Characterizing the angiogenic activity of patients with single ventricle physiology and aortopulmonary collateral vessels

Abstract

Objectives: Patients with single ventricle congenital heart disease often form aortopulmonary collateral vessels via an unclear mechanism. To gain insights into the pathogenesis of aortopulmonary collateral vessels, we correlated angiogenic factor levels with in vitro activity and angiographic aortopulmonary collateral assessment and examined whether patients with single ventricle physiology have increased angiogenic factors that can stimulate endothelial cell sprouting in vitro.

Methods: In patients with single ventricle physiology (n = 27) and biventricular acyanotic control patients (n = 21), hypoxia-inducible angiogenic factor levels were measured in femoral venous and arterial plasma at cardiac catheterization. To assess plasma angiogenic activity, we used a 3-dimensional in vitro cell sprouting assay that recapitulates angiogenic sprouting. Aortopulmonary collateral angiograms were graded using a 4-point scale.

Results: Compared with controls, patients with single ventricle physiology had increased vascular endothelial growth factor (artery: 58.7 ± 1.2 pg/mL vs 35.3 ± 1.1 pg/mL, P < .01; vein: 34.8 ± 1.1 pg/mL vs 21 ± 1.2 pg/mL, P < .03), stromal-derived factor 1-alpha (artery: 1901.6 ± 1.1 pg/mL vs 1542.6 ± 1.1 pg/mL, P < .03; vein: 2092.8 pg/mL ± 1.1 vs 1752.9 ± 1.1 pg/mL, P < .02), and increased arterial soluble fms-like tyrosine kinase-1, a regulatory vascular endothelial growth factor receptor (612.3 ± 1.2 pg/mL vs 243.1 ± 1.2 pg/mL, P < .003). Plasma factors and sprout formation correlated poorly with aortopulmonary collateral severity.

Conclusions: We are the first to correlate plasma angiogenic factor levels with angiography and in vitro angiogenic activity in patients with single ventricle disease with aortopulmonary collaterals. Patients with single ventricle disease have increased stromal-derived factor 1-alpha and soluble fms-like tyrosine kinase-1, and their roles in aortopulmonary collateral formation require further investigation. Plasma factors and angiogenic activity correlate poorly with aortopulmonary collateral severity in patients with single ventricles, suggesting complex mechanisms of angiogenesis.

Keywords: aortopulmonary collateral; congenital heart disease; single ventricle.

Figure 1

Levels of plasma angiogenic factors sampled from the femoral artery and vein in SV-APC (blue triangles) and control patients (circles). All data are represented as mean values and 95% confidence intervals with individual data points overlaid. A, Plasma sFlt-1 levels. B, Plasma, VEGF-A levels. C, Plasma VEGF-C levels. D, Plasma VEGF-D levels. E, Plasma SDF-1a levels. F, Plasma bFGF levels. sFlt-1, soluble fms-like tyrosine kinase 1; VEGF, vascular endothelial growth factor; SDF-1a, stromal derived factor; bFGF, basic fibroblastic growth factor; SV-APC, single ventricle patients with aortopulmonary collateral vessels.

Figure 2

Confocal microscopy images (20x magnification) of fluorescently stained endothelial cell macrospheres incubated with venous (A, B, C, D, E) and arterial (A′, B′, C′, D′, E′) plasma from three SV-APC patients (A, B, & C) and one acyanotic biventricular control patient (D). Patients A & B have greater cell sprouting activity and either increased plasma VEGF or SDF levels (pg/mL) compared to patients C & D. Positive and negative controls with a 100 μm scale bar are shown for reference (E). Overall, there was so significant difference in sprouting activity between SV-APC (blue triangles) and control (circles) patients (F). Data are represented as mean values and 95% confidence intervals with individual data points overlaid. SV-APC, single ventricle patient with aortopulmonary collateral vessels. Abbreviated: Single ventricle patients' plasma display a wide range of angiogenic activity in vitro

Figure 3

Venous plasma VEGF was positively correlated with cell sprouting activity in all 48 patients. VEGF, vascular endothelial growth factor.

Figure 4

Levels of plasma angiogenic factors sampled from the femoral artery (red diamond) and vein (black cross) as well as sprouting activity in all 27 SV-APC patients were correlated with APC severity (i.e. Spicer score). Spearman’s rho (ρ) and p-values are shown for each graph. A, Spicer scoring criteria. B, Plasma VEGF-A levels. C, Plasma sFlt-1 levels. D, Plasma SDF-1a levels. E, Cell sprouting activity. F, A proposed mechanism describing a possible time dependent relationship between angiogenic factor levels and APC formation. The black bar represents when APCs can be visualized with contrast angiography. At time 1, a stimulus for APC formation triggers an upregulation of angiogenic factors. At time 2, APCs are angiographically visible while factors continue to upregulate. At time 3, angiogenic factors have reached their peak level and the initial stimulus for APC formation is extinguished. By time 4, factors have downregulated back to normal levels. Blue dots represent possible time points when various SV-APC patients may have been enrolled, where APCs are visible but factor levels can vary widely. VEGF, vascular endothelial growth factor; sFlt-1, soluble fms-like tyrosine kinase 1; SDF-1a, stromal derived factor. APC, aortopulmonary collateral vessel; SV-APC, single ventricle patient with aortopulmonary collateral vessels.