The relationship between miRNA-26b and connective tissue growth factor in rat models of aortic banding and debanding

Abstract

Background/aims: Connective tissue growth factor (CTGF) is a profibrotic factor implicated in pressure overload-mediated myocardial fibrosis. In this study, we determined the role of predicted CTGF-targeting microRNAs (miRNAs) in rat models of aortic stenosis and reverse cardiac remodeling.

Methods: Minimally invasive ascending aortic banding was performed in 24 7-week-old male Sprague-Dawley rats, which were divided into three groups. The banding group consisted of eight rats that were sacrificed immediately after 6 weeks of aortic constriction. The debanding group underwent aortic constriction for 4 weeks and was sacrificed 2 weeks after band removal. The third group underwent sham surgery. We investigated the expression of CTGF, transforming growth factor-β1 (TGFβ1), and matrix metalloproteinase-2 using ELISA and examined miRNA-26b, miRNA-133a, and miRNA-19b as predicted CTGF-targeting miRNAs based on miRNA databases in 24-hour TGFβ-stimulated and TGFβ- washed fibroblasts and myocardial tissues from all subjects.

Results: CTGF was elevated in 24-hour TGFβ-stimulated fibroblasts and decreased in 24-hour TGFβ-washed fibroblasts. miRNA-26b was significantly increased in TGFβ-washed fibroblasts compared with control and TGFβ-stimulated fibroblasts (p < 0.05). CTGF expression was significantly higher in the banding group than that in the sham and debanding groups. The relative expression levels of miRNA-26b were higher in the debanding group than in the banding group.

Conclusion: The results of our study using models of aortic banding and debanding suggested that miRNA-26b was significantly increased after aortic debanding. The in vitro model yielded the same results: miRNA-26b was upregulated after removal of TGFβ from fibroblasts.

Keywords: Aortic banding; Aortic debanding; Connective tissue growth factor; Fibrosis; miRNA-26b.

Figure 1.

Fibrosis in Rat2 cells was identified on the basis of the expression of collagen I and α-smooth muscle actin (αSMA). Upon transforming growth factor-β (TGFβ) treatment, Collagen I increased from 18 to 24 hours, decreased by 30 hours and then increased again by 42 hours (A). After TGFβ treatment, αSMA continuously increased from 18 to 30 hours and then decreased until 42 hours. In addition, collagen I and αSMA decreased when TGFβ was removed (B). To verify the association between TGFβ-associated fibrosis and the expression of connective tissue growth factor (CTGF), CTGF expression in Rat2 cells was examined by real-time polymerase chain reaction. The expression of CTGF was significantly increased after 24 hours of TGFβ stimulation and decreased from 6 to 24 hours after 24 hours of TGFβ washing (p < 0.05) (C) . All experiments were repeated three times for each group. The bars and error bars indicate the mean ± SD. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Figure 2.

Gross findings using Masson’s trichrome staining. Left ventricles detached from a sham rat (A, D), a rat that underwent the 6-week aortic banding procedure (B, E) and a rat that underwent the aortic debanding procedure (C, F) revealed concentric hypertrophied myocardium and increased global longitudinal strain (GLS). Concentric remodeling was relieved by the debanding operation (relative wall thickness [RWT] = 0.5), but microscopically reactive myocardial fibrosis was not significantly regressed (C, F). Compared with the GLS in the banding group, the GLS in the debanding group was not significantly improved.

Figure 3.

Connective tissue growth factor (CTGF) was significantly higher in the banding group than that in the sham and debanding groups (A). Matrix metalloproteinase-2 (MMP2) and transforming growth factor-β1 (TGFβ1) tended to increase in the banding group and were significantly decreased in the debanding group (B, C). The bars and error bars indicate the mean ± SD.

Figure 4.

To analyze the fibrosis-related microRNAs (miRNAs) associated with the expression of connective tissue growth factor (CTGF), real-time polymerase chain reaction was performed using miRNA-specific primers. miRNA-26b, miRNA-133a, and miRNA-19b were evaluated in vivo and in vitro in the control group, the transforming growth factor-β (TGFβ)-stimulated group (the remodeling group), and the 24-hour TGFβ-washed group (the reverse remodeling group). Among these miRNAs, miRNA-26b was significantly elevated in the TGFβ-washed group (p < 0.05). miRNA-19b was significantly decreased in the TGFβ-stimulated group and the 24-hours TGFβ-washed group compared with the control group (p < 0.05). All experiments were repeated three times for each group (A-C). In vivo, the relative expression level of miRNA-26b was significantly higher in the debanding group than in the banding group (D). However, the relative expression level of miRNA-133a and miRNA-19b did not significantly change in our study (E,F). The bars and error bars indicate the mean ± SD.

Figure 5.

(A) Transfected of fibroblasts with microRNA (miRNA)-26b mimic decreased the relative expression of connective tissue growth factor (CTGF). (B) Stimulation of fibroblasts with transforming growth factor-β1 (TGFβ1) increased the relative expression of CTGF and decreased that of miRNA-26b. After TGFβ1 washing, CTGF expression was decreased, and miRNA-26b expression was increased.