Abstract
Myocardial infarction (MI) is a leading cause of death worldwide, largely because efficient interventions to restore cardiac function after MI are currently lacking. Here, we characterize vascular aberrancies induced by MI and propose to target acquired endothelial cell (EC) changes to normalize vessels and promote cardiac repair after MI. Single-cell transcriptome analyses of MI-associated ECs indicates that ECs acquire mesenchymal gene signatures that result in phenotypic and functional changes and lead to vessel abnormalities. We identify a platelet-derived growth factor (PDGF)–nuclear factor κB (NF-κB)–hypoxia-inducible factor 1-α (HIF-1α) axis that induces Snail expression and mesenchymal phenotypes in ECs under hypoxia, altogether causing aberrant vascularization. EC-specific knockout of platelet-derived growth factor receptor beta (PDGFR-β), pharmacological PDGFR inhibition or nanoparticle-based targeted PDGFR-β small interfering RNA delivery in mice attenuates vascular abnormalities in the infarcted tissue and improves cardiac repair after MI. These findings illustrate a mechanism controlling aberrant neovascularization after ischemia and suggest that targeting PDGF/Snail-mediated endothelial plasticity may offer opportunities for normalizing vasculature and treating ischemic heart diseases.
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Data availability
The RNA-seq data have been deposited in the National Center for Biotechnology Information’s Gene Expression Omnibus under accession no. GSE163772, while the single-cell and single-nuclei RNA-seq analysis of mouse cardiac tissues with MI have been deposited in the same publicly available database under the accession nos. GSE163956 and GSE193290, respectively. The data supporting the findings of this study are available within the paper and its supplementary information. Source data are provided with this paper.
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Acknowledgements
We thank J. Eberwine (Penn Pharmacology), J. Schug (Penn Next-Generation Sequencing Core), A. Stout (Penn CDB Microscope Core) and E. Blankemeyer (Penn Small Animal Imaging Core) for assistance with RNA linear amplification, scRNA-seq, light sheet fluorescence imaging and SPECT analysis, respectively. This work was supported in part by an American Heart Association (AHA) Innovative Project Award no. IPA34170252 (to Y.F.), National Institutes of Health (NIH) grant nos. R01HL155198 (to Y.F. and Y.G.) and R01NS094533, R01NS106108 and R01CA241501 (to Y.F.), American Association for Cancer Research (AACR) Judah Folkman Award (to Y.F.), AHA Scientist Development grant no. SDG9050018 and grant-in-Aid no. GRNT3365002 (to Y.G.) and AHA Predoctoral Fellowship (to D.Z.). L.P. is supported by the Office of the Assistant Secretary of Defense for Health Affairs through the Peer Reviewed Medical Research Program (nos. W81XWH2010042 and W81XWH2010089). M.J.M. acknowledges support from a Burroughs Wellcome Fund Career Award at the Scientific Interface, an NIH Director’s New Innovator Award (no. DP2 TR002776), NIH grant nos. R01CA241661, R37CA244911 and R01DK123049, an Abramson Cancer Center-School of Engineering and Applied Sciences Discovery Grant (no. P30CA016520) and an AACR-Bayer Innovation and Discovery Grant (no. 18-80-44-MITC).
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M.H., F.Y., D.Z. and M.L. designed, performed and analyzed the experiments. D.Z., L.Z. and L.Q. contributed to the scRNA-seq studies. M.L. and S.V.S. contributed to the echocardiogram analysis. H.D. helped tissue histology analysis. R.E. and M.J.M. contributed to nanoparticle production. L.P. helped with the single-nucleus analysis. D.J.R. contributed to the experimental design and discussion. Y.G. and M.L. performed the MI surgery. Y.F. and Y.G. cosupervised the experiments and wrote the manuscript. All authors commented on the manuscript.
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Extended Data Fig. 1 Aberrant vascularization characterizes infarcted cardiac tissue.
Mice were subjected to MI surgery or sham operation. Hearts were excised 3 weeks after surgery. Tissue sections were immunostained with (a) anti-CD31 antibody, (b) anti-NG2 and anti-CD31 antibodies, or (c) anti-collagen IV and anti-CD31 antibodies. a, Representative images in normal tissue and infarct zone are shown (n = 3 mice). Bars indicate 100 µm. b,c, Left, representative images in normal tissue and infarct zone are shown. Right, quantified results (mean ± SEM, n = 3 mice). Statistical analysis by unpaired two-tailed Students’ t test. Bars indicate 100 µm.
Extended Data Fig. 2 Cardiomyocyte-conditioned medium induces FSP-1 and α-SMA expression in ECs.
Mouse cardiac-conditioned medium (CCM) were harvested from primary mouse cardiomyocytes cultured under hypoxia (1% O2). Mouse cardiac microvascular ECs were treated with CCM under normoxia or hypoxia for 2 days. Cell lysates were immunoblotted. These experiments were repeated independently twice with similar results.
Extended Data Fig. 3 Expression of mesenchymal genes in MI-associated ECs.
MI was induced in wild-type mice. Three weeks after MI induction, ECs were isolated from normal LV and MI tissues. RNA was extracted and subjected to RT-PCR analysis (mean ± SEM, n = 5-7 mice, specific n indicated in the graphs). Statistical analysis by unpaired two-tailed Students’ t test. Expression of (a) S100A4 (FSP-1) and Acta2 (α-SMA), and (b) Snai1 (Snail), Snai2 (Slug), and Twist1 was normalized with GAPDH expression levels.
Extended Data Fig. 4 PDGF-AB induces expression of mesenchymal proteins in ECs.
Human cardiac microvascular ECs were treated with 100 ng/ml SCF, PDGF-AB, or HGF for 2 days under hypoxia. Cell lysates were immunoblotted. These experiments were repeated independently twice with similar results.
Extended Data Fig. 5 PDGFR-β knockout inhibits PDGF-AB-induced phosphorylation of Erk1 and Akt1 in ECs and does not affect vascular density in normal hearts.
a, Aortic ECs were isolated from tamoxifen-treated Cdh5-CreERT2;Pdgfrbfl/fl (PDGFR-β-ΔEC) and Pdgfrbfl/fl (control) mice. Cells were treated with 100 ng/ml PDGF-AB for 10 min, followed by immunoblot analysis. These experiments were repeated independently twice with similar results. b,Left ventricles were collected from PDGFR-β-ΔEC and control mice. Cardiac sections were stained with anti-CD31 antibody and analyzed by immunofluorescence. Left, representative images are shown. Scale bar: 200 μm. Right, quantified results (mean ± SEM, n = 6 mice). Statistical analysis by unpaired two-tailed Students’ t test.
Extended Data Fig. 6 PDGFR-β knockout in ECs does not increase vascular density in infarct zone and border zone.
MI was induced in control and PDGFR-β-ΔEC mice. (a,b) 4 or (c) 8 weeks after MI induction, cardiac tissues were collected. Sections were stained with anti-collagen I and anti-CD31 antibodies, followed by immunofluorescence analysis. a, Representative images are shown (n = 6 mice). Scale bar: 100 μm. b,c, Vascular density was quantified (mean ± SEM). b, n = 3-4 mice, specific n indicated in the graphs. c, n = 6 mice. Statistical analysis by two-way ANOVA Fisher’s test.
Extended Data Fig. 7 PDGFR-β knockout reduces vascular density and inhibits Ki67, Snail and FSP-1 expression in MI- associated ECs.
MI was induced in WT and PDGFR-β-ΔEC mice. Hearts were excised 3 weeks after surgery. MI tissue sections were immunostained with (a-c) anti-Ki67 and anti-CD31, (d) anti-vWF and anti-Snail, or (e) anti-CD31 and anti-FSP-1 antibodies. a, Representative immunofluorescence images in infarct zone are shown (n = 3 mice). Scale bar: 100 μm. b,c, quantified results (mean ± SEM, n = 3 mice). b, Density of CD31+ cells. AU, arbitrary unit. c, Ki67 expression in CD31+ cells. Statistical analysis by unpaired two-tailed Students’ t test. d,e, Representative immunofluorescence images in infarct zone are shown (n = 5 mice). Scale bar: 100 μm.
Extended Data Fig. 8 PDGFR-β knockout reduces Snail expression in MI-associated ECs.
MI was induced in WT and PDGFR-β-ΔEC mice. (a,b) 14 and (c,d) 28 days after MI induction, MI tissue sections were collected and subjected to single-nuclei RNA sequencing analysis (total = 4 mice). Uniform manifold approximation and projection (UMAP) analysis of transcriptome gene signature in ECs. (a,c) UMAP analysis of ECs. (b,d) Expression of Snail, Slug, and Ki67 in ECs.
Extended Data Fig. 9 PDGFR-β knockout in ECs alters expression of metabolism-associated genes in myocytes.
MI was induced in WT and PDGFR-β-ΔEC mice. 14 and 28 days after MI induction, MI tissue sections were collected and subjected to single-nuclei RNA sequencing analysis (total = 4 mice). Expression of metabolism-associated genes were analyzed. a, Heatmap of mapped genes. b, Global changes in gene expression at days 14 and 28 after MI induction.
Extended Data Fig. 10 PDGFR inhibition improves cardiac function recovery and promotes tissue repair after MI.
MI was induced in mice, followed by administration with saline or 15 mg/kg crenolanib. a,b, Cardiac function was analyzed by echocardiogram (n = 11 mice). Representative images are shown. a, Long-axis echocardiogram analysis. Scale bar: 2 mm. b, M mode echocardiogram analysis. Scale bar: 2 mm. Arrows indicate myocardial anterior walls. c,d, Heart tissues were harvested 3 weeks after MI induction. Cardiac sections were stained with Masson’s trichrome stain. c, Representative images are shown (n = 4 mice). Scale bar: 500 μm. Arrows indicate epicardium. d, Quantified cardiomyocyte area (mean ± SEM, n = 4-5 mice). Statistical analysis by unpaired two-tailed Students’ t test. e, Heart tissues were harvested 3 weeks after MI induction. Cardiac sections were stained with an anti-CD31 antibody and analyzed by immunofluorescence imaging. Vascular density was quantified (mean ± SEM, n = 5 mice). Statistical analysis by two-way ANOVA.
Supplementary information
Supplementary Video 1.
MI was induced in control and PDGFR-β-ΔEC mice. Mice underwent echocardiography analysis at days 14 and 28 after MI.
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Huang, M., Yang, F., Zhang, D. et al. Endothelial plasticity drives aberrant vascularization and impedes cardiac repair after myocardial infarction. Nat Cardiovasc Res 1, 372–388 (2022). https://doi.org/10.1038/s44161-022-00047-3
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DOI: https://doi.org/10.1038/s44161-022-00047-3
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