1 Department of Pathology, Medical School, University of Manchester and Christie
Hospital, Manchester M13 9PT, UK
2 Department of Biological Sciences, Manchester Metropolitan University,
Manchester, UK
3 University of Manchester Academic Unit of Obstetrics and Gynaecology, St
Mary's Hospital, Manchester M13 0JH, UK
4 Institute Reina Sofia de Investigacion Nefrologica, Department of Physiology
and Pharmacology, Salamanca University, Salamanca, Spain
5 Department of Immunology, Centro de Investigaciones Biologicas, CSIC, 28006
Madrid, Spain
* Author for correspondence (e-mail: mddpscl2{at}fs1.scg.man.ac.uk)
Accepted 7 March 2003
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Summary |
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Key words: Hypoxia, CD105, Endothelial cells, TGFß1, Apoptosis
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Introduction |
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Hypoxia is a common feature of tumour and ischaemic tissues
(Semenza, 2000). If the
cellular oxygen concentration fails to match the requirements of energy
metabolism, a number of genes are triggered to respond to the hypoxic
environment. Hypoxia-inducible genes promote cell survival by expediting
oxygen delivery to the oxygen-deprived tissues (e.g. erythropoietin)
(Wanner et al., 2000
), by
increasing glucose transport (e.g. glucose transporter-1)
(Ebert et al., 1995
), by
raising the levels of glycolytic enzymes (e.g. lactate dehydrogenase A)
(Kambe et al., 1998
) and, most
importantly, by promoting angiogenesis
(Marti and Risau, 1999
;
Yue and Tomanek, 1999
). A
number of angiogenic factors, such as vascular endothelial growth factor
(VEGF) (Mukhopadhyay et al.,
1995
; Schweiki et al.,
1992
), angiopoietin-2
(Mandriota et al., 2000
),
erythropoietin (Huang et al.,
1997
) and fibroblast growth factor
(Sakaki et al., 1995
) are
induced by oxygen-deprivation to facilitate angiogenesis.
Considering the pro-angiogenic function of CD105, we speculated that there might be an association between oxygen deprivation and CD105 gene expression. Should such a relationship exist, it would not only explain the augmented expression of CD105 in tumours and ischaemic tissues but would also have therapeutic implications for many angiogenic diseases. Furthermore, elucidating the function of CD105 under hypoxic stress would improve our understanding of the pathogenesis of several vascular diseases in which CD105 is implicated.
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Materials and Methods |
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To suppress CD105 expression in HDMECs, an antisense approach was applied
as described previously (Li et al.,
2000a). Briefly, the 16-mer antisense phosphorothioate-modified
oligodeoxynucleotide (AS ODN: 5'-ATGCTGTCCACGTGGG-3') and the
scrambled control ODN with the same base composition (SC ODN:
5'-ACTCGTGC-TACGGTGG-3') were synthesised (Applied Biosystems,
Foster City, CA). Subconfluent HDMECs grown in 35 mm petri dishes were
transfected with 0.5 nM/ml of AS or SC ODN plus 8 µg/ml of DMRIE-C
(Invitrogen) in serum-free medium. After overnight incubation, the medium was
replenished with complete growth medium, and incubation was continued for an
additional 48 hours in normoxia. Thereafter the medium was replaced with fresh
complete growth medium, and the cultures were exposed to hypoxia for up to 24
hours.
Analysis of CD105 protein expression
Cell surface expression of CD105 protein in AS-, SC-treated or untreated
cells was quantified by flow cytometry as described previously
(Li et al., 2000a). Briefly,
105 cells per tube were incubated with 50 µl (10 µg/ml in
PBS) of anti-CD105 mAb E9 or pre-immunised mouse serum as a negative control
antibody (10 µg/ml in PBS) on ice for 1 hour and washed twice with cold
PBS. mAb 44G4 to human CD105 (Gougos and
Letarte, 1988
) was also employed for comparison with mAb E9. After
incubation with a fluorescein-labelled
F(a
)2 fragment of rabbit
anti-mouse antibody (1/40; DAKO, Denmark) for 30 minutes on ice, the cells
were washed and resuspended in 0.3 ml of 2% buffered formalin and analysed on
a Becton Dickinson FACScan flow cytometer.
For analysis of CD105 protein by immunoblotting, CD105 protein was
extracted from HDMECs by solubilising 1x107 cells/ml with a
cocktail buffer [0.2% (v/v) Nonidet P-40 in 0.1 M Tris buffer (pH 7.3), 0.5 M
PMSF, 1 mM pepstatin, 0.1 mM leupeptin, 1 mM EDTA] and resolved on 4-7.5%
(w/v) sodium dodecyl sulphate (SDS)-polyacrylamide gel and electrophoretically
transferred onto a PVDF membrane (Millipore)
(Li et al., 2000a). mAb E9
(1:1000; 0.5 µg/ml) in blocking solution was applied to detect CD105
protein, and filters were incubated overnight at 4°C. Finally, the blots
were incubated with horseradish peroxidase (HRP)-conjugated rabbit anti-mouse
IgG (DAKO) for 2 hours at 4°C with shaking. The CD105 protein was
visualised using the enhanced chemiluminescence system (Amersham Biosciences,
UK). As an internal loading control, the blot was stripped and reprobed using
a goat anti-
-actin (Sigma) or mouse anti-CD31 antibody (DAKO) and
detected by HRP-conjugated secondary antibodies. CD105 shed into the medium
was detected by a chemiluminescent ELISA system as described previously
(Wang et al., 1995
;
Li et al., 2000c
).
Analysis of CD105 mRNA by northern blotting
Total RNA from HDMECs was extracted using guanidinium
thiocyanate/phenol/chloroform (Chomczynski
and Sacchi, 1987). RNA samples (15 µg) were denatured and
fractionated in 1% (w/v) agarose/2.8% (v/v) formaldehyde gel and blotted onto
nitrocellulose. The CD105 probe used was a 2.3 kb fragment excised with
EcoRI from the pcEXV-EndoL plasmid
(Lastres et al., 1996
) and
labelled with 32P by random prime labelling. The blot was
hybridised with the 32P-labelled probe and the hybridisation was
revealed by a phosphorimager (Molecular Dynamics), then analysed using Image
Quant software. The blots were rehybridised with 32P-labelled probe
for GAPDH for use as a loading control.
Luciferase reporter gene assay
To determine the level of CD105 promoter activity, plasmid pXP2 harbouring
the 2.8 kb (-2450/+350) CD105 promoter
(Rius et al., 1998) and a
downstream firefly luciferase gene was used for transient transfection of
HDMECs. Internal normalisation was performed by cotransfection of the pXP2
plasmid with CMVßgal, a ß-galactosidase expression vector driven by
the cytomegalovirus (CMV) promoter. Transfection of HDMECs was carried out
using the liposome-mediated gene transfer technique. Briefly, cells were
seeded at 1x105 cells per 35 mm dish and the following day
were transfected with 8 µg/ml of DMRIE-C (Invitrogen) plus 1 µg of
plasmid CMVßgal mixed with 2 µg of pXP2 in serum-free medium.
Twenty-four hours after transfection, the cultures were replenished with
complete medium and placed in hypoxic or normoxic conditions for the
designated time periods. Thereafter, the cells were harvested and the
enzymatic activity determined. Luciferase and ß-galactosidase activities
were measured on a TD-20/20 Luminometer (Promega, WI) and a colorimetric plate
reader (Labsystems), respectively, using kits from Promega (UK).
Cell cycle analysis
Cell cycle distribution was evaluated by propidium iodide staining of
nuclei and flow cytometric analysis (Li et
al., 1997). 1x106 cells were fixed with 2 ml cold
70% (v/v) ethanol in PBS, immediately mixed and slowly agitated for 15 minutes
at room temperature. After two washes with PBS, 0.7 ml of 0.2 mg/ml pepsin
(Sigma) in 2 M HCl was added to the pelleted cells for simultaneous
proteolysis and DNA denaturation at 37°C for 30 minutes. The hydrolysis
was terminated by addition of 2 ml of 1 M Tris (pH 10). Cells were washed
twice and incubated in 0.3 ml PBS containing 10 µg/ml propidium iodide for
15 minutes on ice. To determine the proportion of cells in various phases of
the cell cycle, propidium iodide staining of the DNA in 2x104
nuclei was quantified using a Becton Dickinson FACScan flow cytometer.
Analysis of apoptosis
The terminal deoxynucleotidyltransferase-mediated dUTP end labelling
(TUNEL) assay was used to identify cell apoptosis. Cells were seeded onto
chamber slides (Nunc) at a density of 2x104 cells per 0.5 ml
per well, grown overnight and subjected to hypoxic or normoxic conditions.
Fragmented DNA staining of apoptotic cells was carried out using a commercial
kit (Roche, Mannheim, Germany). Briefly, the cells were rinsed twice with
pre-warmed PBS followed by fixation using 4% paraformaldehyde for 1 hour at
room temperature. The fixed cells were permeabilised by incubation with 0.1%
Triton X-100 in 0.1% sodium citrate for 2 minutes. The cells were rinsed again
with PBS and incubated with 50 µl per sample of TUNEL reaction mixture for
1 hour at 37°C. A negative control was included in each staining wherein
only the labelling solution was added.
TUNEL staining of cells for FACS analysis was carried out using the same kit as described above. The cells were fixed with a freshly prepared paraformaldehyde solution (4% in PBS, pH 7.4) for 1 hour at room temperature. After one wash with PBS, cells were permeabilised using 0.1% Triton X-100 in 0.1% sodium citrate for 2 minutes on ice, followed by two washes with PBS. The permeabilised cells were stained with 50 µl/sample of the TUNEL reaction mixture containing the enzyme or the labelling solution without the enzyme as a negative control and incubated for 1 hour at 37°C in a humidified chamber in the dark. Finally, the cells were washed twice with PBS and resuspended in 0.5 ml PBS for FACS analysis. For quantification of anti- and pro-apoptotic proteins, cells were harvested, fixed and permeabilised as described above. Antibodies to Bcl-2, Bcl-XL, Bax, Mcl-1, caspase-3 and caspase-8 (BD Biosciences) were incubated with 5x105 cells/tube for 1 hour on ice. Pre-immunised rabbit serum or isotype matched mouse immunoglobulins were used as negative controls. The FITC-conjugated secondary antibodies (DAKO) were added and incubated for 30 minutes on ice. Flow cytometric analysis was carried out on a FACScan.
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Results |
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CD105 levels in the blood of patients with breast cancer and those with
atherosclerosis are elevated and associated with disease progression. Thus
CD105 could be a marker for certain clinical conditions
(Blann et al., 1996;
Li et al., 2000c
). In the
present study, CD105 levels were quantified in conditioned medium collected
from cells used for protein and mRNA analysis. The levels of secreted CD105
were increased after 1 hour of oxygen deprivation, peaked at 16 hours in
hypoxic cultures and remained higher than controls at 24 hours
(Fig. 1C). However the
biological implications of the increase in CD105 are unknown. The profile of
secreted CD105 in the oxygen-deprived medium was comparable to cell surface
CD105 observed by flow cytometry and total CD105 estimated by immunoblotting
analysis.
Hypoxia upregulates CD105 mRNA levels and promoter activity
To analyse whether the levels of CD105 transcripts were also affected by
hypoxia, total RNA was fractionated and hybridised with radiolabelled CD105
and GAPDH cDNAs, visualised on a phosphorimager and quantified using a
densitometer. Hypoxia elevated CD105 mRNA levels, which was evident even after
1 hour, compared with normoxic culture
(Fig. 2A). The highest
expression of CD105 mRNA (a threefold increase) occurred at 3 hours. The
relatively weak signal for CD105 mRNA after 24 hours of hypoxic culture was
considered to reflect hypoxia-induced cell cycle arrest and apoptosis, which
was confirmed by propidium iodide staining and a TUNEL assay, as described
below. These low levels of CD105 mRNA were concomitant with high expression
levels of CD105 protein (Fig.
1), in agreement with the high stability of the protein
(Paquet et al., 2001
). To
assess whether the increased CD105 expression in hypoxic conditions was due to
an increased promoter activity, CD105 promoter activity was examined using a
luciferase reporter assay. CD105 promoter activity was significantly induced
by hypoxia, reaching a maximal level after 3-6 hours of hypoxic culture
(Fig. 2B). Longer durations of
hypoxia (more than 16 hours) led to basal levels of the promoter activity,
probably owing to cell cycle arrest and apoptosis.
|
Hypoxia induces cell cycle arrest and apoptosis in HDMECs
Since persistent hypoxia induces alterations in cell cycle and apoptosis in
certain cell types (Carmeliet et al.,
1998), an analysis of cell cycle and apoptosis was performed in
hypoxic HDMECs. Prolonged exposure to hypoxia (24 hours) led to a dramatic
alteration in cell cycle, that is, more cells arrested at the G0/G1 phases and
fewer cells undergoing DNA synthesis (S phase). Moreover, DNA fragmentation
was observed in a considerable proportion of cells (approximately 21% of the
total population blue profile) after 24 hours of hypoxic culture,
implying massive cell apoptosis (Fig.
3A). A TUNEL assay was carried out in parallel on the same batch
of cells to identify cells with fragmented DNA. Under normoxic conditions, few
apoptotic cells were observed. In contrast, in cells grown under hypoxic
conditions DNA fragmentation occurred in a time-dependent manner: prolonged
exposure to oxygen deprivation induced substantial cell apoptosis
(Fig. 3B). The highest
proportion of apoptotic cells was detected after 24 hours of hypoxic culture,
which is consistent with the data obtained by propidium iodide staining.
|
Hypoxia alters expression of pro- and anti-apoptotic markers
To investigate the mechanisms by which hypoxia induces cell apoptosis, the
protein expression of the known anti-apoptotic markers Bcl-2,
Bcl-XL, Mcl-1 and the pro-apoptotic markers Bax, caspase-3 and
caspase-8 were quantified in cells under normoxia or hypoxia for 24 hours. As
shown in Fig. 4,
Bcl-XL and Mcl-1 were significantly suppressed, Bax and Caspase-3
were markedly raised and caspase-8 slightly raised in hypoxia. Although Bcl-2
was elevated in hypoxia, the ratio of Bcl-2 to Bax was lowered from 1.36 in
normoxia to 0.79 in hypoxia. These data suggest that hypoxia induces cell
apoptosis through the downregulation of Bcl-XL and Mcl-1,
upregulation of Bax, caspase-3 and caspase-8 and the lowering of the Bcl-2 to
Bax ratio.
|
Suppression of CD105 gene expression using an antisense approach
To investigate the role of CD105 in HDMECs, CD105 gene expression was
specifically inhibited using an antisense approach. There have been numerous
reports in the literature using the same experimental approach, and the method
of transfection is mild and well-tolerated. Thus, examining the effect of
hypoxia on the transfected cells is considered to be appropriate. In
comparison with HUVECs, in which CD105 was significantly reduced using 0.25
nM/ml of AS ODN plus 5.6 µg/ml of lipofectACE (Invitrogen)
(Li et al., 2000a), HDMECs
were less responsive to such a formulation. However, the efficacy was improved
by using an increased concentration of AS ODN (0.5 nM/ml) plus 8 µg/ml of
DMRIE-C (Invitrogen). FACS and immunoblotting analysis revealed a 55-60%
reduction in CD105 protein levels in the AS-ODN-treated cells after 72 hours
of incubation in normoxia and 24 hours exposure to hypoxia, compared with
SC-ODN-treated (0.5 nM/ml plus 8 µg/ml DMRIE-C) or untreated cells under
the same conditions (Fig.
5A,B). To investigate whether CD105 mRNA was altered by ODN
treatment, northern blotting was carried out. As depicted in
Fig. 5C, CD105 mRNA was
decreased by AS but not by the SC ODN, demonstrating that specific degradation
of CD105 mRNA was one of the mechanisms involved in the antisense effect. The
specificity of the antisense effect was further verified by quantifying the
expression of CD31, vWF, TGFß receptor I and receptor II in the untreated
HDMECs or HDMECs treated with either AS or SC ODN. As determined by FACS and
immunoblotting, these cell membrane proteins were not affected by the ODN
treatment, whereas CD105 protein was significantly reduced by the AS ODN (data
not shown). The selective inhibition of CD105 gene expression by the AS ODN
can also be seen in Fig. 5,
where CD105 but not CD31 protein (A), CD105 but not GAPDH mRNA (C) were
markedly decreased by the AS ODN.
|
Inhibition of CD105 gene expression heightens hypoxia-induced cell
apoptosis
Hypoxia stimulates angiogenesis by upregulating expression of angiogenic
factors but causes apoptosis as well. A balance between the two forces
determines the fate of the cell. To elucidate whether upregulation of CD105 in
HDMECs under hypoxic stress is of functional importance, we quantified cell
apoptosis of the AS-ODN-treated, SC-ODN-treated or untreated HDMECs. Exposure
of untreated cells to hypoxic stress for 24 hours elicited a significant cell
apoptosis, 48.5% under hypoxia in contrast to 4.3% under normoxia. Hypoxia
induced a maximal cell apoptosis in the CD105-depressed out of the three
groups of cells cultured in complete growth medium without exogenously added
TGFß1 or TGFß3. Thus, the geometric mean fluorescence intensity was
139.1±13.3 in untreated, 150.7±9.0 in SC-ODN-treated and
193.1±10.6 in AS-ODN-treated cells (P<0.05, AS-ODN-treated,
compared with either SC-ODN-treated or untreated cells)
(Fig. 6A). The percentage of
apoptotic cells correlates well with the geometric mean fluorescence
intensity, which was 66.5% in CD105-depressed compared with 48.5% in untreated
or 48.2% in SC-ODN-treated cells (P<0.05)
(Fig. 6B). Administration of
the neutralising antibodies to TGFß1 and TGFß3 produced no
significant alteration either in the percentage of apoptotic cells or in their
fluorescence intensity, demonstrating that CD105 is able to act independently
of TGFß in preventing cell apoptosis under hypoxic stress.
|
Hypoxia and TGFß1 synergistically augment cell apoptosis in
CD105-depressed cells
CD105 regulate TGFß1 signalling in endothelial cells and in CD105
transfectants (Lastres et al.,
1996; Letamendia et al.,
1998
; Li et al.,
2000a
) but how CD105 and TGFß1 interact under hypoxic
conditions to control cell apoptosis has not been examined. Therefore we
investigated this issue using the AS-ODN-treated (CD105-deficient) cells, the
SC-ODN-treated and untreated cells. As shown in
Fig. 6, TGFß1 induced a
marginal increase in apoptotic cells in the SC-ODN-treated and the untreated
cells. In contrast, it caused a considerable increase in apoptotic cells in
the CD105-depressed cells. The fluorescence intensity of CD105-deficient cells
was 193.1±10.6 in the absence of TGFß1, 229.7±10.7 at 0.1
ng/ml of TGFß1, and 298.2±11.5 at 10 ng/ml of TGFß1, in
contrast to 150.7±9.0, 157.9±8.7 and 175.6±10.8,
respectively, in SC-ODN-treated cells (P<0.05 or
P<0.01, significant difference between the two groups at each same
concentration of TGFß1) (Fig.
6A). This represents 28.1%, 45.5% and 69.8% increases in
fluorescence intensity in the CD105-deficient cells compared with
SC-ODN-treated cells at the same concentrations of TGFß1. A higher
proportion of apoptotic cells was observed in the CD105-deficient cells
compared with either the SC-ODN-treated or untreated cells at the same
concentration of TGFß1 (Fig.
6B). The percentage of apoptotic cells increased from
48.2±2.6, 51.3±3.0 and 56.9±3.0 in SC-ODN-treated cells
at 0, 0.1 and 10 ng/ml of TGFß1 to 66.5±2.3, 75.7±2.6 and
87.4±2.5 in the AS-ODN-treated cells at the same concentrations of
TGFß1 (P<0.05 or P<0.01, significant difference
between the two groups at each same concentration of TGFß1). These data
demonstrate that CD105 functions as an anti-apoptotic protein in the presence
of TGFß1 under hypoxic stress.
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Discussion |
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The notion that CD105 is associated with angiogenesis initially came from
the observation that a mAb to CD105 reacts most strongly with the endothelium
of tumours but only weakly or not at all with normal tissues
(Wang et al., 1993).
Subsequently, numerous studies using mAbs to CD105 on a broad range of tissues
have provided supportive evidence that CD105 is indeed upregulated in many
types of tissues undergoing angiogenesis
(Kumar et al., 1996
;
Miller et al., 1999
;
Seon and Kumar, 2001
;
Wikstrom et al., 2002
). The
expression of CD105 in blood vessels of breast and lung cancer tissues was
found to be correlated with poor prognosis
(Kumar et al., 1999
;
Tanaka et al., 2001
), which
suggests that CD105 promotes tumour progression. A conclusive demonstration of
the crucial role of CD105 in vascular development came from CD105-knockout
mice, which had severe defects in angiogenesis; the homozygotes died in utero
owing to impaired development of vasculature
(Arthur et al., 2000
;
Bourdeau et al., 1999
;
Li et al., 1999
). In line with
these observations, CD105 expression correlates with activation/proliferation
of tumour endothelial cells (Kumar et al.,
1999
; Miller et al.,
1999
). These findings provide compelling evidence that CD105 is
important for angiogenesis. However, the underlying mechanism of how CD105
promotes angiogenesis is not clear. Existing data support the view that CD105
overexpression in transfectants weakens the effects of TGFß1 and that
these effects might be cell type dependent
(Lastres et al., 1996
;
Letamendia et al., 1998
). In
addition, suppression of CD105 in ECs with antisense ODN in combination with
TGFß1 led to a strong inhibition of angiogenesis
(Li et al., 2000a
). These
observations indicate that CD105 modulates TGFß1 signalling in these
cells and that an adequate level of CD105 in ECs is required for
angiogenesis.
The expression of CD105 is likely to be regulated by factors involved in
angiogenesis and/or vessel remodelling. Here we have demonstrated that CD105
is markedly induced in ECs by exposure to hypoxia. CD105 promoter activity and
mRNA transcription responded rapidly to hypoxia, which suggests that it is
regulated at the transcriptional level. In this regard, a consensus hypoxia
responsive element (HRE) has been recently characterized within the CD105
promoter (Sanchez-Elsner et al.,
2002). The CD105 protein was maintained at high levels until the
end of the experiment. The half-life of CD105 on the cell surface has been
estimated to be 17 hours, measured by metabolic labelling
(Paquet et al., 2001
). Thus,
once it is upregulated, CD105 can remain in the EC, exhibiting a prolonged
effect. The relatively low promoter activity and mRNA level after 24 hours of
hypoxic culture is apparently a consequence of hypoxia-induced cell cycle
arrest and apoptosis. This is in line with previous observations that CD105 is
more strongly expressed in activated/mitotic cells than quiescent cells,
suggesting that it is a proliferation-associated gene. These findings also
highlight the potential value of the CD105 promoter for gene therapy in cancer
and ischaemic diseases (Brekken et al.,
2002
; Velasco et al.,
2001
). Genes driven by the CD105 promoter may be more strongly and
specifically expressed in the oxygen-deprived tissues and exert therapeutic
effects.
Hypoxia is the primary driving force for neovascularisation. The major
angiogenic factors, such as VEGF and bFGF, are upregulated by hypoxia, and
promote angiogenesis and thus improves oxygen supply to the hypoxic tissues
(Mukhopadhyay et al., 1995;
Sakaki et al., 1995
;
Schweiki et al., 1992
). On the
other hand, persistent hypoxic stress induces EC apoptosis
(Carmeliet et al., 1998
;
Hogg et al., 1999
). In this
study we have shown that the pro-apoptotic proteins Bax, caspase-3 and
caspase-8 were elevated and that the anti-apoptotic proteins Bcl-XL
and Mcl-1 were significantly decreased under hypoxic stress. Although
anti-apoptotic Bcl-2 was upregulated, the ratio of Bcl-2 to Bax was
considerably lowered under hypoxic stress compared with normoxia. Therefore,
the mechanisms of hypoxia-induced EC apoptosis largely depend on the lowered
Bcl-2 to Bax ratio, Bcl-XL and Mcl-1 and increased expression of
caspase-3 and caspase-8. These findings are in agreement with previous reports
(Khurana et al., 2002
;
Taraseviciene-Stewart et al.,
2001
; Wang et al.,
2002
).
Because CD105 is strongly expressed in the HDMECs under hypoxic conditions,
we adopted an antisense approach to suppress CD105 expression so that its
function could be specifically addressed. The CD105 protein and mRNA levels
were considerably reduced by the antisense ODN but not affected by the control
SC ODN. Using the CD105-deficient cells and the control cells, an important
function of CD105 has been discovered, which is that it acts as an
anti-apoptotic protein in ECs under hypoxic stress. Such an effect was
observed in the absence of TGFß1 and TGFß3, indicating that CD105
functions beyond its role as a receptor for TGFß1 and TGFß3. In
fact, only approximately 1% of membrane-bound CD105 binds to TGFß1 and
TGFß3 (Cheifetz et al.,
1992). The function of the majority of CD105 that does not bind to
TGFß remains unclear. Our data suggest that the non-TGFß-binding
CD105 in EC plays a self-protective role against apoptotic factors such as
hypoxia. The protective role of CD105 against apoptosis has been further
confirmed using a completely different system murine endothelial cells
from CD105 heterozygous knockout mice (J.M.L.-N., C.L., S.K. et al.,
unpublished). The addition of TGFß1 to control cells under hypoxia
induced a marginal increase in the proportion of apoptotic cells, whereas the
apoptotic action of TGFß1 was considerably increased in the
CD105-depressed cells, demonstrating that the upregulation of CD105 protects
ECs from the apoptotic action of TGFß1. In an in vivo environment, where
both hypoxia and TGFß1 can co-exist, the augmented expression of CD105
may act as an anti-apoptotic force, so as to protect ECs against hypoxia and
against TGFß1-induced apoptosis.
In conclusion, we have demonstrated that hypoxia activates the CD105 promoter and significantly induces its gene expression in human microvascular ECs. The upregulated CD105 exhibits a role in self-protection against hypoxia and TGFß1-induced cell apoptosis, resulting in an enhanced survival ability of EC under hypoxic stress. These findings may lead us to a better understanding of the functions of CD105 in angiogenesis and of the pathogenesis of other CD105-related vascular disorders.
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Acknowledgments |
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