantly different between the two groups. In multivariate analysis, maximum IMT was associated with impaired immediate recall score on Wechsler Memory Scale-revised, independent of the presence of deep white matter hyperintensities on the magnetic resonance imaging scan. CONCLUSIONS: Subclinical carotid atherosclerosis, defined as thickened IMT, could be a marker for early stages of CI, especially for immediate memory recall. The impairment is presumably caused by inducing cerebral microvascular dysfunction in the frontal lobe. Geriatr Gerontol Int 2018; 18: 65-71.

AIM: Long-term high-fat diet (HFD) induces both cardiac remodelling and myocardial dysfunction in murine models. The aim was to assess the time course and mechanisms of metabolic and cardiac modifications induced by short-term HFD in wild-type (WT) mice. METHODS AND RESULTS: Thirty-three WT mice were subjected to HFD (60% fat, n = 16) and chow diet (CD, 13% fat, n = 17). Metabolic and echocardiographic data were collected at baseline and every 5 weeks for 20 weeks. Invasive haemodynamic data and myocardial samples were collected at 5 and 20 weeks. Echocardiographic data included left ventricular (LV) diameters and thickness, and systolic function using radial strain rate (SR). Histological assessment of cardiomyocyte and adipocyte sizes, interstitial fibrosis, and apoptosis index were performed. During follow-up, body weight, and glycaemia levels were higher in HFD than in CD mice, in association with an early adipose tissue remodelling. Despite no difference between both groups in blood pressure and LV mass at 5 weeks, an early LV dysfunction was observed in HFD mice as assessed by radial SR (21 ± 0.8 vs. 27 ± 0.8 unit/s, P < 0.001) and haemodynamic assessment. During follow-up, both groups demonstrated a progressive systolic and diastolic LV dysfunction and remodelling including dilatation and hypertrophy, which were more severe in HFD mice. Compared with CD mice, the early LV impairment in HFD mice was coupled with a higher cardiomyocyte apoptosis level (0.95 vs. 0.02%, P < 0.05) associated with an interstitial fibrosis process (2.3 vs. 0.2%, P < 0.05), which worsen during follow-up. CONCLUSION: The HFD promoted early metabolic and cardiac dysfunctions, and adipose and myocardial tissues remodelling.

BACKGROUND: Type 2 diabetes mellitus (T2DM) may alter cardiac structure and function, but obesity, hypertension (HTN), or aging can induce similar abnormalities. OBJECTIVES: This study sought to link cardiac phenotypes in T2DM patients with clinical profiles and outcomes using cluster analysis. METHODS: Baseline echocardiography and a composite endpoint (cardiovascular mortality and hospitalization) were evaluated in 842 T2DM patients from 2 prospective cohorts. A cluster analysis was performed on echocardiographic variables, and the association between clusters and clinical profiles and outcomes was assessed. RESULTS: Three clusters were identified. Cluster 1 patients had the lowest left ventricular (LV) mass index and ratio between early mitral inflow velocity and mitral annular early diastolic velocity (E/e') ratio, had the highest left ventricular ejection fraction (LVEF), and were predominantly male with the lowest rate of obesity or HTN. Cluster 2 patients had the highest strain and highest E/e' ratio, were the oldest, were predominantly female, and had the lowest rate of isolated T2DM (without HTN or obesity). Cluster 3 patients had the highest LV mass index and volumes and the lowest LVEF and strain, were predominantly male, and shared similar age and rate of obesity and HTN as cluster 1 patients. After follow-up of 67 months (interquartile range: 40 to 87), the composite endpoint occurred in 56 of 521 patients (10.8%). Clusters 2 (hazard ratio: 2.37; 95% confidence interval: 1.15 to 4.88) and 3 (hazard ratio: 2.19; 95% confidence interval: 1.00 to 4.82) had a similar outcome, which was worse than cluster 1. CONCLUSIONS: Cluster analysis of echocardiographic variables identified 3 different echocardiographic phenotypes of T2DM patients that were associated with distinct clinical profiles and highlighted the prognostic value of LV remodeling and subclinical dysfunction.

The abundance of epicardial adipose tissue (EAT) is associated with atrial fibrillation (AF), the most frequent cardiac arrhythmia. However, both the origin and the factors involved in EAT expansion are unknown. Here, we found that adult human atrial epicardial cells were highly adipogenic through an epithelial-mesenchymal transition both in vitro and in vivo. In a genetic lineage tracing the WT1CreERT2+/-RosatdT+/- mouse model subjected to a high-fat diet, adipocytes of atrial EAT derived from a subset of epicardial progenitors. Atrial myocardium secretome induces the adipogenic differentiation of adult mesenchymal epicardium-derived cells by modulating the balance between mesenchymal Wingless-type Mouse Mammary Tumor Virus integration site family, member 10B (Wnt10b)/β-catenin and adipogenic ERK/MAPK signaling pathways. The adipogenic property of the atrial secretome was enhanced in AF patients. The atrial natriuretic peptide secreted by atrial myocytes is a major adipogenic factor operating at a low concentration by binding to its natriuretic peptide receptor A (NPRA) receptor and, in turn, by activating a cGMP-dependent pathway. Hence, our data indicate cross-talk between EAT expansion and mechanical function of the atrial myocardium.

Excessive growth of pulmonary arterial (PA) smooth muscle cells (SMCs) is a major component of PA hypertension (PAH). The calcium-activated neutral cysteine proteases calpains 1 and 2, expressed by PASMCs, contribute to PH but are tightly controlled by a single specific inhibitor, calpastatin. Our objective was to investigate calpastatin during pulmonary hypertension (PH) progression and its potential role as an intracellular and/or extracellular effector. We assessed calpains and calpastatin in patients with idiopathic PAH and mice with hypoxic or spontaneous (SM22-5HTT(+) strain) PH. To assess intracellular and extracellular roles for calpastatin, we studied effects of the calpain inhibitor PD150606 on hypoxic PH in mice with calpastatin overexpression driven by the cytomegalovirus promoter (CMV-Cast) or C-reactive protein (CRP) promoter (CRP-Cast), inducing increased calpastatin production ubiquitously and in the liver, respectively. Chronically hypoxic and SM22-5HTT(+) mice exhibited increased lung calpastatin and calpain 1 and 2 protein levels and activity, both intracellularly and extracellularly. Prominent calpastatin and calpain immunostaining was found in PASMCs of remodeled vessels in mice and patients with PAH, who also exhibited increased plasma calpastatin levels. CMV-Cast and CRP-Cast mice showed similarly decreased PH severity compared with wild-type mice, with no additional effect of PD150606 treatment. In cultured PASMCs from wild-type and CMV-Cast mice, exogenous calpastatin decreased cell proliferation and migration with similar potency as PD150606 and suppressed fibronectin-induced potentiation. These results indicate that calpastatin limits PH severity via extracellular mechanisms. They suggest a new approach to the development of treatments for PH.

Constitutive activation of the mammalian target of rapamycin (mTOR) complexes mTORC1 and mTORC2 is associated with pulmonary hypertension (PH) and sustained growth of pulmonary artery (PA) smooth muscle cells (SMCs). We investigated whether selective mTORC1 activation in SMCs induced by deleting the negative mTORC1 regulator tuberous sclerosis complex 1 gene (TSC1) was sufficient to produce PH in mice. Mice expressing Cre recombinase under SM22 promoter control were crossed with TSC1(LoxP/LoxP) mice to generate SM22-TSC1(-/-) mice. At 8 weeks of age, SM22-TSC1(-/-) mice exhibited PH with marked increases in distal PA muscularization and Ki67-positive PASMC counts, without systemic hypertension or cardiac dysfunction. Marked activation of the mTORC1 substrates S6 kinase and 4E-BP and the mTORC2 substrates p-Akt(Ser473) and glycogen synthase kinase 3 was found in the lungs and pulmonary vessels of SM22-TSC1(-/-) mice when compared with control mice. Treatment with 5 mg/kg rapamycin for 3 weeks to inhibit mTORC1 and mTORC2 fully reversed PH in SM22-TSC1(-/-) mice. In chronically hypoxic mice and SM22-5HTT(+) mice exhibiting PH associated with mTORC1 and mTORC2 activation, PH was maximally attenuated by low-dose rapamycin associated with selective mTORC1 inhibition. Cultured PASMCs from SM22-TSC1(-/-), SM22-5HTT(+), and chronically hypoxic mice exhibited similar sustained growth-rate enhancement and constitutive mTORC1 and mTORC2 activation; both effects were abolished by rapamycin. Deletion of the downstream mTORC1 effectors S6 kinase 1/2 in mice also activated mTOR signaling and induced PH. We concluded that activation of mTORC1 signaling leads to increased PASMC proliferation and subsequent PH development.

Pulmonary artery smooth muscle cell (PA-SMC) proliferation and inflammation are key components of pulmonary arterial hypertension (PAH). Interleukin (IL)-1β binds to IL-1 receptor (R)1, thereby recruiting the molecular adaptor myeloid differentiation primary response protein 88 (MyD88) (involved in IL-1R1 and Toll-like receptor signal transduction) and inducing IL-1, IL-6 and tumour necrosis factor-α synthesis through nuclear factor-κB activation.We investigated the IL-1R1/MyD88 pathway in the pathogenesis of pulmonary hypertension.Marked IL-1R1 and MyD88 expression with predominant PA-SMC immunostaining was found in lungs from patients with idiopathic PAH, mice with hypoxia-induced pulmonary hypertension and SM22-5-HTT(+) mice. Elevations in lung IL-1β, IL-1R1, MyD88 and IL-6 preceded pulmonary hypertension in hypoxic mice. IL-1R1(-/-), MyD88(-/-) and control mice given the IL-1R1 antagonist anakinra were protected similarly against hypoxic pulmonary hypertension and perivascular macrophage recruitment. Anakinra reversed pulmonary hypertension partially in SM22-5-HTT(+) mice and markedly in monocrotaline-treated rats. IL-1β-mediated stimulation of mouse PA-SMC growth was abolished by anakinra and absent in IL-1R1(-/-) and MyD88(-/-) mice. Gene deletion confined to the myeloid lineage (M.lys-Cre MyD88(fl/fl) mice) decreased pulmonary hypertension severity versus controls, suggesting IL-1β-mediated effects on PA-SMCs and macrophages. The growth-promoting effect of media conditioned by M1 or M2 macrophages from M.lys-Cre MyD88(fl/fl) mice was attenuated.Pulmonary vessel remodelling and inflammation during pulmonary hypertension require IL-1R1/MyD88 signalling. Targeting the IL-1β/IL-1R1 pathway may hold promise for treating human PAH.

The activation of the calpain system is involved in the repair process following myocardial infarction (MI). However, the impact of the inhibition of calpain by calpastatin, its natural inhibitor, on scar healing and left ventricular (LV) remodeling is elusive. Male mice ubiquitously overexpressing calpastatin (TG) and wild-type (WT) controls were subjected to an anterior coronary artery ligation. Mortality at 6 wk was higher in TG mice (24% in WT vs. 44% in TG, P < 0.05) driven by a significantly higher incidence of cardiac rupture during the first week post-MI, despite comparable infarct size and LV dysfunction and dilatation. Calpain activation post-MI was blunted in TG myocardium. In TG mice, inflammatory cell infiltration and activation were reduced in the infarct zone (IZ), particularly affecting M2 macrophages and CD4(+) T cells, which are crucial for scar healing. To elucidate the role of calpastatin overexpression in macrophages, we stimulated peritoneal macrophages obtained from TG and WT mice in vitro with IL-4, yielding an abrogated M2 polarization in TG but not in WT cells. Lymphopenic Rag1(-/-) mice receiving TG splenocytes before MI demonstrated decreased T-cell recruitment and M2 macrophage activation in the IZ day 5 after MI compared with those receiving WT splenocytes. Calpastatin overexpression prevented the activation of the calpain system after MI. It also impaired scar healing, promoted LV rupture, and increased mortality. Defective scar formation was associated with blunted CD4(+) T-cell and M2-macrophage recruitment.

AIMS: Krüppel-like factors (KLFs) are a family of transcription factors which play important roles in the heart under pathological and developmental conditions. We previously identified and cloned Klf6 whose homozygous mutation in mice results in embryonic lethality suggesting a role in cardiovascular development. Effects of KLF6 on pathological regulation of the heart were investigated in the present study. METHODS AND RESULTS: Mice heterozygous for Klf6 resulted in significantly diminished levels of cardiac fibrosis in response to angiotensin II infusion. Intriguingly, a similar phenotype was seen in cardiomyocyte-specific Klf6 knockout mice, but not in cardiac fibroblast-specific knockout mice. Microarray analysis revealed increased levels of the extracellular matrix factor, thrombospondin 4 (TSP4), in the Klf6-ablated heart. Mechanistically, KLF6 directly suppressed Tsp4 expression levels, and cardiac TSP4 regulated the activation of cardiac fibroblasts to regulate cardiac fibrosis. CONCLUSION: Our present studies on the cardiac function of KLF6 show a new mechanism whereby cardiomyocytes regulate cardiac fibrosis through transcriptional control of the extracellular matrix factor, TSP4, which, in turn, modulates activation of cardiac fibroblasts.

Aortic dissection and intramural haematoma comprise an aortopathy involving separation of the aortic wall. Underlying mechanisms of the condition remain unclear. Here we show that granulocyte macrophage colony-stimulating factor (GM-CSF) is a triggering molecule for this condition. Transcription factor Krüppel-like factor 6 (KLF6)-myeloid-specific conditional deficient mice exhibit this aortic phenotype when subjected to aortic inflammation. Mechanistically, KLF6 downregulates expression and secretion of GM-CSF. Administration of neutralizing antibody against GM-CSF prevents the condition in these mice. Conversely, administration of GM-CSF in combination with aortic inflammation to wild-type mice is sufficient to induce the phenotype, suggesting the general nature of effects. Moreover, patients with this condition show highly increased circulating levels of GM-CSF, which is also locally expressed in the dissected aorta. GM-CSF is therefore a key regulatory molecule causative of this aortopathy, and modulation of this cytokine might be an exploitable treatment strategy for the condition.

Caveolae and their coat proteins, caveolins (Cav), are cave-like invaginations found in the plasma membrane of a variety of cells. These unique vesicles and their coat proteins, Cavs, have diverse effects on endothelial function, nitric oxide synthesis regulation, signal transduction, cholesterol metabolism, and apoptosis. Animal studies in Cav knockout mice demonstrate the vital role of these structural proteins on endothelial and vascular function. Genetic studies have proposed that beside neoplasia, Cavs may play a role in the development of atherosclerosis, cardiomyopathy, long QT syndrome, pulmonary fibrosis, and muscular dystrophy. The role of Cav expression in atherosclerotic disease is poorly understood and remains controversial. Interestingly, there is emerging evidence between low Cav-1 levels and the vulnerable plaque, which could potentially identify Cav-1 as a novel plaque biomarker. Cavs, through intricate biochemical pathways involving endothelial nitric oxide synthase and mitogen-activated protein kinase, are known to affect the cardiovascular system at multiple levels. In the present review, we aim to highlight the nature and types of caveolae, caveolar signaling mechanisms and regulation, and the pathophysiology of Cavs as it pertains to the cardiovascular system. Ongoing research is needed to clarify the diagnostic and prognostic role of these novel proteins and to determine how the effects of Cavs can translate into clinical medicine.

OBJECTIVES: This study aimed to assess the relation between stent edge restenosis (SER) and the distance from the stent edge to the residual plaque using quantitative intravascular ultrasound. BACKGROUND: Although percutaneous coronary intervention with drug-eluting stents has improved SER rates, determining an appropriate stent edge landing zone can be challenging in cases of diffuse plaque lesions. It is known that edge vascular response can occur within 2 mm from the edge of a bare metal stent, but the distance to the adjacent plaque has not been evaluated for drug-eluting stents. METHODS: A total of 97 proximal residual plaque lesions (plaque burden [PB] >40%) treated with everolimus-eluting stents were retrospectively evaluated to determine the distance from the stent edge to the residual plaque. RESULTS: The SER group had significantly higher PB (59.1 ± 6.1% vs. 51.9 ± 9.1% for non-SER; P = 0.04). Higher PB was associated with SER, with the cutoff value of 54.74% determined using receiver operating characteristic (ROC) curve analysis. At this cutoff value of PB, the distance from the stent edge to the lesion was significantly associated with SER (odds ratio = 2.05, P = 0.035). The corresponding area under the ROC curve was 0.725, and the cutoff distance value for predicting SER was 1.0 mm. CONCLUSION: An interval less than 1 mm from the proximal stent edge to the nearest point with the determined PB cutoff value of 54.74% was significantly associated with SER in patients with residual plaque lesions.

Activation of heme oxygenase-1 (HO-1), a heme-degrading enzyme responsive to a wide range of cellular stress, is traditionally considered to convey adaptive responses to oxidative stress, inflammation and vasoconstriction. These diversified effects are achieved through the degradation of heme to carbon monoxide (CO), biliverdin (which is rapidly converted to bilirubin by biliverdin reductase) and ferric iron. Recent findings have added antiproliferative and angiogenic effects to the list of HO-1/CO actions. HO-1 along with its reaction products bilirubin and CO are protective against ischemia-induced injury (myocardial infarction, ischemia-reperfusion (IR)-injury and post-infarct structural remodelling). Moreover, HO-1, and CO in particular, possess acute antihypertensive effects. As opposed to these curative potentials, the long-believed protective effect of HO-1 in cardiac remodelling in response to pressure overload and type 2 diabetes mellitus (DM) has been questioned by recent work. These challenges, coupled with emerging regulatory mechanisms, motivate further in-depth studies to help understand untapped layers of HO-1 regulation and action. The outcomes of these efforts may shed new light on critical mechanisms that could be used to harness the protective potential of this enzyme for the therapeutic benefit of patients suffering from such highly prevalent cardiovascular disorders.

BACKGROUND: Restenosis after percutaneous coronary intervention (PCI) is still a great concern even in the recent drug-eluting stent (DES) era. As less invasive and sensitive parameter to detect restenosis is needed, this study was aimed to assess whether the clinical implication of temporal change in plasma BNP levels might be a useful indicator of restenosis after DES implantation. METHODS AND RESULTS: 847 consecutive patients who underwent elective PCI using silorimus-eluting sent (SES) between 2005 and 2009 were analyzed. Primary endpoint was subsequent target-lesion revascularization (TLR) after PCI. There was no significant difference in either baseline (TLR+vs. TLR-: 107.2 ± 172.2 vs. 96.2 ± 175.5 pg/mL, P=0.53) or follow-up plasma B-type natriuretic peptide (BNP) levels (TLR+vs. TLR-: 88.6 ± 111.6 vs. 68.5 ± 226.0 pg/mL, P=0.35) between patients with and without subsequent TLR. Conversely, ratio of follow-up to baseline BNP was significantly higher in patients with TLR (TLR+vs. TLR-: 1.55 ± 1.58 vs. 1.07 ± 1.04, P<0.001). Multivariate analysis using logistic regression showed log transformed BNP-ratio was an independent predictor of TLR (adjusted odds ratio (aOR): 1.94, 95%CI: 1.42-2.66, P<0.001). A closer relationship between BNP elevation greater than 2-fold and subsequent TLR was found (aOR: 2.69, 95%CI: 1.27-5.69, P<0.009). Furthermore, propensity score matching analysis showed that the incidence of subsequent TLR was significantly higher in patients with BNP elevation (P<0.001). CONCLUSION: Serial measurement of plasma BNP levels and its change might be a useful approach to predict restenosis in patients without typical chest symptoms receiving SES.

BACKGROUND: Restenosis, a condition in which the lesion vessel renarrows after a coronary intervention procedure, remains a limitation in management. A surrogate biomarker for risk stratification of restenosis would be welcome. B-type natriuretic peptide (BNP) is secreted in response to pathologic stress from the heart. Its use as a biomarker of heart failure is well known; however, its diagnostic potential in ischemic heart disease is less explored. Recently, it has been reported that processed forms of BNP exist in the circulation. We hypothesized that circulating processed forms of BNP might be a biomarker of ischemic heart disease. METHODS: We characterized processed forms of BNP by a newly developed mass spectrometry-based detection method combined with immunocapture using commercial anti-BNP antibodies. RESULTS: Measurements of processed forms of BNP by this assay were found to be strongly associated with presence of restenosis. Reduced concentrations of the amino-terminal processed peptide BNP(5-32) relative to BNP(3-32) [as the index parameter BNP(5-32)/BNP(3-32) ratio] were seen in patients with restenosis [median (interquartile range) 1.19 (1.11-1.34), n = 22] vs without restenosis [1.43 (1.22-1.61), n = 83; P < 0.001] in a cross-sectional study of 105 patients undergoing follow-up coronary angiography. A sensitivity of 100% to rule out the presence of restenosis was attained at a ratio of 1.52. CONCLUSIONS: Processed forms of BNP may serve as viable potential biomarkers to rule out restenosis.

The development of diagnostic biomarkers of acute cardiovascular disease remains an important topic of interest given potential use to aid in early diagnosis. Cardiac biomarkers of ischemia and heart failure have already proven to be clinically useful. Biomarkers of aortic diseases are also needed, especially for life-threatening conditions such as aortic dissection. In this review, we discuss the present status of the development of biomarkers of aortic diseases. Although aortic dissection has been most vigorously pursued, there has also been notable recent progress in biomarkers of aneurysms and inflammatory aortic disease.

The aim of this study was to evaluate the short-term operative results of patients with Marfan syndrome who underwent thoracic or abdominal aortic surgery in a 4-year period in Japan. Data were collected from the Japan Cardiovascular Surgery Database (JCVSD). We retrospectively analyzed the data of 845 patients with Marfan syndrome who underwent cardiovascular surgery between January 2008 and January 2011. Logistic regression was used to generate risk models. The early mortality rate was 4.4% (37/845). Odds ratios (OR), 95% confidence intervals (CI), and P values for structures and processes in the mortality prediction model were as follows: renal insufficiency (OR, 11.37; CI, 3.7234.66; P < 0.001); respiratory disorder (OR, 11.12; CI, 3.20-38.67; P < 0.001); aortic dissection (OR, 13.02; CI, 2.8060.60; P = 0.001); pseudoaneurysm (OR, 11.23; CI, 1.38-91.66; P = 0.024); thoracoabdominal aneurysm (OR, 2.67; CI, 1.22-5.84; P = 0.014); and aorticrupure (OR, 4.23; CI, 1.26-14.23; P = 0.002). The mortality prediction model had a Cindex of 0.82 and a Hosmer-Lemeshow P value of 0.56. In conclusion, this study demonstrated that renal insufficiency and respiratory disorder had great impact on the operative mortality of Marfan patients undergoing cardiovascular surgery. Because patients with aortic dissection or aortic rupture showed high operative mortality, close follow-up to avoid emergency operation is mandatory to improve the operative results. Achieving good results from surgery of the thoracoabdominal aorta was quite challenging, also in Marfan patients.

BACKGROUND: Macrocytosis, as a qualitative abnormality of erythrocytes, has not drawn attention as a prognostic indicator after PCI, while anemia, as a quantitative abnormality of erythrocytes, has been recognized as a predictor of adverse outcomes. The aim of this study was to perform prognostic risk stratification of patients after PCI based on the presence or absence of macrocytosis. METHODS: The clinical records of 941 consecutive patients who underwent PCI at a single institution were retrospectively reviewed. The prognostic implication of macrocytosis was evaluated by univariate and multivariate Cox's proportional hazard regression analysis. RESULTS: There were 130 (13.8%) patients with macrocytosis. A significantly higher all-cause and cardiac mortality, as well as incidence of composite adverse events were observed in the Macrocytic group. Kaplan-Meier analysis also showed a significantly poorer overall survival in patients with macrocytosis. Even after exclusion of anemic patients, this tendency was still observed. Furthermore, macrocytosis was significantly and independently associated with adverse outcomes after PCI (aHR of cardiac death: 3.45, 95%CI: 1.22-9.80, P=0.019). Interestingly, fewer patients with macrocytosis were prescribed statins compared with those without it (33.8% vs. 47.1%, P=0.005). CONCLUSIONS: The results of the study indicate that measuring mean corpuscular volume (MCV) as a qualitative index of erythrocytes might be helpful for a prognostic risk stratification of patients subjected to PCI.

Rapid measurement of B-type natriuretic peptide (BNP) plays a practical role in the diagnosis of congestive heart failure. Analytical evaluation of a new small-footprint immunochromatography reader of BNP (Rapidpia®) was performed and compared with the commercially available SHIONOSPOT® Reader as the index. The new BNP assay had a within-run coefficient of variation (CV) of 9.0% and a between-run CV of 2.1%. Correlations between whole blood and plasma samples and those with the index SHIONOSPOT® Reader were y = 0.93x + 0.88, R2 = 0.98 and y = 1.08x - 6.67, R2 = 0.93, respectively. Based on our findings, the two point-of care (POC) assays for BNP, Rapidpia® and SHIONOSPOT® Reader, showed comparable results.

症例は79歳男性。72歳時3枝病変を認めるも保存的加療となった。2009年労作時胸痛が出現、冠動脈病変の進行と心機能低下を認め、前下行枝に対しバイパス術施行。術直後から持続性/非持続性心室頻拍(VT)が頻発、回旋枝へPCI施行後、Overdrive pacingとニフェカラント/ランジオロールの持続投与にても消失せず。薬物抵抗性のVTと判断、LVマッピング下に電気的焼灼術を2回施行。しかし敗血症を機にVT stormに至り、挿管管理下に再度pacingとニフェカラント/ランジオロール持続点滴を併用し、ICD植込み術を施行。アミオダロン+カルベジロール内服開始したが、点滴薬漸減するとVT出現するためソタロールを追加。以後VT消失したため前医に転院となった。虚血性心筋症の血行再建に伴う難治性VTに対し、アブレーションに加えアミオダロン/ソタロールの併用が奏効した症例を経験したので報告する。(著者抄録)

BACKGROUND: Clinical evidence suggests that intracoronary thrombus formation is associated with a high incidence of late restenosis after successful coronary intervention in patients with myocardial infarction (MI). However, little is known about the mechanism by which intracoronary thrombi play pathological roles. METHODS AND RESULTS: We analysed the cellular constituents of 108 thrombi aspirated from coronary lesions with a thrombectomy device in 62 patients who underwent emergent coronary intervention for the treatment of acute (<24 h) or recent (24-72 h) ST-segment elevation MI (44 men, 18 women, aged 68.0+/-19.3 years). Immunohistological analysis of aspirated thrombotic materials revealed that the content of platelets, as determined by immunostaining for CD42a, had a negative correlation with the time after the onset of chest pain (correlation coefficient -0.683, p<0.01). Immunofluorescent staining for CD34 and breast cancer-resistant protein-1 (bcrp-1) detected primitive cells in intracoronary thrombi. Furthermore, the ratio of CD34-positive cells in intracoronary thrombi had a significant positive correlation with restenosis at follow-up coronary angiography (correlation coefficient 0.76, p=0.01). CONCLUSIONS: The findings of this study indicate that the early accumulation of primitive cells in platelet aggregates may play a role in neointimal growth after successful coronary intervention in patients with acute coronary syndrome.

Abnormal transforming growth factor-beta (TGF-beta) signaling is a critical contributor to the pathogenesis of various human diseases ranging from tissue fibrosis to tumor formation. Excessive TGF-beta signaling stimulates fibrotic responses. Recent research has focused in the main on the antiproliferative effects of TGF-beta in fibroblasts, and it is presently understood that TGF-beta-stimulated cyclooxygenase-2 (COX-2) induction in fibroblasts is essential for antifibroproliferative effects of TGF-beta. Both TGF-beta and COX-2 have been implicated in tumor growth, invasion, and metastasis, and therefore tumor-associated fibroblasts are a recent topic of interest. Here we report the identification of positive and negative regulatory factors of COX-2 expression induced by TGF-beta as determined using proteomic approaches. We show that TGF-beta coordinately up-regulates three factors, heterogeneous nuclear ribonucleoprotein A/B (HNRPAB), nucleotide diphosphate kinase A (NDPK A), and nucleotide diphosphate kinase A (NDPK B). Functional pathway analysis showed that HNRPAB augments mRNA and protein levels of COX-2 and subsequent prostaglandin E(2) (PGE(2)) production by suppressing degradation of COX-2 mRNA. In contrast, NDPK A and NDPK B attenuated mRNA and protein levels of COX-2 by affecting TGF-beta-Smad2/3/4 signaling at the receptor level. Collectively, we report on a new regulatory pathway of TGF-beta in controlling expression of COX-2 in fibroblasts, which advances our understanding of pathophysiological mechanisms of TGF-beta.

Krüppel-like factor 5 (KLF5), originally isolated as a regulator of phenotypic modulation of vascular smooth muscle cells, induces pathological cell growth and is expressed in the neointima. Although induction of KLF5 up-regulates growth factors like platelet-derived growth factor-A chain, how KLF5 actually contributes to vascular remodeling, notably its direct effects on cell proliferation, had been poorly clarified. To investigate the effects of KLF5 on neointimal formation, we at first performed adenoviral overexpression of KLF5 to rats subjected to carotid balloon injury. Neointimal formation and proliferating cell nuclear antigen-positive rate were significantly increased at 14 days after injury in the KLF5-treated animals. At the cellular level, overexpression of KLF5 also resulted in markedly increased cell proliferation and cell cycle progression. As a molecular mechanism, we showed that KLF5 directly bound to the promoter and up-regulated gene expression of cyclin D1, as well as showing specific transactivation of cyclins and cyclin-dependent kinase inhibitors in cardiovascular cells. Conversely, knockdown of KLF5 by RNA interference specifically down-regulated cyclin D1 and impaired vascular smooth muscle cell proliferation. Furthermore, KLF5 attenuated cleavage of caspase-3 under conditions of apoptotic stimulation. Moreover, KLF5-administered animals exhibited a significant decrease in terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling-positive cells in the medial layer, suggesting inhibition of apoptosis in the early phase after denudation. These findings collectively suggest that KLF5 plays a central role in cardiovascular pathologies through direct and specific stimulation of cell growth as well as inhibition of apoptosis.

We show that transcription factor Krüppel-like factor 5 (KLF5), which is important in cardiovascular remodeling, interacts with retinoic acid receptor-alpha (RARalpha) to regulate downstream gene expression. Here, we investigated whether acyclic retinoid (ACR) regulates KLF5 and inhibits vascular remodeling. Co-immunoprecipitation and pull-down binding assay showed that ACR attenuates functional interaction of KLF5 and RARalpha. ACR affects KLF5 functions by regulating transactivation of platelet-derived growth factor A (PDGF-A) chain. ACR may be a new vascular therapy to target KLF5 in cardiovascular pathology.

OBJECTIVES: Acyclic retinoid (ACR) is a synthetic retinoid with a high safety profile that has been pursued with high expectations for therapeutic use in prevention (recurrence) and treatment of malignancies. With the objective of addressing the therapeutic potential in the cardiovasculature, namely neointima formation, effects of ACR on neointima formation and the involved mechanisms were investigated. METHODS AND RESULTS: ACR was administered to cuff-injured mice which showed inhibition of neointima formation. Investigation of involved mechanisms at the cellular and molecular levels showed that ACR induces apoptosis of neointimal cells and this to be mediated by selective induction of retinoic-acid receptor beta (RARbeta) which shows growth inhibitory and proapoptotic effects on smooth muscle cells. CONCLUSION: We show that ACR inhibits neointima formation by inducing RARbeta which in turn inhibits cell growth and induces apoptosis. The retinoid, ACR, may be potentially exploitable for treatment and prevention of neointima formation.

Krüppel-like factor 5 (KLF5) is a transcription factor important in regulation of the cardiovascular response to external stress. KLF5 regulates pathological cell growth, and its acetylation is important for this effect. Its mechanisms of action, however, are still unclear. Analysis in KLF5-deficient mice showed that KLF5 confers apoptotic resistance in vascular lesions. Mechanistic analysis further showed that it specifically interacts with poly(ADP-ribose) polymerase-1 (PARP-1), a nuclear enzyme important in DNA repair and apoptosis. KLF5 interacted with a proteolytic fragment of PARP-1, and acetylation of KLF5 under apoptotic conditions increased their affinity. Moreover, KLF5 wild-type (but not a non-acetylatable point mutant) inhibited apoptosis as induced by the PARP-1 fragment. Collectively, we have found that KLF5 regulates apoptosis and targets PARP-1, and further, for acetylation to regulate these effects. Our findings thus implicate functional interaction between the transcription factor KLF5 and PARP-1 in cardiovascular apoptosis.

An 84-year-old man was referred to the emergency department with severe dyspnea. Based on his physical findings, electrocardiogram, X-ray and echocardiographic findings, congestive heart failure was suspected and drip infusion of prophylactic heparin against intracardiac thrombosis was commenced together with dopamine, nitroglycerin and furosemide. Diuresis occurred and the pulmonary congestion ameliorated remarkably. Starting on the 20th hospital day, the platelet count was gradually reduced (from 256,000 to 55,000 /microl) and the fibrin degradation product concentration rose (27.6 microg/ml). However, prothrombin time was not prolonged (89%), the concentration of antithrombin III was low -normal (69%) and the fibrinogen concentration was high (650 mg/dl). Thus, heparin-induced thrombocytopenia (HIT), rather than disseminated intravascular coagulation (DIC), was suspected. Heparin was withdrawn on the 24th hospital day and replaced by nafamostat mesilate after which the platelet count was restored to 100,000 /microl. Enzyme-linked immunosorbent assay for HIT antibodies was positive. Unfortunately, the patient died from uncontrolled sepsis on the 29th hospital day. At autopsy, platelet-rich thrombi were found in the small pulmonary arteries and intestinal arteries. No evidence of DIC, such as fibrin-rich thrombosis, was observed. This is the first autopsy report of HIT in Japan.

A 52-year-old man was admitted with anemia and slight fever, which he had for the last 2 months. He had undergone replacement of the ascending aorta for acute aortic dissection 10 years previously. Echocardiography demonstrated a flailing thin structure in the anterior wall of the ascending aorta corresponding to the proximal portion of the prosthetic graft. This abnormal echocardiographic finding led us to repeat blood cultivation. We finally detected Enterococcus facium and Staphylococcus epidermidis in his blood sample. We diagnosed this as a graft infection and prepared for surgical re-replacement of the infected graft. While he was waiting for the operation, an infectious aneurysm of a tibialis posterior artery ruptured and an emergency operation was done. Replacement of the infected ascending aorta graft was done thereafter. In surgery, 2-cm-long vegetation was found. It stuck to the graft wall near the former hole used for air removal in the first surgery. The patient recovered fully and left our hospital after 3 months of postoperative antibiotics therapy. This rare case of aortic graft infection long after the original replacement surgery suggests the importance of thorough echocardiographic investigation of prosthetic graft infection as a possible cause of fever of unknown origin.

A 79-year-old female patient, who was initially suspected to have pneumonia, was admitted to the respiratory department of our hospital. She experienced chest pain on the second admission day. Electrocardiography showed ST-segment elevation in leads V3 through V6, and echocardiography revealed hypokinetic left ventricular wall motion. Therefore, myocardial infarction was suspected. She was transferred to the coronary care unit. Heart catheterization was immediately performed. Coronary angiography showed no significant stenotic lesion. Left ventriculography showed regional hypokinesis of the anterior and posterior walls near the base and normokinesis in the apex. Iodine-123-beta-methyl-p-iodophenyl-pentadecanoic acid (123-BMIPP)myocardial single photon emission computed tomography(SPECT) revealed inhomogeneous decrease in uptake, especially in the lateral wall, which did not correspond to any of the coronary artery territories. The echocardiographic asynergy was dramatically resolved after 1 week and the 123I-BMIPP SPECT finding was normal at 3 months. Although the clinical course of this patient was similar to that of tako-tsubo-like left ventricular dysfunction, the shape of her left ventricle was not typical. Left ventriculography showed hypokinesis of the anterior and posterior walls near the base and normokinesis in the apex, appearing like a chestnut rather than a tako-tsubo.

A 69-year-old man was transferred to our hospital because of severe general fatigue and progressive systemic muscle weakness. He had taken 300 mg/day of cibenzoline for his sustained ventricular tachycardia (VT) for years. At the end of June 2001 he began to feel general fatigue, which slowly progressed to systemic muscle weakness and walking disturbance. On 2nd July 2001, he finally could not stand up by himself. He also felt dyspnea. He was transferred to our emergency room, where he developed severe respiratory depression. Acute myocardial infarction was ruled out based on his ECG and blood chemistry data. Serum BUN and creatinine were elevated to 32 and 2.2 mg/dl, respectively, which returned to normal range 2 weeks later. What we did in our ICU were basically a replacement of cibenzoline with mexiletine and mechanical support of ventilation. As his renal function gradually improved, his spontaneous respiration and muscle power were slowly restored. He was discharged on foot after 1 month of hospitalization. His blood cibenzoline content taken 2 days after the cessation of cibenzoline was 959.6 mg/ml that was abnormally elevated. Considering metabolism and excretion for the 2 days between the cibenzoline cessation and the blood sample drawing, his cibenzoline level on the day of admission must have been extraordinary high. We should be aware of the possibility of abrupt overdosage of cibenzoline even in patients with normal kidney function in the event of a transient or an acute renal dysfunction.