Young Bone Marrow Sca-1 Cells Rejuvenate the Aged Heart by Promoting Epithelial-to-Mesenchymal Transition

Abstract

Background: To improve the regenerative capacity of aged individuals, we reconstituted bone marrow (BM) of aged mice with young Sca-1 cells, which repopulated cardiac progenitors and prevented cardiac dysfunction after a myocardial infarction (MI). However, the mechanisms involved were incompletely elucidated. This study aimed to investigate whether young, highly regenerative BM Sca-1 cells exert their cardio-protective effects on the aged heart through reactivation of the epithelial-to-mesenchymal transition (EMT) process. Methods:In vitro, BM Sca-1 cells were co-cultured with epicardial-derived cells (EPDCs) under hypoxia condition; mRNA and protein levels of EMT genes were measured along with cellular proliferation and migration. In vivo, BM Sca-1⁺ or Sca-1^- cells from young mice (2-3 months) were transplanted into lethally-irradiated old mice (20-22 months) to generate chimeras. In addition, Sca-1 knockout (KO) mice were reconstituted with wild type (WT) BM Sca-1⁺ cells. The effects of BM Sca-1 cell on EMT reactivation and improvement of cardiac function after MI were evaluated. Results:In vitro, BM Sca-1⁺ cells increased EPDC proliferation, migration, and EMT relative to Sca-1^- cells and these effects were inhibited by a TGF-β blocker. In vivo, more young BM Sca-1⁺ than Sca-1^- cells homed to the epicardium and induced greater host EPDC proliferation, migration, and EMT after MI. Furthermore, reconstitution of Sca-1 KO mice with WT Sca-1⁺ cells was associated with the reactivation of EMT and improved cardiac function after MI. Conclusions: Young BM Sca-1⁺ cells improved cardiac regeneration through promoting EPDC proliferation, migration and reactivation of EMT via the TGF-β signaling pathway.

Keywords: aging; heart; rejuvenation; stem cells.

Figure 1

BM Sca-1⁺ cells homed to the epicardium and increased proliferation of host epicardial cells after MI. Sca-1⁺ and Sca-1^- bone marrow (BM, 2×10⁶) cells from young (Y) GFP (green fluorescent protein) transgenic mice were used to reconstitute irradiated old (O, 9.5 Gy) wild type mice, generating Y(Sca1⁺)-O and Y(Sca1^-)-O chimeras, respectively. Twelve weeks after BM reconstitution, coronary occlusion was performed to induce myocardial infarction (MI). To detect cell proliferation after MI, BrdU (50 mg/kg) was administered to mice by intraperitoneal injection for 3 consecutive days. Coronary artery ligation was performed 1 day later. (A) Immunofluorescent staining of GFP and WT1 in chimeric hearts at baseline, 3 and 7 days post-MI. (B) Quantification of GFP⁺ in the epicardium, myocardium, and endocardium of chimeric hearts at baseline and in the infarcted region at 3 and 7 days post-MI. (C) Quantification of WT1⁺ cells in the epicardium, myocardium, and endocardium of the infarct area in the chimeric hearts. (D) Quantification of WT1⁺GFP^- or WT1⁺GFP⁺ cells in the infarcted region of the chimeric hearts. (E) Immunofluorescent staining of GFP, WT1, and BrdU in chimeric hearts at baseline, 3 and 7 days post-MI. Yellow arrows indicate GFP, WT1, and BrdU triple-positive cells. White arrows indicate WT1 and BrdU double-positive cells. (F) Quantification of WT1⁺BrdU⁺ (proliferating epicardial cells) cells in the infarcted region of the chimeric hearts at 3 and 7 days post-MI. Quantification of WT1⁺BrdU⁺GFP^- (host-derived proliferating epicardial cells) and WT1⁺BrdU⁺GFP⁺ cells (donor-derived proliferating epicardial cells) in the infarcted region of the chimeric hearts at 3 (G) and 7 (F) days post-MI. n= 6/group, mean ± SD; *P<0.05, **P<0.01. BrdU: bromodeoxyuridine; WT1: wilms tumor 1.

Figure 2

BM Sca-1⁺ cells activated EMT of epicardial cells after MI. Bone marrow (BM; 2×10⁶) Sca-1⁺ and Sca-1^- cells from young (Y) GFP (green fluorescent protein) transgenic mice were used to reconstitute irradiated old (O, 9.5 Gy) wild type mice, generating Y(Sca1⁺)-O and Y (Sca1^-)-O chimeras, respectively. Twelve weeks after BM reconstitution, coronary occlusion was performed to induce myocardial infarction (MI). Quantification of EMT embryonic epicardial genes (A), transcriptional genes (B), mesenchymal genes (C), and epithelial genes (D) at baseline and in the infarcted region at 3 days post-MI by RT-qPCR. (E) Immunofluorescent staining of smooth muscle actin (SMA) at 3 days post-MI. Yellow arrows indicate GFP, WT1, and SMA triple-positive cells (donor-derived epicardial cell obtained the mesenchymal phenotype). White arrows indicate WT1 and SMA double-positive cells (host-derived epicardial cell obtained the mesenchymal phenotype). (F) Immunofluorescent staining of vimentin at 3 days post-MI. (G) Immunofluorescent staining of calponin at 3 days post-MI. (H) Protein levels of SMA, vimentin, and calponin (mesenchymal phenotype) at baseline and in the infarcted region of the chimeric hearts were determined by Western blot and normalized by GAPDH. n=6/group, mean ± SD; **P<0.01.

Figure 3

Co-culture of BM Sca-1⁺ cells with EPDCs under hypoxia conditions increased proliferation and migration of EPDCs. Epicardial-derived cells (EPDCs, abbreviated as E) were isolated and co-cultured with bone marrow (BM) Sca-1⁺ cells or Sca-1^- cells under normoxia and hypoxia (0.1% O₂) conditions. (A) Representative images of EPDCs and immunofluorescent labeling of WT1. (B) Cell proliferation was determined by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. (C, D) Immunofluorescent staining of BrdU and quantification of BrdU⁺ (proliferating epicardial cells) cells. EPDCs, co-cultured with BM Sca-1⁺ cells or Sca-1^- cells under normoxia (Nor) and hypoxia (Hypo) conditions for 72 h, were pulse-chased with BrdU (10 µM) for labeling of proliferative cells. Cell migration was evaluated by the transwell (E, F) and wound-scratch (G, H) assays after co-culture for 24 h. n=6/group, mean ± SD; **P<0.01. BrdU: bromodeoxyuridine.

Figure 4

Co-culture of BM Sca-1⁺ cells with EPDCs under hypoxia conditions activated EMT of EPDCs. Epicardial-derived cells (EPDCs, abbreviated as E) were separately co-cultured with bone marrow (BM) Sca-1^- cells or Sca-1⁺ cells under normoxia and hypoxia (0.1% O₂) conditions for 72 h. Young Sca1⁺ or Sca1^- BM cells (1×10⁵/cm²) in serum-free DMEM medium were plated in the transwell cell culture inserts (1 μm diameter pores). EPDCs (2×10⁴/cm²) were cultured in the lower compartment. Quantification of the expression of EMT transcriptional genes (A), epithelial genes (B) and mesenchymal genes (C) in EPDCs. (D) Immunofluorescent labeling of mesenchymal markers (smooth muscle actin (SMA), vimentin, calponin) and epithelial tight junction protein (ZO-1) in EPDCs. (E) Protein levels of vimentin and calponin in EPDCs were determined by Western blot and normalized by GAPDH. n=6/group, mean ± SD; **P<0.01.

Figure 5

BM Sca-1⁺ cells increased proliferation and migration of EPDCs through TGF-β1 signaling. Epicardial-derived cells (EPDCs, abbreviated as E), bone marrow (BM) Sca-1⁺ and Sca-1^- cells were isolated and subjected to normoxia and hypoxia (0.1% O₂) conditions. (A) Higher expression levels of TGF-β1 mRNA were found in BM Sca-1⁺ cells than in EPDCs and Sca-1^- cells under hypoxia conditions for 72 h. (B) TGF-β1 homodimer secretion in EPDCs, BM Sca-1⁺ and Sca-1^- cell lysate and culture medium under normoxia and hypoxia conditions for 72 h was quantified by ELISA. (C) TGF-β1 mRNA expression was measured in the Y(sca-1⁺)-O and Y(sca-1^-)-O chimeric hearts at baseline and in the infarcted area at 3 days post-MI. Epicardial-derived cells (EPDCs, abbreviated as E) were co-cultured with BM Sca-1^- cells (S^-), Sca-1⁺ cells (S⁺), TGF-β1 (T, 5 ng/mL), Sca-1⁺ cells with TGF-β1 blocking antibody (S⁺+BL, 1 µg/mL) or Sca-1^- cells with TGF-β1 (S^-+T) under hypoxia conditions. The following assays were conducted in EPDCs: (D) MTT assay measured cell proliferation; (E) EPDCs, co-cultured with BM Sca-1⁺ cells or Sca-1^- cells under normoxia and hypoxia conditions for 72 h, were pulse-chased with BrdU (10 µM) for labeling of proliferative cells; (F) transwell and (G) wound-scratch assays measured migration areas and number of EPDCs after co-culture for 24 h, respectively. Insufficiency of Sca1^- cells on EPDC migration can be rescued by TGF-β1, and EPDC migration was restored to a level comparable with that of the Sca-1⁺ cell- or TGF-β1-treated groups. n=6/group, mean ± SD; **P<0.01. BrdU: bromodeoxyuridine; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

Figure 6

BM Sca-1⁺ cells activated EMT of EPDCs through TGF-β1 signaling. Epicardial-derived cells (EPDCs, abbreviated as E) were separately co-cultured with Sca-1^- cells (S^-), Sca-1⁺ cells (S⁺), TGF-β1 (T, 5 ng/mL) or Sca-1⁺ cells with a TGF-β1 blocking antibody (S⁺+BL, 1 µg/mL) under hypoxia (0.1% O₂) conditions for 72 h. Young Sca1⁺ or Sca1^- BM cells (1×10⁵/cm²) in serum-free DMEM medium were plated in the transwell cell culture inserts (1 μm diameter pores). EPDCs (2×10⁴/cm²) were cultured in the lower compartment. EMT transcriptional genes (A), epithelial genes (B) and mesenchymal genes (C) in EPDCs were quantified by RT-qPCR. (D) Immunofluorescent staining of mesenchymal markers (smooth muscle actin, vimentin, calponin) and epithelial tight junction protein (ZO-1) in EPDCs. (E) The protein levels of vimentin and calponin in EPDCs were determined by Western blot and normalized by GAPDH. n=6/group, mean ± SD; **P<0.01.

Figure 7

Reconstitution of Sca-1 KO mice with WT BM Sca-1⁺ cells restored cardiac function after MI. Sca-1 knock-out (KO) mice were reconstituted with wild type (WT) bone marrow (BM) Sca-1⁺ cells (2×10⁶, (Sca-1⁺)-KO) for 3 months, then underwent myocardial infarction (MI). Cardiac function was measured by echocardiography at baseline (before MI), 7, 14, 21, and 28 days after MI in WT, Sca-1 KO and (Sca-1⁺)-KO groups. (A) Representative M-mode echocardiographic images. (B) Fractional shortening. (C) Left ventricular internal end systolic dimension (LVIDs). (D) Left ventricular internal end-diastolic dimension (LVIDd). (E) Eject fraction (EF). (F) Representative images of whole sectioned hearts; H&E and Masson's Trichrome staining in WT, Sca-1 KO and (Sca-1⁺)-KO groups at 28 days after MI. (G) Viable myocardium (identified as red with Trichrome's staining) in the ischemia zone was quantified and expressed as a percentage of the total infarcted area. (H) Scar size thickness is presented as an average of wall thickness measurements taken at the middle and at each edge of the scar area at its thinnest point. (B-E), n=5/group; (G-H), n=6/group; mean ± SD; * vs. WT, P<0.01; # vs. KO, P<0.05.

Figure 8

Reconstitution of Sca-1 KO mice with WT BM Sca-1⁺ cells restored EMT response after MI. Sca-1 knock-out (KO) mice were reconstituted with wild type (WT) bone marrow (BM) Sca-1⁺ cells (2×10⁶, (Sca-1⁺)-KO) for 3 months, then underwent myocardial infarction (MI). Quantification of EMT embryonic epicardial genes (A), transcriptional genes (B), mesenchymal genes (C), and epithelial genes (D) at the infarcted region of WT, Sca-1 KO and (Sca-1⁺)-KO hearts at 3 days post-MI by RT-qPCR. (E) Immunofluorescent staining of smooth muscle actin (SMA), vimentin, and calponin at the infarcted region of WT, Sca-1 KO and (Sca-1⁺)-KO hearts at 3 days post-MI. (F) Protein levels of SMA, vimentin, and calponin at the infarcted region of WT, Sca-1 KO and (Sca-1⁺)-KO hearts were determined by Western blot and normalized by GAPDH. (A-D), n=5/group; (F), n=6/group; mean ± SD; * vs. WT, P<0.01; # vs. KO, P<0.05.