Alternatively spliced vascular endothelial growth factor receptor-2 is an essential endogenous inhibitor of lymphatic vessel growth

Abstract

Disruption of the precise balance of positive and negative molecular regulators of blood and lymphatic vessel growth can lead to myriad diseases. Although dozens of natural inhibitors of hemangiogenesis have been identified, an endogenous selective inhibitor of lymphatic vessel growth has not to our knowledge been previously described. We report the existence of a splice variant of the gene encoding vascular endothelial growth factor receptor-2 (Vegfr-2) that encodes a secreted form of the protein, designated soluble Vegfr-2 (sVegfr-2), that inhibits developmental and reparative lymphangiogenesis by blocking Vegf-c function. Tissue-specific loss of sVegfr-2 in mice induced, at birth, spontaneous lymphatic invasion of the normally alymphatic cornea and hyperplasia of skin lymphatics without affecting blood vasculature. Administration of sVegfr-2 inhibited lymphangiogenesis but not hemangiogenesis induced by corneal suture injury or transplantation, enhanced corneal allograft survival and suppressed lymphangioma cellular proliferation. Naturally occurring sVegfr-2 thus acts as a molecular uncoupler of blood and lymphatic vessels; modulation of sVegfr-2 might have therapeutic effects in treating lymphatic vascular malformations, transplantation rejection and, potentially, tumor lymphangiogenesis and lymphedema (pages 993-994).

Figure 1

Loss of endogenous sVegfr-2, which antagonizes Vegf-c, leads to spontaneous corneal lymphangiogenesis. (a) sVegfr2 mRNA detection (purple-blue) by in situ hybridization in the mouse cornea. Epi, epithelium; Str, stroma. (b) sVegfr-2 immunolocalization (brown) in the mouse cornea using AA21127. Cell nuclei stained blue by hematoxylin. (c) Representative corneal flat mounts of a LeCre/Vegfr2^loxP/loxP mouse (n = 30). Lyve-1⁺ (green) lymphatic and Cd31⁺ (red) Lyve-1^– blood vessels are shown. (d,e) Immunofluorescent detection of Lyve-1⁺ (green) Prox1⁺ (red) lymphatic vessels in corneal whole mounts (d) and cross sections (e) of LeCre/Vegfr2^loxP/loxP mice. (f) Transmission electron microscopy of lymphatic vessel in cornea of LeCre/Vegfr2^loxP/loxP mice. Inset (right). (g) Quantification of vessel area in corneal flat mounts (n = 20). Error bars depict s.e.m. (h) Representative western blot of samples of recombinant sVegfr-2 (rsVegfr-2) incubated with recombinant Vegf-c (rVegf-c) and immunoprecipitated with either anti-Vegfr-2 (3^rd lane from the left) antibody or isotype control IgG (4^th lane from the left) and immunoblotted with anti-Vegf-c antibody. sVegfr-2 and Vegf-c resolution shown on first two lanes from the left. (i) Representative immunoblot of Vegf-c-induced Vegfr-3 phosphorylation of mouse lymphatic endothelial cells. Vegfr-3 re-blot is shown. ϕ, media only. (j) Proliferation of human lymphatic microvascular endothelial cells, quantified by BrdU uptake. (f,j), NS, not significant ;* P < 0.05, Significance by Mann Whitney U test. Error bars depict s.e.m. Scale bars: (a,b,d), 50 μm; (c) 500 μm; (e) 25 μm; left panel of (g), 1 μm; right panel of (g), 200 nm.

Figure 2

sVegfr-2 inhibits reparative corneal lymphangiogenesis and rejection of corneal allografts. (a) Representative western blot, 5 days after suture placement in Vegfr2^loxP/loxP mouse corneas treated with plasmid coding for Cre recombinase (pCre) or empty vector (pNull) (n = 8). Loading control, Gapdh. Bottom panel (arrows) shows Lyve-1⁺ vessels-green 14 days after suture placement. (b) Real-time PCR of sVegfr2 in the Vegfr2^loxP/loxP mouse corneas (n = 4). (c) Quantification of vessel area in corneal flat mounts of Vegfr2^loxP/loxP and Balb/C wild-type (WT) corneas (n = 5-6) and Vegfr2^loxP/loxP mice corneas injected with pCre followed by systemic administration of a Vegfr-3 tyrosine kinase inhibitor (R3TKI) (n = 8). Lyve-1⁺, lymphatic vessel - white bars; Cd31⁺/Lyve-1^–, blood vessel – black bars. (d) Representative corneal flat mounts of C57Bl/6J wild-type mouse corneas. Lyve-1⁺, lymphatic vessel - green;Cd31⁺/Lyve-1^–, blood vessel – red. (e) Quantification of vessel area in flat mounts of C57Bl/6J wild-type mouse corneas (n = 5). Lyve-1⁺, lymphatic vessel - white bars; Cd31⁺/Lyve-1^–, blood vessel – black bars. (f) Kaplan-Meier survival curves showing survival of corneal grafts in BALB/c hosts (n = 10–13). (g) Representative corneal flat mounts of transplanted mouse corneas. Lyve-1⁺, lymphatic vessel - green;Cd31⁺/Lyve-1^–, blood vessel – red. Limbus, bottom dotted-line; Recipient-donor interface, top dotted-line. (h) Quantification of corneal graft vessel area in flat mounts of transplants (n = 5). Lyve-1⁺, lymphatic vessel - white bars;Cd31⁺/Lyve-1^–, blood vessel – black bars. (NS, non-significant; *, P < 0.05; Significance by Mann Whitney U test). Error bars depict s.e.m. Scale bars: (a,d,g), 500 μm.

Figure 3

Endogenous Vegf-c and sVegfr-2 selectively modulate corneal lymphangiogenesis. (a) Representative color photographs of sutured wild-type mouse corneas (top). Representative flat mounts of sutured corneas. Lyve-1⁺, lymphatic vessel - green;Cd31⁺/Lyve-1^–, blood vessel – red. (b) Quantification of corneal vessel area of sutured corneas of wild-type mice (n = 8). Lyve-1⁺, lymphatic vessel - white bars; Cd31⁺/Lyve-1^–, blood vessel – black bars. (c) ELISA quantification of Vegf-c/VEGF-C in wild-type mouse corneas 2 and 4 days after suture placement or implantation of pellets containing 160 ng recombinant human VEGF-C. (d) Immunobloting of sVegfr-2 secreted by transfected human embryonic kidney cells (HEK), Chinese hamster ovary cells (CHO), and mouse corneal epithelial cells (KEPI) under reducing (RED) and non-reducing (NR) conditions. M, marker; lane 1. pNull; lane 2. psVegfr-2 (RED); lane 3. psVegfr-2 (NR). (e) Silver staining of polyacrylamide gel loaded with Vegfr-2/Fc and recombinant sVegfr-2 (rsVegfr-2) and resolved under reducing versus non-reducing conditions. Dim, dimer; Mon, monomer; M, marker. (f) Corneal area occupied by blood vessels (Cd31⁺/Lyve-1^–) following suture injury in wild-type mice. (g) Representative immunoblot of phosphorylated Vegfr-2 (phosVegfr-2) of porcine aortic endothelial cells stably transfected with Vegfr-2 (PAE-KDR). Reblotting for total Vegfr-2 and Vinculin are shown. ϕ, no Vegf-a. (h) Corneal area occupied by blood vessels (Cd31⁺/Lyve-1^–) following pVegf-a injection. NS, not significant; *, P < 0.05; Significance by Mann Whitney U test. n = 4–8. Error bars depict s.e.m.(b,c,f,h). Scale bar: (a), 500μm.

Figure 4

Loss of sVegfr2 in the skin induces lymphatic hyperplasia. (a) sVegfr2 mRNA detection (purple-blue) by in situ hybridization in the mouse skin. (b) sVegfr-2 immunolocalization (brown) in the mouse skin using AA21127. Cell nuclei stained blue by hematoxylin. (c) Immunofluorescence of sVegfr-2 in the skin of newborn mice. Superficial keratin autofluorescence is seen in all instances. Cell nuclei stained with DAPI. Scale bar, 50 μm. (d) Representative whole mount of the ventral skin of P0 mice. Lyve-1⁺ (green) lymphatic and Cd31⁺ (red) Lyve-1^– blood vessels are shown. (e) Skin area occupied by blood vessels (Cd31⁺/Lyve-1^–, black bars) and lymphatic vessels (Lyve-1⁺, white bars) in P0 mice. (f) Representative whole mount immunofluorescent image showing Lyve-1⁺ (green) and Prox1⁺ (red) lymphatic vessels of P0 mice (n = 14). (g) Lymphatic endothelial cell (LEC) density, quantified by number of Prox1⁺ (red) nuclei per 100 μm of lymphatic vessel (Lyve-1⁺, green) length, in P0 mice (n = 12). (h) Quantitative branch point analysis of Lyve-1⁺ lymphatic vessels per unit area (750 μm × 750 μm) is shown (n = 8). Scale bar: (a-d,f), 50 μm; (e,g,h) NS, not significant; *, P < 0.05; Significance by Mann Whitney U test. Error bars depict s.e.m.

Figure 5

sVegfr-2 is produced by blood endothelial cells (BECs) and skin epithelium and circulates in plasma. (a) Representative western blots of mouse plasma immunoprecipitated with an antibody against the amino terminus of Vegfr-2 (N) and immunoblotted with either anti-Vegfr-2 (N; left), anti-sVegfr-2 (C; right) and an antibody against the carboxyl terminus of mbVegfr-2 (C; center). (b) PCR amplification of sVegfr2 mRNA using cDNA derived from mouse lymphatic endothelial cells (LECs) and mouse BECs isolated from the brain (Bend3), Skin (Py4) and pancreas (MS1). Adjacent lane (H2O) shows template negative control. Gapdh was loading control (lower band), (n = 5). (c) ELISA quantification of sVegfr-2 protein in supernatant of blood and lymphatic endothelial cells (n = 3). (d) Immunofluorescence of sVegfr-2 (red) in the pulmonary microvasculature (Cd31⁺ vessel – green). Cell nuclei stained with DAPI. Scale bar, 50 μm. (e) ELISA quantification of sVegfr-2 levels in plasma (n = 16). *, P < 0.05; Significance by Mann Whitney U test. (c,e) Error bars depict s.e.m.

Figure 6

sVEGFR-2 exists in humans and inhibits human lymphangioma cell proliferation. (a) PCR cloning of open-reading-frame of VEGFR2 from human umbilical vein endothelial cells. (b) sVEGFR-2 immunolocalization (brown) in the human cornea using AA21129. Cell nuclei stained blue by hematoxylin. (c,d) Proliferation of lymphatic endothelial cells isolated from two children with lymphangioma, stimulated by VEGF-C and treated with sVEGFR-2 or bovine serum albumin (BSA), quantified by BrdU uptake. (n = 6–9); c, Patient #1 is a 4-month old child; d, Patient #2 is a 10-month-old child. NS, not significant; *, P < 0.05; Significance by Mann Whitney U test. Error bars depict s.e.m.