Alcohol abuse has a negative impact on human health; however, epidemiological studies show that moderate consumption of ethanol (EtOH) reduces the risk of coronary heart disease, sudden cardiac death, and ischemic stroke. The mechanisms for these reductions in cardiovascular disease are not well established. Using cultured coronary artery vascular smooth muscle cells, we found that moderate levels of EtOH (10 and 20 mM) caused dose-related increases in both vascular endothelial growth factor (VEGF) mRNA (Northern blot) expression (1.9- and 2.6-fold) and VEGF protein (ELISA) expression (19 and 68%) compared with control (P < 0.05). EtOH at 0.25 g. kg(-1). day(-1) (7 days) increased VEGF mRNA expression by 1.48-fold over control, and increased vessel length density from 3.9 +/- 0.7 (control) to 6.0 +/- 0.3 mm/mm(2) (P < 0.05) in chick chorioallantoic membrane (CAM). We conclude that moderate levels of ethanol can induce VEGF expression and stimulate angiogenesis in chick CAM. Therefore, the results provide a theoretical basis for speculating that the cardiovascular-protective effects of moderate alcohol consumption may be partly mediated through VEGF-induced angiogenesis.
BACKGROUND The mechanisms by which alcohol consumption causes cancer have not been established due to a lack of experimental studies. METHODS A chick embryo chorioallantoic membrane (CAM) model that bore human fibrosarcoma (HT1080) was used to determine whether the administration of physiologically relevant doses of ethanol could stimulate tumor growth, angiogenesis, metastasis, and vascular endothelial growth factor (VEGF) expression in tumors. HT1080 cells were inoculated onto the “upper CAM” on Day 8, saline or ethanol was administrated at a dose of 0.25g/kg per day on the CAM, and the tumors were harvested on Day 17. VEGF mRNA and protein were determined by Northern blot analysis and enzyme‐linked immunosorbent assay. Intratumoral vascular volume density (IVVD) was determined by point counting on periodic acid–Schiff‐stained sections. Intravasation of HT1080 cells was determined using human‐Alu polymerase chain reaction analysis. The effects of ethanol on VEGF expression and cell proliferation were examined in cultured HT1080 cells. RESULTS Ethanol treatment for 9 days caused a 2.2‐fold increase in tumor volume (867 ± 138 mm3 vs. 402 ± 28 mm3), a 2.1‐fold increase in IVVD (0.021 ± 0.004 mm3/mm3 vs. 0.010 mm3/mm3 ± 0.002 mm3/mm3), and a significant increase in VEGF mRNA or protein expression in tumors compared with a group of control embryos (n = 6 embryos; P < 0.01). Ethanol treatment caused an increase > 8‐fold in the intravasated HT1080 cells in the CAM group compared with the control group (n = 6 embryos; P < 0.01). Physiologically relevant levels of ethanol (10 mM and 20 mM) caused a dose‐related increase in VEGF mRNA and protein expression in cultured HT1080 cells. The ethanol–HT1080‐conditioned media increased the proliferation of endothelial cells, but not of HT1080 cells. CONCLUSIONS The findings suggest that the induction of angiogenesis and VEGF expression by ethanol represents an important mechanism of cancer progression associated with alcoholic beverage consumption. Cancer 2005. © 2004 American Cancer Society.
We tested whether increased endogenous adenosine produced by the adenosine kinase inhibitor GP-515 (Metabasis Therapeutics) can induce vascular endothelial growth factor (VEGF) expression in cultured rat myocardial myoblasts (RMMs). RMMs were cultured for 18 h in the absence (control) and presence of GP-515, adenosine (Ado), adenosine deaminase (ADA), or GP-515 + ADA. GP-515 (0.2-200 microM) caused a dose-related increase in VEGF protein expression (1.99-2.84 ng/mg total cell protein); control VEGF was 1.84 +/- 0.05 ng/mg. GP-515 at 2 and 20 microM also increased VEGF mRNA by 1.67- and 1. 82-fold, respectively. ADA (10 U/ml) decreased baseline VEGF protein levels by 60% and completely blocked GP-515 induction of VEGF. Ado (20 microM) and GP-515 (20 microM) caused a 59 and 39% increase in VEGF protein expression and a 98 and 33% increase in human umbilical vein endothelial cell proliferation, respectively, after 24 h of exposure. GP-515 (20 microM) had no effect on VEGF protein expression during severe hypoxia (1% O(2)) but increased VEGF by an additional 27% during mild hypoxia (10% O(2)). These results indicate that raising endogenous levels of Ado through inhibition of adenosine kinase can increase the expression of VEGF and stimulate endothelial cell proliferation during normoxic and hypoxic conditions.
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