Innate Immunomodulation of Retinal Vascular Development

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Project Summary Retinal blood vessel formation is a highly regulated process that requires multi-cellular crosstalk and interactions. Due to the complexity of retinal vascular development, there are several critical knowledge gaps that need to be addressed. For example, the key glial cell type that regulate retinal vascular development are astrocytes, which lay a template for blood vessel formation. Endothelial cells migrate over the spatially organized astrocytic template to form superficial blood vessels that give rise to three interconnected vascular layers in the mature retina. Disruption of the astrocytic template during development or loss of astrocyte association with blood vessels in adulthood can be detrimental to vascular growth, integrity, and function. The underlying cellular and molecular signaling mechanisms that regulate astrocyte spatial patterning and subsequent organized blood vessel formation during development remain incompletely understood. Elucidating the molecular mechanisms that govern blood vessel development and function is necessary to identify more targeted therapeutic strategies for blinding retinal vascular pathologies, and to identify which critical developmental processes should not be targeted in some contexts, for example retinopathy of prematurity. Our major goal is to delineate the signaling mechanisms that regulate astrocyte template spatial arrangement and vascular network formation. Our preliminary findings strongly support the rationale for the present study. Our RNA-seq data reveal that specific chemokine and complement gene expression levels are elevated during retinal vascular development. Intriguingly, deletion of one of the affected chemokine receptors disrupts astrocyte template formation and microglial recruitment and distribution. On the other hand, deletion of complement components results in an aberrantly dense astrocytic template and dysmorphic excessive tip cell formation. Based on these novel findings, we hypothesize that microglial chemokine signaling and complement activation are critical for normal retinal vascular development. We will refute or validate our hypothesis in the following two Specific Aims: Aim 1: To determine if chemokine signaling recruits microglia to modulate astrocyte template assembly. Aim 2: To define the role of complement receptors in astrocyte template and vascular network formation. We will utilize innovative multiplex RNA/protein based assays, novel ex vivo migration assays, unique reporter mice, and cre/lox animals for cell-specific deletion. We expect that successful completion of the proposed studies will identify novel roles for the innate immune system in regulating highly complex retinal vascular developmental processes. Moreover, further elucidating physiological regulatory mechanisms of vascular development, will also identify future putative therapeutic strategies for retinal vascular pathologies.