Arihiro Kiyosue

A new constitutively active mutant of AMP-activated protein kinase inhibits anoxia-induced apoptosis of vascular endothelial cell

The inhibition of apoptotic changes in vascular endothelial cells is important for preventing vascular damage from hypoxia. AMP-activated protein kinase (AMPK) has recently been identified as playing a role in vascular protection. Although the chemical reagent 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) has been used to stimulate AMPK activity, AICAR has been associated with several nonspecific reactions. We therefore constructed a new constitutively active mutant of AMPKα1 (NcaAMPK), which lacks the autoinhibitory domain in AMPKα1 and in which threonine 172 has been replaced with aspartate. We investigated whether NcaAMPK has an anti-apoptotic effect in vascular endothelial cells under anoxic conditions. NcaAMPK, or green fluorescent protein (GFP) as a control, was overexpressed in human umbilical vein endothelial cells (HUVECs). After HUVECs were incubated for 40 h under normoxic or anoxic conditions, we examined cell viability, caspase 3/7 activity, and expression and phosphorylation levels of apoptosis-related proteins. Cell viabilities under anoxic conditions were improved in NcaAMPK-overexpressing cells. Anoxia increased caspase 3/7 activity, but NcaAMPK reduced this increase significantly. NcaAMPK overexpression increased protein kinase B/Akt Ser473 and endothelial nitric oxide synthase Ser1177 phosphorylation, but pretreatment with the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) did not decrease the viability of NcaAMPK-overexpressing HUVECs. Furthermore, co-expression of a dominant-negative Akt reduced the improvement in cell viability and the suppression of poly (ADP-ribose) polymerase cleavage by NcaAMPK under anoxic conditions. In conclusion, NcaAMPK inhibited anoxia-induced apoptosis in vascular endothelial cells through Akt activation, suggesting that activation of AMPK might protect against ischemic vascular injury.

Prognostic implication of macrocytosis on adverse outcomes after coronary intervention

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 Coxs 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.

Involvement of P2Y12 receptor in vascular smooth muscle inflammatory changes via MCP-1 upregulation and monocyte adhesion

Antiplatelet drugs, frequently used for cardiovascular events with thrombotic involvement, are also regarded as possible promising agents for cardiovascular primary prevention. The roles of P2Y12, an ADP receptor and the target of thienopyridine antiplatelet drugs, are not satisfactorily known in the vascular wall. We investigated the hypothesis that vascular smooth muscle cell (VSMC) P2Y12 is involved in vascular wall inflammatory changes by upregulating monocyte chemoattractant protein-1 (MCP-1) and promoting monocyte adhesion. ADP at 10(-5) M induced a 3.6 ± 0.3-fold upregulation of MCP-1 mRNA in cultured rat VSMCs, which was significantly inhibited by R-138727, the active metabolite of P2Y12 inhibitor prasugrel and siRNAs against P2Y12. ADP also induced MCP-1 protein upregulation, which was diminished by R-138727 and P2Y12 siRNAs. JNK (c-Jun NH2-terminal kinase) inhibition attenuated ADP-induced MCP-1 mRNA and protein upregulation. R-138727 and P2Y12 siRNAs inhibited ADP-induced JNK activation. The reactive oxygen species (ROS) inhibitors N-acetylcysteine (NAC), diphenyleneiodonium (DPI), and Tempol also diminished MCP-1 upregulation and JNK activation induced by ADP. ADP induced MCP-1 promoter activation, which was inhibited by R-138727 and P2Y12 siRNAs. Nuclear factor-κB (NF-κB) consensus sites in the MCP-1 promoter region were involved in this activation. ADP-induced NF-κB pathway activation, examined by a plasmid containing multiple NF-κB sites, was diminished by P2Y12 inhibition. For cellular function analysis, stimulation of VSMC with ADP increased subsequent THP-1 monocyte adhesion. P2Y12 siRNAs and CCR2 antagonism diminished this ADP-induced monocyte adhesion. These data suggested that ADP, via the VSMC P2Y12 receptor, induces vascular inflammatory changes by upregulating MCP-1 and promoting monocyte adhesion.

Hydrogen peroxide regulates osteopontin expression through activation of transcriptional and translational pathways.

Background: Osteopontin expression is increased in numerous diseases with underlying increases in H2O2. Results: H2O2-dependent osteopontin expression is biphasic. Early increases occur through translation via redox-dependent 4EPB-1, whereas late increases require NF-κB- and AP-1-dependent transcription. Conclusion: H2O2-induced osteopontin expression is both translational and transcriptional. Significance: Understanding how osteopontin is regulated is critical for targeting this inflammatory protein in H2O2-dependent pathologies. Recent in vivo studies establish that osteopontin (OPN) expression is hydrogen peroxide (H2O2)-dependent. However, the mechanisms by which H2O2 increases OPN expression remain poorly defined. OPN protein expression increased in an unusual biphasic pattern in response to H2O2. To investigate whether these increases were mediated through transcriptional and/or translational regulation of OPN, smooth muscle cells stimulated with 50 μm H2O2 were used as an in vitro cell system. Early protein increases at 6 h were not preceded by increased mRNA, whereas later increases (18 h) were, suggesting multiple mechanisms of regulation by H2O2. Polyribosomal fractionation assays established that early increases (6 h) in OPN expression were due to increased translation. This increase in translation occurred through phosphorylation of 4E-BP1 at the reactive oxygen species-sensitive Ser-65, which allowed for release and activation of eukaryotic initiation factor eIF4E and subsequent OPN translation. This early increase (6 h) in OPN was blunted in cells expressing a phospho-deficient 4E-BP1 mutant. H2O2 stimulation increased rat OPN promoter activity at 8 and 18 h, and promoter truncation studies established that promoter region −2284 to −795 is crucial for H2O2-dependent OPN transcription. ChIP studies determined that H2O2-dependent transcription is mediated by the reactive oxygen species-sensitive transcription factors NF-κB and AP-1. In conclusion, H2O2 stimulates OPN expression in a unique biphasic pattern, where early increases are translational and late increases are transcriptional.

Aldosterone-induced osteopontin gene transcription in vascular smooth muscle cells involves glucocorticoid response element

Osteopontin (OPN) is known to be one of the cytokines that is involved in the vascular inflammation caused by aldosterone (Aldo). Previous reports have shown that Aldo increases OPN transcripts, and the mechanisms for this remain to be clarified. In this study, we investigated how Aldo increases OPN transcripts in the vascular smooth muscle cells of rats. Aldosterone increased OPN transcripts time-dependently as well as dose-dependently. This increase was diminished by eplerenone, a mineralocorticoid receptor (MR) antagonist. Luciferase promoter assays showed that the OPN promoter deleted to the −1599 site retained the same promoting ability as the full-length OPN promoter when stimulated by 10−7 M Aldo, but the promoter deleted to the −1300 site lost the promoting ability. A glucocorticoid response element (GRE) is located in that deleted region. Luciferase assays of a mutated promoter without the GRE lost the luciferase upregulation, although mutated promoters with the deletion of other consensus sites maintained the promoter activity. The binding of the Aldo–MR complex to the GRE fragment was confirmed by an electrophoretic-mobility shift assay. This is the first report showing that Aldo regulates the transcriptional levels of OPN and inflammatory responses in the vasculature through a specific GRE site in the OPN promoter region.

Plasma neutrophil gelatinase-associated lipocalin predicts major adverse cardiovascular events after cardiac care unit discharge

BACKGROUND Emerging acute kidney injury biomarkers, including neutrophil gelatinase-associated lipocalin (NGAL), have a high potential for predicting worsening renal function. Acute exacerbation of renal dysfunction has a great impact on the outcomes of cardiovascular patients in critical conditions. This study aimed to evaluate whether plasma NGAL can predict the mortality and major adverse cardiovascular events (MACEs) after discharge from the cardiac care unit (CCU). METHODS Patients who were admitted to the CCU of the Tokyo University Hospital were prospectively enrolled (101 patients). Blood and urinary markers, including the blood NGAL, brain natriuretic peptide, creatinine, cystatin C, urinary albumin, N-acetyl-β-d-glucosaminidase, and L-type fatty acid-binding protein, were measured at CCU discharge. The primary outcome was MACEs until at least 6 months after CCU discharge. RESULTS Thirty-five patients experienced MACEs (35%). Multivariate logistic analysis revealed that the plasma NGAL, length of CCU stay, and existence of diabetes and heart failure were independent predicting factors for MACEs. Patients with the highest NGAL at discharge (>75th percentile) showed a significantly higher risk of MACEs than those with the lowest NGAL (<25th percentile) (log-rank test; hazard ratio, 5.15; 95% confidence interval 1.84-18.20; p<0.01). CONCLUSION Plasma NGAL at CCU discharge is a significant prognostic indicator of outcomes at 6 months in critically ill cardiac patients treated in a CCU.

Impact of the Distance from the Stent Edge to the Residual Plaque on Edge Restenosis following Everolimus-Eluting Stent Implantation

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.

Telmisartan Activates Endothelial Nitric Oxide Synthase via Ser1177 Phosphorylation in Vascular Endothelial Cells

Because endothelial nitric oxide synthase (eNOS) has anti-inflammatory and anti-arteriosclerotic functions, it has been recognized as one of the key molecules essential for the homeostatic control of blood vessels other than relaxation of vascular tone. Here, we examined whether telmisartan modulates eNOS function through its pleiotropic effect. Administration of telmisartan to mice significantly increased the phosphorylation level of eNOS (Ser1177) in the aortic endothelium, but administration of valsartan had no effect. Similarly, telmisartan treatment of human umbilical vein endothelial cells significantly increased the phosphorylation levels of AMP-activated protein kinase (Thr172) and eNOS and the concentration of intracellular guanosine 3′,5′-cyclic monophosphate (cGMP). Furthermore, pretreatment with a p38 mitogen-activated protein kinase (p38 MAPK) inhibitor suppressed the increased phosphorylation level of eNOS and intracellular cGMP concentration. These data show that telmisartan increases eNOS activity through Ser1177 phosphorylation in vascular endothelial cells mainly via p38 MAPK signaling.

Nonfasting TG/HDL-C ratio seems a good predictor of MACE in CAD patients with statin therapy. Could it be a treatment target?

Dyslipidemia is one of the well-known risk factors for coronary artery disease (CAD), so researchers have been seeking for effective surrogate markers in order to evaluate the possibility of CAD events in patients with dyslipidemia. Low-density lipoprotein cholesterol (LDL-C) is the most established marker. Not only the possibility is significantly high in patients with high LDL-C, but also treatment intervention in high LDL-C patients by statins was demonstrated to be effective in order to prevent CAD events even in Japanese [1]. However, researchers continue to look for other surrogate markers because dyslipidemic patients even on statin therapy have substantial residual risk of having CAD. Although statins showed preventive effects with statistical differences on cardiovascular events in many studies, risk reduction remained around 20–30% [1]. In that context, high-density lipoprotein cholesterol (HDL-C) and triglyceride (TG) are the candidates to evaluate additional risk among those patients or to be a treatment target. Many studies demonstrated that low HDL-C is a surrogate marker for the high possibility of CAD [2], but three cholesteryl ester transfer protein (CETP) inhibitors (so-called “HDL-C increasing drugs”) did not show any effect on prevention of CAD or lowering mortality (the event study result of the fourth CETP inhibitor was announced at the European Society of Cardiology Congress 2017) [3]. TG has been a debatable issue in this field. High TG was recognized as a risk for CAD already in the 1950s, but the relatively small studies could not show statistical significance of TG as a marker to predict CAD or TG-lowering therapy to prevent CAD. After the year 2000, some meta-analyses successfully showed the

Coronary Artery Perforation During Percutaneous Coronary Intervention in a Patient with a Prior Modified Bentall Procedure

The Bentall procedure is a surgical technique for an ascending aortic or aortic aneurysm in combination with valve disease. A well-known uncommon complication of after the Bentall procedure is coronary artery stenosis related to coronary anastomosis of an interposed graft. We report on a 73-year-old woman who presented with heart failure secondary to graft stenosis of the right coronary artery 6 months after undergoing a modified Bentall procedure. Percutaneous coronary intervention (PCI) was performed and type II coronary artery perforation occurred during PCI of the right coronary artery. We used a perfusion balloon and achieved hemostasis successfully. We report a case of coronary artery perforation that was treated with perfusion balloon during PCI in a patient with a prior modified Bentall procedure. In addition, we present a case series of PCI for ostial coronary stenosis after the Bentall procedure.