VEGF-C gene therapy augments postnatal lymphangiogenesis and ameliorates secondary lymphedema
Young-sup Yoon, … , Jeffrey M. Isner, Douglas W. Losordo
Young-sup Yoon, … , Jeffrey M. Isner, Douglas W. Losordo
Published March 1, 2003
Citation Information: J Clin Invest. 2003;111(5):717-725. https://doi.org/10.1172/JCI15830.
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Article Genetics

VEGF-C gene therapy augments postnatal lymphangiogenesis and ameliorates secondary lymphedema

Abstract

Although lymphedema is a common clinical condition, treatment for this disabling condition remains limited and largely ineffective. Recently, it has been reported that overexpression of VEGF-C correlates with increased lymphatic vessel growth (lymphangiogenesis). However, the effect of VEGF-C–induced lymphangiogenesis on lymphedema has yet to be demonstrated. Here we investigated the impact of local transfer of naked plasmid DNA encoding human VEGF-C (phVEGF-C) on two animal models of lymphedema: one in the rabbit ear and the other in the mouse tail. In a rabbit model, following local phVEGF-C gene transfer, VEGFR-3 expression was significantly increased. This gene transfer led to a decrease in thickness and volume of lymphedema, improvement of lymphatic function demonstrated by serial lymphoscintigraphy, and finally, attenuation of the fibrofatty changes of the skin, the final consequences of lymphedema. The favorable effect of phVEGF-C on lymphedema was reconfirmed in a mouse tail model. Immunohistochemical analysis using lymphatic-specific markers: VEGFR-3, lymphatic endothelial hyaluronan receptor-1, together with the proliferation marker Ki-67 Ab revealed that phVEGF-C transfection potently induced new lymphatic vessel growth. This study, we believe for the first time, documents that gene transfer of phVEGF-C resolves lymphedema through direct augmentation of lymphangiogenesis. This novel therapeutic strategy may merit clinical investigation in patients with lymphedema.

Authors

Young-sup Yoon, Toshinori Murayama, Edwin Gravereaux, Tengiz Tkebuchava, Marcy Silver, Cynthia Curry, Andrea Wecker, Rudolf Kirchmair, Chun Song Hu, Marianne Kearney, Alan Ashare, David G. Jackson, Hajime Kubo, Jeffrey M. Isner, Douglas W. Losordo

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Figure 3

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Increased expression of VEGF-C protein and VEGFR-3 mRNA in the phVEGF-C–...
Increased expression of VEGF-C protein and VEGFR-3 mRNA in the phVEGF-C–transfected ears. (a and b) Western blot of VEGF-C protein from skin. VEGF-C was detected in its 58-kDa (a) and 31-kDa forms (b). VEGF-C protein expression was significantly higher at and around the phVEGF-C–transfected lymphedema skin. Prox, Mid, and Dist represent samples obtained from ear skin proximal to the skin bridge, skin from the bridge itself, and intact ear skin just distal to the skin bridge of the phVEGF-C–transfected ear, respectively. Neg, samples from the skin bridge of saline-injected lymphedema ear. NL, samples from the bridge site of unoperated contralateral ear. (c and d) Using degenerate oligonucleotides, RT-PCR was performed for total RNA extracted from mesentery (Mes), lung, kidney, and LNs. The PCR product (470 bp) from the kidney sample was sequenced. At the protein level, the rabbit (Rb) VEGFR-3 clone displayed 92.9%, 93.6%, and 94.3% identity with human (Hu), bovine (Bo), and mouse (Mo) VEGFR-3, respectively. (e) New primer sets were designed from the sequenced rabbit VEGFR-3 DNA, yielding a single PCR product of 362 bp. (f) Representative semiquantitative RT-PCR showing higher expression of VEGFR-3 in the lymphedema skin transfected with phVEGF-C than in the saline-injected or unoperated skin. (g) Quantification of VEGFR-3 mRNA levels. (*P < 0.001. **P < 0.01). (h and i) The effect of phVEGF-C gene transfer on tyrosyl phosphorylation of VEGFR-3 (h) and VEGFR-2 (i) by immunoprecipitation with anti-phosphotyrosine Ab followed by Western blot analysis with anti–VEGFR-3 or anti–VEGFR-2 Ab’s, respectively. Samples transfected with phVEGF-C revealed similar levels of phosphorylated VEGFR-2 compared with the control groups (saline and LacZ).