Fibronectin is essential for formation of fenestrae in endothelial cells of the fenestrated capillary
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Fibronectin is essential for formation of fenestrae in endothelial cells of the fenestrated capillary Takashi Nakakura 1 & Takeshi Suzuki 2 & Hideyuki Tanaka 1 & Kenjiro Arisawa 1 & Toshio Miyashita 1 & Yoko Nekooki-Machida 1 & Toshiki Kurosawa 3 & Yuma Tega 3 & Yoshiharu Deguchi 3 & Haruo Hagiwara 1 Received: 17 March 2020 / Accepted: 6 August 2020 # Springer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract Endothelial fenestrae are transcellular pores that pierce the capillary walls in endocrine glands such as the pituitary. The fenestrae are covered with a thin fibrous diaphragm consisting of the plasmalemma vesicle–associated protein (PLVAP) that clusters to form sieve plates. The basal surface of the vascular wall is lined by basement membrane (BM) composed of various extracellular matrices (ECMs). However, the relationship between the ECMs and the endothelial fenestrae is still unknown. In this study, we isolated fenestrated endothelial cells from the anterior lobe of the rat pituitary, using a dynabeads-labeled antibody against platelet endothelial cell adhesion molecule 1 (PECAM1). We then analyzed the gene expression levels of several endothelial marker genes and genes for integrin α subunits, which function as the receptors for ECMs, by real-time polymerase chain reaction (PCR). The results showed that the genes for the integrin α subunit, which binds to collagen IV, fibronectin, laminin-411, or laminin511, were highly expressed. When the PECAM1-positive cells were cultured for 7 days on collagen IV-, fibronectin-, laminins-411-, or laminins-511-coated coverslips, the sieve plate structures equipped with probably functional fenestrae were maintained only when the cells were cultured on fibronectin. Additionally, real-time PCR analysis showed that the fibronectin coating was effective in maintaining the expression pattern of several endothelial marker genes that were preferentially expressed in the endothelial cells of the fenestrated capillaries. These results indicate that fibronectin functions as the principal factor in the maintenance of the sieve plate structures in the endothelial cells of the fenestrated capillary. Keywords Fibronectin . Fenestra . Endothelial cell . Rat pituitary . Plasmalemma vesicle–associated protein (PLVAP)
Abbreviations AL Anterior lobe BM Basement membrane BSA Bovine serum albumin DAPI 4′, 6-Diamidino-2-phenylindole Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00441-020-03273-y) contains supplementary material, which is available to authorized users. * Takashi Nakakura [email protected] 1
Department of Anatomy and Cell Biology, Teikyo University School of Medicine, 2-11-1 Kaga Itabashi-Ku, Tokyo 173-8605, Japan
2
Department of Biology, Sapporo Medical University, Sapporo, Japan
3
Laboratory of Drug Disposition and Pharmacokinetics, Faculty of Pharma-Sciences, Teikyo University, Tokyo, Japan
ECM ECMs GP HBT IgG KDR PBS PCR PECAM1 PFA PLVAP PNGase F RT SEM siRNA TBST VEGF-A
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