Plastic Surgery Research Council
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PSRC 60th Annual Meeting
Program and Abstracts

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Th2 Cytokines Inhibit Lymphangiogenesis
Ira L. Savetsky, MD, Swapna Ghanta, MD, Jason C. Gardenier, MD, Jeremy S. Torrisi, BA, Gabriela D. Garcia Nores, MD, Matthew D. Nitti, BA, Geoffrey E. Hespe, BS, Raghu P. Kataru, PhD, Babak J. Mehrara, MD FACS.
Memorial Sloan Kettering Cancer Center, New York, NY, USA.

Purpose: Lymphangiogenesis is the process by which new lymphatic vessels develop in response to a variety of physiologic stimuli including wound healing, inflammation, and tumor metastasis. This process is coordinated by a complex interplay between cytokines and growth factors that promote or inhibit lymphatic endothelial cell (LEC) proliferation, migration, and differentiation. Our group has recently reported that Th2 responses play a key role in the pathology of lymphedema by promoting fibrosis and inhibiting lymphatic function. However, while the evidence supporting a role for T cells and Th2 cytokines as negative regulators of lymphangiogenesis is clear, the effects of Th2 cytokines on isolated LECs remains unknown.
Methods: Human dermal lymphatic endothelial cells (HLECs) were used for all in vitro experiments. In order to study the role of IL4 and IL13 in regulating the proliferation of HLECs, flow cytometry was performed using EDU staining after treatment of HLECS with 50ng/ml of recombinant IL4 or IL13. In order to study lymphatic tubule formation and function, we used a matrigel tubule formation assay to study the ability of HLECs to form connections to nearby cells after exposure to these cytokines. Finally, in order to study the effect of IL4 and IL13 in vivo, we used a well-described model of suture-induced inflammatory corneal neovascularization and treated mice with or without monoclonal neutralizing antibodies against IL4 or IL13.
Results: Exposure of LECs to physiologic doses of recombinant IL4 or IL13, resulted in significant impairment of cellular proliferation. These changes correlated with increased apoptosis and increased cell death. Similarly, after plating HLECs in a matrigel tubule formation assay, the ability for HLECs to form connections to nearby cells was significantly diminished after being exposed to IL4 or IL13. These findings were confirmed in vivo when mice treated with IL4mab or IL13mab after corneal suture placement displayed significantly increased lymphatic vessel area and volume as well as increased Ki67+/LECs compared to isotype controls. There were no differences in blood vessel formation, indicating the specificity of these cytokines on lymphatic vessels. Interestingly, there was a greater increase of LEC proliferation and vessel area in the mice that received IL13mab compared to IL4mab, both of which were greater than isotype controls.
Conclusions: This is the first study that shows the direct effect of IL4 and IL13 on LEC proliferation and apoptosis. We show that IL4 and IL13 in vivo regulate LEC proliferation and function. This is important since we have previously shown that these cytokines play a critical role in the regulation of pathological changes associated with lymphedema and provide a mechanistic explanation for our previous findings.


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