Endothelial Cell-Specific Deletion of P2Y2 Receptor Promotes Plaque Stability in Atherosclerosis-Susceptible ApoE-Null Mice.

OBJECTIVE: Nucleotide P2Y2 receptor (P2Y2R) contributes to vascular inflammation by increasing vascular cell adhesion molecule-1 expression in endothelial cells (EC), and global P2Y2R deficiency prevents fatty streak formation in apolipoprotein E null (ApoE-/-) mice. Because P2Y2R is ubiquitously expressed in vascular cells, we investigated the contribution of endothelial P2Y2R in the pathogenesis of atherosclerosis. APPROACH AND RESULTS: EC-specific P2Y2R-deficient mice were generated by breeding VEcadherin5-Cre mice with the P2Y2R floxed mice. Endothelial P2Y2R deficiency reduced endothelial nitric oxide synthase activity and significantly altered ATP- and UTP (uridine 5'-triphosphate)-induced vasorelaxation without affecting vasodilatory responses to acetylcholine. Telemetric blood pressure and echocardiography measurements indicated that EC-specific P2Y2R-deficient mice did not develop hypertension. We investigated the role of endothelial P2Y2R in the development of atherosclerotic lesions by crossing the EC-specific P2Y2R knockout mice onto an ApoE-/- background and evaluated lesion development after feeding a standard chow diet for 25 weeks. Histopathologic examination demonstrated reduced atherosclerotic lesions in the aortic sinus and entire aorta, decreased macrophage infiltration, and increased smooth muscle cell and collagen content, leading to the formation of a subendothelial fibrous cap in EC-specific P2Y2R-deficient ApoE-/- mice. Expression and proteolytic activity of matrix metalloproteinase-2 was significantly reduced in atherosclerotic lesions from EC-specific P2Y2R-deficient ApoE-/- mice. Furthermore, EC-specific P2Y2R deficiency inhibited nitric oxide production, leading to significant increase in smooth muscle cell migration out of aortic explants. CONCLUSIONS: EC-specific P2Y2R deficiency reduces atherosclerotic burden and promotes plaque stability in ApoE-/- mice through impaired macrophage infiltration acting together with reduced matrix metalloproteinase-2 activity and increased smooth muscle cell migration.

Direct Evidence for P2Y2 Receptor Involvement in Vascular Response to Injury.

OBJECTIVES: Extracellular nucleotide release at the site of arterial injury mediates the proliferation and migration of vascular smooth muscle cells. Our aim was to investigate the role of the P2Y2 nucleotide receptor (P2Y2R) in neointimal hyperplasia. Approach and Results: Vascular injury was induced by the implantation of a polyethylene cuff around the femoral artery in wild-type and P2Y2R-deficient mice (P2Y2R-/-). Electron microscopy was used to analyze monocyte and lymphocyte influx to the intima 36 h after injury. Compared to wild-type littermates, P2Y2R-/- mice exhibited a 3-fold decreased number of mononuclear leukocytes invading the intima (p < 0.05). Concomitantly, the migration of smooth muscle cells was decreased by more than 60% (p < 0.05), resulting in a sharp inhibition of intimal thickening formation in P2Y2R-/- mice (n = 15) 14 days after cuff placement. In vitro, loss of P2Y2R significantly impaired monocyte migration in response to nucleotide agonists. Furthermore, transgenic rats overexpressing the P2Y2R developed accelerated intimal lesions resulting in more than 95% luminal stenosis (p < 0.05, n = 10). CONCLUSIONS: Loss- and gain-of-function approaches established direct evidence for P2Y2R involvement in neointimal hyperplasia. Specific anti-P2Y2R therapies may be used against restenosis and bypass graft failure.

Basal ATP release signals through the P2Y2 receptor to maintain the differentiated phenotype of vascular smooth muscle cells.

BACKGROUND AND AIMS: Vascular smooth muscle cell (VSMC) dedifferentiation contributes substantively to vascular disease. VSMCs spontaneously release low levels of ATP that modulate vessel contractility, but it is unclear if autocrine ATP signaling in VSMCs is critical to the maintenance of the VSMC contractile phenotype. METHODS: We used pharmacological inhibitors to block ATP release in human aortic smooth muscle cells (HASMCs) for studying changes in VSMC differentiation marker gene expression. We employed RNA interference and generated mice with SMC-specific inducible deletion of the P2Y2 receptor (P2Y2R) gene to evaluate resulting phenotypic alterations. RESULTS: HASMCs constitutively release low levels of ATP that when blocked results in a significant decrease in VSMC differentiation marker gene expression, including smooth muscle actin (SMA), smooth muscle myosin heavy chain (SMMHC), SM-22α and calponin. Basal release of ATP represses transcriptional activation of the Krüppel-Like Factor 4 (KFL4) thereby preventing platelet-derived growth factor-BB (PDGF-BB) from inhibiting expression of SMC contractile phenotype markers. SMC-restricted conditional deletion of P2Y2R evoked dedifferentiation characterized by decreases in aortic contractility and contractile phenotype markers expression. This loss was accompanied by a transition to the synthetic phenotype with the acquisition of extracellular matrix (ECM) proteins characteristic of dedifferentiation, such as osteopontin and vimentin. CONCLUSIONS: Our data establish the first direct evidence that an autocrine ATP release mechanism maintains SMC cytoskeletal protein expression by inhibiting VSMCs from transitioning to a synthetic phenotype, and further demonstrate that activation of the P2Y2R by basally released ATP is required for maintenance of the differentiated VSMC phenotype.

P2Y2 receptor-mediated lymphotoxin-α secretion regulates intercellular cell adhesion molecule-1 expression in vascular smooth muscle cells.

The proinflammatory cytokine lymphotoxin-α (LTA) is thought to contribute to the pathogenesis of atherosclerosis. However, the mechanisms that regulate its expression in vascular smooth muscle cells (VSMC) are poorly understood. The ability of exogenous nucleotides to stimulate LTA production was evaluated in VSMC by ELISA. The P2Y(2) nucleotide receptor (P2Y(2)R) agonist UTP stimulates a strong and sustained release of LTA from WT but not P2Y(2)R(-/-) SMC. Assessment of LTA gene transcription by LTA promoter-luciferase construct indicated that LTA levels are controlled at the level of transcription. We show using RNAi techniques that knockdown of the actin-binding protein filamin-A (FLNa) severely impaired nucleotide-induced Rho activation and consequent Rho-mediated LTA secretion. Reintroduction of FLNa in FLNa RNAi SMC rescued UTP-induced LTA expression. In addition, we found that UTP-stimulated LTA secretion is not sensitive to brefeldin A, which blocks the formation of vesicles involved in protein transport from the endoplasmic reticulum to the Golgi apparatus, suggesting that P2Y(2)R/filamin-mediated secretion of LTA is independent of the endoplasmic reticulum/Golgi secretory vesicle route. Furthermore, UTP selectively induces ICAM-1 expression in WT but not SMC expressing a truncated P2Y(2)R deficient in LTA secretion. These data suggest that P2Y(2)R recruits FLNa to provide a cytoskeletal scaffold necessary for Rho signaling pathway upstream of LTA release and subsequent stimulation of ICAM-1 expression on vascular smooth muscle cells.

P2Y2 nucleotide receptors mediate metalloprotease-dependent phosphorylation of epidermal growth factor receptor and ErbB3 in human salivary gland cells.

The G protein-coupled receptor P2Y(2) nucleotide receptor (P2Y(2)R) has been shown to be up-regulated in a variety of tissues in response to stress or injury. Recent studies have suggested that P2Y(2)Rs may play a role in immune responses, wound healing, and tissue regeneration via their ability to activate multiple signaling pathways, including activation of growth factor receptors. Here, we demonstrate that in human salivary gland (HSG) cells, activation of the P2Y(2)R by its agonist induces phosphorylation of ERK1/2 via two distinct mechanisms, a rapid, protein kinase C-dependent pathway and a slower and prolonged, epidermal growth factor receptor (EGFR)-dependent pathway. The EGFR-dependent stimulation of UTP-induced ERK1/2 phosphorylation in HSG cells is inhibited by the adamalysin inhibitor tumor necrosis factor-alpha protease inhibitor or by small interfering RNA that selectively silences ADAM10 and ADAM17 expression, suggesting that ADAM metalloproteases are required for P2Y(2)R-mediated activation of the EGFR. G protein-coupled receptors have been shown to promote proteolytic release of EGFR ligands; however, neutralizing antibodies to known ligands of the EGFR did not inhibit UTP-induced EGFR phosphorylation. Immunoprecipitation experiments indicated that UTP causes association of the EGFR with another member of the EGF receptor family, ErbB3. Furthermore, stimulation of HSG cells with UTP induced phosphorylation of ErbB3, and silencing of ErbB3 expression inhibited UTP-induced phosphorylation of both ErbB3 and EGFR. UTP-induced phosphorylation of ErbB3 and EGFR was also inhibited by silencing the expression of the ErbB3 ligand neuregulin 1 (NRG1). These results suggest that P2Y(2)R activation in salivary gland cells promotes the formation of EGFR/ErbB3 heterodimers and metalloprotease-dependent neuregulin 1 release, resulting in the activation of both EGFR and ErbB3.

Endothelial pannexin 1-TRPV4 channel signaling lowers pulmonary arterial pressure in mice.

Pannexin 1 (Panx1), an ATP-efflux pathway, has been linked with inflammation in pulmonary capillaries. However, the physiological roles of endothelial Panx1 in the pulmonary vasculature are unknown. Endothelial transient receptor potential vanilloid 4 (TRPV4) channels lower pulmonary artery (PA) contractility and exogenous ATP activates endothelial TRPV4 channels. We hypothesized that endothelial Panx1-ATP-TRPV4 channel signaling promotes vasodilation and lowers pulmonary arterial pressure (PAP). Endothelial, but not smooth muscle, knockout of Panx1 increased PA contractility and raised PAP in mice. Flow/shear stress increased ATP efflux through endothelial Panx1 in PAs. Panx1-effluxed extracellular ATP signaled through purinergic P2Y2 receptor (P2Y2R) to activate protein kinase Cα (PKCα), which in turn activated endothelial TRPV4 channels. Finally, caveolin-1 provided a signaling scaffold for endothelial Panx1, P2Y2R, PKCα, and TRPV4 channels in PAs, promoting their spatial proximity and enabling signaling interactions. These results indicate that endothelial Panx1-P2Y2R-TRPV4 channel signaling, facilitated by caveolin-1, reduces PA contractility and lowers PAP in mice.

Binding of the P2Y2 nucleotide receptor to filamin A regulates migration of vascular smooth muscle cells.

The functional expression of the G protein-coupled P2Y(2) nucleotide receptor (P2Y(2)R) has been associated with proliferation and migration of vascular smooth muscle cells (SMCs), two processes involved in atherosclerosis and restenosis. Activation of the P2Y(2)R causes dynamic reorganization of the actin cytoskeleton, which transmits biochemical signals and forces necessary for cell locomotion, suggesting that P2Y(2)Rs may be linked to the actin cytoskeleton. Here, we identified filamin A (FLNa) as a P2Y(2)R-interacting protein using a yeast 2-hybrid system screen with the C-terminal region of the P2Y(2)R as bait. The FLNa binding site in the P2Y(2)R is localized between amino acids 322 and 333. Deletion of this region led to selective loss of FLNa binding to the P2Y(2)R and abolished Tyr phosphorylation of FLNa induced by the P2Y(2)R agonist UTP. Using both time-lapse microscopy and the Transwell cell migration assay, we showed that UTP significantly increased SMC spreading on collagen I (6.8 fold; P < or = 0.01) and migration (3.6 fold; P < or = 0.01) of aortic SMCs isolated from wild-type mice, as compared with unstimulated SMCs. UTP-induced spreading and migration of aortic SMCs did not occur with cells isolated from P2Y(2)R knockout mice. Expression of the full-length P2Y(2)R in SMCs isolated from P2Y(2)R knockout mice restored both UTP-induced spreading and migration. In contrast, UTP-induced spreading and migration did not occur in SMCs isolated from P2Y(2)R knockout mice transfected with a mutant P2Y(2)R that does not bind FLNa. Furthermore, ex vivo studies showed that both ATP and UTP (10 micromol/L) promoted migration of SMCs out of aortic explants isolated from wild-type but not P2Y(2)R knockout mice. Thus, this study demonstrates that P2Y(2)R/FLNa interaction selectively regulates spreading and migration of vascular SMCs.

Interleukin-1beta enhances nucleotide-induced and alpha-secretase-dependent amyloid precursor protein processing in rat primary cortical neurons via up-regulation of the P2Y(2) receptor.

The heterologous expression and activation of the human P2Y(2) nucleotide receptor (P2Y(2)R) in human 1321N1 astrocytoma cells stimulates alpha-secretase-dependent cleavage of the amyloid precursor protein (APP), causing extracellular release of the non-amyloidogenic protein secreted amyloid precursor protein (sAPPalpha). To determine whether a similar response occurs in a neuronal cell, we analyzed whether P2Y(2)R-mediated production of sAPPalpha occurs in rat primary cortical neurons (rPCNs). In rPCNs, P2Y(2)R mRNA and receptor activity were virtually absent in quiescent cells, whereas overnight treatment with the pro-inflammatory cytokine interleukin-1beta (IL-1beta) up-regulated both P2Y(2)R mRNA expression and receptor activity by four-fold. The up-regulation of the P2Y(2)R was abrogated by pre-incubation with Bay 11-7085, an IkappaB-alpha phosphorylation inhibitor, which suggests that P2Y(2)R mRNA transcript levels are regulated through nuclear factor-kappa-B (NFkappaB) signaling. Furthermore, the P2Y(2)R agonist Uridine-5'-triphosphate (UTP) enhanced the release of sAPPalpha in rPCNs treated with IL-1beta or transfected with P2Y(2)R cDNA. UTP-induced release of sAPPalpha from rPCNs was completely inhibited by pre-treatment of the cells with the metalloproteinase inhibitor TACE inhibitor (TAPI-2) or the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002, and was partially inhibited by the MAPK/extracellular signal-regulated kinase inhibitor U0126 and the protein kinase C inhibitor GF109203. These data suggest that P2Y(2)R-mediated release of sAPPalpha from cortical neurons is directly dependent on a disintegrin and metalloproteinase (ADAM) 10/17 and PI3K activity, whereas extracellular signal-regulated kinase 1/2 and PI3K activity may indirectly regulate APP processing. These results demonstrate that elevated levels of pro-inflammatory cytokines associated with neurodegenerative diseases, such as IL-1beta, can enhance non-amyloidogenic APP processing through up-regulation of the P2Y(2)R in neurons.

P2Y2 nucleotide receptor activation up-regulates vascular cell adhesion molecule-1 [corrected] expression and enhances lymphocyte adherence to a human submandibular gland cell line.

Sjögren's syndrome (SS) is a chronic inflammatory autoimmune disease that causes salivary and lacrimal gland tissue destruction resulting in impaired secretory function. Although lymphocytic infiltration of salivary epithelium is associated with SS, the mechanisms involved have not been adequately elucidated. Our previous studies have shown that the G protein-coupled P2Y2 nucleotide receptor (P2Y2R) is up-regulated in response to damage or stress of salivary gland epithelium, and in salivary glands of the NOD.B10 mouse model of SS-like autoimmune exocrinopathy. Additionally, we have shown that P2Y2R activation up-regulates vascular cell adhesion molecule-1 (VCAM-1) expression in endothelial cells leading to the binding of monocytes. The present study demonstrates that activation of the P2Y2R in dispersed cell aggregates from rat submandibular gland (SMG) and in human submandibular gland ductal cells (HSG) up-regulates the expression of VCAM-1. Furthermore, P2Y2R activation mediated the up-regulation of VCAM-1 expression in HSG cells leading to increased adherence of lymphocytic cells. Inhibitors of EGFR phosphorylation and metalloprotease activity abolished P2Y2R-mediated VCAM-1 expression and decreased lymphocyte binding to HSG cells. Moreover, silencing of EGFR expression abolished UTP-induced VCAM-1 up-regulation in HSG cells. These results suggest that P2Y2R activation in salivary gland cells increases the EGFR-dependent expression of VCAM-1 and the binding of lymphocytes, a pathway relevant to inflammation associated with SS.

TLR3 deficiency exacerbates the loss of epithelial barrier function during genital tract Chlamydia muridarum infection.

PROBLEM: Chlamydia trachomatis infections are often associated with acute syndromes including cervicitis, urethritis, and endometritis, which can lead to chronic sequelae such as pelvic inflammatory disease (PID), chronic pelvic pain, ectopic pregnancy, and tubal infertility. As epithelial cells are the primary cell type productively infected during genital tract Chlamydia infections, we investigated whether Chlamydia has any impact on the integrity of the host epithelial barrier as a possible mechanism to facilitate the dissemination of infection, and examined whether TLR3 function modulates its impact. METHOD OF STUDY: We used wild-type and TLR3-deficient murine oviduct epithelial (OE) cells to ascertain whether C. muridarum infection had any effect on the epithelial barrier integrity of these cells as measured by transepithelial resistance (TER) and cell permeability assays. We next assessed whether infection impacted the transcription and protein function of the cellular tight-junction (TJ) genes for claudins1-4, ZO-1, JAM1 and occludin via quantitative real-time PCR (qPCR) and western blot. RESULTS: qPCR, immunoblotting, transwell permeability assays, and TER studies show that Chlamydia compromises cellular TJ function throughout infection in murine OE cells and that TLR3 deficiency significantly exacerbates this effect. CONCLUSION: Our data show that TLR3 plays a role in modulating epithelial barrier function during Chlamydia infection of epithelial cells lining the genital tract. These findings propose a role for TLR3 signaling in maintaining the integrity of epithelial barrier function during genital tract Chlamydia infection, a function that we hypothesize is important in helping limit the chlamydial spread and subsequent genital tract pathology.

The P2Y2 nucleotide receptor is an inhibitor of vascular calcification.

BACKGROUND AND AIMS: Mutations in the 5'-nucleotidase ecto (NT5E) gene that encodes CD73, a nucleotidase that converts AMP to adenosine, are linked to arterial calcification. However, the role of purinergic receptor signaling in the pathology of intimal calcification is not well understood. In this study, we examined whether extracellular nucleotides acting via P2Y2 receptor (P2Y2R) modulate arterial intimal calcification, a condition highly correlated with cardiovascular morbidity. METHODS: Apolipoprotein E, P2Y2R double knockout mice (ApoE-/-P2Y2R-/-) were used to determine the effect of P2Y2R deficiency on vascular calcification in vivo. Vascular smooth muscle cells (VSMC) isolated from P2Y2R-/- mice grown in high phosphate medium were used to assess the role of P2Y2R in the conversion of VSMC into osteoblasts. Luciferase-reporter assays were used to assess the effect of P2Y2R on the transcriptional activity of Runx2. RESULTS: P2Y2R deficiency in ApoE-/- mice caused extensive intimal calcification despite a significant reduction in atherosclerosis and macrophage plaque content. The ectoenzyme apyrase that degrades nucleoside di- and triphosphates accelerated high phosphate-induced calcium deposition in cultured VSMC. Expression of P2Y2R inhibits calcification in vitro inhibited the osteoblastic trans-differentiation of VSMC. Mechanistically, expression of P2Y2R inhibited Runx2 transcriptional activation of an osteocalcin promoter driven luciferase reporter gene. CONCLUSIONS: This study reveals a role for vascular P2Y2R as an inhibitor of arterial intimal calcification and provides a new mechanistic insight into the regulation of the osteoblastic trans-differentiation of SMC through P2Y2R-mediated Runx2 antagonism. Given that calcification of atherosclerotic lesions is a significant clinical problem, activating P2Y2R may be an effective therapeutic approach for treatment or prevention of vascular calcification.

Differential coupling of the P2Y1 receptor to Galpha14 and Galphaq/11 proteins during the development of the rat salivary gland.

UNLABELLED: In rat submandibular gland (SMG), the P2Y1 receptor (P2Y1R) mediates increases in the intracellular calcium concentration, [Ca2+]i that diminish as the animal ages from 1 to 4-6 weeks. However, P2Y1R mRNA levels do not change with age, suggesting that the age-dependent decrease in the [Ca2+]i response to P2Y1R agonists may be due to alterations in the activity of a component of the P2Y1R signalling pathway. OBJECTIVES: To assess whether the decrease in P2Y1R-mediated intracellular calcium signalling in SMG cells as rats age is due to a decrease in P2Y1R coupling to G proteins or to a decrease in the expression of a cognate G protein. DESIGN: SMG cells were isolated from Sprague-Dawley rats. P2Y1R function was assessed by measuring 2-MeSADP-induced increases in [Ca2+]i and ERK1/2 activation. P2Y(1)R-mediated activation of G proteins was determined by the [35S]GTPgammaS binding assay. Gq protein expression was determined by RT-PCR, Northern, and Western analysis. RESULTS: In SMG cells from 1-week-old rats, two bands (52 and 42kDa) were detected using anti-Galpha14 antibody, whereas in SMG cells from 4- to 6-week-old rats only the 42 kDa band was detected. Furthermore, 2-MeSADP-induced GTPgamma35S binding to Galpha14 and Galphaq/11 decreases in SMG cells from 4- to 6-week-old rats as compared to 1-week-old rats. CONCLUSIONS: These findings suggest that the age-dependent decrease in P2Y1R-mediated intracellular calcium signalling in rat SMG cells is due to a loss of 52 kDa Galpha14 and indicate the differential coupling of the P2Y1R to Galpha14 and Galphaq/11 as the gland develops.

P2 receptors in health and disease.

In the past ten years since the discovery and cloning of members of the P2 receptor family, rapid progress has been made in the field regarding the function and pharmacology of different P2 receptor subtypes. This research resulted in identifying these receptors as important players in the pathology of atherosclerosis, cystic fibrosis, neurodegenerative and autoimmune diseases, among other disorders. The signalling mechanisms whereby P2 receptors mediate pathogenesis are not clear in most cases. Future studies in this field will focus on the integration of signalling pathways coupled to P2 receptors and the generation of specific agonists/antagonists for each receptor subtype to provide strategies for the treatment of a variety of diseases.

Deletion of P2Y2 receptor reveals a role for lymphotoxin-α in fatty streak formation.

BACKGROUND: Lymphotoxin alpha (LTα) is expressed in human atherosclerotic lesions and genetic variations in the LTα pathway have been linked to myocardial infarction. Activation of the P2Y2 nucleotide receptor (P2Y2R) regulates the production of LTα. in vitro. We aimed to uncover a potential pathway linking purinergic receptor to LTα-mediated inflammatory processes pivotal to the early stages of atherosclerosis in apolipoprotein E (ApoE(-)(/)(-)) deficient mice. METHODS AND RESULTS: En face immunostaining revealed that P2Y2R and VCAM-1 are preferentially expressed in the atherosclerosis prone site of the mouse aortic sinus. Deletion of the P2Y2R gene suppresses VCAM-1 expression. Compared with ApoE(-)(/)(-) mice, ApoE(-)(/)(-) mice lacking the P2Y2R gene (ApoE(-)(/)(-)/P2Y2R(-)(/)(-)) did not develop fatty streak lesions when fed a standard chow diet for 15weeks. Systemic and CD4(+) T cell production of the pro-inflammatory cytokine lymphotoxin-alpha (LTα) were specifically inhibited in ApoE(-)(/)(-)/P2Y2R(-)(/)(-)mice. Anti-LTα preventive treatment was initiated in ApoE(-)(/)(-)mice with intraperitoneal administration of recombinant human tumor necrosis factor receptor 1 fusion protein (TNFR1-Fc) on 5 consecutive days before the disease onset. Remarkably, none of the TNFR1:Fc-treated ApoE(-)(/)(-)mice exhibited atherosclerotic lesions at any developmental stage. SIGNIFICANCE: ApoE(-)(/)(-) mice deficient in P2Y2R exhibit low endothelial cell VCAM-1 levels, decreased production of LTα and delayed onset of atherosclerosis. These data suggest that targeting this nucleotide receptor could be an effective therapeutic approach in atherosclerosis.

The P2Y2 receptor mediates uptake of matrix-retained and aggregated low density lipoprotein in primary vascular smooth muscle cells.

BACKGROUND AND AIMS: The internalization of aggregated low-density lipoproteins (agLDL) mediated by low-density lipoprotein receptor related protein (LRP1) may involve the actin cytoskeleton in ways that differ from the endocytosis of soluble LDL by the LDL receptor (LDLR). This study aims to define novel mechanisms of agLDL uptake through modulation of the actin cytoskeleton, to identify molecular targets involved in foam cell formation in vascular smooth muscle cells (VSMCs). The critical observation that formed the basis for these studies is that under pathophysiological conditions, nucleotide release from blood-derived and vascular cells activates SMC P2Y2 receptors (P2Y2Rs) leading to rearrangement of the actin cytoskeleton and cell motility. Therefore, we tested the hypothesis that P2Y2R activation mediates agLDL uptake by VSMCs. METHODS: Primary VSMCs were isolated from aortas of wild type (WT) C57BL/6 and.P2Y2R-/- mice to investigate whether P2Y2R activation modulates LRP1 expression. Cells were transiently transfected with cDNA encoding a hemagglutinin-tagged (HA-tagged) WT P2Y2R, or a mutant P2Y2R that unlike the WT P2Y2R does not bind the cytoskeletal actin-binding protein filamin-A (FLN-A). RESULTS: P2Y2R activation significantly increased agLDL uptake, and LRP1 mRNA expression decreased in P2Y2R-/- VSMCs versus WT. SMCs, expressing P2Y2R defective in FLN-A binding, exhibit 3-fold lower LDLR expression levels than SMCs expressing WT P2Y2R, while cells transfected with WT P2Y2R show greater agLDL uptake in both WT and P2Y2R-/- VSMCs versus cells transfected with the mutant P2Y2R. CONCLUSIONS: Together, these results show that both LRP1 and LDLR expression and agLDL uptake are regulated by P2Y2R in VSMCs, and that agLDL uptake due to P2Y2R activation is dependent upon cytoskeletal reorganization mediated by P2Y2R binding to FLN-A.

Functional P2Y2 nucleotide receptors mediate uridine 5'-triphosphate-induced intimal hyperplasia in collared rabbit carotid arteries.

BACKGROUND: Extracellular uridine 5'-triphosphate (UTP) induces mitogenic activation of smooth muscle cells (SMCs) through binding to P2Y2 nucleotide receptors. P2Y2 receptor mRNA is upregulated in intimal lesions of rat aorta, but it is unclear how this G-protein-coupled receptor contributes to development of intimal hyperplasia. METHODS AND RESULTS: This study used a silicone collar placed around rabbit carotid arteries to induce vascular injury and intimal thickening. Collar placement caused rapid upregulation of P2Y2 receptor mRNA in medial SMCs before appearance of neointima. Fura-2 digital imaging of single SMCs was used to measure changes in myoplasmic calcium concentration (Ca(m)) in response to P2Y receptor agonists. In contrast to UDP, activation by UTP or adenosine 5'-triphosphate (ATP) greatly increased Ca(m), which indicates upregulation of functional P2Y2 receptors at which UTP and ATP are equipotent agonists. The number of responsive cells was significantly greater for freshly dispersed SMCs from collared arteries than for controls. Perivascular infusion of UTP (100 micromol/L) within the collar significantly enhanced neointimal development. Intimas that resulted from UTP exposure were infiltrated by macrophages. Moreover, increased expression of osteopontin occurred in response to in situ application of UTP. ATP or UTP also stimulated osteopontin expression in cultured SMCs in a dose-dependent manner. Furthermore, P2Y2 antisense oligonucleotide inhibited osteopontin expression induced by UTP. CONCLUSIONS: These findings indicate for the first time a role for the UTP/ATP receptor, P2Y2, in development of intimal hyperplasia associated with atherosclerosis and restenosis.

Mechanisms for inhibition of P2 receptors signaling in neural cells.

Trophic factors are required to ensure neuronal viability and regeneration after neural injury. Although abundant information is available on the factors that cause the activation of astrocytes, little is known about the molecular mechanisms underlying the regulation of this process. Nucleotides released into the extracellular space from injured or dying neural cells can activate astrocytes via P2 nucleotide receptors. After a brief historical review and update of novel P2 receptor antagonists, this article focuses on recent advancements toward understanding molecular mechanisms that regulate G protein-coupled P2Y receptor signaling. Among P2Y receptor subtypes, the heptahelical P2Y2 nucleotide receptor interacts with vitronectin receptors via an RGD sequence in the first extracellular loop, and this interaction is required for effective signal transduction to activate mitogen-activated protein kinases ERK1/2, to mobilize intracellular calcium stores via activation of phospholipase C, protein kinase C isoforms, and to activate focal adhesion kinase and other signaling events. Ligation of vitronectin receptors with specific antibodies caused an inhibition of P2Y2 receptor-induced ERK1/2 and p38 phosphorylation and P2Y2 receptor-induced cytoskeleton rearrangement and DNA synthesis. Structure-function studies have identified agonist-induced phosphorylation of the C-terminus of the P2Y2 receptor, an important mechanism for receptor desensitization. Understanding selective mechanisms for regulating P2Y2 receptor signaling could provide novel targets for therapeutic strategies in the management of brain injury, synaptogenesis, and neurological disorders.

7-Ketocholesterol induces reversible cytochrome c release in smooth muscle cells in absence of mitochondrial swelling.

OBJECTIVE: 7-Ketocholesterol, a major oxysterol in oxidized low-density lipoproteins in advanced atherosclerotic plaques, induces vascular smooth muscle cell (SMC) death. We investigated whether cytochrome c release participated in SMC death induced by 7-ketocholesterol and whether the processes were reversible. METHODS: SMC cultures derived from the rabbit aorta were exposed to 25 microM 7-ketocholesterol. Cytochrome c and Bax were studied by means of immunofluorescence and immunoblotting, apoptosis by the TUNEL technique and mitochondrial structure by transmission electron microscopy. RESULTS: 7-Ketocholesterol induced rapid upregulation of the proapoptotic protein Bax and its translocation from cytosol into the mitochondria (4 h). This was followed by mitochondrial cytochrome c release (65% at 8 h) into the cytosol, which was almost complete at 16 h. The mitochondria became spherical and ultracondensed, without showing signs of lysis. They clustered around the nucleus and were wrapped by wide cisternae of the rough endoplasmic reticulum. Cytochrome c release was not blocked by the pan-caspase inhibitor zVAD-fmk, in contrast to DNA fragmentation and SMC loss. Interestingly, upon removal of 7-ketocholesterol after 16 h and re-exposure to serum for 24 h, the mitochondrial cytochrome c content, their transmembrane potential and TUNEL labelling normalised and SMC loss decreased. However, none of these cell death markers was rescued when the SMCs had been exposed to the oxysterol for 24 h. CONCLUSION: The results indicate that cytochrome c release during oxysterol-induced SMC apoptosis is not caspase-dependent and occurs as a result of a reversible mitochondrial conformational change rather than swelling and rupture of the outer membrane. The reversibility of these events suggests that the apoptotic cascade could be arrested before a point of no return.

P2Y receptors in the mammalian nervous system: pharmacology, ligands and therapeutic potential.

P2Y receptors for extracellular nucleotides are coupled to activation of a variety of G proteins and stimulate diverse intracellular signaling pathways that regulate functions of cell types that comprise the central nervous system (CNS). There are 8 different subtypes of P2Y receptor expressed in cells of the CNS that are activated by a select group of nucleotide agonists. Here, the agonist selectivity of these 8 P2Y receptor subtypes is reviewed with an emphasis on synthetic agonists with high potency and resistance to degradation by extracellular nucleotidases that have potential applications as therapeutic agents. In addition, the recent identification of a wide variety of subtype-selective antagonists is discussed, since these compounds are critical for discerning cellular responses mediated by activation of individual P2Y receptor subtypes. The functional expression of P2Y receptor subtypes in cells that comprise the CNS is also reviewed and the role of each subtype in the regulation of physiological and pathophysiological responses is considered. Other topics include the role of P2Y receptors in the regulation of blood-brain barrier integrity and potential interactions between different P2Y receptor subtypes that likely impact tissue responses to extracellular nucleotides in the CNS. Overall, current research suggests that P2Y receptors in the CNS regulate repair mechanisms that are triggered by tissue damage, inflammation and disease and thus P2Y receptors represent promising targets for the treatment of neurodegenerative diseases.