Lymphangiogenic growth factor responsiveness is modulated by postnatal lymphatic vessel maturation

Abstract

Lymphatic vessel plasticity and stability are of considerable importance when attempting to treat diseases associated with the lymphatic vasculature. Development of lymphatic vessels during embryogenesis is dependent on vascular endothelial growth factor (VEGF)-C but not VEGF-D. Using a recombinant adenovirus encoding a soluble form of their receptor VEGFR-3 (AdVEGFR-3-Ig), we studied lymphatic vessel dependency on VEGF-C and VEGF-D induced VEGFR-3 signaling in postnatal and adult mice. Transduction with AdVEGFR-3-Ig led to regression of lymphatic capillaries and medium-sized lymphatic vessels in mice under 2 weeks of age without affecting collecting lymphatic vessels or the blood vasculature. No effect was observed after this period. The lymphatic capillaries of neonatal mice also regressed partially in response to recombinant VEGFR-3-Ig or blocking antibodies against VEGFR-3, but not to adenovirus-encoded VEGFR-2-Ig. Despite sustained inhibitory VEGFR-3-Ig levels, lymphatic vessel regrowth was observed at 4 weeks of age. Interestingly, whereas transgenic expression of VEGF-C in the skin induced lymphatic hyperplasia even during embryogenesis, similar expression of VEGF-D resulted in lymphangiogenesis predominantly after birth. These results indicate considerable plasticity of lymphatic vessels during the early postnatal period but not thereafter, suggesting that anti-lymphangiogenic therapy can be safely applied in adults.

Figure 1-6926

AdVEGFR-3-Ig establishes long-term inhibition of VEGF-C in vivo. A: AdVEGFR-3-Ig- or AdLacZ-transduced cells were metabolically labeled and the conditioned media were precipitated with protein A Sepharose. B and C: Metabolically labeled VEGF-C was precipitated with VEGFR-3-Ig produced by AdVEGFR-3-Ig-transduced cells or with the 882 polyclonal VEGF-C antiserum (B) or using serum from nude mice transduced intraperitoneally with AdVEGFR-3-Ig or AdLacZ 1 week after birth and analyzed at the age of 2 or 4 weeks (C). The bound proteins were analyzed by 7.5% (A) or 12% (B and C) SDS-PAGE under reducing conditions. D: Concentration of VEGFR-3-Ig in the circulation of C57BL/6 mice as a function of time after a single intravenous injection of 10 μg of the recombinant protein. Dashed line indicates half of the protein concentration obtained 5 minutes after injection.

Figure 2-6926

Adenovirally encoded soluble VEGFR-3 causes lymphatic vessel regression in mice under 2 weeks of age but has no effect on the blood vasculature. VEGFR-3^+/LacZmice were transduced intraperitoneally with 5 × 10 pfu of AdVEGFR-3-Ig or PBS on day 7 after birth and analyzed 1 week thereafter. The lymphatic vessels were visualized by β-galactosidase staining in the heart (A and B), stomach (C–F), and colon (G and H). The lymphatic and blood vessels in skin sections were visualized by immunohistochemistry using antibodies against the lymphatic-specific marker LYVE-1 (I and J, red) or the pan-endothelial marker PECAM-1 (K and L, red) of AdVEGFR-3-Ig-transduced and control mice, respectively. The vessels with wide lumens indicated with arrows in J and L are lymphatics, which are absent in I and K. Similar results were observed when the mice were injected at day 1 after birth and analyzed at the age of 7 days. Scale bars = 50 μm.

Figure 3-6926

Lymphatic vessels become resistant to inhibition of VEGFR-3 signaling after the first 2 postnatal weeks. β-Galactosidase staining of hearts (A and B), ears (C and D), and tail skin (E and F) and immunohistochemical staining of skin sections using antibodies against LYVE-1 (G and H, red) and PECAM-1 (I and J, red) of VEGFR-3^+/LacZ mice transduced intraperitoneally with AdVEGFR-3-Ig or PBS, respectively, at the age of 2 weeks and analyzed 1 week thereafter. Scale bars = 50 μm.

Figure 4-6926

Lymphatic vessels start to regrow 4 weeks after birth despite neutralizing VEGFR-3-Ig levels in the serum. Fluorescent dextran lymphangiography in the ear skin (A–D) as well as whole-mount LYVE-1 (red) and PECAM-1 (green) staining of the ears (E–H) of nu/numice injected intraperitoneally with AdVEGFR-3-Ig (A–C and E–G) or AdLacZ (D and H) at the age of 1 week and analyzed at the indicated time points. Scale bars: 1 mm (A–D); 500 μm (E–H).

Figure 5-6926

Intraperitoneally injected blocking antibodies against VEGFR-3 or recombinant VEGFR-3-Ig induce a partial regression of lymphatic vessels. A–D:nu/nu mice were injected intraperitoneally with mF4-31C1 blocking antibodies against VEGFR-3 at a dose of 30 mg/kg (A and B) or with PBS (C and D) every 2nd day starting at day 4 after birth. Eight days later the diaphragm (A and C) and the ears (B and D) were analyzed by fluorescent whole-mount staining with LYVE-1 antibodies (red). Similar results were obtained when a dose of 60 mg/kg every day was used. E: Pharmacokinetics of mF4-31C1 in nu/nu mice. F–H:VEGFR-3^+/LacZ mice were either transduced with AdVEGFR-3-Ig at a single dose of 5 × 10 pfu (F) or injected intraperitoneally with recombinant VEGFR-3-Ig proteins (G) or control proteins (H) at a dose of 25 mg/kg once a day starting 1 day after birth and the mesenteric lymphatic vessels were stained with X-gal at the age of 7 days. I and J:nu/numice were injected intraperitoneally with AFL4 blocking antibodies against VEGFR-3 (I) or with control antibodies (J) at a dose of 20 mg/kg once a day starting 1 day after birth and the lymphatic vessels in the stomach were stained in whole mount with VEGFR-3 antibodies at the age of 7 days. Scale bars = 500 μm.

Figure 6-6926

VEGF-D promotes lymphangiogenesis predominantly after birth. A–F: Whole-mount β-galactosidase staining of K14-VEGF-D × VEGFR-3^+/LacZ, K14-VEGF-C × VEGFR-3^+/LacZ, and control VEGFR-3^+/LacZ mice at E14.5. G–L: Immunohistochemical staining for LYVE-1 (red) in the skin sections of K14-VEGF-D and K14-VEGF-C mice as well as in their wild-type littermates at days 1 (G–I) and 7 (J–L) after birth. Note the progression of lymphatic hyperplasia between P1 and P7 in the K14-VEGF-D skin (G and J). Scale bars: 1 mm (A–F); 50 μm (G–L).