Fenestrae Formation in vitro Provides Clues to Their Biogenesis and Molecular Composition

Abstract

Purpose: : Fenestrae are 60 nm in diameter endothelial pores normally found in vascular beds of high blood–tissue exchange, and upregulated in non–fenestrated vessels in association with pathological permeability, for example during diabetic retinopathy. Numerous fenestrae are normally present in the choriocapillaris of the eye to facilitate nutrient and waste exchange between the choroid and the outer retina. The study of fenestrae has been hampered by the lack of unique markers and suitable cell–based models. Our aim was to develop an appropriate in vitro assay to study fenestrae composition and mechanism of formation.

Methods: : A microvascular endothelial cell culture model of fenestrae formation was established using a defined extracellular matrix, and induction agents such as vascular endothelial growth factor (VEGF) or cytoskeleton disruption agents. Fenestrae numbers were assessed using quantitative ultrastructural and light microscopy methods. The protein composition of fenestrae was analyzed using cationic colloidal silica to isolate plasma membranes highly enriched with fenestrae. These proteins were subjected to a proteomic analysis consisting of 2D–gel electrophoresis and mass spectrometry. We validated the resulting proteomic candidates of fenestrae through localization studies in vitro and in vivo and loss of function studies in vitro.

Results: : Our tissue–culture model produced fenestrae at a density up to 5 fenestrae/µm², representing a 100 fold induction when compared to untreated cells. We found that major disruption of actin microfilaments alone was sufficient for induction in our assay, and also necessary for VEGF–mediated fenestrae formation. Moreover, we detected actin filaments and actin binding proteins in fenestrae sieve plates, and perturbing the function of actin binding proteins resulted in deformed fenestrae sieve plates suggesting their functional involvement in fenestrae morphogenesis.

Conclusions: : We have developed a novel assay and discovered new markers that can provide insight into the biogenesis of endothelial fenestrae. Our in vitro assay is a useful tool for further experimental studies on fenestrae biology, while the newly identified protein components of fenestrae may serve as a diagnostic or therapeutic target for pathological permeability.

Keywords: choroid • proteomics