Blood Vessel Endothelial VEGFR-2 Delays Lymphangiogenesis: An Endogenous Trapping Mechanism Links Lymph- and Angiogenesis

Abstract

Purpose: : Angio- and lymphangiogenesis are inherently related processes. However, how blood and lymphatic vessels regulate each other is unknown.

Methods: : VEGF-A (200ng) was implanted in mouse cornea using the micropocket assay. Immunostaining of LYVE-1 and CD31 was performed to quantify angio- and lymphangiogenesis. To examine VEGF-C and VEGFR-2 regulation, VEGF-A-stimulated HUVECs in vitro and VEGF-A-implanted corneas in vivo were harvested and samples were examined using western blotting and immunoprecipitation. anti-R2 mAb- or IgG-coated microspheres (MS) were systemically injected to perform in vivo molecular imaging in corneal angiogenic vessels.

Results: : VEGF-A-induced angiogenesis started upon implantation, while lymphangiogenesis was observed first on day 10, suggesting a significant delay in lymphatic growth (n=8, P<0.01). VEGF-C and VEGFR-2 were significantly upregulated in VEGF-A-implanted corneas. Our new in vivo molecular imaging approach revealed higher VEGFR-2 expression in the angiogenic tips, compared to the angiogenic stalk or normal vessels (n=8, P=0.004). Confocal microscopy showed internalization of the VEGF-C/VEGFR-2 protein complex into the cytoplasm of VEGF-A-treated HUVECs, suggesting a novel clearance mechanism for growth factors during angiogenesis. The areas of high VEGFR-2 expression in the angiogenic tips consistently showed impeded lymphatic growth.

Conclusions: : VEGFR-2-upregulation on VEGF-A-induced angiogenic endothelium traps VEGF-C and reduces its concentration in the extracellular matrix, therewith delaying lymphangiogenesis. Growth factor clearance by receptor-mediated internalization is a new paradigm explaining various characteristics of lymphatics.

Keywords: growth factors/growth factor receptors • vascular endothelial growth factor