Cell-Surface Proteomics to Unravel Cytokine-Induced Endothelial Activation

Endothelial dysfunction is associated with a variety of vascular disease processes, including hypertension, hypercholesterolemia, diabetes and atherosclerosis. The molecular mechanisms that underlie endothelial dysfunction are poorly understood, but are associated with endothelial activation by pro-inflammatory cytokines. Although transcriptional profiling has revealed cytokine-induced regulation of mRNA levels of various proteins, including the cell surface adhesion molecules Vascular Cell Adhesion Molecule 1 (VCAM1) and Intercellular Adhesion Molecule 1 (ICAM1), cell surface alterations at the protein level have remained largely unexplored. Unraveling these cell surface alterations is key to identify novel inflammation-markers and putative therapeutic targets to treat endothelial dysfunction. Therefore, the aim of this study is to probe cytokine-induced cell surface changes on endothelial cells.

We have developed a novel quantitative cell surface proteomics method by combining metabolic labeling and cell surface foot printing. Briefly, Blood Outgrowth Endothelial Cells (BOECs) were metabolically labeled using Stable Isotope Labeling with Amino acids in Cell culture (SILAC) and treated with Interleukin 1β (IL1ß) or Tumor necrosis factor α (TNFα) or mock treated for 24 hours. Lysine residues available on the cell surface were labeled using a non-membrane permeable N -hydroxysuccinimidobiotin label. Pooled cell lysates were processed into peptides, biotinylated peptides were enriched using streptavidin pull-down, desalted using Empore C18 StageTips, subjected to high resolution chromatography, measured at the Orbitrap Fusion Tribrid Mass Spectrometer and analyzed using the MaxQuant and Perseus computational platform.

Using this approach, 2526 biotinylated peptides were identified. The majority of the biotinylated peptides showed a SILAC ratio close to 1, indicating an unaltered cell surface presence. However, for 397 biotinylated peptides, the SILAC ratio was more than 2-fold increased or decreased in the cytokine-stimulated samples and we considered these to be regulated. Gene Ontology (GO)-term enrichment analysis of the corresponding proteins was assessed using BioMart. This analysis revealed a clear enrichment of the GO terms 'extracellular region' and 'extracellular space', supporting the applied approach. Hierarchical clustering of the regulated biotinylated peptides revealed a cluster of proteins with an increased cell surface expression after induction by both IL1ß and TNFα. In addition, two clusters of proteins were identified that show a unique footprint for one of the cytokine-stimulated samples, indicating that IL1ß and TNFα induce specific cell surface alterations. As expected, the most prominent changes were detected in ICAM1 and VCAM1. For ICAM1 6 peptides were identified with SILAC ratios 13.5 ± 1.75 (mean ± standard deviation) upon TNFα and 6.6 ± 2.9 upon IL1ß stimulation. For VCAM1 4 peptides were identified with ratios of 20.8 ± 14.3 (TNFα) and 3.2 ± 0.5 (IL1ß). Secondly, an increased presence of Human Leukocyte Antigen (HLA) and Beta-2-Microglobulin (B2M), part of the self-antigen presenting complex, was detected. Six peptides of HLA were increased on average 4.0 times ± 2.5 (TNFα) and 1.8 ± 0.6 (IL1ß). B2M peptides identified showed comparable values: 3.3 ± 1.1 (TNFα) and 1.7 ± 0.2 (IL1ß). These proteins are present on the cell surface in complex, thereby confirming the validity of our approach. Moreover, all biotinylated peptides identified for ICAM1, VCAM1 and HLA are in the extracellular region, further validating our method. Intriguingly, our approach not only identified the established adhesion molecules ICAM1 and VCAM1, but also a variety of other adhesion molecules involved in cell-cell interactions. More importantly, we also identified increased expression of proteins with other biological functions, including basement membrane proteins, ion channels, tyrosine phosphatase receptors, enzymes and protease inhibitors.

In conclusion, we developed a novel mass spectrometry-based cell surface proteomics method to quantify cytokine-induced cell surface alterations. Our approach not only enabled a detailed quantification at the protein level for established inflammatory cell surface markers, but also revealed a variety of potential novel targets for intervention of endothelial activation and dysfunction.