[Frontiers in Bioscience 5, d619-628, July 1, 2000]

Current Issue

Send correspondence to:

Dr Vicente Andrés,
Instituto de Biomedicina,
Spanish Council for Scientific Research (CSIC)
C/Jaime Roig 11,
46010 Valencia, Spain,

Tel: +34-96-3391752,
Fax: +34-96-3690800,
E-mail: vandres@ibv.csic.es


Vascular smooth muscle, Cell cycle, growth, p21, -27, TGF-beta, Cell signaling, Review


Copyright © Frontiers in Bioscience, 1995


Silvia M. Sanz-González, Enric Poch, Ignacio Pérez-Roger, Antonio Díez-Juan, Carmen Ivorra and Vicente Andrés

Unit of Vascular Biology, Instituto de Biomedicina de Valencia, Spanish Council for Scientific Research, Valencia, Spain.


1. Abstract
2. Introduction
3. Discussion
3.1. Inhibition of VSMC proliferation by CKIs
3.2. Role of p21 and p27 in the control of VSMC growth by extracellular matrix components
3.3. Role of CKIs in nitric oxide-dependent suppression of VSMC proliferation
3.4. p27 as a regulator of the phenotypic response of VSMCs to mitogenic and hypertrophic stimuli
4. Perspectives
5. Acknowledgments
6. References


While quiescence is a defining characteristic of differentiated vascular smooth muscle cells (VSMCs) residing within the medial layer of elastic arteries in the adult organism, mature VSMCs can undergo phenotypic modulation and reenter the cell cycle in response to several physiological and pathological stimuli. Abnormal VSMC proliferation is thought to contribute to the pathogenesis of vascular occlusive lesions, including atherosclerosis, vessel renarrowing after successful angioplasty (restenosis), and graft atherosclerosis after coronary transplantation. Therefore, elucidating the molecular mechanisms limiting VSMC growth is currently the subject of active research. This review will focus on the role of cyclin-dependent kinase inhibitory proteins in the regulation of VSMC proliferation and its implication in intimal lesion formation during the pathogenesis of vascular proliferative diseases.


In the adult organism, the vessel wall in a healthy artery is composed of an outer layer of connective tissue (adventitia), a medial layer of VSMCs (media) and an inner monolayer of endothelial cells (ECs) (intima). Accumulation of cellular and extracellular substances in the space between the EC lining and the underlying VSMCs leads to neointimal lesion formation and the ensuing progressive reduction of arterial patency. According to the response-to-injury hypothesis, atherosclerosis is triggered by different risk factors (hypercholesterolemia, aging, hypertension, smoking and diabetes) that can somehow lead to endothelial dysfunction (1, 2). Studies in hypercholesterolemic animals and in human atherosclerotic specimens have identified three processes involved in the formation of the neointimal lesion once the normal properties of the endothelium have been altered (1, 2): 1) the proliferation of VSMCs, macrophages and possibly lymphocytes; 2) the formation by VSMCs of a connective tissue matrix comprising elastic fibre proteins, collagen and proteoglycans; and 3) the accumulation of lipid and mostly free and esterified cholesterol in the surrounding matrix and the associated cells. Figure 1 shows an example of diet-induced pathological proliferation of medial VSMCs and macrophages within the intimal lesion of hypercholesterolemic rabbits. Numerous observations suggest that VSMCs in atherosclerotic lesions have changed from a contractile to a synthetic state (3), in which they can respond to different growth factors and synthetize extracellular matrix (4). "Activated" VSMCs can also migrate toward the arterial lumen and express abundant levels of novel matrix components and proteases that modify the surrounding matrix. This "growth and synthetic" response of VSMCs contributes to atheroma formation.

Figure 1: Hypercholesterolemia induces abnormal cell proliferation in the aortic arch. Male New Zealand rabbits were fed a control diet (A, B) or a cholesterol-rich diet for 2 months (C, D). Animals received 4 intraperitoneal injections of 5-bromodeoxyuridine (BrdU) during the last 2 days prior to sacrifice to identify proliferating cells. The aortic arch was embedded in paraffin and cut in 5-micron sections for immunohistochemistry using a mouse monoclonal anti-BrdU antibody and an streptavidin-peroxidase detection system. Dark nuclei indicate BrdU-immunoreactive cells. Specimens were countestained with hematoxylin. Arrowheads point to the internal elastic lamina, which marks the boundary between the tunica media (composed of VSMCs and elastic fibers) and the intima (composed of a monolayer of endothelial cells in control arteries). Immunohistochemistry using mouse monoclonal anti-RAM11 antibody (not shown) demonstrated that the intimal lesion at these early time points is mainly composed of macrophages. Note the presence of BrdU-positive VSMCs in the media (C) and macrophages in the intimal lesion (D) of hypercholesterolemic rabbits.

Excessive VSMC proliferation also contributes to restenosis, the recurrence of arterial narrowing at the site of balloon angioplasty that occurs in 20-55% of coronary artery disease patients after successful angioplasty (1, 5, 6). Acute disruption of the protective endothelial lining at the site of angioplasty appears to trigger this aggressive form of atherosclerosis, which is typically characterized by exuberant VSMC hyperplastic response (7-9), extracellular matrix accumulation (10, 11) and local "remodeling" (elastic recoil) of the dilated vessel (12, 13).

Cell cycle progression and cellular proliferation in mammals requires the activation of cyclin-dependent kinases (CDKs) through their association with regulatory subunits called cyclins (14, 15). Different CDK/cyclin holoenzymes are orderly activated at specific phases of the cell cycle. Active CDK/cyclin complexes are presumed to hyperphosphorylate pRb and the related pocket proteins p107 and p130 from mid G1 to mitosis. The interaction among members of the E2F family of transcription factors and individual pocket proteins is a complex regulatory event that determines whether E2F proteins function as transcriptional activators or repressors (16-20). VSMC proliferation in the balloon-injured rat carotid artery is associated with a temporally and spatially coordinated expression of CDK2 and its regulatory subunits, cyclin E and cyclin A (21). Induction of these factors correlated with increased CDK2-, cyclin E- and cyclin A-dependent kinase activity, indicating the assembly of functional CDK2/cyclin E and CDK2/cyclin A holoenzymes in the injured arterial wall. Expression of CDK2 and cyclin E was also detected in human VSMCs within atherosclerotic and restenotic tissue (21-23), suggesting that induction of positive cell-cycle control genes is a hallmark of vascular proliferative diseases.

CDK activity is negatively regulated by specific cell cycle regulators, termed CDK inhibitors (CKIs), which associate with and inhibit the activity of CDKs (15, 24-26). CKIs of the CIP/KIP family (p21, p27 and p57) bind to and inactivate CDK2-containing holoenzymes, while members of the INK4 family (p15, p16, p18, p19) are specific for CDK4- and CDK6-containing holoenzymes. In addition to its inhibitory effect on CDKs, p21 can also inhibit DNA replication through direct interaction with proliferating cell nuclear antigen (PCNA) (27, 28). Separate domains of p21 are involved in the inhibition of CDK and PCNA (29, 30), and reversible phosphorylation at the C-terminal regulatory domain of p21 modulates PCNA binding (31). In the next sections, we will discuss the role of CKIs in the pathogenesis of atherosclerotic cardiovascular diseases.


3.1. Inhibition of VSMC proliferation by CKIs

While proliferating cells are present at all stages of development of atherosclerotic lesions (1, 2), studies with Watanabe heritable hyperlipidemic and hypercholesterolemic fat-fed rabbits have demonstrated an inverse relationship between lesion size (and severity) and the proliferative index in the arterial wall (32-35). These findings suggest that cell proliferation may be a relatively early event in the atherogenic process. Likewise, balloon angioplasty leads to a rapid proliferative response of VSMCs within the media, followed by a second peak of proliferation in neointimal VSMCs which then resume a quiescent phenotype within 2-6 weeks after angioplasty (36-40). Thus, both atherosclerosis and restenosis are characterized by the reestablishment of the quiescent phenotype after the initial burst of proliferation.

Recent studies suggest that p27 and p21 are physiological regulators of VSMC proliferation that contribute to limiting neointimal hyperplasia during arterial repair. Balloon angioplasty in rat and porcine arteries resulted in the induction of p21 and p27 in VSMCs at time points that correlated with reduced CDK2 activity and the decline in VSMC proliferation (41-43). Moreover, overexpression of p27 efficiently blocked mitogen- and c-fos-dependent induction of cyclin A promoter activity in cultured VSMCs (41, 44). Thus, upregulation of p21 and p27 may limit VSMC growth at late time points after angioplasty (Figure 2). In agreement with this hypothesis, Chang et al. (45) and Yang et al. (43) first demonstrated that adenovirus-mediated overexpression of p21 attenuated neointimal thickening in balloon-injured rat and porcine arteries. Likewise, Chen et al. reported that local delivery of adenovirus encoding for p27 at the time of angioplasty reduced neointimal hyperplasia in the rat carotid artery (41). Additional studies by other investigators have corroborated the ability of p21 and p27 to inhibit the development of injury-induced vascular occlusive lesions (46, 47). These studies also showed that overexpression of p16 failed to inhibit neointimal VSMC proliferation (47). Of note is that induction of p27, but not p21, is associated with inhibition of VSMC proliferation in cells stably transfected with PKC delta (48). Whether PKC delta is involved in the upregulation of p27 after angioplasty in vivo remains to be explored.

Figure 2: Role of p21 and p27 in the regulation of VSMC growth after angioplasty. Using several animal models of balloon angioplasty, it has been shown that injury-induced vascular remodeling is characterized by a rapid proliferative response of VSMCs within the media, which migrate towards the arterial lumen and initiate a second wave of proliferation within the intimal lesion. Two to four weeks after angioplasty, VSMC proliferation returns to basal levels. Medial and intimal VSMC proliferation correlates with low level of expression of p21 and p27 and high CDK2 activity. Reduced CDK2 activity and low proliferation at later time points coincides with upregulation of p21 and p27, suggesting that induction of these CKIs may contribute to the reestablishment of the quiescent phenotype. Consistent with this notion, adenovirus-mediated overexpression of p21 and p27 following angioplasty limited intimal thickening (See text for details).

Tanner et al (42) analyzed CKI expression in human coronary arteries ranging from normal to advanced atherosclerosis. Expression of p27 was abundant within normal and atherosclerotic arteries. While p21 was undetectable in normal arteries, its expression was elevated in atherosclerotic tissue. In this same study, p16 could not be detected in normal or atherosclerotic specimens, demonstrating that the CIP/KIP and INK4 families of CKIs have different temporal patterns of expression in VSMCs in balloon-injured arteries and atherosclerotic lesions. More recently, Ihling et al (23) have demonstrated coexpression of type I and II transforming growth factor-beta (TGF-beta) receptors in virtually all cells positive for p27 within human atherosclerotic tissue. In the atherosclerotic specimens, approximately 13% of the nuclei were positive for cyclin E, whereas in the control tissue cyclin E staining was restricted to 0.19% of the cells. Importantly, about 5% of p27-positive cells disclosed cyclin E immunoreactivity. These results suggest that TGF-beta present in human atherosclerotic tissue may mediate its growth suppressive activity through p27-dependent blockade of cyclin E-CDK2 activity.

Internal mammary artery (IMA) bypass grafts have a higher patency than saphenous vein (SV) grafts. Yang et al. (49) examined the growth properties of human VSMCs isolated from IMAs and SVs. Cell outgrowth from explants over a 20-day period and serum-induced increase in cell number over an 8-day period were more pronounced in SV than in IMA of the same patient. These differences in the response to growth stimuli were observed despite functional growth factor receptor expression and MAPK activation in VSMCs isolated from both SVs and IMAs. Platelet-derived growth factor-BB (PDGF-BB) markedly downregulated p27 protein level in SV, but this was much less pronounced in IMA. Thus, sustained p27 expression in spite of growth stimuli may contribute to the resistance to growth of VSMCs from IMA and to the longer patency of arterial versus venous grafts.

Fibroblast growth factor 2 (FGF2 or basic FGF) plays a critical role in the induction of medial VSMC proliferation in balloon-injured arteries (50-52). In marked contrast, neutralizing antibodies to FGF2 failed to inhibit intimal VSMC proliferation after balloon angioplasty (53), and only a small increase in proliferation was seen when FGF2 was added to arteries with existing intimal lesions (50, 52). Attenuated FGF2-dependent proliferation of intimal VSMCs occurred despite a robust activation of the MAPK pathway and induction of positive cell cycle regulators (i. e., cyclin D, cyclin E, CDK2 and CDK4) (52). Interestingly, intimal VSMCs expressed high levels of p15 and p27 compared with medial VSMCs, and FGF2 infusion did not reduce the level of these inhibitors in arteries with established intimal lesion. Collectively, the studies by Yang et al. (49) and Olson et al (52) using different sources of VSMCs suggest that high level of expression of p15 and p27 can attenuate VSMC proliferation in spite of the activation of the MAPK pathway and expression of cell cycle activators.

3.2. Role of p21 and p27 in the control of VSMC growth by extracellular matrix components

Accumulating evidence indicates that specific components of the extracellular matrix (ECM) and integrins are physiological cell-cycle control elements in atherosclerosis and restenosis (54). Neointimal VSMCs within atherosclerotic lesions synthesize novel ECM components and induce the expression of matrix-degrading proteases that remodel the surrounding ECM. For example, matrix-degrading metalloproteinase (MMP) expression is induced within atherosclerotic plaques and after balloon angioplasty (55-58). Moreover, MMP inhibitors repressed VSMC proliferation in vitro and after angioplasty in vivo (59-61). Accordingly, these ECM enzymes have been implicated in the induction of neointimal VSMC hyperplasia during atherosclerosis and restenosis.

Integrins are transmembrane heterodimers that bind to a number of ligands, primarily ECM molecules, and stimulate a variety of transduction pathways (62). One integrin in particular, alphavbeta3, is thought to interact with osteopontin and play a critical role in regulating cellular functions deemed essential for restenosis including migration, ECM invasion and proliferation of VSMCs (63). Alphavbeta3 has been found to be expressed by VSMCs in the intima of diseased human coronary arteries (64) and is upregulated following balloon injury of baboon brachial arteries (65). Further evidence of the importance of this integrin in the pathogenesis of restenosis has been provided by showing that selective alphavbeta3 blockade could potently limit neointimal hyperplasia in animal models of arterial injury (66, 67). Interestingly, it has been suggested that inhibition of alphavbeta3 could constitute a potential mechanism for the beneficial effects on clinical restenosis of abciximab (an inhibitor of platelet glycoprotein IIb/IIIa) in patients undergoing high-risk percutaneous coronary interventions (68).

Changes in collagen content have been well documented in different animal models of atherosclerosis and angioplasty (11, 69, 70). To investigate whether changes in collagen may regulate VSMC proliferation, Koyama et al. studied the growth properties of VSMCs cultured on monomer collagen fibers and on polymerized collagen (71). The rationale for these studies is that polymerized collagen may resemble the scenario of a normal artery composed of quiescent VSMCs, and monomer collagen might mimic the ECM surrounding proliferating VSMCs within atherosclerotic and restenotic plaques. Consistent with this interpretation, mitogen-stimulated VSMCs proliferated in culture dishes coated with monomer collagen, but were arrested in G1 when grown on polymerized collagen. The inhibitory effect of polymerized collagen on VSMC growth appeared to be mediated by alpha2 integrins, and was associated with suppression of p70 S6 kinase and upregulation of p21 and p27. These findings indicate that the ability of VSMCs to respond to growth signals in vitro is highly dependent on changes in specific ECM components through regulation of CKIs. Of note is that lack of proliferation in nonadherent NRK fibroblasts can be linked to an increased association of p21 and p27 to cyclin E-containing holoenzymes (72). Further studies are required to determine whether cell cycle control in the arterial wall is linked in vivo to integrins and ECM components through changes in CKI expression.

The glycoprotein thrombospondin 1 (TSP1) is a component of the ECM synthesized and secreted by activated platelets (73) and a variety of cell types including ECs (74, 75), macrophages (76), fibroblasts (77) and VSMCs (78). TSP1 is a 450 kD homotrimer that interacts with multiple extracellular macromolecules and cell surface receptors, thus exerting a wide range of functions (79, 80). TSP1 can induce EC growth arrest in vitro (81, 82), and inhibits the spontaneous development of angiogenic tube-like structures both in vitro and in vivo (83-85). In marked contrast, TSP1 promotes VSMC proliferation and migration (86, 87), and plays a stimulatory role in platelet activation and aggregation (88, 89). TSP1 expression has been associated with atherosclerotic lesions, acute vascular injury, hypercholesterolemia and hypertension (75, 90-95). Thus, TSP1 may have detrimental effects of the vessel wall (Figure 3). Consistent with this notion, antibody blockade of TSP1 accelerated reendothelialization and reduced neointima formation in balloon-injured rat carotid artery (96). Neutralizing A4.1 anti-TSP1 antibody inhibited CDK2 activity and blocked the induction of S-phase entry which normally occurs in serum-stimulated VSMCs (97). This growth inhibitory effect was associated with a marked induction of total cellular p21 expression and increased level of CDK2-bound p21 in A4.1-treated VSMCs. A4.1 antibody inhibited [3H]-thymidine incorporation in wild-type, but not in p21-deficient mouse embryonic fibroblasts, suggesting that p21 plays an essential role in TSP1-mediated control of cellular proliferation.

Figure 3: Thrombospondin 1 expression is induced in atherosclerotic plaques and restenotic lesions. Schematic showing the deleterious effects of TSP1 accumulation within injured arteries. TSP1 is secreted at the sites of arterial injury by macrophages and T-lymphocytes. In the left part is show the inhibitory effect of TSP1 on endothelial cell (EC) migration and proliferation, which delays reendothelialization after balloon angioplasty. On the other hand, TSP1 stimulates VSMC proliferation and migration, therefore contributing to intimal thickening. Consistent with these harmful effects of TSP1, antibody blockade of TSP1 promotes reendothelialization and reduces intimal lesion development after angioplasty in the rat carotid artery. In vitro experiments have shown that p21 is essential for TSP1-dependent regulation of cellular proliferation (See text for details).

3.3. Role of CKIs in nitric oxide-dependent suppression of VSMC proliferation

Nitric oxide (NO) has critical roles in the maintenance of vascular homeostasis. In addition to its role as a vasodilator, NO inhibits platelet function, leukocyte adhesion to ECs, and VSMC growth (98, 99). Teleologically, the lack of endothelium-derived NO production due to disruption of the protective endothelial lining after balloon angioplasty might be expected to contribute to VSMC hyperplasia. Consistent with this notion, arterial delivery of EC mitogens that accelerated reendothelization also attenuated neointimal hyperplasia after vascular injury (100-102). Studies with endothelial NO sinthase (eNOS)-deficient mice have provided direct evidence for the importance of endothelium-derived NO in vascular response to injury (103, 104). Moreover, high production of NO by intimal VSMCs via inducible NOS (iNOS) may contribute to the restoration of the quiescent phenotype after balloon angioplasty (105, 106). Of note is that NO from VSMCs can reduce eNOS protein expression by ECs via a tumor necrosis factor (TNF) alpha-dependent mechanism (107).

Administration of the NO precursor L-arginine (108-111), or in vivo transfer of NO synthase gene (112-116) inhibited neointimal lesion development in several animal models, including balloon angioplasty, cholesterol-induced atherosclerosis and allograft atherosclerosis. Conversely, inhibition of NO production by treatment with NG-nitro-L-arginine methyl ester (L-NAME) accelerated neointima formation in hypercholesterolemic rabbits (117) and apolipoprotein E-deficient mice (118). These findings are consistent with the observation that resistance of VSMCs to NO contributes to abnormal endothelium-dependent vasodilation during hypercholesterolemia (119), and suggest that NO plays a critical role during the pathogenesis of vascular proliferative diseases.

Recent studies have shed significant insight into the mechanisms involved in the antiproliferative effect of NO. When starvation-synchronized human VSMCs were serum-restimulated, the mRNA and protein levels of p21 were high in early G1 and then rapidly decreased prior to the induction of CDK2 activity (120). Addition of the NO donor S-nitroso-N-acetylpenicillamine (SNAP) to serum-restimulated VSMCs inhibited DNA synthesis assessed by [3H]thymidine incorporation. The antiproliferative effect of SNAP was associated with enhanced and sustained p21 expression, increased amount of CDK2-associated p21 and inhibition of CDK2 activity (120). Moreover, evidence has been presented suggesting that NO-dependent VSMC growth arrest results, at least in part, from the repression of cyclin A gene transcription (121).

The involvement of cGMP in NO-dependent regulation of CKI expression is controversial. Loss of NO responsiveness in aged rats due to the lack of the beta subunit of soluble guanylyl cyclase may contribute to the enhanced intimal thickening in response to injury in old animals (122). Gu et al suggested that NO increases p21 expression by a cGMP-dependent mechanism that includes activation of extracellular signal-regulated kinase (Erk) and p70 S6 kinase (123). In contrast, Sarkar et al. reported that NO inhibition of VSMC proliferation is associated with two distinct and reversible cell cyle arrests, an immediate cGMP-independent S-phase block followed by a shift back in the cell cycle from the G1-S boundary to a quiescent G0-like state (124). Likewise, Tanner et al. suggested that upregulation of p21 in VSMCs treated with the NO donor diethylenetriamineNONOate may occur independent of cGMP (125). It is important to note that diethylenetriamineNONOate did not change p27 expression, whereas a transient increase in p27 in CDK2 immunoprecipitates, without changes in total cellular p27, correlated with the delay in CDK2 activation caused by cGMP in human VSMCs (126). cGMP-elevating agents inhibited EGF-induced VSMC proliferation by a mechanism that appears to involve the repression of Ras-dependent activation of Raf-1 (127). While the above studies clearly suggest a role of p21 and p27 in NO-dependent VSMC growth arrest, additional studies are required to clarify the role of cGMP in this pathway.

Several studies have suggested the contribution of adventitial myofibroblasts to vascular remodeling and intimal lesion formation after experimental angioplasty (128). In this regard, it is noteworthy to point out that NO has been implicated as a potential regulator of the cell cycle in aortic adventitial myofibroblasts through a cGMP-mediated transcriptional mechanism involving the induction of p21 (129).

3.4. p27 as a regulator of the phenotypic response of VSMCs to mitogenic and hypertrophic stimuli

VSMC hypertrophy is associated with cardiovascular disease in elderly and hypertensive individuals. Therefore, a better understanding of the molecular mechanisms underlying the onset of VSMC hypertrophy may have implications for the design of novel therapeutic interventions in cardiovascular disease. Angiotensin II (Ang II) has been shown to stimulate hypertrophy but not hyperplasia of quiescent VSMCs in serum-free media, in spite of increased expression of several protooncogenes and autocrine growth factors (130-133). While both serum and Ang II treatment of quiescent VSMCs led to upregulation of positive cell-cycle regulators, including proliferating cell nuclear antigen, cyclin D1, CDK2 and CDK1, only serum-treated VSMCs induced CDK2 and CDK1 activity (134). Braun-Dullaeus et al. provided compelling evidence implicating p27 as a molecular switch that regulates the phenotypic response of VSMCs to mitogenic and hypertrophic stimuli (134). Their experiments show that Ang II-induced hypertrophy of quiescent VSMCs correlated with sustained expression of p27, unlike serum-dependent cell-cycle reentry of starvation-synchronized cells, which correlated with a marked downregulation of p27 protein level. Importantly, forced overexpression of p27 inhibited serum-stimulated proliferation and induced VSMC hypertrophy. Moreover, inhibition of p27 expression in VSMCs treated with antisense oligodeoxynucleotides increased [3H]-thymidine incorporation and the percentage of S-phase cells in Ang II-treated cultures. These results demonstrate that Ang II treatment of quiescent VSMCs is associated with cell-cycle entry, but hypertrophic rather than hyperplastic growth may prevail by the failure of cells to downregulate p27. In another study, Servant et al. (135) compared the effects of Ang II and the mitogenic factor PDGF-BB on cultured VSMCs. While both factors stimulated the accumulation of G1 cyclins and CDKs, only PDGF-BB activated CDK2 in late G1. Lack of CDK2 activity in Ang II-treated cells correlated with sustained p27 protein level. In contrast, PDGF-BB downregulated p27 expression, and this effect correlated with a reduced rate of synthesis and an increased rate of degradation of p27. Moreover, the reduction in p27 synthesis by PDGF-BB was associated with diminished p27 gene transcription and decreased mRNA accumulation. Collectively, these studies identify p27 as an important regulator of the phenotypic response of VSMCs to mitogenic and hypertrophic stimuli.


Abnormal VSMC hyperplastic and hypertrophic growth play an important role in the pathogenesis of cardiovascular diseases, including atherosclerosis and restenosis. Because of the public health importance and economic impact of these pathological processes, elucidating the regulatory factors and molecular mechanisms that control VSMC growth is currently the subject of active research. In this review, we have discussed the role of CKIs in the regulation of VSMC proliferation. In vitro studies have implicated p27 as a molecular switch that regulates the phenotypic response of VSMCs to mitogenic and hypertrophic stimuli. Moreover, induction of endogenous p21 and p27 at late time points after balloon-angioplasty may contribute to the restoration of the quiescent phenotype during vascular remodeling. Consistent with this notion, adenovirus-mediated overexpression of p21 and p27 inhibited VSMC hyperplasia and prevented arterial narrowing in balloon-injured rat and porcine arteries. In a recent study (136), cDNA array hybridization techniques showed that p21 induces the expression of genes implicated in atherosclerosis, including serum amyloid A (137), connective tissue growth factor (138), and galectin-3 (139). It is therefore essential to continue our efforts to elucidate the molecular mechanisms governing the control of CKI expression in the vessel wall. Ultimately, a thorough understanding of these regulatory networks may lead to the development of novel therapies for the treatment of vascular proliferative diseases in human patients.


Work in the laboratory of V. Andrés is supported in part by grants from the Spanish Dirección General de Educación Superior e Investigación Científica (PM97-0136 and 1FD97-1035-C02-02) and from the American Heart Association, Massachusetts Affiliate (9860022T).


1. Ross, R.: The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature 362, 801-9 (1993)

2. Ross, R.: Atherosclerosis. An inflammatory disease. N. Engl. J. Med. 340, 115-26 (1999)

3. Campbell, G. R. and Campbell, J. H.: The phenotypes of smooth muscle expressed in human atheroma. Ann. N. Y. Acad. Sci. 598, 143-58 (1990)

4. Sjölund, M., Rahm, M., Claesson-Welsh, L., Sejersen, T., Heldin, C.-H. and Thyberg, J.: Expression of PDGF alpha- and beta-receptors in rat arterial smooth muscle cells is phenotype and growth state dependent. Growth Factors 3, 191-203 (1990)

5. Fuster, V., Badimón, L., Badimón, J. J. and Chesebro, J. H.: The pathogenesis of coronary artery disease and the acute coronary syndromes. N. Engl. J. Med. 236, 242-50 (1992)

6. Libby, P., Schwartz, D., Brogi, E., Tanaka, H. and Clinton, S. K.: A cascade model for restenosis: a special case of atherosclerosis progression. Circulation 86 (Supplement), III47-III52 (1992)

7. Libby, P. and Tanaka, H.: The molecular basis of restenosis. Prog. Cardiovasc. Dis. 40, 97-106 (1997)

8. Bauters, C. and Isner, J. M.: The biology of restenosis. Prog. Cardiovasc. Dis. 40, 107-16 (1997)

9. Thyberg, J.: Phenotypic modulation of smooth muscle cells during formation of neointimal thickening following vascular injury. Histol. Histopathol. 13, 871-91 (1998)

10. Schwartz, R. S., Holmes, D. R. and Topol, E. J.: The restenosis paradigm revisited: an alternative proposal for cellular mechanisms. J. Am. Coll. Cardiol. 20, 1284-93 (1992)

11. Strauss, B. H., Chisholm, R. J., Keeley, F. W., Gotlieb, A. I., Logan, R. A. and Armstrong, P. W.: Extracellular matrix remodeling after balloon angioplasty injury in a rabbit model of restenosis. Circ. Res. 75, 650-8 (1994)

12. Post, M. J., Borst, C. and Kuntz, R. E.: The relative importance of arterial remodeling compared with intimal hyperplasia in lumen renarrowing after balloon angioplasty. A study in the normal rabbit and the hypercholesterolemic yucatan micropig. Circulation 89, 2861-21 (1994)

13. Wilensky, R. L., March, K. L., Gradus-Pizlo, I., Sandusky, G., Fineberg, N. and Hathaway, D. R.: Vascular injury, repair, and restenosis after percutaneous transluminal angioplasty in the atherosclerotic rabbit. Circulation 92, 2995-3005 (1995)

14. Nurse, P.: Ordering S phase and M phase in the cell cycle. Cell 79, 547-50 (1994)

15. Morgan, D. O.: Principles of CDK regulation. Nature 374, 131-4 (1995)

16. Helin, K. and Harlow, E.: The retinoblastoma protein as a transcriptional repressor. Trends Cell Biol. 3, 43-6 (1993)

17. Weinberg, R. A.: E2F and cell proliferation: a world turned upside down. Cell 85, 457-9 (1996)

18. Mayol, X. and Graña, X.: The p130 pocket protein: keeping order at cell cycle exit/re-entrance transitions. Front. Biosci. 3, 11-24 (1998)

19. Dyson, N.: The regulation of E2F by pRB-family proteins. Genes Dev 12, 2245-62 (1998)

20. Lavia, P. and Jansen-Durr, P.: E2F target genes and cell-cycle checkpoint control. Bioessays 21, 221-30 (1999)

21. Wei, G. L., Krasinski, K., Kearney, M., Isner, J. M., Walsh, K. and Andrés, V.: Temporally and spatially coordinated expression of cell cycle regulatory factors after angioplasty. Circ. Res. 80, 418-26 (1997)

22. Kearney, M., Pieczek, A., Haley, L., Losordo, D. W., Andrés, V., Schainfield, R., Rosenfield, R. and Isner, J. M.: Histopathology of in-stent restenosis in patients with peripheral artery disease. Circulation 95, 1998-2002 (1997)

23. Ihling, C., Technau, K., Gross, V., Schulte-Monting, J., Zeiher, A. M. and Schaefer, H. E.: Concordant upregulation of type II-TGF-beta-receptor, the cyclin- dependent kinases inhibitor p27Kip1 and cyclin E in human atherosclerotic tissue: implications for lesion cellularity. Atherosclerosis 144, 7-14 (1999)

24. Elledge, S. J. and Harper, J. W.: Cdk inhibitors: on the threshold of checkpoints and development. Curr. Opin. Cell Biol. 6, 847-52 (1994)

25. Peter, M. and Herskowitz, I.: Joining the complex: cyclin-dependent kinase inhibitory proteins and the cell cycle. Cell 79, 181-4 (1994)

26. Graña, X. and Reddy, E. P.: Cell cycle control in mammalian cells: role of cyclins, cyclin dependent kinases (CDKs), growth suppressor genes and cyclin-dependent kinase inhibitors (CKIs) Oncogene 11, 211-9 (1995)

27. Flores-Rozas, H., Kelman, Z., Dean, F. B., Pan, Z., Harper, J. W., Elledge, S. J., O'Donnell, M. and Hurwitz, J.: Cdk-interacting protein 1 directly binds with proliferating cell nuclear antigen and inhibits DNA replication catalyzed by the DNA polymerase delta holoenzyme. Proc. Natl. Acad. Sci. USA 91, 8655-9 (1994)

28. Waga, S., Hannon, G. J., Beach, D. and Stillman, B.: The p21 inhibitor of cyclin-dependent kinases controls DNA replication by interacting with PCNA. Nature 369, 574-7 (1994)

29. Luo, Y., Hurwitz, J. and Massagué, J.: Cell-cycle inhibition by independent CDK and PCNA binding domains in p21Cip1. Nature 375, 159-61 (1995)

30. Chen, J., Jackson, P. K., Kirschner, M. W. and Dutta, A.: Separate domains of p21 involved in the inhibition of Cdk kinase and PCNA. Nature 374, 386-8 (1995)

31. Scott, M. T., Morrice, N. and Ball, K. L.: Reversible phosphorylation at the C-terminal regulatory domain of p21Waf1/Cip1 modulates proliferating cell nuclear antigen binding. J. Biol. Chem. 275, 11529-37 (2000)

32. Spraragen, S. C., Bond, V. P. and Dahl, L. K.: Role of hyperplasia in vascular lesions of cholesterol-fed rabbits studied with thymidine-3H autoradiography. Circ. Res. 11, 329-36 (1962)

33. McMillan, G. C. and Stary, H. C.: Preliminary experience with mitotic activity of cellular elements in the atherosclerotic plaques of cholesterol-fed rabbits studied by labeling with tritiated thymidine. Ann. N. Y. Acad. Sci. 149, 699-709 (1968)

34. Cavallero, C., Turolla, E. and Ricevuti, G.: Cell proliferation in the atherosclerotic plaques of cholesterol-fed rabbits. 1. Colchicine and (3H)thymidine studies. Atherosclerosis 13, 9-20 (1971)

35. Rosenfeld, M. E. and Ross, R.: Macrophage and smooth muscle cell proliferation in atherosclerotic lesions of WHHL and comparably hypercholesterolemic fat-fed rabbits. Arteriosclerosis 10, 680-7 (1990)

36. Stemerman, M. B., Weinstein, R., Rowe, J. W., Maciag, T., Fuhro, R. and Gardner, R.: Vascular smooth muscle cell growth kinetics in vivo in aged rats. Proc. Natl. Acad. Sci. USA 79, 3863-6 (1982)

37. Clowes, A., Reidy, M. and Clowes, M.: Kinetics of cellular proliferation after arterial injury. I: smooth muscle cell growth in the absence of endothelium. Lab. Invest. 49, 327-33 (1983)

38. Clowes, A. W. and Schwartz, S. M.: Significance of quiescent smooth muscle migration in the injured rat carotid artery. Circ. Res. 56, 139-45 (1985)

39. Hanke, H., Strohschneider, T., Oberhoff, M., Betz, E. and Karsch, K. R.: Time course of smooth muscle cell proliferation in the intima and media of arteries following experimental angioplasty. Circ. Res. 67, 651-9 (1990)

40. Geary, R. L., Williams, J. K., Golden, D., Brown, D. G., Benjamin, M. E. and Adams, M. R.: Time course of cellular proliferation, intimal hyperplasia, and remodeling following angioplasty in monkeys with established atherosclerosis. A nonhuman primate model of restenosis. Arterioscler. Thromb. Vasc. Biol. 16, 34-43 (1996)

41. Chen, D., Krasinski, K., Chen, D., Sylvester, A., Chen, J., Nisen, P. D. and Andrés, V.: Downregulation of cyclin-dependent kinase 2 activity and cyclin A promoter activity in vascular smooth muscle cells by p27Kip1, an inhibitor of neointima formation in the rat carotid artery. J. Clin. Invest. 99, 2334-41 (1997)

42. Tanner, F. C., Yang, Z.-Y., Duckers, E., Gordon, D., Nabel, G. J. and Nabel, E. G.: Expression of cyclin-dependent kinase inhibitors in vascular disease. Circ. Res. 82, 396-403 (1998)

43. Yang, Z.-Y., Simari, R. D., Perkins, N. D., San, H., Gordon, D., Nabel, G. J. and Nabel, E. G.: Role of p21 cyclin-dependent kinase inhibitor in limiting intimal cell proliferation in response to arterial injury. Proc. Natl. Acad. Sci. USA 93, 7905-10 (1996)

44. Sylvester, A. M., Chen, D., Krasinski, K. and Andrés, V.: Role of c-fos and E2F in the induction of cyclin A transcription and vascular smooth muscle cell proliferation. J. Clin. Invest. 101, 940-8 (1998)

45. Chang, M. W., Barr, E., Lu, M. M., Barton, K. and Leiden, J. M.: Adenovirus-mediated over-expression of the cyclin/cyclin-dependent kinase inhibitor, p21 inhibits vascular smooth muscle cell proliferation and neointima formation in the rat carotid artery model of balloon angioplasty. J. Clin. Invest. 96, 2260-8 (1995)

46. Ueno, H., Masuda, S., SNishio, S., Li, J. J., Yamamoto, H. and Takeshita, A.: Adenovirus-mediated transfer of cyclin-dependent kinase inhibitor p21 suppresses neointimal formation in the balloon-injured rat carotid arteries in vivo. Ann. N. Y. Acad. Sci. 811, 401-11 (1997)

47. Tanner, F. C., Boehm, M., Akyürek, L. M., San, H., Yang, Z.-Y., Tashiro, J., Nabel, G. J. and Nabel, E. G.: Differential effects of the cyclin-dependent kinase inhibitors p27Kip1, p21Cip1, and p16Ink4 on vascular smooth muscle cell proliferation. Circulation 101, 2022-5 (2000)

48. Fukumoto, S., Nishizawa, Y., Hosoi, M., Koma, H., Yamakawa, K., Ohno, S. and Morii, H.: Protein kinase C delta inhibits the proliferation of vascular smooth muscle cells by suppressing G1 cyclin expression. J. Biol. Chem. 272, 13816-22 (1997)

49. Yang, Z., Oemar, B. S., Carrel, T., Kipfer, B., Julmy, F. and Lüscher, T. F.: Different proliferative properties of smooth muscle cells of human arterial and venous bypass vessels: role of PDGF receptors, mitogen-activated protein kinase, and cyclin-dependent kinase inhibitors. Circulation 97, 181-7 (1998)

50. Lindner, V., Lappi, D. A., Baird, A., Majack, R. A. and Reidy, M. A.: Role of basic fibroblast growth factor in vascular lesion formation. Circ. Res. 68, 106-13 (1991)

51. Lindner, V. and Reidy, M. A.: Proliferation of smooth muscle cells after vascular injury is inhibited by an antibody against basic fibroblast growth factor. Proc. Natl. Acad. Sci. USA. 88, 3739-43 (1991)

52. Olson, N. E., Kozlowski, J. and Reidy, M. A.: Proliferation of intimal smooth muscle cells. Attenuation of basic fibroblast growth factor 2-stimulated proliferation is associated with increased expression of cell cycle inhibitors. J. Biol. Chem. 275, 11270-7 (2000)

53. Olson, N. E., Chao, S., Lindner, V. and Reidy, M. A.: Intimal smooth muscle cell proliferation after balloon catheter injury. The role of basic fibroblast growth factor. Am. J. Pathol. 140, 1017-23 (1992)

54. Assoian, R. K. and Marcantonio, E. E.: The extracellular matrix as a cell cycle control element in atherosclerosis and restenosis. J. Clin. Invest. 98, 2436-9 (1996)

55. Bendeck, M. P., Zempo, N., Clowes, A. W., Gelardy, R. E. and Reidy, M. A.: Smooth muscle cell migration and matrix metalloproteinase expression after arterial injury in the rat. Circ. Res. 75, 539-45 (1994)

56. Galis, S., Sukhova, G. K., Lark, M. V. and Libby, P.: Increased expression of matrix metalloproteinases and matrix degrading activity in vulnerable regions of human atherosclerotic plaques. J. Clin. Invest. 94, 2493-503 (1994)

57. Zempo, N., Kenagy, R. D., Au, T., Bendeck, M., Clowes, M. M., Reidy, M. A. and Clowes, A. W.: Matrix metalloproteinases of vascular wall cells are increased in balloon-injured rat carotid artery. J. Vasc. Surg. 20, 209-17 (1994)

58. Southgate, K. M., Fisher, M., Banning, A. P., Thurston, V. J., Baker, A. H., Fabunmi, R. P., Groves, P. H., Davies, M. and Newby, A. C.: Upregulation of basement membrane-degrading metalloproteinase secretion after balloon injury of pig carotid artery. Circ. Res. 79, 1177-87 (1996)

59. Southgate, K. M., Davies, M., Booth, R. F. G. and Newby, A. C.: Involvement of extracellular matrix degrading metalloproteinases in rabbit aortic smooth muscle cell proliferation. Biochem. J. 288, 93-9 (1992)

60. Zempo, N., Koyama, M., Kenagy, R. D., Lea, H. J. and Clowes, A. W.: Regulation of vascular smooth muscle cell migration and proliferation in vitro and in injured rat arteries by a synthetic matrix metalloproteinase inhibitor. Arterioscler. Throm. Vasc. Biol. 16, 28-33 (1996)

61. Cheng, L., Mantile, G., Pauly, R., Nater, C., Felici, A., Monticone, R., Bilato, C., Gluzband, Y. A., Crow, M. T., Stetler-Stevenson, W. and Capogrossi, M. C.: Adenovirus-mediated gene transfer of the human tissue inhibitor of metalloproteinase-2 blocks vascular smooth muscle cell invasiveness in vitro and modulates neointimal development in vivo. Circulation 98, 2195-201 (1998)

62. Hynes, R. O.: Integrins: versatility, modulation, and signaling in cell adhesion. Cell 69, 11-25 (1992)

63. Panda, D., Kundu, G. C., Lee, B. I., Peri, A., Fohl, D., Chackalaparampil, I., Mukherjee, B. B., Li, X. D., Mukherjee, D. C., Seides, S., Rosenberg, J., Stark, K. and Mukherjee, A. B.: Potential roles of osteopontin and alphavbeta3 integrin in the development of coronary artery restenosis after angioplasty. Proc. Natl. Acad. Sci. U S A 94, 9308-13 (1997)

64. Hoshiga, M., Alpers, C. E., Smith, L. L., Giachelli, C. M. and Schwartz, S. M.: Alpha-v beta-3 integrin expression in normal and atherosclerotic artery. Circ. Res. 77, 1129-35 (1995)

65. Stouffer, G. A., Hu, Z., Sajid, M., Li, H., Jin, G., Nakada, M. T., Hanson, S. R. and Runge, M. S.: Beta3 integrins are upregulated after vascular injury and modulate thrombospondin- and thrombin-induced proliferation of cultured smooth muscle cells. Circulation 97, 907-15 (1998)

66. Choi, E. T., Engel, L., Callow, A. D., Sun, S., Trachtenberg, J., Santoro, S. and Ryan, U. S.: Inhibition of neointimal hyperplasia by blocking alphavbeta3 integrin with a small peptide antagonist GpenGRGDSPCA. J. Vasc. Surg. 19, 125-34 (1994)

67. Srivatsa, S. S., Fitzpatrick, L. A., Tsao, P. W., Reilly, T. M., Holmes, D. R., Jr., Schwartz, R. S. and Mousa, S. A.: Selective alphavbeta3integrin blockade potently limits neointimal hyperplasia and lumen stenosis following deep coronary arterial stent injury: evidence for the functional importance of integrin alphavbeta3 and osteopontin expression during neointima formation. Cardiovasc. Res. 36, 408-28 (1997)

68. Topol, E. J., Califf, R. M., Weisman, H. F., Ellis, S. G., Tcheng, J. E., Worley, S., Ivanhoe, R., George, B. S., Fintel, D., Weston, M. et al.: Randomized trial of coronary intervention with antibody against platelet IIb/IIIa integrin for reduction of clinical restenosis: results at six months. Lancet 343, 881-6 (1994)

69. Karim, M. A., Miller, D. D., Farrar, M. A., Elefheriades, E., Reddy, B. H., Brelan, C. M. and Samarel, A. M.: Histomorphometric and biochemical correlates of arterial procollagen gene expression during vascular repair after experimental angioplasty. Circulation 91, 2049-57 (1995)

70. Coats, W. D., Jr., Whittaker, P., Cheung, D. T., Currier, J. W., Han, B. and Faxon, D. P.: Collagen content is significantly lower in restenotic versus nonrestenotic vessels after balloon angioplasty in the atherosclerotic rabbit model. Circulation 95, 1293-300 (1997)

71. Koyama, H., Raines, E. W., Bornfeldt, K. E., Roberts, J. M. and Ross, R.: Fibrillar collagen inhibits arterial smooth muscle proliferation through regulation of cdk2 inhibitors. Cell 87, 1069-78 (1996)

72. Zhu, X., Ohtsubo, M., Böhmer, R. M., Roberts, J. M. and Assoian, R. K.: Adhesion-dependent cell cycle progression linked to the expression of cyclin D1, activation of cyclin E-cdk2, and phosphorylation of the retinoblastoma protein. J. Cell Biol. 133, 391-403 (1996)

73. Lawler, J., Slayter, H. S. and Coligan, J. E.: Isolation and characterization of a high molecular weight glycoprotein from human blood platelets. J. Biol. Chem. 253, 8609-16 (1978)

74. McPherson, J., Sage, H. and Bornstein, P.: Isolation and characterization of a glycoprotein secreted by aortic endothelial cells in culture: apparent identity with platelet thrombospondin. J. Biol. Chem. 256, 11330-6 (1981)

75. Reed, M. J., Iruela-Arispe, L., O'Brien, E. R., Truong, T., LaBell, T., Bornstein, P. and Sage, E. H.: Expression of thrombospondins by endothelial cells. Injury is correlated with TSP-1. Am. J. Pathol. 147, 1068-80 (1995)

76. Jaffe, E. A., Ruggiero, J. T. and Falcone, D. J.: Monocytes and macrophages synthesize and secrete thrombospondin. Blood 65, 79-84 (1985)

77. Jaffe, E. A., Ruggiero, J. T., Leung, L. K., Doyle, M. J., McKeown-Longo, P. J. and Mosher, D. F.: Cultured human fibroblasts synthesize and secrete thrombospondin and incorporate it into extracellular matrix. Proc. Natl. Acad. Sci. USA 80, 998-1002 (1983)

78. Mumby, S. M., Abbott Brown, D., Raugi, D. and Bornstein, P.: Regulation of thrombospondin secretion by cells in culture. J Cell Physiol. 120, 280-8 (1984)

79. Frazier, W. A.: Thrombospondins. Curr. Opin. Cell. Biol. 3, 792-9 (1991)

80. Asch, A. S., Tepler, J., Silbiger, S. and Nachman, R. L.: Cellular attachment to thrombospondin: Cooperative interactions between receptor systems. J. Biol. Chem. 266, 1740-5 (1991)

81. Bagavandoss, P. and Wilks, J. W.: Specific inhibition of endothelial cell proliferation by thrombospondin. Biochem. Biophys. Res. Comm. 170, 867-72 (1990)

82. Taraboletti, G., Roberts, D., Liotta, L. A. and Giavazzi, R.: Platelet thrombospondin modulates endothelial cell adhesion, motility, and growth: a potential angiogenesis regulatory factor. J. Cell. Biol 111, 765-72 (1990)

83. O'Shea, K. S. and Dixit, V. M.: Unique distribution of extracellular matrix component thrombospondin in the developing mouse embryo. J. Cell. Biol. 101, 2737-48 (1988)

84. Good, D. J., Polverini, P. J., Rastinejad, F., Le Beau, M. M., Lemons, R. S., Frazier, W. A. and Bouck, N. P.: A tumor suppressor-dependent inhibitor of angiogenesis is immunologically and functionally indistinguishable from a fragment of thrombospondin. Proc. Natl. Acad. Sci. USA 87, 6624-8 (1990)

85. Iruela-Arispe, M. L., Bornstein, P. and Sage, H.: Thrombospondin exerts an antiangiogenic effect on cord formation by endothelial cells in vitro. Proc. Natl. Acad. Sci. USA. 88, 5026-30 (1991)

86. Majack, R. A., Cook, S. C. and Bornstein, P.: Control of smooth muscle cell growth by components of the extracellular matrix: Autocrine role for thrombospondin. Proc. Natl. Acad. Sci. USA 83, 9050-4 (1986)

87. Yabkowitz, R. Y., Mansfield, P. J., Ryan, U. S. and Suchard, S. J.: Thrombospondin mediates migration and potentiates platelet-derived growth factor-dependent migration of calf pulmonary artery smooth muscle cells. J. Cell. Physiol. 157, 24-32 (1993)

88. Dixit, V. M., Haverstick, D. M., O'Rourke, K. M., Hennessy, S. W., Grant, G. A., Santoro, S. A. and Frazier, W. A.: Monoclonal antibodies against human thrombospondin inhibit platelet aggregation. Proc. Natl. Acad. Sci. USA. 82, 3472-6 (1985)

89. Tuszynski, G. P., Rothman, V. L., Murphy, A., Siegler, K. and Knudsen, K. A.: Thrombospondin promotes platelet aggregation. Blood 72, 109-15 (1988)

90. Wight, T. N., Raugi, G. J., Mumby, S. M. and Bornstein, P.: Light microscopic immunolocation of thrombospondin in human tissues. J. Histochem. Cytochem 33, 295-302 (1985)

91. Raugi, G. J., Mullen, J. S., Barb, D. H., Okada, T. and Mayberg, M. R.: Thrombospondin deposition in rat carotid artery injury. Am. J. Pathol. 137, 179-85 (1990)

92. Botney, M. D., Kaiser, L. R., Cooper, J. D., Mecham, R. P., Parghi, D., Roby, J. and Parks, W. C.: Extracellualr matrix protein gene expression in atherosclerotic hypertensive pulmonary arteries. Am. J. Pathol. 140, 357-64 (1992)

93. Liau, G., Winkles, J. A., Cannon, M. S., Kuo, L. and Chilian, W. M.: Dietary-induced atherosclerotic lesions have increased levels of acidic FGF mRNA and altered cytoskeletal and extracellular matrix mRNA expression. J. Vasc. Res. 30, 327-32 (1993)

94. Van Zanten, G. H., de Graaf, S., Slootweg, P. J., Heijnen, H. F. G., Connolly, T. M., de Groot, P. G. and Sixma, J. J.: Increased platelet deposition on atherosclerotic coronary arteries. J. Clin. Invest. 93, 615-32 (1994)

95. Roth, J. J., Gahtan, V., Brown, J. L., Gerhard, C., Swami, V. K., Rothman, V. L., Tulenko, T. N. and Tuszynski, G. P.: Thrombospondin-1 is elevated with both intimal hyperplasia and hypercholesterolemia. J. Surg. Res. 74, 11-6 (1998)

96. Chen, D., Asahara, T., Krasinski, K., Witzenbichler, B., Yang, J., Magner, M., Kearney, M., Frazier, W. A., Isner, J. M. and Andrés, V.: Antibody blockade of thrombospondin accelerates reendothelialization and reduces neointima formation in balloon-injured rat carotid artery . Circulation 100, 849-54 (1999)

97. Chen, D., Guo, K., Yang, J., Frazier, W. A., Isner, J. M. and Andrés, V.: Vascular smooth muscle cell growth arrest upon blockade of thrombospondin-1 requires p21Cip1/WAF1. Am. J. Physiol. 277, H1100-H6 (1999)

98. Moncada, S., Palmer, R. M. J. and Higgs, E. A.: Nitric oxide: physiology, pathophysiology and pharmacology. Pharmacol. Rev. 43, 109-42 (1991)

99. Nava, E., Noll, G. and Lüscher, T. F.: Nitric oxide in cardiovascular disease. Ann. Med. 27, 343-51 (1995)

100. Bjornsson, T. D., Dryjski, M., Tluczek, J., Mennie, R., Ronan, J., Mellin, T. N. and Thomas, K. A.: Acidic fibroblast growth factor promotes vascular repair. Proc. Natl. Acad. Sci. USA 88, 8651-5 (1991)

101. Asahara, T., Bauters, C., Pastore, C., Kearney, M., Rossow, S., Bunting, S., Ferrara, N., Symes, J. F. and Isner, J. M.: Local delivery of vascular endothelial growth factor accelerates reendothelization and attenuates intimal hyperplasia in balloon-injured rat carotid artery. Circulation 91, 2793-801 (1995)

102. Van Belle, E., Maillard, L., Tio, F. O. and Isner, J. M.: Accelerated endothelialization by local delivery of recombinant human vascular endothelial growth factor reduces in-stent intimal formation. Biochem. Byophis. Res. Commun. 235, 311-6 (1997)

103. Moroi, M., Zhang, L., Yasuda, T., Virmani, R., Gold, H. K., Fishman, M. C. and Huang, P. L.: Interaction of genetic deficiency of endothelial nitric oxide, gender, and pregnancy in vascular response to injury in mice. J. Clin. Invest. 101, 1225-32 (1998)

104. Rudic, R. D., Shesely, E. G., Maeda, N., Smithies, O., Segal, S. S. and Sessa, W. C.: Direct evidence for the importance of endothelium-derived nitric oxide in vascular remodeling. J. Clin. Invest. 101, 731-6 (1998)

105. González-Fernández, F., López-Farré, A., Rodríguez-Feo, J. A., Farré, J., Guerra, J., Fortes, J., Millás, I., García-Durán, M., Rico, L., Mata, P., de Miguel, L. S. and Casado, S.: Expression of inducible nitric oxide synthase after endothelial denudation of the rat carotid artery - Role of platelets. Circ. Res. 83, 1080-7 (1998)

106. Yan, Z.-q. and Hansson, G. K.: Overexpression of inducible nitric oxide synthase by neointimal smooth muscle cells. Circ. Res. 82, 21-9 (1998)

107. de Frutos, T., de Miguel, L. S., García-Durán, M., González-Fernández, F., Rodríguez-Feo, J. A., Montón, M., Guerra, J., Farré, J., Casado, S. and Lopez-Farré, A.: NO from smooth muscle cells decreases NOS expression in endothelial cells: role of TNF-alpha. Am. J. Physiol. 277, H1317-25 (1999)

108. Schwarzacher, S. P., Lim, T. T., Wang, B., Kernoff, R. S., Neibauer, J., Cooke, J. P. and Yeung, A. C.: Local intramural delivery of L-arginine enhances nitric oxide generation and inhibits lesion formation after balloon angioplasty. Circulation 95, 1863-9 (1997)

109. Cooke, J. P., Singer, A. H., Tsao, P., Zera, P., Rowan, R. A. and Billingham, M. E.: Anti-atherogenic effects of L-arginine in the hypercholesterolemic rabbit. J Clin. Invest. 90, 1168-72 (1992)

110. McNamara, D. B., Bedi, B., Aurora, H., Tena, L., Ignarro, L. J., Kadowitz, P. J. and Akers, D. L.: L-arginine inhibits balloon catheter-induced intimal hyperplasia. Biochem. Biophys. Res. Commun. 193, 291-6 (1993)

111. Hamon, M., Vallet, B., Bauters, C., Wernet, N., McFadden, E. P., Lablanche, J., Dupuis, B. and Bertrand, M. E.: Long-term oral administration of L-arginine reduces intimal thickening and enhances neoendothelium-dependent acetylcholine-induced relaxation after arterial injury. Circulation 90, 1357-62 (1994)

112. von der Leyen, H. E., Gibbons, G. H., Morishita, R., Lewis, N. P., Zhang, L., Nakajima, M., Kaneda, Y., Cooke, J. P. and Dzau, V. J.: Gene therapy inhibiting neointimal vascular lesion: In vivo transfer of endothelial cell nitric oxide synthase gene. Proc. Natl. Acad. Sci. USA 92, 1137-41 (1995)

113. Shears, L. L., II, Kawaharada, N., Tzeng, E., Billiar, T. R., Watkins, S. C., Kovesdi, I., Lizonova, A. and Pham, S. M.: Inducible nitric oxide synthase suppresses the development of allograft arteriosclerosis. J. Clin. Invest. 100, 2035-42 (1997)

114. Janssens, S., Flaherty, D., Nong, Z., Varenne, O., van Pelt, N., Haustermans, C., Zoldhelyi, P., Gerard, R. and Collen, D.: Human endothelial nitric oxide synthase gene transfer inhibits vascular smooth muscle cell proliferation and neointima formation after balloon injury in rats. Circulation 97, 1274-81 (1998)

115. Varenne, O., Pislaru, S., Gillijns, H., Van Pelt, N., Gerard, R. D., Zoldhelyi, P., Van de Werf, F., Collen, D. and Janssens, S. P.: Local adenovirus-mediated transfer of human endothelial nitric oxide synthase reduces luminal narrowing after coronary angioplasty in pigs. Circulation 98, 919-26 (1998)

116. Chen, L., Daum, G., Forough, R., Clowes, M., Walter, U. and Clowes, A. W.: Overexpression of human endothelial nitric oxide synthase in rat vascular smooth muscle cells and in balloon-injured carotid artery. Circ. Res. 82, 862-70 (1998)

117. Cayatte, A. J., Palacino, J. J., Horten, K. and Cohen, R. A.: Chronic inhibition of nitric oxide production accelerates neointima formation and impairs endothelial function in hypercholesterolemic rabbits. Arterioscler. Thromb. 14, 753-9 (1994)

118. Kauser, K., da Cunha, V., Fitch, R., Mallari, C. and Rubanyi, G. M.: Role of endogenous nitric oxide in progression of atherosclerosis in apolipoprotein E-deficient mice. Am. J. Physiol. 278, H1679-H85 (2000)

119. Weisbrod, R. M., Griswold, M. C., Du, Y., Bolotina, V. M. and Cohen, R. A.: Reduced responsiveness of hypercholesterolemic rabbit aortic smooth muscle cells to nitric oxide. Arterioscler. Thromb Vasc. Biol. 17, 394-402 (1997)

120. Ishida, A., Sasaguri, T., Kosaka, C., Nojima, H. and Ogata, J.: Induction of the cyclin-dependent kinase inhibitor p21Sdi1/Cip1/Waf1 by nitric oxide-generating vasodilator in vascular smooth muscle cells. J. Biol. Chem. 271, 10050-7 (1997)

121. Guo, K., Andrés, V. and Walsh, K.: Nitric oxide-induced downregulation of cdk2 activity and cyclin A gene transcription in vascular smooth muscle cells. Circulation 20, 2066-72 (1998)

122. Chen, L., Daum, G., Fischer, J. W., Hawkins, S., Bochaton-Piallat, M.-L., Gabbiani, G. and Clowes, A. W.: Loss of expression of the beta subunit of soluble guanylil cyclase prevents nitric oxide-mediated inhibition of DNA synthesis in smooth muscle cells of old rats. Circ. Res. 86, 520-5 (2000)

123. Gu, M., Lynch, J. and Brecher, P.: Nitric oxide increases p21Waf1/Cip1 expression by a cGMP-dependent pathway that includes activation of extracellular signal-regulated kinase and p70S6k. J. Biol. Chem. 275, 11389-96 (2000)

124. Sarkar, R., Gordon, D., Stanley, J. C. and Webb, R. C.: Cell cycle effects of nitric oxide on vascular smooth muscle cells. Am. J. Physiol. 272, H1810-H8 (1997)

125. Tanner, F. C., Meier, P., Greutert, H., Champion, C., Nabel, E. G. and Lüscher, T. F.: Nitric oxide modulates expression of cell cycle regulatory proteins. A cytostatic strategy for inhibition of human vascular smooth muscle cell proliferation. Circulation 101, 1982-9 (2000)

126. Fukumoto, S., Koyama, H., Hosoi, M., Yamakawa, K., Tanaka, S., Morii, H. and Nishizawa, Y.: Distinct role of cAMP and cGMP in the cell cycle control of vascular smooth muscle cells: cGMP delays cell cycle transition through suppression of cyclin D1 and cyclin-dependent kinase 4 activation. Circ. Res. 85, 985-91 (1999)

127. Yu, S. M., Hung, L. M. and Lin, C. C.: cGMP-elevating agents suppress proliferation of vascular smooth muscle cells by inhibiting the activation of epidermal growth factor signaling pathway . Circulation 95, 1269-77 (1997)

128. Wilcox, J. N., Cipolla, G. D., Martin, F. H., Simonet, L., Dunn, B., Ross, C. E. and Scott, N. A.: Contribution of adventitial myofibroblasts to vascular remodeling and lesion formation after experimental angioplasty in pig coronary arteries. Ann. N. Y. Acad. Sci. 811, 437-47 (1997)

129. Gu, M. and Brecher, P.: Nitric oxide-induced increase in p21Sdi1/Cip1/Waf1 expression during the cell cycle in aortic adventitial fibroblasts. Arterioscler. Thromb. Vasc. Biol. 20, 27-34 (2000)

130. Geisterfer, A. A., Peach, M. J. and Owens, G. K.: Angiotensin II induces hypertrophy, not hyperplasia, of cultured rat aortic smooth muscle cells. Circ. Res. 62, 749-56 (1988)

131. Gibbons, G. H., Pratt, R. E. and Dzau, V. J.: Vascular smooth muscle cell hypertrophy vs. hyperplasia. Autocrine transforming growt factor-beta1 expression determines growth response to angiotensin II. J. Clin. Invest. 90, 456-61 (1992)

132. Naftilan, A. J., Pratt, R. E. and Dzau, V. J.: Induction of platelet-derived growth factor A-chain and c-myc gene expression by angiotensin II in cultured rat vascular smooth muscle cells. J. Clin. Invest. 83, 1419-24 (1989)

133. Naftilan, A. J., Gilliland, G. K., Eldridge, C. S. and Kraft, A. S.: Induction of the protooncogen c-jun by angiotensin II. Mol. Cell. Biol. 10, 5536-40 (1990)

134. Braun-Dullaeus, R. C., Mann, M. J., Ziegler, A., von der Leyen, H. E. and Dzau, V. J.: A novel role for the cyclin-dependent kinase inhibitor p27Kip1 in angiotensin II-stimulated vascular smooth muscle cell hypertrophy. J. Clin. Invest. 104, 815-23 (1999)

135. Servant, M. J., Coulombe, P., Turgeon, B. and Meloche, S.: Differential regulation of p27Kip1 expression by mitogenic and hypertrophic factors: Involvement of transcriptional and posttranscriptional mechanisms. J. Cell Biol. 148, 543-56 (2000)

136. Chang, B. D., Watanabe, K., Broude, E. V., Fang, J., Poole, J. C., Kalinichenko, T. V. and Roninson, I. B.: Effects of p21Waf1/Cip1/Sdi1 on cellular gene expression: implications for carcinogenesis, senescence, and age-related diseases. Proc. Natl. Acad. Sci. U S A 97, 4291-6 (2000)

137. Jensen, L. E. and Whitehead, A. S.: Regulation of serum amyloid A protein expression during the acute-phase response. Biochem. J. 334, 489-503 (1998)

138. Oemar, B. S., Werner, A., Garnier, J. M., Do, D. D., Godoy, N., Nauck, M., März, W., Rupp, J., Pech, M. and Lüscher, T. F.: Human connective tissue growth factor is expressed in advanced atherosclerotic lesions. Circulation 95, 831-9 (1997)

139. Nachtigal, M., Al-Assaad, Z., Mayer, E. P., Kim, K. and Monsigny, M.: Galectin-3 expression in human atherosclerotic lesions. Am. J. Pathol. 152, 1199-208 (1998)