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Lymphatic remodelling in response to lymphatic injury in the hind limbs of sheep

Abstract

Contractile activity in the lymphatic vasculature is essential for maintaining fluid balance within organs and tissues. However, the mechanisms by which collecting lymphatics adapt to changes in fluid load and how these adaptations influence lymphatic contractile activity are unknown. Here we report a model of lymphatic injury based on the ligation of one of two parallel lymphatic vessels in the hind limb of sheep and the evaluation of structural and functional changes in the intact, remodelling lymphatic vessel over a 42-day period. We show that the remodelled lymphatic vessel displayed increasing intrinsic contractile frequency, force generation and vessel compliance, as well as decreasing flow-mediated contractile inhibition via the enzyme endothelial nitric oxide synthase. A computational model of a chain of lymphatic contractile segments incorporating these adaptations predicted increases in the flow-generation capacity of the remodelled vessel at the expense of normal mitochondrial function and elevated oxidative stress within the lymphatic muscle. Our findings may inform interventions for mitigating lymphatic muscle fatigue in patients with dysfunctional lymphatics.

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Fig. 1: Sheep lymphatic anatomy and lymphatic remodelling after surgery.
Fig. 2: Lymphatic function assessed via NIR imaging.
Fig. 3: Isolated vessel contractile response to varying transmural pressure.
Fig. 4: Remodelled vessels exhibited impaired flow-mediated dilation.
Fig. 5: Proteomic analysis of isolated LMCs.
Fig. 6: Structural and mechanical properties of isolated lymphatic vessels.
Fig. 7: Computational simulation of lymphatic chain performance.

Data availability

The authors declare that all data supporting the results in this study are available in the paper and Supplementary Information. Source data for the figures in this study are available on reasonable request. The mass spectrometry proteomics data have been deposited with the ProteomeXchange Consortium via the PRIDE partner repository64 with the dataset identifier PXD014343.

Code availability

Matlab code for the computational modelling and for the analysis of NIR videos is available at https://github.com/LLBB-GT/NBE-2019. Any additional custom codes (LabView and Matlab) used in this study are available from the authors on reasonable request.

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Acknowledgements

The authors acknowledge the following individuals at the at the University of Georgia for their contributions to this study: A. Bullington, for assistance with the care and handling of the sheep; M. Barletta, for conducting and developing the anaesthesia protocol; S. P. Holmes, for conducting the MRI scans. The authors acknowledge the following funding sources that supported this project: Regenerative Engineering and Medicine (REM) Seed Grant (to J.B.D. and J.P.), NIH RO1HL113061 (to J.B.D.) and American Heart Association (AHA) Predoctoral Fellowship 16PRE27390011 (to T.N.).

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T.S.N., Z.N., R.L.G.Jr, J.F.P. and J.B.D. conceived the concept and designed the experiments. T.S.N., Z.N., J.S.T.H., T.L., M.T.C. and M.R. developed the methods. T.S.N., Z.N., J.S.T.H., T.L., M.T. and J.F.P. performed the experiments. T.S.N., Z.N., J.S.T.H., M.R., C.C.C. and L.S. analysed the data. T.S.N., Z.N., J.S.T.H. and J.B.D. prepared and edited the manuscript.

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Correspondence to J. Brandon Dixon.

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Supplementary Video 1

Rotating movie of 3D MRI lymphangiography of the drainage pathways of a sheep prior to surgery. Five independent experiments were performed with similar results.

Supplementary Video 2

Movie demonstrating surgical excision of lymphatic vessel via NIR guidance. Five independent experiments were performed with similar results.

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Nelson, T.S., Nepiyushchikh, Z., Hooks, J.S.T. et al. Lymphatic remodelling in response to lymphatic injury in the hind limbs of sheep. Nat Biomed Eng 4, 649–661 (2020). https://doi.org/10.1038/s41551-019-0493-1

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