Figure 1.
Hypoxia activated cardiac fibroblasts (CFs) to myofibroblasts (MCFs) through TGF-β/Smad2 pathway and increased collagen production.
Cardiac fibroblasts cultured at normoxia (N-CFs) and hypoxia condition (H-CFs), or treated with 10 ng/ml TGF-β1 at normoxia condition 24 hours (N-CFs-TGF-β1) were collected for subsequent experiments. (A) α-SMA mRNA level measured by qRT-PCR of total RNA isolated from CFs. mRNA value of H-CFs and N-CFs-TGF-β1 was normalized to N-CFs. (B) Western blots quantification of α-SMA protein expression in cell lysates from CFs after hypoxia and TGF-β1 pretreatment. The α-SMA ratios normalized to β-actin in both hypoxia and TGF-β1 treated groups were compared with the control group. (C) Representative western blot analysis of α-SMA protein expression in CFs cultured under standard condition was used as a control. Both hypoxia and TGF-β1 treatment for 24 hours increased α-SMA expression. (D) Representative western blot of collagen IαI protein expression in cell lysates from N-CFs, H-CFs and N-CFs-TGF-β1. Both hypoxia and TGF-β1 treatment for 24 hours increased collagen IαI expression. (E) Quantification of collagen IαI western blot in D. The ratios of collagen IαI normalized to β-actin in both hypoxia and TGF-β1 pretreated groups were compared with the normoxia cultured group. (F) Representative western blot of TGF-β1 and phospho-Smad2 protein expression in N-CFs and H-CFs. (G) Quantification of TGF-β1 and phospho-Smad2 western blot in F. The ratio of TGF-β1 and p-Smad2 in H-CFs were compared to normoxia cultured CFs (n = 3, *p<0.05).
Figure 2.
H-MSCsWT reverses hypoxia stimulated collagen production and cardiac fibroblast differentiation into myofibroblasts.
H-MSCsWT and N-MSCsWT co-cultured with H-CFs at normal culture condition 24 hrs. Cardiac fibroblasts from the lower chamber which co-cultured with different preconditioned MSCsWT were collected for subsequent experiments. (A) Representative western blot of α-smooth muscle actin, collagen IαI and SM22α in cellular lysates from cardiac fibroblasts co-cultured with N-MSCsWT and H-MSCsWT. (B) Quantification analysis of western blot in A. (C) Immunofluorescence staining for α-smooth muscle actin in H-CFs co-cultured with N-MSCsWT and H-MSCsWT. (D) Quantification analysis of images presented on (C). (E) Quantitative analysis of the effects of N-MSCsWT and H-MSCsWT on the collagen production induced by hypoxia in cardiac fibroblasts stained by Sirius Red, resolution in 1 mol HCl. Data are the mean ± SEM of three independent experiments. * P<0.05 versus H-CF; #P<0.05 versus H-CF-N-MSCsWT. Scale bars in C represent 50 µm.
Figure 3.
Hypoxia preconditioning upregulated leptin expression of MSCs.
(A) leptin mRNA level in MSCs after normoxia, hypoxia treatment were measured by qRT–PCR. Data are the mean ±SEM of three independent experiments. *P<0.05 versus N-MSCWT.
Figure 4.
H-MSCsWT prevent hypoxia induced cardiac fibroblast activation, but leptin deficit inhibit this effect.
(A) Western blot of α-SMA, collagen IαI and SM22αin cellular lysates from hypoxia-activated cardiac fibroblasts co-cultured with H-MSCsWT and H-MSCsob/ob. Blots were reprobed for α-Tublin as control. (B) Quantification analysis of α-SMA and SM22α protein level expressed as % of controls (N-CFs). (C) Immunofluorescence staining of α-SMA in hypoxia-activated cardiac fibroblasts co-cultured with H-MSCsWT and H-MSCsob/ob. (D) Quantitative analysis of α-SMA immunofluorescence staining presented on (C). (E) Quantitative analysis of collagen protein level expressed as % of controls (N-CFs) in A. (F) Quantitative analysis of hypoxia-activated cardiac fibroblasts co-cultured with H-MSCsWT and H-MSCsob/ob, stained with Sirius Red. (G) Western blot of α-SMA in cellular lysates of hypoxia-activated wide type or leptin deficit db/db cardiac fibroblasts co-cultured with H-MSCsWT. (H) Quantification analysis of α-SMA protein level expressed as % of controls (N-CFs). * P<0.05 versus N-CF; #P<0.05 versus H-CFs-H-MSCs. Scale bars in C represent 50 µm.
Figure 5.
H-MSCsWT reduced infarct size and collagen I expression 7 days post-MI.
(A) Representative Masson's trichrome staining of heart tissue to show the infarct area (blue) 7 days after MI. (B) Representative immunofluorescence staining of collagen I (red) in LV. (C) Quantification of cardiac infarct size in A. (D) Quantification of collagen I percentage of LV in B. n = 5–6, *P<0.05 vs. others.
Figure 6.
H-MSCsWT decrease myofibroblasts in peri-infarction region.
(A) Immunofluorescence staining of myofibroblast (DAPI (blue), α-SMA (red) and FSP (green)) in heart frozen section. Arrows indicate double positively stained myofibroblasts. Scale bars represent 50 µm. (B) Quantification of double positive stained myofibroblasts in A, analyzed by Image Pro 6.0 software.*P<0.05 versus N-MSCs, #P<0.05 versus H-MSCs, n≥5 in each group.
Figure 7.
Leptin deficit hypoxia exposed-MSCs failed to limit cardiac fibroblast activation, through TGF-β/Smad2 and MRTF-A.
(A) Western blot of TGF-β, p-Smad2, MRTF-A and MRTF-B in cellular lysates from hypoxia-activated cardiac fibroblasts co-cultured with H-MSCsWT and H-MSCsob/ob. Blots were reprobed for α-Tublin as a control. (B) Densitometric analysis of protein level expressed as % of controls (N-CFs). (C) Immunofluorescence staining of MRTF-A and DAPI has done in H-CFs from four groups. * P<0.05 versus N-CF; #P<0.05 versus H-CF-H-MSCs. Scale bars in E represent 50 µm.
Figure 8.
H-MSCsWT inhibited hypoxia induced cardiac fibroblast activation via leptin.
Hypoxia induced secretion of TGF-β from cardiac fibroblasts, leading to Smad2 phosphorylation, meanwhile, hypoxia increased expression and nuclear translocation of MRTF-A. These two signaling pathways co-regulated the transcription of myofibroblast target gene α-SMA and collagen IαI, causing the activation of fibroblasts. Our study indicated that H-MSCsWT inhibited the expression of TGF-β, phosphorylation of Smad2 and nuclear translocation of MRTF-A, leading to cardiac fibroblast inactivation, and this protective effect was leptin dependent.