(Circulation. 1999;99:2019-2026.)
© 1999 American Heart Association, Inc.
Basic Science Reports |
Vascular Cell Apoptosis
Cell Type–Specific Modulation by Transforming Growth Factor-ß1 in Endothelial Cells Versus Smooth Muscle Cells
From the Cardiovascular Research Laboratory, Brigham and Women's Hospital, Harvard Medical School, Boston, Mass (M.J.P., G.H.G.); and the Department of Pediatrics, Stanford University Medical Center, Stanford, Calif (L.N.).
Correspondence to Gary H. Gibbons, MD, Brigham and Women's Hospital, Thorn Cardiovascular Research Laboratories-1326, 75 Francis St, Boston, MA 02115. E-mail ggibbons{at}bustoff.bwh.harvard.edu
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Abstract |
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Background—It is postulated that vascular lesion formation and remodeling involves a balance between vascular cell death and cell proliferation. Transforming growth factor-ß1 (TGF-ß1) is a pleiotropic factor expressed within vascular cells that regulates cell growth in a tissue-specific manner. This study tested the hypothesis that the control of vascular cell apoptosis involves cell type–specific regulation by TGF-ß1.
Methods and Results—In response to serum withdrawal, cultured endothelial cells and vascular smooth muscle cells exhibited apoptosis as evidenced by DNA laddering and quantitated by analysis of nuclear chromatin morphology. Addition of TGF-ß1 to endothelial cells in serum-free media further potentiated the induction of apoptosis in a dose-dependent fashion. Moreover, TGF-ß1 promoted endothelial cell death despite the presence of 10% serum. However, endothelial cells plated on collagen I were resistant to TGF-ß1–induced apoptosis. This antiapoptotic influence of the matrix was mimicked by integrin activation with anti-ß1 antibodies and associated with increased expression of the antiapoptotic factor bcl-2. In accord with the hypothesis that the modulation of antiapoptotic gene expression may mediate the effects of TGF-ß1 and ß1 integrins on cell fate, we observed that endothelial cells with constitutive upregulation of bcl-2 remained viable despite exposure to TGF-ß1 in serum-free conditions. In contrast to the proapoptotic effect of TGF-ß1 in endothelial cells, addition of TGF-ß1 to vascular smooth muscle cells in serum-free media inhibited apoptosis.
Conclusions—These findings suggest that the effect of cytokines such as TGF-ß1 on cell fate is contextual and is modulated by cell-matrix interactions in a cell type–specific manner.
Key Words: vasculature • apoptosis • cells
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Introduction |
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The pathogenesis of vascular diseases such as atherosclerosis and restenosis after angioplasty is characterized by the accumulation of cells within the intima. Although studies of the pathogenesis of vascular disease have focused on the role of cell proliferation as a determinant of vessel cellularity, a growing body of evidence suggests that vascular structure and lesion formation is determined by a balance between cell growth and cell death by apoptosis.
The process of apoptosis appears to determine tissue structure in health and disease in a variety of contexts.1 Seminal studies by Cho et al2 have documented that programmed cell death may play a critical role in the ontogeny of the vasculature. Moreover, recent studies of human vascular disease specimens have provided evidence that apoptosis may occur within the context of atherogenesis and restenosis after angioplasty.3 4 5 However, the factors that determine the balance between cell death and cell survival within the vessel wall remain to be characterized.
Studies of human atherosclerotic plaques as well as restenosis lesions after angioplasty have documented transforming growth factor-ß1 (TGF-ß1) expression by vascular cells within lesions.6 Similarly, TGF-ß1 expression within the vasculature is upregulated in the context of diabetes and hypertension in animal models of vascular disease.7 8 TGF-ß1 is postulated to play a mediator role in promoting alterations in vessel structure on the basis of its pleiotropic properties as a modulator of cell growth, differentiation, migration, and matrix production.9 Furthermore, a recent study suggests that alterations in the local abundance of TGF-ß1 in the arterial wall promotes vascular cell transdifferentiation, vascular wall remodeling, arterial lesion growth, and lesion regression associated with apoptosis.10 The biological effect of TGF-ß1 is cell specific and contextual. Given the pleiotropic effects of TGF-ß1 on vascular cell function, the present study tested the hypothesis that TGF-ß1 also modulates the process of vascular cell death by apoptosis. Our findings indicate that TGF-ß1 is a bifunctional modulator of cell death that exerts a proapoptotic influence on endothelial cells and is antiapoptotic for vascular smooth muscle cells (VSMC). These observations have important implications for understanding the role of cytokine growth factors such as TGF-ß1 in the pathogenesis of vascular disease.
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Methods |
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Endothelial Cell Experimental Protocols
Bovine aortic endothelial cells (BAEC) were isolated and characterized as previously described.11 BAEC (passages 6 to 12) were maintained in Dulbecco's modified Eagle's medium (DME) (Gibco Laboratories) supplemented with 10% heat-inactivated calf serum (CS) (Gibco), penicillin (100 U/mL), and streptomycin (100 µg/mL) and incubated in a humidified atmosphere of 95% air/5% CO2. The medium was changed every 2 to 3 days, and cells were routinely passaged by trypsin/EDTA with a split ratio of 1:6. Human umbilical vein endothelial cells (HUVEC) were obtained as primary cultures from Clonetics Corp (San Diego, Calif). HUVEC (passage 2 to 5) were maintained in endothelial growth medium (EGM) containing 10 ng/mL human recombinant epidermal growth factor, 1 µg/mL hydrocortisone, 2% fetal bovine serum (FBS), 50 µg/mL gentamicin, 50 ng/mL amphotericin-B, 12 µg/mL bovine brain extract, and 10 µg/mL heparin. Bovine pulmonary artery endothelial cells (CPAE) were obtained from American Type Culture Collection (Rockville, Md) and maintained (passages 18 to 22) in DME medium 20% FBS (Gibco). For growth and apoptosis studies, HUVEC, BAEC, and CPAE were grown to a near-confluent state on Labtek glass chamber slides (Nunc, Inc).
BAEC DNA Synthesis
BAEC were grown as described above and incubated in serum-free
DME with or without supplementation with human recombinant basic
fibroblast growth factor (bFGF) (50 ng/mL) (Sigma Chemical Co, St
Louis, Mo) in the presence or absence of human recombinant
TGF-ß1 (1 ng/mL) (R&D Systems, Minneapolis,
Minn). 5-bromo-2'-deoxyuridine (BrdU) (Sigma Chemical Co) (5 µg/mL)
was added to each well for a total incubation of 48 hours before
fixation in 70% ethanol. Nuclei incorporating BrdU were detected with
a streptavidin-biotin detection system (Zymed) and the percent positive
nuclei determined by counting 200 total nuclei.
BAEC Population Dynamics
BAEC were grown as described above and incubated for 48 hours in
serum-free DME with or without supplementation with FGF (50 ng/mL) in
the presence or absence of TGF-ß1 (1 ng/mL).
Adherent cells were harvested by trypsinization, and mean cell numbers
were determined with a Coulter counter at baseline and after 48
hours.
Kinetics of BAEC Apoptosis
BAEC were grown as described above and incubated for 8, 18, 24,
or 48 hours in serum-free DME, DME supplemented by FGF (50 ng/mL), or
TGF-ß1 (1 ng/mL). Cells were stained with
fluorescent DNA-binding dyes, harvested, and analyzed
for apoptosis as described below. To define the dose-response
relations, cells were exposed to increasing concentrations of
TGF-ß1 (0, 0.1 0.25, 0.5, 1, and 5 ng/mL) for
48 hours in 10% CS or serum-free DME with or without supplementation
with FGF (50 ng/mL). After 48 hours, the adherent and nonadherent cells
were harvested for in situ end-labeling, extraction of chromosomal DNA,
or stained with fluorescent DNA-binding dyes and
analyzed for apoptosis as described below.
TGF-ß1 Induction of Apoptosis in
HUVEC
HUVEC were grown to a near-confluent state in EGM before
incubation with 1 ng/mL of TGF-ß1. After 24
hours, the adherent and nonadherent cells were harvested and stained
with fluorescent DNA-binding dyes and analyzed for
apoptosis as described below. For collagen coating experiments,
rat tail collagen type I (Collaborative Biomedical Products,
Bedford, Mass) was used according to the manufacturer's specifications
to apply a thin coating (50 µg/mL) on the surface of the glass
chamber slides. The slides were incubated at room temperature for 1
hour before rinsing with PBS. For antibody coating experiments, glass
chamber slides were incubated for 2 hours with 20 µg/mL mouse
anti-human ß1 integrin monoclonal antibody
(clone TS2/16; Endogen, Cambridge, Mass) or control purified mouse IgG
(Sigma) before rinsing with PBS. For collagen and antibody coating
experiments, HUVEC were plated at 1.5x105
cells/well in fully supplemented EGM in the presence of 1 ng/mL
TGF-ß1. After 24 hours, the adherent and
nonadherent cells were harvested and stained with fluorescent
DNA-binding dyes and analyzed for apoptosis as
described below.
bcl-2 Immunoblot
Collagen type I was used to coat tissue culture flasks as
described above. CPAE were plated at 3x106
cells/flask in serum-free medium in the presence or absence of collagen
I. After 24 hours, the adherent and nonadherent cells were harvested
and homogenized in lysis buffer (10 mmol/L Tris, 1%
SDS solution). Protein concentrations were determined by a BCA assay
(Pierce Chemical). Cell lysates (50 µg) were loaded on a 12%
SDS-polyacrylamide gel, electrophoretically transferred to a
nitrocellulose membrane (Hybond ECL, Amersham), and the membrane
stained with india ink to verify equal loading and transfer efficiency.
The membrane was blocked in PBS, 0.1% Tween, 5% nonfat dry milk and
probed with a polyclonal antibody directed against bcl-2
(#492, Santa Cruz Biotechnology, Santa Cruz, Calif) (1:100 dilution)
followed by a streptavidin-biotinylated-horseradish peroxidase–linked
secondary antibody (1:100 dilution) (Dako Corp) and detected with
electrochemical luminescence detection reagents (Amersham) by
autoradiography.
Retroviral Constructs and Infection of Endothelial Cells
Retroviral constructs containing the human bcl-2 gene
under the control of the retroviral long-terminal repeat promoter and
the neomycin resistance gene were generated, titered, and characterized
as previously described.12 For infections of BAEC,
1x106 cells were treated with viral supernatants
added at 12-hour intervals for 3 days. Stable transfectants were
selected by incubation with G418 (1 mg/mL). Medium was changed twice
weekly for 2 weeks, until G418-resistant colonies appeared.
Cultures were subsequently trypsinized and maintained as described
above. Pools of cells were used for subsequent experiments to minimize
differences that might have resulted from viral integration. Expression
of the human bcl-2 protein was confirmed by
immunohistochemical staining with a monoclonal antibody directed
against human bcl-2 (Dako, Carpenteria, Calif) as previously
described.12 The majority of BAEC transfected with
the human bcl-2 vector exhibited positive
immunostaining for bcl-2 (>90%), whereas
BAEC transfected with the control vector containing the neomycin
resistance cassette alone exhibited no detectable staining (data not
shown).
VSMC Experimental Protocols
Rabbit smooth muscle cells (VSMC) were isolated and
characterized as previously described.13 Human umbilical
artery smooth muscle cells were purchased from Clonetics Corp (San
Diego, Calif). VSMC (passages 5 to 9) were maintained in DME medium and
Ham's F12 (DME/F12) (Gibco) supplemented with either 10% (rabbit
VSMC) or 20% (human VSMC) heat-inactivated FBS (Gibco),
penicillin (100 U/mL), and streptomycin (100 µg/mL) and 25
mmol/L HEPES buffer. For each of the following experiments, VSMC were
grown in 10% FBS to a near-confluent state.
VSMC DNA Synthesis
VSMC were grown as described above and incubated in 10% FBS or
serum-free DME in the presence or absence of
TGF-ß1(1 ng/mL). BrdU (5 µg/mL) was added to
each well for a 24-hour incubation before fixation and analysis
as described above.
VSMC Population Dynamics
VSMC were grown as described above and incubated for an
additional 24 hours in 10% FBS or serum-free DME in the presence or
absence of TGF-ß1(1 ng/mL). Mean cell numbers
were determined with a Coulter counter.
TGF-ß1 Inhibition of Apoptosis in
VSMC
VSMC were grown as described above and incubated in serum-free
DME in the presence or absence of TGF-ß1 (0.25,
0.50, or 1 ng/mL) for 24 hours. After 24 hours, the adherent and
nonadherent cells were harvested for extraction of chromosomal DNA or
stained with fluorescent DNA-binding dyes and analyzed
for apoptosis as described below.
Apoptosis Analysis
Microscale Analysis of DNA Fragmentation
DNA was extracted from both adherent and nonadherent cells and
purified DNA was 3'-end labeled and size fractionated by agarose gel
electrophoresis for autoradiography as previously
described.14 15
In Situ 3'-End-Labeling of DNA
Identification of fragmented DNA in situ was performed by
collecting adherent and nonadherent cells through trypsinization and
centrifugation and fixing in 4% buffered formaldehyde.
In situ labeling of 3'-OH ends was performed by an adaptation of a
previously described procedure.16 Specimens in which the
terminal deoxynucleotide transferase was omitted served as
a negative control, and a DNase-treated specimen served as a positive
control.
Quantitative Analysis of Apoptosis by
Fluorescent Microscopy
As an additional assay of cell death by apoptosis, we
used fluorescent DNA-binding dyes to define nuclear chromatin
morphology as a quantitative index as previously described by our
laboratory.14 Counts are expressed as apoptotic
nuclei/total nuclei to obtain percent apoptotic nuclei.
The apoptotic index obtained is a cross-sectional and not a cumulative assessment at the time of cell harvest and may be an underestimate of the total apoptotic cell population. We have observed a high concordance between rates of apoptosis observed with this method compared with assays of subdiploid apoptotic populations with fluorescence-activated cell sorter analysis and phase-contrast time-lapse video microscopy.
Statistical Analysis
Experiments were performed in triplicate except as indicated and
repeated a minimum of 3 times. Data are presented as mean±SD.
Statistical analyses were performed by ANOVA or unpaired
2-tailed Student's t test.
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Results |
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Endothelial Cell Growth Kinetics: Response to bFGF Versus TGF-ß1
In accordance with previous reports,17 the initial set of experiments characterized the effects of serum deprivation, bFGF, and TGF-ß1 on cell growth and kinetics. Under serum-free conditions, we observed a net decrease in cell number over a 48-hour period. The addition of bFGF prevented the net cell loss induced by serum withdrawal and effectively maintained cellularity at baseline levels (Figure 1
). However, the administration of
TGF-ß1 potentiated the net cell loss induced by
serum withdrawal. Moreover, TGF-ß1 induced a
net cell loss compared with baseline even in the presence of bFGF
(Figure 1). We hypothesized that both serum withdrawal and
TGF-ß1 administration result in a net decrease
in cell number by inducing cell death in addition to the well-described
effects on inhibiting cell cycle progression.
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Endothelial Cell Apoptosis: Induction by
Serum Withdrawal and TGF-ß1
To test the hypothesis that TGF-ß1 induces
endothelial cell loss by apoptosis, we used 3
complementary assays. In DNA extracts obtained from
endothelial cells maintained in medium containing 10%
serum, there was little evidence of apoptosis by DNA laddering.
In contrast, DNA extracted from cells exposed to serum-free conditions
for 48 hours exhibited extensive cleavage of DNA in the characteristic
internucleosomal pattern. The addition of bFGF inhibited
apoptosis, whereas the administration of
TGF-ß1 further enhanced this process of cell
death (Figure 2). The finding that the
net cell loss after exposure to TGF-ß1 or serum
withdrawal was due to apoptotic death was further confirmed by
analysis of nuclear chromatin morphology with
fluorescence microscopy with the use of a DNA-binding dye
(Hoechst 33342) as well as by the in situ end-labeling assay (data not
shown). All 3 techniques were concordant in documenting
apoptosis in response to serum withdrawal and
TGF-ß1 administration.
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The process of apoptosis was quantitated by the nuclear
chromatin morphology assay. Cells maintained in 10% serum have a low
basal apoptotic index of 
2%. The induction of
apoptosis in response to serum withdrawal was detectable above
baseline within 18 hours, achieved a plateau within 24 hours, and was
sustained for up to 48 hours (Figure 3).
The administration of TGF-ß1 (1 ng/mL) caused a
significant increase in the percentage of apoptotic cells noted
at these time points. Indeed, picomolar concentrations of
TGF-ß1 induced apoptosis in a
dose-dependent manner under serum-free conditions (Figure 4).
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On the basis of the observation that bFGF effectively reverses the
induction of apoptosis in response to serum withdrawal, we
assessed the effect of bFGF supplementation on
TGF-ß1–induced apoptosis. Although
bFGF administration substantially reduced the baseline level of
apoptosis, cells treated with TGF-ß1
continued to undergo cell death by apoptosis in a
dose-dependent manner despite coincubation with bFGF. To further
confirm the proapoptotic effect of
TGF-ß1, we assessed the response in the
presence of 10% serum. Although multiple mitogens and putative
survival factors were present under these conditions, exposure to
TGF-ß1 induced endothelial cell
death in a dose-dependent manner (Figure 4
). To confirm the
broad applicability of this observation, we also assessed the effect of
TGF-ß1 on human umbilical vein
endothelial cells and confirmed that responses observed
in bovine aortic cells could be replicated in
endothelial cells from another species and vascular bed
in the presence of fully supplemented growth medium (11%±0.7% [1
ng/mL TGF-ß1], n=6, vs 4%±0.6% [vehicle
control], n=6; P<0.001).
Modulation of TGF-ß1-Induced Apoptosis:
Cell-Matrix Interactions
We were intrigued by the observation that survival factors
present within serum failed to abolish
TGF-ß1–induced apoptosis. Given that
the response to pleiotropic cytokines is often contextual, we
tested the hypothesis that the proapoptotic influence of
TGF-ß1 is modulated by factors within the
microenvironment such as matrix proteins. Indeed, we observed that
plating HUVEC on collagen I markedly attenuated the induction of
apoptosis in response to TGF-ß1 (1
ng/mL) as compared with cells plated on glass (19%±3.5%[ glass] vs
2%±0.6% [collagen], n=6; P<0.001). As an additional
set of control experiments, we confirmed previous observations that
plating on collagen failed to modulate other
TGF-ß1–induced cellular responses such as the
inhibition of DNA replication (data not shown).
To further characterize this countervailing antiapoptotic
influence of the cell substratum, we tested the hypothesis that the
enhanced cell survival on collagen I was mediated by
ß1 integrins that bind to this matrix protein.
We observed that HUVEC plated on immobilized control IgG
exhibited a similar induction of
TGF-ß1–induced apoptotic death when
compared with cells plated on glass (19%±3.5% [glass] vs 17%±2%
[IgG], n=6; P>0.05). In contrast, HUVEC plated on
immobilized anti-ß1 integrin
antibodies18 proved to be resistant to the
induction of apoptosis in response to
TGF-ß1 administration (1 ng/mL) (17%±2%
[IgG] vs 7%±2.2% [anti-ß1 integrin],
n=6; P<0.001) (Figure 5).
Thus activation of ß1 integrins by collagen I
within the microenvironment activates a survival signal that
can prevent TGF-ß1–induced apoptotic
death.
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It is intriguing that we observed a more potent inhibition of
TGF-ß1–induced apoptosis by
cell-matrix interactions compared with peptide growth factors. We
hypothesized that the survival-promoting effects of the collagen matrix
was mediated by the upregulation of the antiapoptotic gene
bcl-2. In contrast to the relatively low levels of
bcl-2 expression observed while plated on tissue culture
plastic, plating on collagen I markedly increased the expression of
bcl-2 (Figure 6). To confirm
that the upregulation of bcl-2 observed in response to
matrix-integrin interactions is sufficient to inhibit
TGF-ß1–induced death, we examined the response
of BAEC stably transfected with the bcl-2 gene.
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Although neomycin-selected BAEC control cells remained susceptible to
TGF-ß1–induced cell death, BAEC with
constitutive upregulation maintained cell viability despite expose to
TGF-ß1 in serum-free conditions (2%±1.3%
[serum free] vs 4%±1.2% [TGF-ß1];
P>0.05) (Figure 7). Taken
together, these findings suggest that the proapoptotic effect
of TGF-ß1 is modulated by matrix-integrin
interactions involving the upregulation of bcl-2.
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Given that TGF-ß1 is a cytokine factor produced by both endothelial cells and VSMC, we examined whether TGF-ß1 had similar effects on VSMC apoptosis regulation.
VSMC Growth Kinetics: Response to TGF-ß1
We initially confirmed the effect of serum deprivation
and TGF-ß1 administration on cell growth
kinetics as previously defined.19 The
administration of TGF-ß1 (1 ng/mL)
significantly inhibited DNA replication in VSMC grown in the presence
of 10% serum (29%±1.2% [10% serum] vs 21%±1.0%
[TGF-ß1]; P<0.001) and
potentiated the decrease in cell cycle progression induced by serum
withdrawal over a 24-hour period (18%±0.4% [serum withdrawal] vs
13%±0.3% [TGF-ß1];
P<0.001).
To further define the growth kinetics, changes in cell number were
assessed in parallel with changes in DNA replication. In the presence
of 10% serum, as compared with baseline cell counts, the
near-confluent cells continued to accumulate over a 24-hour period,
whereas cells exposed to serum withdrawal exhibited a significant net
decrease in cell number. In response to TGF-ß1
administration, we observed an apparent paradox. Although
TGF-ß1 inhibited cell cycle progression in
VSMC, the administration of TGF-ß1 actually
increased cellularity by preventing the net cell loss that accompanied
serum withdrawal (Figure 8). Given this
discordance between the inhibitory effect of
TGF-ß1 on DNA replication and the net increase
in cell number, we hypothesized that TGF-ß1
preserved cellularity by preventing VSMC death.
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VSMC Apoptosis: Effect of TGF-ß1
VSMC grown to near confluence maintained in 10% serum exhibited
little evidence of apoptosis, as determined by the gel
electrophoresis assay for DNA fragmentation (data not shown). In
contrast, DNA extracted from cells exposed to serum withdrawal for 24
hours had substantial DNA fragmentation that was markedly attenuated by
the administration of TGF-ß1 (Figure 9). These results assayed by gel
electrophoresis were verified and quantitated by examining nuclear
chromatin morphology with fluorescent microscopy. In contrast
to the proapoptotic effect observed with
endothelial cells, TGF-ß1 at
picomolar concentrations prevented the death of VSMC triggered by serum
withdrawal (Figure 10). To confirm the
broad applicability of this observation, the effect of
TGF-ß1 on human VSMC was also determined. In
response to serum withdrawal, we observed an increase in
apoptotic frequency from 2%±0.4% (n=9) to 23%±2.1% (n=6)
as compared with serum controls (P<0.001). Exposure to
TGF-ß1 (1 ng/mL) significantly inhibited
apoptosis in human VSMC (11%±0.8% [serum
withdrawal+TGF-ß1], n=6, vs 23%±2.1%
[serum withdrawal], n=6; P<0.001). These findings suggest
that although TGF-ß1 exerts a similar
antiproliferative effect on both endothelial cells and
VSMC, there are distinct cell type–specific regulatory mechanisms that
govern programmed cell death in these vascular cells.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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It has been postulated that vascular structure and lesion formation is determined in part by a balance between cell proliferation and cell death by apoptosis. Indeed, recent studies of human vascular specimens and animal models suggest that apoptosis is involved in the pathogenesis of vascular disease.3 4 5 20 The cytokine factors that regulate apoptosis within the vessel wall remain to be further characterized. The present study provides the first evidence that the pleiotropic growth factor TGF-ß1 is a bifunctional modulator of vascular cell apoptosis. TGF-ß1 is an inducer of endothelial cell death, whereas it prevents VSMC death. In addition, we are intrigued by the observation that the proapoptotic effect of TGF-ß1 can be abrogated by countervailing signals induced by cell-matrix interactions. These in vitro observations suggest that the regulation of apoptosis within an intact vessel by cytokine growth factors such as TGF-ß1 may be dependent on the cell type as well as the local cellular context.
The specificity of the effect of TGF-ß1 on cell death is substantiated by several lines of evidence including DNA laddering, nuclear chromatin morphology, and in situ end labeling. The specificity of the response was further evidenced by the fact that the effect of TGF-ß1 was dose dependent and observed within the picomolar range. These picomolar concentrations of TGF-ß1 are similar to stimulated levels in response to shear stress or angiotensin II.11 13 In addition, the observation that TGF-ß1 had opposing effects on different cell types indicates that the administration of TGF-ß1 did not have a nonspecific toxic effect. Finally, additional studies in human vascular cells documented that the bifunctional response was not limited to the bovine or rabbit cell isolates that were the primary focus of this study. The effects of TGF-ß1 on vascular cell apoptosis were therefore cell type specific and not species specific. Overall, our findings in vascular cells are consistent with previous reports in hepatic cells that have shown that TGF-ß1 has the capacity to induce apoptosis.21 During the preparation of this report, 2 reports confirmed the observation that TGF-ß1 induces endothelial cell apoptosis.22 23 We have further extended these findings by documenting that the proapoptotic effect is contextual and that engagement of ß1 integrins by matrix proteins such as collagen I can abrogate the induction of apoptosis in response to TGF-ß1. Furthermore, our findings in an in vitro model appear to be supported by data from a recent in vivo study in which transient overexpression of TGF-ß1 induces vascular lesion growth followed by vascular cell apoptosis during TGF-ß1 withdrawal and lesion regression.
It is intriguing that our findings on cell type specificity in the regulation of cell death are similar to previous observations that have documented cell type–specific effects of TGF-ß1 in the regulation of cell growth.17 24 25 26 In addition, the observation that the response to TGF-ß1 is contextual indicates that the endothelial cell fate is dependent on a variety of proapoptotic and antiapoptotic mediators present within the microenvironment. Further studies are necessary to characterize the actual role of apoptosis in vascular remodeling and lesion formation in vivo.
The regulation of apoptosis in vascular cells remains poorly characterized. Studies in other cell systems suggest that a balance of proapoptotic versus antiapoptotic factors such as bax and bcl-2 may be involved.27 28 Given our observation that the regulation of apoptosis in vascular cells is cell type specific, it is clear that pathways defined in other cell systems may not be directly applicable to elucidating the regulation of apoptosis in vascular cells. It is noteworthy that we observed a dissociation between the regulation of cell growth and cell death by TGF-ß1. Although TGF-ß1 inhibited cell cycle progression in both endothelial and vascular smooth muscle cells, we documented diametric effects on the regulation of apoptosis. This observation suggests that these cellular signaling pathways regulating growth and death may be divergent in vascular cells.
The pathway that links TGF-ß1 receptor activation with cell death remains to be clarified. It was recently reported that TGF-ß1 stimulation results in decreased bcl-2 protein levels in endothelial cells.22 We demonstrated that microenvironmental cues such as the cell substratum can modulate the proapoptotic influence of TGF-ß1 on endothelial cell fate. We observed that engagement of ß1 integrins promotes an antiapoptotic signal that can override the proapoptotic effect of TGF-ß1. Furthermore, plating on collagen I results in increased expression of bcl-2 in association with an antiapoptotic effect. Moreover, forced expression of bcl-2 prevents both endothelial cell death in response to serum withdrawal and TGF-ß1. Taken together, these findings suggest that endothelial cell fate is determined in part by the modulation of bcl-2 expression by the countervailing influence of proapoptotic factors such as TGF-ß1 and antiapoptotic mediators such as ß1 integrins. Further studies are necessary to clarify the role of other antiapoptotic members of the bcl-2 family in endothelial cells29 as well as the receptor-mediated signaling pathways that determine the expression of apoptosis regulatory genes. Similarly, further investigation is necessary to define the cellular mediators involved in the regulation of VSMC fate.
In summary, the present study has documented that TGF-ß1 is an inducer of endothelial cell apoptosis, whereas it prevents VSMC death. The effect of TGF-ß1 on cell fate is contextual and is modulated by cell-matrix interactions mediated by ß1 integrins. This observation further extends the mechanisms by which this pleiotropic factor can modify vascular structure. The cell type specificity of the effect of TGF-ß1 on cell viability has implications for understanding the role of this cytokine factor in the process of vascular remodeling and intimal lesion formation. Further investigation is necessary to elucidate the molecular basis of the bifunctional effects of TGF-ß1 on vascular cell apoptosis and characterize the actual role of apoptosis as a determinant of vascular structure in vivo.
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Acknowledgments |
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This work was supported by the Baxter Foundation, the American Heart Association, and National Institutes of Health grant HL-48638. Dr Gibbons is a Pew Biomedical Research Scholar. Dr Pollman is a recipient of a California Affiliate American Heart Association Postdoctoral Fellowship Award. Dr Naumovski is an Amgen/American Society of Hematology Scholar.
Received August 1, 1998; revision received November 17, 1998; accepted December 7, 1998.
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