Inhibition of Notch Signaling Promotes the Differentiation of Epicardial Progenitor Cells into Adipocytes

Figures

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  Figure 1. EAT located at the left and right atrial–ventricular groove in postnatal 8-week-old mouse hearts. (a, b) Oil red O staining showed that EAT located at the atrial–ventricular groove in mouse. The boxed regions were shown at higher magnification in A' and B'. (c, d) H&E staining showed that EAT was present in the atrial–ventricular groove underneath the epicardium. The boxed regions were shown at higher magnification in C' and D'. (e, f) Immunofluorescence staining for adipocyte marker perilipin-1 showed the epicardial adipocytes were present in the right and left atrial–ventricular groove. The boxed regions were shown at higher magnification in E' and F'. (g–l) The genetic lineage tracing methods were used to trace the EPCs. Immunofluorescence staining for adipocyte markers perilipin-1 (red) and FABP4 (red) was partly colocalized with the lineage marker EYFP (green) in the same section.

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  Figure 2. EPCs migrated out from the edge of embryonic mouse heart. (a) The cobblestone-like EPCs grew from the edge of the embryonic ventricular tissue after 24 hours' culture. (b) The cobblestone-like EPCs continued growing from the edge of the embryonic ventricular tissue after 48 hours' culture. (c) Cobblestone-like EPCs were observed after removing the ventricular tissue. (d, e) Immunofluorescence staining showed all cultured EPCs expressed Tbx18 and Wt1.

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  Figure 3. Notch signaling and its target genes in EPCs were regulated by the treatment of jagged-1 and DAPT for 2 days. (a, b) The mRNA expression of Notch signaling and its target genes were significantly upregulated with the treatment of jagged-1 for 2 days compared with the control. (c, d) The mRNA expression of Notch signaling and its target genes were significantly downregulated with the treatment of DAPT for 2 days compared to the control. The p values were calculated by t-test. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 for pairwise comparison. (e) Immunofluorescence staining showed that activated Notch-1 was significantly upregulated with jagged-1 treatment while decreased with DAPT treatment. (f) Immunofluorescence staining showed that Hes-1 was significantly upregulated with jagged-1 treatment while decreased with DAPT treatment. The p values were calculated by ANOVA. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 for pairwise comparison among the groups.

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  Figure 4. Inhibition of Notch signaling promoted the adipogenic differentiation of EPCs. (a) The mRNA expression levels of adipocyte-related genes adiponectin, FABP4, leptin, LPL, and perilipin-1 were determined by RT-PCR after EPCs cultured in adipogenic induction medium alone, with jagged-1, or with DAPT for 7 days. (b, c) The adipocyte markers FABP-4 and perilipin-1 were determined by immunofluorescence staining after EPCs were cultured in adipogenic induction medium alone, with jagged-1, or with DAPT for 7 days. (d) Lipid droplet accumulation was detected by Oil red O staining after EPCs cultured in adipogenic induction medium alone, with jagged-1, or with DAPT for 21 days. The p values were calculated by ANOVA. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 for pairwise comparison among the groups.

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  Figure 5. The dynamic changes of Notch signaling in mouse epicardium. (a) The dynamic changes of Jagged-1 in mouse epicardium at different ages. Jagged-1 was mainly expressed in the heart of E11.5 and neonatal mice. (b) The dynamic changes Notch-1 in mouse epicardium at different ages. Notch-1 expression in the mouse epicardium gradually decreased from E11.5 to postnatal 8-week-old mice. (c) The dynamic changes of NICD (activated Notch-1) in epicardium in mice at different ages. NICD was found in epicardium at E11.5 and neonatal mouse but not found in epicardium at postnatal 2-, 4-, and 8-week-old mice.

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  Figure 6. DAPT treatment upregulated the transcription factor PPAR-γ. (a, b) The crucial adipogenic transcription factors PPAR-γ and the C/EBP-α were determined by immunofluorescence staining after EPCs were cultured in adipogenic induction medium alone, with jagged-1 or with DAPT for 7 days. (c) The mRNA expression levels of PPAR-γ and C/EBP-α were determined by RT-PCR. (d, e) The adipocyte markers FABP-4 and perilipin-1 were determined by immunofluorescence. EPCs were cultured in adipogenic induction medium with DAPT alone or combined with PPAR-γ antagonists. (f) Lipid droplet accumulation was determined by Oil red O staining. EPCs were cultured in adipogenic induction medium with DAPT alone or combined with PPAR-γ antagonists. (g) The mRNA expression levels of adipocyte-related genes FABP4, perilipin-1, leptin, LPL, and adiponectin were determined by RT-PCR. The p values were calculated by t-test between 2 groups and by ANOVA among 3 groups. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 for pairwise comparison among the groups.

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  Figure 7. DAPT treatment reduced the differentiation of EPCs into smooth muscle cells. (a, b) Jagged-1 treatment increased the expression of smooth muscle cell specific marker α-SMA and Myh11, while DAPT reduced their expression. (c) Jagged-1 and DAPT treatment did not affect the expression of fibroblast specific marker periostin.