Investigation of hypoxia and hyperglycaemia mediated DNA damage in human endothelial cells.

Abstract

Chronic complications of diabetes mellitus are a pathological consequence of unregulated levels of blood glucose leading to disorders of the vascular system. When investigating the vascular complications the changes in the endothelium are of particular significance as they are central to vascular function. The endothelium actively regulates vascular function by regulating permeability, vascular tone and inflammatory responses. Certain endothelium derived molecules, such as nitric oxide, act as vasodilators as well as exhibiting anti-platelet, anti-proliferative, permeability decreasing and anti-inflammatory properties (Kawashima et al. 2004). Changes to the molecular processes or injury to the endothelium lead to endothelial dysfunction and the initiation and progression of certain diseases such as the development of atherosclerosis as well as micro and macro-angiopathy (De Caterina et al. 2000). In the present study, we examine the effect of glucose and oxygen on endothelial DNA damage derived within 24h of incubation and the cytoprotective effect of the flavonoid antioxidant, silymarin, and two of its constituents, taxifolin and silibinin. Results show a significant increase in endothelial DNA damage in response to glucose and hypoxia which appears to be additive. These findings are supported by a significant rise in mitochondrial ractive oxygen species (ROS) production within 6h of exposure, while mitochondrial number, morphology and HUVEC size stay the same. A change in superoxide radical production in hypoxic conditions alone emphasises the important contribution of hypoxia in the pathogenesis of endothelial damage. Further investigation reveals a role for HIF-1a in the development of endothelial DNA damage which is hypoxia-dependent. Attempts to counteract the observed cytotoxicity to endothelial cells using the flavonoid antioxidant, silymarin, revealed that silymarin is a potent cytoprotective agent for hypoxia-induced DNA damage. This appears to be due to its strong mitochondrial radical scavenging activity. Further analysis into the mechanism of silymarin reveals that the impurity, taxifolin, possesses more potent cytoprotective activity than the formerly known active component silibinin. Taken together, the results strongly emphasise the important contribution of hypoxia in the early development of endothelial changes, which have the potential to accumulate to vascular complications in diabetes since its cytotoxic effect appears to be additive to that of glucose. Flavonoid antioxidants, such as silymarin and taxifolin, show promising potential to prevent or slow the progression of these changes