Snail as a potential target molecule in cardiac fibrosis: paracrine action of endothelial cells on fibroblasts through snail and CTGF axis - PubMed (original) (raw)
Snail as a potential target molecule in cardiac fibrosis: paracrine action of endothelial cells on fibroblasts through snail and CTGF axis
Sae-Won Lee et al. Mol Ther. 2013 Sep.
Abstract
Ischemia/reperfusion (I/R) injury to myocardium induces death of cardiomyocytes and destroys the vasculature, leading to cardiac fibrosis that is mainly mediated by the transdifferentiation of fibroblasts to myofibroblasts and the collagen deposition. Snail involvement in fibrosis is well known; however, the contribution of Snail to cardiac fibrosis during I/R injury and its underlying mechanisms have not been defined. We showed that I/R injury to mouse hearts significantly increases the expression of Snail. An in vitro hypoxia/reoxygenation (Hy/Reoxy) experiment showed that the cell source of Snail induction is endothelial cells rather than cardiac fibroblasts (cFibroblasts) or cardiomyoblasts. When Snail was overexpressed in endothelial cells, they underwent endothelial-to-mesenchymal transition (EndMT) but showed very poor capacity for collagen synthesis. Instead, reoxygenation- or Snail overexpression-mediated EndMT-like cells noticeably stimulated transdifferentiation of fibroblasts to myofibroblasts via secretion of connective tissue growth factor (CTGF). The injection of a peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist, a selective Snail inhibitor, remarkably suppressed collagen deposition and cardiac fibrosis in mouse I/R injury, and significantly improved cardiac function and reduced Snail and CTGF expression in vivo. Our findings suggested a new mechanism of cell-to-cell communication between EndMT-like cells and fibroblasts for fibrosis induction and implicated Snail as a potential target molecule in cardiac fibrosis after I/R injury.
Figures
Figure 1
Snail is induced by I/R injury in mouse heart. (a) Male C57BL/6 mice underwent I/R surgery by the occlusion of the LAD following reperfusion. (b) Detection of Snail protein (brown) in endocardial (Ba) and myocardial (Bb) regions. Many nuclei were positive for Snail (red arrows), whereas the other nuclei were negative (blue triangles). No signals in the IgG control group. Low magnification: ×12.5; high magnification: ×400 (n = 4 each). (c) Myocardial proteins at 7 days after I/R were immunoblotted with anti-Snail antibody. Snail was upregulated in I/R group (n = 4). I/R, ischemia/reperfusion; IgG, immunoglobulin G; LAD, left anterior descending artery; LV, left ventricle; RV, right ventricle.
Figure 2
Hy/Reoxy stimulates the expression of Snail in endothelial cells, but not in CFibroblasts or myoblasts. (a) Three cell types – endothelial cells (HUVEC), cFibroblast, and cardiomyoblasts (H9C2) – were cultured under Hy for 16 hours and then reoxygenated for 2 hours (HR) (n = 3; *P < 0.05). (b) Immunohistochemical staining for Snail and VE-cad in ischemia/reperfusion cardiac tissue at postoperative day 7. Capillary endothelial cells are positive for VE-cad staining (reddish brown; arrows). Most Snail staining was observed in the nuclei of capillary endothelial cells (reddish brown; arrows). Original magnification ×200; scale bar: 50 µm. (c) Colocalization of Snail immunofluorescence (red) with VE-cad (green). Original magnification: ×630; scale bar: 10 µm. (d) PPAR-γ agonist (10 µmol/l) significantly diminished the Snail upregulation caused by Hy/Reoxy in HUVEC (n = 3; *P < 0.05). cFibroblast, cardiac fibroblast; DAPI, 4′,6-diamidino-2-phenylindole; Hy/Reoxy, hypoxia/reoxygenation; HUVEC, human umbilical vein endothelial cell; Hy, hypoxia; N, normoxia; NS, nonsignificant; PPAR-γ, peroxisome proliferator-activated receptor-γ VE-cad, vascular endothelial cadherin.
Figure 3
Snail overexpression promotes transition from endothelial cells toward mesenchymal ones (EndMT). (a) HUVEC was transfected with pSnail for 48 hours. Snail overexpression reduced the endothelial markers, CD31, VE-cad, and vWF, while increased the mesenchymal markers α-SMA and SM22α Quantitative graphs for Western blotting bottom, n = 3). (b) Phase bright imaging of HUVEC with Snail overexpression showed change in cell morphology from round cells in cobblestone arrangement to elongated spindle-shaped cells characteristic of EndMT. Magnification: ×100. Representative images are shown. Snail-overexpressing HUVEC showed (c) increased migration (16 hours) and (d) retarded tube formation on Matrigel (10 hours). Magnification: ×40. Con, nontransfected HUVEC. All experiments were repeated three times independently. HUVEC, human umbilical vein endothelial cell; SMA, smooth muscle actin; VE-cad, vascular endothelial cadherin; vWF, von Willebrand factor.
Figure 4
CM from endothelial-to-mesenchymal transition-like cells, after Hy/Reoxy or Snail overexpression, activate cFibroblasts to myofibroblasts. (a) ELISA for collagen type I in CM. The amount of collagen from HUVEC was much lower than that from cFibroblasts or H9C2 cardiomyoblasts (n = 3). (b) Schematic illustration of CM preparation and treatment. CM from various conditions (i, ii, iii conditions) it was treated to cFibroblast for 3 days. In parallel with CM treatment, cFibroblasts themselves were incubated under normoxia (3 days) or Hy/Reoxy conditions (hypoxia 16 hours plus reoxygenation 32 hours, total 3 days). (c) Phenotype switches from cFibroblasts to myofibroblasts by HR CM. Myofibroblasts display strong and fibrous α-SMA fluorescence (green). Nucleus for DAPI (blue). No fluorescence signal in the isotype IgG group. Magnification: ×100 (n = 3). (d) Snail was highly expressed in HUVEC via the lentiviral vector. (e) CM from Snail-overexpressing HUVEC (Snail CM) stimulated the formation of α-SMA filaments in cFibroblasts (green). Magnification: ×200 (n = 4). (f–i) The induction of Snail in HUVEC or in HMVEC-C by Hy/Reoxy was effectively blocked by siRNA against Snail (siSnail). (f,h) Western blotting for Snail (n = 3). (g,i) Snail knock-down with siSnail abolished the effect of HR CM on α-SMA fiber formation (green) in cFibroblasts. Magnification: ×400. siCon indicates control siRNA (n = 3). HR, cFibroblasts under Hy/Reoxy conditions; HR CM, CM from Hy/Reoxy HUVEC; Mock CM, CM from Mock-overexpressing HUVEC; Nor, cFibroblasts under normoxia; Nor CM, cFibroblasts treated with CM from normoxic HUVECs. α-SMA, α-smooth muscle actin; cFibroblast, cardiac fibroblast; CM, conditioned media; ELISA, enzyme-linked immunosorbent assay; HMVEC-C, human cardiac microvascular endothelial cell; HUVEC, human umbilical vein endothelial cell; HR, hypoxia/reoxygenation; IgG, immunoglobulin G; siRNA, small interfering RNA.
Figure 5
CTGF is a downstream target of Snail and mediates the activation of cFibroblasts to myofibroblasts. (a,b) Regulation of profibrosis factors by Snail. (a) Only CTGF expression was increased by Snail overexpression among several key factors that encourage the transition of fibroblasts to myofibroblasts. Quantification graph (bottom; n = 3). (b) CTGF expression was induced by HR in HUVEC and in HMVEC-C (n = 3). (c) ELISA for CTGF protein in culture supernatant (n = 4). (d) Schematic time table of Lenti-Snail transduction and siCTGF transfection. (e) Enhanced expression of CTGF by Snail overexpression in HUVEC was totally blocked by siCTGF. Quantitative graphs (bottom, n = 3). Silencing of (f) CTGF or (g) CTGF-neutralizing antibody (3 µg/ml) abolished the effect of CM from Snail-overexpressing HUVEC or HMVEC-C on the emergence of myofibroblasts (green; α-SMA) (n = 3). AGT, angiotensinogen; cFibroblast, cardiac fibroblast; CM, conditioned media; CTGF, connective tissue growth factor; ELISA, enzyme-linked immunosorbent assay; HMVEC-C, human cardiac microvascular endothelial cell; HR, hypoxia/reoxygenation; HUVEC, human umbilical vein endothelial cell; IgG, immunoglobulin G; N, normoxia; NS, nonsignificant; siCon, control small interfering RNA; siRNA, small interfering RNA; SMA, smooth muscle actin; TGF-β1, transforming growth factor-β1.
Figure 6
Snail inhibition reduces cardiac fibrosis and improves cardiac function after I/R injury. (a–c) Paraffin serial sections of mouse heart 4 weeks after I/R were analyzed by MT staining for cardiac fibrosis and immunohistochemistry for collagen type I (reddish brown). In MT staining, collagenous tissues are blue, whereas muscle fibers and the other tissue are red. Cardiac fibrosis induced by I/R injury was significantly reduced by PPAR-γ agonist. Magnification: ×400. Quantitative graph of (b) fibrotic area and (c) collagen+ area using ImageJ program is shown. (d,e) Echocardiographic results at 4 weeks after surgery. Left ventricular (LV) function was improved in PPAR-γ agonist-treated mice (n = 11) than in vehicle (DMSO)-treated mice (n = 8). For LV contractility, LV EF and LV FS were significantly improved. For dimension reduction, LVESD was decreased by PPAR-γ agonist. Sham-operated animals (n = 5). *P < 0.05, **P < 0.05. (f) Immunohistochemical staining of Snail and CTGF in adjacent thin sections from I/R heart at postoperative day 7. Strong Snail expression in nucleus and CTGF in cytosolic area were observed (arrows). The induction of Snail and CTGF was remarkably reduced by PPAR-γ agonist, the selective Snail inhibitor. Low magnification: ×20; high magnification: ×400. (g) CTGF expression was highly stimulated by Hy/Reoxy only in endothelial cells, and it was reduced by a Snail inhibitor, the PPAR-γ agonist (10 µmol/l) (n = 3). (h) Scheme of cardiac fibrosis after I/R injury. Hy/Reoxy induced Snail overexpression in endothelial cells and thus induced an EndMT-like process; the loss of endothelial markers and the gain of mesenchymal ones. EndMT-like cells secreted profibrotic cytokine CTGF, which might influence neighboring cardiac fibroblasts to differentiate into active myofibroblasts, resulting in collagen deposition and cardiac fibrosis. cFibroblast, cardiac fibroblast; CTGF, connective tissue growth factor; DMSO, dimethyl sulfoxide; EF, ejection fraction; EndMT, endothelial-to-mesenchymal transition; FS, fractional shortening; HUVEC, human umbilical vein endothelial cell; HR, hypoxia/reoxygenation; I/R, ischemia/reperfusion; LVEDD, left ventricular end-diastolic dimension; LVESD, left ventricular end-systolic dimension; MT, Masson's Trichrome; PPAR-γ, peroxisome proliferator-activated receptor-γ.
References
- Prasad A, Stone GW, Holmes DR, Gersh B. Reperfusion injury, microvascular dysfunction, and cardioprotection: the “dark side” of reperfusion. Circulation. 2009;120:2105–2112. - PubMed
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