(Circulation. 2000;102:III-269.)
© 2000 American Heart Association, Inc.
Myocardial Protection and Vascular Biology |
Vacuolar H+-ATPase Plays a Crucial Role in Growth and Phenotypic Modulation of Myofibroblasts in Cultured Human Saphenous Vein
From the Department of Thoracic and Cardiovascular Surgery (H.O., T.Y., Y.N., R.H., H.F., H.N., M.K., H.K., M.O., H.I.), Kansai Medical University, Moriguchi, Japan; and Department of Surgery (II) (T.O.), and Laboratory of Biochemistry (S.O.), Department of Molecular and Cell Biology, Faculty of Pharmaceutical Science, Kanazawa University, Kanazawa, Japan.
Correspondence to Hajime Otani, MD, Department of Thoracic and Cardiovascular Surgery, Kansai Medical University, 10-15 Fumizono-cho, Moriguchi City, Osaka 570, Japan. E-mail otanih{at}takii.kmu.ac.jp
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Abstract |
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Background—The molecular mechanism of neointimal hyperplasia after vein graft surgery remains elusive. Vacuolar H+-ATPase (V-ATPase) is involved in intracellular trafficking and may play a crucial role in neointimal cell growth.
Methods and Results—Cultured human saphenous vein segments
developed neointimal formation within 10 days.
Neointimal cells were positive for vimentin and 
-smooth
muscle actin but negative for desmin, which is indicative of
myofibroblasts. Those myofibroblasts were found to have originated from
periadventitial fibroblasts, which upregulated the expression of 16-kDa
proteolipid of V-ATPase before proliferation and phenotypic modulation.
Neointimal myofibroblast growth and survival were highly
sensitive to inhibition of V-ATPase by bafilomycin A1
(BA1), because the incorporation of
[3H]thymidine into the myofibroblasts was significantly
inhibited by nanomolar concentrations of BA1 and
apoptotic cell death was induced by a similar concentration
range of BA1. In contrast, endothelial
cells and differentiated smooth muscle cells were resistant to
apoptosis by BA1.
Conclusions—These results suggest that V-ATPase plays a crucial role in growth and phenotypic modulation of myofibroblasts that contributes to neointimal formation in cultured human saphenous vein.
Key Words: vacuolar H+-ATPase • myofibroblasts • neointima • apoptosis • veins
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Introduction |
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Neointimal hyperplasia is known to occur in saphenous vein (SV) grafts after CABG. This vascular remodeling may affect graft patency.1 2 3 Better understanding of the pathophysiology of neointimal hyperplasia in vein grafts could provide valuable information for the development of a strategy to treat vein graft disease. Cellular events that emerge downstream from the activation of growth factor receptors have been targets for extensive research. Despite accumulating knowledge of signal transduction pathways and regulatory proteins for cell cycle progression,4 effector systems responsible for the growth and phenotypic modulation of smooth muscle cells (SMCs) have been poorly understood.
We previously showed that vacuolar-type H+-ATPase (V-ATPase) may be involved in neointimal formation and medial thickening in cultured human SV segments.5 V-ATPases have molecular structures distinct from the mitochondrial F1F0 (F-type) ATPases and the gastric E1E2 (P-type) ATPases.6 V-ATPases are composed of catalytic V1-domain and membrane-embedded channel V0-domain.7 8 Although the exact function of each subunit is not completely elucidated, the 16-kDa subunit (hydrophobic proteolipid [16 kDaPL]) is considered to be the principal component of the V0 membrane channel sector, which is representative of a highly conserved family of polypeptides implicated in diverse transport functions in eukaryotic cells.9 10 V-ATPases reside on the membranes of acidic organelles such as synaptic vesicles, chromaffin granules, platelet-dense granules, secretary granules, lysosomes, and the trans-Golgi network, and maintain acidic environment by pumping protons with the use of the energy of ATP hydrolysis.11 12 The acidic pH within such organelles is proposed to be responsible for a wide variety of important cellular functions such as endocytosis, exocytosis, and intracellular trafficking, as well as for cell growth and differentiation.13 14 Thus, in the present study, we extended our previous work to address the questions of whether the expression of V-ATPase is upregulated in proliferating vascular cells and whether enhanced expression of V-ATPase is associated with growth and phenotypic modulation of these cells. The results of the present study suggest that overexpression of V-ATPase may be involved in the growth and phenotypic modulation of myofibroblasts that contribute to neointimal formation in cultured human SV segments.
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Methods |
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Tissue Culture Procedures
Segments of SV were obtained from 24 patients (mean age 67 years, age range 40 to 82 years, 7 women) who were undergoing elective CABG. Informed consent was obtained from the patients according to the ethical permission required by the Ethical Committee of Kansai Medical University. The veins were dissected with minimal handling, and
2-cm
segments were taken immediately after the harvest without distention.
Organ culture procedures were performed as described in the previous
study.5 Briefly, a 1-cm segment of SV was incubated in
RPMI 1640 medium containing 30% FBS and penicillin/streptomycin (100
µg/mL each) and incubated in a 5% CO2–95%
air incubator at 37°C. Bafilomycin A1
(BA1; Sigma Chemical Co), a V-ATPase
inhibitor, was dissolved in dimethyl sulfoxide (DMSO) and
diluted with tissue culture medium to yield desired concentrations and
0.1% DMSO. Control cultures were performed with medium that contained
0.1% DMSO. This concentration of DMSO had no significant effect on
neointimal formation in our pilot study. To trace the
translocation of proliferating cells, cultured SV segments were
administered with 50 µmol/L 5-bromo-2'-deoxyuridine (BrdU)
between 2 and 3 days in culture, and the cultivation was terminated at
various stages as indicated in the text. In additional sets of
experiments, SV segments were incubated with 50 µmol/L BrdU for
the final 2 days of culture to identify the proliferating cells at
various cultivation stages. At the end of the cultivation, the SV
segments were fixed with 10% formaldehyde in 0.1 mol/L phosphate
buffer, pH 7.2.
Immunohistochemistry
The formaldehyde-fixed and paraffin-embedded SV segments were
cut into 4-µm-thick sections and deparaffinized with a graded series
of xylene and ethanol solutions. Immunostaining of 16
kDaPL was performed as described previously.15 Briefly,
the sections were incubated with proteinase K
(Boehringer-Mannheim Biochemica) at a concentration of 40
µg/mL for 10 minutes at 37°C. After being washed in PBS, the
sections were incubated in absolute methanol that contained 0.3%
hydrogen peroxide and incubated with 10% normal goat serum at a 1:30
dilution for 30 minutes at room temperature. The rabbit anti-16 kDaPL
antisera obtained according to a previously described
method16 were applied at the predetermined optimal
dilution and incubated at 4°C overnight. The sections were washed in
PBS and incubated for 2 hours at room temperature with tetrarhodamine
isothiocyanate (TRITC)–labeled goat anti-rabbit IgG (DAKO Japan). In
each immunostain run, the primary antisera were replaced
with nonimmune normal rabbit serum (DAKO) as negative controls. The
specificity of immunostaining was confirmed by a
competitive inhibition test with the synthetic peptide; primary
antisera were mixed with the synthetic peptide (1 µg/mL), followed by
the immunostaining. The fluorescence was viewed
with a confocal laser microscope (Fluoview; Olympus Tokyo).
Immunostaining for BrdU and cell marker proteins (von
Willebrand factor, vimentin, –smooth muscle actin
[-SMA], and desmin) was performed as described
previously.5 For double
immunofluorescence experiments, sections were
incubated first with primary antibodies as described earlier and
stained with fluorescein isothiocyanate (FITC)- or
TRITC-conjugated secondary antibodies.
Immunohistochemical detection of apoptotic cells was carried out using an in situ nick end-labeling method (TUNEL) as described previously.5
Cell Culture Procedures
The freshly prepared SV segments obtained from an additional 6
patients (mean age 63 years, age range 47 to 75 years, 1 woman) were
tissue cultured as described here. At 1 week after the cultivation, a
glass coverslip was placed on the intimal surface of the SV
segment so as to introduce the outgrowth of the intimal cells. After 14
days in culture, the glass coverslip was removed, and the cells were
further cultured in RPMI medium that contained 10% FBS until
confluence. The cells were passaged through trypsinization on 0.25%
trypsin in PBS, and 1x105 cells/mL was seeded
onto the dishes. Confluent cultures were obtained in 5 to 7 days. Only
1-passaged cells were used in the study to avoid possible alterations
of their phenotype.
Immunocytochemistry
The cells were fixed in absolute methanol at -20°C for 10
minutes, and immunostaining for 16 kDaPL and TUNEL was
performed as described earlier with FITC-labeled secondary antibodies.
The number of TUNEL-positive cells was counted on 10 fields on each
dish at a magnification of x600. The percentage of TUNEL-positive
cells was calculated by dividing the number of TUNEL-positive cells by
the total number of cells.
[3H]Thymidine Labeling
The confluent cells passaged through trypsinization were
reseeded onto a 96-well microtiter plate at the concentration of
5x103 cells per well. After 24 hours, incubation
in RPMI medium that contained 10% FBS and 1 µCi of
[3H]thymidine was added to each well, and the
cells were incubated for an additional 24 hours. Then, the cells were
harvested for measurement of [3H]thymidine
incorporation.
Statistical Analysis
All numerical data are presented as mean±SD. One-way
ANOVA and Scheffé’s multiple comparison test were used to
compare the multigroup variables. A value of P<0.05 was
considered to be statistically significant.
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Results |
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Translocation of Proliferating Cells
Our recent study demonstrated that the human SV segments developed significant neointimal formation within 14 days in culture.5 Neointimal hyperplasia occurred on the entire intimal surface, although the degree of intimal hyperplasia was not uniform over each specimen. In the present study, we first investigated the origin of the neointimal cells in these SV segments by tracing proliferating cells labeled with BrdU. It was quite infrequent that the cells in SV segments incorporated BrdU for the first 2 days in culture; therefore, BrdU was administered between 2 and 3 days in culture to trace the proliferating cells. When SV segments were treated with BrdU at a concentration of 50 µmol/L for 48 hours at this stage, BrdU-labeled cells were found predominantly in the periadventitial region (Figure 1A
). A
number of BrdU-labeled cells were translocated to the media and the
intima after 6 days in culture (Figure 1B). The majority of the
neointimal cells were labeled with BrdU after 10 days in
culture (Figure 1C), indicating that these
neointimal cells were daughter cells that predominantly
originated from the periadventitial cells that had incorporated BrdU
between 2 and 3 days in culture.
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5 experiments at each cultivation
stage. a indicates adventitia; m, media; and i, intima (magnification
x200).
Expression of 16 kDaPL in Proliferating Cells
We hypothesized that the 16 kDaPL subunit of V-ATPase may be
upregulated in the proliferating vascular cells. Therefore, spatial and
temporal distributions of 16 kDaPL-positive cells were examined in the
BrdU-labeled SV segments to elicit the relationship between 16 kDaPL
expression and proliferation. None of the cells in the freshly prepared
SV segments (n=8) showed detectable expression of 16 kDaPL before
culture. However, overexpression of 16 kDaPL occurred in the
periadventitial cells within 2 days in culture when no BrdU uptake was
observed in these cells (Figure 2A).
After 3 days in culture, some 16 kDaPL-positive cells incorporated BrdU
(Figure 2B). All of the BrdU-labeled cells were positive for 16
kDaPL. After 7 days in culture, the BrdU-labeled cells in the intima
and the media were positive for 16 kDaPL (Figure 2C). The 16
kDaPL–positive cells were distributed predominantly in the intima
after 14 days in culture, and the majority of these cells were also
labeled with BrdU (Figure 2D).
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Intracellular Localization of 16 kDaPL
Because immunohistochemical staining for 16 kDaPL did not provide
images that were sufficiently fine for resolution of the intracellular
localization of 16 kDaPL, immunocytochemical studies were performed on
the neointimal cells outgrown onto a glass coverslip. The
neointimal cells with extensive growth potential after 14
days in culture showed a distinct immunoreactivity pattern (Figure 3A). The immunostaining
was strongly enhanced in the perinuclear region. In contrast, the
neointimal cells made quiescent for 48 hours with the
medium containing 0.2% FBS lost this immunostaining
pattern (Figure 3B).
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Phenotypic Modulation of Proliferating Cells
Because it has been suggested that the neointimal
cells are myofibroblasts that had been derived from fibroblastic cells
via phenotypic modulation,17 18 we investigated whether
the proliferating cells underwent phenotypic modulation during
translocation from the periadventitia to the intima. The BrdU-labeled
cells in the periadventitia after 3 days in culture lacked
immunostaining for -SMA (Figure 4A). On the contrary, SMCs that expressed
-SMA lacked BrdU labeling. However, the BrdU-labeled cells located
in the intima became expressed with -SMA after 7 days in culture
(Figure 4B). Expression of -SMA in the BrdU-labeled cells was
enhanced after 14 days in culture (Figure 4C). These findings
suggest that the BrdU-labeled cells that had been devoid of -SMA at
the early stage in culture acquired this SMC-specific protein during
translocation from the periadventitia to the intima.
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Effect of BA1 on Growth and Phenotypic Modulation
of Myofibroblasts
The role of V-ATPase in growth and phenotypic modulation was
further investigated with BA1, a selective
inhibitor of V-ATPase. The freshly isolated SV segments
showed SMC layers in the media, which were negative for vimentin but
positive for -SMA and desmin (Figures 5A and 5E). These SV segments also
contained fibroblastic cells with negative
immunostaining for -SMA and desmin but positive
immunostaining for vimentin. These fibroblasts were
predominantly distributed in the periadventitial region.
Myofibroblasts, which have been characterized by positive
immunostaining for vimentin and -SMA but negative
immunostaining for desmin,19 were not
found in the freshly isolated SV segments. In contrast, both vimentin-
and -SMA–positive cells were distributed uniformly in the
neointima after 14 days in culture (Figure 5B). The
neointimal cells were exclusively negative for desmin
(Figure 5F), indicating that the neointimal cells
expressed a phenotypic feature consistent with myofibroblasts.
It should be noted that there were few periadventitial cells that were
positive for only vimentin, and a considerable number of myofibroblasts
were localized in the periadventitia (Figures 5C and 5G).
In the media, the majority of -SMA–positive cells were also
positive for desmin, indicating that after 14 days in culture, the SV
segments retained SMCs of a differentiated phenotype in the
media. The SV segments cultured in the presence of 10 nmol/L
BA1 for 14 days showed an immunoreactivity
pattern similar to that observed in the freshly isolated SV segments.
-SMA was colocalized with desmin but not with vimentin in layers of
the medial cells (Figures 5D and 5H). Vimentin-positive
nonmuscle cells were distributed between the medial SMC layers and in
the adventitia, as were observed in the freshly isolated SV segments.
Immunohistochemically detectable myofibroblasts were not found in these
BA1-treated SV segments.
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BA1 Induces Apoptosis Selectively in
Myofibroblasts
These observations suggest that myofibroblasts may be eliminated
selectively with BA1 treatment in the cultured SV
segments. Therefore, the susceptibility to BA1 of
myofibroblasts, endothelial cells, and differentiated
SMCs was compared with the use of specific cytoskeletal marker proteins
and TUNEL. When 14-day cultured SV segments were treated with 10 nmol/L
BA1 for 48 hours, massive apoptosis
occurred in the neointimal myofibroblasts (Figure 6A). In contrast, the same concentration
of BA1 did not induce apoptosis in the
endothelial cells after 4 days in culture when
myofibroblasts that translocated to the media underwent massive
apoptosis (Figure 6B). Moreover, desmin-positive SMCs in
these SV segments were also free from TUNEL staining (Figure 6C).
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Dose-Response Effect of BA1 on
[3H]Thymidine Labeling and Apoptosis in Cultured
Myofibroblasts
Cultured cells were exclusively positive for vimentin and -SMA
but negative for desmin, indicating a pure population of myofibroblast
(not shown). To characterize the mechanism for
BA1-induced inhibition of neointimal
formation, the effect of BA1 on growth activity
and apoptotic cell death was investigated with the use of
cultured myofibroblasts. [3H]Thymidine
incorporation in cultured myofibroblasts was significantly inhibited
with as little as 1 nmol/L BA1 (Figure 7A). BA1 at a
concentration of 10 nmol/L completely abrogated
[3H]thymidine incorporation into
myofibroblasts.
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The dose-response effect of BA1 on myofibroblast
apoptosis was also examined in cultured myofibroblasts.
BA1 induced significant myofibroblast
apoptosis at a concentration of >1 nmol/L (Figure 7B
).
Treatment with BA1 at a concentration of 50
nmol/L for 48 hours induced apoptosis in >80% of cultured
myofibroblasts. These results suggest that
BA1-induced inhibition of neointimal
formation is attributed to both growth arrest and apoptotic
cell death of myofibroblasts.
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Discussion |
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The origin of cells that constitute the neointimal lesion after vascular injury and vein graft implantation has been a matter of debate. Neointimal cells have been characterized as myofibroblasts that are proposed to derive from the adventitial fibroblasts as a result of wound-healing response.17 18 20 The results of the present study are consistent with a potential role of the adventitial fibroblasts in neointimal formation and suggest further that V-ATPase may play a crucial role in the growth and phenotypic modulation of myofibroblasts in cultured human SV segments. By tracing BrdU-labeled cells, we were able to analyze the translocation and phenotypic modulation of proliferating cells in the SV segments. The majority of BrdU-labeled cells first appeared in the periadventitial region after 3 days in culture, migrating from the media toward the intima by day 6 and proliferating in the intima thereafter. Double immunofluorescence confocal laser microscopy with an antibody against 16 kDaPL of V-ATPase in the BrdU-labeled SV segments revealed that overexpression of 16 kDaPL first appeared in the periadventitial cells within 2 days in culture, when no incorporation of BrdU into these cells was found. However, some of the 16 kDaPL–positive cells incorporated BrdU after 3 days in culture. Thereafter, the proliferating cells in the intima and the media were exclusively positive for 16 kDaPL. These findings suggest that the neointimal cells originated from the periadventitial cells that upregulated 16 kDaPL and incorporated BrdU at the early stage in culture.
Enhanced expression of 16 kDaPL in the periadventitial cells was found
before BrdU uptake and phenotypic modulation. Enhanced expression of 16
kDaPL in the periadventitial cells occurred within 2 days in culture,
whereas the BrdU-labeled cells did not emerge until 3 days in culture.
Furthermore, the BrdU-labeled cells lacked -SMA after 3 days in
culture. The BrdU-labeled cells located in the intima and the media
modestly expressed -SMA after 7 days in culture. Finally, the
neointimal cells labeled with BrdU acquired abundant
-SMA after 14 days in culture. In contrast, the medial SMCs that
constitutively expressed -SMA and desmin lacked BrdU-labeling. Thus,
these differentiated SMCs were thought to be devoid of proliferative
activity and appeared to retain the same phenotype during the
culture. The facts that overexpression of 16 kDaPL in the
periadventitial fibroblasts preceded BrdU uptake and a phenotypic
change to myofibroblasts and that BA1 prevented
the emergence of myofibroblasts indicate that overexpression of
V-ATPase may be a prerequisite for transformation of the fibroblasts to
a more synthetic and proliferative phenotype.
The mechanism of inhibition of neointimal formation by BA1 can be attributed to both growth arrest and apoptotic cell death of myofibroblasts. BA1-induced inhibition of [3H]thymidine incorporation and induction of apoptosis in cultured myofibroblasts occurred at a nanomolar concentration range. Moreover, the TUNEL study in cultured SV segments demonstrated that compared with endothelial cells and differentiated SMCs, myofibroblasts were prone to undergo apoptosis in response to treatment with BA1. These observations suggest that the susceptibility of vascular cells to inhibition of V-ATPase may be inversely related to the differentiation status of SMCs. A similar conclusion was drawn in the previous study with various cells, demonstrating that cell transformation toward the dedifferentiated phenotypes increases sensitivity to BA1.21
To our knowledge, this report is the first to provide the immunohistochemical evidence for overexpression of V-ATPase subunit in proliferating cells. However, physiological significance of enhanced expression of V-ATPase in the perinuclear region in myofibroblasts remains unclear. The perinuclear region consists of various organelles such as lysosomes, endosomes, secretory granules, coated vesicles, and the trans-Golgi network, all of which are known to contain V-ATPase. Proton pumping into these organelles is thought to be important in the maintenance of cytosolic pH, especially when proton production is increased through enhanced metabolic demand as a result of proliferation and migration. Accommodation of intracellular pH is indeed crucial for cell survival. Intracellular acidosis after hypoxia, ischemia, and irradiation has been shown to induce apoptosis.22 23 24 Another important function of V-ATPase in eukaryotic cells is the facilitation of growth factor recycling and reutilization, thereby increasing mitogenic potential of growth factors. Exposure to low pH within endosomes activates dissociation of ligands and receptors such as insulin and epidermal growth factor after receptor-mediated endocytosis.25 26 27 Moreover, full mitogenic activity of basic fibroblast growth factor has been shown to require internalization of the growth factor or the growth factor complex into the acidic granules by endocytosis, followed by translocation to the nucleus.28 V-ATPase may also be involved in the stimulation of molecular transport across the organelle membranes. Unlike F-ATPases in mitochondria, which synthesize ATP at the expense of proton motive force, the vacuolar systems in eukaryotic cells are energized primarily by V-ATPase that functions as an ATP-dependent proton pump to generate proton motive force.12 Electrochemical proton gradient across the membranes is used to drive the coupled transport of essential ions as well as macromolecules that are usually impermeable through the vacuolar membranes. Thus, overexpression of V-ATPase may be necessary for certain proliferating cells, such as myofibroblasts, to increase translational activity and molecular trafficking. Perturbation of these processes by V-ATPase inhibition could induce growth arrest and cell death. Further investigation is warranted to explore an exact role of V-ATPase in the regulation of cell growth and survival.
In conclusion, the results of the present study suggest that V-ATPase may be involved in the growth and phenotypic modulation of myofibroblasts that contribute to neointimal formation in cultured human SV. Future studies are required to clarify whether the same mechanism takes place in a wide variety of vascular proliferative disorders and whether inhibition of V-ATPase represents an effective adjunct of treatment for postangioplasty restenosis, vein graft disease, and allovasculopathy.
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Acknowledgments |
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We gratefully acknowledge the technical assistance of Aya Kobayashi.
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