Brain natriuretic peptide is released from human astrocytoma cell line U373MG under hypoxia: a possible role in anti-apoptosis.

We have recently shown that the plasma brain-type natriuretic peptide (BNP) level is elevated in acute ischemic stroke patients, but the origin and role of BNP remain unclear. We investigated whether human astrocytes secrete BNP under hypoxia, and if so, what signaling pathway is involved, and what is the role of BNP. Human astrocytoma cell line U373MG was exposed to hypoxia by Anaeropack. BNP gene expression was increased by 3.9±2.5- and 6.5±2.9-fold at 12 and 24 h after hypoxia respectively (n=6, both P<0.05) and was associated with twofold increase in BNP protein at 24 h. BNP release in the culture media (pg/mg protein) was elevated from 1.8±1.8 under normoxia to 13.5±7.8 after 24-h hypoxia (n=9, P<0.01 versus normoxia). Western blot revealed the tyrosine4¹5 phosphorylation of tyrosine kinase c-Src under hypoxia. Treatment of the cells with tyrosine kinase inhibitor PP1 abolished hypoxia-induced increase in BNP expression and release. In the cells exposed to hypoxia, caspase activity and apoptosis, measured by annexin-V-propidium iodide kit, were increased at 24 h compared with the cells under normoxia (n=5, both P<0.05). It was further increased in the cells transfected with siRNA for natriuretic peptide precursor B (n=6, both P<0.05) and reversed by administration of exogenous BNP (P<0.01). In conclusion, hypoxia increased BNP expression and release in human astrocytoma cell line U373MG through c-Src activation. BNP may play a pivotal role in anti-apoptosis of astrocytes under hypoxia.

Coupling factor 6-induced prostacyclin inhibition is enhanced in vascular smooth muscle cells from spontaneously hypertensive rats.

OBJECTIVES: Coupling factor 6 (CF6) attenuates the endothelial generation of prostacyclin. However, the role of CF6 in the resistance arteriole that is directly related to vascular tone is not determined yet. We investigated the effect of endogenous and exogenous CF6 on prostacyclin generation in cultured vascular smooth muscle cells (VSMCs). METHODS AND RESULTS: We cultured resistance arteriole VSMCs from the mesenteric artery network of spontaneously hypertensive rats (SHRs, n = 8) and Wistar-Kyoto rats (WKY, n = 8) by enzymatic method. The gene expression of CF6 was higher by 76 +/- 24% in SHR-derived VSMCs compared with WKY rat-derived VSMCs (P < 0.05) concomitant with the reduced degradation rate of CF6 mRNA. The release of CF6 in SHRs was higher than that in WKY rats (11.0 +/- 0.8 vs. 3.8 +/- 0.4 pg/microg protein, P < 0.05). Prostacyclin generation was attenuated in mesenteric arteriolar VSMCs from SHRs compared with those from WKY rats, but it was restored by neutralization of CF6 with its antibody. Exogenous administration of CF6 suppressed arachidonic acid release in a dose-dependent manner, and it was greater in SHRs than in WKY rats. Pretreatment with PP1, an inhibitor of tyrosine kinase c-Src, or receptor blockers such as ADP, efrapeptin, and an antibody to beta-subunit of ATP synthase blocked CF6-induced decrease in prostacyclin generation. CONCLUSION: These data suggest that CF6 suppresses prostacyclin generation in resistance arteriole VSMCs in an autocrine or paracrine fashion, and it is enhanced in SHRs by the overproduction of CF6 and the hyperresponsiveness to CF6.

Coupling factor 6 enhances Src-mediated responsiveness to angiotensin II in resistance arterioles and cells.

AIMS: Coupling factor 6 (CF6) induces hypertension by attenuating the endothelial generation of prostacyclin. However, intracellular signalling of CF6 in the resistance arteriole vascular smooth muscle cells (VSMCs) that are directly related to vasoconstriction has not been determined. Here we investigated the direct effect of exogenous CF6 on Ca2+ signalling in cultured VSMCs and the in vivo role of endogenous CF6 in the genesis of hypertension using CF6 transgenic (TG) mice. METHODS AND RESULTS: CF6 induced a monophasic increase in the intracellular free Ca2+ concentration ([Ca2+]i) through nifedipine-sensitive Ca2+ channels in A7r5 cells, a cell line of VSMCs, and enhanced the angiotensin II-induced spike phase of [Ca2+]i to a greater degree in VSMCs derived from spontaneously hypertensive rats (SHRs). In the mesenteric arterioles obtained from CF6-TG mice that manifested hypertension, angiotensin II-induced vasoconstriction was enhanced, compared with wild-type mice, and its enhancement was abolished by an anti-CF6 antibody. Pre-treatment with PP1, a tyrosine kinase c-Src inhibitor, blocked CF6-induced increase in Ca2+ signalling in VSMCs and vasoconstriction in TG mice. The receptor of CF6 was F1 motor of adenosine triphosphate (ATP) synthase with a higher affinity in SHRs. CF6 decreased intracellular pH via activation of ATPase activity and led to c-Src activation to a greater degree in SHR-derived VSMCs. CONCLUSION: CF6 causes hypertension by directly enhancing Ca2+ signalling in VSMCs and vasoconstriction in the mesenteric arteriolar network via c-Src activation.

Elevated plasma brain natriuretic peptide levels independent of heart disease in acute ischemic stroke: correlation with stroke severity.

We tested the hypothesis that plasma brain natriuretic peptide (BNP) levels are elevated in patients with acute cerebrovascular diseases (CVD) independent of heart disease, and reflect CVD severity. After careful evaluations for heart disease, the study included 79 consecutive patients with CVD without any evidence of heart disease admitted within 48 h after onset (71+/-10 years), and 26 control subjects without CVD (CT, 67+/-12 years). Ischemic stroke subtypes were defined by the TOAST classification. Large-artery atherosclerosis (LAA, n=27), small-artery occlusion (SAO, n=27), and intracerebral hemorrhage (ICH, n=25) were included. The plasma BNP levels were measured at admission and 1 month later. Stroke severity and brain infarct volume were evaluated. There were no significant differences in the clinical profiles including echocardiographic parameters among the groups. The plasma BNP level (pg/mL) upon admission was higher in LAA (70.6+/-53.9) than in SAO (38.2+/-28.4) and CT (28.5+/-19.9) (both p<0.05). The level in ICH (47.3+/-28.6) was not significantly different from that in CT. The BNP level in ischemic stroke was positively correlated with the NIH Stroke Scale (NIHSS) (rho=0.42, p<0.05) and infarct volume (r=0.34, p<0.05). Brain infarct volume and NIHSS were independent contributors to the plasma BNP level in ischemic stroke. One month later, the BNP level was significantly decreased and was similar in all CVD groups. The plasma BNP level transiently increased in patients with LAA independently of heart disease, and reflected infarct volume and the severity of acute ischemic stroke.

Coupling factor 6 downregulates platelet endothelial cell adhesion molecule-1 via c-Src activation and acts as a proatherogenic molecule.

Coupling factor 6 (CF6), a component of ATP synthase, suppresses the generation of prostacyclin and nitric oxide (NO). Platelet endothelial cell adhesion molecule-1 (PECAM-1) is involved in shear-induced NO production. To investigate the linkage between the actions of CF6 and PECAM-1, we examined the effects of CF6 on PECAM-1 expression and shear-mediated NO release, comparatively with those of angiotensin II (AngII). Treatment of human umbilical vein endothelial cells (HUVEC) and aortic endothelial cells (HAEC) with CF6 at 10(-7)M or AngII at 10(-7)M for 24h suppressed PECAM-1 gene and protein expression. CF6 or AngII activated c-Src at 15 min in HUVEC, and blockade of c-Src with PP1, its specific inhibitor, restored them. Efrapeptin, an inhibitor of ATPase, attenuated CF6-induced suppression of PECAM-1 gene expression by blockade of acidification, whereas superoxide dismutase or apocinin, an inhibitor of NADPH oxidase, blocked AngII-induced suppression of PECAM-1. Exposure of the cells to shear stress at 25 dynes/cm(2) for 30 min enhanced phosphorylation of eNOS at Ser(1177) and NO release. Pretreatment with CF6 or AngII for 24h attenuated them in HUVEC and HAEC. These suggest that CF6 downregulates PECAM-1 expression via c-Src activation and attenuates shear-induced NO release presumably by suppressing eNOS phosphorylation.

Efficacy and safety of low-dose pioglitazone after primary coronary angioplasty with the use of bare metal stent in patients with acute myocardial infarction and with type 2 diabetes mellitus or impaired glucose tolerance.

Thiazolidinediones (TZDs) have beneficial effects on markers of cardiovascular risk in patients with type 2 diabetes mellitus (DM). This study aimed to investigate the efficacy and safety of low-dose pioglitazone (15 mg per day) in patients with acute myocardial infarction (AMI) and type 2 DM or impaired glucose tolerance (IGT) treated with coronary angioplasty using bare metal stent (BMS). In 56 patients, pioglitazone was orally administered for 6 months after stenting (pioglitazone group). The incidence of in-stent restenosis (ISR) and left ventricular end-diastolic volume index (LVEDVI) at acute phase and 6 months after stenting in these patients were retrospectively compared with those in the other 37 patients (control group) treated without pioglitazone. No adverse events including death, emergency bypass surgery, and reinfarction, occurred in any patients in the hospital. There was no congestive heart failure (CHF) during a follow-up period in the pioglitazone group. At 6 months after stenting, the overall angiographic ISR rate was significantly lower in the pioglitazone group than in the control group (28.6% vs 48.6%, P = 0.049). In patients with hemoglobin A1c (HbA1c) <7.0% at follow-up, the ISR rate was also significantly lower in the pioglitazone group than in controls (21.3% vs 44.8%, P = 0.03). Delta-LVEDVI (defined as follow-up LVEDVI minus acute LVEDVI) was similar between the pioglitazone group and control group (0.13 vs 5.16 ml/m(2), P = 0.482). Low-dose pioglitazone seems to have a potential to reduce ISR and does not adversely affect LV remodeling after AMI treated with coronary angioplasty using BMS in patients with type 2 DM or IGT.