Diphtheria toxin–mediated ablation of lymphatic endothelial cells results in progressive lymphedema
Jason C. Gardenier, … , Sagrario Ortega, Babak J. Mehrara
Jason C. Gardenier, … , Sagrario Ortega, Babak J. Mehrara
Published September 22, 2016
Citation Information: JCI Insight. 2016;1(15):e84095. https://doi.org/10.1172/jci.insight.84095.
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Research Article Inflammation

Diphtheria toxin–mediated ablation of lymphatic endothelial cells results in progressive lymphedema

Abstract

Development of novel treatments for lymphedema has been limited by the fact that the pathophysiology of this disease is poorly understood. It remains unknown, for example, why limb swelling resulting from surgical injury resolves initially, but recurs in some cases months or years later. Finding answers for these basic questions has been hampered by the lack of adequate animal models. In the current study, we used Cre-lox mice that expressed the human diphtheria toxin receptor (DTR) driven by a lymphatic-specific promoter in order to noninvasively ablate the lymphatic system of the hind limb. Animals treated in this manner developed lymphedema that was indistinguishable from clinical lymphedema temporally, radiographically, and histologically. Using this model and clinical biopsy specimens, we show that the initial resolution of edema after injury is dependent on the formation of collateral capillary lymphatics and that this process is regulated by M2-polarized macrophages. In addition, we show that despite these initial improvements in lymphatic function, persistent accumulation of CD4+ cells inhibits lymphangiogenesis and promotes sclerosis of collecting lymphatics, resulting in late onset of edema and fibrosis. Our findings therefore provide strong evidence that inflammatory changes after lymphatic injury play a key role in the pathophysiology of lymphedema.

Authors

Jason C. Gardenier, Geoffrey E. Hespe, Raghu P. Kataru, Ira L. Savetsky, Jeremy S. Torrisi, Gabriela D. García Nores, Joseph J. Dayan, David Chang, Jamie Zampell, Inés Martínez-Corral, Sagrario Ortega, Babak J. Mehrara

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Figure 4

Lymphatic regeneration after DT administration is associated with macrophage infiltration and M2 differentiation.

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Lymphatic regeneration after DT administration is associated with macrop...
(A) Representative triple immunofluorescence localization of F4/80 (top), CD206 (middle), and LYVE-1 (bottom) in the hind limbs of control (normal) and DT-treated mice 1 and 3 weeks after DT administration (scale bar: 100 μm). (B) Quantification of macrophage (F4/80+) infiltration at various time points following lymphatic ablation with DT. (C) Quantification of lymphatic vessel density over time in control and DT-treated mice. (D) Quantification of F4/80+ and CD206+ dual positive cells in hind limb tissues over time. (E) Real-time quantitative PCR for VEGF-A, VEGF-C, and LYVE-1 in cell-sorted CD11b+ cells harvested from hind limb tissues before (control) and 1 or 3 weeks after DT administration (*P < 0.01). (F) ELISA for VEGF-A, VEGF-C, IL-4, and IL-13 in hind limb tissues harvested from control and DT-treated animals 1 and 3 weeks following DT administration (*P < 0.05). 2-tailed Student’s t test.