Proteomic perspectives on endothelial responses

In this thesis, we explored dynamic endothelial cell (EC) responses to external stimuli in the context of hemostasis and inflammation. Chapter 1 introduces the active roles of the endothelium, a single-cell layer lining all blood vessels. Due to its boundary position between blood and tissue, the endothelium is vital for nutrient exchange, waste removal, and cell transmigration. Additionally, ECs play key roles in regulating coagulation and inflammation. Disruption in these regulatory functions leads to endothelial dysfunction, which underlies numerous vascular disorders. Given that blood vessels vary in form and are present across all tissues, endothelial cells are heterogenous. Chapter 2 investigates this heterogeneity by comparing proteomes of in vitro cultured ECs from different organs. While some variation existed, it was not organ-specific. All ECs responded similarly to the inflammatory cytokine Interferon-gamma (IFNγ), suggesting endothelial identity is shaped more by microenvironmental factors than by EC intrinsic organ-specific traits. Chapters 3 and 4 delve into cytokine-induced inflammation in ECs. Chapter 3 identifies Tumor Necrosis Factor (TNFα) and IFNγ as inducing the strongest proteomic responses. Using multi-omics integration of transcriptome, proteome, phosphoproteome, and secretome, we demonstrated that both cytokines have system-wide effects and, when combined, produce a unique, synergistic inflammatory response in ECs. Chapter 4 evaluates inhibition of this synergistic inflammation. Inhibition of IKK2 and STAT3 reduced responses to TNFα and IFNγ, while JAK1 inhibition selectively blocked the IFNγ response. Both inhibitors suppressed the synergistic inflammatory protein response and reduced inflammation-induced von Willebrand Factor (VWF) release. This provides mechanistic insight into targeting synergistic endothelial inflammation in vascular disorders. Chapters 5 to 7 focus on EC-mediated hemostasis. Chapter 5 examines the signal induction of thrombin and histamine, which trigger VWF release and increase vessel permeability. Both stimuli activated similar signaling pathways such as the MAPK axis and tight-junction proteins. However, distinct phosphorylation events suggested stimulus-specific outcomes on tight-junction regulation. Chapter 6 addresses von Willebrand Disease (VWD), characterized by deficient or non-functional VWF and impaired primary hemostasis. In many VWD cases, no genetic mutation in the VWF gene is found. We used proteomic profiling of ECFCs (Endothelial Colony Forming Cells) from patients to explore defects in VWF secretion. Although significant heterogeneity among patient-derived ECFCs complicated pinpointing specific defects, the study identified proteins of interest involved in VWF regulation. In Chapter 7, we investigated the role of MADD (MAP kinase-activating death domain protein), a trafficking protein in VWD. Patients with MADD mutations had significantly reduced protein levels, resulting in impaired VWF secretion, identifying MADD as a novel contributor to VWD pathophysiology. Chapter 8 discusses five major themes from this thesis: endothelial heterogeneity, regulation of inflammation (including synergistic responses), proteins influencing VWF dynamics, the value of ECFCs in disease modeling, and the future of proteomics in endothelial biology. We conclude by posing unresolved questions and proposing future experimental directions.