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Lymphoangiocrine signals promote cardiac growth and repair

Abstract

Recent studies have suggested that lymphatics help to restore heart function after cardiac injury1,2,3,4,5,6. Here we report that lymphatics promote cardiac growth, repair and cardioprotection in mice. We show that a lymphoangiocrine signal produced by lymphatic endothelial cells (LECs) controls the proliferation and survival of cardiomyocytes during heart development, improves neonatal cardiac regeneration and is cardioprotective after myocardial infarction. Embryos that lack LECs develop smaller hearts as a consequence of reduced cardiomyocyte proliferation and increased cardiomyocyte apoptosis. Culturing primary mouse cardiomyocytes in LEC-conditioned medium increases cardiomyocyte proliferation and survival, which indicates that LECs produce lymphoangiocrine signals that control cardiomyocyte homeostasis. Characterization of the LEC secretome identified the extracellular protein reelin (RELN) as a key component of this process. Moreover, we report that LEC-specific Reln-null mouse embryos develop smaller hearts, that RELN is required for efficient heart repair and function after neonatal myocardial infarction, and that cardiac delivery of RELN using collagen patches improves heart function in adult mice after myocardial infarction by a cardioprotective effect. These results highlight a lymphoangiocrine role of LECs during cardiac development and injury response, and identify RELN as an important mediator of this function.

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Fig. 1: Lymphatics are required for embryonic heart growth.
Fig. 2: Lymphatics are required for CM proliferation and survival.
Fig. 3: LEC-secreted RELN promotes CM proliferation and survival.
Fig. 4: RELN improves neonatal and adult cardiac function after myocardial infarction.

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Data availability

All data from the manuscript are available from the corresponding author on request. RNA-seq raw data have been deposited to the Gene Expression Omnibus (GEO) repository with accession number GSE158504Source data are provided with this paper.

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Acknowledgements

This work was supported by NIH grant (RO1HL073402-16) to G.O., AHA grant (18CDA34110356) to X.L., 5T32HL134633 to W.M., FPU grant from the Spanish Ministry of Education, Culture and Sports and EMBO Short-Term Fellowship to E.D.C., Leducq TNE-17CVD and RD16/0011/0019 (ISCIII) from the Spanish Ministry of Science, Innovation, and Universities to M.T., NIH T32 GM008061 to C.L., HL63762, and NS093382 to J.H. We thank G. M. Rune and B. Brunne for the Reln+/− strain. RNA-seq work was supported by the Northwestern University NUSeq Core Facility. We thank the Robert H. Lurie Cancer Center Flow Cytometry facility supported by NCI CCSG P30 CA060553 for their invaluable assistance. Flow Cytometry Cell Sorting was performed on a BD FACSAria SORP system and BD FACSymphony S6 SORP system, purchased through the support of NIH 1S10OD011996-01 and 1S10OD026814-01. Imaging work was performed at the Northwestern University Center for Advanced Microscopy supported by NCI CCSG P30 CA060553 awarded to the Robert H Lurie Comprehensive Cancer Center. Spinning disk confocal microscopy was performed on an Andor XDI Revolution microscope, purchased through the support of NCRR 1S10 RR031680-01. Proteomics services were performed by the Northwestern Proteomics Core Facility supported by NCI CCSG P30 CA060553 awarded to the Robert H Lurie Comprehensive Cancer Center, instrumentation award (S10OD025194) from NIH Office of Director, and the National Resource for Translational and Developmental Proteomics supported by P41 GM108569. We thank the George M. O’Brien Kidney Research Core Center (NU GoKidney, supported by a P30 DK114857 award from NIDDK) for the use of the Echocardiography machine. The myocardial infarction surgeries were performed by the comprehensive Transplant Center Microsurgery Core, partially supported by NIH NIAID P01AI112522. We thank J. Jin and P. Liu for help with the ELISA reagents and data analysis, R. Ma for help with DNA polyploidy analysis, A. Shi for the MEF2C antibodies, M. Dellinger for the Prox1-creERT2 mice and H. Ardehali for the Myh6-cre mice. We specially thank B. Sosa-Pineda for advice and suggestions and P. Ruiz-Lozano for sharing her expertise in the preparation of the collagen patches.

Author information

Authors and Affiliations

Authors

Contributions

X.L. and G.O. designed the experiments and analysed the data. X.L. performed most of the experiments and data analysis. E.D.C. performed the neonate myocardial infarction and acquired data. T.T. and J.H. provided the Reln conditional mouse strain and generated some of the conditional crosses. X.G. helped with the generation, isolation and data analysis of Reln conditional embryos. C.L. and E.T. provided valuable advice with the neonate myocardial infarction and Echo data protocols. Z.J. and L.B. generated the Vegfr3kd/kd embryos and analysed that data. M.O.-B. and W.M. helped with the generation of mouse lines, histology and discussions. H.K. and P.B. generated the iPS cell-derived CMs. T.B. helped with the primary cell culture experiments and qPCR analysis. O.C. helped to obtain and generate some of the mutant strains. M.T. provided valuable experimental advice and critical reading of the manuscript. X.L. and G.O. wrote the manuscript.

Corresponding author

Correspondence to Guillermo Oliver.

Ethics declarations

Competing interests

J.H. is a shareholder of Reelin Therapeutics and a co-inventor on a pending US patent application filed by his institution (UT Southwestern; application number 15/763,047 and publication number 20180273637, title “Methods and Compositions for Treatment of Atherosclerosis”; Inventors: J.H., Y. Ding, X. Xian, L. Huang, C. Mineo, P. Shaul and L. Calvier). This patent application covers no aspects of the current manuscript. Findings regarding the potential applications and methods for using RELN to treat cardiac diseases are the subject of provisional patent application (US63/091,558) owned by Northwestern University and list X.L. and G.O. as inventors. All other authors declare no competing interests.

Additional information

Peer review information Nature thanks Kristy Red-Horse and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 E17.5 Prox1ΔLEC/ΔLEC hearts lack LECs and have a reduced number of CMs.

a, Whole-mount immunostaining with anti-PROX1 antibody shows that cardiac lymphatics are missing in E17.5 Prox1ΔLEC/ΔLEC hearts (TAM injected at E13.5 and E14.5). Squared areas are shown in larger magnification in the adjacent images. n = 3 embryos per group from two litters. b, Co-immunostaining of E17.5 control and Prox1ΔLEC/ΔLEC heart sections with anti-α-actinin and F-actin antibodies show that cardiac muscle is not affected in Prox1ΔLEC/ΔLEC embryos (TAM injected at E13.5 and E14.5). n = 3 per group. c, Flow cytometry analysis shows reduced CM numbers in E17.5 Prox1ΔLEC/ΔLEC hearts (TAM injected at E13.5 and E14.5). d, Hoechst 33342 labelling shows no significant differences in CMs ploidy between control and Prox1ΔLEC/ΔLEC hearts. n = 3 (control) and n = 4 (Prox1ΔLEC/ΔLEC) embryos from the same litter used in c and d. Data are mean ± s.e.m. **P = 0.001, unpaired two-tailed Student’s t-test. n.s., not significant. eg, The percentage of multinucleated CMs in E17.5 mutant hearts is increased (e, f), and no global differences in CM size were detected (e, g) after CM dissociation and overnight plating. n = 3 embryo per genotype from the same litter. White arrows indicate CMs and yellow arrows indicate a bi-nucleated CM (g). The average cell size was calculated from 25 cTnC+ CMs per culture (1 whole heart per culture; 3 cultures per genotype). n = 75 (control CMs) and n = 75 (Prox1ΔLEC/ΔLEC CMs). Data are mean ± s.e.m. *P = 0.023, unpaired two-tailed Student’s t-test. Scale bars, 500 μm (a), 25 μm (b, e). Flow cytometry gating strategy is included in Supplementary Fig. 10.

Source Data

Extended Data Fig. 2 CM proliferation is reduced in E17.5 Prox1ΔLEC/ΔLEC hearts.

a, EdU labelling shows an overall reduction in the number of EdU+ cells in sections of E17.5 Prox1ΔLEC/ΔLEC hearts. Dashed boxes indicate the corresponding areas of the heart that are shown at higher magnification in be. be, Immunostaining results show the presence of PROX1+LYVE1+ cardiac lymphatics (white arrows) in sections of control hearts (b, c), and lack of lymphatics in Prox1ΔLEC/ΔLEC hearts (d, e). Yellow arrows indicate LYVE1+ PROX1 macrophages. n = 3 embryos per genotype from three separate litters. f, CM proliferation is reduced in the myocardium of the left ventricle (LV) area, the right ventricle (RV) area and the septum. n = 4 embryos per genotype from three separate litters. At least three images per region and three separate regions per heart were quantified. Data are mean ± s.e.m. *P = 0.01, **P = 0.003, 0.006 and 0.02 (top); ***P = 0.0001, **P = 0.004, 0.002 and 0.005 (middle); **P = 0.001, 0.001, *P = 0.01 and **P = 0.002 (bottom), unpaired two-tailed Student’s t-test. g, Immunostaining with antibodies against vimentin (fibroblasts), PECAM1 (blood endothelial cells), CD68 (macrophages), SIX2 (nephron progenitors) or HNF4A (hepatocytes) together with EdU labelling (white arrows) shows no differences in proliferation in those cell types between E17.5 Prox1ΔLEC/ΔLEC and control hearts (TAM injected at E13.5 and E14.5). P values by unpaired two-tailed Student’s t-test. n = 3 embryos per genotype from three separate litters. Control are TAM-treated cre embryos and cre+;Prox1+/+ littermates. Data are mean ± s.e.m. Scale bars, 200 μm (a), 100 μm (be), 25 μm (g).

Source Data

Extended Data Fig. 3 Vegfr3kd/kd embryos lack cardiac lymphatics and have smaller hearts.

a, Bright-field images of whole E17.5 Vegfr3kd/kd and wild-type embryos and hearts. Quantification of organ weight (heart, liver and kidney) relative to body length indicates that the heart is smaller and the liver and kidney have comparable sizes between Vegfr3kd/kd and control embryos. n = 10 (WT) and n = 8 (Vegfr3kd/kd). Embryos are from three different litters. *P = 0.019. b, LYVE1 whole-mount immunostaining shows that ventral and dorsal sides of the heart lack lymphatics in Vegfr3kd/kd embryos. n = 3 per genotype. cf, Co-immunostaining using antibodies against cell proliferation markers (EdU, pH3, Ki67 and auroraB) and antibodies against CM markers (cTnC, PROX1, α-actinin and/or MEF2C) shows reduced CM proliferation in Vegfr3kd/kd hearts compared to wild-type hearts at E17.5. Arrows indicate representative proliferating CMs. g, Quantification shows significantly reduced percentage of EdU+ and Ki67+ CMs and significantly reduce number of pH3+ and auroraB+ CMs in Vegfr3kd/kd hearts compared to controls. n = 4 embryos per genotype from three separate litters. **P = 0.005 (EdU), **P = 0.001 (Ki67, pH3), *P = 0.02 (auroraB). h, Active CASP3+ immunostaining shows increased CM apoptosis (white arrows) in Vegfr3kd/kd hearts compared to wild-type hearts at E17.5. Right, quantitative data showing significantly increased percentage of active caspase-3+ CMs (PROX1+) in Vegfr3kd/kd hearts compared to wild-types. n = 4 embryos per genotype from three separate litters. *P = 0.032. i, Co-immunostaining with antibodies against vimentin, PECAM1, CD68, SIX2 and HNF4A, together with EdU labelling shows comparable proliferation of cardiac fibroblasts, blood endothelial cells and macrophages, and of nephron progenitors and hepatocytes between wild-type and Vegfr3kd/kd embryos at E17.5. White arrows indicate EdU+ proliferating cells. Right, quantification of the proliferation for each cell type. n = 3 embryos per genotype from three separate litters. Data are mean ± s.e.m. P values were determined by unpaired two-tailed Student’s t-test. Scale bars, 1 mm (a), 500 μm (b), 25 μm (cf, h), 25 μm (i). Lower magnification images for ce and h are included in Supplementary Fig. 4.

Source Data

Extended Data Fig. 4 Heart size and CM proliferation is normal in E17.5 Prox1ΔLEC/+ embryos and E14.5 Prox1ΔLEC/ΔLEC embryos.

a, Bright-field images of whole embryos and hearts show no difference in heart size in E17.5 Prox1ΔLEC/+ embryos (TAM injected at E13.5 and E14.5). White arrows indicate oedema in the Prox1ΔLEC/+ embryo. b, Whole-mount immunostaining shows that LYVE1+ cardiac lymphatics are present in both dorsal and ventral sides of Prox1ΔLEC/+ hearts. Lymphatics are less branched (arrows). c, Cardiac lymphatic density is significantly reduced on the ventral surface of the heart but not on the dorsal one in Prox1ΔLEC/+ embryos. This difference may be because cardiac lymphatics on the dorsal side and the ventral side originate from two different lineages during embryonic development. n = 3 embryos per genotype from three separate litters. *P = 0.027. d, Heart size is normal in E17.5 Prox1ΔLEC/+ embryos. n = 13 (controls) and n = 9 (Prox1ΔLEC/+) embryos from three separate litters. e, Quantification of the immunostaining analysis shows no significant differences in CM proliferation between E17.5 Prox1ΔLEC/+ hearts and controls, as indicated by the percentage of EdU+ and Ki67+ CMs and the number of pH3+ and auroraB+ CMs. n = 4 embryos per genotype from three separate litters. Controls are TAM-treated cre embryos and cre+;Prox1+/+ littermates. f, Bright field images of whole embryos and hearts show no difference in cardiac size between E14.5 wild-type and Prox1ΔLEC/ΔLEC embryos (TAM injected at E10.5 and E11.5). White arrows indicate severe oedema. n = 6 embryos per genotype from two separate litters. Control embryos are TAM-treated cre embryos and cre+;Prox1+/+ littermates. g, Whole-mount staining of skin shows efficient PROX1 deletion as indicated by the lack of PROX1+ or NRP2+ lymphatics at E14.5 in Prox1ΔLEC/ΔLEC embryos. n = 3 embryos per genotype from same litter. h, Co-immunostaining against cell proliferation markers (EdU, Ki67, pH3 and auroraB) together with CM markers (cTnC, PROX1, α-actinin and/or MEF2C). Quantification of those immunostainings shows no differences in CM proliferation between wild-type and Prox1ΔLEC/ΔLEC hearts at E14.5. Squares indicate proliferating CMs. n = 3 embryos per genotype from the same litter. Data are mean ± s.e.m. P values determined by unpaired two-tailed Student’s t-test. Scale bars, 1 mm (a, f), 500 μm (b), 25 μm (g, h).

Source Data

Extended Data Fig. 5 Pathways related to cell cycle are downregulated in E17.5 Prox1ΔLEC/ΔLEC embryos and LEC-conditioned medium promotes CM proliferation and survival in vitro.

a, Gene set expression analysis shows downregulation of cell cycle pathways and upregulation of cell death pathways in Prox1ΔLEC/ΔLEC hearts. n = 4 per genotype from the same litter. b, qPCR analysis confirmed the upregulation of pro-apoptotic genes (Bcl2l11, Pdcd4, Trp53inp1, Stat1 and P21 (also known as Cdkn1a)) and downregulation of cell cycle related genes (Cdc6, E2f1, Pcna, Mcm5 and Ccne2) in Prox1ΔLEC/ΔLEC hearts. n = 3 per genotype from the same litter. TAM was injected at E13.5 and E14.5. Control embryos are TAM-treated cre embryos and cre+;Prox1+/+ littermates. *P = 0.02 (Bcl2l11), **P = 0.001 (Pdcd4), 0.005 (Trp53inp1), *P = 0.01 (Stat1), 0.03 (P21), 0.04 (Cdc6), 0.02 (E2f1), 0.01 (Pcna), 0.02 (Mcm5) and 0.03 (Ccne2). c, Co-immunostaining against the proliferation marker Ki67 and the CM markers α-actinin and PROX1 shows that LEC-conditioned medium increases primary CM proliferation. Arrows indicate proliferating CMs. Percentage of CM proliferation was quantified by the number of Ki67+ PROX1+ CMs relative to total number of PROX1+ CMs. n = 3. **P = 0.001. d, Co-immunostaining against the apoptotic marker active CASP3+ and the CM markers α-actinin and PROX1 shows reduced primary CM apoptosis upon LEC-conditioned medium treatment under CoCl2-induced hypoxia. Arrows indicate apoptotic CMs. Percentage of apoptotic CMs was quantified by the number of active CASP3+ CMs relative to PROX1+ CMs. n = 3. **P = 0.003. Data are mean ± s.e.m. P values were determined by unpaired two-tailed Student’s t-test Scale bar, 25 μm (c, d).

Source Data

Extended Data Fig. 6 E17.5 Reln−/− embryos develop smaller hearts.

a, qPCR analysis shows reduced Reln expression in E17.5 Prox1ΔLEC/ΔLEC hearts (TAM injected at E13.5 and E14.5). n = 3 embryos per genotype from the same litter. Control embryos are TAM treated cre embryos and cre+;Prox1+/+littermates. *P = 0.014. b, qPCR analysis validates the expression of candidates from the LECs secretome (Serpine1, Fn1, Reln, Hspg2, Mmrn1, Lama4, Fstl1 and Thbs1). Experiments were repeated three times using different batches of LECs. Gene expression is normalized as a fold change relative to 100× Gapdh. c, RELN protein can be detected in three different batches of LEC-conditioned medium and the relative RELN level is quantified by ELISA according to the absorbance value at 450 nm (A450 nm). d, e, Immunostaining of sections of E17.5 wild-type hearts shows RELN is highly expressed in cardiac lymphatics of the epicardium and myocardium. Some blood vessels in the heart express low levels of RELN (e, arrows). n = 3 wild-type embryos. f, Immunostaining of E17.5 control and Prox1ΔLEC/ΔLEC heart sections with antibodies against RELN and LYVE1 shows that cardiac lymphatics and RELN are absent in Prox1ΔLEC/ΔLEC hearts (TAM injected at E13.5 and E14.5). n = 3 embryos per genotype from the same litter. Control embryos are TAM-treated littermate cre and cre+; Prox1+/+embryos. g, Representative bright-field images show smaller hearts in E17.5 Reln−/− embryos. h, Quantifications of organ weight (heart, liver and kidney) relative to body length indicate that hearts are smaller in E17.5 Reln−/− embryos compared to controls. n = 7 (WT) and n = 6 (Reln−/−) embryos from three separate litters. *P = 0.03. i, Whole-mount immunostaining shows that cardiac lymphatic development is normal in Reln−/− embryos. n = 3 embryos per genotype from two separate litters. Data are mean ± s.e.m. P values were determined by unpaired two-tailed Student’s t-test. Scale bars, 25 μm (df), 1 mm (g), 500 μm (i).

Source Data

Extended Data Fig. 7 RELN is efficiently deleted in RelnΔLEC/ΔLEC cardiac-associated lymphatics.

a, Immunostaining of E17.5 control and RelnΔLEC/ΔLEC heart sections with antibodies against RELN and LYVE1 confirms that RELN is deleted from cardiac lymphatics in RelnΔLEC/ΔLEC hearts (TAM injected at E13.5 and E14.5). n = 3 embryos per genotype from two separate litters. Control embryos are TAM-treated cre embryos and cre+;Reln+/+embryos. b, Co-immunostaining with antibodies against vimentin, PECAM1, CD68, SIX2 and HNF4Α, together with EdU labelling shows comparable proliferation of cardiac fibroblasts, blood endothelial cells and macrophages, and of nephron progenitors and hepatocytes between controls and E17.5 RelnΔLEC/ΔLEC hearts (TAM injected at E13.5 and E14.5). White arrows indicate EdU+ proliferating cells. Right, quantification of the proliferation for each cell type. n = 3 embryos per genotype from two separate litters. Control embryos are TAM-treated cre and cre+;Reln+/+ littermates. Data are mean ± s.e.m. P values determined by unpaired two-tailed Student’s t-test. Scale bar, 25 μm.

Source Data

Extended Data Fig. 8 Cardiac size is reduced in E17.5 β1ΔCM/+;Reln+/− embryos.

a, qPCR analysis shows efficient Reln knockdown in LECs after siRNA treatment. n = 3. Data are mean ± s.e.m. *P < 0.05, unpaired two-tailed Student’s t-test. b, Representative western blot of primary CMs cultured with DMEM, conditioned medium from CMs treated with short interfering RNA (siRNA) against Reln (siReln) or control siRNA (siCtrl), or with conditioned medium plus integrin-β1 blocking antibody overnight. The addition of the LEC-conditioned medium (siCtrl group) to primary CMs increased DAB1, FAK, AKT and ERK activities. These activities are reduced when cultured CMs are treated with RELN-deficient LEC-conditioned medium or with LEC-conditioned medium with integrin-β1 blocking antibody. Experiments were repeated three times. Data are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, two-way ANOVA followed by Bonferroni test. c, Ki67 quantification of immunostained cultured cells (similar to Extended Data Fig. 5c) shows that addition of the LEC-conditioned medium (siCtrl group) to cultured primary CMs improves CM proliferation and this effect is partially abolished in CMs treated with Reln-deficient (siReln) LEC-conditioned medium or with LEC-conditioned medium containing integrin-β1 blocking antibody. Percentage of CM proliferation was quantified by the number of Ki67+ PROX1+ CMs relative to total numbers of PROX1+ CMs. n = 3. Data are mean ± s.e.m. **P < 0.01, two-way ANOVA followed by Bonferroni test. d, Quantification of cultured CMs immunostained with active CASP3+ shows that the addition of the LEC-conditioned medium (siCtrl group) to primary CMs protects them from apoptosis and this effect is partially abolished in CMs treated with Reln-deficient LEC-conditioned medium or with LEC-conditioned medium with integrin-β1 blocking antibodies. Percentage of apoptotic CMs was quantified by the number of active CASP3+ CMs relative to PROX1+ CMs. n = 3. Data are mean ± s.e.m. *P < 0.05, **P < 0.01, two-way ANOVA followed by Bonferroni test. e, Representative western blot of primary CMs after treatment with RELN-conditioned medium from RELN- transfected cells, or conditioned medium from mock-transfected cells (control) or RELN-conditioned medium with integrin-β1 blocking antibody (ab) shows that RELN treatment increases DAB1, FAK, AKT and ERK activities in primary CMs, and these activities are reduced by adding the integrin-β1 blocking antibody. n = 3. Data are mean ± s.e.m. *P < 0.05; **P < 0.01 by one-way ANOVA followed by Tukey’s test. f, Bright-field images show no difference in embryo size at E17.5 among control, Reln+/−, β1ΔCM/+ and β1ΔCM/+;Reln+/− embryos. Quantification of organ weight (heart, liver and kidney) relative to body length indicates that hearts are smaller in E17.5 β1ΔCM/+;Reln+/− embryos. n = 9 (control), n = 7 (Reln+/−), n = 6 (β1ΔCM/+) and n = 6 (β1ΔCM/+;Reln+/−) embryos from three separate litters. Data are mean ± s.e.m. *P = 0.015, one-way ANOVA followed by Tukey’s test. g, Whole-mount immunostaining using LYVE1 antibodies shows normal cardiac lymphatic development in control, β1ΔCM/+, β1ΔCM/+;Reln+/− and Reln+/− embryos. n = 3 embryos per genotype from three separate litters. Scale bars, 1 mm (f), 500 μm (g). For western blot source data, see Supplementary Figs. 8 and 9. Exact P values included in Source Data.

Source Data

Extended Data Fig. 9 RELN promotes CM proliferation and survival through Itgb1 signalling.

a, Co-immunostaining using cell proliferation markers (EdU, Ki67, pH3 and auroraB) together with CM markers (cTnC, PROX1, α-actinin and/or MEF2C) shows reduced CM proliferation in β1ΔCM/+;Reln+/− hearts at E17.5. Arrows indicate proliferating CMs. Quantification in the bottom panel shows reduced proliferation in E17.5 β1ΔCM/+;Reln+/− hearts, as indicated by the percentage of EdU+ and Ki67+ CMs and the number of pH3+ and auroraB+ CMs. n = 4 embryos per genotype from three separate litters. *P = 0.022 (EdU), 0.029 (Ki67), ***P = 0.0001 (pH3) and *P = 0.033 (auroraB). b, Active CASP3+immunostaining shows increased CM apoptosis in β1ΔCM/+;Reln+/− hearts at E17.5, as quantified by the percentage of active CASP3+ CMs relative to PROX1+ CMs. Arrows indicate apoptotic CMs. n = 4 embryos per genotype from three separate litters. Control embryos are cre embryos and cre+;β1+/+ littermates. *P = 0.01. c, Co-immunostaining with antibodies against vimentin, PECAM1, CD68, SIX2 and HNF4A, together with EdU labelling shows comparable proliferation of cardiac fibroblasts, blood endothelial cells and macrophages, and of nephron progenitors and hepatocytes between controls and E17.5 β1ΔCM/+;Reln+/− embryos. White arrows indicate EdU+ proliferating cells. Right, quantification of the proliferation analysis for each of cell type. n = 3 embryos per genotype from three separate litters. Control are cre embryos and cre+;β1+/+ littermates. Data are mean ± s.e.m. P values determined by unpaired two-tailed Student’s t-test. Scale bars, 25 μm. Lower magnification images for a and b are included in Supplementary Fig. 5.

Source Data

Extended Data Fig. 10 RELN expression is developmentally downregulated, but is upregulated in newly formed cardiac lymphatics after myocardial infarction.

a, Immunostaining with RELN, PROX1 and PECAM shows RELN is highly expressed in cardiac lymphatics in the epicardium and myocardium nearby the base of the heart at E17.5. RELN expression level is gradually downregulated during development from P2 to P14. n = 3 hearts per stage. Arrows indicate PROX1+ cardiac lymphatics. b, qPCR analysis using sorted cardiac lymphatics shows Reln levels are drastically downregulated in cardiac LECs during development. n = 3. Reln relative level from each experiment is presented as fold changes relative to E17.5. Data are mean ± s.e.m. **P = 0.009 (P2 versus E17.5), 0.004 (P7 versus E17.5), 0.001 (P14 versus E17.5) by one-way ANOVA followed by Tukey’s test. c, Immunostaining shows RELN expression is highly upregulated in the newly formed cardiac lymphatics in wild-type P7 pups (myocardial infarction was performed at P2). Notably, the pre-existing cardiac lymphatics in the non-infarcted area express low levels of RELN. Reln−/− hearts completely lack RELN expression in both, newly formed cardiac lymphatics and pre-existing lymphatics. Arrows indicate cardiac lymphatics. n = 3 hearts per group. d, Immunostaining against the pan-endothelial marker PECAM1 and the lymphatic marker LYVE1 shows normal lymphangiogenesis in wild-type and Reln−/− hearts 21 days after myocardial infarction (myocardial infarction performed at P2). n = 3 hearts per group. Data are mean ± s.e.m. P values determined by unpaired two-tailed Student’s t-test. e, EdU labelling shows no differences in LECs proliferation in wild-type and Reln−/− hearts 21 days after myocardial infarction (myocardial infarction performed at P2). n = 3 hearts per group. Data are mean ± s.e.m. P values determined by unpaired two-tailed Student’s t-test. Arrow indicates EdU+ LECs. Scale bars, 100 μm (d), 25 μm (a, c, e).

Source Data

Extended Data Fig. 11 RELN improves cardioprotection in neonates and adult mice after myocardial infarction.

ad, Co-immunostaining using cell proliferation markers (EdU, Ki67, pH3 and auroraB) together with the CM markers PROX1, α-actinin or MEF2C shows decreased CM proliferation in the border of the infarcted area of Reln−/− hearts at P7. Arrows indicate proliferating CMs.  n = 4 mice per group. e, Immunostaining using active CASP3+ shows increased CM apoptosis in the infarcted area of Reln−/− hearts at P7. Arrows indicate apoptotic CMs in the section. n = 4 mice per group. f, Immunostaining against the cell proliferation markers EdU, Ki67 and pH3 together with the CM markers MEF2C or cTnC shows no differences in CM proliferation in the infarcted areas between control patch or RELN patch treated hearts 7 days after myocardial infarction. Arrows indicate proliferating CMs. n = 4 hearts per group. g, Immunostaining using active caspase-3 shows reduced CM apoptosis in the infarcted area of RELN patch-treated hearts. Arrows indicate apoptotic CMs. n = 4 mice per group. Arrows indicate apoptotic CMs. Scale bars, 25 μm. Lower magnification for ac, e and g are included in Supplementary Fig. 3.

Supplementary information

Supplementary Figures 1-10

This file contains Supplementary Figures 1-5 showing lower magnification images for CM proliferation and apoptosis; Supplementary Figures 6-9 showing source data for Western blots; and Supplementary Figure 10 showing gating strategy for flow cytometry.

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Liu, X., De la Cruz, E., Gu, X. et al. Lymphoangiocrine signals promote cardiac growth and repair. Nature 588, 705–711 (2020). https://doi.org/10.1038/s41586-020-2998-x

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