© 1999 American Heart Association, Inc.
Vascular Biology |
Autocrine FGF-2 Is Responsible for the Cell Density–Dependent Susceptibility to Apoptosis of HUVEC
A Role of a Calpain Inhibitor–Sensitive Mechanism
From the National Cardiovascular Center Research Institute, Fujishirodai 5-7-1, Suita, Osaka 565-8565, Japan.
Correspondence to Kentaro Shimokado, MD, National Cardiovascular Center Research Institute, Fujishirodai 5-7-1, Suita, Osaka 565-8565, Japan. E-mail kshimoka{at}res.ncvc.go.jp
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Abstract |
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Abstract—To elucidate the factors affecting endothelial susceptibility to apoptosis, we studied the effects of cell density on endothelial cell apoptosis induced by deprivation of serum and fibroblast growth factor-2 (FGF-2/basic FGF). On deprivation, more cells became apoptotic in a dense culture (5x102 cells/mm2) than in a sparse culture (1x102 cells/mm2) of human umbilical vein endothelial cells. FGF-2, hepatocyte growth factor, and vascular endothelial cell growth factor, but not insulin-like growth factor-I, decreased apoptosis in the dense culture to a level similar in the sparse culture. An anti–FGF-2 antibody significantly increased the apoptosis in the sparse culture, suggesting that the sparse culture was resistant to apoptosis because of the greater autocrine production of FGF-2. Western blot analysis and metabolic labeling revealed that the sparse culture has, in fact, more FGF-2 than the dense culture. The steady state level of mRNA for FGF-2 was not significantly different between the dense and sparse cultures. Among a panel of inhibitors for 2 major cytoplasmic proteolytic enzymes, calpain inhibitors increased FGF-2 in the dense culture, but proteasome inhibitors did not. Our findings demonstrate that cell density affects endothelial survival by regulating autocrine FGF-2 production through a calpain inhibitor–sensitive mechanism.
Key Words: apoptosis • growth factors • cytoplasmic neutral protease • endothelial integrity • endothelial cells
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Introduction |
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Regulation of endothelial loss and regeneration is important to maintain the integrity of the endothelial monolayer in the vascular system. Endothelial denudation induces migration and proliferation of the endothelial cells, and as a consequence, the endothelial monolayer is partly or fully restored.1 Autocrine fibroblast growth factor-2 (FGF-2) plays an important role in recovering from the endothelial denudation as shown by in vitro and in vivo experiments2 3 Apoptosis was recently found to be another mode of endothelial cell loss.4 Endothelial apoptosis is induced by a wide variety of factors, including deprivation of growth factor,5 oxidized LDL,6 ionizing radiation,7 nitric oxide,8 lipopolysaccharide,9 ceramide,10 and tumor necrosis factor-
.11 However, only a few factors
affecting the susceptibility of endothelial
apoptosis have been elucidated.12 Recently we reported that oxidized LDL and oxysterols induce endothelial apoptosis by increasing cellular ceramide.13 During the study, we noticed that sparse endothelial cells are resistant to oxidized LDL–induced apoptosis (data not shown). Similar resistance to oxidative stress in proliferating endothelial cells was reported by others.14 In this study, we attempted to confirm the effect of the cell density on susceptibility to apoptosis and elucidate the underlying mechanisms. We found that sparse cells are more resistant to apoptosis than dense cells, mainly due to the greater amount of FGF-2 associated with the cell surface. A novel mechanism is involved in the cell density–dependent regulation of cellular FGF-2 content.
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Methods |
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Dulbecco's modified Eagle Medium (DMEM) was purchased from Nissui Pharmaceutical Co, Ltd and fetal calf serum (FCS) from GIBCO BRL. Recombinant human FGF-2, human FGF-1/acid FGF, and human vascular endothelial growth factor (VEGF) were purchased from Pepro Tech Inc; recombinant human hepatocyte growth factor (HGF) from Beckton Dickinson Labware; and recombinant human insulin-like growth factor-I (IGF-I) from Genzyme Diagnostic. Antibovine FGF-2 monoclonal antibody (Types I and II)15 were purchased from Upstate Biotechnology Inc. Carbobenzoxy-leucyl-leucyl-leucinal (MG132),16 and carbobenzoxy-leucyl-leucinal17 were purchased from the Peptide Institute. Lactacystin was kindly provided by Dr S. Omura (The Kitasato Institute).18 [L-3-trans-Ethoxycarbonyloxirane-2-Carbonyl]-leucine(3-Methylbuthyl)amide (E64d)19 were provided by Dr K. Suzuki (Tokyo University, Japan) and cDNA for human FGF-2 by Dr D.B. Rifkin (New York University, New York).20
Cells
Human umbilical vein endothelial cells (HUVEC)
were obtained from the umbilical cord and characterized as reported
previously.21 Cells were cultured in DMEM supplemented
with penicillin (100 U/mL), streptomycin (100 µg/mL), 20% FCS, and
10 ng/mL FGF-2 and used for the experiment between passages 2 and 6. In
some experiments, FGF-1 was used in place of FGF-2. Bovine aortic
endothelial cells (BAEC) was isolated from the calf
aorta and cultured in DMEM supplemented with penicillin, streptomycin,
and 10% FCS.
Detection of DNA Ladder
Cells were seeded at 5x102
cells/mm2 in a 9-cm culture dish (a dense
culture) or 1x102
cells/mm2 in a 15-cm culture dish (a sparse
culture) and cultured for 24 hours. The medium was changed to DMEM
containing 0.1% bovine serum albumin and various
concentrations of FGF-2 and cultured further for 48 hours. DNA was
prepared, and fragmented DNA was detected with the TACS
apoptotic DNA laddering kit (TREVIGEN) according to the
manufacturer's instructions.22
Quantitative Analysis of Apoptotic Cells
HUVEC were seeded in gelatin coated 8 well-chamber slides (NUNC,
Inc) at 5x102 cells/mm2
(a dense culture) or 1x102
cells/mm2 (a sparse culture) and cultured in the
DMEM containing 20% FCS and 10 ng/mL of FGF-2 for 48 hours before the
medium was changed to DMEM containing 0.1% bovine serum
albumin. The cells were cultured in this serum-free medium for
the indicated period and then stained by 0.05%
4',6-diamidino-2-phenyliodole, as reported previously.13
The apoptotic cells that had characteristic fragmented nuclei
were counted in 9 high-power fields under a fluorescent
microscope (approximately 100 to 200 cells/well). The percentage of
apoptotic cells was calculated as the number of
apoptotic cells/the number of total cellsx100%. Assays were
conducted in triplicate or more and repeated at least twice.
Western Blot Analysis
For Western blot analysis, 6x106
cells were lysed with lysis buffer (20 mmol/L Tris-HCl, 2 mol/L
NaCl, 3 mmol/L EDTA, 0.3% sodium dodecylsulfate (SDS), 1%
Triton X-100, 1 mmol/L DTT, 0.5 µg/mL leupeptin, and 20 U/mL
Trasylol).20 The concentration of NaCl was adjusted to 0.5
mol/L with NaCl-free lysis buffer. The samples containing the same
amount of protein, which turned out to represent the same
numbers of cells, were incubated with heparin-Sepharose CL-6B beads
(Pharmacia Biotech) for 2 hours at 4°C. The beads were washed 3 times
with the lysis buffer containing 0.5 mol/L NaCl and electrophoresed on
15% SDS polyacrylamide gels under a reducing condition.
Electroblotting and detection with antibovine FGF-2 monoclonal antibody
were conducted as reported previously using the ECL system (Amersham
International).23 Protein concentrations were determined
with a DC protein assay kit (BioRad Laboratories). To rule out
the potential contamination of exogenous into endogenous
FGF-2, HUVEC were cultured in DMEM supplemented with 10 ng/mL FGF-1 and
10 U/mL heparin in place of FGF-2 at least for 2 passages before use in
some experiments. HUVEC grew well in this medium, similarly to cells in
FGF-2–containing medium.
Metabolic Labeling
The sparse and dense cultures of HUVEC were incubated with a
mixture of 35S-methionine and
35S-cystein (35S 0.5
mCi/mL, DuPont) in methionine-cysteine free DMEM containing 20% FCS
and 10 ng/mL FGF-2.24 The concentrations of
35S were adjusted to 0.5 mCi/mL in the media.
Cell surface extract was prepared by washing 107
cells twice with 20 mmol/L Tris-HCl, 2 mol/L NaCl, pH 4.0. Total
cell lysate was prepared as described above. The cell surface extract
and total cell lysate were incubated with antibovine FGF-2 monoclonal
antibody (Type II) coupled with protein G-Sepharose (Pharmacia
Biotech). In some experiments, cell lysate and surface extract were
incubated with heparin-Sepharose CL-6B instead of anti–FGF-2-Sepharose
for 2 hours at 4°C. The beads were washed twice with lysis buffer and
separated on 15% SDS-polyacrylamide gel under a reducing
condition. The gel was analyzed using a Fuji BAS 2000
Bioimazing analyzer (Fuji Film).
Northern Blot Analysis
Total cellular RNA was prepared using RNeasy (QIAGEN) according
to the manufacturer's instructions. Messenger RNA was prepared from
total cellular RNA with Oligotex-dT30 (Nippon Roche Ltd).
Northern blot analysis was conducted as reported previously
with 32P-labeled cDNA for human FGF-2 as a
probe.20 25 The autoradiogram was
analyzed by a Fuji BAS 2000 Bioimazing analyzer. Part
of the experiments was repeated with BAEC.
Statistical Analysis
Statistical analysis was performed using the Student's
t test.
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Results |
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Cell Density–Dependent Endothelial Apoptosis
A higher percentage of HUVEC became apoptotic in a dense culture (5x102 cells/mm2) than in a sparse culture (1x102 cells/mm2) when serum and FGF-2 were removed from the culture medium. This effect of cell density was demonstrated by both DNA ladder formation (Figure 1A
) and
counting apoptotic cells under a fluorescent microscope
(Figure 1B). Although cells gradually became apoptotic
even at low cell density, significantly more cells became
apoptotic in the dense culture at all time points tested. By 72
hours, a sizable number of cells had detached from the culture dish,
and reliable quantitative analysis became difficult
thereafter.
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, dense culture) or 1x102 cells/mm2
(•, sparse culture) in gelatin-coated 8 well–chamber slides
and cultured for 48 hours. The medium was changed to DMEM free of FCS
and FGF-2 at time 0. Apoptosis at times 0, 6, 12, 24, and 48
hours was quantified by counting cells that revealed characteristic
fragmented-nuclei by fluorescent microscopy. The percentage of
apoptotic cells was calculated as the number of
apoptotic cells/the number of total cells x100%. Each point
represents mean±SD of 6 values except at 72 hours (triplicate
values). The lines indicate regression curves between apoptotic
cell and incubation time; Y=0.250X+1.364 (r=0.99,
P<0.001) for the dense culture; Y=0.119X-0.623
(r=0.99, P<0.001) for the sparse
culture. The asterisks denote significant increases
(P<0.01, t test) in percentage of
apoptotic cells over the value obtained with the sparse culture
at each time point.
FGF-2 prevented cells from apoptosis even in the absence of
FCS. Apoptotic cells decreased in a dose-dependent fashion from
20% of the total cells in the absence of FGF-2% to 5% in the
presence of 20 ng/mL FGF-2 in the dense culture (Figure 2A). The anti-apoptotic effect of
FGF-2 was confirmed by DNA ladder formation (Figure 2B). A
higher concentration did not further decrease the apoptosis. In
the sparse culture, exogenous FGF-2 also tended to reduce the
apoptosis, but the reduction was not statistically significant.
Among other endothelial growth
factors,26 27 28 VEGF and HGF reduced the apoptosis
but to a lesser extent than FGF-2. IGF-I did not inhibit the
apoptosis significantly at 50 ng/mL (Figure 2C). Our
findings demonstrated that VEGF and HGF, as well as FGF-2, act as
survival factors for endothelial cells, whereas IGF-I,
another endothelial growth factor, does not.
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), HGF (
), and IGF-I (
) or absence of
growth factors. Each point represents mean±SD of 6 values. The
asterisks denote significant increases (P<0.01,
t test) in percentage of apoptotic cells over
the value without growth factor. 
and 
indicate significant
increase (P<0.01, t test) over FGF-2 at
5 ng/mL (
The Resistance of a Sparse Culture to Apoptosis Is Due to a
Greater Amount of FGF-2 Associated With the Cell Surface
Because FGF-2 is produced by human vascular
endothelial cells,24 we studied the role
of autocrine FGF-2 on density-dependent apoptosis by using a
neutralizing antibody against FGF-2 (Upstate Biotechnology Inc, Type
I).15 The antibody significantly increased
apoptosis in the sparse cell culture, whereas control IgG did
not, suggesting that the resistance to apoptosis in the sparse
culture was partly due to autocrine production of FGF-2 by
sparse HUVEC (Figure 3).
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To confirm this notion, we compared the amounts of cellular FGF-2
protein in the dense and sparse culture by Western blot
analysis. In accordance with previous
reports,20 29 we detected only 18 kDa form in the cell
surface extract and both 18 and 22.5 kDa forms in the whole cell
extract. The amount of 18 kDa FGF-2 in the cell surface extract (Figure 4A) and the total cell lysate (Figure 4B) was greater in the sparse culture than in the dense culture
at the 4 time points tested (0, 6, 12, and 24 hours after the
deprivation). The amount of 18 kDa FGF-2 was largest at
time 0 and gradually decreased thereafter, suggesting that synthesis of
18 kDa FGF-2 requires serum components. Contrary to the18 kDa form,
22.5 kDa FGF-2 was not detected until 6 hours after the
deprivation and was increased at the high cell density
(Figure 4B).
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To rule out any potential contamination of exogenous into
endogenous FGF-2 in Western blot analysis, we
conducted the same experiments using HUVEC cultured with FGF-1 and
heparin in place of FGF-2. A specific anti–FGF-2 that did not
recognize FGF-1 revealed a greater amount of FGF-2 in the sparse
culture than in the dense culture (Figure 4C
), confirming the
greater amount of endogenous FGF-2 associated with the
sparse cells.
For quantitative analysis, we conducted a
metabolic labeling study. Although the majority of
synthesized FGF-2 was present inside the cell, a significant
portion of synthesized FGF-2 was detected in the surface extract
(6.7±2.5% of FGF-2 in total cell lysate, the mean±SD of 3
independent experiments conducted in duplicate). Both in the cell
surface extract (Figure 5A) and in the
total cell lysate (Figures 5B and 5C), the amount of
35S-labeled FGF-2 was greater in the sparse
culture than in the dense culture (1.7±0.20-fold increase in total
cell lysate, the mean±SD of 3 independent experiments).
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Mechanisms for Cell Density-Dependent Regulation of Amount of
Cellular FGF-2
Next we studied the mechanisms for cell density–dependent
decrease in FGF-2 content. First we studied the steady state level of
mRNA for FGF-2. As reported previously, 2 major transcripts of 6.6 and
3.7 kb were detected by cDNA for human FGF-2 (Figure 6A).24 There was no
difference in the amount of mRNA for FGF-2 between the sparse and the
dense culture either before or after the deprivation
(Figure 6B) in HUVEC. Contrary, the amount of mRNA for FGF-2 was
greater in the dense culture than in the sparse culture in BAEC (Figure 6C), agreeing with a previous report.30 These
findings suggested that FGF-2 increased in the sparse culture of HUVEC
by post-transcriptional mechanisms.
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Recent studies showing that cytoplasmic proteases regulate the
rapid turnover of many functional proteins31 32 lead us to
study the effects of a panel of inhibitors of 2 major
cytoplasmic proteases (proteasome and calpain) on the cell
density–dependent decrease in FGF-2. Among the protease
inhibitors tested, specific inhibitors of
calpain, E64d19 and
carbobenzoxy-leucyl-leucinal,17 increased FGF-2 content in
the dense culture (Figures 7A and 7B).
Carbobenzoxy-leucyl-leucyl-leucinal (MG132),16 which
inhibits both calpain and proteasome, also increased the FGF-2 content
significantly in the dense culture (Figure 7B). However,
lactacystin,18 a specific inhibitor of
proteasome, did not affect the cellular FGF-2 content (Figure 7B). These findings were confirmed under different experimental
conditions with FGF-2 or FGF-1 as a growth supplement and by Western
blot and metabolic labeling. Our findings suggest that
calpain or an enzyme sensitive to calpain inhibitors
decreased FGF-2 in the dense culture.
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Discussion |
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In this article, we reported that the sparse culture of human vascular endothelial cells is resistant to apoptosis as compared with the dense culture. This resistance is mainly due to a greater production of FGF-2 by the sparse culture of HUVEC than by the dense culture. This conclusion was based on the following 3 sets of evidence: prevention of apoptosis by exogenous FGF-2 (Figure 2
), reversal of the
anti-apoptotic state in the sparse culture by anti–FGF-2
antibody (Figure 3), and greater production of 18 kDa
FGF-2 by the sparse culture (Figures 4 and 5). We also
revealed that a calpain inhibitor–sensitive enzyme is
involved in this cell density–dependent regulation of FGF-2
production. Autocrine FGF-2 plays an important role in the
wound healing of the endothelial monolayer by promoting
endothelial proliferation and
migration.1 2 The current study suggests that autocrine
FGF-2 may play an important role in this process also by increasing the
resistance to apoptosis and preventing further cell loss. Our
study also extends the notion of previous investigations that exogenous
FGF-2 acts as not only a growth factor but a survival
factor33 34 and demonstrates that
endothelial cells change their own susceptibility to
apoptosis by producing this and potentially other autocrine
growth factors. Although HUVEC produces the 18 and 22.5 kDa forms of FGF-2 from a single gene transcript by using 2 different start codons for translation,35 the 18 kDa form is responsible for the resistance to apoptosis of the sparse cells. This notion was supported by 3 sets of evidence. First, nonpermeable anti–FGF-2 antibody abolishes the resistance to apoptosis in the sparse culture, demonstrating that the cell surface FGF-2 is involved. Second, in accordance with previous reports,20 24 only the 18 kDa form is detected in the cell surface extract, whereas both 18 and 22.5 kDa forms are found in the total cell lysate. Finally, the amount of only 18 kDa form but not 22.5 kDa form is decreased at the high cell density.
An unusual finding in our study was that cell density-dependent FGF-2 production was not mediated by changing the steady state level of FGF-2 mRNA. This notion is different from most previous studies, which reported a positive or negative correlation between the cell density and the steady-state level of FGF-2 mRNA. For example, wounding of a confluent monolayer of bovine aortic endothelial cells increased gene the transcript and protein of FGF-2.2 36 Using long-term culture systems, the initial low cell density was found to be correlated with a greater amount of FGF-2 mRNA and protein in various cell types such as an astrocytoma cell line.37 On the other hand, FGF-2 mRNA was increased in bovine coronary artery endothelial cells 2 days after confluence.30
One potential cause of the discrepancy between previous studies and our
study is the origin of the endothelial cells used for
the experiments. We demonstrated that the steady state level of the
mRNA was affected by the cell density in BAEC under the same conditions
at which that of HUVEC was not affected (Figure 6C). Therefore,
our finding with HUVEC could not be repeated with
endothelial cells of other species or of other parts of
the vascular tree. Another potential cause of the discrepancy is that
the amount of FGF-2 mRNA could be affected by factors other than the
cell density. A predominant part of the increase in FGF-2 mRNA in
wounding experiments was attributed to growth factors and protease
released on wounding rather than the cell density
itself.38 Long-term culture systems might be affected by
accumulation of the extracellular matrix of growing cells. Because our
experimental conditions were free from these factors, we detected the
effect of cell density itself and revealed the presence of a
post-transcriptional mechanism that regulates the FGF-2 protein level
in a cell density–dependent manner.
Specific inhibitors for cytosolic neutral proteases enabled us to identify the role of these enzymes in density-dependent regulation of FGF-2 production. E64d is a calpain inhibitor that is widely used to demonstrate the physiological and pathophysiological roles of this enzyme.39 40 Carbobenzoxy-leucyl-leucyl-leucinal (MG132) is a potent inhibitor of proteasome, but it also inhibits calpain to the same extent.16 31 41 Carbobenzoxy-leucyl-leucynal has recently been shown to be highly specific for calpain; it inhibits calpain at a 1000 times lower concentration than it inhibits proteasome both in vitro and in vivo.17 They are membrane permeable, inhibit activity of not only purified enzymes but also the activity in cultured cells, and affect cellular functions mediated by these enzymes.16 39 40 41 Lactacystin is a specific inhibitor of proteasome18 and has been used to investigate proteasome function in the cell.41 Our findings that all inhibitors for calpain, but not a specific inhibitor for proteasome, increased FGF-2 in dense cells strongly suggest that calpain or an enzyme closely related to calpain is involved in the density-dependent decrease in FGF-2 protein.
Calpains are a family of calcium-activated neutral proteases present ubiquitously in mammalian tissues (m-calpain and µ-calpain) including vascular smooth muscle cells and endothelial cells.42 43 44 Calpain degrades a wide variety of substrate, including cytoskeletal proteins, protein kinases, transcription factors, precursors of a cytokine, and calpain itself, and plays a role in various cellular functions that include cytoskeletal reorganization, cell adhesion, gene expression, and intracellular signal transduction.32 43 Although the precise mechanism by which calpain regulates the amount of FGF-2 is not clear, the current study revealed a new functional role of calpain: modulation of cell proliferation and survival by regulating the production of autocrine growth factor.
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Acknowledgments |
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This study was supported by Special Coordination Funds from the Science and Technology Agency of Japan and a grant from the Ministry of Health and Welfare (to K.S.). We thank Drs H. Kato, M. Harada-Shiba, T. Miyata, T. Saido, and K. Suzuki for helpful discussion and H. Sugita for excellent technical assistance.
Received July 28, 1998; accepted April 23, 1999.
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