Lymphedema: Determining the Role of Angiogenesis

Purpose: Lymphedema increases the size of the affected site, first by accumulation of protein-rich fluid, followed by deposition of adipose and fibrous tissue. Because increasing tissue mass requires neovascularization, we hypothesized that the progression of lymphedema might be angiogenesis-dependent.

Methods: Lymphedematous tissue was collected prospectively from six patients with primary lymphedema undergoing resection between 2007 and 2009. Comparisons, lymphedematous and normal tissue, as well as between severe and minor/moderate stage disease were made. Immunohistochemistry using CD31, á-SMA, and D2-40 was used to quantify microvessel, vascular smooth muscle cells (vSMC), and lymphatic vessel density, respectively. Proliferative indices were determined using Ki-67. Quantitative real time-PCR (qRT-PCR) was used to evaluate m-RNA expression of vascular endothelial growth factor (VEGF), angiopoietins 1 and 2 (Ang-1, -2), thrombospondin-1 (TSP-1), and hypoxia inducible factor-1á (HIF-1á). Zymography was used to analyze matrix metalloproteinase (MMP) activity.

Results: Lymphedema tissue was resected from the lower extremity (n=3) or male genitalia (n=3). Median age was 21 years (range 1.5-50). Microvessel (1.1% ± 0.8) and vSMC (3.0% ± 1.2) density in lymphedema was increased compared to control tissue (0.3% ± 0.4 and 0.7% ± 0.8, respectively) (p=0.02). Lymphatic vessel density (0.2% ± 0.3) and proliferative index (0.1% ± 0.1) did not differ from control tissue (p=0.9). Ang-1 and 2 were increased (6.6 and 4.4 fold, respectively) compared to control tissue (p<0.05); TSP-1 was not elevated (p=0.7). VEGF, HIF-1á, and MMP-2,-9 were elevated 4-11 times in severe lymphedema compared to minor/moderate stage.

Conclusions: Lymphedematous tissue exhibits increased vasculature and pro-angiogenic factors. Angiogenesis may play a role in the evolution of lymphedema.