Effects of rosuvastatin on expression of angiotensin-converting enzyme 2 after vascular balloon injury in rats

Abstract

Objective: To investigate the effects and mechanisms of rosuvastatin on angiotensin -converting enzyme 2 (ACE2) in the process of neointimal formation after vascular balloon injury in rats, and to explore the effects of ACE2 and rosuvastatin in restenosis.

Methods: Thirty-six Wistar rats were randomly allocated into three groups: control group (n = 12), surgery group (n = 12), and statin group (n = 12). Aortic endothelial denudation of rats was performed using 2F balloon catheters. At days 14 and 28 after injury, aortic arteries were harvested to examine the following. Intimal thickening was examined by hematoxylin and eosin staining. We measured angiotensin II (Ang II) and angiotensin 1-7 (Ang-[1-7]) levels by a radioimmunological method or enzyme-linked immunosorbent assay. Protein and mRNA expression of ACE2 and Ang II type 1 receptor (AT1) were investigated by immunohistochemistry, Western blots, and Reverse transcriptase-polymerase chain reaction (RT-PCR). We measured changes in proliferating cell nuclear antigen (PCNA) by immunohistochemistry. The level of phosphorylated extracellular signal regulated kinase 1/2 (P-ERK1/2) was evaluated by Western blotting.

Results: Proliferation of vascular smooth muscle cells (VSMC) and intimal thickening were higher at day 14 after vascular balloon injury in the surgery group compared with the control group. Proliferation of VSMC was decreased by day 28 after injury, while intimal thickening continued. With rosuvastatin treatment, the extent of VSMC proliferation and intimal thickening was reduced at day 14 and 28 after injury. Ang II and P-ERK levels were significantly increased, Ang-(1-7) levels were significantly decreased, mRNA and protein expressions of ACE2 were significantly decreased, and AT1 expression was significantly increased at days 14 and 28 after vascular balloon injury in the surgery group compared with the control group. PCNA expression was higher in the surgery group than in the control group, and it was significantly decreased after being given rosuvastatin. Expression of ACE2 mRNA and protein, and Ang-(1-7) levels were significantly increased, while AT1 expression and levels of Ang II and P-ERK were significantly decreased in the statin group compared with the surgery group.

Conclusions: Expression of ACE2 mRNA and protein is decreased in the process of intimal thickening after balloon injury. The inhibitory effect of rosuvastatin on intimal thickening is related to upregulation of ACE2, an increase in Ang-(1-7), downregulation of AT1, and activation of the P-ERK pathway.

Keywords: Angiotensin converting enzyme 2; Balloon injury; Extracellular signal regulated kinase; Rosuvastatin.

Figure 1.. Effects of rosuvastatin on histological changes at day 14 after balloon injury.

1: intima. 2: media. Pathological sections of arterial lesions by HE staining showed that the intima and vessel walls were thin in the control group (A), but the intima was thick and there were more proliferated VSMCs than in the surgery group (B). In the rosuvastatin group (C), the intima was thinner and there were less proliferated VSMCs than in the surgery group (× 200). VSMCs: vascular smooth muscle cells.

Figure 2.. Ang II and Ang-(1–7) levels in the aorta.

At days 14 (A) and 28 (B), surgery group rats showed higher plasma Ang II levels and lower Ang-(1–7) levels compared with the control group. Pretreatment with rosuvastatin decreased plasma Ang II and increased Ang-(1–7) levels. Values are expressed as mean ± SE (n = 6). *P < 0.05, **P < 0.01 vs. the control group; ^#P<0.05, ^##P<0.01 vs. the surgery group. Ang II: angiotensin II; Ang-(1–7): angiotensin 1–7.

Figure 3.. ACE2 and AT₁ mRNA levels.

Expression of ACE2 (A) and AT₁ (B) mRNA are shown at day 14 after injury; (C) ACE2 and AT₁ mRNA at days 14 and 28 after injury. ACE2 mRNA expression was decreased and AT₁ mRNA was increased after injury, but ACE2 mRNA was increased and AT₁ mRNA was decreased after rosuvastatin treatment. Values are expressed as mean ± SE (n = 6). *P < 0.05, **P < 0.01 vs. the control group; ^#P < 0.05, ^##P < 0.01 vs. the surgery group. 1: control group; 2: surgery group; 3: statin group; 4: marker. ACE2: angiotensin -converting enzyme 2; AT₁: Ang II type 1 receptor.

Figure 4.. ACE2 and AT1 protein levels by Western blot.

(A): ACE2 and AT₁ levels shown by Western blot at day 14 after injury; (B): ACE2 and AT₁ protein levels shown by Western blot at days 14 and 28 after injury. ACE2 levels were decreased and AT₁ levels were increased after injury, but ACE2 levels were increased and AT₁ levels were decreased after rosuvastatin treatment. Values are expressed as mean ± SE (n = 6).*P < 0.05, **P < 0.01 compared with the control group; ^#P < 0.05, ^##P < 0.01 compared with the surgery group. ACE2: angiotensin -converting enzyme 2; AT1: Ang II type 1 receptor.

Figure 5.. ACE2 (A) and AT₁ (B) expression in the control group, surgery group and statin group.

ACE2 and AT₁ protein are expressed in the membrane and cytoplasm (brown-yellow particles) in vascular endothelial cells and smooth muscle cells. ACE2 protein expression was lower (brown granules were significantly lower) and AT₁ protein expression was higher (brown granules were significantly higher) in the surgery group compared with the control group. After rosuvastatin treatment, ACE2 expression was significantly higher and AT₁ expression was significantly lower than in the surgery group. ACE2: angiotensin -converting enzyme 2; AT₁: Ang II type 1 receptor.

Figure 6.. PCNA expression in VSMCs 14 days after aortic injury (× 400).

(A): Control group; B: surgery group; C: statin group. Black lines show the neointima and red arrows indicate a brown positive nucleus. PCNA expression was significantly higher in the surgery group than in the control group, but it was lower in the statin group. PCNA: proliferating cell nuclear antigen; VSMCs: vascular smooth muscle cells.

Figure 7.. Expression of P-ERK1/2 by Western blot at day 14 after injury.

(A) Western blot showing P-ERK expression; (B) P-ERK protein levels. P-ERK1/2 protein expression was higher in the surgery group than in the control group, and it was significantly lower than in the statin group. Values are expressed as mean ± SE (n = 6).*P<0.05, **P<0.01 compared with the control group; ^#P < 0.05, ^##P < 0.01 compared with the control group. P-ERK: phosphorylated extracellular signal regulated kinase.