eNOS inhibition of proliferation: a role for p21Sdi1/Cip1/Waf1 and p27kip1

See article by Sato et al. [12] (pages 697–706) in this issue. It is over 10 years since NO donors were shown to reduce proliferation of vascular smooth muscle cells (VSMC) [1]. More recently, increase in vessel wall thickness and hyperplasia were shown to occur following ligation of the carotid artery in NO synthase (eNOS) knockout mice suggesting NO to be a negative regulator of vascular cell proliferation [2]. Due to the short half-life and limited solubility of NO, NO synthase (NOS) is often used as a way of enhancing local NO production within the vessel wall, with potential therapeutic effects in vascular proliferative disorders. In addition to inhibiting VSMC proliferation, NOS also causes vasodilation, reduces platelet aggregation and adhesion as well as inhibiting leucocyte adhesion to the endothelium [3]. Thus it has been proposed as a useful inhibitor of neointimal hyperplasia, the hallmark of restenosis following angioplasty and stenting. Indeed, the last few years have seen a plethora of studies investigating gene transfer of NOS to various vascular systems for potential therapeutic benefit. One of the early studies showed that eNOS gene transfer using HVJ virus–liposome complexes delivered to balloon injured rat carotid arteries inhibited neointimal formation via a reduction in BrdU incorporation [4]. Other subsequent studies have utilised adenoviral vectors and have shown similar effects on rat VSMC in vitro and in vivo in the rat balloon arterial injury model [5]. Inhibition of pig VSMC proliferation in vitro and a reduction in luminal narrowing after coronary angioplasty in pigs has also been demonstrated [6]. Thus, promising results have been obtained in various animal models both in vitro and in vivo. Despite this, the mechanism of action of eNOS in reducing neointimal hyperplasia is not fully understood. One possible mechanism of action of eNOS is via apoptosis. Apoptosis has been shown to occur following balloon angioplasty where it precedes cell proliferation [7]. Indeed antisense to bcl-x, and ribozymes to bcl-2, both anti-apoptotic genes, increased apoptosis and inhibited neointimal hyperplasia in experimental models of balloon injury [8,9]. An alternative mechanism to explain the effects of eNOS would be via cell cycle arrest. Cyclins are responsible for the progression of cells through various stages of the cell cycle via the formation of complexes with cyclin-dependent kinases (CDK) and their activation at specific stages of the cell cycle. Cyclin-dependent kinase inhibitors (CKIs) inactivate such complexes and lead to cell cycle arrest at G1. p27 and p21 are examples of CKIs that inhibit cyclin E and cyclin A kinase activity. p27 is constitutively expressed in normal arteries, decreases following balloon injury and increases during the later stages of remodelling when proliferation decreases and increased procollagen and TGFβ is observed [10]. Expression of p27 therefore, is inversely correlated with VSMC proliferation. p21 on the other hand, is not detected in normal arteries but along with p27 it is upregulated in the later processes of arterial repair [10]. Indeed, adenovirus-mediated over-expression of p21 was shown to decrease VSMC proliferation and neointimal formation in a rat carotid model of balloon injury [11]. A study in this issue of Cardiovascular Research focuses on the mechanism of eNOS inhibition of VSMC proliferation in an attempt to dissect out whether apoptosis or cell cycle arrest is predominant [12]. The authors transfected porcine VSMC with an adenoviral vector encoding eNOS. Transduction was confirmed via immunohistochemistry, western blotting and diaphorase staining with functional assessment of recombinant protein determined by increased levels of cGMP. The authors subsequently detected a delay in cell cycle progression and a rise in p27 and p21 in eNOS transduced but not control VSMC, in keeping with a mechanism of action of eNOS via cell cycle arrest. However, the authors failed to show an induction of apoptosis using annexin V staining and DNA ladders. A similar finding was observed by Holm et al. [13], using l-arginine as an NO precursor in a rat carotid injury model and analysis by PCNA and TUNEL, they concluded that NO upregulation was associated with a decrease in VSMC proliferation and not an increase in apoptosis. This failure to induce apoptosis would correlate with NO's action of maintaining an intact endothelial barrier since induction of endothelial cell apoptosis would tend to promote a pro-inflammatory environment and hinder rather than inhibit restenosis. The findings of the study reported in this issue [12] are in partial agreement with those of Tanner et al. [14] who earlier this year showed that p21 was enhanced following treatment with an NO donor and this correlated with a reduction in cyclin A, both findings preceding onset of G1 arrest. In addition, Ishida et al. [15] demonstrated induction of p21 in VSMC following treatment with NO donors and this correlated with cdK2 expression. However, in the study by Tanner et al. [14] and a separate study by Sharma et al. [16], no significant rise in p27 was detected using either NO donors or eNOS transfer to VSMC suggesting that p27 may not be a target of NO mediated inhibition of proliferation. It is possible that p27 and p21 have different patterns of expression and regulation as previously suggested by Tanner et al. [10] and the timing of analysis may be crucial in determination of the effects on CKI expression following NO induction. In summary, the study reported in this issue of Cardiovascular Research strongly suggests that the mechanism of action of inhibition of VSMC proliferation induced by eNOS is via induction of the CKIs, p27 and p21. Further studies evaluating these mechanisms in vivo are awaited before the inhibitory effect of eNOS on vascular neointimal hyperplasia can be fully elucidated. My thanks go to Dr Nadim Malik for his critical reading of this article.