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Regular Articles |
From the Departments of Pathology *
and
Neurosurgery,¶
National Cardiovascular Center,
Suita City, Japan; Licensing Business
Development,
R&D Operations, Dainippon
Pharmaceutical Co. Ltd., Suita City, Japan; the Department of
Neuroanatomy,
Kanazawa University School of
Medicine, Kanazawa City, Japan; the Department of
Pathology,||
Osaka Rosai Hospital, Sakai City, Japan; the
Department of Pathology,
Osaka University
Graduate School of Medicine, Suita City, Japan; and the Department of
Physiology and Cellular Biophysics,**
College of
Physicians and Surgeons, Columbia University, New York, New York
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Ischemia-induced perturbation of vascular function is associated with a range of blood vessel disorders. For example, decreased arterial wall oxygen tension occurs in atherosclerotic lesions.9,10 In this context, we have demonstrated expression of a 150-kd oxygen-regulated protein (ORP150) in atherosclerotic lesions, as well as its capacity to buttress cellular viability under hypoxic conditions, especially in the presence of otherwise toxic levels of modified low density lipoprotein.11 In atherosclerotic lesions, re-oxygenation may also trigger a range of mechanisms. For example, cycles of ischemic stress and re-oxygenation in atherosclerotic lesions may promote macrophage generation of pro-inflammatory mediators, such as interleukin-1, thereby contributing to remodeling of the vessel wall.12
These considerations led us to examine the expression of RA301/Tra2ß in human vascular tissues, balloon-injured rat carotid arteries, and cultured vascular cells. Our results demonstrate expression of RA301/Tra2ß in vascular smooth muscle cells (VSMCs) of neointimal lesions. Analysis of cultured VSMCs showed that hypoxia/re-oxygenation stimulated expression of RA301/Tra2ß and proliferation, both of which seemed to be related based on their parallel inhibition by diphenyl iodonium (DPI), PD98059, and antisense oligonucleotides for RA301/Tra2ß. Because RA301/Tra2ß functions as a sequence-specific RNA-binding protein, its expression in re-oxygenated vessels might be important for posttranscriptional regulation of multiple genes important in the vascular stress response.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Twenty specimens of human tissues were obtained from autopsy cases within 1 to 4 hours of death (see text for the description of patients). Tissue samples for RNA extraction were stored at -80°C until use, and those for in situ hybridization and immunohistochemistry were fixed with 4% paraformaldehyde in 0.1 mol/L phosphate buffer, pH 7.0, and embedded in paraffin. Certain samples for immunohistochemical analysis were fixed in 10% buffered formaldehyde and embedded in paraffin. Tissue samples from the rat arterial injury model were excised and fixed in 10% buffered formaldehyde and embedded in paraffin. The rat carotid balloon angioplasty model was performed as described.13 Serial sections (3 µm) were cut from either rat or human vascular tissues for further study.
In Situ Hybridization
A RA301/Tra2ß BglII fragment corresponding to 292 to 531 (240 bp)5 was subcloned into the BamHI site of the pBluescript SK(-) vector (Stratagene Inc., La Jolla, CA) and sequencing was performed.14 After linearization of the plasmid, digoxigenin-labeled single-stranded RNA was synthesized by T7 and T3 RNA polymerase with digoxigenin-UTP and unlabeled ATP, GTP, and CTP. Details of the in situ hybridization protocol have been described.15 Hybridization was performed at 50°C for 16 hours, and signals were detected using a Nucleic Acid Detection kit (Boehringer Mannheim Biochemicals, Mannheim, Germany). Controls included hybridization with sense probe, RNase treatment before hybridization, and omission of either the antisense RNA probe or the antibody to digoxigenin.
Immunohistochemistry
Proliferating cells were identified with a mouse monoclonal
antibody to proliferating cell nuclear antigen (PCNA) (Santa Cruz
Biotechnology Inc., Santa Cruz, CA). Smooth muscle cells and
macrophages were identified with mouse monoclonal antibodies 1A4 and
PG-M1 (anti-CD68) (DAKO SA, Glostrup, Denmark). Endothelial cells were
identified with goat polyclonal antibody to mouse PECAM-1 (Santa Cruz
Biotechnology Inc.) as described.16
For detection of
RA301/Tra2ß and PCNA antigens, sections were microwaved for 10
minutes and incubated in
H2O2 (0.3%) in methanol
for 30 minutes, followed by washing in phosphate-buffered saline (PBS)
(0.1 mol/L) and incubation with normal mouse serum (1%) for 30 minutes
at room temperature (to block nonspecific binding of the antibody).
Slides were incubated with rabbit anti-RA301/Tra2ß polyclonal
antibody (40 µg/ml) or mouse anti-PCNA monoclonal antibody (5
µg/ml) for 18 hours at 4°C. Sites of primary antibody binding were
detected by incubating slides with horseradish peroxidase-conjugated
anti-rabbit IgG or anti-mouse IgG followed by reaction with the
substrate, 3,3'-diaminobenzidine tetrahydrochloride. As a negative
control, either preimmune rabbit or mouse serum was used instead of the
antiserum. Antibody specificity was confirmed as described
previously.5
For the detection of 
-smooth muscle actin
or CD68 or PECAM-1, sections were treated as the same method as
described above except for the microwave treatment. Co-expression of
RA301/Tra2ß and PCNA was confirmed with confocal laser-scanning
microscopy (Olympus LSM-GB200; Olympus, Tokyo, Japan) as
described.17
Fluorescein isothiocyanate-conjugated swine
anti-rabbit IgG and R-phycoerythrin-conjugated goat anti-mouse IgG
(DAKO SA) were used as the secondary antibodies. The excitation
wavelength is 488 nm for fluorescein isothiocyanate and 515 nm for
R-phycoerythrin.
Cell Culture and Conditions for Hypoxia/Re-Oxygenation
Rat VSMCs were isolated by enzyme digestion from thoracic aortae of 12-week-old Sprague-Dawley rats as described.18 VSMC populations obtained by this method were grown to the fifth passage in Dulbecco’s modified Eagle’s medium (Life Technologies, Inc., Grand Island, NY) supplemented with 10% fetal calf serum. Human aortic endothelial cells were purchased from Kurabo (Osaka, Japan) and cultured according to previously described methods.19 Human mononuclear phagocytes were cultured as described.11 Cells were plated at a density of 2 x 104 cells/cm2, and exposed to hypoxia using an incubator attached to a hypoxia chamber that maintained a humidified atmosphere with low oxygen tension (pO2 = 12 to 14 torr; Coy Laboratory Products, Ann Arbor, MI) as described.19 After exposure to hypoxia, cells were returned to ambient atmosphere. Oxygen tension in the medium was measured using a blood gas analyzer (ABL-2; Radiometer, Sweden). Cell viability was assessed by morphological criteria, trypan blue exclusion, lactate dehydrogenase (LDH) release, and general protein synthesis was measured by the incorporation of 3H-leucine to trichloroacetic acid-precipitable material.4 Cell proliferation was evaluated by measuring the change in cell number using a Coulter counter (Coulter Electronic, Hialeah, FL) and by the dimethylthiazoldiphenyl tetrazolium bromide (MTT) assay.
Northern Blot Analysis
Total RNA was extracted from the indicated tissue of human autopsy cases as described.20 After purification of total RNA, its quality was judged by Northern analysis and hybridization with a human ß-actin probe. RNA samples that failed to show a distinct ß-actin band were excluded from further experiments. For Northern blotting, total RNA (10 µg) was fractionated by agarose gel (1%) electrophoresis, transferred to Hybond N+ membrane (Amersham International, Buckinghamshire, UK) and then hybridized with a 32P-dCTP-labeled rat RA301/Tra2ß cDNA probe. After hybridization, the membrane was washed and signals were detected by autoradiography. Total RNA was processed from cultured cells using the same procedure.
Western Blot Analysis
Detection of RA301/Tra2ß antigen in cultured vascular cells was
performed as described previously.5
In brief, either
cultured rat VSMCs, human aortic endothelial cells, or human
mononuclear phagocytes were exposed to hypoxia for 36 hours, followed
by re-oxygenation. At the indicated time points, cells were harvested
and lysed in PBS (pH 7.4) containing Nonidet P-40 (1%), sodium dodecyl
sulfate (0.5%), and ethylenediaminetetraacetic acid (5 mmol/L). After
measurement of protein content,21
5 µg of protein was
subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(10%) under reducing condition, transferred to polyvinylidene fluoride
membranes (Immobilon-P; Millipore Corporation, Bedford, MA), and
incubated with either rabbit anti-RA301/Tra2ß polyclonal antibody (5
µg/ml)5
or mouse anti-PCNA monoclonal antibody (0.5
µg/ml; Santa Cruz Biotechnology Inc.). Sites of binding of primary
antibody were detected with either anti-rabbit IgG or anti-mouse IgG,
both conjugated to alkaline phosphatase (Sigma, St. Louis, MO). Where
indicated, cultured rat VSMCs were maintained under serum-free
conditions for 48 hours. After the addition of either platelet-derived
growth factor-BB (PDGF-BB) (10 ng/ml; Pepro Tech EC, London, UK) or
angiotensin II (100 µg/ml; Peptide Institute, Osaka, Japan), cultures
were further maintained in normoxia for up to 48 hours. Cells were then
harvested at the indicated time points, followed by Western blot
analysis as described above.
Detection of Superoxide Anion (O2-) in Re-Oxygenated VSMCs
O2- was measured by
lucigenin-based chemiluminescence by adding bis N-methyl
acridinium nitrate (lucigenin, 0.2 mg/ml;
Sigma)/CaCl2 (10 mmol/L) to hypoxic VSMCs
(107
cells) suspended in PBS at the time of
re-oxygenation. Chemiluminescence was detected using a luminometer
(BRL-301; Aloka, Tokyo, Japan), after equilibration of the cell
suspension with 5% CO2/room air.22
Effects of DPI, PD98059, and RA301/Tra2ß Antisense Oligonucleotide on Re-Oxygenated VSMCs
Rat VSMCs (5 x 104
cells/well) were
exposed to hypoxia for 36 hours, followed by re-oxygenation. Either
DPI, PD98059, or sense/antisense oligonucleotide for
RA301/tra2ß5
was added to culture medium 30 minutes
before re-oxygenation. After re-oxygenation, cells were maintained in
normoxia for 4 hours and then lysed in PBS containing Nonidet P-40
(1%) and sodium dodecyl sulfate (0.5%), as described above. Protein
extracts (10 µg/lane) were then subjected to Western blot analysis
using either rabbit anti-RA301/Tra2ß polyclonal antibody or mouse
anti-human inducible 70-kd heat-shock protein (HSP70i) monoclonal
antibody (0.2 µg/ml; Amersham International, Buckinghamshire, UK)
as described previously.23
Where indicated, protein
extracts were subjected to Western blot analysis using either rabbit
anti-ERK1/2 polyclonal antibody for the detection of total p42/44
mitogen-activated protein kinase, or anti-active MAPK polyclonal
antibody or anti-active c-Jun N-terminal kinase (JNK) antibody for the
detection of phosphorylated p42/44 MAP kinase or phosphorylated JNK
(Promega, Madison, WI).
Cellular proliferation and viability were also assessed in response to
the above agents. VSMCs were plated in 24-well plates
(104
cells/well) and exposed to hypoxia for 36
hours. Cells were then re-oxygenated in the presence of DPI, PD98059,
or sense/antisense oligonucleotide for RA301/Tra2ß. Cultures were
maintained for an additional 48 hours in normoxia and cell
proliferation was quantified using the MTT (Sigma Chemical Co.) rapid
colorimetric assay, as described.24
In brief, MTT (100
µl of 5 mg/ml solution of MTT in PBS) was added to all wells for the
final 4 hours of re-oxygenation. Cells were lysed with 0.02 mol/L
HCl/isobutanol, and optical density at 520 nm was determined. Where
indicated, DPI was added to the culture at the indicated time points
before or after re-oxygenation.
Cell viability was assessed by the release of LDH activity into culture
supernatants using a commercially available kit (Wako Chemicals,
Japan). Cultured rat VSMCs plated in 24-well plates
(104
cells/well) were exposed to hypoxia for
36 hours and re-oxygenated in the presence of either DPI or PD98059 at
the indicated concentrations. Cells were further maintained for 48
hours under normoxic condition, followed by the measurement of LDH
activity in the supernatant as described in the manual of the kit.
In the above experiments, sense and antisense oligonucleotides for RA301/Tra2ß were used:5 sense, 3'-CGA CAG CGA GTA CTG AGG CC-5' and antisense, 5'-GCT GTC GCT CAT GAC TCC GG-3'.
Statistical Analysis
Where indicated, statistical analysis was performed by multiple comparison analysis (Neuman-Keul’s analysis) after analysis of variance.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human arteries harvested postmortem from 20 cases were analyzed by
Northern blotting for expression of RA301/Tra2ß. Among them,
representative cases are shown in Figure 1
. Human spleen was used as a positive
control. Transcripts for RA301/Tra2ß were obviously observed in an
aorta with atheromatous plaques from a 58-year-old male who died of
cerebral hemorrhage because of hypertension and had a past history of
hypercholesterolemia (lane 8), in an atherosclerotic coronary artery of
a 63-year-old male who died of gastric cancer/hepatocellular carcinoma
and had a past history of hypertension and angina pectoris (lane 9),
and in an atherosclerotic aorta of a 69-year-old male who died of
cardioembolic brain infarction (lane 10).
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In situ hybridization and immunohistological studies were
performed to further analyze the expression of RA301/Tra2ß in the
above vascular samples. A coronary artery from a 63-year-old male
(Figure 1
, lane 10) who had a past history of hypertension and angina
pectoris was studied first (Figure 2A
displays hematoxylin and eosin (H&E) staining for orientation).
In situ hybridization with the antisense RA301/Tra2ß probe
showed the presence of RA301/Tra2ß transcripts (Figure 2, B and E)
in
proliferating smooth muscle cells based on co-localization with PCNA
(Figure 2C)
and -smooth muscle actin (Figure 2D)
. To ensure the
color reaction of known positive cells for anti-PCNA antibody, sections
of normal human colon were stained with anti-PCNA antibody or preimmune
serum (Figure 2, H and I)
(Figure 2G
displays H&E stain for
orientation.) In addition, transcripts for RA301/Tra2ß in an aorta
from a 58-year-old patient with hypertension and hypercholesterolemia
(above; Figure 1
, lane 8) were shown to be present in the intima, most
likely in smooth muscle cells (data not shown). And plasma cells,
visualized in another portion of this atherosclerotic aorta near the
vasa vasorum, showed RA301/Tra2ß mRNA, consistent with its function
as an RNA splicing factor (data not shown).
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The effect of acute arterial injury on RA301/Tra2ß expression
was studied using the rat carotid-artery balloon injury model.
Transcripts for RA301/Tra2ß were up-regulated by day 2, and remained
increased through day 7 (Figure 3A
;
arrowheads indicate the splicing variants observed in arterial injury).
Immunohistochemical analysis of instrumented rat carotid arteries
showed increased expression of RA301/Tra2ß by day 3 (Figure 3B
, panel
F; note that uninjured rat carotid artery showed no RA301/Tra2ß
staining, Figure 3B
, panel E), reaching a maximum by day 7 (Figure 3B
,
panel G), and thereafter falling off (Figure 3B
, panel H).
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;
panels J, K, and L). The localization of RA301/Tra2ß antigen within
cells appeared confined to nuclei, consistent with the results of
previous studies.8,25,26
Double staining of injured artery
(day 7) showed the overlapping of RA301 and PCNA signals (Figure 3B
,
panels N to P; H&E staining is shown in panel M for orientation).
Furthermore, the expression pattern of RA301/Tra2ß antigen paralleled
the distribution reported for cells displaying either thymidine
incorporation or BrdU uptake or PCNA immunogenicity after arterial
injury in animals.13,27-29
On day 3 after the injury
(panel F), RA301/Tra2ß antigens were scattered throughout the media
and adventitia, whereas in panels G and H (on days 7 & 14),
RA301/Tra2ß antigens were shown mainly in neointima, but partly in
media. Cells expressing RA301/Tra2ß antigens mainly consisted of
smooth muscle cells, but partly of endothelial cells on the luminal
surface and adventitial cells. In Figure 3B
, we showed intimal and
medial components of uninjured and injured arteries. Effect of Re-Oxygenation on the Induction of RA301/Tra2ß in Vascular Cells
Subconfluent VSMCs subjected to hypoxia displayed diminished cell
growth (not shown), consistent with our previous results in cultured
endothelial cells.30
However, on subsequent
re-oxygenation, there was a burst of cell proliferation (Figure 4A)
accompanied by induction of PCNA
antigen (Figure 4B)
, a well-known marker of cell
proliferation.31
In cultured VSMCs, re-oxygenation also
triggered a marked increase in RA301/Tra2ß message within 1 hour that
was sustained for up to 24 hours (Figure 4D)
and followed by an
increase in RA301/Tra2ß antigen (Figure 4C)
. In contrast to the
results shown in the injured artery, VSMCs in cultured condition
basically expressed the lower Mr variant and
re-oxygenation mainly increased the lower Mr
variant, whereas the higher Mr variant was
observed 1 to 4 hours after the re-oxygenation (Figure 4D
, indicated by
arrowheads). The expression of RA301/Tra2ß was also observed when
subconfluent cultured endothelial cells were exposed to
hypoxia/re-oxygenation, accompanied by accelerated proliferation (data
not shown). In contrast, exposure of blood-derived mononuclear
phagocytes to hypoxia/re-oxygenation failed to induce RA301/Tra2ß
antigen and had no effect on cell proliferation (not shown). These data
provided further support for a link between proliferation of vascular
cells and induction of RA301/Tra2ß. Consistent with this concept,
cultured VSMCs incubated with platelet-derived growth factor isoform BB
(PDGF-BB) under serum-free conditions demonstrated increased cell
growth (not shown)32
and induction of RA301/Tra2ß
(Figure 5)
. Addition of angiotensin II,
which causes hypertrophy of VSMCs but not proliferation,33
failed to induce RA301 antigen in cultured VSMCs (Figure 5)
.
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To investigate the mechanism of RA301 induction in cultured VSMCs
after re-oxygenation, the generation of superoxide anion
(O2-) was assessed by
chemiluminescence. Exposure of cultured VSMCs to hypoxia followed by
re-oxygenation caused the production of
O2-, which was completely
inhibited by pre-incubation of hypoxic VSMCs with DPI, an inhibitor of
NADPH oxidase (Figure 6A)
. Because DPI
also blocks nitric oxide synthase, experiments were performed in an
arginine-free condition. However, production of
O2- by re-oxygenated VSMCs was
not altered in the absence of arginine (data not shown), suggesting
involvement of an NAPDH oxidase-like enzyme. Suppression of
O2- in re-oxygenated VSMCs with
DPI also inhibited expression of RA301/Tra2ß antigen in a
dose-dependent manner (Figure 6B
, i). In contrast, the level of HSP70i
antigens was not altered by hypoxia/re-oxygenation, and DPI had no
effect on HSP70i expression (Figure 6B
, i). These data indicate that
generation of O2- in cultured
VSMCs could serve as a trigger for expression of RA301, similar to what
we observed previously in cultured astrocytes.5
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, it completely blocked the expression of
RA301/Tra2ß (Figure 6B
, ii). These data suggested that the pathway
regulating expression of RA301/Tra2ß in re-oxygenated VSMCs involved
activation of the MAP kinase cascade. Suppression of VSMC Proliferation by the Inhibition of RA301/Tra2ß Expression
To determine whether RA301/Tra2ß had a causative role in cellular proliferation associated with hypoxia/re-oxygenation, we further investigated whether its expression was suppressed by the addition of a specific RA301/Tra2ß antisense oligonucleotide.
Addition of the antisense oligonucleotide to hypoxic/re-oxygenated VSMC
cultures demonstrated dose-dependent inhibition of the RA301/Tra2ß
antigen (Figure 6B
, iv), whereas the sense oligonucleotide was without
effect at the same concentrations (Figure 6B
, iii). The apparent
specificity of antisense RA301/Tra2ß oligonucleotide was suggested by
its lack of effect on HSP70i expression (Figure 6B
, iv).
Next, we examined the relationship between proliferation of VSMCs
subject to hypoxia/re-oxygenation and generation of
O2- and activation of the MAP
kinase pathway. The same approximate concentration range of DPI and
PD98059 that suppressed RA301/Tra2ß expression in
hypoxic/re-oxygenated VSMCs also inhibited proliferation (Figure 6C
,
i). Furthermore, the effect of DPI was only observed when it was added
to hypoxic VSMC cultures before or simultaneously with re-oxygenation,
but not 60 minutes thereafter (Figure 6D
, i). Thus, for DPI to inhibit
cell proliferation, it had to be present before generation of
O2- occurred early during the
re-oxygenation period (Figure 6A)
. It is important to note that cell
lysis with extrusion of cytosolic contents, such as LDH, into the
medium did not occur during these experiments performed in the presence
of DPI and PD98059.
To specifically examine the contribution of RA301/Tra2ß expression to
VSMC proliferation in this setting, cultures subject to
hypoxia/re-oxygenation were exposed to either antisense or sense
RA301Tra2ß oligonucleotides (Figure 6D
, ii). In a dose-dependent
manner, only the antisense oligonucleotide inhibited cellular
proliferation with a maximal effect of 70% by a concentration of 50
µmol/L. To further understand the role of RA301/Tra2ß, Western blot
analysis was performed in cultured VSMCs re-oxygenated in the presence
of antisense oligonucleotide. The suppression of RA301/Tra2ß was
without effect on either cellular p42/44 MAP kinase content or
activation of c-Jun N-terminal kinase (Figure 7, A and C)
. In contrast, the amount of
activated p42/44 MAP kinase remain elevated in the presence of
antisense oligonucleotide 6 hours after re-oxygenation, although
activated p42/44 MAP kinase was not detected in the presence of sense
oligonucleotide (Figure 7B)
. Further, suppression of RA301 in culture
suppressed the re-oxygenation-mediated increase of PCNA antigen,
compared with cultures treated with sense oligonucleotide (Figure 7D)
.
These data suggest that RA301/Tra2ß participates in the proliferation
of VSMCs in the downstream of the MAP kinase pathway.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently, two human homologs of Tra2, Tra237
and
Tra2ß,25,26
have been identified. Surprisingly, RA301 is
completely identical to human Tra2ß. Furthermore, both Tra2 and
Tra2ß splice pre-mRNA in a sequence-specific manner.8
Because mechanisms of sexual differentiation are not conserved between
mammals and flies, these data suggest that human Tra2 proteins have
more general functions in RNA processing than their Drosophila
homologs. In fact, RA301/Tra2ß is associated with the stress response
to hypoxia/re-oxygenation5
and has also been shown to be
up-regulated in response to nerve injury.7
Our current study using human vascular tissues extends these observations by indicating enhanced expression of RA301/Tra2ß transcripts and antigens in VSMCs at lesional sites, especially in those cells likely to be in a proliferative mode. In human lesions, however, proliferation in VSMCs occurs infrequently.38 To further examine the role of RA301/Tra2ß in VSMC replication, we therefore applied a balloon injury model of rat carotid artery, where proliferation of VSMCs are more prominent, although this extrapolation has limitations because other mechanisms are proposed in this model distinct from those operating in human lesions.39 Although there is the growing importance of the adventitial compartment in neointimal formation of arterial injury model, we could not show the precise effect of adventitial cells on the neointimal expression of RA301/Tra2ß. But our data showing the up-regulation of RA301/Tra2ß by PDGF in cultured VSMCs might pose some suggestions as described.40
Consistent with the increase in RA301/Tra2ß antigen in injured vascular lesions, the results obtained from cultured VSMCs support this association of RA301/Tra2ß with cell growth. First, the pathway mediating increased expression of RA301/Tra2ß in hypoxia/re-oxygenation seems to result from generation of O2- and, most likely, their subsequent activation of the MAP kinase cascade. Thus, addition of DPI to hypoxic cultures before re-oxygenation (when the burst of O2- formation was observed) suppressed, in parallel, RA301/Tra2ß expression and cell proliferation. However, PD98059 acted more distally, although it was without effect on O2- production, it also blocked proliferation and RA301/Tra2ß expression. Finally, RA301/Tra2ß antisense oligonucleotide selectively suppressed expression of RA301/Tra2ß in VSMCs subject to hypoxia/re-oxygenation, and decreased proliferation and induction of PCNA antigen. Although experiments with antisense can be difficult to interpret, putting together these different lines of evidence, including the lack of effect of RA301/Tra2ß sense oligonucleotide, it seems logical to conclude that RA301/Tra2ß is likely to contribute to mechanisms leading to increased cellular proliferation during the re-oxygenation period. That RA301/Tra2ß might be more generally involved in growth of VSMCs was suggested by its up-regulation in the presence of the potent smooth muscle cell mitogen PDGF, but not by angiotensin II that induces hypertrophy not proliferation. Furthermore, increased expression of RA301/Tra2ß was observed in cultured aortic endothelial cells, which also demonstrate a proliferative response to hypoxia/re-oxygenation, but not in mononuclear phagocytes, which do not proliferate under these conditions.
Taken together, these data lead us to propose that RA301/Tra2ß may be a common denominator of cellular proliferation in a variety of settings, especially those associated with vascular disease. Although considerable additional studies will be required to gain insight into underlying mechanisms and to extrapolate these data in vivo, it is possible that RA301/Tra2ß represents a new target for control of vascular cell proliferation in vivo.
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Acknowledgements |
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Footnotes |
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Supported in part by the Research Grants for Cardiovascular diseases (10C-1) from the Ministry of Health and Welfare, Japan.
Accepted for publication February 6, 2001.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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