Expression of nephrin by human pancreatic islet endothelial cells

Abstract

Aims/hypothesis: The islet microcirculation has morphological characteristics resembling those of renal glomeruli. Transcription of the nephrin gene, a highly specific barrier protein of the slit diaphragm of podocyte foot processes, has been reported in the pancreas, although its cellular localisation and function remain to be defined. In this study, we purified and characterised microvascular endothelial cells (MECs) isolated from human islets and investigated the expression and distribution of nephrin on these cells. Methods: Human islet MECs were extracted and purified using anti-CD105-coated immunomagnetic beads and their endothelial characteristics were confirmed by expression of classical endothelial markers and basal high-level expression of intercellular adhesion molecule-1 and TNF-α-inducible vascular cell adhesion molecule-1. Nephrin expression was assessed by immunofluorescence, flow cytometric analysis and western blotting on cell lysates, as well as by RT-PCR. Results: Immunofluorescence studies detected nephrin in a fine, punctate, diffuse pattern on cultured islet MECs, and also in human pancreatic islet sections. In both cases nephrin colocalised with endothelial markers. TNF-α treatment induced a marked reduction and redistribution of the protein in one or multiple aggregates. Nephrin expression was confirmed by flow cytometry, western blotting and RT-PCR studies. In contrast, nephrin could not be detected at the protein or mRNA level in human macro-and microvascular cells from other sites. Conclusions/interpretation: Nephrin is expressed at protein and mRNA levels in islet microendothelium, supporting the hypothesis that islet MECs exhibit distinctive morphological characteristics that indicate functional specialisation of potential pathophysiological importance.

Activin Receptor-Like Kinase 5 Inhibition Reverses Impairment of Endothelial Cell Viability by Endogenous Islet Mesenchymal Stromal Cells

“…Our identification of islet cell populations with overlapping gene expression profiles has implications for optimizing procedures used for isolating and characterizing islet ECs in the future. For example, using endoglin-coated immunomagnetic beads for extracting ECs from human islets has been described [30] and applied to several studies of islet EC function [31][32][33][34]. As described by the authors, cells isolated using this technique must be used at early passages due to possible contamination with nonendothelial, endoglin-expressing cells, consistent with our identification of two endoglin-expressing cell populations in human islets.…”

Endoglin (CD105) is not a specific selection marker for endothelial cells in human islets of Langerhans

“…Macro-and microvascular endothelial cells (ECs) are certainly CD105-positive, but this accessory receptor is also highly expressed by a range of other cell types including bone marrow-and tissue-derived mesenchymal stromal cell (MSC)-like populations. In accordance with this, we have recently isolated from human islets, and expanded in vitro, a population of CD105-expressing cells that are clearly not MECs, but which share phenotypic characteristics with other cells that have been immuno-isolated on the basis of CD105 expression and identified as islet MECs for in vitro studies [1][2][3][4][5].…”

“…In this study, as in several previous studies [2][3][4][5], the MECs were expanded in vitro from a population that had been isolated from dispersed human islet cells using anti-CD105 immunobeads. CD105 (also known as endoglin) is a membrane glycoprotein that acts with transforming growth factor (TGF)-β receptors as an auxiliary binding protein for members of the TGF-β family, including activin, TGF-β1/β3 and bone morphogenic proteins.…”

Primary and immortalised human pancreatic islet endothelial cells: phenotypic and immunological characterisation

“…Importantly, the fenestrated islet microvascular endothelium not only facilitates rapid release of insulin into the circulation, but also appears to play a role in fine-tuning blood glucose sensing and regulation [12,[16][17][18]. Moreover, some studies support the possibility that the islet microvasculature participates in sensing the environment of the islets and generating signals to induce the growth and maintenance of the islets in response to physiological stimuli, such as insulin resistance [17][18][19]. Specific markers of the islet microvasculature are α-1 proteinase inhibitor (Api) [11] and nephrin, a highly specific barrier protein [19], both of which are absent in other microvascular endothelial districts.…”