The Effect Of Lymphatic Proliferation In Extremity Trauma And Aberrant Wound Healing
Chase A. Pagani, BA1, Cori Booker, PhD1, Alec Bancroft, BS1, Nicholas Livingston, BS1, Geoffrey Hespe, MD2, Johanna Nunez, MD1, Michael Dellinger, PhD1, Benjamin Levi, MD1.
1University of Texas Southwestern, Dallas, TX, USA, 2University of Michigan, Ann Arbor, MI, USA.
PURPOSE: Traumatic injury results in alterations to tissue organization requiring a highly orchestrated process for proper healing. Following traumatic injury, muscle, tendon, vasculature, nerves, and bone are destroyed resulting in impaired regeneration. There is a paucity of studies investigating the role of lymphatic vasculature network plays in tissue healing following traumatic injury. This is surprising considering the role that the lymphatic system plays in the normal inflammatory response and managing resulting edema which is critical in preventing infection and initiating the healing cascade. Alterations in this process can result in aberrant healing seen following traumatic injury. Utilizing a reliable and reproducible method of traumatic injury, we investigated the role of lymphatic vessels in and aberrant wound healing utilizing novel bioinformatic techniques and 2- and 3-dimensional confocal imaging modalities.
METHODS: We utilized a murine model of traumatic injury resulting in formation of heterotopic ossification (HO). We performed a 30% total body surface area burn to the dorsum of the mouse and a concurrent Achilles’ tenotomy (Burn/tenotomy; BT) resulting in fully formed HO at the Achilles’ injury site nine weeks following injury. C57B6 and Prox1-eGFP mice labeling lymphatic endothelial cells were injured. To visualize lymphatic endothelium prior to injury, uninjured hindlimbs were harvested and processed utilizing the PEGASOS method for tissue clearing. Hindlimbs were harvested prior to injury, one, three, and nine weeks post-injury for histology. Tissue was also harvested from uninjured mice, one, and six weeks post-injury for single-cell RNA-sequencing (scRNA). Separately, we performed the BT injury in chylous ascites (Chy) mice characterized by dysfunctional lymphatic system due to an inactivating mutation in Vegfr3, a cell-surface receptor critical for the growth and survival of lymphatic endothelium. Mice were harvested nine weeks post-injury to assess the impact on HO formation.
RESULTS: Reconstructed 3-D confocal microscopy images of cleared uninjured whole-mount hindlimb tissue stained for lymphatic endothelial cell markers CD31 and LYVE1 show lymphatic vessels at the injury site (A). Sections from uninjured Achilles’ tendon and one, three, and nine weeks post-injury from Prox1-eGFP mice co-stained with LYVE1 show lymphatics in tendon peritenon, however, no lymphatic vessels within the tendon prior to injury (B). Following injury, lymphatics endothelium is disrupted, characterized by no positive Prox1-eGFP staining following injury. Interestingly, Prox1-eGFP+ cells are found within the Achilles’ tendon and Prox1-eGFP/LYVE1 double-positive cells forming a lumen are found nine weeks post-injury within the Achilles’ tendon (B; n=3/group, p*<0.5). ScRNA shows expression of lymphatic endothelial growth factor Vegfc following injury by mesenchymal progenitor cells (MPCs), while receptors for the ligand are expressed by the lymphatic endothelial cell cluster (C, D). VEGFR3 mutation leads to increased variability of HO formation following injury (E; n=4/6/group).
CONCLUSION: These findings highlight the disruption and regeneration of lymphatic vessels following extremity trauma. For the first time, we propose a mechanism for MPC-lymphatics interaction that drives lymphatic regeneration following extremity injury through VEGFC-VEGFR3 driven lymphangiogenesis.
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