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 6

Lymphatic regeneration requires infiltration of macrophages.

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Lymphatic regeneration requires infiltration of macrophages. (A) Represe...
(A) Representative images of hind limbs following DT administration in control (PBS liposomes) and macrophage-depleted (clodronate liposome) animals (n = 5/group). (B) Quantification of increase in foot diameter from baseline in control and macrophage-depleted DT treated animals over time (*P < 0.001). (C) Representative H&E-stained cross sections of the distal hind limb harvested 4 weeks after DT administration in control and macrophage-depleted animals. Fibroadipose tissue of the hypodermis is outlined with dotted black lines and marked with an asterisk (scale bar: 1 mm). (D) Quantification of fibroadipose deposition as a percentage of the total cross-sectional area of the distal hind limb (*P < 0.001). (E) Left: Representative NIR imaging of control (top) and macrophage-depleted (bottom) animals 4 weeks after DT administration (n = 5/group). Middle and right: Whole mount dual immunofluorescence localization of LYVE-1 (middle) and LYVE-1/CD11b (right), demonstrating lymphatic vessels in the distal hind limbs of control (top) but not macrophage-treated (bottom) animals. Note the diminutive and hypoplastic appearance of lymphatics in macrophage-depleted mice (scale bar: 200 μm). (F) Quantification of dermal lymphatic vessels in hind limb tissue sections in control and macrophage-depleted animals 4 weeks after DT treatment (*P < 0.001). (G) Quantification of F4/80+ cells in hind limb tissue sections in control and macrophage-depleted animals 4 weeks after DT treatment (*P < 0.001). (H) ELISA for VEGF-A in hind limb protein from control and depleted mice at 3 weeks (*P < 0.01). (I) ELISA for VEGF-C in hind limb protein from each group of mice (*P < 0.01). 2-tailed Student’s t test.