Identification of cell surface markers to differentiate rat endothelial and fibroblast cells using lectin arrays and LC-ESI-MS/MS

Abstract

Vascular endothelial cells located at the inner surface of blood vessels are a key component in angiogenesis and are employed as a primary cell type in the study of angiogenesis. These endothelial cells are, however, easily contaminated with fibroblast cells, which are located in proximity to the endothelial cells, during their isolation from tissue. It is thus important to find markers to distinguish the two cell types. In the present work, lectin arrays were prepared using aldehyde-terminated self-assembled monolayers (SAMs) and utilized to explore cell surface carbohydrate expression patterns on endothelial and fibroblast cells. It was found that the lectins Griffonia simplicifolia II (GS II) and Ulex europaeus agglutinin I (UEA I) selectively bind to rat fibroblast cells and not to rat endothelial cells. GS II-binding glycoproteins on fibroblast cells, which are potential cell surface markers to differentiate endothelial and fibroblast cells, were captured on a GS II lectin column and analyzed by LC-ESI-MS/MS. Six candidate cell surface glycoproteins were identified. Differential expression was confirmed by Western blot analysis for two of these proteins, lysosome-associated membrane glycoprotein-1 and transmembrane glycoprotein NMB.

Figure 1

Optical images of endothelial and fibroblast cell binding to the lectin-modified substrates by phase contrast microscopy. A total of 1 × 10⁶ endothelial cells and fibroblast cells, respectively, were added to lectin-modified substrates and captured by each lectin depending on the carbohydrate expression on the cell surfaces. Bar: 500 μm, 4× magnification objective lens used.

Figure 2

Optical images representing fibroblast cell binding specificity for GS II. Endothelial cells (1 × 10⁶) expressing green fluorescent protein (GFP) were captured on GS I-modified substrate and imaged using both phase contrast microscopy and fluorescence microscopy (excitation, 494 nm; emission, 518 nm). (A). Fibroblast cells (5 × 10⁵) and endothelial cells (5 × 10⁵) expressing GFP were mixed and captured on (B) GS I and (C) GS II-modified surfaces. Bar: 500 μm, 4× magnification objective lens used.

Figure 3

SDS-PAGE gel image of GS II bound proteins obtained from the fibroblast cell membrane protein fraction. A delipidated membrane protein fraction was obtained from a 5 × 10⁸ fibroblast cell lysate and loaded on a GS II lectin column followed by elution of the bound proteins using GS II inhibiting sugar.

Figure 4

Western blot analysis for LAMP-1 and GPNMB (EC, endothelial cells; Fb, Fibroblast cells).