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Cardiac lymphatics are heterogeneous in origin and respond to injury

Abstract

The lymphatic vasculature is a blind-ended network crucial for tissue-fluid homeostasis, immune surveillance and lipid absorption from the gut. Recent evidence has proposed an entirely venous-derived mammalian lymphatic system. By contrast, here we show that cardiac lymphatic vessels in mice have a heterogeneous cellular origin, whereby formation of at least part of the cardiac lymphatic network is independent of sprouting from veins. Multiple Cre–lox-based lineage tracing revealed a potential contribution from the putative haemogenic endothelium during development, and discrete lymphatic endothelial progenitor populations were confirmed by conditional knockout of Prox1 in Tie2+ and Vav1+ compartments. In the adult heart, myocardial infarction promoted a significant lymphangiogenic response, which was augmented by treatment with VEGF-C, resulting in improved cardiac function. These data prompt the re-evaluation of a century-long debate on the origin of lymphatic vessels and suggest that lymphangiogenesis may represent a therapeutic target to promote cardiac repair following injury.

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Figure 1: Spatiotemporal development of the murine cardiac lymphatic vasculature.
Figure 2: Incomplete contribution of Tie2+ venous-derived LECs indicates a novel non-venous contribution to the developing cardiac lymphatics.
Figure 3: Vav1+, Pdgfrβ+ and Csf1r+ lineages contribute LECs to the developing cardiac lymphatics.
Figure 4: Myocardial infarction induces a significant cardiac lymphangiogenic response that can be enhanced by VEGF-C-stimulation to promote functional improvement.

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Acknowledgements

This work was funded by the Wellcome Trust (L.K.), EU FP7 Marie Curie ITN (CardioNet; M.M.) and the British Heart Foundation (S.N., J.M.V., M.R., K.D., S.B., C.A.C., P.R.R.). We thank M. Fruttiger for providing the Pdgfb-creERT2 line, J. Pollard for providing the Csf1r-creER mouse line, K. Alitalo for providing the Vegfr3lacZ/+ mouse line, T. Makinen for providing Pdgfrb-cre embryos and the transgenic services at the University of Oxford for re-deriving the Prox1fl/+ mouse line from RIKEN (accession number CDB0482K). We are grateful to B. Vernay and A. Eddaoudi for assistance with confocal microscopy and flow cytometry, respectively.

Author information

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Authors

Contributions

L.K., S.N. and J.M.V. carried out all experiments (except myocardial infarction surgeries and MRI scanning), analysed the data and contributed figures for the manuscript. K.D., S.B., M.M. and M.R. performed all MI surgeries and post-processed the hearts in preparation for immunofluorescence. F.M. provided the RIKEN Prox1-floxed mice. S.N. and C.A.C. performed blinded-MRI scanning and analysed the data. P.R.R. established the hypotheses and analysed the data. J.M.V. and P.R.R. supervised the studies and co-wrote the manuscript. All authors declare no conflicting or competing interests.

Corresponding author

Correspondence to Paul R. Riley.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Molecular characterization of the murine cardiac lymphatic vasculature.

aj, Whole-mount DAB staining of hearts (n = 3 per time point) with the lymphatic marker VEGFR-3 revealed cardiac lymphatic vessels first sprout from the region of the sinus venosus, on the dorsal side of the heart at E14.5 (a, white box, enlarged in b). At E16.5, ventrally the first small vessels arose between the atria (c), while the main dorsal vessels spread inferiorly from the sinus venosus at the inflow region of the heart (d). At E18.5 the network appears similar with little expansion (e, f). From birth (P0) lymphatic vessels branch and expand onto the ventral epicardial surface of the heart such that by P10 the network has expanded markedly, coincident with cardiac growth (g, h). Consistent with the systemic lymphatic vasculature, cardiac lymphatic vessels are fully developed by P15 (i, j), with no difference in vessel density at later stages (data not shown). kn, Whole-mount DAB staining of E17.5 hearts with the lymphatic marker Prox1 (n = 4) further confirmed extensive spread of the sprouting lymphatics inferiorly from the outflow tract region (k) and sinus venosus, at the inflow region of the heart (l). White inset boxes in k and l are shown in m and n, respectively, highlighting the punctate nuclear expression of Prox1 in coronary lymphatics. ov, Whole-mount confocal imaging of E17.5 hearts (n = 4) stained with VEGFR-3, Prox1 and Lyve-1 confirmed co-labelling of coronary lymphatic vessels. Note that while at this developmental stage VEGFR-3 is restricted to LECs (p, t), Prox1 is also expressed in the underlying myocardium (o, s) and Lyve-1 labels tissue-resident macrophages (q, u). Scale bars: a, 750 μm; b, 300 μm; c, d, 750 μm; e, f, 1 mm; g, h, 2 mm; i, j, 2.5 mm; k, l, 400 μm; m, n, 200 μm; o, s, 100 μm.

Extended Data Figure 2 Cardiac lymphatic vessels do not emerge from the developing coronary vasculature.

ao, Whole-mount confocal imaging of hearts stained with Emcn (vessels) and Prox1 (lymphatics) revealed sprouting of Prox1+ lymphatics from extra-cardiac tissue neighbouring the sinus venosus on the dorsal side of the developing heart at E12.5–13.5 (a, d), but no Prox1+ LECs were observed budding from Emcn+ coronary vessels (c and f; white arrowheads in b and e highlight Prox1+ LECs). Prox1+ lymphatics had reached the sinus venosus by E13.5 (white arrow in f) and the outflow tract, on the ventral side of the heart by E14.5 (white arrow in h); no Prox1+ LECs were observed emerging from Emcn+ vessels on the ventral side at E14.5 (gi). Background-like labelling on the ventricular surface in c, f and i reflects Prox1 expression in the developing myocardium. Between E15.5–17.5, Prox1+ lymphatics aligned with Emcn+ coronary veins but no contribution of Prox1+ LECs was observed (j, l and n, white boxes enlarged in k, m and o, respectively; n = 5 hearts analysed per time point). Scale bars: a, 550 μm; c, f, i, 250 μm; d, g, 750 μm; j, l, n, 400 μm; k, m, o, 200 μm.

Extended Data Figure 3 The common cardinal vein contributes LECs that migrate towards the sinus venosus and outflow tract of the developing heart.

ac, Whole-mount confocal analysis of E10.5 embryos stained with Emcn or Prox1 and VEGFR-3 revealed Prox1/VEGFR-3+ LECs emerging along the common cardinal vein (a, red box enlarged in b; white box enlarged in c) migrating towards the sinus venosus (white arrowheads in c; n = 3 embryos). df, Whole-mount DAB staining revealed Prox1+ LECs migrating towards the outflow tract, on the ventral surface of the developing heart at E12.5 (d, white inset box enlarged in e; alternative lateral view in f; white arrowheads indicate migrating LECs; n = 4 embryos). ba, branchial arch; ccv, common cardinal vein; fl, forelimb; h, heart; isv, inter-somitic vessel; la, left atrium; lv, left ventricle; oft, outflow tract; paa, pharyngeal artery arch; ra, right atrium; rv, right ventricle. Scale bars: a, 1 mm; b, 500 μm; c, 200 μm; d, 600 μm; e, 400 μm; f, 300 μm.

Extended Data Figure 4 Tie2-Cre efficiently labels the developing cardinal vein and partial contribution of Pdgfb+-derived LECs indicates a non-venous contribution to the developing cardiac lymphatics.

ad, Tie2-Cre;R26R-eYFP lineage tracing revealed recombination and labelling of the cardinal vein and jugular lymph sacs at E10.5 (a; n = 3 embryos analysed). Plane of section to capture jugular lymph sacs is shown in b. White inset box in a is shown at higher magnification and demarcated by GFP (c) and Emcn (d) co-staining. eo, Schematic (e) to show how embryos were generated by breeding Pdgfb-CreERT2 mice with either R26R-tdTomato (fl) or R26R-mTmG (mo) reporter mice and then being injected with 4-hydroxytamoxifen (4-OHT) at E9.5, before venous sprouting. Whole-mount confocal analysis of E17.5 hearts (n = 4) stained with Lyve-1 revealed incomplete tdTomato recombination in cardiac lymphatic vessels (f). Both Pdgfb+ (gi) and Pdgfb (jl; mo) lymphatic vessels were observed, highlighted by the dotted green outlines (g, j, m), indicating a combined Pdgfb+ endothelial origin and Pdgfb non-venous source for the cardiac LECs. Scale bars: a, 200 µm; b, 1.5 mm; c, d, 50 µm; f, 400 µm; e, l, o, 100 µm.

Extended Data Figure 5 Neither the pro-epicardial organ, cardiac mesoderm nor cardiac neural crest contribute LECs to the developing heart and dermal lymphatics are not derived from the Vav1+ lineage.

al, Lineage tracing using WT1-CreERT2;R26R-eYFP (4-hydroxytamoxifen injected at E9.5; a–c), Mesp1-Cre;R26R-eYFP (df), Nkx2.5-Cre;R26R-EYFP (gi) and Wnt1-Cre;R26R-eYFP (jl; n = 3 hearts analysed per lineage trace) showed no YFP recombination in cardiac lymphatic vessels as marked by Prox1 or Lyve-1, suggesting that neither the pro-epicardial organ/epicardium, cardiac mesoderm (early or late) or cardiac neural crest, respectively, contribute LECs to the developing cardiac lymphatics. mo, Embryos generated by breeding Vav1-Cre with R26R-tdTomato reporter mice were subject to whole-mount confocal analysis of E17.5 dorsal skin preparations (n = 4 Vav1-tdTomato+ embryos analysed). tdTomato epifluorescence (m) and Prox1 immunostaining (n) revealed a lack of Vav1-Cre recombination in dermal lymphatic vessels (highlighted by the green dotted lines, m) and a lack of overlap of tdTomato with Prox1 and Lyve-1 expression (o; all Prox1+ nuclei assessed across 5 fields of view per embryonic skin; n = 4 skins in total). pr, Higher magnification of inset white box (n) revealed that tdTomato+ cells (p) did not overlap with Prox1+ nuclei in the lymphatic vessels (q) (white arrowhead highlights tdTomato+/Prox1 cell in r). Scale bars: al, 100 µm; mo, 100 µm; pr, 50 µm.

Extended Data Figure 6 The Vav1+ lineage does not contribute to LECs emerging from the common cardinal or jugular veins but contributes to VEGF-C induced LECs emerging from yolk sac explants.

ad, Vav1-Cre;R26R-tdTomato lineage tracing revealed no recombination nor labelling of the nascent LECs budding from common cardinal vein endothelium in E10.0 embryos (a) as confirmed by co-staining for Emcn, Prox1 and tdTomato fluorescence. White inset box in a is highlighted in enlarged panels (bd; arrows indicate Prox1+ (blue) LECs delaminating from the common cardinal vein b, c). In the sinus venosus region Vav1+ cells were evident but lacked Prox1 expression, excluding an LEC identity (d). eh, Vav1-Cre;R26R-eYFP lineage tracing revealed no recombination nor labelling of LECs forming the jugular lymph sacs in E12.5 embryos (e) as confirmed by co-staining for GFP, Emcn and Prox1. White inset box in e highlighted by individual GFP (f), Emcn (g) and Prox1 (h) staining (n = 3 embryos analysed per time-point). ccv, common cardinal vein; jls, jugular lymph sac; jv, jugular vein; sv, sinus venosus. i, j, Representative staining for Prox1 and native tdTomato fluorescence of ex vivo cultures of explanted Vav1-Cre;R26R-tdTomato conceptuses at E8.0, including the intact yolk sac (i) and outgrowth of tdTomato+ cells (j). Explants were cultured with 100 ng ml−1 of recombinant VEGF-C(C156S)28 (R&D Systems), a potent selective lymphangiogenic cue that only signals via VEGFR-3 (i). kz, High-resolution images of the specification of tdTomato+/Prox1+ LECs (indicated by white inset boxes) in the yolk sac explants (kn) and in the surrounding cellular outgrowth (oz) was observed (tdTomato+ in red; Prox1+ in blue; single and merged channels shown). Co-staining was confirmed by z-stack reconstructions for each four high-resolution panel set (n, r, v, z); n = 6 explants analysed. Scale bars: a, e, 50 μm; bd and fh, 12.5 μm; i, 100 μm; j, 50 μm; m, q, u, y, 15 μm.

Extended Data Figure 7 Prox1 knockdown results in significantly decreased Vegfr3 and Lyve1 and Tie2-Cre;Prox1fl/fl mutant embryos exhibit superficial vascular defects whereas Vav1-Cre;Prox1fl/fl mutants have a normal systemic vasculature.

a, Prox1 targeting via floxed excision of exon 1 and 2, results in EGFP expression thus labelling targeted cells. b, E17.5 hearts from either Tie2-Cre;Prox1fl/+ control embryos or Tie2-Cre; Prox1fl/fl mutants were grouped and digested to create a single-cell suspension for FACS. A total of 100,000 GFP+ cells were collected for each sample group. ce, Relative gene expression was determined by qRT–PCR and revealed significantly decreased Prox1 (c; 0.59 fold), Vegfr3 (d; 0.39 fold) and Lyve1 (e; 0.22 fold) expression; n = 5 hearts per sample group, analysed in triplicate; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. All graphs are mean ± s.e.m. Statistical test used was Student’s t-test. fi, Dissection of Tie2-Cre;Prox1fl/+ heterozygous (f; n = 6) and Tie2-Cre;Prox1fl/fl mutant (g, h, i; n = 9) littermate embryos at E17.5 revealed gross vascular anomalies in the double-floxed mutants (three examples shown in gi), with evidence of ectopic surface blood vessels (g, ectopic vessels highlighted by black arrowheads), a disrupted vascular network (h; black arrowheads indicate blood-filled superficial vessels) and either haemorrhaging (i; bleeding foci highlighted by black arrowheads) or blood-filled lymphatics, compared to littermate Prox1fl/+ controls (f). jm, Vav1-Cre;Prox1fl/+ heterozygous (j, k; n = 5) and Vav1-Cre;Prox1fl/fl mutants (l, m; n = 8) revealed no obvious systemic vessel defects. Scale bars: g, i, m, 100 μm.

Extended Data Figure 8 The emergence of cardiac lymphatics at E14.5 is disrupted in Tie2-Cre;Prox1fl/fl mutant hearts, which are dysmorphic and exhibit elevated apoptosis of LECs, however, mutant embryos recover with normal cardiac lymphatics at post-natal stages.

ac, GFP+-targeted and Lyve-1+ LECs emerged from the base of the heart in the atrioventricular region at E14.5 in control Tie2-Cre;Prox1fl/+ hearts (LECs highlighted by white arrowheads in a and b; n = 3 hearts analysed). df, In mutant Tie2-Cre;Prox1fl/fl hearts (n = 7) the GFP+ network was absent (d) and Lyve-1 only detected tissue resident macrophages with an absence of lymphatics at the inflow base of the heart (arrows in e, f). g, h, Coronary vessels, as determined by whole mount CD31 staining, were comparable between control Tie2-Cre;Prox1fl/+ (g) and Tie2-Cre;Prox1fl/fl hearts (h). i, j, Haematoxylin and eosin staining of paraffin-embedded E17.5 hearts revealed that Tie2-Cre;Prox1fl/fl mutants (j; n = 3 analysed) were grossly smaller compared to control hearts (i), with lack of extension of the ventricles towards the apex, smaller chambers and thickening of the ventricular free wall (j). Normal membranous septation of the mutant ventricle (white asterix) and valve leaflet formation (white arrowhead in j) indicate normal endocardial cushion development. k, l, Whole-mount confocal imaging of hearts stained with GFP, cleaved caspase-3 and Prox1 revealed an increase in apoptotic cells within the termini of mutant coronary lymphatic vessels (white arrowheads in magnified panels), compared to control hearts, supporting the requirement for Prox1 in LEC identity and maintenance. n = 3 hearts analysed for histology and immunostaining. mp, Whole-mount VEGFR-3 immunostaining of hearts isolated at P7 revealed that Tie2-Cre;Prox1fl/+ heterozygotes (m, n) and Tie2-Cre;Prox1fl/fl mutants (o, p) have an equivalent normal cardiac lymphatic vasculature (n = 3 hearts analysed per genotype). As such the lymphatic hypoplasia and disruption of the vessel network, evident in mutant hearts at E17.5 (Extended Data Fig. 10), is rescued during the later stages of development and neonatal period. qx, In Vav1-Cre;Prox1fl/fl hearts (n = 4) there was evidence of an initial formation of the cardiac lymphatics on both ventral (q, s) and dorsal (u, w) surfaces and the coronary vessels were unaffected (r, t, v, x). Scale bars: f, h, x, 400 μm; j, 1 mm; k, 400 μm; l, 50 μm; m, 500 μm.

Extended Data Figure 9 Prox1 is essential for Tie2+- and Vav1+-derived cardiac lymphatics.

ad, In control Tie2-Cre;Prox1fl/+ mice at E17.5 (n = 6) there was an extensive lymphatic network on both the dorsal and ventral surfaces, as indicated by whole-mount VEGFR-3 immunostaining (a, b), whereas the lymphatic vessels were virtually absent in mutant Tie2-Cre;Prox1fl/fl hearts (c, d; n = 9); a few vessels evident on the dorsal surface was consistent with LECs arising from a non-Tie2-targeted source (c, white arrowheads). Tie2-Cre;Prox1fl/fl mutant hearts were dysmorphic relative to controls (compare c, d with a, b). e, f, GFP+ staining indicated targeting of Prox1 in Tie2-Cre;Prox1fl/+ mice (e) and an expansive Lyve-1+ lymphatic network (f). g, h, Prox1 immunostaining confirmed expression in LECs in heterozygote controls (g; inset box shown at higher magnification in h). Co-expression of GFP/Lyve-1 and Prox1 was evident in LECs in addition to Lyve-1/Prox1 double-positive cells not targeted by Tie2-Cre (h, white arrowheads). il, In contrast, Tie2-Cre;Prox1fl/fl mutant hearts revealed an absence of the GFP+ lymphatic network with only a minor contribution of Lyve-1 LECs evident at the base of the heart on the dorsal surface (i, j; white arrowhead in j highlights retained Lyve-1+ LECs), which were Prox1+ (k, l). mp, On the ventral surface there was complete absence of GFP and Lyve-1+ lymphatic vessels (m); Lyve-1 staining was retained in tissue-resident macrophages (n). Loss of LECs correlated with a loss of Prox1 (o, p). qt, In control Vav1-Cre;Prox1fl/+ mice at E17.5 (n = 5) there was evidence of appropriate targeting of GFP+ LECs (q) and an extensive lymphatic network on the dorsal surface as indicated by Lyve-1 (r). Prox1 expression was retained (s), which at higher resolution revealed co-expression of GFP+/Lyve-1+/Prox1+ in a subpopulation of LECs, consistent with the lineage trace data (Fig. 3a–d; white inset box in s shown at higher magnification in t). ux, In Vav1-Cre;Prox1fl/fl mutant hearts (n = 8) there was equivalent GFP+ targeting (u) and a Lyve-1+ network (v) with retained Prox1 expression (w, x). y, z, Specific loss of Lyve-1 staining (y) correlated with loss of Prox1 and GFP-targeting (z, left and right panels, respectively, highlighted by white arrowheads). Mosaic levels of Prox1 knockdown accounted for examples of isolated LECs that, despite GFP-targeting, remained Lyve-1+ (white arrows in y, z). Scale bars: d, g, l, p, s, x, 400 μm; h, t, 40 μm; y, 5 μm.

Extended Data Figure 10 Prox1 knockdown in Tie2-Cre;Prox1fl/fl mutants results in a hypoplastic and disrupted lymphatic plexus.

af, Relative to Tie2-Cre;Prox1fl/+ control hearts at E17.5 (ac), GFP+ lymphatic vessels were thinner and the network truncated along the short axis, having failed to appropriately extend and remodel in Tie2-Cre;Prox1fl/fl hearts with partial knockdown of Prox1 (df; see Extended Data Fig. 7c; n = 4 hearts per genotype; representative regions indicated by white inset boxes in a, d). gl, Higher magnification of the lymphatic plexus in Tie2-Cre;Prox1fl/+ control (gi) and Tie2-Cre;Prox1fl/fl mutants (jl) were captured for AngioTool analyses. AngioTool tracing in red of GFP+ vessels and blue for branch points (gl), enabled quantitative assessment of vessel parameters. mo, The mutant lymphatic vessels were significantly shorter in overall length (m), more truncated and disorganized with an increased total number of end points (n). Mutant vessels were also significantly reduced in diameter, being thinner on average, compared to controls (o). Scale bars: c, f, 400 μm; l, 30 mm; All graphs show mean ± s.e.m. Student’s t-test; *P ≤ 0.05; **P ≤ 0.001 (n = 4 hearts analysed per genotype).

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Klotz, L., Norman, S., Vieira, J. et al. Cardiac lymphatics are heterogeneous in origin and respond to injury. Nature 522, 62–67 (2015). https://doi.org/10.1038/nature14483

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