(Received for publication, October 24, 1994; and in revised form, August 31, 1995)
From the
Hypoxia is the principal factor that causes angiogenesis. These
experiments were conducted to explore how it induces the proliferation
of vascular cells, a key step in angiogenesis. Human umbilical vein
endothelial cells and bovine retinal pericytes were grown in controlled
atmosphere culture chambers containing various concentrations of
oxygen. The numbers of both endothelial cells and pericytes increased
significantly under hypoxic conditions; the O concentrations that achieved maximal growth promotion were 10%
for endothelial cells and 2.5% for pericytes. Quantitative reverse
transcription-polymerase chain reaction analysis revealed that mRNAs
coding for the secretory forms of vascular endothelial growth factor
(VEGF), a mitogen specific to endothelial cells, were present in both
endothelial cells and pericytes and that their levels increased
significantly in the two cell types as the atmospheric O
concentration decreased. The two genes for VEGF receptors, kinase
insert domain-containing receptor (kdr) and fms-like
tyrosine kinase 1 (flt1), were found to be constitutively
expressed in endothelial cells, and their relative mRNA levels were
ranked in that order. On the other hand, only flt1 mRNA was
detected in pericytes under hypoxic conditions. Furthermore, most
antisense oligodeoxyribonucleotides complementary to VEGF mRNAs
efficiently inhibited DNA synthesis in endothelial cells cultured under
hypoxic conditions. These results indicate that autocrine and paracrine
VEGFs may take part in the hypoxia-induced proliferation of endothelial
cells.
Angiogenesis is a process by which new vascular networks are formed from pre-existing capillaries(1) . Physiologically, it is essential for embryogenesis, development, ovulation, corpus luteum formation, and wound repair. In addition, it occurs during the progression of various pathological conditions such as cancers, diabetic retinopathy, rheumatoid arthritis, and occlusive vascular diseases, e.g. neovascularization is needed by solid tumors to access sufficient nutrients and oxygen for growth(1, 2) . To determine how angiogenesis is induced under these circumstances is therefore important for clarifying the pathogenesis, prevention, and treatment of such diseases as well as for understanding the basis of the physiological processes involved.
A decrease in tissue oxygen concentrations has been considered as the leading cause of angiogenesis(3) . However, the mechanisms underlying the induction of angiogenesis by hypoxia are still poorly understood. Using primary cultured vascular cells, we have been investigating the biochemical basis underlying various vascular functions and disturbances(4, 5, 6, 7) . In this study, we employed a hypoxic culture system and examined how low oxygen tensions affect the proliferation of endothelial cells and pericytes, a key step in angiogenesis; the latter cell type is the microvascular constituent encircling the endothelium, which we showed plays important roles in the growth, function, and damage of endothelial cells(4, 5) . The first part of this paper describes the accelerated growth of both endothelial cells and pericytes caused by hypoxia.
The following parts of this paper deal with the
molecular mechanism underlying the hypoxia-induced proliferation of the
vascular cells. Recently, there has been an explosive growth in
knowledge regarding angiogenic growth
factors(8, 9, 10) , including vascular
endothelial growth factor (VEGF), ()an endothelial
cell-specific mitogen. This factor was initially identified in the
conditioned medium of bovine pituitary follicular stellate cells (11, 12) , and its expression was subsequently shown
to increase in human gliomas under hypoxic
conditions(13, 14) . The presence of VEGF in vascular
cells, including endothelial cells(15) , smooth muscle
cells(16) , and mesangial cells(17) , has also been
noted. Here we show that endothelial cells and pericytes per se can produce secretory forms of VEGF in response to hypoxia. We
also determined the VEGF receptor subtypes expressed in these two cell
types. Furthermore, a functional relationship between the vascular VEGF
system and hypoxia-driven endothelial cell growth was tested by
manipulating VEGF gene expression with antisense DNA.
Figure 1:
Effect of hypoxia on the growth of
endothelial cells (A) and pericytes (B). The number
of viable cells is indicated on the ordinate. The culture
period after cell attachment is indicated on the abscissa.
O tension was changed on day 0 from 20% to the
concentrations indicated. Each point represents the mean for triplicate
experiments; vertical bars show standard deviation when larger
than the symbol. *, p < 0.05;**, p < 0.01
compared with the number of cells cultured under 20% O
(Student's t test).
, 20%;
, 10%;
, 5%;
, 2.5% O
.
Figure 2:
Quantitative RT-PCR analysis of VEGF
mRNAs. A, schematic representation of four alternative
splicing products of the human VEGF gene. Boxes indicate open
reading frames. Arrows indicate primers for RT-PCR
amplification. Bars indicate probes for Southern
hybridization. Expected sizes of RT-PCR products are indicated on the right. B and C, titration curves of RT-PCR products.
Poly(A) RNA from U251 cells was used as the template.
Signal intensities of RT-PCR products are expressed as arbitrary
logarithm values of radioactivities and plotted against template
amounts (B) and against cycle numbers (C).
Radioactivities were measured with a Fujix BA100 BioImage analyzer.
, VEGF;
,
-actin. D, RT-PCR analysis of VEGF
mRNA. Thirty nanograms of poly(A)
RNAs from
endothelial cells (EC), pericytes, and U251 cells, which had
been incubated under various oxygen tensions, underwent quantitative
RT-PCR analysis. The products were electrophoresed on 2% agarose gel,
transferred onto nylon membranes, and hybridized with
P-end-labeled probes specific to VEGF (upper
panels) and
-actin (lower panels) mRNAs. PCR
amplification for the latter was performed for 15 cycles. Bars indicate size markers in base pairs. Endothelial cell and pericyte
blots were exposed for 12 h, and the U251 blot for 6 h. RT(-), the reaction without reverse
transcriptase.
Fig. 2(B and C) shows titration curves of
RT-PCR products for determining the quantitative range in which the
reactions proceeded exponentially. Poly(A) RNA from
U251 cells, a human glioma cell line, was used as a standard template;
this cell line has been known to produce high amounts of
VEGF(13, 14) . Signal intensities of the products
obtained with U251 poly(A)
RNA were plotted as
functions of template amount and cycle number. The products increased
linearly up to 50 ng (Fig. 2B) and up to 30 cycles (Fig. 2C); hence, we chose 30-ng templates and 25
cycles as the conditions for VEGF mRNA analysis.
As shown in Fig. 2D, poly(A) RNAs from endothelial
cells and pericytes gave signals at 486 and 618 bp, which corresponded
to mRNAs for VEGF
and VEGF
, respectively,
as did U251 poly(A)
RNA. The levels of the two mRNA
species increased significantly in these cells as the atmospheric
O
concentration decreased from 20 to 0%. The sum of the
486- and 618-bp band intensities was strongest in anoxic cultures; in
endothelial cells and pericytes, it was 8- and 9-fold higher than
respective normoxic cultures when standardized with the signal
intensities of
-actin mRNA as an internal control. On the other
hand, signals for VEGF
and VEGF
mRNAs were
not detected in either the vascular cells or the glioma cells
throughout these experiments.
Figure 3:
Quantitative RT-PCR analysis of VEGF
receptor mRNAs. A, schematic representation of human VEGF
receptor mRNAs. Boxes indicate open reading frames. Arrows and bars indicate primers and probes, respectively, for flt1, kdr, and flt4 mRNA detections.
Expected sizes of RT-PCR products are indicated on the right.
B, specific detection of flt1, kdr, and flt4 mRNAs. RT-PCR products yielded with each set of primers were
specifically recognized by the corresponding probe. Numbers on
the left indicate lengths of the products in base pairs. C and D, titration curves of RT-PCR products.
Poly(A) RNA from endothelial cells was used as the
template. Signal intensities of RT-PCR products are expressed as the
arbitrary logarithm values of radioactivities and plotted against
template amounts (C) and against cycle numbers (D).
Radioactivities of hybridization bands were measured with a Fujix BA100
BioImage analyzer.
, flt1;
, kdr;
,
-actin. E, RT-PCR analysis of VEGF receptor
mRNAs in endothelial cells. Thirty nanograms of poly(A)
RNA from endothelial cells incubated under the indicated O
concentrations was amplified by RT-PCR and hybridized with
P-end-labeled probes specific to flt1 and kdr (upper panels) and
-actin (lower panels)
mRNAs. PCR amplification for the latter was performed for 15 cycles.
The flt1 blot was exposed for 32 h, and the kdr blot
for 1 h. RT(-), the reaction without reverse
transcriptase. F, RT-PCR analysis of VEGF receptor mRNAs in
pericytes. Thirty nanograms of poly(A)
RNA from
pericytes incubated under the indicated O
concentrations
was amplified by RT-PCR and hybridized with
P-end-labeled
probes specific to flt1 (upper panel) and
-actin (lower panel) mRNAs. PCR amplification for the latter was
performed for 15 cycles. The film was exposed for 7
days.
As shown in Fig. 3E, endothelial cells were found to contain mRNAs
for flt1 and kdr. Between the two, the intensities of
the hybridization signals were much stronger for kdr mRNA than
for flt1 mRNA. In contrast with VEGF mRNAs, the levels of kdr and flt1 mRNAs were essentially unchanged when
atmospheric O tensions were lowered.
On the other hand,
in pericytes, neither of the two mRNA species was detected under
normoxic conditions. However, as shown in Fig. 3F, the
1098-bp band corresponding to flt1 mRNA was visible in
pericytes grown at 2.5% O and became clearly marked at 0%. kdr mRNA remained undetected at 2.5 and 0% O
even
when the template amount and the cycle number were raised to 100 ng and
35 cycles, respectively (data not shown).
Figure 4:
Effect
of antisense oligodeoxyribonucleotides on DNA synthesis in endothelial
cells. A, dose dependence. After endothelial cells had been
cultured for 24 h in the presence of the indicated concentrations of
antisense (hatched columns) or sense (black columns)
oligodeoxyribonucleotides, [H]thymidine was added
to a final concentration of 1 µCi/ml, and the cells were further
cultured for 4 h. The culture was carried out under 10% O
in RPMI 1640/Medium 199 (1:1) supplemented with 15% FBS, but
without endothelial cell growth supplement or heparin.
H
radioactivity incorporated is expressed as disintegrations/minute. Columns represent the means of triplicate experiments. Bars indicate standard deviations.**, p < 0.01
compared with the value for the culture without
oligodeoxyribonucleotides (Student's t test). The
control without oligodeoxyribonucleotides is also shown (open
column). B and C, time courses under 10 and 20%
O
, respectively. Four hours after the addition of
[
H]thymidine, 10 µM oligodeoxyribonucleotide was administered, and
H
radioactivity incorporation into endothelial cell DNA was assayed at
the indicated time points. Each point represents the mean of triplicate
experiments. Vertical bars show standard deviation when larger
than the symbol.**, p < 0.01 compared with the value
without oligodeoxyribonucleotides (Student's t test).
and
, cumulative DNA synthesis without
oligodeoxyribonucleotides;
and
, with antisense
oligodeoxyribonucleotides;
and
, with sense
oligodeoxyribonucleotides.
Fig. 4B shows the time course of endothelial cell
synthesis of DNA in response to the antisense oligodeoxyribonucleotide
under 10% O. DNA synthesis began to decrease as early as 4
h after the oligodeoxyribonucleotide administration. The inhibition of
DNA synthesis with similar kinetics was also noted under 20% O
(Fig. 4C).
Figure 5:
Effect of antisense
oligodeoxyribonucleotides on VEGF synthesis in endothelial cells.
Endothelial cells were incubated with
[S]methionine in the presence or absence of the
indicated concentrations of antisense or sense
oligodeoxyribonucleotides, lysed, and immunoprecipitated as described
under ``Experimental Procedures.'' A, fluorogram of
total labeled proteins. Proteins were electrophoresed on a 12%
SDS-polyacrylamide gel under reducing conditions. Ten-microliter
aliquots of cell lysate were loaded per lane. Bars on the left indicate molecular mass markers in kilodaltons. B, immunoprecipitates. Immunoreacted materials that had been
prepared from 8.0
10
dpm of lysate each except for
pericytes (4.0
10
dpm) were electrophoresed under
the same conditions as described for A. Specific
immunoprecipitates were marked at 22 and 18 kDa. Note that pericytes
synthesized VEGF, as did U251 cells.
Figure 6:
Inhibition of endothelial cell DNA
synthesis by different antisense oligodeoxyribonucleotide species
against various regions of VEGF mRNA. Each oligodeoxyribonucleotide (10
µM) was added to the endothelial cell culture, and
[H]thymidine incorporation was determined as
described in the legend to Fig. 4. Values are expressed as the
means of triplicate experiments and relate to the mean of
H
radioactivities incorporated in cultures without
oligodeoxyribonucleotides (2.0
10
dpm). Hatched and shaded columns indicate the values for antisense
oligodeoxyribonucleotides, and open columns indicate those for
sense oligodeoxyribonucleotides. Vertical bars indicate
standard deviation. * and**, p < 0.05 and p <
0.01, respectively, compared with both the value without
oligodeoxyribonucleotides and that for the respective sense control
(Student's t test). The regions to which antisense
oligodeoxyribonucleotides correspond are shown by horizontal bars over the schematic diagram of the VEGF mRNA. Boxes indicate the coding regions; solid lines indicate the
untranslated regions. Italic numbers above the schematic
diagram indicate nucleotide numbers beginning at the cap
site(26) . AS, the antisense oligodeoxyribonucleotide
corresponding to the region around the initiator
codon.
The process of angiogenesis is thought to consist of the following four steps: 1) proteolytic degradation of the basement membrane, 2) migration of endothelial cells, 3) proliferation of endothelial cells, and 4) tube formation. The final step is completed when new capillaries are covered with pericytes(1, 2) . In this study, we have focused on how the proliferation of endothelial cells and pericytes is affected by hypoxia, the leading cause of angiogenesis.
This study has confirmed
that low atmospheric O tensions can result in the
stimulation of the proliferation of both human umbilical vein
endothelial cells and bovine retinal pericytes. The O
concentrations that induced maximal growth promotion were 10% for
endothelial cells and 2.5% for pericytes. This is comparable with
earlier reports that human umbilical vein endothelial cells grew at the
greatest rate under 7.5% O
(37) and bovine brain
microvascular pericytes under 3% O
(38) .
This
study has also demonstrated for the first time that mRNAs coding for
VEGF, a potent endothelial cell mitogen, are present not only in
endothelial cells per se, but also in pericytes, and that
their level is significantly elevated in both cell types as atmospheric
O concentrations decrease. Moreover, it is VEGF
and VEGF
, the secretory forms of VEGF, that are
coded for by the mRNAs expressed in the vascular cells. VEGF is a
growth factor known to be present in a variety of tissues, including
ovary (39, 40) and malignant
tumors(41, 42, 43, 44) , where
angiogenesis takes place. Its expression has been shown to be localized
around necrotic foci in brain tumors (45) and to be enhanced by
O
depletion in glioblastoma cell
cultures(13, 14) . Iizuka et
al.(46) , using an RT-PCR analysis similar to that
conducted in this study, recently showed that VEGF mRNA levels are
elevated during hypoxia in human osteosarcoma cells. From these
observations, VEGF has been regarded as the principal angiogenic factor
under ischemic and hypoxic conditions. Our finding that vascular cells per se can express the VEGF gene in response to hypoxia would
seem, therefore, to have significant implications in angiogenesis.
Vascular VEGF may participate in the process of angiogenesis in
autocrine and paracrine manners.
In general, the actions of growth
factors are mediated by receptors on their target
cells(47, 48) . In this study, we have also identified
VEGF receptor subtypes expressed in endothelial cells and pericytes.
Endothelial cells were found to constitutively express the two genes
for flt1 and kdr, regardless of the atmospheric
O tensions. The rank order of mRNA abundance was kdr
flt1, suggesting that Kdr is the major VEGF receptor
species expressed in human umbilical vein endothelial cells. On the
other hand, only flt1 mRNA was detected in pericytes under
hypoxic conditions. The difference in the mode of flt1 gene
expression between the two cell types is of interest in understanding
the regulation of VEGF receptor gene expression. Transcripts of the kdr gene were not detected in pericytes; given the relatively
low level of the transcript as determined by Northern blot analysis
with human kdr cDNA (data not shown) and the lack of bovine
sequence, it was unfortunate that RT-PCR could not be used to evaluate
the level of the kdr transcript.
The results obtained
indicate that vascular endothelial cells per se possess a
system for triggering their own growth. In endothelial cells, VEGF
expression was inducible by low O concentrations, whereas
receptor expressions were constitutive. This may be an indication that
ligand expression is a rate-limiting step in the putative autocrine
actions of VEGF, as in the case of the keratinocyte growth factor
system in dermal wound healing(49) . We therefore manipulated
VEGF expression with antisense oligodeoxyribonucleotides to test the
functional role of the VEGF system. Antisense oligodeoxyribonucleotides
complementary to the 5`-region of VEGF mRNA, encompassing the initiator
codon, were found to cause a dose- and time-dependent inhibition of DNA
synthesis in endothelial cells cultured under hypoxic conditions (Fig. 4), probably through a blockage of the translation of VEGF
mRNA (Fig. 5). We also conducted control experiments with
additional antisense oligodeoxyribonucleotide species against different
regions of VEGF mRNA, including the 5`- and 3`-untranslated regions and
the VEGF open reading frame, the majority of which (6 of 10) could also
efficiently inhibit endothelial cell synthesis of DNA (Fig. 6);
arrests of the ribosome transition (50) and an RNase H-like
activity-driven degradation of target mRNA (51) would account
for the antisense DNA action. The results could be regarded as evidence
that vascular VEGF is causally related to the hypoxia-induced
proliferation of endothelial cells. (
)
In light of these
findings, we propose a model for the mechanism of the hypoxia-induced
proliferation of vascular cells (Fig. 7). When the local oxygen
concentration is lowered, VEGF gene expression would be induced in
endothelial cells and in pericytes to produce secretory forms of VEGF.
VEGF in turn may act on Kdr and Flt1 receptors on endothelial cells in
autocrine and paracrine manners, thereby causing the proliferation of
endothelial cells, which may lead to angiogenesis. Basal amounts of
vascular VEGF synthesized in normoxic states may promote the
maintenance of microvascular homeostasis, as suggested by our
observation that the antisense VEGF oligodeoxyribonucleotide could
modify endothelial cell DNA synthesis under 20% O (Fig. 4C). According to this model, we can also
suggest a possible approach for the prevention of angiogenesis.
Interruptions of the series of biochemical events at certain steps
might halt the process of angiogenesis, e.g. antisense DNA/RNA
against VEGF mRNA may have therapeutic potential in the treatment of
proliferative angiopathies or tumors. In support of the above-mentioned
model, Aiello et al.(53) recently reported that
ocular VEGF levels were abnormally high in a large population of
patients with actively proliferative diabetic retinopathy, but dropped
following successful treatment.
Figure 7: A possible mechanism of hypoxia-induced proliferation of vascular endothelial cells and pericytes. EC, endothelial cells.
The model proposed was based on in vitro observations with cultivated endothelial cells and
pericytes. Although it can partially explain how hypoxia causes
endothelial proliferation, the involvement of other growth factors must
also be taken into account when considering the processes that would
occur in vivo. In addition to VEGF, acidic fibroblast growth
factor, basic fibroblast growth factor (bFGF), epidermal growth factor,
transforming growth factor-, transforming growth factor-
(TGF-
), and platelet-derived growth factor B (PDGF-B) have been
implicated in
angiogenesis(54, 55, 56, 57) . Among
them, bFGF, TGF-
, and PDGF-B seem particularly important because
they can be produced by endothelial cells themselves, thus possessing
potential autocrine/paracrine activities. bFGF is a mitogen for a wide
variety of cell types, playing diverse roles in vascular and nervous
systems as well as in connective tissues(58) . Concerning
vascular functions, bFGF has been reported to stimulate not only
endothelial cell mitosis and chemotaxis, but also tube formation.
Furthermore, it can induce collagenases and plasminogen activator,
which would promote degradation of the basement membrane of the
parental vessels(59) . These observations indicate that bFGF
may be related to all the steps of angiogenesis. However, the
expression of this growth factor has been shown not to be influenced by
hypoxia(60) . TGF-
inhibits the proliferation of
endothelial cells in culture (61) , but is known to induce new
capillary formation in vivo(56) . This apparently
paradoxical effect of TGF-
may be explained by the fact that this
factor is chemotactic for macrophages and causes them to release
angiogenic factors(56) . However, TGF-
expression has also
been shown not to be affected by hypoxia(60) . PDGF-B is a
major serum mitogen for mesenchymally derived cells. Since PDGF-B is
not only released by platelets, but also secreted by cells involved in
inflammatory responses, it has been suggested to play a role in wound
healing(10) . Although PDGF-B was previously thought to be
devoid of mitogenic activity on endothelial cells, Funa et al.(62) have recently demonstrated that functional PDGF-B
receptors are expressed on hyperplastic capillary endothelial cells in
malignant glioma, suggesting that autocrine PDGF-B has a role in the
proliferation of endothelial cells. In contrast to bFGF and TGF-
,
hypoxia-induced up-regulation of the PDGF-B gene has also been reported (60) . Available evidence thus suggests that the major
autocrine/paracrine growth factors involved in the control of
endothelial cell growth under normoxic conditions would be bFGF, VEGF,
and PDGF-B. Under hypoxic conditions, induced VEGF and PDGF-B would
mainly account for the endothelial proliferation.
We have shown
previously that pericytes can restrict the replication of co-cultured
endothelial cells and suggested TGF- and heparan sulfate as the
candidate regulatory molecules(4) . We also have shown that
endothelium-dependent stimulation of pericyte growth is mediated mainly
by endothelin-1(5) , a hypoxia-inducible mitogen(63) .
In the present study, pericytes were demonstrated to express the VEGF
gene in response to hypoxia, as do endothelial cells. Therefore,
pericytes may be regarded as cells that can exert both negative and
positive growth effects on their microvascular counterpart. Although
the role of pericytes during angiogenesis is poorly understood, it is
likely that hypoxia would turn them predominantly mitogenic, thereby
promoting the growth of endothelial cells together with that of
pericytes through synergistic actions of VEGF, PDGF-B, and
endothelin-1. Whether VEGF in fact stimulates pericyte replication,
however, remains to be established because of the following
observations. Midy and Plouët (64) reported
that VEGF stimulated the migration, but not mitosis, of bovine
osteoblasts, a cell type of the same mesenchymal origin as pericytes.
Vascular smooth muscle cells, the equivalent of pericytes in larger
vessels, have also been reported not to respond to VEGF(16) .
Seetharam et al.(65) showed that transfection of flt1 cDNA failed to confer responsiveness to VEGF on NIH3T3
cells.