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Finny Monickaraj, Sampathkumar Rangasamy, Andrea Cabrera, Paul McGuire, Arup Das; Role of Notch3 Signaling in Pericyte Survival: A Novel Pathway of Blood Retinal Barrier Alteration in Diabetic Retinopathy.. Invest. Ophthalmol. Vis. Sci. 2020;61(7):724.
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© ARVO (1962-2015); The Authors (2016-present)
Pericyte loss from the retinal microcirculation is the classic histological finding of early diabetic retinopathy in the human retina. Based on the transcriptome analysis done on pericytes isolated from diabetic mice, here we further examined the role of Notch3 receptor - a survival factor that was downregulated in the pericytes from diabetic mice, and its impact on pericyte dysfunction, pericyte dropout and subsequently blood retinal barrier alteration.
Human retinal pericytes (HRP) were treated with 500ug of advanced glycation end-product (AGE) for 96 hours, Notch3 and scramble siRNA. Gene expression analysis of autophagy and inflammatory genes was done using realtime PCR. Protein expression of Notch3 and PDGFRb were done using western blot method. In vitro endothelium monolayer permeability was measured on human retinal endothelial cells (HREC) using Electric Cell-substrate Impedance Sensing (ECIS) method.
Pericytes treated with 500ug AGE for 96 hrs. showed a significant decrease in the mRNA expression of Notch3 (p<0.027) compared with untreated cells (Ctrl) which was similar to the data we obtained from the RNA-Seq analysis. HRPs knockdown for Notch3 using siRNA showed significant increase in pro inflammatory genes (Ang2, CCL2, ICAM1, VEGF, MMP2) (p<0.01) compared with cells treated with scramble siRNA. Likewise the Notch3 siRNA cells showed significantly increased expression of autophagy genes (ATG5, BECN1, LC3B and p62) (p<0.05) compared with cells treated with scramble siRNA. The protein expression of PDGFRb, an important molecule in pericyte development, was significantly decreased in Notch3 siRNA cells (p<0.05). The in vitro monolayer permeability assay showed a significant decrease in resistance which implies increased permeability in HRECs treated with conditioned medium (CM) from HRPs treated with Notch3 siRNA cells compared with CM from scramble siRNA cells.
Our results indicate that there is a potential role for Notch3 leading to dysfunctional pericytes followed by alteration in the pericyte-endothelial interaction and alteration of the blood retinal barrier and vascular leakage in diabetes.
This is a 2020 ARVO Annual Meeting abstract.
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