Differential Effects of the Cyclin-Dependent Kinase Inhibitors p27Kip1, p21Cip1, and p16Ink4 on Vascular Smooth Muscle Cell Proliferation
Background—The cyclin-dependent kinase inhibitors (CKIs) have different patterns of expression in vascular diseases. The Kip/Cip CKIs, p27Kip1 and p21Cip1, are upregulated during arterial repair and negatively regulate the growth of vascular smooth muscle cells (VSMCs). In contrast, the Ink CKI, p16Ink4, is not expressed in vascular lesions. We hypothesized that a variation in the inactivation of cdk2 and cdk4 during the G1 phase of the cell cycle by p27Kip1, p21Cip1, and p16Ink4 leads to different effects on VSMC growth in vitro and in vivo.
Methods and Results—The expression of p27Kip1 and p21Cip1 in serum-stimulated VSMCs inactivated cdk2 and cdk4, leading to G1 growth arrest. p16Ink4 inhibited cdk4, but not cdk2, kinase activity, producing partial inhibition of VSMC growth in vitro. In an in vivo model of vascular injury, overexpression of p27Kip1 reduced intimal VSMC proliferation by 52% (P<0.01) and the intima/media area ratio by 51% (P<0.005) after vascular injury and gene transfer to pig arteries, when compared with control arteries. p16Ink4 was a weak inhibitor of intimal VSMC proliferation in injured arteries (P=NS), and it did not significantly reduce intima/media area ratios (P=NS), which is consistent with its minor effects on VSMC growth in vitro.
Conclusions—p27Kip1 and p21Cip1 are potent inhibitors of VSMC growth compared with p16Ink4 because of their different molecular mechanisms of cyclin-dependent kinase inhibition in the G1 phase of the cell cycle. These findings have important implications for our understanding of the pathophysiology of vascular proliferative diseases and for the development of molecular therapies.
Arterial vascular smooth muscle cells (VSMCs) are normally quiescent, proliferate at low indices (<0.05%), and stay in the G0/G1 phase of the cell cycle.1 After vascular injury, VSMCs are stimulated to divide in response to mitogens, and they exit the G1 phase and enter the S phase. Progression through G1 and entry into the S phase is regulated by the formation and activation of cyclin/cyclin-dependent kinase (CDK) complexes, predominantly cyclin D-cdk4/6 and cyclin E-cdk2.2 Cyclin-dependent kinase inhibitors (CKIs) are naturally occurring gene products that inhibit the cyclin-CDK activity leading to G1 arrest.3
The CKIs p27Kip1 and p21Cip1 inactivate the cyclin-CDK complexes in the G1 phase leading to cell cycle arrest, and thus function in growth regulation and wound repair. p27Kip1 is constitutively expressed in normal arteries, is downregulated after arterial injury, becomes upregulated during the later phases of arterial repair, and is inversely correlated with VSMC proliferation.4 A deficiency of p27Kip1 in mice leads to benign hyperplasia in multiple organs, but it does not directly produce tumors.5 p16Ink4 is not expressed in normal or injured arteries, but deletions or mutations of p16Ink4 have been described in human malignancies.6 The molecular mechanisms accounting for the differences in the expression and function of p27Kip1, p21Cip1, and p16Ink4 in blood vessels are not known.
We hypothesized that differences in the activation of cdk2 and cdk4 by p27Kip1, p21Cip1, and p16Ink4 would lead to differential effects on VSMC growth. To test this hypothesis, we examined the effects of p27Kip1, p21Cip1, and p16Ink4 on cdk2 and cdk4 activity, G1 growth arrest, and VSMC proliferation in vitro and in vivo.
Cell Culture and Adenoviral Infection
Primary porcine aortic VSMCs were isolated by an explantation method. Recombinant replication-defective adenoviral vectors encoding human p27Kip1, p21Cip1, p16Ink4, and a control vector, AdΔE1, were constructed in an E1A/E3-deleted adenoviral plasmid containing a cytomegalovirus promoter and bovine growth human polyadenylation signal.7 Adenoviral infection of VSMCs was performed at a multiplicity of infection of 1000.
Cell Cycle Analysis, Immunoprecipitation, and Kinase Assay
VSMCs were cultured and infected with adenoviral vectors as previously described.7 Cell cycle distribution was analyzed for DNA content using propidium iodide in flow cytometry. Cdk2 was immunoprecipitated with a rabbit polyclonal antibody, with histone H1 as a substrate. Cdk4 was immunoprecipitated with a goat polyclonal antibody, with Rb as a substrate.8
In Vivo Gene Transfer
p27Kip1 and p16Ink4 were expressed in pig femoral arteries by adenoviral gene transfer.7 Control arteries were either infected only with AdΔE1 vectors or were not infected (n=10 in each group). Proliferation indices were measured 7, 14, and 21 days after gene transfer using an immunohistochemical analysis of bromodeoxyuridine incorporation. Intima and media areas were measured 21 days after gene transfer using an image analysis system.7 Animal experiments were performed in accordance with institutional animal care guidelines.
Effect of CKIs on VSMC Proliferation and Kinase Activity
We first examined the effect of p27Kip1, p21Cip1, and p16Ink4 expression on the proliferation of VSMCs in vitro. Overexpression of p27Kip1 and p21Cip1 resulted in the complete inhibition of proliferation in serum-stimulated VSMCs, and p16Ink4 induced only a partial inhibition (Figure 1A⇑); all had equivalent levels of transfection efficiency (P<0.0001, p27Kip1 and p21Cip1 versus p16Ink4 and controls). To determine the phase of the cell cycle in which the VSMCs were arrested, the cells were stained with propidium iodide, and the DNA content was analyzed by flow cytometry. p27Kip1, p21Cip1, and p16Ink4 promoted G1 arrest compared with AdΔE1-infected and noninfected VSMCs (Figure 1B⇑).
To determine the mechanism by which the Kip/Cip and Ink CKIs induced G1 arrest in VSMCs, CDK activity was examined by immunoprecipitation of cdk2 or cdk4 complexes and determination of kinase activity using histone H1 or glutathione S-transferase-Rb as a substrate. Expression of p27Kip1 or p21Cip1 completely inhibited cdk2 activity; in contrast, p16Ink4 only partially inhibited this CDK (Figure 1C⇑). p27Kip1, p21Cip1, and p16Ink4 inhibited cdk4 activity. The degree of inhibition of cdk4 was comparable among the 3 CKIs (Figure 1D⇑). Thus, p27Kip1 or p21Cip1 abrogated the activity of both cdk4 and cdk2, whereas p16Ink4 completely inactivated only cdk4.
Function of CKIs In Vivo
We next determined the function of the CKIs in an animal model of vascular injury. p27Kip1 and p16Ink4 were expressed in balloon-injured pig femoral arteries by adenoviral gene transfer. The effects of gene expression on cell proliferation were measured 7, 14, and 21 days after injury and gene transfer. p27Kip1 expression reduced intimal cell proliferation by 52% compared with AdΔE1 arteries at 7 days (5.23±0.06% versus 10.85±1.04%, respectively; P<0.001; Figure 2A⇑). In contrast, no differences existed in the number of intimal-replicating cells between p16Ink4 and AdΔE1 groups (12.47±0.86% versus 10.85±1.04%, respectively; P=NS; Figure 2A⇑). Double-label immunohistochemistry demonstrated that the bromodeoxyuridine-positive cells were α-actin–positive, which identified them as VSMCs (data not shown). The inhibition in cell proliferation by p27Kip1 persisted to 14 and 21 days compared with control arteries (51.9% and 44.5% reductions, respectively; P<0.01).
This reduction of cellular proliferation in p27Kip1-expressing arteries was associated with an inhibition of intima formation 21 days after injury and gene transfer (Figure 2B⇑). p16Ink4 overexpression did not significantly limit the development of a neointima (intima/media area ratios: p27Kip1, 0.41±0.04; p16Ink4, 0.67±0.05; AdΔE1, 0.72±0.04; noninfected, 0.85±0.05; n=10 arteries in each group; P<0.001 for p27Kip1 versus p16Ink4, AdΔE1, and noninfected cells). The p27Kip1 data are consistent with the reduction in cell proliferation neointima formation previously reported for p21Cip1.7 9 Representative photomicrographs of arterial cross-sections at 21 days are shown in Figure 2C⇑. These findings demonstrate that p27Kip1 significantly inhibits VSMC growth in vitro and in vivo when compared with p16Ink4.
The major findings in this study are that the Kip/Cip and Ink CKIs differentially regulate cdk2 and cdk4 in VSMCs; this, in turn, leads to differences in the inhibition of VSMC proliferation in vitro and in vivo. The expression of p27Kip1 and p21Cip1 in VSMCs inactivated cdk2 and cdk4, whereas p16Ink4 inhibited only cdk4 activity. In vivo, p27Kip1 significantly inhibited intimal cell proliferation and the development of a neointima after vascular injury, whereas p16Ink4 expression did not lead to a reduction in cell proliferation or neointima formation.
This different pattern of CKI inactivation of the CDKs suggests varied biological roles for p27Kip1 and p21Cip1 compared with p16Ink4 in VSMCs. p27Kip1 was initially characterized as an inhibitor of cyclin E/cdk2 phosphorylation.10 11 p27Kip1 and p21Cip1 are upregulated in several animal models of wound repair. p27Kip1 is constitutively expressed in normal arteries, is downregulated after arterial injury, and is upregulated during the later phases of arterial repair; thus, it is inversely correlated with VSMC proliferation.4 p21Cip1 is not detected in normal arteries but, like p27Kip1, it is upregulated in the later phases of arterial repair. p16Ink4 is expressed only transiently in injured arteries and is not detected in atherosclerotic or nonatherosclerotic human coronary arteries. The overexpression of p27Kip1 in injured rat carotid arteries also results in a reduction in VSMC proliferation and intimal hyperplasia. This effect of p27Kip1 in rat arteries is likely due to the downregulation of the cyclin A promoter.12 Indeed, a deficiency of p27Kip1 in mice leads to benign cellular hyperplasia in multiple organs, predominantly the endocrine organs, but these mice do not develop tumors.5
p16Ink4 was initially cloned from a human cancer cell line.13 Data from cancer cell lines suggest p16Ink4 induces early G1 arrest and the attenuation of growth14 and that cell cycle arrest by p16Ink4 is partly due to the induction of p21Cip1 and the inhibition of cdk2.15 The differences in results between these published data and the present study likely reflect differences in cell types. The partial inhibition of VSMC growth by p16Ink4 was not due to lower numbers of cells transfected; indeed, previous studies suggest that >90% of cells are transfected at a multiplicity of infection of 500. Deletions and/or mutations of p16Ink4 have been described in human carcinomas and lymphomas.6 Likewise, p16Ink4−/− mice develop epithelial and mesenchymal tumors in the postnatal period.16 However, p16Ink4 is not expressed in either normal or injured arteries,4 and abnormalities of blood vessels have not been observed in the p16Ink4-null mice. These findings suggest that p16Ink4 regulation of cyclin D/cdk4 and its effects on the phosphorylation of Rb may be linked to tumorigenesis but not arterial wound repair.
The CDK inhibitors p27Kip1, p21Cip1, and p16Ink4 exert differential effects on VSMC proliferation due to the inhibition of cdk2 activity by p27Kip1 and p21Cip1 but not by p16Ink4. These differences in molecular mechanisms have important implications for our understanding and treatment of vascular diseases.
F.C.T. was supported by the Swiss Society of Internal Medicine, Ciba Geigy Jubiläums Stiftung, and the Swiss National Science Foundation.
- Received January 11, 2000.
- Revision received February 25, 2000.
- Accepted February 29, 2000.
- Copyright © 2000 by American Heart Association
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