Capillary Fenestrae Formation, Structure and Function

Abstract

Abstract: : Purpose: The choriocapillaris is responsible for nutrient and waste exchange with the retinal pigment epithelium and the outer neural retina; deterioration of this vascular bed is a morphological feature present in a number of human retinal degenerations. Presumably to facilitate blood–tissue exchange, endothelial cells of the choriocapillaries form numerous 60nm–diameter plasma membrane pores called fenestrae. The study of fenestrae has been limited due to their low abundance in cultured endothelial cells and the lack of specific molecular markers. The aim of this study was to generate in vitro and in vivo models to facilitate the study of the fenestrae. Methods: We have established a tissue–culture model in microvascular endothelial cells where diaphragmed fenestrae were rapidly induced in quantities sufficient for cell biological and proteomic analyses. This model was used to study the effects of extracellular matrix composition, VEGF signaling cascades and the cytoskeleton on fenestrae formation. Assessment of fenestrae appearance and abundance was achieved through quantitative SEM and wholemount TEM assays, as well as a semi–quantitative light microscopy assay based on the diaphragm marker PV–1. We have also optimized methods to visualize and quantify choriocapillary fenestrae in vivo using ultrastructural and light microscopic methods. Results: A peak induction of 10 fenestrae/µm² was achieved in our tissue–culture model. This represents more than 1000–fold greater density of fenestrae than previously attained using in vitro models. Using this assay, we found that remodeling of the actin cytoskeleton is necessary and sufficient for fenestrae biogenesis. In a light microscopy assay we have shown that upon fenestrae induction PV–1, a marker of the diaphragms of both fenestrae and caveolae, segregated away from the caveolae marker caveolin–1, and primarily accumulated in thin fenestrated areas of the cell. Conclusions: We have developed methods and molecular markers that have facilitated dissection of the pathways controlling fenestrae formation. These tools will assist in determining the importance of various peptide growth factors and the cytoskeleton in fenestrae structure and function, and the relevance of fenestrae in pathological vascular permeability and chorio–retinal disease.

Keywords: cell membrane/membrane specializations • imaging/image analysis: non–clinical • vascular cells