VEGF-C gene therapy augments postnatal lymphangiogenesis and ameliorates secondary lymphedema
J. Clin. Invest. Young-sup Yoon, et al. 111:717 doi:10.1172/JCI15830 [Go to this article.]

Figure 3
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).