Articles |
From the Unité 441 d'Athérosclérose de l'Institut National de la Santé et de la Recherche Médicale, Pessac, France, and the Centre de Recherches sur les Maladies Vasculaires Périphériques, Association Claude Bernard, Hôpital Broussais, Paris, France (L.C.).
Correspondence to Dr Claude Desgranges, Unité 441 d'Athérosclérose de l'INSERM, Avenue du Haut-Lévêque, 33600 Pessac, France. E-mail claude.desgranges{at}bordeaux.inserm.fr
| Abstract |
|---|
|
|
|---|
Key Words: nucleotides purinoceptors rats smooth muscle cells balloon injury
| Introduction |
|---|
|
|
|---|
In this study, we describe the cloning and sequencing of a rat P2Y2 cDNA isolated from a rat aortic SMC cDNA library. This cDNA was used to study by Northern blot the expression of P2Y2 mRNA both in rat aortic media and in cultured rat aortic SMCs. Furthermore, the P2Y2 mRNA was detected by using in situ approaches on SMCs and endothelial cells of normal rat aorta and was found at a high level in the cells of intimal thickenings of balloon catheterized rat aortas.
| Methods |
|---|
|
|
|---|
-32P]dCTP (3000 Ci/mmol),
[
-35S]dATP (>1000 Ci/mmol), terminal
deoxynucleotidyl transferase, and Hybond
N+ membranes from Amersham; DMEM, phosphate
buffer saline, fetal calf serum, trypsin-EDTA, PDGF BB, and reverse
transcriptase from Gibco-BRL; Taq polymerase from Bioprobe;
proteinase K, Tween 20, angiotensin II, diaminobenzidine,
and triethanolamine from Sigma. Oligonucleotides were
synthesized by Eurogentec S.A.
Cultured SMCs
Cultures of rat aortic SMCs were obtained and maintained as
previously described.17 Cultured SMCs from 15-day
intimal thickenings18 were provided by Pr G.
Gabbiani and Dr M.L. Bochaton-Piallat (Université de
Genève, Switzerland). The rat aorta SMC line A10 was obtained
from American Type Culture Collection.
For proliferative studies, SMCs and A10 cells were seeded in DMEM containing 10% fetal calf serum at a cell density of 6x104 cells/cm2 in 24-well tissue culture plates (Falcon), made quiescent by a 24-hour incubation in serum-free DMEM, and then counted by using a Coulter counter ZM after a 48-hour incubation in the specific medium to test.
For P2Y2 mRNA studies, cultured SMCs from adult rat aorta were used between passages 5 and 14. IT15 cells and rat embryo aorta SMCs, respectively, were used at passages 9 and 6. SMCs plated at 4x104 cells/cm2 in 75-cm2 flasks (Nunc) were generally made quiescent by incubation in serum-free medium before RNA extraction. In some experiments, quiescent SMCs were then incubated with either DMEM containing 10% FCS, angiotensin (1 µmol/L) or PDGF (10 ng/ml) for various periods.
Polymerase Chain Reaction, cDNA Screening, and Sequencing
cDNAs were synthesized by reverse transcription from total
RNA extracted from cultured rat aortic SMCs according to classic
procedures. A P2Y2 cDNA fragment was amplified
from these cDNA by PCR by using a 35-cycle program (94°C for 1
minute, 60°C for 30 seconds, and 72°C for 1 minute) in the presence
of two oligonucleotide primers (1 µmol/L)
corresponding to positions 426 to 455 and 788 to 817 of the mouse
P2u sequence,14 and 50 U/mL
Taq polymerase. After PCR, the reaction products were
resolved on 2% agarose gels, and the expected amplified fragment was
purified and ligated into the plasmid vector pBluescript SK(-) for DNA
sequencing. The cloned PCR product was used to screen a rat aortic
SMC cDNA library made in
gt11.19 Phages from
one plaque giving a strong hybridization signal were submitted to an
additional screening. cDNA from this clone was purified and cloned into
the EcoRI site of the Bluescript plasmid. DNA sequencing was
performed by the dideoxy chain termination method using a T7 polymerase
sequencing kit (Pharmacia). GenBank was consulted for
nucleotide sequence homology by using the Fasta
program.
Northern Blots
Generally, 30 µg of total RNA from various cells and tissues
were electrophoresed in 1% agarose gel and transferred onto Hybond
N+ membranes, which were hybridized with the rat
P2Y2 cDNA labeled with
[
-32P]dCTP as previously
described.19
Arterial Injury
Aortas from adult male Wistar rats (250 to 300 g) were
denuded from their endothelium according to the method
of Tiell et al,20 by using a 2F Fogarty balloon
catheter (Edwards Laboratories) moved from the femoral artery up to the
diaphragm. The balloon was inflated with air to a pressure of 800
mm Hg and drawn into the abdominal aorta. This operation was repeated
twice more. The iliac artery was subsequently ligated after balloon
withdrawal.
In Situ Hybridization and Immunohistochemistry
Normal or balloon-injured aortas were fixed in situ by 4%
paraformaldehyde perfusion, then embedded in paraffin.
For in situ hybridization, serial 8-µm sections were collected on
Biobond (British BioCell International) coated slides, and after
deparaffination and rehydration, were treated with proteinase K (1
µg/mL) in 0.1 mol/L Tris-HCl (pH 8) containing 50 mmol/L EDTA.
After another paraformaldehyde fixation and washing,
the slides were incubated for 5 minutes in a medium containing 0.1%
triethanolamine and 0.25% acetic anhydride. Sections were
prehybridized in a hybridization buffer: 4x SSC, 5xDenhardt's
solution, and 0.1% N-lauroylsarcosine. Antisense and sense
riboprobes were generated from the 1.7-kb cDNA of rat
P2Y2 receptor by in vitro transcription in the
presence of digoxigenin-UTP by using the DIG RNA labeling mix
(Boehringer Mannheim) and following the manufacturer's
instructions. Probes were diluted at 1 ng/µL in hybridization buffer,
and 35 µL of this hybridization mix were applied to each section.
Hybridization was performed overnight at 50°C in a humid chamber.
Sections were washed for 5 minutes in 5xSSC, twice in 50% formamide,
and then for 2 hours in 2xSSC buffer at 55°C. Slides were treated
with RNase A for 15 minutes at 37°C and rinsed in 2xSSC for 2x15
minutes. To detect the P2Y2 specific hybrids, the
slides were incubated for 90 minutes at 37°C with an antidigoxigenin
antibody conjugated to alkaline phosphatase 1:500 diluted in buffer
containing 100 mmol/L Tris-HCl (pH 7.5) and 150 mmol/L NaCl.
After the slides were washed four times in Tris-buffered saline, they
were incubated with 337.5 µg/mL nitro blue tetrazolium salt and 175
µg/mL 5-bromo-4-chloro-3-indolyl phosphate in the staining buffer:
100 mmol/L Tris-HCl (pH 9.5), 100 mmol/L NaCl, 50 mmol/L
MgCl2, and 1 mmol/L levamisole. Staining was
allowed to develop generally overnight in the dark and was stopped by
three washes in the staining buffer. Slides were dehydrated, mounted in
DePeX Gurr (BDH) and observed under a light microscope. The percentage
of cells expressing P2Y2 receptor was defined by
the ratio of P2Y2 mRNA-positive cells to the
total cell number determined by counting nuclei of a parallel section
stained with hemalum, either in the normal media or in intimal
thickenings present at the luminal edge of each aorta section. At
least four sections were observed for each aorta, and three aortas were
studied for each time after balloon injury. For in situ hybridization
of cultured cells, SMCs were seeded directly on glass slides, fixed
with paraformaldehyde, and then directly used for
hybridization as described for histological slides.
For immunohistochemical analysis, serial sections were treated
with the following primary antibodies: a mouse monoclonal anti-smooth
muscle
-actin (Sigma Chemical Co) diluted at 1:400, a rabbit
polyclonal anti-von Willebrand factor (Sigma Chemical Co)
diluted at 1:600, and a mouse monoclonal anti-PCNA (Novo Castra)
diluted at 1:200. Detection of complexed primary antibodies was
achieved by using either biotinylated anti-mouse or anti-rabbit
secondary antibodies (Amersham) and the streptavidin biotinylated
horseradish peroxidase complex (Amersham). The final complex was
visualized by treatment with 0.5 µg/mL diaminobenzidine. Antibody
dilutions and washes were performed in PBS buffer containing 0.2%
Tween 20 and 0.5% bovine serum albumin. Controls were carried
out without primary antibodies. The percentage of PCNA-positive cells
on total cell number found in the same intimal thickenings was
determined, and results are expressed as mean percentage±SD of the
mean on six distinct intimal lesions for each time after
angioplasty.
| Results |
|---|
|
|
|---|
|
P2Y2 mRNA Expression in Normal Rat Aorta
Using the rat P2Y2 cDNA, we detected a 3-kb
mRNA by Northern blot analysis in the media of normal adult rat
aortas by Northern blot analysis (Fig 2
).
|
Tissue localization of P2Y2 mRNA was explored by
in situ hybridization on cross sections of adult rat aortas, by using a
digoxigenin-UTP-labeled rat P2Y2 riboprobe. Cells
expressing P2Y2 mRNA were detected both in the
media and intima of normal aortas. In the media, highly positive cells
were disseminated throughout this layer without apparent preferential
localization (Fig 3A
). The percentage of
these highly positive cells represented <25% of total
medial cells. As expected, these cells were identified as SMCs because
they expressed smooth muscle
-actin (Fig 3C
). In the intima, nearly
all the endothelial cells, identified by the presence
of von Willebrand factor (Fig 3D
), demonstrated a high
expression of P2Y2 mRNA (Fig 3A
).
|
In contrast with adult rat aorta, medial cells from the aorta of rat
fetuses on gestational day 19 demonstrated a high expression of
P2Y2 mRNA (Fig 3E
). At those times, SMCs already
expressed
-actin (Fig 3G
) and demonstrated a high degree of
proliferation detected by PCNA expression (Fig 3H
).
P2Y2 transcripts were also detected in
endothelial cells and in periarterial
embedding tissue of fetus aortas. An identical pattern was found in
aortas of 1-day-old newborn rats (not shown).
P2Y2 mRNA Expression in Injured Aorta of Adult
Rats
P2Y2 mRNA detection by in situ hybridization
was also performed on balloon-injured aortas 3, 8, and 20 days after
endothelial denudation. On day 3 after balloon injury,
the luminal surface of the aorta was free of cells. SMCs from the inner
medial layer, which are known to proliferate at that time in the rat
carotid model,22 did not particularly demonstrate
a high P2Y2 expression. At 8 days after balloon
injury, the intimal thickening consists of two to three cell layers and
was still evolving since 28.4±2.1% of cells were PCNA-positive (Fig 4C
). At that time, the percentage of
P2Y2 mRNA-positive cells found in the intimal was
high in comparison with the underlying media, reaching 90% of the
total neointimal cells (Fig 4A
). A high percentage of cells
expressing P2Y2 mRNA at a high level was also
found in nonevolving intimal thickenings 20 days after injury at a time
when there was 0.7±0.1% of PCNA-positive cells (Fig 4E
and 4G
).
Whatever the time elapsed since injury, most neointimal
cells had a smooth muscle origin because they expressed smooth muscle
-actin (Fig 4D
and 4H
). At those times, the cells covering the
intimal lesions were probably of smooth muscle origin because they did
not express von Willebrand factor (not shown) but were positive
for smooth muscle
-actin (Fig 4D
and 4H
). P2Y2
mRNA expression of these cells located on the luminal surface of
intimal thickenings was higher than in other intimal cells, both at 8
and 20 days after balloon injury (Fig 5
).
|
|
P2Y2 mRNA Expression in Cultured Rat Aortic
SMCs
Using the rat P2Y2 cDNA, we also detected a
3-kb mRNA by Northern blot analysis in secondary cultures of
SMCs derived from adult rat aorta and from 19-day rat fetus aorta (Fig 6
). Analysis of cultured SMCs by
in situ hybridization demonstrated that the P2Y2
mRNA was present in all the cells (Fig 7
). In contrast, this mRNA was not found
in the A10 rat aortic SMC line (Fig 6
), confirming previous
observations.15 The lack of
P2Y2 expression in these cells correlated with
the absence of potentiation of proliferation by the preferential
P2Y2 agonist UTP, which normally increased
mitogenesis of cultured SMCs in basal medium
(Table
). P2Y2 mRNA
were found at a faintly higher level in cultured SMCs from 15-day
intimal thickenings than in cultured SMCs from the media of normal
adult aorta (Fig 6
). This expression was not significantly different
between G0 quiescent SMCs and exponentially
growing SMCs, and neither PDGF nor angiotensin II induced
long-lasting modulation of P2Y2 mRNA expression
(Fig 6
).
|
|
|
| Discussion |
|---|
|
|
|---|
Modulation of P2Y2 Expression in Aortic SMCs
In situ hybridization studies show that only a part of medial SMCs
of adult rat aorta express P2Y2 mRNA. However,
our previous studies demonstrated that freshly dissociated aortic SMCs
were responsive at 97% to extracellular UTP.23
This apparent discordance between these two results may be explained by
the fact that all medial SMCs express the P2Y2
receptor at the cell surface, but only a limited percentage of them
demonstrate a P2Y2 mRNA level sufficient to be
detectable by in situ hybridization. Another possibility is that
effectively only some medial SMCs expressed the
P2Y2 receptor; the remaining cells expressed
another kind of P2 receptor such as the P2Y6
receptor, which is also activated by UTP and UDP and whose mRNA
has been described recently in rat aortic SMCs together with that of
P2Y2 mRNA.24 An alternate
explanation for the high expression of P2Y2 in
dissociated SMCs is that the process of dissociation itself upregulates
expression of this receptor.
A large proportion of cells of intimal thickenings demonstrated a high P2Y2 expression. According to this and other studies, the majority of these cells have a smooth muscle origin,25 26 and the percentage of macrophages found in rat intimal thickenings did not exceed 25% of intimal cells,27 28 29 demonstrating that the majority of intimal cells expressing the P2Y2 receptor have a SMC origin. At this time, it is difficult to explain the extension of P2Y2 expression to nearly all the SMCs of intimal thickenings. A possible explanation may be that the level of P2Y2 expression in intimal SMCs is sufficiently high to be detected by in situ hybridization. This high expression could be related to the new phenotypic status demonstrated by SMCs in intimal thickenings. Indeed, in the rat, intimal cells of balloon-induced arterial lesions are known to be partially dedifferentiated SMCs,25 26 and their phenotypic status has been compared with that of newborn rat aortic SMCs.30 In this point of view, our study demonstrates that medial cells of rat embryo aorta also demonstrate, as do intimal thickening SMCs, a high P2Y2 expression. Moreover, our study demonstrates that rat aortic SMCs in culture, which are also submitted to a dedifferentiation process,25 express the P2Y2 mRNA. Lastly, the P2Y2 expression of cultured SMCs from intimal thickening is higher than that of normal media. Taken together, these results suggest that the generalization of P2Y2 expression to the entire cell population of intimal thickenings is closely associated with a poorly differentiated phenotype of SMCs. Although proliferation operates in embryo aorta and in early intimal thickenings, it is difficult to strictly associate the increase in P2Y2 expression and the proliferative process, since this expression remains high even at a time when proliferation has stopped in intimal thickenings. These hypotheses are strengthened by the fact that P2Y2 expression of exponentially growing SMC in culture is not significantly increased in comparison with quiescent SMCs. Moreover, the P2Y2 expression was not modulated by factors involved in the genesis of intimal thickenings such as angiotensin II or PDGF.1
Physiological Significance of High
P2Y2 Expression in Intimal Thickenings
The significance of the strong P2Y2
expression observed in balloon-induced intimal lesions is not clear.
Nevertheless, this strong expression could reflect an increased
reactivity to extracellular nucleotides with consequent
modulation of proliferation or vasoreactivity. Indeed, extracellular
nucleotides, particularly ATP and UTP, have been shown to
induce cell cycle progression and proliferation of cultured
arterial SMCs3 4 5 6 7 and to induce a
vasoconstriction in the absence of endothelial
cells.11 12 13 Since both neointimal
hyperplasia and vasoconstrictive remodeling have been
found to be involved in postangioplasty
restenosis,1 30 31 32 our data suggest that
extracellular nucleotides might play a significant role in
this process, at least as long as the functional
endothelial cells, which control intimal
thickening33 34 and nucleotide
vasorelaxant effects,35 36 are not
regenerated.
The increased P2Y2 receptor expression in the neointima may by itself be sufficient to enhance the local effects of extracellular nucleotides on SMC proliferation. Although the expression of other P2 receptors has been described at the arterial SMC level,7 24 37 38 the P2Y2 receptor seems to be more specifically involved in the response of SMCs to ATP and UTP39 and particularly in the potentiation of proliferation by these extracellular nucleotides.6 7 This hypothesis is strengthened by the demonstration that UTP did not increase SMC proliferation of P2Y2-deficient A10 SMCs. The effects of extracellular nucleotides is not only dependent on the nature and of the number of P2 receptors present on target cells, but also on the local concentrations of these nucleotides. Although in vivo modulation of extracellular nucleotides has not yet been demonstrated, various in vitro experiments suggest that extracellular nucleotides may be released both by blood and vascular cells when exposed to various physicochemical conditions (stress, hypoxia, and exposure to various other factors2 40 41 ), which may be found during the angioplasty process.
The expression of P2Y2 receptor remains high in intimal thickenings even at a time when proliferating cells have considerably decreased, suggesting that this receptor may be involved in other processes. SMC P2Y2 receptors are involved in the nucleotide-induced constriction of normal arteries.11 12 13 Long-lasting alterations of the vasomotricity after endothelial denudation, resulting in increased sensitivity to vasoconstrictive substances, have previously been demonstrated.35 36 It appears that like other receptors of vasoconstrictive factors such as angiotensin II,42 endothelin,43 PDGF,44 or thrombin45 which are overexpressed in neointima, P2Y2 receptors may play an important role in controlling the vasoactive properties of pathological arteries, particularly in chronic constriction at the lesion site, which may be one of the processes leading to postangioplasty restenosis.31 32 Moreover, P2Y2 receptors may be involved in cell recruitment in the neointima, since they have been demonstrated to mediate the nucleotide-induced expression of chemoattractant proteins for both SMCs and monocytes.7
The presence of P2 purinoceptors on endothelial cells involved in the endothelium-dependent relaxation induced by extracellular nucleotides had first been suggested on the basis of physiological experiments.46 47 Further studies have confirmed this hypothesis by demonstrating P2y (P2Y1) and P2Y2 subtype responses in endothelial cells,48 and more recently by Northern blot detection of P2Y2 mRNA in cultured rat artery endothelial cells.21 Our study demonstrates for the first time in situ P2Y2 mRNA expression in endothelial cells of normal rat aorta. In this study, we found that neointimal cells recovering the luminal surface of rat intimal lesions demonstrate a high P2Y2 expression, suggesting a high reactivity to extracellular nucleotides and consequently a possible modification of nucleotide-mediated vasomotricity (contraction versus relaxation) of pathological arteries.
Although these results remain to be extended to human atherosclerotic and restenotic lesions, the demonstration of a high expression of the P2Y2 purinoceptor in rat neointimal cells suggests that extracellular nucleotides may contribute via the P2Y2 receptor to the genesis and evolution of these arterial lesions and/or to the modulation of vascular tone of pathological arteries.
| Selected Abbreviations and Acronyms |
|---|
|
| Acknowledgments |
|---|
Received April 18, 1997; accepted July 29, 1997.
| References |
|---|
|
|
|---|
This article has been cited by other articles:
![]() |
N. Yu, L. Erb, R. Shivaji, G. A. Weisman, and C. I. Seye Binding of the P2Y2 Nucleotide Receptor to Filamin A Regulates Migration of Vascular Smooth Muscle Cells Circ. Res., March 14, 2008; 102(5): 581 - 588. [Abstract] [Full Text] [PDF] |
||||
![]() |
S. Jalvy, M.-A. Renault, L. Lam Shang Leen, I. Belloc, A. Reynaud, A.-P. Gadeau, and C. Desgranges CREB Mediates UTP-Directed Arterial Smooth Muscle Cell Migration and Expression of the Chemotactic Protein Osteopontin via Its Interaction with Activator Protein-1 Sites Circ. Res., May 11, 2007; 100(9): 1292 - 1299. [Abstract] [Full Text] [PDF] |
||||
![]() |
M. P. Abbracchio, G. Burnstock, J.-M. Boeynaems, E. A. Barnard, J. L. Boyer, C. Kennedy, G. E. Knight, M. Fumagalli, C. Gachet, K. A. Jacobson, et al. International Union of Pharmacology LVIII: Update on the P2Y G Protein-Coupled Nucleotide Receptors: From Molecular Mechanisms and Pathophysiology to Therapy Pharmacol. Rev., September 1, 2006; 58(3): 281 - 341. [Abstract] [Full Text] [PDF] |
||||
![]() |
W. P. Robinson III, C. D. Douillet, P. M. Milano, R. C. Boucher, C. Patterson, and P. B. Rich ATP stimulates MMP-2 release from human aortic smooth muscle cells via JNK signaling pathway Am J Physiol Heart Circ Physiol, May 1, 2006; 290(5): H1988 - H1996. [Abstract] [Full Text] [PDF] |
||||
![]() |
S. Bagchi, Z. Liao, F. A. Gonzalez, N. E. Chorna, C. I. Seye, G. A. Weisman, and L. Erb The P2Y2 Nucleotide Receptor Interacts with {alpha}v Integrins to Activate Go and Induce Cell Migration J. Biol. Chem., November 25, 2005; 280(47): 39050 - 39057. [Abstract] [Full Text] [PDF] |
||||
![]() |
J. M. Camden, A. M. Schrader, R. E. Camden, F. A. Gonzalez, L. Erb, C. I. Seye, and G. A. Weisman P2Y2 Nucleotide Receptors Enhance {alpha}-Secretase-dependent Amyloid Precursor Protein Processing J. Biol. Chem., May 13, 2005; 280(19): 18696 - 18702. [Abstract] [Full Text] [PDF] |
||||
![]() |
J. Shen, C. I. Seye, M. Wang, G. A. Weisman, P. A. Wilden, and M. Sturek Cloning, Up-Regulation, and Mitogenic Role of Porcine P2Y2 Receptor in Coronary Artery Smooth Muscle Cells Mol. Pharmacol., November 1, 2004; 66(5): 1265 - 1274. [Abstract] [Full Text] [PDF] |
||||
![]() |
C. I. Seye, M. W.M. Knaapen, D. Daret, C. Desgranges, A. G. Herman, M. M. Kockx, and H. Bult 7-Ketocholesterol induces reversible cytochrome c release in smooth muscle cells in absence of mitochondrial swelling Cardiovasc Res, October 1, 2004; 64(1): 144 - 153. [Abstract] [Full Text] [PDF] |
||||
![]() |
C. I. Seye, N. Yu, F. A. Gonzalez, L. Erb, and G. A. Weisman The P2Y2 Nucleotide Receptor Mediates Vascular Cell Adhesion Molecule-1 Expression through Interaction with VEGF Receptor-2 (KDR/Flk-1) J. Biol. Chem., August 20, 2004; 279(34): 35679 - 35686. [Abstract] [Full Text] [PDF] |
||||
![]() |
D. Erlinge Extracellular ATP: a central player in the regulation of vascular smooth muscle phenotype. Focus on "Dual role of PKA in phenotype modulation of vascular smooth muscle cells by extracellular ATP" Am J Physiol Cell Physiol, August 1, 2004; 287(2): C260 - C262. [Full Text] [PDF] |
||||
![]() |
Y Huang, K Salu, X Liu, S Li, L Wang, E Verbeken, J Bosmans, and I De Scheerder Methotrexate loaded SAE coated coronary stents reduce neointimal hyperplasia in a porcine coronary model Heart, February 1, 2004; 90(2): 195 - 199. [Abstract] [Full Text] [PDF] |
||||
![]() |
M.-A. Renault, S. Jalvy, I. Belloc, S. Pasquet, S. Sena, M. Olive, C. Desgranges, and A.-P. Gadeau AP-1 Is Involved in UTP-Induced Osteopontin Expression in Arterial Smooth Muscle Cells Circ. Res., October 3, 2003; 93(7): 674 - 681. [Abstract] [Full Text] [PDF] |
||||
![]() |
C. I. Seye, N. Yu, R. Jain, Q. Kong, T. Minor, J. Newton, L. Erb, F. A. Gonzalez, and G. A. Weisman The P2Y2 Nucleotide Receptor Mediates UTP-induced Vascular Cell Adhesion Molecule-1 Expression in Coronary Artery Endothelial Cells J. Biol. Chem., June 27, 2003; 278(27): 24960 - 24965. [Abstract] [Full Text] [PDF] |
||||
![]() |
<