Original article
Inhibition of intimal hyperplasia after stenting by over-expression of p15: A member of the INK4 family of cyclin-dependent kinase inhibitors

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Abstract

We evaluated the role of p15Ink4, a member of the INK4 family of CDK inhibitors on vascular smooth muscle cells (VSMCs) proliferation, cell cycle progression and intimal hyperplasia after stenting. Aortic VSMCs transduced with either adenovirus encoding for p15Ink4 or β-galactosidase were assessed for DNA synthesis, cell cycle progression, and pRb phosphorylation. Rabbit carotid arteries were stented and treated with peri-adventitial delivery of saline or adenovirus encoding for p15Ink4 or β-galactosidase. p15Ink4 transgene and protein expression were evaluated at 24 h and 72 h, respectively. In-stent cell proliferation was evaluated by BrdU at day 7. Histomorphometric analysis of in-stent intimal hyperplasia was performed at 10 weeks. Human p15Ink4 DNA was detected in transduced VSMCs at 24 h. p15Ink4 over-expression reduced VSMCs DNA synthesis by 60%. Cell cycle progression was inhibited, with a 30% increase in G1 population accompanied by inhibition of pRb phosphorylation. Human p15Ink4 transgene was identified in transduced stented arteries but not in control arteries. p15Ink4 immunostaining was increased and cell proliferation significantly reduced by 50% in p15Ink4 transduced arteries. Intimal cross-sectional area (CSA) of p15Ink4-treated group was significantly lower than the β-gal treated and non-transduced groups (p = 0.008). There were no differences in the intimal or medial inflammatory response between groups. p15Ink4 over-expression blocks cell cycle progression leading to inhibition of VSMCs proliferation. Peri-adventitial delivery of p15Ink4 significantly inhibits in-stent intimal hyperplasia.

Research highlights

►We examined the in vitro and in vivo effects of p15Ink4 (a member of the INK4 family of CDK inhibitors) over-expression on smooth muscle cell proliferation, cell cycle progression and intimal hyperplasia after stenting. ►Over-expression of p15Ink4 results in marked inhibition of SMCs DNA synthesis and proliferation. Cell cycle progression was inhibited accompanied by inhibition of pRb phosphorylation. ►In vivo, intimal hyperplasia was significantly decreased in the p15Ink4-transduced stented arteries as compared to controls.

Introduction

Vascular smooth muscle cell (VSMC) proliferation is a critical event in the mechanism of restenosis after arterial balloon injury and stenting. After arterial injury, vascular VSMCs are stimulated to proliferate in response to several growth factors and mitogens. This stimulation causes the cells to exit the G0 phase and progress through the G1 phase into the S phase with subsequent mitosis at the M phase leading to cell division [1]. The critical progression from G1 into S phase is regulated by the G1 cyclin/cyclin-dependent kinase (CDK) complexes [2]. Endogenous inhibitors of the cyclin/CDK complexes (CKIs) regulate the activity of CDKs. Two groups of CKIs are known to be important in vascular biology: (1) the CIP/KIP family, which includes p21, p27, and p57 and (2) the INK4 family, which includes p15, p16, p18, and p19 [3]. Interestingly, p27Kip1 was found to play a critical role in mediating the antiproliferative and anti-migratory properties of Rapamycin [4], [5] and alterations in all INK4 genes were also found to be associated with several hematological malignancies [6]. A potential strategy to block VSMCs proliferation and prevent restenosis is to over-express specific CKI genes in the arterial wall. Chang et al. [7] have initially shown that adenovirus-mediated over-expression of a member of the CIP/KIP cell cycle inhibitors, p21, inhibits growth factor-stimulated VSMC proliferation in vitro by efficiently arresting VSMCs in the G1 phase of the cell cycle. In addition, it was demonstrated that localized arterial transduction with a p21-encoding adenovirus at the time of balloon angioplasty significantly reduced neointimal hyperplasia in the rat carotid artery model of restenosis.

Support for the above came from Yang et al. [8] demonstrating that the induction of p21 in porcine arteries following balloon injury results in inhibition of intimal cell proliferation. Later on, this group has demonstrated that p27 and p21 (CIP/KIP members) are both potent inhibitors of VSMCs growth, while p16 (INK4 member) was only a weak inhibitor [9], [10].

To further evaluate the role of INK4 family members on restenosis, we studied the effects of p15Ink4 on VSMCs proliferation, cell cycle progression and in-stent intimal hyperplasia.

Section snippets

p15 expression in quiescent and proliferating VSMC

Rat aortic SMCs (Catalogue #CRL-2018, ATCC) were initially grown to 100% confluence in 10% fetal calf serum[FCS]/Dulbecco Modified Eagle's medium [DMEM] media with 0.2 mg/ml G418. Cells were then either grown in 0.25% FCS/DMEM media for 24 h to induce quiescence or in 10% FBS/DMEM until approximately 80% confluence. Cells were harvested for flow cytometry or Western blot analysis. For flow cytometry, 500,000 cells were resuspended in 50 μl of phosphate buffered saline [PBS] with 2% calf serum and

In vitro

There was a modest increase in p15Ink4 protein expression in quiescent compared to proliferating rat aortic smooth muscle cells (Fig. 1). Flow cytometry analysis confirmed the reduction of number of VSMCs in S1 phase from 15.8–9.8% in 10% and 0.25% FCS, respectively.

At 24 h after cell transduction, an approximately 300 bp human p15Ink4 DNA fragment was identified in p15-transduced cells but not in β-gal transduced (Fig. 2) and non-transduced cells (data not shown). Over-expression of p15Ink4

Discussion

The cell cycle is an important target to prevent VSMCs proliferation and restenosis after arterial injury. The effects on VSMCs proliferation of the Kip/Cip CKIs compared to INK group of inhibitors have been evaluated by Tanner et al. [10]. The CDK inhibitor p27Kip1 was shown to be constitutively expressed in normal arteries, down regulated after arterial injury, and upregulated again during the later phases of arterial repair; the temporal expression of p27Kip1 was thus inversely correlated

Acknowledgments

We gratefully acknowledge the excellent technical work of the Pathology and Laboratory Medicine Research Service Group at Mount Sinai Hospital and the University Health Network, Toronto, Canada.

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    This work was supported by a generous donation from the Koschitzky Family.

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