Role of Factor VIII in Angiogenesis, Vessel Stability and the Regulation of Extracellular Matrix Proteins

Introduction: The factor VIII (FVIII) gene (F8) located on the X chromosome is critical for coagulation. Hemophilia A (HA) is a rare X-linked disorder leading to deficient or dysfunctional FVIII resulting in impaired hemostasis. Patients with HA have shown diminished macrovascular and microvascular endothelial function suggestive of endothelial cell (EC) dysfunction. A limited understanding of the role of FVIII on EC function hinders the optimization of treatment outcomes in patients with HA.

Aims: To investigate the role of FVIII in maintaining EC function and its impact on gene regulation in ECs.

Methods: Blood outgrowth endothelial cells (BOECs) were isolated from HA patients and healthy donors. HA BOECs were transduced with a lentiviral vector (LV) carrying the B-domain deleted FVIII (LV-FVIII). The transcriptomic profiles of healthy, HA and LV-FVIII HA BOECs were assessed by RNA-sequencing technology. Gene set enrichment analysis (GSEA) and gene ontology (GO) analysis were performed to analyze differentially expressed genes. Proteomic analysis was performed to investigate the proteins modulated by FVIII. The role of FVIII in EC function was investigated using tubulogenesis, migration, and permeability assays in BOECs. In vivo angiogenesis and vessel permeability were assessed in adult NOD-scid IL2Rg^null (NSG) mice and NSG-HA mice. Angiogenic potential was evaluated by intradermal implantation of Matrigel® plugs, and vessel permeability was assessed using Evans Blue dye injection. Recombinant (r)FVIII (Nuwiq®, simoctocog alfa) was added to both in vitro and in vivo experiments to investigate the influence of exogenous FVIII treatment on EC function. The role of FVIII in regulating signaling pathways involved in angiogenesis, migration and EC function was also investigated.

Results: BOECs from patients with HA showed significantly decreased tubulogenesis and migration, and higher permeability in vitro vs healthy and LV-FVIII HA BOECS (p < 0.0001). LV-FVIII HA BOECs showed similar EC function to healthy BOECs. Addition of simoctocog alfa to HA BOECs significantly increased tubulogenesis and migration, and decreased permeability vs HA BOECs without added rFVIII (p < 0.0001). NSG-HA mice showed decreased angiogenesis and higher vessel permeability compared with NSG mice, which was improved after the addition of rFVIII. Addition of rFVIII induced phosphorylation of focal adhesion kinase (FAK) and Src along with downstream targets of EC function (AKT, mTOR and p38). RNA-sequencing showed significantly downregulated genes involved in extracellular matrix (ECM) composition, including nidogen-2 (NID2) (fold change: -4.9; p < 0.005) in HA BOECs vs healthy BOECs (p < 0.05). GSEA and GO analysis identified pathways corresponding to vascular development, cell migration, regulation of cell adhesion and ECM organization that were downregulated in HA BOECs compared with healthy BOECs. Downregulated genes and pathways were significantly restored following FVIII transduction (p < 0.05). These data were reinforced by the proteomic analysis confirming an enhanced expression of proteins involved in the tubulogenic process and ECM stability when FVIII is expressed.

Conclusions: Downregulation of genes involved with ECM composition, including NID2, a FVIII-regulated gene encoding a glycoprotein crucial for endothelial basement membrane stability, may contribute to impairment of EC function in HA. Restoration of EC function by F8 gene transduction highlights the importance of FVIII for vessel stability. Loss of EC function in vitro and in vivo can be restored with rFVIII treatment which is accompanied by activation of the FAK/Src signaling pathways in EC. These results further support the use of exogenous FVIII in HA. Overall, our findings suggest that, in addition to coagulation, FVIII plays an important role in the regulation of EC function and vessel stability.