Abstract 210: Hyperglycemia Results in an Exaggerated Response to Ischemia in Human Dermal Fibroblasts Through MyD88 Dependant Toll-Like Receptor 4 Activation
Introduction: Foot ulceration is a multifactorial, frequent and challenging complication of diabetes. There is increasing recognition of diabetes as a pro-inflammatory condition and evidence that toll-like receptor 4 (TLR4) activation is involved in its systemic pathogenesis and the subsequent impairment of wound healing, however the mechanism remains unclear. We aim to study the effect of simulated diabetic-ischaemic conditions on TLR4 function in dermal fibroblasts.
Method: Human fibroblasts were cultured at physiological glucose concentration (5.5mM) and subsequently exposed to glucose concentrations from 0mM to 25mM for 24 hours. Identical samples were placed within a hypoxic chamber for 8 hours. Cell lysate protein and supernatant were harvested and western blot assays for TLR4, MyD88 and cleaved Caspase 3 were undertaken. Migration was assessed by scratch wound assay. The effects of MyD88 and TRIF inhibitory peptides and TLR4 neutralising-antibody and antagonist on fibroblast migration were assessed in the 25mM glucose groups.
Results: Hypoxic conditions led to an increase in TLR4 protein expression. This effect was significantly increased (p<0.05) in very high glucose concentrations (25mM), and resulted in increased apoptosis (cleaved caspase3) and IL-6 release. Hypoxic conditions resulted in impaired fibroblast migration, particularly at high glucose concentrations (p<0.05). Inhibition with a MyD88 inhibitory-peptide and a TLR4 neutralising antibody and antagonist ameliorated the effects of high glucose and ischaemia (p<0.05).
Conclusion: Hypoxia leads to increased TLR4 protein expression and this effect is exaggerated by hyperglycaemia. This results in an increase in cellular apoptosis. The migration of fibroblasts in hypoxia was disproportionately impaired in the very high glucose treatment groups. TLR4 and MyD88 inhibition resulted in significantly improved fibroblast migration under these conditions. We conclude that inflammatory processes mediated via the MyD88-dependent TLR4 pathway results in reduced migration of fibroblasts and may contribute to impaired wound healing, under conditions commonly found in diabetic patients with concomitant ischaemia.
Author Disclosures: M.J. Portou: None. R. Yu: None. X. Shi-wen: None. D. Abraham: None. G. Hamilton: None. D. Baker: None. J. Tsui: None.
- © 2014 by American Heart Association, Inc.