Breast cancer-related lymphoedema (BCRL) is a chronic swelling of the arm that occurs in approximately 25 % of patients treated for breast cancer. Previous studies have shown that the interstitial protein concentration in the swollen arm is reduced (Bates et al. 1993). There is also evidence to suggest that lymphangiogenesis may occur (Mellor et al. 2000) and that dermal angiogenesis occurs in the swollen arm (Mellor et al. 2002). One hypothesis that would be consistent with these findings is upregulation of VEGF-C. This ligand for VEGFR-2 and VEGFR-3 has been shown to be angiogenic and lymphangiogenic in vivo and in vitro. VEGF-C also increases the hydraulic conductivity (Lp) of single capillaries measured in vivo (Hillman et al. 2001). To test this hypothesis, 5 mm biopsies were excised from the arms of BCRL patients (n = 7) and normal subjects (n = 5) under local anaesthesia (2 % lignocaine, intradermal injection). Local ethics committee approval was obtained and informed, written consent was given by all subjects. Biopsies were fixed in 10 % buffered formalin for 36 h before being dehydrated, cleared and embedded in paraffin wax. Sections (5 µm) were dewaxed, rehydrated and subjected to microwave treatment (800 W) for 10 min in Tris-EDTA buffer, pH 9. Sections were incubated with either 1.14 µg ml-1 VEGF-C antibody (Santa Cruz SC-7133) or normal goat IgG (Zymed, negative controls) overnight at 4 °C before detection with 2 µg ml-1 biotinylated anti-goat secondary antibody using ‘Elite’ avidin-biotin enzyme complex and diaminobenzidine substrate (VectorLabs). The primary antibody recognised the carboxyl terminus of the VEGF-C propeptide, which is cleaved before secretion. Only the cellular form is therefore detected. Analysis was performed using NIH Image 1.62. Images were converted using a ‘Gold’ look-up table to allow densitometric analysis of positive brown staining. The VEGF-C intensity was determined from the difference between the gold scale densities of the VEGF-C antibody and non-specific IgG. The density of VEGF-C staining observed in the dermis and epidermis of the skin from patients with lymphoedema was significantly greater than in skin from normal subjects (P < 0.0005 and P < 0.02, respectively, Mann-Whitney U test). These results indicate that VEGF-C is upregulated in the skin of patients with BCRL.
This work was supported by The Wellcome Trust (62951).