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. 2024 Oct 1;109(10):3391-3397.
doi: 10.3324/haematol.2024.285089.

Factor VIII promotes angiogenesis and vessel stability regulating extracellular matrix proteins

Cristina Olgasi  1 , Alessia Cucci  2 , Ivan Molineris  3 , Simone Assanelli  2 , Francesca Anselmi  3 , Chiara Borsotti  2 , Chiara Sgromo  2 , Andrea Lauria  3 , Simone Merlin  2 , Gillian E Walker  2 , Salvatore Oliviero  4 , Antonia Follenzi  5
Affiliations

Factor VIII promotes angiogenesis and vessel stability regulating extracellular matrix proteins

Cristina Olgasi et al. Haematologica. .
No abstract available

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Figures

Figure 1.
Figure 1.
Factor VIII restores the defective function of hemophilia A blood outgrowth endothelial cells through the focal adhesion kinase/SRC proto-oncogene pathway involvement. (A) Tubulogenic assay. Quantitative analysis of the number of nodes, junctions, branches, and total length of tubule networks using control (C)-blood outgrowth endothelial cells (BOEC) (N=4), hemophilia A (HA)-BOEC (N=4), or HA-BOEC transduced with a lentiviral vector (LV) carrying the F8 transgene (LV-FVIII HA-BOEC) (N=4). The BOEC (5x104) were placed on top of the Matrigel® and incubated overnight. ImageJ Angiogenesis software was used to quantify the number of nodes, junctions, branches, and total length of acquired images. Migration assay. Indirect measurement of cell migration through crystal violet staining elution of BOEC. BOEC were plated into the upper compartment of an 8-μm pore size Transwell at a density of 105 cells per well in serum-free medium, while the lower compartment was filled with complete medium and incubated overnight. Migrated cells were stained with crystal violet that was eluted with acetic acid and quantified using a Victor spectrophotometer at 590 nm. Permeability assay. Permeability was measured by the extravasation of FITC-dextran across a monolayer of BOEC (8x104 cells/well) cultured on a 0.1% gelatin-coated Transwell (3 μm pore) until confluence was reached. At the end of the culture, 5 μg/mL of FITC-conjugated 40 kDa dextran was added to the upper chamber and the fluorescence of the lower chamber was measured in the medium using the Victor spectrophotometer (490 nm [excitation]/520 nm [emission]). Fluorescence readings were normalized to dextran permeability in Transwell inserts without cells. (B) Western blot analysis of whole cell lysate from HA-BOEC treated for 15 min with or without 1 IU/mL of B-domain-deleted recombinant human factor VIII (rhFVIII) and stained with antibodies against pFAK, total FAK, pSrc, total Src, pAKT, total AKT, pmTOR, total mTOR, pp38, total p38, pERK, and total ERK. Tubulin was used as a loading control. (C) Quantification of increased phosphorylation of FAK, Src, AKT, mTOR, and p38 expressed as fold-change relative to untreated control. (D) Western blot analysis of whole cell lysate from HA-BOEC incubated for 15 min in the presence or absence of rhFVIII (1 IU/mL) or rhVEGF (50 ng/mL) and stained with antibody against phosphor vascular endothelial growth factor receptor 2 (VEGFR2) and pERK. Tubulin was used as a loading control. (E) Quantitative analysis of the number of nodes, junctions, branches, and total length of tubule networks performed on untreated HA-BOEC or HA-BOEC treated with 1 μM FAK inhibitor. All cells were incubated in the presence or absence of 1 IU/mL of B-domain-deleted rhFVIII overnight. Indirect measurement of cell migration by elution of crystal violet staining in HA-BOEC. Permeability assay results calculated on the extravasation of FITC-dextran through an intact monolayer of BOEC. Data in (A) and (E) are expressed as mean ± standard deviation. Statistical analysis was performed by a one-way analysis of variance test (****P<0.0001; ***P<0.001; **P<0.01; *P<0.05, NS: not significant). A.U.: arbitrary units; OD: optical density; FAKi: focal adhesion kinase inhibitor.
Figure 2.
Figure 2.
Factor VIII boosts the in vivo angiogenic potential of endothelial cells and reduces the impaired vessel permeability in hemophilia A micee. (A) Immunofluorescence analysis conducted on Matrigel® plugs to detect green fluorescence protein (GFP)-positive control (C-) blood outgrowth endothelial cells (BOEC), hemophilia A (HA)-BOEC), HA-BOEC transduced with a lentiviral vector (LV) carrying the F8 transgene (LV-FVIII HA-BOEC) and HA-BOEC treated with B-domain-deleted recombinant human factor VIII (rhFVIII). These were assessed for vessel formation within Matrigel® plugs transferred intradermally into NOD-scid IL2Rgnull (NSG) (NSG-HA) mice (9 animals for each condition). Matrigel® plugs with cells were prepared by re-suspending 2x106 BOEC in Matrigel®. The mix was implanted intradermally into 8-week-old NSG or NSG-HA mice. For FVIII stimulation, 3 IU/mL of B-domain-deleted rhFVIII was added to Matrigel® and 2 IU of rhFVIII were injected within the Matrigel® plug every 2 days. After 10 days, the plugs were removed, fixed, and embedded in Killik OCT for histological analysis. Scale bar= 100 μm. (B) Quantification of vessel density in the transplanted plugs and measurement of vessel diameter of samples described in (A). (C) Immunofluorescence staining showing murine CD31 (red) and αSMA (green) on vessels formed within Matrigel® plugs harvested from 8-week-old NSG mice (N=6), NSG-HA mice (N=6), NSG-HA mice injected with LV-FVIII (N=3), and NSG-HA mice treated in situ with rhFVIII (N=6). For FVIII stimulation, 3 IU/mL of B-domain-deleted rhFVIII was added to Matrigel® and 2 IU of rhFVIII were injected within the Matrigel® plug every 2 days. After 10 days, the plugs were removed, fixed, and embedded in Killik OCT for histological analysis. Scale bar= 50 μm. (D) Quantification of vessel density and measurement of vessel diameter in Matrigel® plugs of the samples described in (C). (E) Representative images showcasing Evans blue dye extravasation in the interstitial tissue of 8-week-old NSG mice, NSG-HA mice, NSG-HA mice injected with LV-FVIII and NSG-HA mice treated with rhFVIII. The total number of animals used for each condition was six except for NSG-HA mice injected with LVFVIII, for which four mice were used. For FVIII delivery, 4 IU/mice of B-domain deleted rhFVIII were injected into the tail vein every 2 days for 20 days. NSG-HA mice were also tail vein-injected with 5x108 transducing unit (TU)/mouse of LV-FVIII. At the end of the experiment, Evans blue solution was injected into the tail vein. After 15 min, mice were killed, and the extravasation was visualized in the interstitial space under the skin of the mice. Data in (B) and (D) are expressed as mean ± standard deviation. Each dot represents the quantification of one image. Statistical analysis was performed by a one-way analysis of variance test (****P<0.0001). Animal studies were approved by the Animal Care and Use Committee at UPO (Italian Health Ministry Authorization numbers 492/2016-PR and DBO64.5).
Figure 3.
Figure 3.
Factor VIII regulates the expression of genes related to the extracellular matrix such as nidogen 2 (NID2). (A) Heatmap showing the expression pattern of genes modulated by factor VIII (FVIII). The upper panel shows upregulated genes in hemophilia A (HA) blood outgrowth endothelial cells (BOEC) versus control BOEC (C-BOEC) and rescued in HA-BOEC transduced with a lentiviral vector (LV) carrying the F8 transgene (LV-FVIII HA-BOEC) and vice versa in the lower panel. (B, C) Volcano plots showing differentially expressed genes in HA-BOEC versus C-BOEC (B) and LV-FVIII HA-BOEC versus HA-BOEC (C). Genes are classified as upregulated if they have a log-fold change >1 and P value <0.01, and as downregulated if they have a log-fold change <-1 and P value <0.01. (D) Left panel. Western blot analysis of NID2 expression levels in C-BOEC (N=4), HA-BOEC (n=3), and LV-FVIII HABOEC (N=3). An anti-vinculin antibody was used to confirm equal loading. Right panel. Western blot analysis of NID2 expression in HA-BOEC treated or not with B-domain-deleted recombinant human FVIII (rhFVIII) in the presence or absence of 1 μM defactinib (a FAK inhibitor) for 48 h. An anti-tubulin antibody was used to confirm equal loading. (E) Genome browser view showing the average RNA-sequencing signal profile of the NID2 gene in C-BOEC (N=4), HA-BOEC (N=3), and LV-FVIII HA-BOEC (N=3). RNA sequencing was performed in the Illumina sequencer NextSeq 500. Sequencing reads were aligned to the human reference genome (version GRCh38.p13) using STAR v2.7.7a0 5 (with parameters –outFilterMismatchNmax 999 –outFilterMismatchNoverLmax 0.04) and providing a list of known splice sites extracted from GENCODE comprehensive annotation (version 32). Expression counts were then analyzed using the edgeR package.
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