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Fibronectin is essential for formation of fenestrae in endothelial cells of the fenestrated capillary

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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 laminin-511, 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.

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Abbreviations

AL:

Anterior lobe

BM:

Basement membrane

BSA:

Bovine serum albumin

DAPI:

4′, 6-Diamidino-2-phenylindole

ECM:

Extracellular matrix

ECMs:

Extracellular matrices

GP:

Guinea pig

HBT:

0.1% Triton X-100/HEPES buffer

IgG:

Immunoglobulin G

KDR:

Kinase insert domain receptor

PBS:

Phosphate-buffered saline

PCR:

Polymerase chain reaction

PECAM1:

Platelet endothelial cell adhesion molecule 1

PFA:

Paraformaldehyde

PLVAP:

Plasmalemma vesicle–associated protein

PNGase F:

Peptide-N-glycosidase F

RT:

Room temperature

SEM:

Scanning electron microscopy

siRNA:

Small interfering RNA

TBST:

Tris-buffered saline containing 0.1% Tween 20

VEGF-A:

Vascular endothelial growth factor-A

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Acknowledgments

We would like to thank Editage (www.editage.com) for English language editing.

Funding

This work was supported in part by the JSPS KAKENHI (C) Grant Number JP19K07257, a research grant from the Takeda Science Foundation, Hokuto Foundation for Bioscience, and Teikyo University.

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Authors and Affiliations

  1. Department of Anatomy and Cell Biology, Teikyo University School of Medicine, 2-11-1 Kaga Itabashi-Ku, Tokyo, 173-8605, Japan

    Takashi Nakakura, Hideyuki Tanaka, Kenjiro Arisawa, Toshio Miyashita, Yoko Nekooki-Machida & Haruo Hagiwara

  2. Department of Biology, Sapporo Medical University, Sapporo, Japan

    Takeshi Suzuki

  3. Laboratory of Drug Disposition and Pharmacokinetics, Faculty of Pharma-Sciences, Teikyo University, Tokyo, Japan

    Toshiki Kurosawa, Yuma Tega & Yoshiharu Deguchi

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  1. Takashi Nakakura
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Correspondence to Takashi Nakakura.

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This study was approved by the Laboratory Animal Ethics Committee established at Teikyo University (Tokyo, Japan) and conducted according to its guidelines. The document ID of the approval is 17-008. This article does not contain any studies with human participants.

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Supplemental Fig. 1

Immunofluorescence images showing PLVAP localization in PECAM1-positive cells cultured on iMatrix-411 and iMatrix-511, which are the product names of laminin coating agent. Signals of PLVAP (a-d, green) and F-actin (a’-d’, red) in PECAM1-positive cells cultured for 3 days (a-a”’, c-c”’) and 7 days (b-b”’, d-d”’) on iMatrix-411-coated (a-a”’, b-b”’) or iMatrix-511-coated (c-c”’, d-d”’) cover slips were observed. Nuclei were counterstained with DAPI (blue). Panels a”'-d”' show the enlarged photographs of the regions surrounded by a white line in the panels a”-d”. Bars: 10 μm (a-d, a’-d’, a”-d”), 5 μm (a”’-d”’). (PNG 1758 kb)

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Nakakura, T., Suzuki, T., Tanaka, H. et al. Fibronectin is essential for formation of fenestrae in endothelial cells of the fenestrated capillary. Cell Tissue Res 383, 823–833 (2021). https://doi.org/10.1007/s00441-020-03273-y

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