Abstract
Purpose :
Vascular endothelial growth factor-A (VEGF-A) orchestrates endothelial cell (EC) phenotypic heterogeneity to drive vessel formation; however, we are only beginning to understand how VEGF-A signaling coordinates pericyte (PC) investment. Here, we identify how VEGF-A signaling regulated by VEGF-A decoy receptor Flt-1 (VEGFR-1) affects PC coverage in an in vivo model of angiogenesis, thereby guiding the pursuit of therapeutic targets for sight-threatening diseases. We hypothesize that a VEGF-A signaling gain-of-function, through loss of Flt-1, reduces PC coverage along forming retinal vessels.
Methods :
We dissected, stained, imaged, and analyzed postnatal day 7 mouse retinas (N=3) that underwent EC-specific conditional deletion of Flt-1. Presence of a conditional reporter signal, TdTomato, was presumed to correlate with Flt-1 loss. Neural/glial antigen-2 (NG2) antibodies labeled PCs, and platelet-endothelial cell adhesion molecule-1 (PECAM-1) antibodies marked ECs. PC coverage of retinal vessels and TdTomato signals were visualized by confocal. ImageJ facilitated mosaic analysis of (i) TdTomato normalized to PECAM-1 and (ii) NG2 relative to PECAM-1 in regions containing high/low TdTomato. Initial statistical analysis included paired two sample t-tests.
Results :
There was a significant difference in the overall TdTomato between areas of high and low TdTomato signal (p-value=0.0025); these differences likely reflect a change in localized Flt-1 deletion within selected regions. PC coverage was significantly higher in regions with high TdTomato and presumed increased Flt-1 deletion compared to areas assumed to lack substantial Flt-1 deletion (i.e., low TdTomato) (p-value=0.035).
Conclusions :
In contrast to our initial hypothesis, mosaic analysis of retina areas presumed to contain widespread Flt-1 deletion showed increased PC coverage. VEGF-A signaling, likely elevated by Flt-1 loss, may increase PC investment in remodeling retinal vessels. This observation demonstrates that mis-regulated VEGF-A signaling may aberrantly shift PC coverage, thus, dysregulating vascular growth. Diseases such as diabetic retinopathy are characterized by abnormal VEGF-A signaling and vascular growth, fueling blindness. By investigating context-dependent changes in vascular components, new therapeutic targets may be identified for sight-threatening diseases.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.