Beneath the surface: Cellular responses in the vasculature. Cellular responses in the vasculature

Abstract

The surface of cells contains specific proteins that mediate responses to extracellular stimuli. In this thesis we use mass spectrometry to study cellular responses in the vasculature. In addition to whole cell proteomics, we developed a cell surface proteomics method that enabled us to detect functional changes in cell surface proteins. We primarily focused on endothelial cells, which line the vasculature, and macrophages, which are responsible for the clearance of pathogens and cell debris. We studied how endothelial cells respond to pro-inflammatory cytokines TNFα and IL-1β in a temporal fashion. Although these molecules activate distinct receptors, their effect on endothelial cells is strikingly similar. This response entails a limited number of early responsive and a great number of late responsive proteins. Many cytokine-responsive proteins were associated with the ECM, thereby emphasizing the role of the endothelial basement membrane in inflammation. We also detected a cytokine-induced conformational change in ITGA5, which may have been the result of activation of this molecule. In addition, we investigated the effect of blood flow on the function of endothelial cells. The elongated morphology of these cells in flow conditions was associated to a drastic remodeling of the subendothelial basement membrane compared to the polygonal shaped cells in static conditions. The ECM reorganization included quantitative and spatial changes in laminin-integrin networks. Furthermore, we studied the development of monocytes into macrophages, dendritic cells and osteoclasts. Although these cell types serve quite distinct functions, their development was characterized by a group of proteins that changed in a remarkably similar manner for the different cell types. Remarkably, the development of osteoclasts was highly similar to the other cell types, although their morphology, in which multiple cells are fused together, is very distinct. Finally, we studied the uptake of hemostatic protein Von Willebrand Factor by macrophages. We found that two distinct receptors, MSR-1 and LRP-1, can contribute to VWF-uptake. However, MSR-1 mediated uptake is facilitated by LRP-1. The research described in this thesis expands our knowledge of the vasculature and elucidates mechanisms involved in cellular responses therein.