Biology of Vascular Endothelial Growth Factor C in the Morphogenesis of Lymphatic Vessels

Abstract

Because virtually all tissues contain blood vessels, the importance of hemevascularization has been long recognized in regenerative medicine and tissue engineering. However, the lymphatic vasculature has only recently become a subject of interest. Central to the task of growing a lymphatic network are lymphatic endothelial cells (LECs), which constitute the innermost layer of all lymphatic vessels. The central molecule that directs proliferation and migration of LECs during embryogenesis is vascular endothelial growth factor C (VEGF-C). VEGF-C is therefore an important ingredient for LEC culture and attempts to (re)generate lymphatic vessels and networks. During its biosynthesis VEGF-C undergoes a stepwise proteolytic processing, during which its properties and affinities for its interaction partners change. Many of these fundamental aspects of VEGF-C biosynthesis have only recently been uncovered. So far, most-if not all-applications of VEGF-C do not discriminate between different forms of VEGF-C. However, for lymphatic regeneration and engineering purposes, it appears mandatory to understand these differences, since they relate, e.g., to important aspects such as biodistribution and receptor activation potential. In this review, we discuss the molecular biology of VEGF-C as it relates to the growth of LECs and lymphatic vessels. However, the properties of VEGF-C are similarly relevant for the cardiovascular system, since both old and recent data show that VEGF-C can have a profound effect on the blood vasculature.

Keywords: A disintegrin and metalloproteinase with thrombospondin motifs 3; VEGF receptors; collagen and calcium binding EGF domains 1; growth factors; lymphatic vessels; lymphedema; tissue engineering; vascular endothelial growth factor C.

Figure 1

VEGFs and VEGF receptors (VEGFRs). Each of the five mammalian VEGFs [PlGF, VEGF-A to -D], the viral VEGF-E, and the snake venom VEGF-F interacts specifically with a certain subset of the three VEGFRs. VEGF-C_C156S is an engineered vascular endothelial growth factor C (VEGF-C) variant that interacts predominantly with VEGF receptor 3 (VEGFR-3) (Joukov et al., 1998). VEGFs that interact with all three receptors do not naturally exist, but have been engineered (Jeltsch et al., 2006). VEGF receptor 1 (VEGFR-1) and VEGF receptor 2 (VEGFR-2) are expressed on blood vascular endothelial cells (BECs), while VEGFR-2 and VEGFR-3 are expressed on lymphatic endothelial cells. VEGFR-3 is the primary mitogenic receptor for lymphatic endothelium, while VEGFR-2 is the primary mitogenic receptor for blood vascular endothelium. Exclusive to higher primates is the appearance of a short splice isoform of VEGFR-3 (VEGFR-3s) (Pajusola et al., ; Borg et al., ; Hughes, 2001). Signaling pathways activated by VEGFR-3s are partially distinct from those activated by the long splice isoform (VEGFR-3l), since it lacks some of the phosphorylation sites required for mediator docking (e.g., for Shc-Grb2) (Fournier et al., ; Dixelius et al., 2003). The dotted arrows from VEGF-D indicate heterogeneous binding patterns. While mature human VEGF-D can activate VEGFR-2, this seems not to be the case for mouse VEGF-D (Baldwin et al., 2001), and consequently, VEGF-D function could have diverged since the evolutionary divide some 60–65 million years ago (O’Leary et al., 2013). Additionally, human VEGF-D can selectively lose its affinity for VEGFR-3 after proteolytic processing (Leppanen et al., 2011).

Figure 2

Biosynthesis and activation of vascular endothelial growth factor C (VEGF-C). VEGF-C is produced as an inactive propeptide. Proprotein convertases such as furin, PC5, or PC7 cleave between the VEGF homology domain and the C-terminal silk homology domain resulting in pro-VEGF-C. The silk homology domain is not removed by this cleavage, but remains covalently connected via cysteine bridges to the rest of pro-VEGF-C (Joukov et al., 1997b). Pro-VEGF-C is able to bind VEGFR-3, but does not activate it (Jeltsch et al., 2014). The second proteolytic cleavage by A disintegrin and metalloproteinase with thrombospondin motifs 3 (ADAMTS3) removes both terminal domains resulting in mature, active VEGF-C. Cleavage by ADAMTS3 results in the major form of the mature VEGF-C, which is nine amino acids shorter compared to the minor form, which is likely a product of plasmin cleavage (Joukov et al., ; Baldwin et al., ; Jeltsch et al., 2014). Three N-glycosylation sites are found in VEGF-C (shown in green). Alternative names for different VEGF-C forms are given in brackets. The band pattern of VEGF-C produced from a full-length cDNA resolved by SDS-PAGE depends on the expressing cell line, expression levels and the antibody used for immunoprecipitation and/or Western blotting. 3T3 fibroblasts produce almost exclusively pro-VEGF-C. In high-level-expressing CHO cells, a significant fraction of the secreted protein can remain unprocessed. Among the most efficiently processing cells are 293 cells, but pro-VEGF-C still represents the majority of the VEGF-C protein.

Figure 3

Alignment of human, mouse, and rat vascular endothelial growth factor C (VEGF-C)/D with human VEGF-A. The sequences of the active, mature VEGF-C/D are boxed gray. Proteolytic cleavage sites and enzymes (if known) are indicated in blue colors. The signal peptide is boxed green. The eight conserved cysteines of the PDGF/VEGF signature (Muller et al., 1997) are boxed yellow and intra- and intermolecular disulfide bridges are indicated by black connecting lines. VEGF-C/D-specific conserved cysteine residues are boxed in orange. The two asterisks denote the only two amino acid residues that are different between fully processed mouse and human VEGF-C. Cysteine 156, which is mutated to serine in the VEGFR-3-monospecific variant VEGF-C_C156S, participates in the intermolecular cystine bridge (Joukov et al., 1998). When mature VEGF-C is produced from a truncated cDNA, the single cysteine 137 remains unpaired decreasing protein stability (Anisimov et al., ; Leppanen et al., 2011). When pairing with cysteine 156, cysteine 137 interferes with intermolecular disulfide bond formation and protein folding, explaining the observation of significant amounts of single-linked dimers, non-covalent VEGF-C dimers and VEGF-C monomers (Joukov et al., ; Jeltsch et al., ; Chiu et al., 2014). Above the alignment, a heat map indicates the areas of highest divergence, deduced from a more comprehensive alignment of VEGF-A, -C, and -D. The C-terminal domains of VEGF-C/D are not shown.

Figure 4

Vascular endothelial growth factor C (VEGF-C) induces specifically the growth of the lymphatic vasculature. Whole-mount LYVE-1 staining of mouse ears 2 weeks after adenoviral transduction with VEGF-C (A) and LacZ (B). AdVEGF-C induces hyperplasia of and neo-sprouting from the lymphatic vasculature. Arrows indicate lymphatic sprouting. Bar, 100 µm.

Figure 5

Works that give important insights into vascular endothelial growth factor C (VEGF-C) and its function grouped according to topic. Publications about the use of VEGF-C specifically in lymphatic tissue engineering are not included since this list tries to highlight the elemental scientific insights on which tissue engineering can build.