Erythroblast transformation-specific 2 correlates with vascular smooth muscle cell apoptosis in rat heterotopic heart transplantation model

Abstract

Background: Cardiac allograft vasculopathy (CAV) decreases the long-term survival of heart transplantation recipients. Vascular smooth muscle cell (VSMC) apoptosis is an important pathological feature of CAV. Erythroblast transformation-specific 2 (Ets-2), as a transcription factor, participates in cell apoptosis and plays an important role in organ transplantation.

Methods: Hearts from Wistar-Furth (WF:RT1u) rats were heterotopically transplanted into Lewis (Lew:RT1(l)) rats without immunosuppression. Additional syngeneic heterotopic cardiac transplantations were performed in Lewis rats. HE staining was used to identify CAV. Ets-2 expression was examined by western blot. Ets-2 tissue location was examined by immunohistochemical assay and double immunostaining. Cleaved caspase 3 expression was detected by western blot. Co-localization of Ets-2 and cleaved caspase 3 was detected by double immunostaining. Ets-2, p53, cleaved caspase 3 and Bcl-xl expression in rat VSMC line A7R5 was examined after Ets-2 siRNA transfection. TUNEL assay was applied to detect A7R5 apoptosis with or without ETS-2 siRNA transfection. Immunoprecipitation was performed to explore the interaction between Ets-2 and p53.

Results: Ets-2 expression decreased in the allograft group but had no obvious change in the isograft group. Meanwhile, the phenomenon of CAV was observed in the allograft group and there is neointima formation in the isograft group which is not obvious compared with allograft group. Additionally, Ets-2 expression was opposite to VSMC apoptosis in the allograft group. In vitro, Ets-2 siRNA transfection in A7R5cells resulted in enhanced cell apoptosis. Finally, Ets-2 interacted with p53.

Conclusions: Ets-2 might inhibit VSMC apoptosis via p53 pathway. The results further elucidate the molecular mechanism of VSMC apoptosis after heart transplantation during CAV and provide theoretical basis for seeking new specific drug targets for CAV prevention and treatment.

Keywords: Cardiac transplantation; cardiac allograft vasculopathy (CAV); erythroblast transformation-specific 2 (Ets-2); vascular smooth muscle cell apoptosis (VSMC apoptosis).

Figure 1

The average survival rate of heterotopic heart transplantation. Isograft survival rate was significantly higher than that of allografts (isograft median survival time >175 days vs. allograft median survival time of 21±2.0 days; n=9 in each group, *P<0.001, log-rank test).

Figure 2

HE staining of allografts and isografts. (A) Allografts and isografts at day 0 (a,f); allografts and isografts at day 7 (b,g); allografts and isografts at day 14 (c,h); allografts and isografts at day 21 (d,i); allografts and isografts at day 28 (e,j); scale bar =100 µm; (B) the bar chart demonstrated intimal thickness in isograft and allograft groups by densitometry; the data are mean ± SEM of three independent experiments, *P<0.05. HE, hematoxylin and eosin.

Figure 3

Erythroblast transformation-specific 2 (Ets-2) expression in allografts and isografts. (A,B) Ets-2 protein level was detected by western blot; (C) the bar chart demonstrated the ratio of Ets-2 to β-actin, the data are mean ± SEM of three independent experiments, *P<0.05; (D) immunohistochemistry identified tissue location of Ets-2 in isografts and allografts; scale bar =100 µm; the bar chart demonstrated the percentage of Ets-2 positive cells; the data are mean ± SEM of three independent experiments, *P<0.05; (E) the co-localization of Ets-2 and α-SMA was determined by immunofluorescent at day 21 following heart transplantation; the bar chart demonstrated the percentage of co-localized cells; the data are mean ± SEM of three independent experiments, *P<0.05. VSMC, vascular smooth muscle cell.

Figure 4

Erythroblast transformation-specific 2 (Ets-2) is associated with VSMC apoptosis after heart xenotransplantation. (A) Western blot was probed for cleaved caspase 3 and procaspase 3 in allografts and isografts; (B) the bar chart demonstrates the ratio of cleaved caspase 3 to β-actin; the data are mean ± SEM of three independent experiments, *P<0.05; (C) double immunofluorescence staining is for cleaved caspase 3 and Ets-2 in isografts and allografts at day 21 post-transplant; the bar chart demonstrated the percentage of co-localized cells; the data are mean ± SEM of three independent experiments, *P<0.05. VSMC, vascular smooth muscle cell.

Figure 5

Erythroblast transformation-specific 2 (Ets-2) inhibits vascular smooth muscle cell (VSMC) apoptosis after heart transplantation. (A) Ets-2 expression is examined by western blot after Ets-2 siRNA transfection; (B) the bar chart indicated the ratio of Ets-2 to β-actin by densitometry; (C) TUNEL staining. Data are represented as mean ± SEM from three independent experiments. *P<0.05, compared with control siRNA group; (D) western blot analysis revealed p53, cleaved caspase 3 and Bcl-xl expression in Ets-2 depletion cells; (E) the bar chart indicated the ratio of p53, cleaved caspase 3 and Bcl-xl o β-actin by densitometry; (F) reciprocal immunoprecipitation of Ets-2 and p53 was performed in A7R5 cells.