Role of Factor VIII As a Regulator of Angiogenesis and Promoter of Endothelial Barrier Stability

Introduction: Hemophilia A (HA) is a rare bleeding disorder caused by the absence or dysfunction of factor VIII (FVIII). Spontaneous bleedings are a clinical manifestation of HA resulting in hemarthroses and intracranial hemorrhages in serious cases. Attenuated macrovascular and microvascular endothelial function has been observed in animal models and patients with HA suggesting a dysfunction of endothelial cells (ECs). ECs can be isolated from peripheral blood as blood outgrowth endothelial cells (BOECs) which can be used as a model to study endothelial function. Current treatment options for HA include prophylaxis with recombinant FVIII (rFVIII) products or non-factor therapy, such as the bispecific antibody emicizumab, to prevent bleeds. However, the impact of rFVIII and emicizumab on EC function and barrier stability remains unknown.

Aims: To elucidate the effect of different rFVIII products and emicizumab on the maintenance of EC function and barrier stability.

Methods:BOEC isolation and culture: BOECs were isolated from healthy donors and patients with HA. HA BOECs were treated in vitro with the rFVIII products simoctocog alfa (Nuwiq®), efmoroctocog alfa (Elocta®), rurioctocog alfa pegol (Adynovate®), damoctocog alfa pegol (Jivi®), octocog alfa (Advate®), or emicizumab (Hemlibra®). FVIII to EC binding: The binding of rFVIII to BOEC membranes was assessed by TriCEPS^TM technology and flow cytometry. EC function: Function was evaluated with proliferation, migration, tubulogenic and permeability assays. Additional factor IX and X were added (100 ng/mL) to EC functional assays with emicizumab. In vivo vessel permeability: Permeability in vivo was evaluated in NOD/SCID γ-Null HA (NSG-HA) mice using Evans Blue dye injection. In vivo angiogenesis: Angiogenesis was evaluated in vivo in NSG-HA mice injected with Geltrex containing the rFVIII products simoctocog alfa or efmoroctocog alfa.

Results: HA ECs showed significantly reduced migration, impaired tubulogenesis and increased permeability in vitro compared with healthy ECs (p < 0.05). EC function significantly improved following the addition of simoctocog alfa (p < 0.05). Treatment with simoctocog alfa resulted in higher EC proliferation compared with efmoroctocog alfa and emicizumab. The addition of rFVIII, with the exception of efmoroctocog alfa, significantly improved EC migration and permeability (p < 0.05). Treatment with emicizumab did not have a noticeable effect on EC migration or permeability. The addition of simoctocog alfa also resulted in the highest number of endothelial junctions and nodes, and the largest total lengths compared with the other tested rFVIII products. In vivo vessel permeability assays showed a notable reduction of dye extravasation in NSG-HA mice treated with simoctocog alfa and efmoroctocog alfa, with a larger reduction in mice treated with simoctocog alfa compared with efmoroctocog alfa. Finally, in vivo murine angiogenesis assays showed new vessel formation in the Geltrex plug containing rFVIII, whereas no vessel formation was seen with the untreated Geltrex plug.

Conclusion: BOECs from patients with HA demonstrate impaired function which can be restored with rFVIII, indicating possible extra-coagulative role of FVIII. Investigating the potential extra-coagulative role of FVIII could be crucial to understanding the key molecular targets at the cellular level which impair EC function in patients with HA. Knowledge of the possible effect of different rFVIII products and non-factor therapies on EC function can be used to optimize therapeutic approaches, which in turn may result in safer and more efficient treatment of HA.

Winge:Octapharma: Current Employment. Follenzi:Sernova: Research Funding; Sparks: Research Funding.