Relaxin inhibits cardiac fibrosis and endothelial-mesenchymal transition via the Notch pathway - PubMed (original) (raw)
Relaxin inhibits cardiac fibrosis and endothelial-mesenchymal transition via the Notch pathway
X Zhou et al. Drug Des Devel Ther. 2015.
Erratum in
- Erratum: Relaxin inhibits cardiac fibrosis and endothelial-mesenchymal transition via the Notch pathway [Corrigendum].
[No authors listed] [No authors listed] Drug Des Devel Ther. 2017 Apr 10;11:1159. doi: 10.2147/DDDT.S130751. eCollection 2017. Drug Des Devel Ther. 2017. PMID: 28435227 Free PMC article.
Abstract
Background: Relaxin (RLX) can prevent cardiac fibrosis. We aimed to investigate the possible mechanism and signal transduction pathway of RLX inhibiting cardiac fibrosis.
Methods: Isoproterenol (5 mg·kg(-1)·d(-1)) was used to establish the cardiac fibrosis model in rats, which were administered RLX. The cardiac function, related targets of cardiac fibrosis, and endothelial-mesenchymal transition (EndMT) were measured. Transforming growth factor β (TGF-β) was used to induce EndMT in human umbilical vein endothelial cells, which were pretreated with RLX, 200 ng·mL(-1), then with the inhibitor of Notch. Transwell cell migration was used to evaluate cell migration. CD31 and vimentin content was determined by immunofluorescence staining and Western blot analysis. Notch protein level was examined by Western blot analysis.
Results: RLX improved cardiac function in rats with cardiac fibrosis; it reduced the content of collagen I and III, increased the microvascular density of the myocardium, and suppressed the EndMT in heart tissue. In vitro, RLX decreased the mobility of human umbilical vein endothelial cells induced by TGF-β, increased the expression of endothelial CD31, and decreased vimentin content. Compared to TGF-β and RLX co-culture alone, TGF-β + RLX + Notch inhibitor increased cell mobility and the EndMT, but decreased the levels of Notch-1, HES-1, and Jagged-1 proteins.
Conclusion: RLX may inhibit the cardiac fibrosis via EndMT by Notch-mediated signaling.
Keywords: Notch; endothelial to mesenchymal transition; myocardial fibrosis; relaxin; transforming growth factor β.
Figures
Figure 1
Effect of RLX on cardiac function index in an isoproterenol (Iso)-induced myocardial fibrosis rat model. Notes: All experiments were performed in three repetitions. Data are mean ± SEM. **P<0.01 vs control, #P<0.05, ##P<0.01 vs Iso. Iso, 5 mg·kg−1·d−1; low-, middle-, and high-dose RLX at concentrations of 0.2, 2, and 20 μg·kg−1·day−1, respectively. Abbreviations: RLX, relaxin; LVSP, left-ventricular mean systolic pressure; LVEDP, left-ventricular end diastolic pressure; NS, normal saline; SEM, standard error of the mean; +dp/dtmax, the maximum pressure rise rate of left ventricle; -dp/dtmax, the maximum pressure drop rate of left ventricle.
Figure 2
Effect of RLX on fibrosis of the rat heart. Notes: (A) Hematoxylin and eosin staining of the LV myocardium. (B, C) Myocardial collagen areas by Masson staining. (D) Quantification of protein content of types I and III collagen. All experiments were performed in three repetitions. Data are mean ± SEM, **P<0.01 vs control, ##P<0.01 vs Iso; ££P<0.01 vs Iso. Abbreviations: RLX, relaxin; NS, normal saline; Iso, isoproterenol; LV, left ventricular; SEM, standard error of the mean.
Figure 3
Effect of RLX on mean MVD in rat heart. Notes: Von Willebrand factor was used as a marker and blood vessels were counted at ×200 magnification. The number of microvessels was calculated as the MVD in five randomly selected non-repeating visual fields. Data are mean ± SEM. **P<0.01, #P<0.05, ##P<0.01 vs Iso. All experiments were performed in three repetitions. Abbreviations: MVD, microvascular density; RLX, relaxin; NS, normal saline; Iso, isoproterenol; SEM, standard error of the mean.
Figure 4
Effect of RLX on endothelial to EndMT in rat heart. Notes: Immunofluorescence staining of α–smooth muscle actin (α-SMA) and CD31 to evaluate the EndMT in the fibrotic heart. Green fluorescence represents α-SMA; red fluorescence represents CD31. Data are mean ± SEM. **P<0.01, ##P<0.01 vs Iso. All experiments were performed in three repetitions. Abbreviations: EndMT, endothelial–mesenchymal transition; RLX, relaxin; NS, normal saline; Iso, isoproterenol; SEM, standard error of the mean.
Figure 5
Transwell assay of migration of HUVECs. Notes: Cells in five unrepeated visual fields were counted. All experiments were performed in three repetitions. Data are mean ± SEM. *P<0.05 vs control, #P<0.05 vs TGF-β, £P<0.05 vs TGF-β + RLX (200 ng·mL−1). Abbreviations: HUVECs, human umbilical vein endothelial cells; TGF-β, transforming growth factor β; RLX, relaxin; DAPT, N-[N-(3,5-difluorophenacetyl)-1-alanyl]-S-Phenylglycine t-butyl ester; SEM, standard error of the mean.
Figure 6
Immunofluorescence staining of CD31 and vimentin to evaluate the EndMT in HUVECs. Notes: (A) Green fluorescence represented CD31 (magnification ×400). (B) Green fluorescence represents vimentin. Blue fluorescence represented the nucleus of the cells (magnification ×200). All experiments were performed in three repetitions. (C) and (D) show the mean density of CD31 and vimentin, respectively. Data are mean ± SEM. *P<0.05, **P<0.01 vs control, #P<0.05, ##P<0.01 vs TGF-β, £P<0.05 vs TGF-β + RLX (200 ng·mL−1). Abbreviations: EndMT, endothelial–mesenchymal transition; HUVECs, human umbilical vein endothelial cells; TGF-β, transforming growth factor β; RLX, relaxin; DAPT, N-[N-(3,5-difluorophenacetyl)-1-alanyl]-S-Phenylglycine t-butyl ester; SEM, standard error of the mean.
Figure 7
Dual immunofluorescence staining of CD31 and vimentin to evaluate the EndMT in HUVECs. Notes: CD31 demonstrated red fluorescence and vimentin demonstrated green fluorescence (magnification ×400). All experiments were performed in three repetitions. Data are mean ± SEM. **P<0.01 vs control, ##P<0.01 vs TGF-β, ££P<0.01 vs TGF-β + RLX (200 ng·mL−1). Abbreviations: RLX, relaxin; TGF-β, transforming growth factor β; HUVECs, human umbilical vein endothelial cells; EndMT, endothelial–mesenchymal transition; DAPT, N-[N-(3,5-difluorophenacetyl)-1-alanyl]-S-Phenylglycine t-butyl ester; SEM, standard error of the mean.
Figure 8
Western blot analysis of the expression of CD31 and vimentin in HUVECs. Notes: All experiments were performed in three repetitions. Data are mean ± SEM. **P<0.01 vs control, #P<0.05, ##P<0.01 vs TGF-β, £P<0.05 vs TGF-β + RLX (200 ng·mL−1). Abbreviations: HUVECs, human umbilical vein endothelial cells; RLX, relaxin; TGF-β, transforming growth factor β; DAPT, N-[N-(3,5-difluorophenacetyl)-1-alanyl]-S-Phenylglycine t-butyl ester; SEM, standard error of the mean.
Figure 9
Western blot analysis of Notch cell signaling pathway. Notes: The expression of Notch-1 and its downstream proteins HES-1 and Jagged-1 were examined, and the inhibitor of Notch-1 DAPT was added. Blank control; negative control (DAPT); TGF-β-induced group; TGF-β + RLX (200 ng·mL−1); TGF-β + RLX (200 ng·mL−1) + DAPT; all experiments were performed in three repetitions. Data are mean ± SEM. **P<0.01 vs control, ##P<0.01 vs TGF-β, &&P<0.01 vs TGF-β + RLX (200 ng·mL−1). Abbreviations: RLX, relaxin; TGF-β, transforming growth factor β; DAPT, N-[N-(3,5-difluorophenacetyl)-1-alanyl]-S-Phenylglycine t-butyl ester; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SEM, standard error of the mean.
References
- Zeisberg EM, Tarnavski O. Endothelial to mesenchymal transition contributes to cardiac fibrosis. Nat Med. 2007;13:952–961. - PubMed
- Widyantoro B, Emoto N, Nakayama K, et al. Endothelial cell-derived endothelin-1 promotes cardiac fibrosis in diabetic hearts through stimulation of endothelial-to-mesenchymal transition. Circulation. 2010;121(22):2407–2418. - PubMed
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