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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:3602-3610.)
© 1997 American Heart Association, Inc.

Articles

Overexpression of the P2Y₂ Purinoceptor in Intimal Lesions of the Rat Aorta

Cheikh Ibra Seye; Alain-Pierre Gadeau; Danièle Daret; Françoise Dupuch; Philippe Alzieu; Loïc Capron; ; Claude Desgranges

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

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Abstract Extracellular nucleotides, particularly ATP, are involved in the modulation of arterial vasomotricity via P2 purinoceptors present on smooth muscle and endothelial cells. These nucleotides could also be implicated in the smooth muscle cell hyperplasia observed in intimal lesions. In this study, we tried to define the potential role of the P2Y₂ (P_2u) purinoceptor by studying its expression in normal and balloon-injured rat aortas. The cloning of a rat P2Y₂ cDNA from a rat smooth muscle cell cDNA library made it possible to study P2Y₂ expression both by Northern blot and in situ hybridization. Northern blot experiments indicated that P2Y₂ mRNA was present in rat medial aortic smooth muscle and in cultured rat aortic smooth muscle cells. In situ hybridization indicated that P2Y₂ mRNA was present in endothelial cells of the intima and in some smooth muscle cells scattered throughout the media of adult rat aortas, while almost all medial smooth muscle cells of rat embryo aorta expressed this receptor. In contrast with adult aortic media, the majority of neointimal smooth muscle cells found in aortic intimal lesions either 8 or 20 days after balloon injury were positive for P2Y₂ mRNA. Moreover, a subpopulation of neointimal cells localized at the luminal surface could be identified by a higher P2Y₂ expression than the underlying neointimal smooth muscle cells. These data showing a strong expression of the P2Y₂ purinoceptor in the neointima of injured arteries suggest that extracellular nucleotides may be involved, via this receptor, in the intimal hyperplasia and/or chronic constriction observed at the lesion site, and consequently in the restenotic process.

Key Words: nucleotides • purinoceptors • rats • smooth muscle cells • balloon injury

	Introduction

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Intimal accumulation of arterial SMCs is an important feature in vascular diseases and is believed to contribute to the development of the intimal lesions found in atherosclerosis and postangioplasty restenosis.¹ However, the factors involved in the induction of the processes leading to this accumulation, ie, migration and proliferation, have only been partially identified on the basis of in vitro and in vivo experiments. Extracellular ATP, which is probably released in vivo from various blood or arterial cell types in a manner similar to that described in vitro,² could play an important role in activation of the SMC proliferative process, since this nucleotide has been reported to induce the mitogenic activation of vascular SMCs in culture.^{3 4 5} Because this effect is also induced by UTP,^{6 7} it could be hypothesized that this action is mediated via a common receptor to both ATP and UTP. Indeed, the effects of extracellular ATP are mediated through its binding to extracellular receptors, termed P2 purinergic receptors.^{8 9} On the basis of pharmacological experiments, three purinergic receptors have previously been suggested to be present in vascular smooth muscle; one of these receptors, the P_2u purinoceptor, also termed nucleotide receptor¹⁰ or P2Y₂ receptor according to the new developing classification,⁸ is activated by UTP in addition to ATP, and has been found to induce smooth muscle contraction.^{11 12 13} This receptor has recently been cloned from mouse neuroblastoma cells,¹⁴ rat alveolar cells,¹⁵ and human airway epithelial cells,¹⁶ which has led to the detection of P2Y₂ mRNA in various tissues such as spleen, kidney, liver, heart, lung, and skeletal muscle.

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

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Materials
The sources of the products were as follows: [-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 P_2u 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

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Cloning of Rat P2Y₂ cDNA
Reverse transcription-PCR amplification was performed on total RNA isolated from cultured aortic SMCs by using oligonucleotide primers corresponding to positions 426 to 455 and 788 to 817 of the mouse P2Y₂ sequence,¹⁴ giving a 390-bp fragment. Sequence analysis of this fragment revealed a 98% identity with the corresponding mouse sequence. This PCR product was used to screen a cDNA library of cultured rat aortic SMCs. One clone carrying an insert of 1.7 kb was isolated and sequenced. The nucleotide sequence of this clone revealed a 1122-bp open reading frame, with a 141-bp 5'-untranslated region and a 424-bp 3'-untranslated region (Fig 1). The rat coding sequence is 94% identical to that of mouse P2Y₂¹⁴ and 84% to that of the human sequence.¹⁶ The rat SMC coding sequence differs only by 4 nucleotides in comparison with the recently reported rat alveolar type II cell P2Y₂ sequence.¹⁵ These differences were confirmed by sequencing a PCR fragment containing the four suspected differences and by restriction analysis. Indeed, these four differences introduced four restriction sites (Age I, Alu I, Bgl I, and HaeIII), which are not present in the rat alveolar cell sequence. A recently reported rat P2Y₂ sequence²¹ fitted well with our sequence, thus confirming the differences found with the rat alveolar cell P2Y₂ cDNA sequence.¹⁵ The rat SMC P2Y₂ open reading frame encodes a putative 374-residue protein. This protein fitted well with the structure of a 7-transmembrane domain receptor. The translated rat aortic SMC P2Y₂ sequence had two potential N-linked glycosylation sites of the NXS/T type in the N-terminal extremity.

View larger version (54K): [in this window] [in a new window]   Figure 1. Nucleotide and deduced amino acid sequences of the cloned SMC P2Y2 purinoceptor. The seven putative transmembrane domains are underlined. Potential sites of N-glycosylation NXS/T are indicated by asterisks, and amino acids differing from the rat alveolar cell sequence15 are marked by black dots. The SMC P2Y2 nucleotide sequence is available from GenBank under accession number U56839.

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).

View larger version (42K): [in this window] [in a new window]   Figure 2. Northern blot analysis of P2Y2 mRNA expression in rat aortic SMCs. Thirty micrograms of total RNA from adult rat aortic media (Aorta) and cultured adult rat aortic SMCs (aSMC) were subjected to electrophoresis and transfer as described in "Methods." The blots were first probed with labeled P2Y2 cDNA, then, after dehybridization, with a labeled 18S probe.

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).

View larger version (113K): [in this window] [in a new window]   Figure 3. In situ detection of P2Y2 mRNA in aorta of adult and embryonic rats. Cross sections of thoracic aortas of adult (A to D) and 19-day embryonic (E to H) rats were hybridized with antisense (A and E) or sense (B and F) P2Y2 riboprobes and treated as described in "Methods" to reveal P2Y2 mRNA hybrids. Serial sections were used for immunodetection of smooth muscle -actin (C and G), von Willebrand factor (D), or PCNA (H). Arrowhead indicates endothelial cells. Magnification x40.

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).

View larger version (111K): [in this window] [in a new window]   Figure 4. In situ detection of P2Y2 mRNA in injured rat aorta. Cross sections of 8-day (A to D) or 20-day (E to H) balloon-injured rat aortas were hybridized with antisense (A and E) or sense (B and F) P2Y2 probes as in the legend to Fig 3. C and G, PCNA immunodetection. D and H, smooth muscle -actin immunodetection. Small and large arrowheads indicate the luminal edge of the aorta and the internal elastic lamina, respectively. Magnification x40.

View larger version (103K): [in this window] [in a new window]   Figure 5. Overexpression of P2Y2 mRNA in cells at the luminal edge of the rat aortic neointima. Cross sections of 8-day (A) or 20-day (B) balloon-injured rat aortas were hybridized with antisense P2Y2 probe as in the legend to Fig 4, except that the staining was developed for only 3 hours instead of overnight as in Figs 4A and 4E. Magnification x40.

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).

View larger version (66K): [in this window] [in a new window]   Figure 6. P2Y2 mRNA expression in cultured rat aortic SMCs. Thirty micrograms of total RNA from cultured adult rat aortic SMCs (aSMC) made quiescent (Quiesc) or proliferating (Prolif), cultured rat embryo aortic SMCs (eSMC), cultured SMCs from experimental intimal thickening 15 days after rat aorta injury (IT15), and cultured rat aortic SMCs from the A10 cell line (A10) were subjected to electrophoresis and transfer as described in "Methods." The blots were first probed with labeled P2Y2 cDNA, and then, after dehybridization, with a labeled 18S probe (top). mRNA of quiescent SMCs stimulated for various times by angiotensin II or PDGF was similarly analyzed for P2Y2 expression (bottom).

View larger version (109K): [in this window] [in a new window]   Figure 7. In situ detection of P2Y2 mRNA in cultured rat aortic SMCs. P2Y2 mRNA expression in quiescent cultured aortic SMCs grown on glass slides was studied by hybridization with antisense or sense (inset) P2Y2 probes. Magnification x20 with a 50% reduction for the inset.

View this table: [in this window] [in a new window]   Table 1. Influence of Extracellular UTP on Proliferation of Cultured SMCs That Do or Do Not Express the P2Y2 Receptor

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The use of a rat P2Y₂ cDNA probe isolated from a rat aortic SMC cDNA library has allowed us to demonstrate the expression of P2Y₂ mRNA in normal and injured rat aortas both by Northern blot and in situ hybridization techniques. In normal adult aorta, P2Y₂ mRNA was found in the endothelial cell lining, while a sustained expression of this receptor was detected in about 25% of SMCs disseminated in all the media. In contrast, the P2Y₂ mRNA was detected in all medial SMCs of fetal aortas and in most of the cells of intimal lesions obtained after aortic ballooning, with an overexpression in cells lining the lumen, both 1 and 3 weeks after injury.

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 SMCs^{3 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 thickening^{33 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 factors^{2 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 P_2y (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

 

DMEM = Dulbecco's modified Eagle's medium PCR = polymerase chain reaction PCNA = proliferating cell nuclear antigen PDGF = platelet-derived growth factor SMC = smooth muscle cells

	Acknowledgments

 
This work was supported by grants from l'Institut National de la Santé et de la Recherche Médicale, from Ministère de l'Enseignement Supérieur et de la Recherche (93C.0071), and from Etablissement Public Régional d'Aquitaine (93.03062) and by fellowships from Ministère de l'Education Nationale du Sénégal (to C. Seye). The authors also thank Pr G. Gabbiani and Dr M.L. Bochaton-Piallat for the gift of IT15 cells, and Isabelle Belloc for her excellent technical assistance.

Received April 18, 1997; accepted July 29, 1997.

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