Differential Receptor Binding and Regulatory Mechanisms for the Lymphangiogenic Growth Factors Vascular Endothelial Growth Factor (VEGF)-C and -D

Abstract

VEGF-C and VEGF-D are secreted glycoproteins that induce angiogenesis and lymphangiogenesis in cancer, thereby promoting tumor growth and spread. They exhibit structural homology and activate VEGFR-2 and VEGFR-3, receptors on endothelial cells that signal for growth of blood vessels and lymphatics. VEGF-C and VEGF-D were thought to exhibit similar bioactivities, yet recent studies indicated distinct signaling mechanisms (e.g. tumor-derived VEGF-C promoted expression of the prostaglandin biosynthetic enzyme COX-2 in lymphatics, a response thought to facilitate metastasis via the lymphatic vasculature, whereas VEGF-D did not). Here we explore the basis of the distinct bioactivities of VEGF-D using a neutralizing antibody, peptide mapping, and mutagenesis to demonstrate that the N-terminal α-helix of mature VEGF-D (Phe93–Arg108) is critical for binding VEGFR-2 and VEGFR-3. Importantly, the N-terminal part of this α-helix, from Phe93 to Thr98, is required for binding VEGFR-3 but not VEGFR-2. Surprisingly, the corresponding part of the α-helix in mature VEGF-C did not influence binding to either VEGFR-2 or VEGFR-3, indicating distinct determinants of receptor binding by these growth factors. A variant of mature VEGF-D harboring a mutation in the N-terminal α-helix, D103A, exhibited enhanced potency for activating VEGFR-3, was able to promote increased COX-2 mRNA levels in lymphatic endothelial cells, and had enhanced capacity to induce lymphatic sprouting in vivo. This mutant may be useful for developing protein-based therapeutics to drive lymphangiogenesis in clinical settings, such as lymphedema. Our studies shed light on the VEGF-D structure/function relationship and provide a basis for understanding functional differences compared with VEGF-C.

Role of Matrix Metalloproteinases in Angiogenesis and Cancer

“…To reverse these adverse physicochemical changes, VEGF-C and VEGF-D are synthesized by the activation of VEGFR-3/2, triggering a rise in diameter and density of the peritumoral lymphatic vessels, favoring the propagation of tumor cells toward sentinel lymph nodes (40,41). It has been shown that the inhibition of these factors by the use of antibodies decreases lymphogenesis and metastasis in nearby ganglia (42)(43)(44). Then, angiogenesis maintains a constant and permanent supply of nutrients for cancer cells that leads to tumor growth.…”

Application of microscale culture technologies for studying lymphatic vessel biology

“…Loss of VEGFR‐3 function results in the regression of lymphatic vessels with mutated VEGFR‐3 being an important predictor for diseases associated with lymphatic dysfunction . Moreover, while both VEGF‐C and VEGF‐D signal through VEGFR‐3, distinct receptor‐binding specificities and biological activities are emerging between these two VEGF variants . Presumably, these findings will have important implications for therapeutic targeting of VEGF‐C and/or VEGF‐D for controlling lymphangiogenesis.…”

“…[36][37][38] Moreover, while both VEGF-C and VEGF-D signal through VEGFR-3, distinct receptorbinding specificities and biological activities are emerging between these two VEGF variants. 39,40 Presumably, these findings will have important implications for therapeutic targeting of VEGF-C and/or VEGF-D for controlling lymphangiogenesis.…”

Potential lymphangiogenesis therapies: Learning from current antiangiogenesis therapies—A review

“…11 The interplay between complex factors such as the structural differences between VEGF-C and VEGF-D and their expression levels, 114 the proteolytic cleavage of these factors, 39,115 and the tissue of origin all impact the binding affinities of VEGF-C and VEGF-D to VEGFR2 and VEGFR3 and thus impact the regulation of lymphangiogenesis. 114,116 Because VEGF-C is considered the main ligand for lymphangiogenesis, this review will continue discussing the role of VEGF-C; however, the role of VEGF-D in human disease is just beginning to be better understood. 117 In a recent study, Zhong et al introduced a Prox1-GFP/Flt1-DsRed mouse model that allows for live imaging of angiogenic and lymphangiogenic processes in the cornea and other organs.…”