Investigating histidine-rich glycoprotein and T cell-specific adaptor as potential biomarkers and therapeutic targets

Sammanfattning: The endothelial cell (EC), the most important cell type in blood vessels, lines the vessel wall and provides vessel integrity. EC function is tightly regulated, and its dysregulation is a key element in many diseases including cardiovascular disease, cancer and several diseases of the eye. This thesis investigates the prognostic and therapeutic potential of two proteins: histidine-rich glycoprotein (HRG) and T cell-specific adaptor protein (TSAd). HRG is an abundant hepatocyte-derived protein, involved in many biological processes including hemostasis and fibrinolysis, inflammation, and angiogenesis. TSAd is an adapter protein downstream of vascular endothelial growth factor (VEGF) receptor 2, required for VEGF-A induced vascular permeability. In Paper I HRG-based therapy is tested in glioma using an HRG-encoding non-replicating adenovirus vector delivered orthotopically in the GL261 mouse glioma model. HRG treatment results in reduced tumor growth and increased vessel perfusion. Further mechanistic analysis reveals that stanniocalcin 2 (STC2) is a binding partner of HRG on the surface of inflammatory cells. Paper II investigates the potential of HRG as a prognostic biomarker in mature B cell lymphomas using tissue microarrays of human lymphoma samples. RNAscope is employed to identify tumor cell expression of HRG, and complementing immunostainings reveal that high HRG expression is a marker of improved overall survival for patients with marginal zone lymphoma, independent of age, stage and sex. In Paper III the interaction of HRG and STC2 is characterized further using the human histiocytic lymphoma cell line U937 that can differentiate towards a macrophage-like cell type after stimulation with vitamin D3. The bioactivity of recombinant HRG and STC2 is ensured by testing their effects on phagocytosis in U937 cells.  Quartz crystal microbalance analysis reveals that HRG binds STC2 with high affinity in a conformation dependent manner. Paper IV describes a high throughput screen for a small chemical compound capable of blocking VEGF-induced vascular permeability by binding TSAd. Screening of approximately 22000 compounds results in the discovery of a lead compound that binds TSAd and blocks VEGF-induced permeability in an ex-vivo assay.In summary, the papers presented in this thesis describe different strategies to investigate the role of HRG and TSAd on ECs and how this information can be applied therapeutically. The results confirm the importance of EC biology in disease, and the clinical potential HRG and TSAd as therapeutics or as biomarkers.