Proceedings of The Physiological Society
University of Edinburgh (2011) Proc Physiol Soc 25, C12 and PC12
Zinc deficiency results in apoptosis of vascular smooth muscle cells in vivo
K. Allen-Redpath1, O. Ou2, J. H. Beattie2, I. Kwun3, G. F. Nixon1
1. School of Medical Sciences, University of Aberdeen, Aberdeen, United Kingdom. 2. Rowett Institute of Nutrition and Health, University of Aberdeen, Aberdeen, United Kingdom. 3. Dept. of Food Science and Nutrition, Andong National University, Andong, Korea, Republic of.
Dietary zinc deficiency, which affects one third of the world’s population, has been associated with a role in the development of cardiovascular diseases although the causes of this are unclear. The aim of this study is to elucidate the effect of a zinc deficient environment on vascular smooth muscle cell (VSMC) function. Male Lister hooded rats (8 weeks old) were maintained for 2 weeks on either zinc adequate diets (35 mg/kg/day) or zinc deficient diets (3 mg/kg/day). Plasma was obtained intravenously following terminal anaesthesia with sodium pentobarbital. There was a significant reduction in plasma zinc levels in rats fed a zinc deficient diet compared to the zinc adequate diet as assessed by atomic absorption spectrophotometry (35 mg/kg/day- 158 ± 4 μg/ml, 3 mg/kg/day- 80 ± 8 μg/ml, n=7, mean ± s.e.m., ANOVA p<0.05). Aortae were dissected from zinc adequate and zinc deficient rats and a Tunel assay to detect apoptosis was performed on fixed sections. There was a significant increase in the number apoptotic VSMC in aortic sections from zinc deficient rats compared to the zinc adequate rats (35 mg/kg/day- 3 ± 2 apoptotic cells; 3 mg/kg/day- 39±3 apoptotic cells, n=3, p<0.05). A protein closely associated with apoptosis is the Bcl-2-associated death promoter (BAD) and over expression of this protein can lead to apoptosis. BAD was over-expressed in the VSMC of aorta from rats on a 2 week zinc deficient diet compared to a zinc adequate diet (n=3 individual aortae) as assessed by immunolocalization with confocal microscopy. In parallel in vitro experiments, primary cultured rat aortic VSMC were maintained for 24 hours in 10% plasma recovered from rats on either a 2 week zinc adequate or 2 week zinc deficient diet. Similar to in vivo experiments, there was a significant increase in the number of apoptotic cells in cultured aortic VSMC maintained in plasma from zinc deficient rats compared to the control (35 mg/kg/day plasma- 2 ± 1 apoptotic cells, 3 mg/kg/day plasma- 32 ± 9 apoptotic cells, n=3, p<0.05). Similar to aorta in vivo, over-expression of BAD was observed in vitro on cultured rat primary VSMC treated with zinc deficient plasma (n=3). In further experiments, cultured aortic VSMC were incubated for 24 hours in medium with 10% foetal bovine serum (FBS) which had reduced zinc (zinc concentration 1 μM) compared to normal FBS (Zinc 12 μM). Cells incubated with zinc-reduced FBS revealed similar levels of apoptosis and BAD expression compared to cells incubated in normal FBS. In conclusion, a zinc deficient diet in vivo induces apoptosis in VSMC from blood vessels probably partly via an increase in the pro-apoptotic protein BAD. This pro-apoptotic effect can be induced by the plasma from zinc-deficient animals however is not a direct effect of the zinc deficiency itself. Such mechanisms may be important in the development of cardiovascular disease in dietary zinc deficiency.
Where applicable, experiments conform with Society ethical requirements