Regulation of cell cycle entry by PTEN in smooth muscle cell proliferation of human coronary artery bypass conduits

Abstract

Proliferation of smooth muscle cells (SMCs) is the key event in the pathogenesis of intimal hyperplasia (IH) leading to coronary artery bypass graft (CABG) occlusion. The saphenous vein (SV) conduits are often affected by IH, while the internal mammary artery (IMA) conduits remain remarkably patent. SMC proliferation is mediated by the cell cycle, under the control of cyclin-dependent kinases (cdks), cdk-inhibitors and the retinoblastoma protein (Rb). Early passage of the SMCs through the cell cycle involves crossing the non-reversible G(1) checkpoint, the restriction (R) point. In this study, we investigated the effect of mitogenic insulin-like growth factor (IGF)-1 stimulation on the R-point and its relationship with the phosphorylation of Rb protein and the cdk inhibitors p21 and p27 in SV and IMA SMCs. We observed no change in the R-point following IGF-1 activation in either SV or IMA SMCs. However, Rb-phosphorylation occurred much earlier and was quantitatively greater in SV SMCs than IMA. Overexpression of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) in SV SMCs followed by IGF-1 activation significantly decreased the expression of cyclin E and pRb and induced p27 expression in SV SMCs, while, pRb levels were markedly decreased and p27 levels were significantly increased in IMA SMCs. Silencing the PTEN gene by siRNA transfection of IMA SMCs significantly induced the expression of pRb and inhibited p27 expression, while, the expression levels of cyclin E, pRb, p21 and p27 were unaffected by the silencing of PTEN in SV SMCs. These results demonstrate that the PTEN plays a critical role in regulating cell cycle entry. Therefore, overexpression of PTEN possibly by means of gene therapy could be a viable option in regulating the cell cycle in SV SMCs in the treatment of vein graft disease.

1

Effect of IGF-1 stimulation on the determination of R-point based on cyclin E expression. Cyclin E expression marks the R-point, which occurs at G1/S transition. Thus, cyclin E accumulation is used to assess the R-point. SV (left panel) and IMA (right panel) SMCs were stimulated with IGF-1 (100 ng/ml) over a time course of 0–5 hrs. Cyclin E levels were determined by Western blotting and the results are graphically represented as densitometric analysis of five individual samples of SV and IMA cells. (**P < 0.001)

2

Phosphorylation of Retinoblastoma protein (ser 608) following IGF-1 activation. SV and IMA SMCs were stimulated with IGF-1 (100 ng/ml) over a time course of 0–24 hrs. Phosphorylation of retinoblastoma protein was determined by Western blotting and densitometric analysis of five individual samples of SV and IMA cells is represented graphically. The phosphorylation of Rb is shown with relation to the restriction (R) point. (*P < 0.001; *P < 0.05)

3

Effect of IGF-1 activation on the expression of cdk inhibitor p21. SV and IMA SMCs were treated with IGF-1 (100 ng/ml) over a time-period extending from 0–24 hrs. p21 was determined by Western blotting and densitometric analysis of five individual samples of SV and IMA cells is represented graphically. GAPDH was used as loading control. There was no significant difference between any of the time points.

4

Effect of IGF-1 activation on the expression of cdk inhibitor p27. SV and IMA SMCs were treated with IGF-1 (100 ng/ml) over a time-period extending from 0–24 hrs. p27 was determined by Western blotting and the densitometric analysis of five individual samples of SV and IMA cells is represented graphically. GAPDH was used as loading control. There was no significant difference between any of the time-points.

5

The effect of IGF-1 activation on cell cycle proteins in pORF9-hPTEN-transfected SV SMCs. SV cells were transiently transfected with PTEN vector to overexpress the protein. Transfected SV SMCs were treated with IGF-1 (100 ng/ml) for 5 hr or 12 hrs. Expression levels of cyclin E (A), pRb, p21 and p27 (B) were determined by Western blotting and the densitometric analysis of five individual samples of SV cells (C) is represented graphically. (*P < 0.01 compared with control group. #P < 0.01 compared with IGF-1 + empty vector).

6

The effect of IGF-1 activation on cell cycle proteins in pORF9-hPTEN-transfected IMA SMCs. IMA cells were transiently transfected with PTEN vector to overexpress the protein. Transfected IMA SMCs were treated with IGF-1 (100 ng/ml) for 5 hrs or 12 hrs. Expression levels of cyclin E (A), pRb, p21 and p27 (B) were determined by Western blotting and the densitometric analysis of five individual samples of IMA cells (C) is represented graphically. (*P < 0.01 compared with control group. #P < 0.01 compared with IGF-1 + empty vector).

7

Effect of PTEN gene silencing on the expression of cyclin E, phosphorylation of Rb protein, p21 and p27 in SV SMCs. The transfected cells were stimulated with IGF-1 (100 ng/ml). Expression levels of cyclin E (A) and pRb, p21 and p27 (B) were determined by Western blotting and the densitometric analysis of five individual samples of SV cells (C) is represented graphically. (*P < 0.01 compared with control group. #P < 0.01 compared with IGF-1 + empty vector).

8

Effect of PTEN gene silencing on the expression of cyclin E, phosphorylation of Rb protein, p21 and p27 in IMA SMCs. The transfected cells were stimulated with IGF-1 (100 ng/ml). Expression levels of cyclin E (A), pRb, p21 and p27 (B) were determined by Western blotting and the densitometric analysis of five individual samples of IMA cells (C) is represented graphically. (*P < 0.01 compared with control group. #P < 0.01 compared with IGF-1 + empty vector).