Abnormalities in the Von Willebrand-Angiopoietin Axis Contribute to Dysregulated Angiogenesis and Angiodysplasia in Children With a Glenn Circulation

Abstract

Children with a bidirectional superior cavopulmonary (Glenn) circulation develop angiodysplasia and pulmonary arteriovenous malformations (AVMs). The von Willebrand factor (vWF)-angiopoietin axis plays a major role in AVM formation in multiple diseases. We observed derangements in global angiogenic signaling, vWF metabolism, angiopoietins, and in vitro angiogenesis in children with a Glenn circulation versus controls and within Glenn pulmonary versus systemic circulations. These findings support the novel hypothesis that abnormalities in the vWF-angiopoietin axis may dysregulate angiogenesis and contribute to Glenn pulmonary AVMs. The vWF-angiopoietin axis may be a target to correct angiogenic imbalance in Glenn patients, for whom no targeted therapy exists.

Keywords: ADAMTS-13, a disintegrin and metalloproteinase thrombospondin (motif) #13; AVM, arteriovenous malformation; EBM, endothelial basal media; EGM, endothelial growth media; Glenn; HUVEC, human umbilical vein endothelial cell; IVC, inferior vena cava; LVAD, left ventricular assist device; PA, pulmonary artery; SVC, superior vena cava; angiogenesis; angiopoietin; arteriovenous malformation; vWF, von Willebrand factor; von Willebrand factor.

Graphical abstract

Figure 1

von Willebrand Factor Metabolism Is Abnormal in Patients With a Glenn Circulation (A) In an enzyme-linked immunosorbent assay (ELISA), a disintegrin and metalloproteinase thrombospondin (motif) #13 (ADAMTS-13) was significantly higher in children with a Glenn circulation than controls. (B) In an ELISA, plasma von Willebrand factor (vWF) antigen was modestly lower in children with Glenn circulation than controls. (C) Representative gel electrophoresis for vWF showed a different pattern of high-molecular-weight (HMW) vWF multimers and vWF fragments in children with a Glenn circulation than controls. The top blot (green box) showed modestly decreased HMW vWF multimers in Glenn patients versus controls. The bottom blot (blue box) showed increases in multiple vWF fragment bands, especially bands at approximately 460, 365, and 310 kDa. (D,E) HMW vWF length and density were significantly lower in children with a Glenn circulation than controls. (F) The density of vWF degradation fragments trended toward an increase in children with a Glenn circulation compared to controls. ANOVA = analysis of variance; IVC = inferior vena cava; PA = pulmonary artery.

Figure 2

Plasma Angiopoietin Levels Are Abnormal in Patients With a Glenn Circulation (A) In an ELISA-based quantitative protein microarray, angiopoietin-1 was significantly lower in children with a Glenn circulation than controls. Within Glenn patients, angiopoietin-1 was significantly lower in PA plasma versus IVC plasma. (B) Angiopoietin-2 was significantly higher in children with a Glenn circulation than controls. Abbreviations as in Figure 1.

Figure 3

Endothelial Cell Proliferation Is Abnormal in Patients With a Glenn Circulation (A) In a 5-ethynyl-2 deoxyuridine (EdU) uptake experiment, endothelial cell proliferation was significantly greater with Glenn IVC and PA plasma than with control plasma. A trend toward a difference in proliferation was observed with IVC versus PA plasma. (B to D) Characteristic micrographics of endothelial cell proliferation with control plasma and Glenn IVC and PA plasma are shown. Abbreviations as in Figure 1.

Figure 4

Endothelial Cell Tubule Formation is Abnormal in Patients With a Glenn Circulation (A,B) In a Matrigel assay, endothelial cell hub and tubule counts were significantly lower with Glenn IVC and PA plasma than with control plasma. Glenn PA plasma significantly increased endothelial cell hub and tubule counts versus Glenn IVC plasma. (C to E) Characteristic micrographics of endothelial cells grown with control plasma and Glenn IVC and PA plasma are shown. Large endothelial hubs composed of many adherent cells and lower tubule counts were noted with plasma from Glenn IVC and PA plasma but not control plasma. Abbreviations as in Figure 1.

Figure 5

Angiopoietin-1 Is Low in Glenn Patients With Macroscopic Pulmonary AVMs In Glenn patients with confirmed pulmonary AVMs, angiopoietin-1 levels were lower in the IVC than in patients without AVMs. Abbreviations as in Figure 1.