Fig 1: Changing composition of the nonmyocyte fraction with advancing age.Nonmyocyte cells were isolated from hearts of animals postnatal day 1, 4 weeks, 14 weeks, 24 weeks, and 1 year of age and subjected to flow cytometry to determine the endothelial and cardiac fibroblast population at these time points. (A–D) Flow cytometry demonstrating fraction of nonmyocytes staining for (A) CD31 (endothelial), (B) PDGFRa, (C) Thy1.2, and (D) MEFSK4, and (E–H). Quantitation of (E) CD31, (F) PDGFRa, (G) Thy1.2, and (H) MEFSK4 expressing cells as a fraction of the entire nonmyocyte population isolated from the hearts at those time points. Postnatal day 1 value was compared with each value at 4 weeks, 14 weeks, 24 weeks, and 1 year, respectively. ***P < 0.0001, ns = P > 0.05. Data are represented as the mean ± SD. Data analysis was performed by 1-way ANOVA with multiple-comparisons correction.
Fig 2: Periostin+ myofibroblasts are derived from the Tcf21 lineage.(a) Schematic representation of four different lineage-specific Cre-expressing mouse lines crossed with a LacZ-expressing reporter in the Rosa26 locus, further crossed with mice containing a periostin promoter transgene-driving ZsGreen. (b) Experimental scheme to lineage trace from each of four different Cre-expressing mouse lines at baseline and after MI injury, harvested 1 week later. The Myh11CreERT2/+ mice required tamoxifen treatment for 2 weeks before MI injury to generate traced cells, and the tamoxifen was removed 3 days before MI surgery. (c,d) Quantification and representative images of lineage-traced cells (red, for LacZ) and ZsGreen from the periostin transgene from the MI region of the heart. LacZ was detected with an antibody (n=4–6 hearts, >20 sections each were quantified with >100 total ZsGreen+ cells counted). (e) Schematic representation of three different Cre-expressing knock-in mouse lines shown in g–i crossed with the eGFP expressing reporter in the Rosa26 locus. (f) Experimental scheme to lineage trace from each of three different Cre-expressing mouse lines shown in g–i at baseline and after MI injury. (g–i) Quantification of immunohistochemistry analysis for vimentin, aSMA, CD31, CD45 and FSP1 that also co-labelled as lineage-traced cells from LysMCre, Myh11CreERT2 and Cdh5Cre alleles (n=3 hearts, >20 sections were quantified, n>200 cells counted for each of the indicated genotypes). All error bars in the figure represent s.e.m.
Fig 3: Periostin+ myofibroblasts derive from Tcf21 resident fibroblasts.(a) Schematic representation of the PostnMCM mouse crossed with a Rosa26-eGFP reporter mouse (R26-eGFP) for lineage tracing, which was further crossed with the Tcf21LacZ knock-in mouse line. (b) Experimental scheme to lineage trace periostin-expressing myofibroblasts in vivo for 1 week with tamoxifen treatment immediately after MI injury. (c) Representative histological section from an MI region of the heart of a PostnMCM/+; R26-eGFP mouse that also contained the Tcf21LacZ allele. The section is only stained for x-gal activity (LacZ expression), and Tcf21+ expanded fibroblasts appear around the demarked injured region. (d) Same scheme as in c except that immunohistochemistry was used to detect LacZ (Tcf21 current expression, red staining) and periostin lineage-traced cells in green. The yellow arrows show a few rare transitional cells that express both periostin and Tcf21. Nuclei are stained in blue (n=4 hearts). (e) Thermogram of gene expression patterns from RNAseq of representative individual cells from the hearts of PostnMCM/+; R26-eGFP or Tcf21MCM/+; R26-eGFP mice. (e,f) Cells were negatively sorted for CD31 and CD45 and were either Tcf21 lineage traced (eGFP+) and sorted from uninjured hearts (yellow bars in f) or from the MI region 7 days after injury as `activated'. As another control periostin lineage-traced cells were collected from the MI region of the heart 7 days after injury for comparison. A population of total interstitial cells were used as a control, which were negatively sorted for CD31 and CD45 from the remote region of the heart. Data produced from a total of 185 cells isolated from three mice in each group in e, and a subset is shown in f. Error bars represent s.e.m.
Fig 4: Overexpression of Sox17 in ECs induces EC proliferation and regeneration. Mixture of 50 µg plasmid with 100 µl liposomes was injected i.v. 3 h after LPS challenge (12 mg/dose i.p.) in wild-type mice. This plasmid has a Flag-tag added to the N-terminus of Sox17 protein coding region and expression is under the regulation of a mouse Cdh5 promoter. a Confocal microscopy of flag staining with CD31 and DAPI co-staining for nuclei in lung cryo-sections from mice receiving a control vector or a Sox17-construct to over-express Sox17. Scale bar = 50 µm (original panel) and 20 µm (enlarged panel). n = 6. OE, overexpression. b Co-localization coefficient for the fraction of Flag in CD31+ cells assesses the transgene expression in the endothelium. The Pearson correlation coefficient is significantly increased in Sox17-overexpressing mice compared to control mice. n = 6. c Western blot analysis and its quantification d showed a significant increase in the flag and Cyclin E1 expression in the pulmonary endothelial cells of mice with 3 days of Sox17 overexpression compared to vector mice. n = 3. e Quantification of BrdU+ nuclei in each field of 425 µm2 area in lung cryo-sections from vector-overexpressing and Sox17-overexpressing mice. n = 5 per group and 6 technical replicates per sample. Slides are co-stained with CD31-AF594, BrdU-AF488, and DAPI. Both groups show increased BrdU+ ECs at day 3 post-LPS as compared to baseline and the response was significantly greater in mice in which ECs overexpressed Sox 17. f Lung transvascular albumin permeability pre-LPS and post-LPS challenge in mice overexpressing endothelial Sox17 and control mice. n = 5. Mice overexpressing Sox17 in ECs showed significantly reduced vascular leakiness post-LPS when compared to control mice. g Survival curve of LPS challenge in control mice and mice over-expressing Sox17 in the endothelium. n = 11 per group. At this lethal dose of LPS (20 mg/kg), the death rate for control mice is 60% while for Sox17-overexpressed mice is 10%. h Model. LPS induces tissue hypoxia due to local oxygen depletion by infiltrating activated neutrophils, thereby stabilizing HIF-1a resulting in upregulation Sox17 expression and Sox17 mediated expression of Cyclin E1. This activates cell cycle re-entry and EC proliferation, and restoration of endothelial integrity. **P < 0.01 and ***P < 0.001. Data are shown as mean ± SEM. Analysis was performed using two-way ANOVA with Bonferroni post-tests for (d–f) and Log-rank (Mantel-Cox) test for (g)
Fig 5: Dietary fatty acids can directly enhance growth factor sensitivity to promote adipose tissue progenitor proliferation. (A) Cd36 mRNA expression in Lin-CD29+CD34+Sca1+ or CD31+ cells sorted by FACS from gWAT of mice fed CD or HFD for 1 week (n = 4 samples/group, each pooled from 2 to 3 mice). (B) Frequency of EdU+ Lin-Sca1+ cells in wildtype (Wt) or Cd36-knockout mice after one week of CD, HFD, and R-HFD with EdU-containing drinking water, determined by flow cytometry (n = 4 mice/group). (C–E) Relative frequency of EdU+ cycling (S/G2/M) Lin-Sca1+ cells in primary cultures from wild type (C,E)) or Wt/Cd36-KO (D) gWAT following treatment with fatty acids (250 µM C16:1, C18:1, C16:0; 100 µM C20:4, C20:5, C22:6) and IGF-1, insulin (50 ng/ml), leptin (20 ng/ml) or GLP-1 (100 nM) as indicated for 16 hours in the presence of EdU [relative to Vehicle (Veh), n = 3–7 independent biological replicates/group, each pooled from >2 mice, except for C20:4, C20:5 and C22:6 (technical replicates, descriptively)]. C16:1: palmitoleic acid, C18:1: oleic acid, C16:0: palmitic acid, C20:4 AA: arachidonic acid, C20:5 EPA: eicosapentaenoic acid, C22:6 DHA: docosahexaenoic acid. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 vs. CD (A,B) or Vehicle (C–E) and #P < 0.05, ##P < 0.01, ###P < 0.001 vs. Lin-Sca1+ (A) or Control (C,E) [2-way ANOVA, posthoc Tukey (A,B) or Holm-Sidak (C–E)].
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