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Lymph Node Transplantation Generates Spontaneous Lymphatic Reconnection and Restoration of Lymphatic Flow
Seth Z. Aschen, BS, Gina T. Farias-Eisner, BS, Daniel A. Cuzzone, MD, Swapna Ghanta, MD, Nicholas J. Albano, BS, Walter J. Joseph, BS, Ira L. Savetsky, MD, Jason C. Gardenier, MD, Babak J. Mehrara, MD.
MSKCC, New York, NY, USA.
Although lymph node transplantation has been shown to improve lymphatic function in patients with lymphedema, the mechanisms regulating lymphatic vessel reconnection and the functional status of lymph nodes (LNs) remains poorly understood. In this study we developed a novel reporter mouse that allowed us to determine the origin of lymphatic endothelial cells (LECs) and vessels forming functional connections with transferred LNs.
We developed tamoxifen-induced Flt4-Cre/LacZ-LoxP lymphatic reporter mice to examine the lineage of lymphatic vessels infiltrating transferred LNs. The expression of Cre recombinase was driven by the promoter for the lymphatic specific gene vessel endothelial growth factor receptor 3 (VEGF-R3). These mice served as LN transplant recipients from donor wild-type C57/B6 mice. Five days prior to sacrifice, animals received daily doses of tamoxifen to activate Cre expression. Lymphatic function following LN transfer was analyzed with lymphoscintigraphy (LS) using Tc99 and ferritin injections. The anatomic localization of lymphatic vessels and high endothelial venules (HEVs) was analyzed by co-staining for LYVE-1 (specific to lymphatics) and MECA32 (specific to HEVs).
Reporter mice were specific and highly sensitive in identifying lymphatic vessels, such that tamoxifen treatment resulted in high-level expression of Cre recombinase with more than 90% of lymphatic vessels (LYVE-1+) demonstrating B-gal staining. Transferred LNs demonstrated near-normal levels of Tc99 uptake 28 days after transfer as compared to control (non-operated) contralateral lymph nodes. This uptake corresponded to a massive infiltration of recipient mouse lymphatics with putative connections to donor lymphatics, as evidenced by anastomoses between B- gal+/LYVE-1+ (i.e., recipient) and B-gal /LYVE-1+ (i.e., donor) vessels. This spontaneous reconnection was associated with high level expression of lymphangiogenic cytokines (VEGF-C) in the perinodal fat and infiltrating lymphatics. Newly formed lymphatic channels in transferred lymph nodes were in close anatomic proximity to HEVs (demonstrated by LYVE-1 and MECA32 co-staining) with histologic evidence of interstitial fluid exchange between lymphatics and HEVs. Importantly, we found that T and B cell populations in the lymph node were preserved suggestive of preserved lymph node function.
The lymphatic reporter mice we developed show a high fidelity for identifying lymphatic vessels. Using these mice we found that transferred lymph nodes have rapid infiltration of functional host lymphatic vessels and spontaneous return of interstitial fluid drainage function. These findings suggest that even in the absence of exogenous growth factors, transferred LNs are capable of generating functional lymphatic connections with recipient lymphatic vessels and that this process is related to endogenous expression of VEGF-C in the perinodal fat and infiltrating lymphatics. Additionally, considering the finding that LECs and HEVs exist in close proximity in transferred LNs, it is possible that an exchange of interstitial fluid occurs between these vessels, thereby increasing lymphatic flow and drainage. Lastly, the transferred LNs remain immunologically competent because T and B cell populations are maintained.
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