Abstract
Diabetic retinopathy (DR) threatens the vision of a third of diabetic patients. Progression of the disease is attributed to the dropout of pericytes, a cell type that enwraps and stabilizes the microvasculature. In tandem with this presumptive pericyte dropout, there is enriched formation of pericyte-like cells that bridge capillaries, along with acellular bridging structures assumed to be remnants of collapsed or regressed vessels and previously classified as acellular capillaries, string vessels, and basement membrane bridges. Together, these observations and assertions point to unanswered questions about the origin, functional significance, longevity, and reversibility of these cellular and acellular cross-capillary bridges. One such question pertains to whether these bridges are formed by migrating pericytes that deposit basement membrane or from remnants of regressed vessels. Additionally, given the increased prevalence of both bridging structures in diabetic conditions, the time frame for their development and possibility of reversibly tuning their frequencies remains unknown.
We investigate these questions by pairing detailed quantification of vascular and perivascular morphological features across various cytokine perturbations, along with cell type specific ablation of a gene associated with migration. An image analysis package, REAVER, is developed to quantify various aspects of vessel architecture, and exhibits superior accuracy and precision compared to competing vascular image analysis packages across all metrics examined. The development of CIRCOAST, a novel hypothesis test for changes in spatial colocalization of structures in biomedical images, allows for characterization of colocalization between a cell population and blood vessel networks across changes in cell or vessel density where ordinary statistics fail. We validate this hypothesis test by demonstrating that a cell type that can assume pericyte-like characteristics, adipose-derived stem cells (ASCs), has enriched colocalization with the retinal vasculature when injected into the eye in a DR model. Our findings implicate active cellular homing as a potential means for enhancing the ASC’s observed therapeutic capacity.
Using these tools, we show that pericyte bridges, not endothelial cells, colocalize with basement bridges; both are enriched by cell-specific knockout of KLF4 and reversibly enriched with elevation of Ang-2, PDGF-BB, and blood sugar. The omission of pericyte bridges, falsely assumed to be endothelial structures, from pericyte counts in retinal digests suggests that previous studies have exaggerated the role of pericyte loss in DR progression. In vivo imaging of corneal limbal vessels demonstrates pericyte migration off-vessel, implicating pericyte movement in formation of pericyte bridges and pathogenesis of DR. Earlier events in disease progression are thought to be superior avenues for intervention since preventative treatments avoid the need to regenerate damaged tissue. With the demonstration of these novel cell behaviors, pericyte bridges could serve as a viable therapeutic target in hyperglycemia.
