Cellular Biology |
From the Laboratorio di Patologia Vascolare, Istituto Dermopatico dellImmacolata, Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy, and the Dipartimento di Scienze Morfologico-Biomediche (L.T.), Sezione di Anatomia e Istologia, Università di Verona, Verona, Italy.
Correspondence to Daniela DArcangelo, Laboratorio di Patologia Vascolare, Istituto Dermopatico dellImmacolata, Istituto di Ricovero e Cura a Carattere Scientifico, via dei Monti di Creta, 104, 00167 Rome, Italy. E-mail d.darcangelo{at}idi.it
| Abstract |
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Key Words: acidosis apoptosis endothelium ischemia growth factors
| Introduction |
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| Materials and Methods |
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Acidification Protocol
Culture dishes or Boyden apparatuses were placed in
airtight modular incubator chambers (Forma Scientific) infused for 20
minutes with either 5% CO2/95% air or 20%
CO2/80% air to achieve pH 7.40±0.02 and pH
7.0±0.05, respectively. Chambers were placed in an incubator at 37°C
for the duration of the experiment.
Apoptosis Assessment
Fluorescence-activated cell sorter (FACS)
analysis was carried out with cells stained with propidium
iodide (50 µg/mL) by use of a FACScan (Becton Dickinson) and
CellQuest software as reported.14 Apoptosis was
also analyzed by Cell Death Detection ELISA
(Boehringer-Mannheim), terminal
deoxynucleotidyl transferasemediated dUTP nick
end-labeling (TUNEL) assay,15 and Hoechst 33258 dye
nuclear staining.16
Preparation of BAEC Conditioned Medium
BAECs in complete medium (1x106 cells per
100-mm dish) were grown for 3 days, then medium was replaced with
serum-free DMEM, and dishes were placed in airtight chambers. After 48
hours, conditioned medium (CM) was collected.
Migration Assay
Migration was assessed in modified Boyden
chambers.17 Medium without (control) or with 10% FCS was
used as a chemoattractant, and cells were incubated for 5 hours at
37°C in a 5% CO2 or 20%
CO2 atmosphere. In other experiments, either BAEC
CM or serum-free medium was used with or without an anti-bFGF
neutralizing antibody (100 ng/mL, R&D) or with the denatured antibody
(10 minutes, 95°C).
Differentiation on Matrigel
BAEC differentiation on Matrigel (Collaborative Research) was
performed as described.18 Capillary-like structures were
quantified by counting internodal points formed in 6 fields per
dish.
RT-PCR Experiments
Total cellular RNA was isolated from BAECs grown in serum-free
medium either at pH 7.4 or pH 7.0 for 48 hours by use of TRIzol reagent
(GIBCO Life Technologies). RNA was converted to cDNA by reverse
transcription (RT) with the Superscript Preamplification System (Life
Technologies). Polymerase chain reaction (PCR) was performed for 10,
15, 20, 25, and 30 cycles. Sequences of the primers were as follows:
for VEGF (250-bp product), upper 5'-TCATGGATGTCTATCAG-3',
lower 5'-TGCTCTAGGAAGCTCAT-3'; for bFGF (220-bp product),
upper 5'-TCAAGTTACAACTTCAAGCAG-3', lower
5'-TATACTGCCCAGTTCGTTTC-3'. PCR conditions for VEGF and bFGF
amplification were as follows: denaturation at 94°C for 1 minute;
annealing at 52°C and 56°C, respectively, for 1 minute; and
extension at 72°C for 1 minute.
Actinomycin D chase studies were performed as described10 to determine mRNA stability on BAECs maintained at either pH 7.4 or pH 7.0 for 48 hours, followed by additional 1 to 8 hours with actinomycin D (5 µg/mL).
Detection and Quantification of bFGF into BAEC CM
CM was concentrated 30-fold through a 10-kDa cutoff (Centriprep
10), and then proteins (40 µg) were subjected to SDS-PAGE under
reducing conditions and electroblotted. bFGF was detected with 0.2
µg/mL antibody to human recombinant fibroblast growth factor-2
(
FGF-2, Santa Cruz Biotechnology Inc) and detected with the ECL
detection system (Amersham Life Technologies); 200 µL of concentrated
CM (100-fold) was used in the ELISA (R&D).
Electrophoretic Mobility Shift Assay
Double-strand oligonucleotide containing an
HIF-1 consensus binding site (40 ng) was labeled with Klenow Enzyme
(Boehringer) for 30 minutes at 37°C with the use of 25 µCi
[
-32P]dATP. Nuclear extracts19
were incubated on ice with probe alone (HIF-1), with double-strand
competitor oligonucleotides (HIF-1 or mutated HIF-1,
100-fold nuclear excess), with an affinity-purified antiarylic
hydrocarbon nuclear receptor translocator (ARNT) antibody (
ARNT, 1
µg) or with a nonspecific antibody, antiarylic hydrocarbon receptor
complex (AHRC) antibody (
AHRC).
Statistical Analysis
Continuous variables were analyzed by the Student
t test and 1-way ANOVA, along with post hoc
Student-Newman-Keuls tests when appropriate. Data are expressed
as mean±SE. A value of P
0.05 was considered statistically
significant.
An expanded Materials and Methods section is available online at http://www.circresaha.org.
| Results |
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50%
(Figure 2A
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These findings suggest that the lower cell number
observed in medium with 10% FCS at pH 7.0 versus pH 7.4, shown in the
proliferation assay depicted in Figure 1A
, was due to diminished
proliferation rate rather than to increased cell death. This conclusion
is also supported by the lower number of detached cells in the
supernatant of BAECs grown at pH 7.0 than at pH 7.4 in 10% FCS and
also in serum-free medium (not shown).
Taken together, the results of Figures 1
and 2
indicate
that acidosis slows down BAEC proliferation and protects these cells
from apoptosis.
Effect of Acidosis on BAEC Migration
The results depicted in Figure 3
show that 10% FCS was a powerful chemoattractant for BAECs and that
acidosis markedly inhibited BAEC migration in response to FCS. A
decrease of basal migration was also observed in acidotic conditions in
the absence of a chemoattractant in the lower chamber of the Boyden
apparatus.
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Effect of Acidosis on BAEC Differentiation
The effect of acidosis on BAEC differentiation on
Matrigel20 was examined in 0% and in 10% FCS. BAECs
developed a diffuse network of capillary-like structures in 10% FCS at
pH 7.4. (Figure 4B
), and this effect was
strongly inhibited at pH 7.0 (Figure 4D
). In 0% FCS, BAECs
failed to differentiate regardless of pH. The average results from the
above experiments are reported in Figure 4E
.
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Effect of BAEC CM on BAEC Proliferation and Migration
BAECs were grown for 48 hours in serum-free conditions either at
pH 7.0 or at pH 7.4. Thereafter, CM was collected and tested on BAEC
proliferation assays carried out for 48 hours at pH 7.4. CM from BAECs
grown at pH 7.0 enhanced BAEC proliferation, and cell number after 48
hours was 183±8% of control versus 121±5% of control for CM from
BAECs grown at pH 7.4 (Figure 5
). In
addition, CM from BAECs grown at both pH levels stimulated BAEC
migration, but with different potency; CM from BAECs grown at pH 7.0
induced the migration of 102±4 cells per field, and CM from BAECs
grown at pH 7.4 induced the migration of 61±2 cells per field. These
results show that BAEC CM enhances BAEC proliferation as well as
migration and that these effects are more marked when CM is obtained
from cells grown under acidotic conditions.
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Characterization of BAEC CM
The mitogenic activity of CM produced under acidotic
conditions was evaluated in a 48-hour proliferation assay; it was
abolished by treatment at 100°C for 5 minutes and reduced by 33±6%
after 15 minutes at 65°C (not shown). Furthermore, the
mitogenic effect of CM produced under acidotic conditions
was not modified by ultrafiltration with a 10-kDa cutoff, indicating
that a proteic factor(s) responsible for the mitogenic
activity had a molecular size >10 kDa (not shown).
Effect of Acidosis on VEGF and bFGF
Semiquantitative PCR amplification of
reverse-transcribed mRNAs derived from BAECs grown at pH 7.4 or pH 7.0
showed that VEGF and bFGF mRNA levels in acidotic conditions were
2-fold higher than those at pH 7.4 (Figures 6A
and 6B
). This effect was not due to
increased mRNA stability, as assessed by actinomycin D chase studies
(Figure 6C
). Furthermore, the amount of bFGF in CM from BAECs
cultured at pH 7.0 for 48 hours was 2.8-fold higher than the amount of
bFGF in CM from BAECs cultured at pH 7.4 (Figure 6D
). This
increase was confirmed by Western blot analysis (Figure 6E
). The presence and the activity of bFGF in BAEC CM were
confirmed in migration assays. Figure 7
shows that the addition of an anti-bFGF antibody to the CM inhibited
the chemotactic activity of CM from BAECs grown at pH 7.4 and pH 7.0 by
34±1% and 41±4%, respectively (P<0.005). The results
are in agreement with the data in Figure 6D
showing enhanced
bFGF secretion at pH 7.0. Because bovine VEGF antibodies are not
commercially available, it was not possible to detect VEGF in BAEC CM,
and VEGF-mediated modulation of BAEC migration could not be
determined.
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Effect of Acidosis on HIF-1
HIF-1, a heterodimer formed by HIF-1
and ARNT, is a
transcription factor involved in hypoxia-induced VEGF
gene-enhanced expression. In hypoxic conditions, it is possible to
detect nuclear HIF-1 bound to its 32P-labeled
consensus binding site (ACGTG) by electrophoretic mobility shift assay,
with use of a specific antibody. The experiments reported in the
present study investigated the effect of acidosis on HIF-1. Figure 8
shows that HIF-1 is not induced in
acidotic cells, because there was no supershift with an anti-ARNT
affinity-purified antibody. As a positive control, BAECs were treated
with 100 µmol/L CoCl2 for 24 hours; this
treatment mimics hypoxia by reducing heme oxygen binding. Under
these conditions, HIF-1 induction was observed. In contrast, HIF-1 was
not supershifted by
AHRC. These results exclude the possibility that
the effect of acidosis to enhance VEGF mRNA may be due to an
HIF-1dependent mechanism.
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| Discussion |
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The protective effect of acidosis on endothelial cell apoptosis is a new finding and is in agreement with prior results that have shown that extracellular acidosis protects primary neurons as well as p53+/+ and p53-/- mouse embryo fibroblasts from serum deprivationinduced apoptotic death.25 In the present study, the mechanism by which acidosis protects endothelial cells from apoptosis was not elucidated. However, because acidification per se increases endothelial cell Ca2+,4 the protective effect of acidosis was unrelated to inhibition of the Ca2+ overload, whereas enhanced production of VEGF and bFGF may have played a role in the inhibition of apoptotic cell death.26 27
Acidosis-induced increase of VEGF and bFGF mRNA is of interest because
an increase in VEGF mRNA has also been described in hypoxic cells and
was related to the activation of HIF-1 and enhanced VEGF mRNA
stability.10 HIF-1 is a transcription factor that is
considered to be a key regulator of the cell hypoxia response
pathway because it activates the transcription of a variety of
genes that help cells survive in hypoxic conditions. Because
hypoxia leads to intracellular acidosis,8 it was
hypothesized that acidosis may also modulate HIF-1 activity. However,
the results of the experiment depicted in Figure 8
show no
activation of HIF-1 in BAECs at pH 7.0. Furthermore, we found no effect
of acidosis on VEGF mRNA stability. Thus, the enhanced VEGF and bFGF
mRNA expression reported in the present study is compatible with
increased gene transcription and may have been due to activation of
unidentified pH-sensitive transcription factor(s).
The results of the present study suggest that acidification may inhibit new blood vessel formation and must be reconciled with the results of other studies that have shown that ischemia in vivo triggers an angiogenic response.6 7 A possible explanation for this apparent discrepancy is related to the enhancement of bFGF and VEGF expression by acidosis. The induction of these growth factors may increase their storage in the extracellular matrix and set the stage for enhanced angiogenesis on return to normal pH. Because most patients with coronary artery28 29 and peripheral vascular disease have normal resting blood flow and develop ischemia in response to exercise, it is possible that acidosis during transient ischemia may enhance growth factor secretion and protect cells from apoptosis. On restoration of blood flow and return to normal pH, the growth factors stored in the extracellular matrix would be available to trigger an angiogenic response and, by this mechanism, would account for the ability of ischemia to induce neovascularization.
In summary, acidification inhibits those endothelial functions that are required for neovascularization to occur and protects endothelial cells from apoptosis. Additional studies will be necessary to characterize the molecular mechanisms by which pH modulates endothelial cell function and gene expression as well as the pathophysiological relevance of these findings.
| Acknowledgments |
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Received September 16, 1999; accepted November 22, 1999.
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C. Gaetano, A. Catalano, B. Illi, A. Felici, S. Minucci, R. Palumbo, F. Facchiano, A. Mangoni, S. Mancarella, J. Muhlhauser, et al. Retinoids Induce Fibroblast Growth Factor-2 Production in Endothelial Cells via Retinoic Acid Receptor {{alpha}} Activation and Stimulate Angiogenesis In Vitro and In Vivo Circ. Res., March 2, 2001; 88 (4): e38 - e47. [Abstract] [Full Text] [PDF] |
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L. Xu and I. J. Fidler Acidic pH-induced Elevation in Interleukin 8 Expression by Human Ovarian Carcinoma Cells Cancer Res., August 1, 2000; 60(16): 4610 - 4616. [Abstract] [Full Text] |
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M. Clauss and W. Schaper Vascular Endothelial Growth Factor : A Jack-of-All-Trades or a Nonspecific Stress Gene? Circ. Res., February 18, 2000; 86(3): 251 - 252. [Full Text] [PDF] |
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