BACKGROUND: Lymphedema is common incurable condition that occurs commonly after lymph node dissection. In the US it is most often encountered following axillary lymph node dissection (ALND) for breast cancer and is thus commonly present in breast reconstruction patients. We have previously shown that lymphatic regeneration following wounding is hindered by scar and the pro-fibrotic molecule TGF-B1. We have also shown TGF-B1 directly inhibits lymphangiogenesis and lymphatic endothelial cell (LEC) function via an unknown mechanism. Therefore the purpose of these studies was to evaluate the mechanism of TGF-B1 inhibition of lymphatic regeneration during wound healing, as well the utility of TGF-B1 blockade in ameliorating this process.
METHODS: LECs were cultured in the presence or absence of TGF-B1 in order to evaluate the in vitro effects of this factor. Proliferation, matrigel tubule formation, rtPCR, and immunocytochemistry were performed. We used an animal model in order to determine the in vivo effects of TGF-B1 and TGF-B1 blockade on lymphatic regeneration in vivo. Briefly, circumferential full thickness skin excisions and microsurgical ligation of the deep lymphatics were performed on the tails of mice. The excision site was covered with a collagen gel and treated with vehicle, recombinant TGF-B1, dominant negative TGF-B receptor II adenovirus (DN-TBRII), or control LacZ virus. Lymphatic regeneration and function were evaluated using tail volume measurements, lymphoscintigraphy, and immunohistochemistry at various time points.
RESULTS: Culture of LECs with TGF-B1 resulted in significantly impaired cellular proliferation and tubule formation. rtPCR and immunocytochemistry demonstrated that TGF-B1 caused significantly decreased expression of the LEC transcription factor Prox-1 with associated decreased expression of LEC markers and interestingly an increased expression of blood endothelial cell (BEC) markers. In vivo experiments demonstrated that compared to vehicle controls, the addition of TGF-B1 resulted in nearly 40% greater increase in tail volumes from baseline at 6 weeks following surgery , impaired lymphatic transport (p<0.001), decreased LEC proliferation, impaired lymphatic capillary regeneration, and in lymphatic fibrosis at all time points. Conversely, blockade of TGF-B1 function using the TBRII virus resulted in approximately 40% less increase in tail volumes from baseline, improved lymphatic transport, and increased LEC proliferation and recruitment. The addition of LacZ virus demonstrated effective transfection, but no significant differences from vehicle control. Immunofluorescence demonstrated that TGF-B1 supplementation resulted in increased colocalization of BEC and LEC markers, as well as an overall decrease in lymphatic vessel numbers.
CONCLUSIONS: We have shown that TGF-B1 is a potent inhibitor of lymphatic regeneration. The mechanism for this effect appears to be increased fibrosis, as well as downregulation of Prox-1 expression and an accompanying LEC to BEC phenotypic change. TGF-B1 blockade was effective in alleviating these deleterious effects, suggesing novel approaches for treating and preventing post-operative lymphedema.