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 1

FLT4-Cre-DTR enables selective ablation of capillary and collecting lymphatics.

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FLT4-Cre-DTR enables selective ablation of capillary and collecting lymp...
(A) Schematic diagram of the inducible Cre-recombinase system. Downstream of FLT4, there is an internal ribosome entry site (IRES) gene, followed by Cre recombinase and estrogen receptor type 2 (ERT2), which induces genetic recombination to place the floxed DTR gene downstream of FLT4 in response to tamoxifen administration. (B) Immunofluorescence staining of the ear skin shows colocalization of DTR with initial lymphatics (LYVE-1+) and collecting lymphatics (CD31+ with patchy α-SMA+ coverage; scale bar: 50 μm). (C) Representative flow cytometry dot plots for LECs (CD45–PODO+CD31+) and BECs (CD45–PODO–CD31+) in inguinal (i.e., subcutaneous) lymph nodes harvested 24 hours after i.p. DT administration. PODO, podoplanin. (D) Quantification (%) of LECs and BECs from flow cytometry of inguinal lymph nodes 24 hours after i.p. DT administration (n = 4–5 animals/group; LECs, *P < 0.05 and BECs, P = NS). (E) Representative whole mount immunofluorescence staining of ear skin localizing podoplanin and α-SMA in control and DT-treated mice. Note virtually complete ablation of capillary and collecting lymphatics. (F) Top row: Gross images of popliteal lymph nodes harvested 1 week after subcutaneous DT injection in the hind limb, with resultant lymph node hemorrhage. Bottom row: Representative photomicrographs of LYVE-1 immunofluorescence localization in popliteal lymph nodes 1 week after hind limb DT injection, showing destruction of the subcapsular and medullary lymphatics (scale bar: 400 μm). (G) Top row: Representative lymphoscintigraphy heat maps and peak nodal uptake of technetium-99m (99mTc) in the popliteal lymph nodes (yellow dotted circles) of control versus DT after unilateral hind limb injection (injection site is marked by arrows). (H) Quantification of the peak nodal uptake of 99mTc in the popliteal lymph node after DT administration for control and 1, 9, and 52 weeks (n = 4/group; *P < 0.05). Note the decreased peak nodal uptake following DT administration. 2-tailed Student’s t test, ANOVA with post hoc comparison tests.