The molecular mechanisms of diabetes mediated impaired wound healing and the development of therapeutic strategies.
Gadad, Pramod C.
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Increased levels of blood glucose are associated with the vascular complications of diabetes. Microvascular complications lead to delayed wound healing in patients suffering from diabetes. Hypoxia and hyperglycaemia characterise a wound environment of a person with diabetes. Angiogenesis is central to restore the supply of oxygen and nutrients to the wounded tissue. Endothelial cell migration is central to angiogenesis which is aided by hypoxia and attenuated by hyperglycaemia. However, the molecular mechanisms underlying the disruption to angiogenesis of diabetic wounds are not completely understood. The effect of hypoxia and/or high glucose concentration on the endothelial cell migration in vitro was studied and an anti-oxidant, silymarin formulated as freeze dried wafer discs was tested for its beneficial effect. A radial migration and a wound healing assay were developed, validated and used to assess the effect of hypoxia and/or high glucose concentration on the migration of human endothelial cells of dermal origin. Circular and semi-circular monolayers of endothelial cells were used for the measurement of the migration by radial migration and wound healing assay respectively. Net migration was calculated by subtracting the radii at a specified time point from that measured at time zero. The migration was studied under normal (20%) or below (5%) normal oxygen tension in combination with normal (5mM) or high (20mM) glucose concentration. Endothelial cells were treated with an anti-proliferative agent, intracellular signal inhibitors and silymarin. Results demonstrated that hypoxia and high glucose concentration have opposing effects of increase (p<0.001) and decrease (p<0.001) respectively on the migration of endothelial cells. The results of the wound healing assay revealed that re-endothelialisation occurs faster (p<0.001) than endothelialisation. The effects of hypoxia and high glucose concentration appeared to be mediated via PI3K-Akt and PKCβII pathways respectively. Further investigations revealed the possibility of HIF-1α being involved in both the pathways. High glucose concentration-induced decrease in cell migration was successfully restored (p<0.001) by the use of an anti-oxidant silymarin. This could be due to anti-oxidant activity of silymarin on glucose-induced overproduction of reactive oxygen species. Silymarin formulated as freeze dried wafer discs, sterilised by gamma irradiation was successful in retaining its effect (p<0.001) against the high glucose impaired cell migration compared to control wafers. In conclusion, delayed wound healing due to disrupted endothelial cell migration was reaffirmed to be due to elevated glucose concentration. Silymarin was successful in restoring glucose-induced attenuation of cell migration. Freeze dried wafers show promising potential as a topical application for the treatment of chronic wounds for people with diabetes.