Genotoxic Stress and Activation of Novel DNA Repair Enzymes in the Endothelial Cells and in the Retina in Diabetes
Abstract Number: 1002-P
Authors: CHUNYAN WANG, BIJU GEORGE, SHALI CHEN, BIAO FENG, SUBRATA CHAKRABARTI
Institutions: London, ON, Canada
Results: Introduction: Mammalian Excision Repair Cross Complimenting-1 (ERCC1) and ERCC-4 (a.k.a Xeroderma Pigmentosum complementation group F) are important nucleotide excision repair (NER) enzymes, which excise 5' end of the damaged DNA. ERCC1 plays important role in aging, glucose metabolism and oxidative stress. ERCC1 and 4 has interactive relationship with Poly (ADP-Ribose) polymerase (PARP), another DNA repair protein, upregulated in diabetic complications. We studied the role of ERCC1 and ERCC4 in glucose induced extracellular matrix (ECM) protein production in the endothelial cells and in the retina.
Methods: Human Umbilical Vein endothelial cells (HUVEC's) were grown with low (5mM) and high glucose (25mM) concentrations. The cells were further subjected siRNA transfection against ERCC1 and XPF, PARP blocker ABA(5mM), p300 (histone acetylase, activated by DNA damage) blocker curcumin(20mM). In parallel, retinas from STZ diabetic rats after 1 month of follow up with or without treatment with curcumin (p300 blocker) and ABA were examined. qRT-PCR were performed to quantify transcripts for fibronectin(FN) PARP and p300. Furthermore, western blot of nuclear proteins were done for ERCC1 and ERCC4 protein levels. Oxidative stress markers were analysed.
Results: ERCC1 and ERCC4 mRNA and protein levels were significantly higher in the HUVECs in high glucose, compared to low glucose and in the retina of diabetic rats compared to controls. Such upregulations were associated with increased oxidative stress and augmented p300 and FN mRNA production. ERCC1 and 4 silencing (in vitro), ABA and curcumin treatment (both in vitro and in vivo) significantly reduced glucose induced upregulation of ERCC1, ERCC4, FN, PARP and p300 mRNA and oxidative stress.
Conclusion: Data from this study indicate that glucose induced upregulation of ERCC1 and 4 leads to increased FN production via a p300 dependent pathway in the endothelial cells and in the retina is diabetes. ERCC1 and ERCC4 play important roles in the development of diabetic retinopathy