P2Y Receptors Research Articles

Efficiency of platelet aggregation with different sensitivity of cyclooxygenase to non-steroid anti-inflammatory drugs in patients with nephrolithiasis

Objective: to establish the dependence of thrombocyte (Tc) aggregation efficiency on crosstalk-signaling pathways associated with stimulation of TR-receptor and purine P2-receptors, with different cyclooxygenase (COX) activity against the background of non-steroidal anti-inflammatory drugs (NSAIDs) administration in patients with nephrolithiasis (NLT). Material and methods. The study was prospective and included 60 patients with NLT who received non-steroid anti-inflammatory drugs (NSAIDs) for analgesia as part of lithokinetic therapy (LKT). The cohort of patients was divided into two groups, with effective (group 1, n=30) and ineffective (group 2, n=30) COX inhibition. The activity of TP receptors, purine P2 receptors (P2X1 and P2Y receptors) of platelets was assessed on a ChronoLog analyzer (USA). Agonists (ATP, ADP and Arachidonic acid) were used at EC50 and EC10 concentrations. Results. In the 1st group, after 72 hours of LCT, the activity of the TR receptor, P2 receptors of Tc decreased to the level of hyporeacti-vity. The regulation of the compensatory reaction of Tc in response to hematuria was provided through the synergism of the P2 receptors, and the TP receptor and the P2X1 receptor. In 2nd group hyperreactivity of the TP receptor and P2 receptors persisted. Optimal modulation of the compensatory reaction of Tc was provided by the synergism of the TP receptor and P2Y receptors. Conclusion. The efficiency of Tc aggregation during NSAIDs administration is determined by intracellular signalling associated with TR receptor and P2 receptors.

Machine learning-aided search for ligands of P2Y6 and other P2Y receptors.

The P2Y6 receptor, activated by uridine diphosphate (UDP), is a target for antagonists in inflammatory, neurodegenerative, and metabolic disorders, yet few potent and selective antagonists are known to date. This prompted us to use machine learning as a novel approach to aid ligand discovery, with pharmacological evaluation at three P2YR subtypes: initially P2Y6 and subsequently P2Y1 and P2Y14. Relying on extensive published data for P2Y6R agonists, we generated and validated an array of classification machine learning model using the algorithms deep learning (DL), adaboost classifier (ada), Bernoulli NB (bnb), k-nearest neighbors (kNN) classifier, logistic regression (lreg), random forest classifier (rf), support vector classification (SVC), and XGBoost (XGB) classifier models, and the common consensus was applied to molecular selection of 21 diverse structures. Compounds were screened using human P2Y6R-induced functional calcium transients in transfected 1321N1 astrocytoma cells and fluorescent binding inhibition at closely related hP2Y14R expressed in CHO cells. The hit compound ABBV-744, an experimental anticancer drug with a 6-methyl-7-oxo-6,7-dihydro-1H-pyrrolo[2,3-c]pyridine scaffold, had multifaceted interactions with the P2YR family: hP2Y6R inhibition in a non-surmountable fashion, suggesting that noncompetitive antagonism, and hP2Y1R enhancement, but not hP2Y14R binding inhibition. Other machine learning-selected compounds were either weak (experimental anti-asthmatic drug AZD5423 with a phenyl-1H-indazole scaffold) or inactive in inhibiting the hP2Y6R. Experimental drugs TAK-593 and GSK1070916 (100µM) inhibited P2Y14R fluorescent binding by 50% and 38%, respectively, and all other compounds by < 20%. Thus, machine learning has led the way toward revealing previously unknown modulators of several P2YR subtypes that have varied effects.

Abstract 5416: Purinergic P2Y6 receptor antagonists as potential anti tumor agents

Abstract Introduction: Cancer places a tremendous strain on society in both developed and economically less developed nations. It remains the most recurring and deadliest disease despite rapid advances in treatment modalities. The major contributors are the prevalence of known risk factors, urbanization, population growth and ageing. Cancer resurgence depends on the organ in which the tumor first appears, immune response, patient’s characteristics, disease stage and tumor microenvironment (TME). Among the several inflammatory molecules found within the TMEs, the levels of adenosine triphosphate (ATP) has been found to be high. In healthy tissue, the concentration of ATP in the extracellular space (eATP) is negligible and does not accumulate significantly due to the activity of ectonucleotidases. However, tissue injury, cellular stress and chemotherapy mediated cell death can increase the levels of ATP in the TME. The eATP exerts its effect mainly through purinergic P2 receptors expressed by most malignant cells. We have previously shown that eATP promoted migration and invasion of cancer cells in vitro through increased expression of the enzyme cyclooxygenase 2 (COX-2) (Sharma et al., 2021). We showed that eATP mediated COX-2 expression is through P2 receptors (Akter et al., 2021). Accordingly, we hypothesized that blocking the P2 receptors would abolish the COX-2-mediated migration of tumor cells and tested the same in an animal model. Methodology: Tumors were generated in 8-15-week-old male C57BL/6 mice (20-25 g weight) through subcutaneous injection of syngeneic EL4 lymphoma cells in the left flank of the mice. When tumors were palpable around day 10, saline or P2 receptor, antagonists were directly injected into the center of the tumor and mice were divided into three groups: saline (vehicle control), suramin (broad-spectrum P2 receptor antagonist), and MRS 2578 (P2Y6 receptor-specific antagonist). Drugs were chosen based on prior in vitro research and a vernier caliper was used to assess the growth of the tumor every day. Behavioral tests were performed on day 8 (prior to P2 receptor injections) and days 11, 14 and 17 post-tumor injection. Mice were continuously monitored for their health and were sacrificed on day 18 with deep CO2 anesthesia and cervical dislocation. Results: We found that suramin-injected tumors had a substantial reduction in tumor volume when compared to saline-injected ones. Intra-tumoral injections of P2 receptor antagonists such as MRS-2578 also resulted in similar outcomes as suramin. In tumors treated with P2 receptor antagonists, the expression of COX-2, cell cycle proteins and epithelial-to-mesenchymal (EMT) markers, which aid in metastasis, were significantly downregulated, indicating a suppressive effect. Therefore, we propose that P2 receptor-dependent anti-inflammatory drugs (PBAIDs) could be considered a potential alternative therapeutic option for preventing cancer recurrence. Citation Format: Khagendra Ghimeray, Shilpa Sharma, Ravi Shankar Akundi. Purinergic P2Y6 receptor antagonists as potential anti tumor agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5416.

Extracellular Purine Metabolism-Potential Target in Multiple Sclerosis.

The purinergic signaling system comprises a complex network of extracellular purines and purine-metabolizing ectoenzymes, nucleotide and nucleoside receptors, ATP release channels, and nucleoside transporters. Because of its immunomodulatory function, this system is critically involved in the pathogenesis of multiple sclerosis (MS) and its best-characterized animal model, experimental autoimmune encephalomyelitis (EAE). MS is a chronic neuroinflammatory demyelinating and neurodegenerative disease with autoimmune etiology and great heterogeneity, mostly affecting young adults and leading to permanent disability. In MS/EAE, alterations were detected in almost all components of the purinergic signaling system in both peripheral immune cells and central nervous system (CNS) glial cells, which play an important role in the pathogenesis of the disease. A decrease in extracellular ATP levels and an increase in its downstream metabolites, particularly adenosine and inosine, were frequently observed at MS, indicating a shift in metabolism toward an anti-inflammatory environment. Accordingly, upregulation of the major ectonucleotidase tandem CD39/CD73 was detected in the blood cells and CNS of relapsing-remitting MS patients. Based on the postulated role of A2A receptors in the transition from acute to chronic neuroinflammation, the association of variants of the adenosine deaminase gene with the severity of MS, and the beneficial effects of inosine treatment in EAE, the adenosinergic system emerged as a promising target in neuroinflammation. More recently, several publications have identified ADP-dependent P2Y12 receptors and the major extracellular ADP producing enzyme nucleoside triphosphate diphosphohydrolase 2 (NTPDase2) as novel potential targets in MS.

Crosstalk between purinergic receptor P2Y11 and chemokine receptor CXCR7 is regulated by CXCR4 in human macrophages

P2Y11 is a G protein-coupled ATP receptor that activates IL-1 receptor (IL-1R) in a cyclic AMP dependent manner. In human macrophages, P2Y11/IL-1R crosstalk with CCL20 as a prime target is controlled by phosphodiesterase 4 (PDE4), which mediates breakdown of cyclic AMP. Here, we used gene expression analysis to identify activation of CXCR4 and CXCR7 as a hallmark of P2Y11 signaling. We found that PDE4 inhibition with rolipram boosts P2Y11/IL-1R-induced upregulation of CXCR7 expression and CCL20 production in an epidermal growth factor receptor dependent manner. Using an astrocytoma cell line, naturally expressing CXCR7 but lacking CXCR4, P2Y11/IL-1R activation effectively induced and CXCR7 agonist TC14012 enhanced CCL20 production even in the absence of PDE4 inhibition. Moreover, CXCR7 depletion by RNA interference suppressed CCL20 production. In macrophages, the simultaneous activation of P2Y11 and CXCR7 by their respective agonists was sufficient to induce CCL20 production with no need of PDE4 inhibition, as CXCR7 activation increased its own and eliminated CXCR4 expression. Finally, analysis of multiple CCL chemokines in the macrophage secretome revealed that CXCR4 inactivation and CXCR7 activation selectively enhanced P2Y11/IL-1R-mediated secretion of CCL20. Altogether, our data establish CXCR7 as an integral component of the P2Y11/IL-1R-initiated signaling cascade and CXCR4-associated PDE4 as a regulatory checkpoint.

The functional role of P2 purinergic receptors in the progression of gastric cancer.

Studies have confirmed that P2 purinergic receptors (P2X receptors and P2Y receptors) expressed in gastric cancer (GC) cells and GC tissues and correlates with their function. Endogenous nucleotides including ATP, ADP, UTP, and UDP, as P2 purinergic receptors activators, participate in P2 purinergic signal transduction pathway. These activated P2 purinergic receptors regulate the progression of GC mainly by mediating ion channels and intracellular signal cascades. It is worth noting that there is a difference in the expression of P2 purinergic receptors in GC, which may play different roles in the progression of GC as a tumor promoting factor or a tumor suppressor factor. Among them, P2 × 7, P2Y2 and P2Y6 receptors have certain clinical significance in patients with GC and may be used as biological molecular markers for the prediction of patients with GC. Therefore, in this paper, we discuss the functional role of nucleotide / P2 purinergic receptors signal axis in regulating the progression of GC and that these P2 purinergic receptors may be used as potential molecular targets for the prevention and treatment of GC.

Purinergic Astrocyte Signaling Driven by TNF-α After Cannabidiol Administration Restores Normal Synaptic Remodeling Following Traumatic Brain Injury

Traumatic brain injury (TBI) is a prevalent form of cranial trauma that results in neural conduction disruptions and damage to synaptic structures and functions. Cannabidiol (CBD), a primary derivative from plant-based cannabinoids, exhibits a range of beneficial effects, including analgesic, sedative, anti-inflammatory, anticonvulsant, anti-anxiety, anti-apoptotic, and neuroprotective properties. Nevertheless, the effects of synaptic reconstruction and the mechanisms underlying these effects remain poorly understood. TBI is characterized by increased levels of tumor necrosis factor-alpha (TNF-α), a cytokine integral for the modulation of glutamate release by astrocytes. In the present study, the potential of CBD in regulating aberrant glutamate signal transmission in astrocytes following brain injury, as well as the underlying mechanisms involved, were investigated using immunofluorescence double staining, enzyme-linked immunosorbent assay (ELISA), western blot analysis, hematoxylin and eosin (H&E) staining, Nissl staining, transmission electron microscopy, and RT-qPCR. In this study, we examined the impact of CBD on neuronal synapses, focusing on the TNF-α-driven purinergic signaling pathway. Specifically, our research revealed that CBD pretreatment effectively reduced the secretion of TNF-α induced by astrocyte activation following TBI. This reduction inhibited the interaction between TNF-α and P2Y1 receptors, leading to a decrease in the release of neurotransmitters, including Ca2+ and glutamate, thereby initiating synaptic remodeling. Our study showed that CBD exhibits significant therapeutic potential for TBI-related synaptic dysfunction, offering valuable insights for future research and more effective TBI treatments. Further exploration of the potential applications of CBD in neuroprotection is required to develop innovative clinical strategies.

High-mobility group box 1 increases platelet surface P2Y12 and platelet activation in sickle cell disease.

Thrombosis and inflammation are intimately linked and synergistically contribute to the pathogenesis of numerous thromboinflammatory diseases, including sickle cell disease (SCD). While platelets are central to thrombogenesis and inflammation, the molecular mechanisms of crosstalk between the 2 remain elusive. High-mobility group box 1 (HMGB1) regulates inflammation and stimulates platelet activation through Toll-like receptor 4. However, it remains unclear whether HMGB1 modulates other thrombotic agonists to regulate platelet activation. Herein, using human platelets, we demonstrate that HMGB1 significantly enhanced ADP-mediated platelet activation. Furthermore, inhibition of the purinergic receptor P2Y12 attenuated HMGB1-dependent platelet activation. Mechanistically, we show that HMGB1 stimulated ADP secretion, while concomitantly increasing P2Y12 levels at the platelet membrane. We show that in SCD patients, increased plasma HMGB1 levels were associated with heightened platelet activation and surface P2Y12 expression. Treatment of healthy platelets with plasma from SCD patients enhanced platelet activation and surface P2Y12, and increased sensitivity to ADP-mediated activation, and these effects were linked to plasma HMGB1. We conclude that HMGB1-mediated platelet activation involves ADP-dependent P2Y12 signaling, and HMGB1 primes platelets for ADP signaling. This complementary agonism between ADP and HMGB1 furthers the understanding of thromboinflammatory signaling in conditions such as SCD, and provides insight for therapeutic P2Y12 inhibition.

Purinergic signaling promotes premature senescence

Extracellular ATP activates P2 purinergic receptors. Whether purinergic signaling is functionally coupled to cellular senescence is largely unknown. We find that oxidative stress induced release of ATP and caused senescence in human lung fibroblasts. Inhibition of P2 receptors limited oxidative stress-induced senescence, while stimulation with exogenous ATP promoted premature senescence. Pharmacological inhibition of P2Y11 receptor (P2Y11R) inhibited premature senescence induced by either oxidative stress or ATP, while stimulation with a P2Y11R agonist was sufficient to induce cellular senescence. Our data show that both extracellular ATP and a P2Y11R agonist induced calcium (Ca++) release from the endoplasmic reticulum (ER) and that either inhibition of phospholipase C or intracellular Ca++ chelation impaired ATP-induced senescence. We also find that Ca++ that was released from the ER, following ATP-mediated activation of phospholipase C, entered mitochondria in a manner dependent on P2Y11R activation. Once in mitochondria, excessive Ca++ promoted the production of reactive oxygen species in a P2Y11R-dependent fashion, which drove development of premature senescence of lung fibroblasts. Finally, we show that conditioned medium derived from senescent lung fibroblasts, which were induced to senesce through the activation of ATP/P2Y11R-mediated signaling, promoted the proliferation of triple-negative breast cancer cells and their tumorigenic potential by secreting amphiregulin. Our study identifies the existence of a novel purinergic signaling pathway that links extracellular ATP to the development of a protumorigenic premature senescent phenotype in lung fibroblasts that is dependent on P2Y11R activation and ER-to-mitochondria calcium signaling.

Effects of pharmacological interventions on mortality in patients with Takotsubo syndrome: a report from the SWEDEHEART registry.

Takotsubo syndrome (TS) is a heart condition mimicking acute myocardial infarction. TS is characterized by a sudden weakening of the heart muscle, usually triggered by physical or emotional stress. In this study, we aimed to investigate the effect of pharmacological interventions on short- and long-term mortality in patients with TS. We analysed data from the SWEDEHEART (the Swedish Web System for Enhancement and Development of Evidence-based care in Heart disease Evaluated According to Recommended Therapies) registry, which included patients who underwent coronary angiography between 2009 and 2016. In total, we identified 1724 patients with TS among 228263 individuals in the registry. The average age was 66±14years, and 77% were female. Nearly half of the TS patients (49.4%) presented with non-ST-elevation acute coronary syndrome, and a quarter (25.9%) presented with ST-elevation myocardial infarction. Most patients (79.1%) had non-obstructive coronary artery disease on angiography, while 11.7% had a single-vessel disease and 9.2% had a multivessel disease. All patients received at least one pharmacological intervention; most of them used beta-blockers (77.8% orally and 8.3% intravenously) or antiplatelet agents [aspirin (66.7%) and P2Y12 inhibitors (43.6%)]. According to the Kaplan-Meier estimator, the probability of all-cause mortality was 2.5% after 30days and 16.6% after 6years. The median follow-up time was 877days. Intravenous use of inotropes and diuretics was associated with increased 30day mortality in TS [hazard ratio (HR)=9.92 (P<0.001) and HR=3.22 (P=0.001), respectively], while angiotensin-converting enzyme inhibitors and statins were associated with decreased long-term mortality [HR=0.60 (P=0.025) and HR=0.62 (P=0.040), respectively]. Unfractionated and low-molecular-weight heparins were associated with reduced 30day mortality [HR=0.63 (P=0.01)]. Angiotensin receptor blockers, oral anticoagulants, P2Y12 antagonists, aspirin, and beta-blockers did not statistically correlate with mortality. Our findings suggest that some medications commonly used to treat TS are associated with higher mortality, while others have lower mortality. These results could inform clinical decision-making and improve patient outcomes in TS. Further research is warranted to validate these findings and to identify optimal pharmacological interventions for patients with TS.

The use of cangrelor in a complex vascular patient: A case report

Medical decision-making surrounding high risk surgical procedures requires extensive consideration about the potential risks and benefits to the patient, including implications for concomitant medications and therapies. Managing cardiovascular risk in patients undergoing non-cardiac surgery is essential for safe and effective patient care. In instances where cardiac revascularization is needed prior to surgery, antiplatelet medication is also needed which can complicate future surgical procedures. This case report describes a patient who underwent percutaneous coronary intervention with drug eluting stent placement, who also needed urgent treatment for expanding thoracic abdominal aortic aneurysm (TAAA). Standard practice for endovascular repair of a TAAA includes placement of a lumbar drain to decrease the risk of spinal cord ischemia, however dual antiplatelet therapy is contraindicated. Cangrelor is the only intravenous platelet P2Y12 receptor inhibitor currently available. The use of Cangrelor, a short-acting P2Y12 inhibitor, was successfully utilized as a bridge in the setting of a patient requiring dual antiplatelet therapy (DAPT) and further surgical intervention. This medication may improve outcomes for this subset of patients.

Cangrelor - Expanding therapeutic options in patients with acute coronary syndrome.

Cangrelor is the only intravenous P2Y12 receptor antagonist. It is an adenosine triphosphate analog that selectively, directly, and reversibly binds to the platelet P2Y12 receptors exerting its antiaggregatory effect. Cangrelor is characterized by linear, dose-dependent pharmacokinetics and rapid onset of action providing potent platelet inhibition exceeding 90%. Cangrelor is rapidly metabolized by endothelial endonucleotidase; thus, its half-life is 2.9 to 5.5 min, and its antiplatelet effect subsides within 60 to 90 min. Data originating from three pivotal cangrelor trials (CHAMPION PLATFORM, CHAMPION PCI, and CHAMPION PHOENIX) indicate that cangrelor reduces the risk of periprocedural thrombotic complications during percutaneous coronary intervention at the expense of mild bleedings. Its unique pharmacological properties allow it to overcome the limitations of oral P2Y12 receptor inhibitors, mainly related to the delayed and decreased bioavailability and antiplatelet effect of these agents, which are often observed in the setting of acute coronary syndrome. Subgroups of patients who could theoretically benefit the most from cangrelor include those in whom pharmacokinetics and pharmacodynamics of oral P2Y12 receptor antagonists are most disturbed, namely patients with ST-segment elevation myocardial infarction, those treated with opioids, with mild therapeutic hypothermia, or in cardiogenic shock. Cangrelor could also be useful if bridging is required in patients undergoing surgery. According to the current guidelines cangrelor may be considered in P2Y12 receptor inhibitor-naïve patients undergoing percutaneous coronary intervention in both acute and stable settings.

Competitive Analysis of the Binding Affinity of Montelukast, Zafirlukast and Gemilukast to CysLTR1, P2Y12 and PPAR-γ and their Possible Cardioprotective Effect: Using in silico Methods

Background Asthma is a very common respiratory disorder, affecting more than 360 million people worldwide. It is a chronic inflammatory disorder of the airways with the symptoms of shortness of breath, coughing, chest tightness, wheezing, and sometimes chest pain. Leukotrienes play an important role in bronchoconstriction during the allergen or exercise-induced acute asthma attack. Aim The study aims to predict the interactions between leukotriene antagonist drugs and CysLT receptor-1 (CysLTR1), P2Y12 and peroxisome proliferator-activated receptor gamma (PPAR-γ) on a competitive basis. The study also has the objective of understanding the cardioprotective roles of the drugs. Introduction Asthma is strongly linked to the development of acute coronary syndrome by the leukotriene-induced activation of CysLTR1, platelet aggregation and thrombosis by activating P2Y12. PPAR-γ is considered to show benefits against atherosclerosis, diabetes, hypertension, obesity and dyslipidaemia, which are risk factors for the development of cardiovascular disorders. Leukotriene receptor inhibitors act with these three types of receptors to show therapeutic effects. Materials and Methods To predict the possible interactions between the drugs and the receptors, the study has used in silico methods. Results and Discussion Montelukast, Zafirlukast and Gemilukast are potential antagonists of CysLTR1 and P2Y12. They are also responsible for the upregulation of PPAR-γ. Thus, these drugs show a cardioprotective role in asthma-induced cardiac disorders. Conclusion A competitive in silico study of Montelukast, Zafirlukast and Gemilukast to predict their binding to CysLTR1, P2Y12 &amp; PPAR-γ revealed that Montelukast is more effective than the other two drugs for showing a cardioprotective role.

Comparison of the Efficacy and Safety of Cyp2c19 Gene-guided Oral Administration of Different P2Y12 Inhibitors in Elderly Patients with Acute Coronary Syndrome

Objective To explore the survival prognosis and safety analysis of the cyp2c19 gene-guided ACS elderly patient group using Tegretol and Clopidogrel. Methods Three hundred and eighty-five ACS patients aged ≥65 years were selected, tested for polymorphisms in the cyp2c19 gene, and divided into two groups according to the oral P2Y12 receptor inhibitor: 320 patients in the clopidogrel group and 165 patients in the tegretol group. Patients were followed up for 1 year after discharge from the hospital, with the primary end point being a composite end point of adverse cardiovascular events including cardiac death, nonfatal infarction, or absence of stroke, and the secondary end point including unstable angina, in-stent stenosis, target vessel revascularization, and all-cause mortality, as well as bleeding events. Results The 1-year rate of ischemic events was lower in patients treated with tegretol compared with the clopidogrel group [28.5% (47/165) versus 26.3% (84/320), P = 0.599], and the difference in the 1-year rate of BARC 2, 3, and 5 events between the two groups was not statistically significant [6.0% (10/165) versus 3.4% (11/320), P = 0.179]. P = 0.179]. Conclusion In elderly patients with cyp2c19-directed ACS, there was no significant difference in survival prognosis, ischemic events, or bleeding risk between tegretol and clopidogrel at 1 year.

The Effect of Clopidogrel and Ticagrelor on Human Adipose Mesenchymal Stem Cell Osteogenic Differentiation Potential: In Vitro Comparative Study.

Ticagrelor (TICA) and clopidogrel (CLP) are extensively used antiplatelet drugs that act by antagonizing the P2Y12 receptors that are found on platelets in addition to bone cells. Aim. The purpose of this study was to investigate the effect of clopidogrel and ticagrelor on stem cells osteogenic differentiation in vitro. Methods. Human adipose-derived mesenchymal stem cells (hAd-MSCs) were divided into (1) control group, (2) osteogenic group (osteo group), (3) clopidogrel group (CLP group), and (4) ticagrelor group (TICA group). The osteogenic differentiation potential was determined by mineralization nodule formation using Alizarin Red S staining, measuring ALP enzyme activity by alkaline phosphatase assay. Quantitative determination for osteogenic markers included osteocalcin (OC); runt-related transcription factor 2 (RUNX2) performed using western blot; osteoprotegerin (OPG) using enzyme-linked immunosorbent assay (ELISA) and inflammatory markers; and tumor necrosis factor (TNF-α) and interleukin-6 (IL-6) measured using real-time polymerase chain reaction quantitative (RT-PCR) and ELISA. Results. In comparison to all study groups, the TICA group showed significant increase in the mineralized extracellular matrix, ALP enzyme activity, and bone markers expression as RUNX2 (P < 0.0001), OC, and OPG (P < 0.05). The expression of IL-6 and TNF-α was determined by RT-qPCR and ELISA techniques. TICA and CLP significantly decreased both markers compared to the control group. The TICA group showed statistically significant lower levels of both markers (P < 0.0001) than the CLP and control groups via the ELISA technique. Conclusion. TICA may possess a positive effect on hAd-MSCs osteogenic differentiation compared to CLP.

Characterization of two distinct immortalized endothelial cell lines, EA.hy926 and HMEC-1, for in vitro studies: exploring the impact of calcium electroporation, Ca2+ signaling and transcriptomic profiles

BackgroundDisruption of Ca2+ homeostasis after calcium electroporation (CaEP) in tumors has been shown to elicit an enhanced antitumor effect with varying impacts on healthy tissue, such as endothelium. Therefore, our study aimed to determine differences in Ca2+ kinetics and gene expression involved in the regulation of Ca2+ signaling and homeostasis, as well as effects of CaEP on cytoskeleton and adherens junctions of the established endothelial cell lines EA.hy926 and HMEC-1.MethodsCaEP was performed on EA.hy926 and HMEC-1 cells with increasing Ca2+ concentrations. Viability after CaEP was assessed using Presto Blue, while the effect on cytoskeleton and adherens junctions was evaluated via immunofluorescence staining (F-actin, α-tubulin, VE-cadherin). Differences in intracellular Ca2+ regulation ([Ca2+]i) were determined with spectrofluorometric measurements using Fura-2-AM, exposing cells to DPBS, ionomycin, thapsigargin, ATP, bradykinin, angiotensin II, acetylcholine, LaCl3, and GdCl3. Molecular distinctions were identified by analyzing differentially expressed genes and pathways related to the cytoskeleton and Ca2+ signaling through RNA sequencing.ResultsEA.hy926 cells, at increasing Ca2+ concentrations, displayed higher CaEP susceptibility and lower survival than HMEC-1. Immunofluorescence confirmed CaEP-induced, time- and Ca2+-dependent morphological changes in EA.hy926’s actin filaments, microtubules, and cell–cell junctions. Spectrofluorometric Ca2+ kinetics showed higher amplitudes in Ca2+ responses in EA.hy926 exposed to buffer, G protein coupled receptor agonists, bradykinin, and angiotensin II compared to HMEC-1. HMEC-1 exhibited significantly higher [Ca2+]i changes after ionomycin exposure, while responses to thapsigargin, ATP, and acetylcholine were similar in both cell lines. ATP without extracellular Ca2+ ions induced a significantly higher [Ca2+]i rise in EA.hy926, suggesting purinergic ionotropic P2X and metabotropic P2Y receptor activation. RNA-sequencing analysis showed significant differences in cytoskeleton- and Ca2+-related gene expression, highlighting upregulation of ORAI2, TRPC1, TRPM2, CNGA3, TRPM6, and downregulation of TRPV4 and TRPC4 in EA.hy926 versus HMEC-1. Moreover, KEGG analysis showed upregulated Ca2+ import and downregulated export genes in EA.hy926.ConclusionsOur finding show that significant differences in CaEP response and [Ca2+]i regulation exist between EA.hy926 and HMEC-1, which may be attributed to distinct transcriptomic profiles. EA.hy926, compared to HMEC-1, displayed higher susceptibility and sensitivity to [Ca2+]i changes, which may be linked to overexpression of Ca2+-related genes and an inability to mitigate changes in [Ca2+]i. The study offers a bioinformatic basis for selecting EC models based on research objectives.

Electroacupuncture Alleviates Pain by Suppressing P2Y12R-Dependent Microglial Activation in Monoarthritic Rats.

Electroacupuncture (EA) effectively improves arthritis-induced hyperalgesia and allodynia by repressing spinal microglial activation, which plays a crucial role in pain hypersensitivity following tissue inflammation. However, the mechanism by which EA suppresses spinal microglial activation in monoarthritis (MA) remains unclear. In the present study, a rat model of MA was established through unilateral ankle intra-articular injection of complete Freund's adjuvant (CFA). The relationship among P2Y12 receptor (P2Y12R) expression, spinal microglial activation, and EA analgesia was investigated using quantitative real-time PCR (qRT‒PCR), western blotting, immunofluorescence (IF), and behavioral testing. The results found that EA treatment at the ipsilateral "Huantiao" (GB30) and "Yanglingquan" (GB34) acupoints markedly attenuated pain and spinal microglia M1 polarization in MA rats. In particular, P2Y12R expression was significantly increased at the mRNA and protein levels in the spinal dorsal horn in MA rats, whereas EA treatment effectively repressed the MA-induced upregulation of P2Y12R. IF analysis further revealed that most P2Y12R was expressed in microglia in the spinal dorsal horn. Pharmacological inhibition of P2Y12R by its antagonist (AR-C69931MX) decreased MA-induced spinal microglial activation and subsequent proinflammatory cytokine production. Consequently, AR-C69931MX significantly intensified the anti-pain hypersensitive function of EA in MA rats. Taken together, these results demonstrate that EA alleviates MA-induced pain by suppressing P2Y12R-dependent microglial activation.

Gender characteristics of hematuria during lithokinetic therapy in patients with nephrolithiasis

Purpose. To study gender characteristics of the severity of hematuria and compensatory mechanisms of the proaggregant component of hemostasis in patients with nephrolithiasis when prescribing litokinetic therapy (LCT), including nonsteroidal anti-inflammatory drugs (NSAIDs). Material and methods. The prospective study included 60 patients (group 1 – 30 men; group 2 – 30 women) with imaging signs of the presence of stones in the urinary tract. For 7 days, patients underwent standard LCT, including NSAIDs, an α1A-blocker (tamsulosin) and antibiotics. In vitro, the activity of the TP receptor for TxA2 and purine P2Y receptors (P2Y1 and P2Y12) was studied on a platelet suspension after 24, 48, 72 hours, 5 and 7 days. Platelet aggregation was assessed using the turbidimetric method using a ChronoLog analyzer (USA). Results. At the hospitalization stage, in both groups (before the introduction of NSAIDs), hyperreactivity of the TP-receptor was observed, and in men the activity of the TP receptor was lower, and hematuria (p&lt;0.05) was higher than in women. The pharmacokinetics of NSAIDs in men was characterized by inhibition of cyclooxygenase (COX) in two time periods – 72 hours and 7 days, as a result of which the synthesis of TхA2 in platelets decreased and hematuria increased 4.4 times (p&lt;0.001) compared with the hospitalization stage. In women, COX inhibition occurred within 5 days; At the same time, the preservation of normal reactivity of the TP-receptor and purine P2Y receptors ensured the implementation of a compensatory platelet response, limiting the severity of hematuria. Conclusion. Analysis of the mechanisms of gender differences in hematuria in nephrolithiasis is important for developing a personalized medicine strategy when prescribing NSAIDs.

Abstract WP309: UDP-Glucose Exacerbates Neuronal Apoptosis via Its Receptor P2y14 After Subarachnoid Hemorrhage in Rats

Introduction: Subarachnoid hemorrhage (SAH) is a severe subtype of stroke that can lead to poor outcomes. Recent studies reported that uridine diphosphate glucose (UDP-G), one of the intermediates in glucose/glycogen metabolism, can be released from damaged cells and proceed with pathologic conditions by activating the purinergic P2Y14 receptor. This study aimed to elucidate the role of brain UDP-G/P2Y14 signaling after SAH. Hypothesis: The inhibition of the brain UDP-G/P2Y14 signaling pathway would attenuate neuronal apoptosis and neurological deficits after SAH in rats. Methods: A total of 200 adult male Sprague Dawley rats were used. Endovascular perforation method was used to induce SAH. The time course changes of CSF UDP-G, brain protein levels of UDP-G converting enzyme UDP-G pyrophosphorylase-2 (UGP2) and P2Y14 receptors, as well as the brain cell types expressing P2Y14 were examined. A selective P2Y14 receptor inhibitor PPTN hydrochloride or exogenous UDP-G was administered intranasally 1 hour after SAH. SAH grading, neurological evaluation, immunofluorescence, TUNEL staining, Nissl staining, western blot, and liquid chromatography-mass spectrometry were performed. Results: Endogenous CSF UDP-G and brain UGP2 were increased at 3 hours and 6 hours after SAH. The protein level of the brain P2Y14 receptor was not changed temporally and was mainly expressed in neurons 24 hours after SAH. PPTN hydrochloride decreased but exogenous UDP-G aggravated the number of TUNEL-positive neurons, brain protein levels of proapoptotic molecules such as cleaved caspase 3, and modified Garcia neurological score at 24 hours after SAH. Early inhibition of P2Y14 receptor signaling by PPTN hydrochloride preserved the numbers of Nissl staining identified surviving neurons in the hippocampus, amygdala, and cortex with improved water maze learning performance and depressive-like behavior at 1-month post-SAH. Conclusion: Brain UDP-G/P2Y14 receptor signaling activation plays a detrimental role after SAH, as a part of glucose metabolic pathology at the tissue level. P2Y14 receptor inhibitor reduced neuronal apoptosis and improved neurological deficits after SAH, which may serve as potential therapeutics in treating patients with SAH.

Abstract A044: P2Y2 signaling disruption reduces ATP-dependent calcium elevation and actin localization to promote breast tumor cell dissemination

Abstract The tumor microenvironment and wound healing after injury both contain extremely high concentrations of the extracellular signaling molecule, adenosine triphosphate (ATP) compared to normal tissue. P2Y2 receptor, an ATP-activated purinergic receptor, is typically associated with pulmonary, endothelial, and neural cell signaling. Here we report the importance of ATP-dependent P2Y2 signaling in breast epithelial cells and how it is altered in metastatic breast cancer. In response to ATP activation, P2Y2 receptor signaling causes an increase of intracellular calcium (Ca2+) in non-tumorigenic breast epithelial cells, while their tumorigenic and metastatic counterparts have significantly reduced Ca2+ responses. The non-tumorigenic cells respond to increased Ca2+ with actin polymerization and localization to cell edges, while the metastatic cells remained unaffected. The increase in intracellular Ca2+ after ATP stimulation was blunted using a P2Y2 antagonist, which also prevented actin mobilization and caused cell dissemination from spheroids in non-tumorigenic breast epithelial cells. Furthermore, the lack of Ca2+ concentration changes and actin mobilization in the metastatic breast cancer cells could be due to reduced P2Y2 expression, which correlates with poorer overall survival in breast cancer patients. This study elucidates rapid changes that occur after elevated intracellular Ca2+ in breast epithelial cells and how metastatic cancer cells have adapted to evade this cellular response. Citation Format: Makenzy Mull, Stephen Pratt, Keyata Thompson, David Annis, Julia Ju, Megan Stemberger, Liron Boyman, Michele Vitolo, W. Jonathan Lederer, Stuart Martin. P2Y2 signaling disruption reduces ATP-dependent calcium elevation and actin localization to promote breast tumor cell dissemination [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A044.