Purchase this article with an account.
Joseph F. Arboleda-Velasquez, Alexandra M. James, Patricia A. D'Amore; Notch-mediated Cell-cell Interactions In The Pathophysiology Of Diabetic Retinopathy. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5423.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Diabetic retinopathy (DR) is among the most common and devastating complications of diabetes. Clinical and experimental observations indicate that interactions between endothelial cells (ECs) and pericytes are central to the integrity of the retinal vasculature. Loss of pericytes in DR is temporally associated with endothelial dysfunction and destabilization of the vasculature. Evidence from studies in animal models and from knowledge of human pathology demonstrates an essential role for the Notch pathway, a fundamental cell signaling mechanism crucial to cell fate decisions in vascular development and homeostasis. We propose that Notch signaling, which depends upon juxtaposition of cellular membranes, is disrupted in diabetes leading to microvascular dysfunction.
We have established a cell culture system to investigate whether pericyte-EC interactions are mediated by the Notch receptor and its ligands, and to study whether these interactions are affected by culture conditions that resemble a diabetic environment. In this system, primary cultures of bovine retinal pericytes (BRPs) were transfected using nucleofection technology (Amaxa) with a Notch signaling-sensitive TP1-luciferase reporter 24 hrs prior to co-culture with cells expressing the Notch ligand Delta 1 (DLL1) in high/normal glucose conditions.
We found induction of Notch signaling upon culturing the BRPs with DLL1-expressing cells compared with that of cells co-cultured with control cells (9.91 fold increase, p<0.001), which was reduced with the addition of a gamma-secretase inhibitor, Compound E, by 84.7% (p<0.001). Transfection of BRPs with the glycosyltransferase Lunatic Fringe, which is known to modulate Notch signaling, increased Notch signaling by 57.61% (p<0.005). Lastly, we found evidence for Notch signaling misregulation in BRPs cultured under high-glucose conditions (43.96% increase, p<0.05), compared with that of the osmolarity control.
We have established an in vitro system to study the effect of high glucose on Notch signaling using co-cultures. The methods that we have developed promise to reveal new signaling pathways and cellular targets involved in the pathophysiology of a prevalent cause of blindness.
This PDF is available to Subscribers Only