Preserving vascular stability: FVIII's influence on endothelial cells

Hemophilia A (HA) is a genetic bleeding disorder caused by the deficiency of coagulation factor VIII (FVIII) and its severity is categorized based on FVIII residual activity: severe (< 1%), moderate (1-5%), and mild (5-40%). Current treatment primarily consists of frequent FVIII infusions, aiming to prevent spontaneous hemorrhages. These bleeding episodes are HA patients’ hallmarks, but they also manifest hemarthrosis and spontaneous intracranial hemorrhages (ICHs) whose exact cause remains unclear. Furthermore, HA patients exhibit endothelial dysfunction and a pro-inflammatory environment, suggesting the potential involvement of FVIII in maintaining endothelial cell (EC) homeostasis. To explore the role of FVIII in endothelial stability, we investigated the functionality of blood outgrowth endothelial cells obtained from both healthy (C-BOECs) and severe HA individuals (HA-BOECs). HA-BOECs showed a reduced tubulogenic capacity, decreased migratory ability, and a higher permeability than C-BOECs. Interestingly, when HA-BOECs were transduced with a lentiviral vector (LV) carrying FVIII or when treated with recombinant FVIII, they displayed rescued functions. To corroborate these data, we also performed CRISPR/Cas9 knockout of F8 in C-BOECs showing a significantly decreased in vitro functionality. For the first time, we found that FVIII binds to integrin βeta1 (ITGB1) activating focal adhesion kinase (FAK) and its downstream effectors in ECs. Moreover, both transcriptomic and proteomic analysis demonstrate that BOECs expressing FVIII result in a higher expression of genes and proteins related to angiogenesis, and extracellular matrix organization. Specifically, we focused on nidogen 2, involved in the endothelial basement membrane stability, whose expression was dependent to FVIII-ITGB1-FAK pathway. Subsequently, we found a drastic reduction in vascular density in HA brains compared to WT ones, and, importantly, long-term LV-FVIII injected HA mice significantly improved brain vessel formation. These preliminary data indicate that FVIII plays a critical role in the brainvasculature development. In conclusion, we explored the intricate effects of FVIII on ECs demonstrating its direct role in preserving the physiological functions of these cells. In HA, where FVIII levels are reduced or absent, disruptions in endothelial signaling occur, potentially leading to vessel fragility. This study is central to formulating precise therapeutic strategies for HA patients.