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(The FASEB Journal. 2001;15:2162-2170.)
© 2001 FASEB

Mutated p21/WAF/CIP transgene overexpression reduces smooth muscle cell proliferation, macrophage deposition, oxidation-sensitive mechanisms, and restenosis in hypercholesterolemic apolipoprotein E knockout mice

GIANLUIGI CONDORELLI*,{dagger}1, JOYCE K. AYCOCK*,{dagger}, GIACOMO FRATI*,{dagger} and CLAUDIO NAPOLI{ddagger},§

* Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA;
{dagger} II Faculty of Medicine, IRCCS Neuromed, ‘La Sapienza’ University of Rome, Italy;
{ddagger} Department of Medicine, Federico II University of Naples, Italy; and
§ Department of Medicine-0682, University of California, San Diego, California 92093, USA

1Correspondence: Kimmel Cancer Center, TJU 233 S. 10th St., Philadelphia, PA 19107, 1907, USA. E-mail: gianluigi.condorelli{at}mail.tju.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We have investigated whether by introducing a mutated p21 cyclin-dependent kinase inhibitor through a standard type 5 adenovirus (Ad), it would be possible to interfere with restenosis in hypercholesterolemic apolipoprotein E knockout mice. Restenosis is a clinically relevant, undesired effect of percutaneous transluminal coronary angioplasty (PTCA). A critical event underlying restenosis is smooth muscle cell (SMC) proliferation leading to neointimal formation and vessel reocclusion. Recent data demonstrated that it is possible to reduce restenosis by introducing various genes blocking the cell cycle through Ad vectors. Nonetheless, most experiments were conducted in the healthy carotid artery of rat, which is far from the condition of human disease. Therefore, we investigated whether antiproliferative or proapoptotic genes affect restenosis in a model of atherosclerosis closer to clinical settings. Ad-mutated(m)-p21WAF/CIP1 transgene overexpression induces a significant reduction of restenosis in hypercholesterolemic apolipoprotein E knockout mice subjected to injury of common carotid artery. This was associated with reduced SMC density and proliferation, macrophage deposition, and oxidation-sensitive mechanisms. Treatment with p21/WAF also enhanced TUNEL positivity of arterial cells. We show that in an experimental model of atherosclerosis, braking the cell proliferation through increased vascular apoptosis and reduced oxidation-sensitive signal transduction and macrophage accumulation can significantly ameliorate the deleterious effects of vascular injuries similar to those that occur during PTCA and related procedures.—Condorelli, G., Aycock, J. K., Frati, G., Napoli, C. Mutated p21/WAF/CIP transgene overexpression reduces smooth muscle cell proliferation, macrophage deposition, oxidation-sensitive mechanisms, and restenosis in hypercholesterolemic apolipoprotein E knockout mice.


Key Words: gene therapy • apoptosis • cell cycle • apo E knockout


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
DESPITE CONSIDERABLE PROGRESS in recent years, pharmacological therapies have not provided a complete solution for common cardiovascular problems, including restenosis postpercutaneous transluminal coronary angioplasty (PTCA) (1) . Optimal drug dosage reproducing plasma concentrations achieved in animal studies established that this approach would often be too toxic to administer, especially when given chronically. In sharp contrast to drug treatments, local gene therapy can extend naturally the presence of the beneficial agent to weeks and, in many cases, longer periods (1 , 2) .

Smooth muscle cell (SMC) proliferation is a critical biological event in determining arterial reocclusion after PTCA (1) . During stretch, growth factors are released by platelets and white blood cells; prothrombotic factors trigger the cell cycle in SMCs of the tunica media of the arteries, switching from G1 to S phase (3) . Recently, the control of SMC proliferation by blocking genes involved in the cell cycle was shown to be a tool with potentially relevant clinical applications (1 , 4) . Besides the cell cycle and apoptosis (programmed cell death), it was also been shown to be a critical phenomenon in determining the extent of restenosis (reviewed in refs 3 , 4 ). Indeed, inhibition of antiapoptotic genes worsens restenosis in the uninvolved vessels of the rat model of angioplasty.

Although the predictive value of the data obtained from the study of animal models for restenosis is limited, encouraging signs are emerging from the development of murine transgenic models of atherosclerosis (reviewed in refs 5 , 6 ). The apolipoprotein E knockout mouse holds promise as a useful animal model in the study of vascular interventions, since lesion distribution and histology are similar to those of human disease (5 , 6) . Thus, the primary utility of atherosclerotic mouse models appears to be in the study of specific components of the response to arterial injury, such as the control of cell cycle progression of instrumented diseased arteries (5) . It is still debated whether hypercholesterolemia influences per se the luminal loss and restenosis after PTCA (7 8 9 10 11) . In the presence of concomitant hypercholesterolemia, restenosis can reach higher severity, and is refractory to cholesterol-lowering drugs or apheresis (12 , 13) . Moreover, there is no doubt that hypercholesterolemia induces an impairment of endothelium-dependent relaxation at sites where angioplasty had previously been performed (7 8 9 10 11 12 13) . Thus, apolipoprotein E knockout hypercholesterolemic mice may also help us to study the effects of hypercholesterolemia and atherosclerosis on pathophysiologic mechanisms involved in restenosis.

The control of the cell cycle by inhibitors of proliferation through gene therapy was shown to directly affect restenosis after PTCA. Introduction of genes acting on the cell cycle machinery such as the universal inhibitor of cyclin-dependent kinase (CDK) p21 (14 , 15) and genes blocking the transcription factors of the E2F family, such as the retinoblastoma-like genes (16 17 18) (pRB and p-RB-like molecules), has proved to be efficient at this task. Most of these experiments were performed in the rat carotid artery model (i.e., in healthy vessels of normocholesterolemic animals); the efficacy of such an approach in atherosclerotic mouse models that would be closer to the human disease status is still unknown. The goal of the present study was to determine the effects of mp21/WAF/CIP1 transgene overexpression on restenosis in the hypercholesterolemic apolipoprotein E knockout mouse model.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Adenoviral production and transduction
The generation of mp21/WAF adenovirus (type 5), a gift from Dr. El-Deiry (University of Pennsylvania, PA), has already been described in detail (19) . The mp21/WAF used is a human carboxyl-terminal truncated form (1–341) (Fig. 1 ). Such a mutation increases the biological effects of mp21/WAF, since it is able to induce growth arrest by interacting with cyclin-dependent kinase 2 (CDK2) and apoptosis in tumor cell lines unresponsive to p53 (19) . Propagation and purification replication-defective mp21 and ß-gal adenoviruses were performed according to the standard procedures, as described (18) .



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  		Figure 1. Adenoviral construct in pJM17 composed of the CMV promoter, p21/WAF carboxyl-terminal truncated form 1–341 and poly(A).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animal studies were carried out according to the Guidelines of the American Heart Association for Accreditation of Laboratory Animal Care and complied with the Guide for the Care and Use of Laboratory Animals (NIH publication no. 86–23, 1985). Male apolipoprotein E knockout mice (Jackson Labs, West Grove, PA) in a C57BL background were fed a classical Western-type atherogenic diet (21% fat by weight, 0.15% by weight cholesterol, and 19.5% by weight casein; #88137, Harlan/Teklad, Madison, WI) from 6–8 wk to 20 wk of age (6) . We selected only male animals in order to avoid gender-related effects. All mice included in this study were healthy with no apparent defects. After anesthesia with ketamine (80 mg/kg body weight, intraperitoneal (i.p.) Ketaset) and xylazine (5 mg/kg body weight, i.p. AnaSed), the carotid artery was exposed (midline neck incision) under light microscope and ligated distally (6–0 silk ties). According to the procedure proposed by Manka et al. (20) , arterial injury was induced by a microcatheter introduced with a 0.014-inch flexible angioplasty guidewire into the common carotid artery (21) . Eccentric plaques (mild to moderate intimal thickening) are often present at this site, which reduce the lumen of the vessel of 30–35% (125–150 µm thickness of lesions). Our preliminary experiments and the work of others (20 , 21) revealed that this experimental procedure resulted in a completely denuded surface covered by platelets 30 min after injury and significant neointima formation and medial thickening 14 days after injury.

After vessel injury (n=8 as a control group that received arterial injury alone), a segment of the common carotid artery of 6–7 mm was isolated through vascular clamps, and 25 µl of adenovirus mp21 (Ad-mp21; 1x109) (n=8) or Ad-ßgal (ßgalactosidase viral vector, 1x109 cfu) (n=6) was instilled for 25 min of incubation (18) . The vector-containing medium was withdrawn; after removal of the wire, the normal anterograde blood flow was reestablished, a cotton-tipped applicator was applied to tamponade bleeding, and the skin was closed with three or four surgical staples (18) . Topical antibiotic ointment was then applied. The contralateral common carotid artery served as a noninjured control vessel. All animals survived until the anticipated time of death without bleeding or infection. Animal health and weight were monitored throughout treatment. A blood sample was obtained from the right ventricle to measure cholesterol and for measurement of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) by standard procedures.

Morphometry, immunohistochemistry, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL), and Western blot analysis
At the time of death (lethal dose of sodium pentobarbital or methoxyflurane 14 days after injury), the animals were reanesthetized and a 24-gauge needle was placed in the left to achieve in situ perfusion fixation of the carotid arteries at physiological pressure (100 mm Hg), with phosphate-buffered paraformaldehyde (4%, 0.1 mol/l, pH 7.3) for histology and normal saline for immunohistochemistry assessed by computer-assisted imaging analysis (6 , 21 22 23) . Histomorphometric parameters were performed by two investigators separately and in a blinded manner. Both common carotid segments (transfected and nontransfected) were then immersed in 4% paraformaldehyde.

Atherosclerotic carotid lesions were determined by oil red O-stained sections using computer-assisted imaging analysis (6 , 21 22 23) . After stepwise dehydration with graded alcohols, specimens were embedded in epoxy-araldite resin. Arteries were serially sectioned in 15–20 slices (3–5 µM) with a rotating diamond-coated saw (Leica, Germany). Every second slide was stained with hematoxylin and eosin for morphometry. Some sections were stained with toluidine blue. The length of the external elastic lamina, the area confined by the internal elastic lamina, and the cross-sectional neointimal area were measured by morphometry (18 , 24) . The percent area of stenosis was then calculated; the vessel injury score was also determined (21) . In additional sections, atherosclerotic carotid artery lesions were determined by oil red O-stained sections using computer-assisted imaging analysis (6 , 22 23 24) . To determine whether Ad-mp21 affected SMC migration or proliferation in injured lesions, total cell number and the number of proliferating cells in the neointima of the arterial cross sections were determined for each animal by computer-assisted imaging cell counting after immunohistochemistry of serial sections with {alpha}-actin monoclonal mouse antibody (Dako-M0858; Carpinteria, CA) and antibody against proliferating cell nuclear antigen (PCNA, 1:250 dilution; clone PC10-Dako; the adjacent medial layer of vessels served as positive control) (6 , 18 , 21 22 23) . Macrophage-derived foam cells were stained by the F4–80 antibody (1:500 dilution; Accurate Chemical and Scientific, Westbury, NY). Vascular smooth muscle cells (VSMCs) were counted only if they stained for VSMC {alpha}-actin and the cell nucleus was visible. The mean percentage of VSMCs relative to total cells was determined for each animal. To determine whether the PCNA-positive cells were derived from VSMCs, immunohistochemical double staining (21 , 24) with PCNA and VSMC {alpha}-actin was performed. On the double-labeled slides, each field was scored for the number of PCNA-positive nuclei associated with cytoplasm positive for VSMC. The number of intimal VSMCs and total cell nuclei were counted in eight nonoverlapping fields. The ratio of the total number of double-labeled cells to total number of PCNA-labeled cells was used to indicate percentages of VSMC proliferative activity in the arterial wall. Additional experiments were performed using F4–80/PCNA double staining. Epitopes recognized by the primary antibody were detected by an avidin-biotin-peroxidase method (6 , 21 , 22 , 24) . Negative controls were prepared by substitution of preimmune serum for the primary antibody.

mp21 transgene overexpression (band of 14 Kd) (19) was detected in carotid sections by Western blot analysis (24 , 25) performed 2 and 7 days after arterial injury and by immunohistochemistry using the F-5 mouse monoclonal antibody specific for p21 and non-cross-reactive with p27 (Santa Cruz, Biotech. Inc., CA).

Finally, apoptotic cells were detected in situ on carotid artery cross sections using a modified TUNEL essentially as described previously in detail (26) . Briefly, cross sections were dewaxed, rehydrated, and incubated in 20 µg/ml proteinase K (Pharmacia Biotech, Uppsala, Sweden) for 1 h. Endogenous peroxidase was blocked by incubation in 3% hydrogen peroxidase for 5 min. Fragmented DNA were nick end-labeled with a mixture of terminal deoxynucleotidyl transferase (TdT) (21.5 U/section; Sigma, Steinheim, Germany) in a TdT buffer (Sigma) for 90 min at 37°C. The reaction was stopped by 15 min incubation in 0.5 M EDTA. Detection was made by streptavidin-conjugated peroxidase, followed by 15 min incubation in aminoethyl carbazole. The sections were counterstained with hematoxylin and calculated as TUNEL-positive cells/total number of nuclei. In negative control experiments, TdT was omitted from the labeling mixture and no staining was detected.

Statistical analysis
The inhibitory effects on SMC proliferation and restenosis induced by treatment with Ad-p21 and controls were compared using the Kruskal-Wallis test. Intimal cell density and SMC content of lesions from animals in treatment and control groups were compared using nonparametric ANOVA analysis with adjustment for multiple measurements from each animal.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Apolipoprotein E knockout mice
We enhanced common carotid atherosclerosis by feeding apolipoprotein E knockout mice a diet containing 0.15% cholesterol for 12–14 wk. The median (range) common carotid plaque area in an additional eight control mice killed at baseline (20 wk of age) was 0.126 mm2 (0.092–0.178). Those lesions were eccentric, and no occlusive or circumferential plaques were observed. The cholesterol levels in treatment and reference groups were not different. Indeed, median cholesterol levels at the time of death were 751 (range 542–878, n=8), 723 (561–864, n=6), and 728 mg/dl (558–866, n=8) in the Ad-mp21-treated group, Ad-ßgal-treated group, and control mice (arterial injury alone), respectively (P=0.89, NS). In a subset of mice, cholesterol levels were similar before and after gene transfer treatment. Median ± SD levels of AST (AST/SGOT) and ALT (ALT/SGOT) at the time of death were 172 ± 27 and 148 ± 25 (n=8), 165 ± 23 and 134 ± 28 (n=6), and 56 ± 4* and 46 ± 5* U/l (n=8) in the Ad-mp21, Ad-ßgal, and control (balloon injury alone) groups, respectively (*P<0.03 vs. both Ad-mp21 and Ad-ßgal-treated mice). Liver toxicity, as reflected by the elevation in AST and ALT serum levels in the Ad-treated groups, was relatively modest and probably due to the medium range of the virus titer. The median weights after treatment were 32 g (31 32 33 34 35 36 37) , 33 g (24 25 26 27 28 29 30 31 32 33 34) , and 35g (26 27 28 29 30 31 32 33 34 35 36 37 38 39) for the Ad-mp21, Ad-ßgal, and control groups (P=0.73, NS).

Effects of mp21/WAF/CIP transgene overexpression in the arterial wall
Western blot analysis clearly demonstrated a quantitative increase in mp21 protein expression in injured arteries from Ad-mp21-treated mice but not in those of Ad-ßgal-treated or control mice (Fig. 2 , upper lane). Similarly, immunohistochemistry confirmed increased F5-postive staining in injured arteries from Ad-mp21-treated mice (Fig. 2 , lower lane).



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  		Figure 2. Upper lane: Western blot analysis for transgene expression of the p21-truncated deletion mutant form 1–341 (14 Kd) in the carotid artery wall. A) Control Ad-ßgal at 2 days; B) Ad-mp21WAF/CIP1 2 days after arterial injury; C) Ad-mp21WAF/CIP1 at 7 days after arterial injury. Lower lane: immunohistochemistry with the same antibody. A) Control healthy contralateral carotid; B) injured carotid artery + Ad-ßgal 7 days after arterial injury; C) injured carotid artery + Ad-mp21WAF/CIP1 7 days after arterial injury.

Effects of arterial injury on intimal thickening
Upon histomorphologic analysis, restenosis after vessel injury of atherosclerotic arteries reflects migration and proliferation of vascular SMCs and accumulation of monocyte/macrophages. In contrast, these alterations were not seen in the control contralateral uninjured carotid artery. Hypercholesterolemia can exacerbate restenosis; in fact, wild-type mice have lesser degree of restenosis than apolipoprotein E knockout mice (neointima/media ratio of 68±11% greater than that of wild-type mice, P=0.0041; Fig. 3 A, B). Massive vascular cell proliferation (Fig. 3C ) concomitant to neointimal formation and SMC proliferation at the site of lesion was evident in control mice after arterial injury (Fig. 4 B, Table 1 , and Fig. 5 ). Moreover, SMC proliferation was associated with collagen I deposition (data not shown). This phenomenon was associated with intima remodelling and was significantly reduced by Ad-mp21 (Fig. 3D , Table 1 ). Consistently, the neointimal/media global area (N/M ratio), neointimal cells, and cell densities were significantly reduced by the Ad-mp21 transgene expression compared with controls (Table 1) . Accordingly, the residual percentage of lumen stenosis was significantly reduced (Table 1) . The arterial injury scores were similar among groups (Table 1) . Thus, differences were not ascribed to a different degree of arterial injury. These effects clearly demonstrate the beneficial actions of Ad-mp21 on the development of restenosis in the concomitant presence of hypercholesterolemia.



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  		Figure 3. Upper lane: photomicrographs of mouse carotid arteries 14 days after arterial injury. A) Histological appearance of carotid artery of ApoE-deficient mouse (x220). B) Wild-type (x220). Middle lane: C) Apo E-deficient untreated mouse (Ad-ßgal) stained for PC10 antibody (x250). D) ApoE deficient-treated mouse (Ad-mp21WAF/CIP1) (x250). Lower lane: macrophage accumulation at the same time point. E) untreated Apo E-deficient mouse (Ad-ßgal) (x650). F) Apo E-deficient-treated mouse (Ad-mp21WAF/CIP1) (x650). See Materials and Methods for further details.



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  		Figure 4. A) Double antibody staining with the macrophage antibody F4–80 developed by alkaline phosphatase and anti-PC10 developed by immunoperoxidase in carotid artery 14 days after injury (x650). B) Double antibody staining with the smooth muscle cell antibody M0858 developed by alkaline phosphatase and anti-PC10 developed by immunoperoxidase in carotid artery 14 days after injury (x720). Arrows indicate double-stained macrophage (A) or SMC (B) cells. Purple is alkaline phosphatase and red/brown is immunoperoxidase. C) TUNEL microsections of mouse carotid artery 14 days after arterial injury in Ad-ßgal-treated mice (x580). D) TUNEL microsections of mouse carotid artery 14 days after arterial injury in Ad-mp21WAF/CIP1-treated mice (x580). The latter treatment increased the number of apoptotic cells.


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  		Table 1. Effects of mp21 transgene overexpression on restenosis in apolipoprotein E knockout mice (mean ± SE)



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  		Figure 5. A) Percentage of lumen restenosis after vessel injury in control (dotted bar), Ad-ßgal-treated (vertical lanes bar), or mp21WAF/CIP1-treated (horizontal lanes bar) apoE arteries, respectively. B) Number of neointimal cells in control apoE arteries (dot bar), Ad-ßgal-treated (vertical lanes bar), or mp21WAF/CIP1-treated (horizontal lane bar) arteries. Asterisk indicates significant difference between control or Ad-ßgal-treated vs. mp21WAF/CIP1-treated arteries. No significance was found between control and Ad-ßgal-treated arteries. See also Table 1 .

Macrophage deposition and oxidation-sensitive mechanisms
Based on immunostaining for the macrophage marker F4–80, macrophage deposition at the site of injury (periluminal and near the internal elastic lamina) was not significantly inhibited by Ad-ßgal (-5±3% of reduction in serial arterial sections stained for macrophages; P=0.878 vs. control mice subjected to arterial injury alone, NS), but was significantly reduced in Ad-mp21-treated mice (-36±8% of reduction in serial arterial sections, P=0.0034 vs. control mice; Fig. 3E , F ). Consistent with the paucity of macrophages in the lesions of Ad-mp21-treated mice was the reduction of cholesterol ‘clefts’ that reflect lipid accumulation and foam cell formation. Double-label immunohistochemistry in serial carotid sections for macrophages revealed a marked reduction in macrophage proliferation (-41±10% of F4–80/PC10 double positive-stained sections vs. controls in the Ad-mp21WAF/CIP1-treated mice, P=0.0095), indicating that both types of cells were reduced by Ad-mp21WAF/CIP1 at the site of lesion (Fig. 4A ). Similarly, oxidation-specific epitope expression in carotid arteries was not changed by Ad-ßgal (8±5% of increment in serial arterial sections stained with the MDA-2 antibody; P=0.611 vs. control mice subjected to injury alone, NS), but was significantly reduced by Ad-mp21WAF/CIP1-mediated gene transfer (-19±5% of reduction in serial arterial sections stained with the MDA-2 antibody, P=0.032 vs. control mice).

Ad-mp21WAF/CIP1-induced apoptosis in the arterial wall
Restenotic lesions of ApoE Ad-ßgal-treated mice contained fewer apoptotic TUNEL-positive cells than carotid atherosclerotic lesions of Ad-mp21WAF/CIP1-treated mice (5±4% vs. 18±6%, P=0.0018). With regard to cell type, the lower frequency of apoptotic cells observed in restenotic tissue was attributable to SMCs and macrophages. For all lesions analyzed, significant inverse correlations were observed between the density of SMCs and the frequency of apoptosis (r=-0.78, P<0.001) as well as the density of macrophages (r=-0.69, P<0.002). Ad-mp21WAF/CIP1 gene transfer significantly increased the rate of TUNEL-positive cells attributable to SMCs and macrophages at the site of injury vs. Ad-ßgal-treated mice (Fig. 4C , D ; from 15±4% to 35±11%, P<0.01). SMCs and macrophages displayed chromatin condensation by electron microscopy localized to the edges of the nuclear membrane, cytoplasmic condensation, nuclear shrinking, and fragmentation, all morphological characteristics of cells undergoing apoptosis (26) .


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
SMC proliferation and macrophages deposition at the site of injury are involved in the pathogenesis of restenosis and vascular remodeling after PTCA (reviewed in refs 1 2 3 4 ). The present study demonstrates for the first time that treatment with Ad-mp21WAF/CIP1 induces a significant reduction in the degree of restenosis in hypercholesterolemic apolipoprotein E knockout mice. This phenomenon was related to reduced cell density and intimal thickening, SMC proliferation, macrophage deposition, and oxidation-sensitive mechanisms. Moreover, mp21/WAF enhances TUNEL positivity of intimal cells, which may reduce cell proliferation at the site of injury.

Thus, mp21/WAF/CIP1 can affect the restenosis process in the atherosclerotic carotids of apolipoprotein E knockout mice. This observation is remarkable because it provides the opportunity to test whether inhibitors of the cell cycle affect the progression of restenosis in these hypercholesterolemic animals. Either apolipoprotein E knockout mice or LDL receptor-deficient mice develop atherosclerotic lesions similar to those of humans (6) . Currently, only limited information on the effects of p21 in restenosis is available. It was previously shown that p21 can block SMC proliferation in vitro and decrease restenosis in the rat model of healthy carotid injury (14 , 15 , 18) . However, this model is distant from the clinical condition of PTCA in atherosclerotic coronary cases (5) . The physiological and pathophysiological effects of p21 are multiple. Besides regulating the cell cycle by interacting with CDK and inhibiting cell proliferation (reviewed in ref 27 ), p21 expression is up-regulated after oxidative stress induced by hydrogen peroxide (28) , ultraviolet rays, and ionizing radiations (29) by both p53-dependent and independent pathways (28) . Previous work has indicated overexpression of wild-type p21/WAF alone in some tumor cell lines is not able to block cell proliferation (19) . Nevertheless, a mutated form of mp21/WAF that is able to interact with cyclins (but not with PCNA) markedly affects the cell cycle in cells insensitive even to the cytostatic and apoptotic effects of p53 (19) . Thus, since mp21/WAF overexpression can deeply affect both the cell cycle and cellular response to stress conditions, this molecule represents an invaluable tool for assessing whether blocking the cell cycle together with inducing apoptosis can control restenosis in an extreme model of oxidative stress such as the apolipoprotein E knockout mice (6) .

In the present study, we found a high rate of mp21-transgene expression in the hypercholesterolemic apolipoprotein E knockout mouse with concentrations of the virus titer in the medium range (1x109). This is consistent with the evidence that high concentrations of the virus (i.e., >3x1010) have been associated with a toxic response in rat carotids (30) and the rabbit iliac artery (31) . Liver toxicity was lower to that observed when C57BL mice were transfected with 1–2 x 1010 adenoviral particles (32) . Our findings also agree with previous data that demonstrated greater transgene expression in the arteries from spontaneously atherosclerotic than normal rabbits (33 , 34) and in atherosclerotic vs. normal monkeys (33) . Advanced, complicated human atherosclerotic plaques demonstrated a similar efficiency of recombinant gene expression; areas of plaque rupture and thrombus are sites of predilection for expression of recombinant genes (2 , 35) . However, a study of patients with chronic leg ischemia showed that in areas of lipid-rich atheroma, the efficiency of intravascular gene transfer may be reduced (36) . It was recently demonstrated that adenovirus-mediated overexpression of tissue inhibitor of metalloproteinase 1 reduces atherosclerotic lesions in apolipoprotein E knockout mice (37) . Taken together, these studies demonstrate the feasibility of gene transfer to atherosclerotic arteries, but also highlight potential barriers to adenoviral gene delivery; further studies are needed to understand the possible clinical role of gene transfer in the restenosis post-PTCA of human atherosclerotic coronaries (2) . Encouraging results come from a recent study by the Finnish Group at the University of Kuopio (38) that demonstrated the feasibility of gene transfer in patients with coronary heart disease perfused with vascular endothelial growth factor complexed with liposomes at the time of PTCA. Finally, the growing body of evidence of the latest years indicates that adeno-associated virus could have potential clinical applications and long-term benefits (39) .

Restenosis can involve multiple factors that control inflammation, cell proliferation and migration, cholesterol metabolism, and interactions between cells, blood, and matrix (1 2 3 4) . It seems that modulation of SMC proliferation is also a critical phenomenon in the acute vascular injury in the atherosclerotic artery. Our results are consistent with this assumption. Although the present study cannot provide conclusive evidence for causality, the combined findings provide strong support for the hypothesis that transgene mp21 expression in the intima contributes to the reduction of restenosis through a significant reduction of SMC proliferation at the site of arterial injury in the apolipoprotein E knockout mouse. We also found that Ad-mp21/WAF/CIP1 overexpression affects restenosis by decreasing macrophage deposition in the injured artery, thus reducing the release of macrophage-derived growth factors and possibly the degree of oxidation-sensitive mechanisms. An antioxidant that reduces oxidation and atherogenesis, vitamin E reduced restenosis in cholesterol-fed rabbits (40) . Therefore, antioxidants may have a therapeutic role in the prevention of restenosis during the concomitant presence of hypercholesterolemia. Increased macrophage deposition was also observed recently in a similar experimental model of femoral restenosis (41) , in other experimental models (42 43 44) , in patients with primary lesions that developed restenosis after coronary atherectomy (45) , and after PTCA and/or stenting (46 , 47) . These effects were associated with an increased rate of apoptotic TUNEL-positive cells during Ad-mp21WAF/CIP1WAF overexpression. Decreases in programmed cell death may contribute to restenotic hyperplasia by prolonging the life span of intimal cells (48) . Ad-mp21WAF/CIP1WAF also induced apoptosis in cancer cell growth (19) . Sodium salicylate inhibits vascular SMC proliferation by up-regulation of p21WAF (49) . Thus, our data support the concept that the degree of restenosis can be affected via mechanisms that involve increased vascular cell apoptosis, oxidation-sensitive signaling transduction, and macrophage-dependent pathways.

The cell cycle of arterial cells can be affected by radiation therapy. Indeed, intravascular brachytherapy may help to reduce restenosis after PTCA (1 , 50) . Some beneficial effects of brachytherapy on restenosis could be attributable to the enhanced rate of apoptosis in the vascular wall; nevertheless, unpredictable biological effects of radiation and other technical problems may limit the extensive use of this new therapeutic strategy. Major limitations of the animal models used to study restenosis have included their relatively large size and the inability to study the effects of hypercholesterolemia (5) . Although the clinical predictive value of the data obtained from the study of animal models for restenosis may be limited, there are encouraging signs from transgenic mouse models of atherogenesis such as the apolipoprotein E knockout mouse (5) . Although the degree of neointimal hyperplasia was variable among mice, we have chosen to study restenosis in the carotid artery with respect to the femoral artery (41 , 51) because early atherosclerotic lesions are virtually absent in the femoral artery of apolipoprotein E knockout mice (6) . By using both wild-type and the apolipoprotein E-deficient mouse, we were able to determine the specific effects of hypercholesterolemia on restenosis after arterial injury. Other benefits to using this model come from the recent possibility of noninvasive in vivo magnetic resonance imaging of injury-induced neointima formation in the carotid artery of the apolipoprotein E knockout mouse (52) .

Factors other than vascular cell proliferation such as adhesion molecules, leukocyte infiltrates, and matrix composition likely also influence the development of restenosis in humans. Further studies may therefore provide insight to understanding pathophysiological mechanisms on the main signaling pathways and molecular cross-talk involved in the protective action of Ad-mp21WAF/CIP1; however, our results suggest that controlling both the cell cycle and apoptosis is critical for the treatment of restenosis in the common concomitant clinical presence of hypercholesterolemia. The recent introduction of coated stents and stent-based gene therapy (53) may offer the promise that new joint therapeutical strategies will be available in the clinical scenario.


   ACKNOWLEDGMENTS
 
This manuscript is dedicated in memory of Dr. Russel Ross. The present studies were supported by funds: 1% from the Italian Ministry of Health, Associazione Italiana Ricerca sul Cancro (A.I.R.C.), the American Heart Association to G.C, and grants ISNIH 99.56980 and M.U.R.S.T.; 40%/96.06183636 to C.N. The p21 adenovirus was generously provided by Dr. Wafik El-Deiry. We are indebted Dr. Carlo M. Croce for his continuous encouragement, Dr. PierPaolo Claudio for advice on adenovirus propagation, and Dr. Yang Lee for invaluable technical assistance with imaging analysis.

Received for publication January 10, 2001. Revision received June 6, 2001.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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