|Program and Abstracts
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A Nonsurgical, Lifelong Animal Model Lymphedema Through Toxin-mediated Lymphatic Ablation
Jason C. Gardenier, MD, Gina T. Farias-Eisner, MD, Raghu P. Kataru, PhD, Ira L. Savetsky, MD, Jeremy S. Torrisi, BA, Gabriela D. Garcia Nores, MD, Matthew D. Nitti, BA, Geoffrey E. Hespe, BS, Babak J. Mehrara, MD.
Memorial Sloan Kettering Cancer Center, New York, NY, USA.
Lymphedema affects up to 6 million patients and is progressive, with few effective therapies. In order to develop new pharmacologic and surgical therapies animal models are needed, but current models are inadequate because they all resolve in just a few months and the inflammatory signature of the disease is confounded by surgical inflammation from infection and wound healing. The purpose of these experiments was therefore to create a non-surgical, chronic, progressive mouse model of lymphedema.
We created Cre-lox mice that express the human diphtheria toxin receptor (DTR) selectively on lymphatic endothelial cells (LECs) using a lymphatic-specific promoter (FLT4). While mice are normally insensitive to diphtheria toxin (DT) this human DTR binds DT avidly, and allows tissue-specific, selective ablation of the target cells. Genotyping was performed to confirm successful genetic transfer and expression of transgene exclusively on capillary and collecting lymphatics was confirmed histologically. We the ablated the hindlimb lymphatics using a peripheral injection of DT and analyzed lymphedema development at various time points up to one year post ablation.
We found that the DTR gene was expressed selectively in capillary/collecting lymphatics at high levels (>90% of cells) and independent of animal age. Systemic administration (IP) of DT resulted in a dose-dependent depletion of LECs with the highest dose resulting in over 80% loss after 24 hours. This treatment was highly toxic resulting in lethality from intestinal necrosis and sepsis. In contrast, administration of DT in the subcutaneous tissues of the hind-limb resulted in ablation of the lymphatic tree in that limb without systemic toxicity. Similar to the clinical scenario, we found that mice injected with DT had initial limb swelling that decreased over an 8-week period, however, this swelling then returned and became progressively worse over the ensuing months and lasting as long as 1 year post-injection. The initial improvement in swelling correlated with expansion of dermal lymphatic vessels, however, these vessels failed to form normal-appearing collecting vessels at any time. In addition, we found that accumulation of macrophages in the early time periods following lymphatic ablation played a critical role in expansion of dermal lymphatics. Histological analysis of tissues from lymphedematous legs demonstrated histological changes that are consistent with lymphedema including fibroadipose hyperplasia, dermal and epidermal thickening, collagen deposition, leukocyte infiltrate and CD4+ helper T cell infiltration.
We have created the first life-long, non-surgical model of secondary limb lymphedema via DT-mediated lymphatic ablation. This model is innovative because it avoids the confounding inflammation associated with surgical or radiation models and enables us to analyze the mechanisms regulating lymphatic function in a variety of settings. It also allows us to test the durability of pharmacologic and surgical therapies for lymphedema, in that we will be able to see if the disease recurs after withdrawal of therapy.
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