(M-500) Regeneration of collecting lymphatic vessels following injury

Introduction:: The lymphatic system is key for tissue fluid homeostasis, immune surveillance, and lipid and macromolecule transport. The lymphatic system relies on the contractile activity of collecting lymphatic vessels to effectively transport lymph (2). Collecting lymphatic vessels have specialized lymphatic muscle cells (LMCs) that drive lymphatic contractions and intraluminal valves to ensure unidirectional lymph propulsion (2). Most lymphatic endothelial cells (LECs) arise embryonically from the cardinal vein, a process that is guided by lymphangiogenic factors such as VEGFC (3, 4). Such lymphangiogenic pathways are known to substantially decrease in adults (5, 6).

In clinical settings, the lymphatic vasculature is routinely damaged due to trauma, cancer surgery and therapy, and secondary to infection. Such lymphatic damage and subsequent pumping dysfunction cause lymphedema, characterized by tissue swelling, chronic inflammation, fibrosis, and recurrent infection. Lymphedema is prevalent among cancer survivors, but unfortunately, there is no cure or even FDA-approved drug for lymphedema, and therapeutic options are limited.

Despite its importance, the regeneration and repair of collecting lymphatic vessels remain poorly understood, and the mechanisms underlying such regeneration and repair responses remain largely unexplored. Toward this end, we sought to answer whether collecting lymphatics vessels regenerate/repair following collecting vessel injury. We developed a novel surgical mouse model to study lymphatic regeneration and showed that collecting vessels that have had a segment resected can regenerate sufficiently following fibrin implantation to restore lymphatic flow to the draining lymph node. Further, we found that the CCL5-CCR5 axis play an important role in lymphatic regeneration.


Materials and Methods:: Mice. C57BL/6 mice, and αSMA-DsRed (C57BL/6), PROX1-eGFP, CCL5-KO mice (C57BL/6, JAX), were used in this study. Animal protocols were approved by the Institutional Animal Care and Use Committees (IACUC) at MGH.

Lymphatic injury surgical procedure. Mice were anesthetized with administration of ketamine (10 mg/kg) and xylazine (100 mg/kg). To gain access to two saphenous lymphatic vessels—the afferent collecting lymphatic vessels to the popliteal lymph node—that run along the saphenous vein, a skin incision distal to the knee in the mouse hindlimb was made using a scalpel. A small segment of the popliteal lymphatic vessel (1-2 mm) was excised using a microsurgical scissor and under a stereomicroscope. For the gel implantation studies, ~10 µL of unpolymerized fibrin gel was added to the injury site by pipette. Once the procedure was completed, the incision site was sutured.

Near-infrared imaging. The lymphatic near-infrared imaging (NIR) was used to assess lymphatic pumping and regeneration in-vivo. Mice were anesthetized with ketamine/xylene. 5μL of Dextran (10K), Flamma® 774, was injected intradermally into the dorsal aspect of the mouse paw. Longitudinal imaging was performed at week 2, and 4 post-injury to assess lymphatic regeneration. Unpaired t-test was used to test significant differences between frequency of injured and gel implantation groups (*p < 0.05, **p < 0.001).

Proteome profiler cytokine array. Proteome profiler cytokine array (R&D systems, Minneapolis, MN) was used to screen relative levels of inflammatory cytokines following lymphatic injury and gel implantation according to the manufacturer’s protocol.


Results, Conclusions, and Discussions:: Fibrin gel implantation promotes collecting vessel regeneration and restores lymphatic transport to the draining lymph node. To investigate whether fibrin scaffolding promotes lymphatic regeneration following lymphatic vessel injury, we established a novel mouse model of lymphatic vessel injury in the hindlimb where 1-2 mm sections of the saphenous collecting lymphatic vessels were surgically resected without damaging the adjacent blood vessels (Figure 1a). A representative NIR image shows reconnection of injured lymphatic vessels and lymph rerouting to the dermal lymphatics (Figure 1b). Confocal imaging reveals lymphatic vessel sprouting at the site of vessel injury, as indicated by PDPN staining of collecting lymphatic vessels and αSMA staining of both lymphatic and blood vessel muscle cells. (Figure 1c). Next, we compared the lymph drainage between the control leg, the injured leg without gel implantation, and the injured leg with gel implantation by quantifying the accumulation of FITC-Dextran in the popliteal draining node and the inguinal lymph node(Figure 1d-e). Injuring collecting lymphatic vessels impedes lymph transport to the draining popliteal node. However, in the injured + gel group, the popliteal to inguinal fluorescence ratio is approximately one, corresponding to the average values of 16.89±3.24 (a.u.) and 16.81±2.99 (a.u.) for popliteal and inguinal nodes, respectively, suggesting that lymph drainage to the popliteal lymphatic vessels is restored after gel implantation.

Fibrin gel implantation results in increased tissue CCL5 following lymphatic vessel injury and genetic ablation of CCL5 inhibits regeneration. To determine which cytokines were upregulated as a result of lymphatic injury and gel implantation, we conducted a semi-quantitative cytokine array analysis on day 10 following lymphatic surgery and fibrin gel implantation. Interestingly, the array suggested that CCL5 increased 31.6-fold after injury plus gel implantation, compared to a 9.6-fold increase for injury and a 10.3-fold increase for gel plus sham surgery, when normalized to the sham surgery group (Figure 2a). According to NIR data from week 4 after injury (Figure 2b), mice lacking CCL5 are less likely than control mice to recover their proximal filling (filling of lymphatic vessels proximal to the injury site) following gel implantation (p=0.0498, Fisher's Exact test).


Acknowledgements (Optional): :

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