Abstract
Lymphatic vessels play important roles in fluid drainage and in immune responses, as well as in pathological processes including cancer progression and inflammation. While the molecular regulation of the earliest lymphatic vessel differentiation and development has been investigated in much detail, less is known about the control and timing of lymphatic vessel maturation in different organs, which often occurs postnatally. We investigated the time course of lymphatic vessel development on the pleural side of the diaphragmatic muscle in mice, the so-called submesothelial initial diaphragmatic lymphatic plexus. We found that this lymphatic network develops largely after birth and that it can serve as a reliable and easily quantifiable model to study physiological lymphangiogenesis in vivo. Lymphangiogenic growth in this tissue was highly dependent on vascular endothelial growth factor receptor (VEGFR)-3 signaling, whereas VEGFR-1 and -2 signaling was dispensable. During diaphragm development, macrophages appeared first in a linearly arranged pattern, followed by ingrowth of lymphatic vessels along these patterned lines. Surprisingly, ablation of macrophages in colony-stimulating factor-1 receptor (Csf1r)-deficient mice and by treatment with a CSF-1R-blocking antibody did not inhibit the general lymphatic vessel development in the diaphragm but specifically promoted branch formation of lymphatic sprouts. In agreement with these findings, incubation of cultured lymphatic endothelial cells with conditioned medium from P7 diaphragmatic macrophages significantly reduced LEC sprouting. These results indicate that the postnatal diaphragm provides a suitable model for studies of physiological lymphangiogenic growth and maturation, and for the identification of modulators of lymphatic vessel growth.Electronic supplementary materialThe online version of this article (doi:10.1007/s10456-016-9523-8) contains supplementary material, which is available to authorized users.
Highlights
Lymphatic vessels play important roles in physiological tissue fluid homeostasis, immune surveillance and dietary lipid uptake, as well as in a range of pathological conditions including chronic inflammation, wound healing and cancer progression [15, 37]
We found that development of the diaphragmatic lymphatic plexus largely occurs postnatally and that it represents a sensitive model to study the regulation of lymphangiogenesis in vivo
Based on the results of our study, combined with recent reports [14, 26], we propose five parameters that are quantifiable and that allow a detailed and reproducible evaluation of lymphatic vessel development in the diaphragm: the average area covered by LYVE-1? lymphatic vessels, the number of branches, the number of vessel loops, the average branch length and the average lymphatic vessel diameter
Summary
Lymphatic vessels play important roles in physiological tissue fluid homeostasis, immune surveillance and dietary lipid uptake, as well as in a range of pathological conditions including chronic inflammation, wound healing and cancer progression [15, 37]. Despite these important roles, only few mediators of lymphatic vessel development and function have been identified. Angiogenesis (2016) 19:513–524 endothelial cells (LECs), which express VEGFR-3, from the cardinal vein These cells migrate toward a VEGF-C gradient and form a lymphatic vessel plexus, which becomes remodeled from embryonic day (E) 14.5 on, and matures until postnatal time points [28, 31, 40]. The time course of lymphatic vessel development can vary in different organs, and for many organs, the exact time course is still unknown
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