Lingwei Wang

P2 Receptor Expression Profiles in Human Vascular Smooth Muscle and Endothelial Cells.

P2 receptors mediate the actions of the extracellular nucleotides ATP, ADP, UTP, and UDP, regulating several physiologic responses including cardiac function, vascular tone, smooth muscle cell (SMC) proliferation, platelet aggregation, and the release of endothelial factors. P2 receptor characterization has been hampered by the lack of selective antagonists. The aim of the current study was to investigate the mRNA and protein expression of P2X and P2Y receptors in human SMC and in endothelial cells (EC). Smooth muscle cells were obtained from human mammary artery and EC from human umbilical vein. Using real-time PCR, the authors established quantitative mRNA assays. Protein expression was studied using Western blotting with recently developed antibodies. The P2X1 receptor was highly specific for human SMC, while the P2X4 was the highest expressed receptor in EC. The P2Y2 receptor was present in both SMC and EC. UTP-mediated effects in these cells are likely to be mediated by P2Y2 and not P2Y4 receptors since the latter had considerably lower expression. The P2Y6 receptor was expressed in both SMC and EC. The P2Y1 and surprisingly the P2Y11 receptors were the most abundantly expressed P2Y receptors in the endothelium. Overall, Western blotting confirmed the mRNA findings in most aspects, and most interestingly, indicated oligomerization of the P2Y1 receptor that may be important for its function. In conclusion, P2X1, P2Y2, and P2Y6 are the most expressed P2 receptors in SMC and are thus probably mediating the contractile and mitogenic actions of extracellular nucleotides. The P2X4, P2Y11, P2Y1, and P2Y2 are the most expressed P2 receptors in EC, and are most likely mediating release of nitric oxide, endothelium-dependent hyperpolarizing factor (EDHF), and t-PA induced by extracellular nucleotides. These findings will help to direct future cardiovascular drug development against the large P2 receptor family.

ADP Receptor P2Y12 Is Expressed in Vascular Smooth Muscle Cells and Stimulates Contraction in Human Blood Vessels

Objective—ADP plays an important role in platelet aggregation by activating P2Y12 receptors. We assessed the hypothesis that P2Y12 receptors are expressed in vascular smooth muscle cells (VSMC). Methods and Results—P2Y12 receptor mRNA was found to have a high expression among the P2 receptors in human VSMC, significantly higher than the other 2 ADP receptors (P2Y1 and P2Y13, real-time polymerase chain reaction). Western blots gave a band of 50 kD, similar to that in platelets. To unmask a P2Y12 receptor-mediated vasoconstriction by simulating the in vivo situation, vessels were precontracted to a submaximal level. 2-MeSADP stimulated contractions in vessel segments from internal mammary artery (IM), IM branches and small veins (Emax=15±6% of 60mmol/L K+ contraction, pEC50=5.6±0.6, Emax=21±1%, pEC50=6.8±0.1, and Emax=48±9%, pEC50=6.6±0.4). The selective P2Y12 antagonist AR-C67085 blocked 2-MeSADP contractions. The contraction was not reduced in patients using clopidogrel, a drug inhibiting ADP-induced platelet aggregation by blocking the P2Y12 receptor. This may be explained by the high instability of the active clopidogrel metabolite that never reaches the systemic circulation. Conclusion—ADP acting on P2Y12 receptors not only is important for platelet activation but also stimulates vasoconstriction. Stable drugs with antagonistic effects on P2Y12 receptors, affecting both platelets and VSMC, could be of double therapeutic benefit in their prevention of both thrombosis and vasospasm.

P2 receptor mRNA expression profiles in human lymphocytes, monocytes and CD34+ stem and progenitor cells

BackgroundExtracellular nucleotides (ATP, ADP, UTP and UDP) exert a wide range of biological effects in blood cells mediated by multiple ionotropic P2X receptors and G protein-coupled P2Y receptors. Although pharmacological experiments have suggested the presence of several P2 receptor subtypes on monocytes and lymphocytes, some results are contradictory. Few physiological functions have been firmly established to a specific receptor subtype, partly because of a lack of truly selective agonists and antagonists. This stimulated us to investigate the expression of P2X and P2Y receptors in human lymphocytes and monocytes with a newly established quantitative mRNA assay for P2 receptors. In addition, we describe for the first time the expression of P2 receptors in CD34+ stem and progenitor cells implicating a potential role of P2 receptors in hematopoietic lineage and progenitor/stem cell function.ResultsUsing a quantitative mRNA assay, we assessed the hypothesis that there are specific P2 receptor profiles in inflammatory cells. The P2X4 receptor had the highest expression in lymphocytes and monocytes. Among the P2Y receptors, P2Y12 and P2Y2 had highest expression in lymphocytes, while the P2Y2 and P2Y13 had highest expression in monocytes. Several P2 receptors were expressed (P2Y2, P2Y1, P2Y12, P2Y13, P2Y11, P2X1, P2X4) in CD34+ stem and progenitor cells.ConclusionsThe most interesting findings were the high mRNA expression of P2Y12 receptors in lymphocytes potentially explaining the anti-inflammatory effects of clopidogrel, P2Y13 receptors in monocytes and a previously unrecognised expression of P2X4 in lymphocytes and monocytes. In addition, for the first time P2 receptor mRNA expression patterns was studied in CD34+ stem and progenitor cells. Several P2 receptors were expressed (P2Y2, P2Y1, P2Y12, P2Y13, P2Y11, P2X1, P2X4), indicating a role in differentiation and proliferation. Thus, it is possible that specific antibodies to P2 receptors could be used to identify progenitors for monocytes, lymphocytes and megakaryocytes.

ADP Acting on P2Y13 Receptors Is a Negative Feedback Pathway for ATP Release From Human Red Blood Cells

Red blood cells may regulate tissue circulation and O2 delivery by releasing the vasodilator ATP in response to hypoxia. When released extracellularly, ATP is rapidly degraded to ADP in the circulation by ectonucleotidases. In this study, we show that ADP acting on P2Y13 receptors on red blood cells serves as a negative feedback pathway for the inhibition of ATP release. mRNA of the ADP receptor P2Y13 was highly expressed in human red blood cells and reticulocytes. The stable ADP analogue 2-MeSADP decreased ATP release from red blood cells by inhibition of cAMP. The P2Y12 and P2Y13 receptor antagonist AR-C67085 (30 &mgr;mol/L), but not the P2Y1 blocker MRS2179, inhibited the effects of 2-MeSADP. At doses where AR-C67085 only blocks P2Y12 (100 nmol/L), it had no effect. AR-C67085 and the nucleotidase apyrase increased cAMP per se, indicating a constant cAMP inhibitory effect of endogenous extracellular ADP. 2-MeSADP reduced plasma ATP concentrations in an in vivo pig model. Our results indicate that the ATP degradation product ADP inhibits ATP release by acting on the red blood cell P2Y13 receptor. This negative feedback system could be important in the control of plasma ATP levels and tissue circulation.

Positive Inotropic Effects by Uridine Triphosphate (UTP) and Uridine Diphosphate (UDP) via P2Y2 and P2Y6 Receptors on Cardiomyocytes and Release of UTP in Man During Myocardial Infarction

The aim of this study was to examine a possible role for extracellular pyrimidines as inotropic factors for the heart. First, nucleotide plasma levels were measured to evaluate whether UTP is released in patients with coronary heart disease. Then, inotropic effects of pyrimidines were examined in isolated mouse cardiomyocytes. Finally, expression of pyrimidine-selective receptors (a subgroup of the P2 receptors) was studied in human and mouse heart, using real time polymerase chain reaction, Western blot, and immunohistochemistry. Venous plasma levels of UTP were increased (57%) in patients with myocardial infarction. In electrically stimulated cardiomyocytes the stable P2Y2/4 agonist UTPγS increased contraction by 52%, similar to β1-adrenergic stimulation with isoproterenol (65%). The P2Y6-agonist UDPβS also increased cardiomyocyte contraction (35%), an effect abolished by the P2Y6-blocker MRS2578. The phospholipase C inhibitor U73122 inhibited both the UDPβS and the UTPγS-induced inotropic effect, indicating an IP3-mediated effect via P2Y6 receptors. The P2Y14 agonist UDP-glucose was without effect. Quantification of mRNA with real time polymerase chain reaction revealed P2Y2 as the most abundant pyrimidine receptor expressed in cardiomyocytes from man. Presence of P2Y6 receptor mRNA was detected in both species and confirmed at protein level with Western blot and immunohistochemistry in man. In conclusion, UTP levels are increased in humans during myocardial infarction, giving the first evidence for UTP release in man. UTP is a cardiac inotropic factor most likely by activation of P2Y2 receptors in man. For the first time we demonstrate inotropic effects of UDP, mediated by P2Y6 receptors via an IP3-dependent pathway. Thus, the extracellular pyrimidines (UTP and UDP) could be important inotropic factors involved in the development of cardiac disease.

Quantification of ADP and ATP receptor expression in human platelets.

Summary.  The mechanism of ADP‐mediated platelet activation has been difficult to unravel due to the large number of receptors for extracellular nucleotides (P2 receptors). mRNA levels in circulating platelets are very low, but have been shown to be translationally active. By optimizing mRNA extraction and using real time (RT)‐PCR we were able to establish a protocol for highly sensitive platelet mRNA quantification in human regular blood samples. In platelets from healthy volunteers, only P2X1, P2Y1 and P2Y12 were found in significant levels, with the following order of expression: P2Y12 >> P2X1 > P2Y1. Other P2 receptors (P2Y2, P2Y4, P2Y6, P2Y11, P2Y13, P2X4, P2X7) had very low expression. As a control measurement to exclude contamination, P2 receptors in buffy coat were quantified but had a completely different profile. Incubation in vitro revealed a more rapid degradation rate for P2X1 receptor mRNA than for P2Y1 and P2Y12, indicating that the level of P2X1 may be relatively higher in newly released platelets and in megacaryocytes. In conclusion, we have developed the first protocol for quantifying mRNA expression in human platelets limiting the P2 receptor drug development targets to P2Y12, P2Y1 and P2X1. Furthermore, the method could be used to study platelet expression for any gene in human materials.

Increased Mitogenic and Decreased Contractile P2 Receptors in Smooth Muscle Cells by Shear Stress in Human Vessels With Intact Endothelium

Objective—We investigated the role of shear stress in regulating P2 receptors in human umbilical vein. Methods and Results—Using a novel, computerized, biomechanical perfusion model, parallel vessel segments were randomized to simultaneous perfusion under high (25 dyn/cm2) or low (<4 dyn/cm2) shear stress at identical mean perfusion pressure (20 mm Hg) for 6 hours. In the endothelium, no significant P2 receptor mRNA differences were found. In smooth muscle cells (SMCs), high shear stress decreased P2X1 receptors, whereas P2Y2 and P2Y6 receptors were upregulated. These findings were consistent at the mRNA level (real-time reverse transcription–polymerase chain reaction), protein level (Western blot), and morphologically (immunohistochemistry). The changes were more pronounced in the subintimal layer of the media. Conclusions—Our findings suggest that shear stress might regulate gene expression in SMCs more than in the endothelium in intact vessels. Decreased expression of the contractile P2X1 receptor could lead to reduced vascular tonus and increased blood flow. Because P2Y2 and P2Y6 receptors stimulate growth and migration of SMCs, increased expression of these receptors could promote vascular remodeling induced by shear stress. The pattern of upregulation of mitogenic P2Y receptors and downregulation of contractile P2X1 receptor is similar to changes seen in the phenotypic shift from contractile to synthetic SMCs.

Transcriptional down-regulation of the platelet ADP receptor P2Y12 and clusterin in patients with systemic lupus erythematosus

Summary.  Cardiovascular complications are common in systemic lupus erythematosus (SLE) and myocardial infarctions are the leading cause of increased mortality. The ADP receptor P2Y12 plays a central role in platelet activation and the P2Y12 blocker clopidogrel reduces the incidence of cardiovascular events. Clusterin, a complement inhibitory protein suggested to be involved in the pathogenesis of SLE, has been found recently in a microarray study to be expressed at very high levels in platelets. Using a new protocol for mRNA quantification in platelets we set out to study if gene expression is altered in SLE patients compared with a healthy control group. Quantitative assay based on real‐time PCR was used to measure mRNA expression, Western blot for P2 receptor protein expression and PFA‐100 for platelet aggregation. The P2Y12 receptor expression was decreased in SLE compared to the controls (P < 0.05), while expression of P2Y1 and P2X1 were unaltered. These findings were consistent at the protein level. The clusterin mRNA expression was very high. However, SLE patients had significantly lower levels than controls (P < 0.05). Platelet aggregation was similar in both groups. It may be suggested that a decreased level of P2Y12 receptors could represent a protective response in SLE against thrombotic complications. Lowered clusterin levels could be involved in the pathogenesis of SLE due to decreased protective effects. These findings could help to achieve a better understanding of the platelet function in SLE and serve as a guide for further research and drug use.

Increased platelet purinergic sensitivity in peripheral arterial disease--a pilot study.

Peripheral arterial disease (PAD) is associated with platelet hyperaggregability as well as an increase in morbidity and mortality from myocardial infarction (MI) and stroke. Purinergic signaling has been shown, both experimentally and clinically, to play an important role in the activation of platelets. Platelets express three different purinergic receptors: P2Y1, P2Y12 and P2X1. We assessed the hypothesis that the hyperaggregability associated with PAD is partly due to an increased P2 receptor expression at the transcriptional and/or translational level. Patients with PAD (n = 8) and controls (n = 8) were studied. Using a high-resolution channelyzer, platelet shape change (PSC) was assessed by measuring the median platelet volume (MPV). The fall in free platelet count following the addition of ADP was also assessed. Real-time PCR was used to quantify the mRNA expression and Western blots to quantify the protein expression of P2 receptors in platelets. The median (and range) fall in free platelet count after adding ADP was significantly (P = 0.02) greater for patients [11% (5–24); n = 8] than for controls [0.5% (0–10); n = 8] despite using a lower concentration of ADP for the patient samples. The MPV did not differ significantly. The mRNA levels for the three P2 receptors were similar in PAD patients and controls. Western blot detected no significant differences in protein expression between these groups. Thus, platelets from PAD patients show an increased activation after stimulation with ADP (even though all patients were on aspirin). This hyperactivity was neither due to an obvious up-regulation of the mRNA levels nor to altered protein levels of P2 receptors in the platelets. It is suggested that the increased sensitivity to ADP in PAD is caused by post-receptor mechanisms.