Regulation of lymphangiogenesis in the diaphragm by macrophages and VEGFR-3 signaling

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.

Defective lymphatic valve development and chylothorax in mice with a lymphatic-specific deletion of Connexin43

“…In addition, Cx43 expression in thymic regulatory T cell precursors enhances the production of Foxp3 + regulatory T cells (Kuczma et al 2011), a population of cells which were recently shown to modulate lymphedema and promote lymphatic vessel function (Gousopoulos et al, 2016). During development, lymphangiogenesis in the diaphragm is regulated by macrophages, which are closely associated with the sprouting tips of lymphatic vessels (Ochsenbein et al, 2016). However, macrophages associated with lymphangiogenesis are typically LYVE-1 positive (Kim et al, 2007; Ochsenbein et al, 2016) and therefore Cx43 in those macrophages may be deleted in Cx43 ΔLEC mice, but this remains to be determined.…”

“…During development, lymphangiogenesis in the diaphragm is regulated by macrophages, which are closely associated with the sprouting tips of lymphatic vessels (Ochsenbein et al, 2016). However, macrophages associated with lymphangiogenesis are typically LYVE-1 positive (Kim et al, 2007; Ochsenbein et al, 2016) and therefore Cx43 in those macrophages may be deleted in Cx43 ΔLEC mice, but this remains to be determined. Cx43 could also play a role in chemokine signaling during lymphatic vessel development, with this pathway being altered in Cx43 −/− mice but not Cx43 ΔLEC mice.…”

Regulation of Embryonic and Postnatal Development by the CSF-1 Receptor

“…Csf1 op/op mice survive for longer periods than Csf1r −/− mice, and the functional consequences of this mutation have been studied in more detail. Csf1 op/op mice have reduced weight, low growth rate, extensive skeletal abnormalities, hearing, vision and olfactory deficits, abnormal intestinal organization, Paneth cell deficiency, infertility, reduced mammary gland development, altered angiogenesis and lymphangiogenesis and altered neurogenesis (Kondo et al, 2013; Ochsenbein et al, 2016; reviewed in Arnold & Betsholtz, 2013; Chitu, Caescu, et al, 2015; Chitu et al, 2016; Harvey & Gordon, 2012; Pollard, 2009; Pollard & Stanley, 1996). CSF-1 regulates tissue development through its action on both hematopoietic (macrophages and osteoclasts) and nonhematopoietic cells.…”

Quantitative measurement of lymphatic function in mice by noninvasive near-infrared imaging of a peripheral vein

“…Finally, we tested the potential of the assay in an anatomical location of the mouse where it is difficult to visualize the lymphatic outflow using in vivo optical methods. We chose the peritoneal cavity, as many different outflow routes have been described including uptake through stomata in the diaphragm and by omental lymphatics of the abdomen (19,(24)(25)(26). After i.p.…”