Diphtheria toxin–mediated ablation of lymphatic endothelial cells results in progressive lymphedema
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
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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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 2

Subcutaneous injection of DT leads to development of chronic lymphedema that models clinical lymphedema temporally, histologically, and radiographically.

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Subcutaneous injection of DT leads to development of chronic lymphedema ...
(A) Representative photographs of mouse hind limbs at 1, 9, and 52 weeks following DT injection as compared with normal (i.e. nontreated) mice. (B) Quantification of increasing foot diameter from baseline after DT administration over the course of the study (n = 5–6/group). (C) Quantification of fibroadipose area for histological sections from 1, 3, 6, 9, 18, and 52 weeks (*P < 0.001). (D) Quantification of scar index (collagen I:collagen III) from foot histological sections for each time point (*P < 0.001). (E) Left panels: NIR lymphangiography in human hands showing normal uptake (left) and dermal backflow with loss of lymphatics in the setting of lymphedema (right). Right panels: NIR lymphangiography of normal (left) and lymphedematous mouse hind limbs (52 weeks after lymphatic ablation; right). Note the similarity of the pattern to clinical disease. (F) Top row: Representative photomicrograph showing immunofluorescence staining of collecting lymphatic vessels (podoplanin+, green; α-SMA+, red) in normal human skin and lymphedematous skin (scale bar: 40 μm). Note the sclerosed collecting vessel in the lymphedematous tissue. Bottom row: Representative photomicrograph showing immunofluorescent staining of collecting lymphatic vessels (podoplanin+; green and α-SMA+; red) in normal mouse hind limb skin and DT treated skin (scale bar: 40 μm). Note decreased luminal area and increased wall thickness in the lymphedematous tissue. (G) Quantification of collecting lymphatic vessel area at different time points after lymphatic ablation (*P < 0.001). (H) Quantification of α-SMA thickness at different time points after lymphatic ablation (*P < 0.001). (I) Representative photomicrographs localizing CD4 and podoplanin in distal mouse hind limb tissues in age-matched normal and DT-treated animals (52 weeks after DT; scale bar: 100 μm). (J) Quantification of CD4+ cells in control and DT-treated mice 52 weeks after lymphatic ablation (*P < 0.001). (K) ELISA for TGF-β1, TNF-α, and IFN-γ in hind limb tissues harvested from control an DT-treated mice 1 and 3 weeks after DT injection (*P < 0.01). 2-tailed Student’s t test.