Correlation between von Willebrand Factor and the PI3K/Akt signalling pathway in the control of angiogenesis

Introduction Angiodysplasia is the most common vascular abnormality in the gastrointestinal tract and a hallmark of pathological angiogenesis associated with von Willebrand disease (VWD). VWD arises from mutations in the von Willebrand factor (VWF) gene, leading to qualitative or quantitative defects in the VWF protein. VWF is a plasma glycoprotein mainly known for its function in hemostasis, but recently also a role in angiogenesis was described. VEGF and Ang-2 signaling seem to be involved in increased angiogenesis in VWF knock-down cells, however, the mechanism by which VWF modulates angiogenesis is incompletely understood. Thus, we strive to identify signaling pathways downstream of VEGF and Ang-2, which are affected by VWF. Here, we focused on PI3K/Akt signaling; a downstream pathway of the Ang-2 receptor Tie-2.

Method The study was conducted in cultured human umbilical vein endothelial cells (HUVECs), in which a VWD phenotype was mimicked by siRNA knock-down of VWF. Cell proliferation was determined by the WST-1 assay, cell migration by a scratch wound-healing assay, tube formation by a Matrigel assay, and protein expression by Western blotting (WB). The PI3K/Akt pathway was investigated by incubation with wortmannin (5 µM for 24 h), an inhibitor of PI3K. Akt phosphorylation was determined by WB employing an antibody specific for phosphorylated Ser473 in Akt.

Results We confirmed that HUVECs, in which VWF was downregulated (84 ± 6.89 %) by siRNA ([Fig.1a], si-VWF), react to treatment with vascular endothelial growth factor (VEGF; 25 ng/ml) by showing enhanced cell proliferation (1.79 ± 0.086 fold vs. 1.3 ± 0.164 fold) (Fig.1B, si-VWF, green bar) and cell migration (1.54 ± 0.084 fold) compared to controls. Moreover, Matrigel assay data revealed that downregulation of VWF enhanced tube formation of ECs in a VEGF-independent manner. In response to treatment with wortmannin, cell proliferation ([Fig.1b], si-VWF, red bar) and tube formation were reduced to normal levels in si-VWF cells and migration was inhibited to the same extent in control and si-VWF cells. Therefore, PI3K inhibition abolished both the VEGF dependent and independent effects of VWF knock-down. Interestingly, reduced VWF expression was further accompanied by an upregulation of Akt phosphorylation, a downstream target of PI3K.

Fig. 1 Enhanced proliferation of VWF knock-down cells can be reversed by PI3K inhibition.; (A) Western blotting (upper panel) and densitometric analysis of VWF expression (lower panel) shows that siRNA-VWF (50 nM) reduced VWF expression 30 h after transfection by 83%. (B) After siRNA transfection, HUVECs were exposed to Wortmannin (5 µM) (blue bars), VEGF (25 ng/ml) (green bars) or VEGF plus Wortmannin (red bars) for 24 h, n=3.

Conclusion The data of the present study indicate the PI3K/Akt pathway to be involved in VWF-regulated angiogenesis. Targeting the PI3K/Akt axis may offer a new therapeutic maneuver for the treatment of angiodysplasia.

Publication History

Article published online:
20 February 2023