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(Circulation. 1998;98:2883-2890.)
© 1998 American Heart Association, Inc.

Basic Science Reports

Restoration of E2F Expression Rescues Vascular Endothelial Cells From Tumor Necrosis Factor-–Induced Apoptosis

Ioakim Spyridopoulos, MD; Nicole Principe, BS; Kevin L. Krasinski, BA; Shu-hua Xu, MS; Marianne Kearney, BS; Meredith Magner, BS; Jeffrey M. Isner, MD; Douglas W. Losordo, MD

From the Department of Medicine, Division of Cardiovascular Research, St. Elizabeth's Medical Center, Boston, Mass.

Correspondence to Douglas W. Losordo, MD, Department of Medicine, Division of Cardiovascular Research, St. Elizabeth's Medical Center, 736 Cambridge St, Boston, MA 02135. E-mail dlosordo{at}opal.tufts.edu

	Abstract

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Background—Normally, quiescent endothelial cells (EC) line the inner surface of arteries and protect against thrombosis and neointimal growth. A variety of noxious stimuli, including balloon angioplasty, may compromise EC integrity, thereby initiating proliferation and triggering the local release of cytokines, including tumor necrosis factor- (TNF-).

Methods and Results—In vivo blockade of TNF- using a soluble receptor molecule results in accelerated reendothelialization at sites of balloon angioplasty, suggesting an important physiological role of TNF- in attenuating regrowth of endothelium after balloon angioplasty. Our studies reveal that TNF-, an apoptosis-inducing cytokine, induces G1 cell-cycle arrest in proliferating EC. Quiescent EC are relatively immune to TNF-induced apoptosis versus proliferating EC, which display repression of the E2F transcription factor coincident with TNF-induced apoptosis and cell-cycle arrest. We also show that in this setting, E2F overexpression exerts a survival effect in proliferating EC and restores cell-cycle progression, in direct contrast to results of prior reports, which revealed that deregulated expression of E2F in normally cycling cells induces apoptosis.

Conclusions—These data demonstrate that TNF-induced apoptosis is highly dependent on cell-cycle activity and that E2F can function as survival factor under certain conditions.

Key Words: apoptosis • cells • endothelium • tumor necrosis factor • angioplasty

	Introduction

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Under normal conditions, the endothelial cells (EC) that line the arterial wall are contact-inhibited for growth.¹ In contrast, injury to the artery, such as abrasion of the vessel during balloon angioplasty, results in EC proliferation, which is driven by locally released cytokines.² Tumor necrosis factor- (TNF-) is a pleiotropic cytokine that has been shown to mediate inflammatory, proliferative, cytostatic, and cytotoxic effects in a variety of cell types, including EC.^{3 4 5 6 7 8} TNF- secretion by inflammatory cells has been widely studied; however, TNF- has also been shown to be expressed by mast cells⁹ and vascular smooth muscle cells in human atherosclerosis¹⁰ and restenosis,¹¹ with increased expression after balloon injury in multiple animal models.^{12 13} TNF- is capable of exerting widely ranging "activating" influences on EC, including induction of inflammatory responses¹⁴ and influencing angiogenesis,^{15 16} but it has also been shown to be capable of inducing programmed cell death.^{17 18 19} We have previously verified that TNF- exposure of endothelial cells results in typical features of apoptosis, including ultrastructural changes seen by electron microscopy, DNA strand breaks manifest as DNA laddering on gel electrophoresis, and positive TUNEL staining, as well as the induction of caspases.^{17 18} Given the demonstrated potential for TNF- to induce EC apoptosis and its enhanced expression at sites of balloon injury, we were interested in evaluating the effects of this cytokine on proliferating EC.

First, the potential physiological role of TNF- in the arterial response to injury was evaluated by in vivo blockade of TNF-, which results in accelerated reendothelialization after balloon angioplasty. These results suggest that TNF- acts to delay endothelial regrowth at sites of balloon injury. To examine the mechanisms of the effect of TNF- on EC recovery, in vitro studies were then performed; these reveal that TNF- induces apoptosis in proliferating EC to a much greater extent than in quiescent cells. TNF--induced EC apoptosis was shown to be accompanied by cell-cycle arrest in G₁phase, whereas EC in G₁ were shown uniquely vulnerable to TNF-–mediated apoptosis. The pivotal role of the E2F transcription factor in TNF-–induced EC apoptosis was shown (1) by the loss of E2F expression and activity induced by TNF-, and (2) by studies revealing that overexpression of E2F rescues the EC phenotype, inhibiting apoptosis and restoring cell-cycle progression.

	Methods

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Rat Carotid Injury Model
TNF-–Soluble Receptor Treatment
Male Sprague-Dawley rats (Charles River Labs, Wilmington, Mass) were divided into 2 groups. The treatment group consisted of 16 animals receiving intraperitoneal injections of TNF-soluble receptor (TNFsr) (generously supplied by Dr Michael B. Widmer, Immunex, Seattle, Wash) 2.5 mg/kg with the first dose administered before balloon injury (day 0) and subsequent doses every third day (days 3, 6, 9, and 12). The control group consisted of 15 animals receiving intraperitoneal injection of human IgG (Sigma) in an equivalent dose.

The TNFsr used was a recombinant fusion protein consisting of 2 ligand binding regions from the human p80 (TNFR2) receptor linked to the Fc region of human IgG1(human IgG was therefore used as the control). This soluble receptor molecule previously has been shown to neutralize human, rodent, and rabbit TNF- in vitro and in vivo.^{20 21} The dosage used (2.5 mg/kg IP every 3 days) was chosen on the basis of previously published studies and a pilot series performed in our laboratory.

Balloon Injury
All rats underwent balloon denudation of the carotid artery, as previously described.^{22 23 24}

Evaluation of Reendothelialization
Reendothelialization was assessed by staining with 0.5 mL 0.5% Evans blue dye (Sigma ), as previously described.^{22 24}

Endothelial-Cell Culture and Reagents
Human umbilical vein endothelial cells (HUVEC) were isolated from umbilical cord vein using enzymatic methods, as previously described.^{17 18} Cells in passages 3 to 5 were used for these experiments. Bovine aortic endothelial cells (BAEC) were isolated as previously described^{17 18} and maintained in DMEM containing 10% FCS, 100 U/mL streptomycin/penicillin, and 50 µg/mL gentamycin. Unless otherwise indicated, all experiments were performed on both HUVEC and BAEC.

Human recombinant TNF- was purchased from R&D Systems and, if not otherwise stated, was used at a concentration of 40 ng/mL. This dose was chosen on the basis of previously published reports demonstrating induction of EC apoptosis at this concentration; dose-response curves, which showed efficient induction of apoptosis within a reasonable time frame; and our own published experience.^{17 18}

Antibodies
Polyclonal antibodies for cyclins A, B, D1, E2F1, cdk2, cdc2, cdk4, and poly-ADP-ribose polymerase (PARP) were purchased (Sigma). Monoclonal antitubulin antibody was purchased from Calbiochem.

Adenoviral Constructs and Infection
Construction of the Ad-p16, Ad-p21, and Ad-ß-galactosidase recombinant adenoviruses used in this study was previously described.²⁵ Isolated recombinant viruses were identified by both restriction digestion and Western analysis. Viral stocks of Ad-E2F1 and Ad-CMV, generously supplied by Dr J.R. Nevins, Duke University, were prepared as previously described.²⁶

BAEC and HUVEC were plated at equal densities (200 000 cells per 100-mm-diameter plate) and infected for 12 hours in 10% FCS-containing media. Cells were harvested for Western analysis or fixed in 70% ethanol for DNA staining and subsequent flow-cytometric analysis. Efficiency of infection was evaluated by immunostaining of Ad-ß-galactosidase–infected cells, which revealed ß-galactosidase expression in >90% of cells (versus 0% of Ad-CMV or uninfected cells). All adenovirus experiments were repeated a minimum of 3 times.

Proliferation Assay
CellTiter 96 Aq_ueous nonradioactive MTS cell-proliferation assay (Promega) was used to assess cell viability and proliferation as previously described.¹⁷

Whole-Cell Extracts
Cells were washed 3 times in cold phosphate-buffered saline and then lysed for 30 min at 4°C in lysis buffer containing 50 mmol/L Tri-HCl (pH 8.0), 2 mmol/L EDTA (pH 8.0), 150 mmol/L NaCl, 0.5% Nonidet P-40, and the following protease inhibitors: 0.5 mmol/L/L PMSF, 1 µg/mL leupeptin, and 0.5 µg/mL pepstatin A. After centrifugation at high speed, the supernatant was collected and protein content of all samples was determined using the Bio-Rad protein assay with -globulin as a standard.

In Vitro Histone H1 Kinase Assay
EC were synchronized by 48 hours of serum starvation. At the time point "0 hours," the media were changed to a high concentration of serum alone or serum plus TNF-. Cells were harvested at various times after release from starvation. Forty µg of the whole-cell extracts was precleaned with protein A–agarose beads (Boehringer Mannheim) and immunoprecipitated with either anti-cdk2 or anti-cyclin A (Santa Cruz) polyclonal antibody overnight at 4°C. The pellets were washed twice in lysis buffer and then 3 times in kinase buffer (25 mmol/L Tris [pH 7.6], 5 mmol/L MgCl₂, and 0.5 mmol/L dithiothreitol) and incubated in 30 µL of kinase assay solution (5 µg of histone H1, 10 µmol/L ATP, and 2.5 µCi of [-³²P] ATP, in kinase buffer) for 30 minutes at 30°C. The mixtures were boiled for 5 minutes, loaded onto an SDS–12% polyacrylamide gel, and exposed to Amersham x-ray film after electrophoresis. Kinase activity was quantified by scintillation counting of the excised band from the gel. Protein extracts from detached EC served as an additional negative control. (Loss of anchorage in other cell types results in loss of kinase activity.)

Western Blot Analysis
Electrophoresis was performed on 10% or 12% SDS-polyacrylamide gels using 40 µg of protein per lane, as previously described.^{17 18}

Electrophoretic Mobility Shift Assay
Electrophoretic mobility shift assay was performed as previously described.²⁷ Synthetic oligonucleotides containing the putative E2F binding site were end-labeled with [-³²P] ATP using T4 polynucleotide kinase (Promega), and 400 000 cpm of probe was incubated with 25 µg of nuclear extract and binding buffer (Promega). An unlabeled competitor was added before the binding site probe to verify binding specificity.

Transient Transfection Assays
BAEC were transiently transfected with luciferase reporter promoter constructs pGL2-basic, Promega) containing 5' E2F1 sequence from -211 to +64 (generously supplied by Dr William G. Kaelin, Dana Farber Cancer Institute, Boston, Mass) using Lipofectamine reagent (GIBCO Laboratories). Transfection mixtures contained 15 µg of Lipofectamine and 5 µg of reporter vector in Opti-MEM. Twenty-four hours after Lipofectamine-mediated transfection in 100-mm dishes, cells were trypsinized, pooled, and transferred to 150 mm dishes to avoid contact inhibition (which could potentially alter E2F1 promoter activity). After allowing the cells to attach, the medium was replaced with either standard culture medium or standard culture medium plus TNF- 40 ng/mL. Cells were harvested after 24 hours and assayed for luciferase activity with the Berthold Lumat LB9501 luminometer. Bars represent the activity of 3 independent transfections (mean±SEM) normalized to the phosphatase activity produced by a cotransfected internal control plasmid (pSVAPAP).

Flow Cytometry
Flow cytometric analysis for quantification of apoptosis was performed as previously described.¹⁷

DNA Synthesis
To measure DNA synthesis, 15 000 endothelial cells per 35-mm dish were starved for 48 hours in MEM with 0.5% FCS. Growth media (10% FCS in DMEM and [³H]thymidine, 3 µCi/mL) were added at selected times up to 44 hours. Adherent cultures were fixed with 1 mL of 10% TCA, lysed in 0.25N NaOH, and then harvested. The amount of [³H]thymidine incorporated was determined by liquid scintillation counting. Each sample was done in triplicate, and the data were presented as mean±SEM of the replicates per assay.

Data Analysis
ANOVA was used to evaluate statistical significance of differences between experimental groups (with 3 or more groups); the Newman-Keuls method was applied to analyze differences between individual means. The Student t test was used to evaluate the differences between 2 experimental groups. Statistical significance was assigned when P<0.05.

	Results

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In Vivo Blockade of TNF- Using a Soluble Receptor Accelerates Reendothelialization of Injured Arteries
A soluble TNF- receptor was used to directly antagonize TNF- in vivo. This molecule, a recombinant fusion protein consisting of 2 ligand binding regions from the human p80 (TNFR2) receptor linked to the Fc region of human IgG1, previously has been shown to neutralize human, rodent, and rabbit TNF- in vitro and in vivo.^{20 21}

Blockade of TNF- resulted in significantly accelerated reendothelialization of the injured carotid artery segments compared with control (IgG-treated) arteries (Figure 1A and 1B). These results provide support in vivo for a significant negative effect of TNF- on proliferating endothelial cells at sites of arterial injury. To characterize the mechanisms responsible for the negative effect of TNF- on proliferating EC in vivo, a series of in vitro studies was performed.

View larger version (49K): [in this window] [in a new window]   Figure 1. In vivo blockade of TNF- accelerates reendothelialization after balloon angioplasty. A, Examples of arteries harvested 1 week after denudation injury. Evans blue staining identifies segments of each artery that have not been recovered by endothelium. The TNFsr-treated artery (bottom) has a significantly larger area of recovered endothelium, identified as the white area on each specimen, than the control artery (top). B, Reendothelialization after balloon angioplasty is accelerated by TNF- blockade. When harvested 1 week after injury, TNFsr-treated arteries have a significantly increased total area (left) and percentage area (right) of reendothelialization compared with the placebo-treated controls. In control and treatment groups, n=8; error bars=SEM. This difference persisted to 2 weeks (P<0.05 and P<0.03 for TNFsr- and placebo-treated arteries, respectively; data not shown).

Proliferating Endothelial Cells are Susceptible to TNF-–Induced Apoptosis and G₁ Arrest
Studies defining the inflammatory and activating influences of TNF- on EC typically have used confluent EC cultures to appropriately simulate the environment of an intact arterial surface.^{6 14 28 29} Given the demonstrated potential of TNF- to induce EC apoptosis,^{17 19 30} its enhanced expression at sites of balloon angioplasty,^{12 13} and the acceleration of reendothelialization observed after in vivo blockade of TNF-, we were interested to evaluate the effects of this cytokine on proliferating EC.

We found that apoptosis, defined by 2 independent methods, was marked in EC under proliferating conditions, whereas quiescent EC were relatively immune to TNF-–induced apoptosis (Figure 2A and 2B). We previously verified bona fide apoptosis in TNF-–treated EC by a variety of methods.^{17 18} Here we also show evidence for caspase activation manifest as PARP cleavage (Figure 2A, bottom). Exposure to TNF- also inhibited normal entry into S phase (Figure 2C) accompanied by an increase in the percentage of EC in G₁ phase (Figure 2D). Thus, TNF- induced apoptosis preferentially in proliferating EC while simultaneously inducing G₁ arrest. To evaluate whether the susceptibility of proliferating EC was tied to the cell-cycle arrest induced by TNF-, we synchronized EC in G₁, S, or G₂/M phase before TNF exposure to observe the effect on TNF-–induced apoptosis.

View larger version (30K): [in this window] [in a new window]   Figure 2. A, top, TNF-–induced EC apoptosis is increased in proliferating cells. HUVEC were rendered quiescent by starving for 48 hours in 0.3% serum while proliferating cells were maintained in high serum. Both groups were subjected to TNF- for 36 hours. Apoptosis was quantified by flow cytometric analysis (fluorescence-activated cell sorting [FACS]) after propidium iodide staining, as described in Methods. Bottom, Apoptosis, which we previously verified under these conditions by multiple assays,17 was also verified here by identifying PARP cleavage. PARP is a substrate for caspase-3. Immunoblotting identifies intact PARP under control conditions with cleavage into its apoptotic fragment after TNF- exposure of HUVEC. B, Reduction of apoptosis in quiescent EC occurs in a range of TNF- concentrations. The colorimetric MTS assay was used to determine cell death.17 Quiescent cells (solid black bars) develop less cell death than proliferating cells (shaded bars; P<0.001). Results are the mean±SEM of 6 experiments. C and D, TNF-–induced cell death is accompanied by G1 cell-cycle arrest. C, Thymidine incorporation was measured after serum restimulation of synchronized BAEC. TNF--treated cells showed a 3.6-fold reduction in thymidine uptake (mean±SEM from triplicate experiments) at the time of anticipated S-phase entry and again at 24 hours. A 3-fold reduction was still apparent after adjusting for the cell number (parallel plate, data not shown. D, Quiescent BAEC (180 000 cells per 10-cm plate) were serum-stimulated (so that the majority of cells would enter S phase 16 hours later). Analysis of adhesive cells by FACS revealed a significant increase in the G1 population in TNF-–treated cells when compared with untreated EC at both 16 and 24 hours. Results are mean±SEM of 3 experiments with 104 data points per experiment.

Endothelial Cells Arrested in G₁ Phase Are Susceptible to TNF-–Mediated Apoptosis, Whereas Those Arrested in S or G₂/M Phase Are Protected
BAEC cultures were infected with replication-defective adenoviral constructs expressing either ß-galactosidase (as a control for the effects of adenoviral infection), or the cdk inhibitors p16^INK4 or p21^CIP1/WAF1. The reporter/control vector had no effect on cell-cycle progression (Figure 3A). As expected, infection with either Ad-p16 or Ad-p21 resulted in a marked accumulation of EC in G₁ phase but did not alone induce EC apoptosis (Figure 3B). Subsequent TNF- exposure of both the p16^INK4- and p21^CIP1/WAF1-infected cells led to a significant increase in TNF--mediated apoptosis. Moreover, the potentiation of TNF--induced apoptosis was proportionate to the increase in the percentage of cells arrested in G₁ phase(Figure 3B and 3C). Thus, increasing the G₁ population enhanced the ability of TNF- to induce programmed endothelial-cell death.

View larger version (31K): [in this window] [in a new window]   Figure 3. TNF-–induced apoptosis in endothelial cells is cell cycle–dependent. Proliferating BAEC were infected under high-serum conditions for 24 hours with either 500 multiplicity of infection (MOI) of Ad-ß-galactosidase control vector or increasing MOI of Ad-p16 or Ad-p21. Alternatively, EC were pretreated with aphidicolin (Aph, 0.1 µg/mL) to induce S-phase arrest or nocodazole (Noc, 0.04 µg/mL) to arrest EC in G2/M. A, Bar graphs show examples of cell-cycle analysis by FACS performed 24 hours after infection with adenovirus. Expression of the p16 and p21 cyclin-dependent kinase inhibitors efficiently arrest EC in G1 phase, whereas infection with Ad-ß-galactosidase (b-Gal) had no effect on cell-cycle progression. For each condition, 104 data points were analyzed. B, Quantification of apoptosis by FACS of BAEC infected with the indicated adenovirus and exposed to control culture medium alone (left) or with the addition of TNF- (right). Overexpression of p21 results in a dose-dependent increase in TNF--mediated apoptosis that corresponds in magnitude to the increase in G1 population. Apoptosis induced by TNF- in EC overexpressing ß-galactosidase (ß-Gal) is identical to that in uninfected control cells. C, Degree of TNF-–induced EC apoptosis corresponds to relative size of G1 population. After pretreatment with ß-galactosidase or p16 adenovirus (Ad-ßgal or Ad-p16), aphidicolin, or nocodazole subconfluent BAEC were subjected to TNF- for an additional 36 hours. Apoptosis was quantified by flow cytometry (mean±SEM). Proliferating BAEC exposed to TNF- were used as the reference and the percent change in apoptosis under each treatment is shown. In each case, the degree of apoptosis corresponds to the relative size of the G1 population.

In contrast, TNF-–induced apoptosis was diminished by pretreatment with aphidicolin, which increased the percentage of cells in S phase while decreasing the G₁ population by almost 30% (Figure 3C). Similarly, nocodazole pretreatment, arresting cells in G₂/M, reduced TNF-–induced apoptosis by >50% (coincident with a significant reduction of the G₁ population). In both cases, TNF-–induced apoptosis was significantly attenuated and the magnitude of inhibition coincided with the relative reduction of the G₁ population, thus providing further evidence that G₁ was the vulnerable period for TNF-–induced EC apoptosis.

Because the transcription factor E2F is known to regulate the expression of several genes important for entry into and completion of S phase and has been linked to apoptosis in certain settings,^{31 32 33 34 35 36 37} we next evaluated its expression in EC after TNF- exposure.

E2F Activity and Expression are Downregulated in Endothelial Cells Exposed to TNF-
Immunoblotting revealed downregulated expression of E2F1 in TNF-—treated cells (Figure 4), accompanied by loss of E2F binding activity. At least part of the regulation of E2F1 expression occurs at the transcriptional level, as revealed by analysis of E2F1 promoter activity, which is repressed in TNF-–exposed EC (Figure 4).

View larger version (34K): [in this window] [in a new window]   Figure 4. Silencing of E2F links apoptosis and cell-cycle arrest induced by TNF-. A, Proliferating HUVEC were maintained in control medium alone or with the addition of TNF-. Expression of E2F protein by EC is decreased at 16 and 24 hours after TNF- exposure. Expression of cdk2 and cdk4 is maintained at control levels despite TNF- exposure of HUVEC; cdk2 and cdk4 are included here as controls for protein loading. B, Electrophoretic mobility shift assays were performed using an E2F binding site oligonucleotide probe and extracts from control or TNF--treated BAEC. E2F binding activity is decreased in extracts from cells that have been exposed to TNF- C, Proliferating subconfluent BAEC were transfected with reporter promoter constructs containing the E2F promoter sequence see Methods. Transfected cells were exposed to control medium alone or with medium plus TNF-. E2F promoter activity is reduced by 75% in EC exposed to TNF-. Results represent mean±SEM of 3 experiments.

E2F activity is critical for the expression of various genes important for G₁/S transition and S phase, including DNA polymerase-, thymidine kinase, cyclin E, and thymidilate synthase. In addition, E2F is also known to regulate the expression of cyclin A, which begins in late G₁ phase. To provide evidence that the loss of E2F expression resulted in the corresponding loss of activity of its downstream targets, we evaluated cyclin A expression in EC after TNF- exposure.

Cyclin A Expression and Kinase Activity Are Repressed by TNF-
Expression of cyclin A was repressed in TNF-—treated EC (Figure 5A). Moreover, cyclin A–associated kinase activity was diminished in TNF-–treated EC at time points corresponding to S-phase entry and increased kinase activity in control cells (Figure 5B), verifying the loss of function of the cyclin A holoenzyme, which is required for successful entry into and completion of S phase.^{38 39} Cyclin D1 (Figure 5A), an early G1 cyclin, and cyclin-dependent kinases cdk2 and cdk4 (Figure 4A), none of which are regulatory targets of E2F, demonstrated consistent levels of expression in EC despite TNF- treatment. To further define the functional significance of loss of E2F expression and activity, we next evaluated whether restoration of E2F function could rescue the EC phenotype from TNF--induced cell-cycle arrest and apoptosis.

View larger version (41K): [in this window] [in a new window]   Figure 5. Silencing of E2F results in loss of cyclin A protein and kinase activity. A, Expression of cyclin A is markedly downregulated 12 and 24 hours after TNF- (TNF) exposure of HUVEC. Expression of cyclin D1 is unchanged in control versus TNF-–treated cells and is included as a loading control. B, Cyclin A–dependent kinase activity is markedly attenuated by TNF-exposure. In vitro kinase assays, using histone H1 as the substrate, were performed as described. TNF- exposure of HUVEC (solid bars) results in markedly reduced cyclin A–associated kinase activity 16 and 48 hours after release from serum starvation. Histone H1 kinase activity was quantified by scintillation counting of the individual bands excised from the gel, and bar graphs represent the actual measurements from the pictured gel. Sx indicates floating cells, included as an additional control group.

E2F Overexpression Attenuates TNF-–Mediated EC Apoptosis
We used a replication-defective adenovirus directing overexpression of E2F1 to determine whether restoration of E2F function would rescue the EC phenotype. Infection of BAEC with Ad-E2F resulted in a marked and dose-dependent 2.6-fold decrease in TNF--induced EC apoptosis (Figure 6A) while progression into S phase was restored (Figure 6B). Increased expression of E2F1 in the Ad-E2F infected cells was confirmed by immunoblotting, which also discloses decreased E2F1 expression in control adenovirus–infected EC exposed to TNF- (Figure 6C). Furthermore, cyclin A expression, which was repressed by TNF- exposure in all other cells, was restored in the Ad-E2F infected EC despite TNF- exposure (Figure 6C). Expression of cdc2 was also maintained at the level of normally cycling EC, implying that (in the E2F overexpressing cells) the cell cycle was progressing despite TNF- exposure. Thus, our studies demonstrate that TNF- is capable of inducing cell-cycle arrest in proliferating EC by a mechanism involving loss of E2F activity, culminating in programmed cell death. Moreover, adenovirus-mediated overexpression of E2F promotes cell-cycle progression and rescues EC from TNF-induced apoptosis.

View larger version (30K): [in this window] [in a new window]   Figure 6. Restoration of E2F expression restores cell-cycle progression and abrogates TNF-–induced EC apoptosis. BAEC were plated at equal densities and infected for 12 hours in 10% FCS-containing media. Cells were allowed to grow for an additional 24 hours before addition of TNF-. Results are mean±SEM of 3 duplicate experiments. A, Restoration of E2F expression abrogates TNF-–induced EC apoptosis. Apoptosis was quantified after 60 hours of TNF- exposure by FACS. Ad-E2F infection results in dose-dependent inhibition of TNF-–induced EC apoptosis. B, Thymidine incorporation assays reveal restoration of cell-cycle progression in E2F overexpressing EC. In the absence of TNF-, Ad-E2F infected cells (750 MOI) show equal amounts of thymidine uptake after serum stimulation compared with noninfected cells. When treated with TNF-, the reduction in S phase seen in control cells is significantly reversed by E2F overexpression at 16 hours (58% of control in the E2F-overexpressing cells versus 28% in the noninfected cells; P=0.0006) and at 24 hours (55% versus 29%; P=0.0066). C, Western blot analysis from control BAEC, Ad-CMV (750 MOI) infected, Ad-p16 infected (500 MOI), and Ad-E2F infected (750 MOI) EC. E2F expression is > 10-fold higher after Ad-E2F infection in both control and TNF-–exposed EC. Cyclin A expression is restored to control levels in Ad-E2F infected cells despite TNF- treatment, compared with marked repression of cyclin A expression by TNF- in all other cells. In addition, cdc2 expression is also maintained in E2F infected cells. Analysis of tubulin expression was used as a loading control.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Vascular endothelial-cell apoptosis has been shown to be induced by a variety of stimuli, including ionizing radiation, cholesterol oxides, deprivation of growth factors, loss of adhesion, and TNF- exposure.^{17 19 30 40 41 42 43 44} The mechanisms by which TNF- induces apoptosis^{45 46 47 48} and the pathways that mediate protection from TNF-–triggered programmed cell death^{7 49 50} have been the subject of intense scrutiny, primarily in the context of cancer therapy, the immune system, and developmental biology. Similarly, the effect of TNF- on cell-cycle progression has been examined but exclusively in tumor and transformed cell lines.^{51 52} The present data, derived from primary cultures of vascular endothelial cells, provide the first mechanistic link between TNF-–mediated programmed cell death and cell-cycle regulation. In addition, the acceleration of endothelial recovery at sites of arterial injury by in vivo blockade of TNF- provides further suggestive evidence that the in vitro observations described herein may bear directly on the biology of the vessel wall.

The ability of E2F to facilitate S-phase entry is not surprising and has been previously shown in other cell types.^{31 32} The survival effect resulting from E2F overexpression, however, is in direct contrast to earlier reports^{32 33 34} and to recent findings in E2F knockout mice,^{35 36} although others have recently described apoptosis induced by loss of E2F function in tumor cells.³⁷ The disparity between our results and these earlier studies may be the result of several factors but most probably stems from the unique mechanism of TNF-–induced apoptosis in EC. TNF induces both apoptosis and G₁ arrest in EC (these appear to result from silencing of E2F activity and subsequent loss of cyclin A expression). The cell-cycle arrest of EC induced by TNF- is shown to be functionally important because endothelial cells in G₁ phase are uniquely susceptible to TNF-–induced apoptosis, whereas EC in other phases of the cell cycle are relatively immune. Restoration of E2F expression in TNF-–treated EC reinstates cyclin A expression, promotes transition through G₁ to S phase, and abrogates TNF-–mediated apoptosis. Our results suggest that mechanisms governing cell-cycle regulation and survival are tightly linked in EC and also define a previously unrecognized role for the cell cycle–regulated transcription factor E2F, that of a putative survival factor for EC under stress.

	Acknowledgments

 
We thank Drs J.R. Nevins and J. DeGregori (Duke University) for generously supplying the Ad-E2F and Ad-CMV constructs, Dr William G. Kaelin (Dana Farber Cancer Institute) for the E2F1 reporter promter constructs, Dr Michael B. Widmer (Immunex Corp) for generously supplying the TNFsr, and Drs Vicente Andres, Kenneth Walsh, and Roy C. Smith for helpful discussions.

Received May 21, 1998; revision received August 7, 1998; accepted August 20, 1998.

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