Inhibition of ERK or Akt ameliorates intimal hyperplasia via up-regulation of Cx37 and down-regulation of Cx43 in balloon injury rat model

Abstract

Background: Connexins (Cxs) are reported to participate in atherosclerosis associated intimal hyperplasia (IH), while their function involved in the balloon injury (BI) induced IH and restenosis is less reported.

Methods: Forty-eight male Sprague-Dawley rats were randomly assigned to not injured (NI) group and BI group, which were further administrated with ERK-inhibitor U0216 and Akt-inhibitor MIK2206. Western blot and RT-PCR were utilized to detect the expression of Cx30, Cx37, Cx40, and Cx43 at 6 hours, 24 hours, 7 days, and 14 days post-surgery. H&E staining and related intima area, media area, and luminal area measurement were applied to indicate neointima formation and IH. ERK and Akt phosphorylation levels and proliferating cell nuclear antigen (PCNA) immunostaining were also detected.

Results: Among the four Cxs detected, Cx37 showed down-regulated, and Cx43 showed up-regulated temporal expression pattern in BI rats with confirmed neointima formation. Up-regulated p-ERK (P<0.01) and p-Akt (P<0.01) can be detected in BI rats compared with NI rats. Meanwhile, U0216 and MIK2206 can significantly reduce Cx43 expression and increase CX37 expression accompanied with reduced neointima formation and PCNA staining (P<0.05 or P<0.01) in BI rats.

Conclusions: ERK or Akt inhibition can alleviate BI-induced IH via up-regulation of Cx37 and down-regulation of Cx43.

Keywords: Akt; Cx37; Cx43; ERK; intimal hyperplasia (IH).

Figure 1

Temporal expression of connexins in the rat carotid artery after balloon injury. 48 male rats were divided into two groups, BI group (n=24) that accepted balloon injury and NI group (n=24) without injury as the control. And every 6 rats of the two groups were sacrificed at 6 hours, 24 hours, 7 days and 14 days after BI surgery for assessments. (A) Western blotting analysis of four kinds of connexins. Protein levels of Cx30, Cx37, Cx40, and Cx43 were analyzed, and β-actin was utilized as a control. (B) Quantification of Western blotting data. Connexin/β actin of BI groups was normalized against that of NI groups. **, P<0.01; ***, P<0.001. (C) Analysis of mRNA levels of the connexins by qRT-PCR. The mRNA levels of connexins were first normalized to β-actin mRNA levels, and the values were further normalized to the value of the respective 6-hour group for comparison. Normalized mRNA levels of Cx30 and Cx37 of BI groups were shown in the left-panel, and those of Cx40 and Cx43 were shown in the right panel. **, P<0.01; ***, P<0.001, versus 6 h groups. Bar graph indicates mean ± SEM. BI, balloon injury; NI, not injured.

Figure 2

Neointima formation was enhanced after balloon injury. Each group (N=6) of BI and NI rats were sacrificed on day 14 for assessment. (A) Representative H&E staining photomicrographs of cross-sections of the left common carotid arteries. L, lumen; M, media; I, intima; N, neointima. (B) Quantification of the intima area. (C) Quantification of the media area. (D) Quantitative analysis of the luminal area. (E) Quantitation of the intima-to-media (I/M) ratio. **, P<0.01; BI rats versus NI rats. Bar graph indicates mean ± SEM. BI, balloon injury; NI, not injured.

Figure 3

Phosphorylation levels of ERK and Akt correlate to protein levels of Cx37 and Cx43 in the balloon injury rat model. Each group (N=6) of NI, BI, BI treated by MIK2206 or U0216 rats were sacrificed on day 14 for assessment. (A) Western analysis of phosphorylation levels of Akt and ERK. (B) Quantification of the Akt and ERK phosphorylation levels. (C) Western analysis of changes of Cx37 and Cx43 protein levels by treatment of ERK and Akt inhibitor. (D,E) Quantitative analysis of change of Cx37 and Cx43 protein levels. **, P<0.01; ***, P<0.001. Bar graph indicates the mean ± SEM. BI, balloon injury; NI, not injured.

Figure 4

Inhibition of ERK and Akt reduced neointima formation and cell proliferation in the balloon-injured rat model. (A) Representative H&E staining photomicrographs of carotid arteries for NI rats and untreated, MK2206-treated, and U0126-treated BI rats on day 14 surgery. Magnification 200×. (B) Quantification of the intima-to-media ratio. (C) Representative PCNA immunostaining 14 days post carotid ligation in NI, BI, MK2206-treated BI and U0126-treated BI rats. Magnification 200×. (D) Quantitative analysis of the luminal area. (E) Quantitative assessment of PCNA positive area over the total carotid area. *, P<0.05; **, P<0.01, versus BI rats. ^#, P<0.05; ^##, P<0.01, versus NI rats. Bar graph indicates the mean ± SEM. BI, balloon injury; NI, not injured; PCNA, proliferating cell nuclear antigen.