Ca2+ signals in human umbilical endothelial cells derived from pregnancy with fetal growth restriction associated with hypertensive disorder.

Fetal growth restriction associated with hypertensive disorders of pregnancy (FGR-HDP) is a prevalent pathology with a higher risk of perinatal morbimortality. In this condition, placental insufficiency and endothelial dysfunction serve key roles. The present prospective cohort study monitored 11 patients with an FGR-HDP and 15 with full-term normotensive pregnancies and studied post-natal intracellular calcium concentration ([Ca2+]i) signals in human umbilical vein endothelial cells (HUVECs). Small fetuses with placental insufficiency were identified using fetal biometry with Doppler velocimetry. Mean gestational age and birth weight were 31.8±4.1 weeks and 1,260±646 g for FGR-HDP and 39.2±0.8 weeks and 3,320±336 g for normal births, respectively. Abnormal umbilical artery Doppler waveforms were found in 64% of neonates with FGR-HDP. A significant percentage (86%) of FGR newborns were admitted to the neonatal intensive care unit at Gustavo Fricke hospital, Viña del Mar, Chile, with one case of death after birth. [Ca2+]i signals were measured by microfluorimetry in Fluo-3-loaded HUVECs from primary cultures. Altered [Ca2+]i signals were observed in HUVECs from FGR-HDP, where the sustained phase of ATP-induced [Ca2+]i responses was significantly reduced compared with the normotensive group. Also, the [Ca2+]i signals induced with 10 mM Ca2+ after depletion of internal Ca2+ stores were significantly higher. The present study provides a better comprehension of the role of altered cytosolic Ca2+ dynamics in endothelial dysfunction and an in vitro model to assess novel therapeutic approaches for decreasing or preventing complications in FGR-HDP.

Purinergic Activation of Store-Operated Calcium Entry (SOCE) Regulates Cell Migration in Metastatic Ovarian Cancer Cells.

Store-operated calcium entry (SOCE) is an important process in calcium signaling. Its role in physiological and pathological events is well recognized. However, in cancerous systems, the importance of SOCE in relation to the degree of cancer aggressiveness, as well as its regulation by ligands such as purinergic molecules, are not well documented. This study aimed to characterize a differential effect of the P2Y2 receptor (promoted by UTP of 10 µM and inhibited by ARC118925XX of 1 µM) on intracellular calcium response between metastatic (SKOV-3) and non-metastatic (CAOV-3) ovarian cell lines in conditions of normal (1.5 mM) and zero extracellular calcium concentration. The sustained calcium influx observed exclusively in SKOV-3 cells was associated with the presence of SOCE (promoted by thapsigargin (74.81 ± 0.94 ΔF) and sensitive to 2-APB (20.60 ± 0.85 ΔF)), whereas its absence in CAOV-3 cells (26.2 ± 6.1 ΔF) was correlated with a low expression of ORAI1. The relevance of SOCE in metastatic SKOV-3 cells was further corroborated when 2-APB significantly inhibited (40.4 ± 2.8% of covered area) UTP-induced cell migration (54.6 ± 3.7% of covered area). In conclusion, our data suggest that SOCE activation elicited by the P2Y2 receptor is involved in the aggressiveness of ovarian cancer cells.

P2Y2-P2X7 receptors cross-talk in primed mesenteric endothelial cells upregulates NF-κB signaling favoring mononuclear cell adhesion in schistosomiasis.

Schistosomiasis is an intravascular infectious disease that impacts over 200 million people globally. In its chronic stage, it leads to mesenteric inflammation with significant involvement of monocytes/macrophages. Endothelial cells lining the vessel lumens play a crucial role, and mount of evidence links this disease to a downregulation of endoprotective cell signaling favoring a primed and proinflammatory endothelial cell phenotype and therefore the loss of immunovascular homeostasis. One hallmark of infectious and inflammatory conditions is the release of nucleotides into the extracellular milieu, which, in turn, act as innate messengers, activating purinergic receptors and triggering cell-to-cell communication. ATP influences the progression of various diseases through P2X and P2Y purinergic receptor subtypes. Among these receptors, P2Y2 (P2Y2R) and P2X7 (P2X7R) receptors stand out, known for their roles in inflammation. However, their specific role in schistosomiasis has remained largely unexplored. Therefore, we hypothesized that endothelial P2Y2R and P2X7R could contribute to monocyte adhesion to mesenteric endothelial cells in schistosomiasis. Using a preclinical murine model of schistosomiasis associated with endothelial dysfunction and age-matched control mice, we showed that endothelial P2Y2R and P2X7R activation increased monocyte adhesion to cultured primary endothelial cells in both groups. However, a distinct upregulation of endothelial P2Y2R-driven canonical Ca2+ signaling was observed in the infected group, amplifying adhesion. In the control group, the coactivation of endothelial P2Y2R and P2X7R did not alter the maximal monocyte adhesion induced by each receptor individually. However, in the infected group, this coactivation induced a distinct upregulation of P2Y2R-P2X7R-driven canonical signaling, IL-1ß release, and VCAM-1 expression, with underlying mechanisms involving inflammasome and NF-κB signaling. Therefore, current data suggest that schistosomiasis alters endothelial cell P2Y2R/P2X7R signaling during inflammation. These discoveries advance our understanding of schistosomiasis. This intricate interplay, driven by PAMP-triggered endothelial P2Y2R/P2X7R cross-talk, emerges as a potential key player in the mesenteric inflammation during schistosomiasis.

Resveratrol and Quercetin as Regulators of Inflammatory and Purinergic Receptors to Attenuate Liver Damage Associated to Metabolic Syndrome.

Nonalcoholic fatty liver disease (NAFLD) is considered a manifestation of metabolic syndrome (MS) and is characterized by the accumulation of triglycerides and a varying degree of hepatic injury, inflammation, and repair. Moreover, peroxisome-proliferator-activated receptors (PPARs) play a critical role in the pathophysiological processes in the liver. There is extensive evidence of the beneficial effect of polyphenols such as resveratrol (RSV) and quercetin (QRC) on the treatment of liver pathology; however, the mechanisms underlying their beneficial effects have not been fully elucidated. In this work, we show that the mechanisms underlying the beneficial effects of RSV and QRC against inflammation in liver damage in our MS model are due to the activation of novel pathways which have not been previously described such as the downregulation of the expression of toll-like receptor 4 (TLR4), neutrophil elastase (NE) and purinergic receptor P2Y2. This downregulation leads to a decrease in apoptosis and hepatic fibrosis with no changes in hepatocyte proliferation. In addition, PPAR alpha and gamma expression were altered in MS but their expression was not affected by the treatment with the natural compounds. The improvement of liver damage by the administration of polyphenols was reflected in the normalization of serum transaminase activities.

P2Y2 receptor activation promotes esophageal cancer cells proliferation via ERK1/2 pathway.

Esophageal cancer is a prominent worldwide illness that is divided into two main subtypes: esophageal squamous cell carcinoma and esophageal adenocarcinoma. Mortality rates are alarming, and the understanding of the mechanisms involved in esophageal cancer development, becomes essential. Purinergic signaling is related to many diseases and among these various types of tumors. Here we studied the effects of the P2Y2 receptor activation in different types of esophageal cancer. Esophageal tissue samples of healthy controls were used for P2Y2R expression quantification. Two human esophageal cancer cell lines Kyse-450 (squamous cell carcinoma) and OE-33 (adenocarcinoma) were used to perform in vitro analysis of cell proliferation, migration, adhesion, and the signaling pathways involved in P2Y2R activation. Data showed that P2Y2R was expressed in biopsies of patients with ESCC and adenocarcinoma, as well as in the two human esophageal cancer cell lines studied. The RT-qPCR analysis demonstrated that OE-33 cells have higher P2RY2 expression than Kyse-450 squamous cell line. Results showed that P2Y2R activation, induced by ATP or UTP, promoted esophageal cancer cells proliferation and colony formation. P2Y2R blockage with the selective antagonist, AR-C 118925XX, led to decreased proliferation, colony formation and adhesion. Treatments with ATP or UTP activated ERK 1/2 pathway in ESCC and ECA cells. The P2Y2R antagonism did not alter the migration of esophageal cancer cells. Interestingly, the esophageal cancer cell lines presented a distinct profile of nucleotide hydrolysis activity. The modulation of P2Y2 receptors may be a promising target for esophageal cancer treatment.

Increased Purinergic Responses Dependent on P2Y2 Receptors in Hepatocytes from CCl4-Treated Fibrotic Mice.

Inflammatory and wound healing responses take place during liver damage, primarily in the parenchymal tissue. It is known that cellular injury elicits an activation of the purinergic signaling, mainly by the P2X7 receptor; however, the role of P2Y receptors in the onset of liver pathology such as fibrosis has not been explored. Hence, we used mice treated with the hepatotoxin CCl4 to implement a reversible model of liver fibrosis to evaluate the expression and function of the P2Y2 receptor (P2Y2R). Fibrotic livers showed an enhanced expression of P2Y2R that eliminated its zonal distribution. Hepatocytes from CCl4-treated mice showed an exacerbated ERK-phosphorylated response to the P2Y2R-specific agonist, UTP. Cell proliferation was also enhanced in the fibrotic livers. Hepatic transcriptional analysis by microarrays, upon CCl4 administration, showed that P2Y2 activation regulated diverse pathways, revealing complex action mechanisms. In conclusion, our data indicate that P2Y2R activation is involved in the onset of the fibrotic damage associated with the reversible phase of the hepatic damage promoted by CCl4.

Caveolin-1 Regulates P2Y2 Receptor Signaling during Mechanical Injury in Human 1321N1 Astrocytoma.

Caveolae-associated protein caveolin-1 (Cav-1) plays key roles in cellular processes such as mechanosensing, receptor coupling to signaling pathways, cell growth, apoptosis, and cancer. In 1321N1 astrocytoma cells Cav-1 interacts with the P2Y2 receptor (P2Y2R) to modulate its downstream signaling. P2Y2R and its signaling machinery also mediate pro-survival actions after mechanical injury. This study determines if Cav-1 knockdown (KD) affects P2Y2R signaling and its pro-survival actions in the 1321N1 astrocytoma cells mechanical injury model system. KD of Cav-1 decreased its expression in 1321N1 cells devoid of or expressing hHAP2Y2R by ~88% and ~85%, respectively. Cav-1 KD had no significant impact on P2Y2R expression. Post-injury densitometric analysis of pERK1/2 and Akt activities in Cav-1-positive 1321N1 cells (devoid of or expressing a hHAP2Y2R) revealed a P2Y2R-dependent temporal increase in both kinases. These temporal increases in pERK1/2 and pAkt were significantly decreased in Cav-1 KD 1321N1 (devoid of or expressing a hHAP2Y2R). Cav-1 KD led to an ~2.0-fold and ~2.4-fold decrease in the magnitude of the hHAP2Y2R-mediated pERK1/2 and pAkt kinases' activity, respectively. These early-onset hHAP2Y2R-mediated signaling responses in Cav-1-expressing and Cav-1 KD 1321N1 correlated with changes in cell viability (via a resazurin-based method) and apoptosis (via caspase-9 expression). In Cav-1-positive 1321N1 cells, expression of hHAP2Y2R led to a significant increase in cell viability and decreased apoptotic (caspase-9) activity after mechanical injury. In contrast, hHAP2Y2R-elicited changes in viability and apoptotic (caspase-9) activity were decreased after mechanical injury in Cav-1 KD 1321N1 cells expressing hHAP2Y2R. These findings support the importance of Cav-1 in modulating P2Y2R signaling during mechanical injury and its protective actions in a human astrocytoma cell line, whilst shedding light on potential new venues for brain injury or trauma interventions.

Purinergic Cooperation Between P2Y2 and P2X7 Receptors Promote Cutaneous Leishmaniasis Control: Involvement of Pannexin-1 and Leukotrienes.

The release of damage-associated molecular patterns, including uridine triphosphate (UTP) and adenosine triphosphate (ATP) to the extracellular milieu is a key component of innate immune response to infection. Previously, we showed that macrophage infection by the protozoan parasite Leishmania amazonensis-the etiological agent of cutaneous leishmaniasis-can be controlled by ATP- and UTP-mediated activation of P2Y and P2X7 receptors (activated by UTP/ATP and ATP, respectively), which provided comparable immune responses against the parasite. Interestingly, in context of Leishmania amazonensis infection, UTP/P2Y triggered apoptosis, reactive oxygen species, and oxide nitric (NO) production, which are characteristic of P2X7 receptor activation. Here, we examined a possible "cross-talk" between P2Y2 and P2X7 receptors, and the requirement for pannexin-1 (PANX-1) in the control of L. amazonensis infection in mouse peritoneal macrophages and in vivo. UTP treatment reduced L. amazonensis parasite load, induced extracellular ATP release [which was pannexin-1 (PANX-1) dependent], and triggered leukotriene B4 (LTB4) production in macrophages. UTP-induced parasite control was blocked by pharmacological antagonism of P2Y2 or P2X7 receptors and was absent in macrophages lacking P2X7 or PANX-1. In addition, ATP release induced by UTP was also inhibited by PANX-1 blocker carbenoxolone, and partially reversed by inhibitors of vesicle traffic and actin cytoskeleton dynamics. In vivo, UTP treatment reduced footpad and popliteal lymph node parasite load, and the lesion in wild-type (WT) mice; fact not observed in P2X7-/- mice. Our data reveal that P2Y2 and P2X7 receptors cooperate to trigger potent innate immune responses against L. amazonensis infection.

P2Y2 purinergic receptors are highly expressed in cardiac and diaphragm muscles of mdx mice, and their expression is decreased by suramin.

INTRODUCTION: In Duchenne muscular dystrophy (DMD) and in the mdx mouse model of DMD, the lack of dystrophin leads to increased calcium influx and muscle necrosis. Patients suffer progressive muscle loss, and cardiomyopathy is an important determinant of morbidity. P2 purinergic receptors participate in the increased calcium levels in dystrophic skeletal muscles. METHODS: In this study, we evaluated whether P2 receptors are involved in cardiomyopathy in mdx mice at later stages of the disease. RESULTS: Western blotting revealed that P2Y2 receptor levels were upregulated (54%) in dystrophic heart compared with a normal heart. Suramin reduced the levels of P2Y2 to almost normal values. Suramin also decreased heart necrosis (reduced CK-MB) and the expression of the stretch-activated calcium channel TRPC1. CONCLUSIONS: This study suggests that P2Y2 may participate in cardiomyopathy in mdx mice. P2-selective drugs with specific actions in the dystrophic heart may ameliorate cardiomyopathy in dystrophinopathies. Muscle Nerve 55: 116-121, 2017.