Plastic Surgery Research Council
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PSRC 60th Annual Meeting

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Macrophages Regulate Tissue Fibrosis In Lymphedema
Swapna Ghanta, MD, Daniel A. Cuzzone, MD, Nicholas J. Albano, BS, Seth Z. Aschen, BS, Ira L. Savetsky, MD, Jason C. Gardenier, MD, Walter J. Joseph, III, BS, Babak J. Mehrara, MD.
MSKCC, New York, NY, USA.

Purpose: Lymphedema is a morbid condition that is characterized by fibrosis and progressive lymphatic dysfunction. We have previously shown that modulation of inflammatory responses not only decreases fibrosis but also prevents the onset of lymphedema suggesting that tissue remodeling and lymphatic function are related. In previous studies, we have found that lymphedema is associated with massive increases in the number of infiltrating macrophages. This is important because macrophages are known to be critical regulators of lymphangiogenesis and tissue remodeling implying that these cells may play a crucial role in regulating the link between these processes in lymphedema. The purpose of these experiments was, therefore, to use conditional ablation of macrophages after lymphatic injury to determine how these cells contribute to fibrosis and lymphatic function.
Methods: Wild-type C57B6 mice were lethally irradiated and then reconstituted with bone marrow cells harvested from transgenic mice that express the simian diphtheria receptor (DTR) under the regulation of Cd11b (a macrophage specific gene). Mosaic mice created in this manner express the simian DTR gene on circulating/bone marrow derived macrophages enabling selective depletion of these cells after administration of minute amounts of diphtheria toxin. Bone marrow transplanted mice were allowed to recover and then underwent surgical ablation of the superficial and deep lymphatic vessels of the mid-portion of the tail to induce lymphedema. Experimental animals underwent macrophage depletion using DT beginning either immediately after surgery for 3 weeks, or for 3 weeks after lymphedema had become established (i.e. 3 weeks postoperatively). Control animals were treated with PBS. Tissue fibrosis and lymphatic function were then analyzed using histology, lymphoscintigraphy, and immunohistochemistry.
Results: Treatment of mosaic-DTR mice with DT was well tolerated and resulted in significant depletion of macrophages systemically (>60% depletion in peripheral lymph nodes and tissues; p<0.05) as compared with controls. However, we did not note a significant decrease in overall inflammation as analyzed by CD45 staining (all leukocytes). Interestingly, we found that depletion of macrophages immediately after surgery improved lymphatic function as analyzed by Tc99 lymphoscintigraphy (p<0.001). In contrast, depletion of macrophages after lymphedema had become established (i.e. 3-weeks after surgery) was associated with increased fibrosis and impaired Tc99 lymph node uptake. Interestingly, there was no significant change in other hallmarks of lymphedema, such as adipose deposition or T-cell infiltration.
Conclusions: Our findings suggest that macrophages play an important role in the regulation of tissue fibrosis and lymphatic dysfunction in a mouse model of lymphedema. The finding that macrophage depletion immediately after surgery has different effects from depletion after lymphedema has become established suggests that these effects are complex, temporally regulated, and modulated by other tissue responses. Understanding these temporal changes is important for developing novel therapies for lymphedema and is a long-term goal of our lab.


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