Alcohol enhances oxysterol-induced apoptosis in human endothelial cells by a calcium-dependent mechanism

Controversy exists about the net effect of alcohol on atherogenesis. A protective effect is assumed, especially from the tannins and phenolic compounds in red wine, owing to their inhibition of low density lipoprotein (LDL) oxidation. However, increased atherogenesis occurs in subjects with moderate to heavy drinking habits. The purpose of this study was to investigate the influence of alcohol in combination with oxysterols on the endothelium. Cultured human arterial endothelial cells (HAECs) served as an in vitro model to test the cellular effects of various oxysterols. Oxysterols (7beta-hydroxycholesterol, 7-ketocholesterol, and cholesterol-5,6-epoxides), which are assumed to be the most toxic constituents of oxidized LDL, induced apoptosis in HAECs through calcium mobilization followed by activation of caspase-3. Ethanol, methanol, isopropanol, tert-butanol, and red wine all potentiated oxysterol-induced cell death up to 5-fold, paralleled by further induction of caspase-3. The alcohol effect occurred in a dose-dependent manner and reached a plateau at 0.05% concentration. Alcohol itself did not affect endothelial cell viability, nor did other solvents such as dimethyl sulfoxide mimic the alcohol effect. So far as the physiologically occurring oxysterols are concerned, this effect was apparent only for oxysterols oxidized at the steran ring. The possibility of alcohol facilitating the uptake of oxysterols into the cell was not supported by the data from an uptake study with radiolabeled compounds. Finally, alcohol in combination with oxysterols did cause a dramatic increase in cytosolic calcium influx. Blockage of calcium influx by the calcium channel blocker aurintricarboxylic acid or the calcium chelator ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid abrogated the alcohol-mediated enhancement of oxysterol toxicity. We describe for the first time a mechanistic concept explaining possible adverse effects of alcohol in conjunction with physiologically occurring oxysterols on atherogenesis.     

Estradiol feedback effects on the alpha-subunit mRNA in the sheep pituitary gland: correlation with serum and pituitary luteinizing hormone concentrations.

The effects of estradiol feedback on pituitary luteinizing hormone (LH) content, serum LH concentration, and in vitro-translated alpha subunit was examined in the ewe. Three animal models were used representing positive, negative, and no estradiol feedback. Two experiments were carried out: (i) anestrous ewes were treated acutely with five Silastic estradiol implants to induce a LH surge (positive feedback) and (ii) ovariectomized ewes were treated chronically with an estradiol implant (negative feedback) or were not treated (no feedback). Pituitary RNA was prepared and translated in a cell-free system; the alpha subunit was identified by immunoprecipitation and NaDodSO4/PAGE. cpm/microgram of RNA and immunoprecipitated growth hormone and prolactin were used to evaluate possible differences in RNA translational efficiencies among the treatment groups. In experiment 1, significantly higher amounts of the alpha subunit were observed in animals exhibiting an estradiol-induced LH surge than in normal anestrous ewes (P less than 0.001). Examination of values from individual animals suggested a correlation between the stage of the LH surge, pituitary LH, and translated alpha subunit. In experiment 2, the amount of alpha subunit observed in animals exposed to chronic estradiol negative feedback was significantly less (P less than 0.005) than that in the untreated ovariectomized animals (no feedback) and no different from that in intact anestrous ewes. These results suggest that both the negative and the positive feedback effects of estradiol include regulation of the amount of alpha-subunit mRNA.     

Time course of smooth muscle cell proliferation in the intima and media of arteries following experimental angioplasty.

Smooth muscle cell (SMC) proliferation is known to be an important factor for the development of restenosis after percutaneous transluminal coronary angioplasty. To determine the time course of intimal and medial SMC proliferation and morphological changes after experimental angioplasty, an intimal atheroma was produced with repeated weak electrical stimulations in the right carotid artery of 45 male New Zealand White rabbits. Angioplasty was subsequently performed in 35 rabbits, and the proliferative responses were analyzed with histomorphological and immunohistological criteria at 3, 7, 14, 21, 28, and 42 days after intervention. A hemodynamic relevant stenosis after angioplasty was found in eight (23%) of 35 dilated arteries. In five rabbits the stenosis was due to a mural thrombus, and in three animals restenosis was caused by intimal SMC proliferation. In all dilated arteries the intimal wall thickness increased from 13 +/- 5 intimal cell layers (after electrical stimulation) to 33 +/- 14 cell layers during 28 days after angioplasty (p less than 0.05). Later than 4 weeks after angioplasty, no additional increase of intimal thickening occurred. Application of bromodeoxyuridine 18 and 12 hours before excision of the vessels allowed determination of the percent of cells undergoing DNA synthesis in the intima and media using monoclonal antibody against bromodeoxyuridine. SMCs were identified by alpha-actin staining. Immunohistological quantification of intimal SMC proliferation showed a maximum of cells undergoing DNA synthesis within the first 7 days after angioplasty (p less than 0.01). In contrast, medial proliferation of SMCs was delayed and showed a small but significant increase 21 days after dilatation (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

High-dose etoposide and cyclophosphamide without bone marrow transplantation for resistant hematologic malignancy

Seventy-five patients with resistant acute leukemia or lymphoma received high-dose cyclophosphamide and etoposide to explore the activity of this combination in resistant hematologic malignancies, and to determine the maximum doses of these drugs that can be combined without bone marrow transplantation. Etoposide was administered over 29 to 69 hours by continuous infusion corresponding to total doses of 1.8 g/m2 to 4.8 g/m2. Cyclophosphamide, 50 mg/kg/d, was administered on 3 or 4 consecutive days total 150 to 200 mg/kg ideal body weight). At all dose levels myelosuppression was severe but reversible. Mucosal toxicity was dose-limiting with the maximum tolerated dose level combining etoposide 4.2 g/m2 with cyclophosphamide 200 mg/kg. Continuous etoposide infusion produced stable plasma levels that were lower than would be achieved after administration by short intravenous infusion, and this could explain our ability to escalate etoposide above the previously reported maximum tolerated dose. There were 28 complete (35%) and 12 partial (16%) responses. Median duration of complete response (CR) was 3.5 months (range 1.1 to 20+). Seventeen of 40 patients (42%) with acute myelogenous leukemia (AML) achieved CR, including 6 of 20 (30%) with high-dose cytosine arabinoside resistance. We conclude that bone marrow transplantation is not required after maximum tolerated doses of etoposide and cyclophosphamide. This regimen is active in resistant hematologic neoplasms, and the occurrence of CR in patients with high-dose cytosine arabinoside-resistant AML indicates a lack of complete cross-resistance between these regimens.     

Quantification of myocardial ischemia and infarction with single photon emission computed tomography

To evaluate the feasibility of ²⁰¹Tl single photon emission computed tomography (SPECT) for quantitative detection of myocardial infarction and ischemia, scintigraphic studies were related to angiographic findings. In study A infarct sizes with SPECT were compared with the angiographic infarct sizes of 30 patients. A linear correlation was found for the % infarct of the left ventricular circumference between both methods (r=0.73; P< 0.001; mean infarct size 20.7%±10.5% (angio) vs 19.8%±12.9% (SPECT), mean±SD). Furthermore, a significant inverse correlation between scintigraphic infarct size and left ventricular ejection fraction (r=-0.87, P< 0.001) was obtained. In study B exercise/rest ²⁰¹Tl SPECT was used for quantification of myocardial ischemia. Forty-three patients underwent both stress ²⁰¹Tl SPECT and biplane exercise left ventriculography. Ischemia was expressed as % defect size of the left ventricular circumference. Sensitivity and specificity for detection of ischemia were 96% and 100% respectively with stress SPECT. Extent of myocardial ischemia correlated significantly with both methods (r=0.63; SPECT defect=1.0 angiographic ischemia +2%; P< 0.001). The regression followed the line of identity and the mean sizes of ischemia were identical (SPECT 12.2±7.6% vs 14.6±12.4% ventriculography, mean±SD) demonstrating the agreement of both methods. However, there was some intraindividual variance between the scintigraphic and the angiographic study. The sensitivity and specificity in single regions with SPECT were lower compared to the global test results. The correlation between the non invasive SPECT and the ventriculography in detection of myocardial infarction and ischemia indicates the clinical value of ²⁰¹Tl SPECT for diagnosis of coronary heart disease.Parts of the results were presented at the 58th sessions of the American Heart Association at Washington, DC (1985)