P2Y11 Agonism Prevents Hypoxia/Reoxygenation- and Angiotensin II-Induced Vascular Dysfunction and Intimal Hyperplasia Development.

Vascular dysfunction in cardiovascular diseases includes vasomotor response impairments, endothelial cells (ECs) activation, and smooth muscle cells (SMCs) proliferation and migration to the intima. This results in intimal hyperplasia and vessel failure. We previously reported that activation of the P2Y11 receptor (P2Y11R) in human dendritic cells, cardiofibroblasts and cardiomyocytes was protective against hypoxia/reoxygenation (HR) lesions. In this study, we investigated the role of P2Y11R signaling in vascular dysfunction. P2Y11R activity was modulated using its pharmacological agonist NF546 and antagonist NF340. Rat aortic rings were exposed to angiotensin II (AngII) and evaluated for their vasomotor response. The P2Y11R agonist NF546 reduced AngII-induced vascular dysfunction by promoting EC-dependent vasorelaxation, through an increased nitric oxide (NO) bioavailability and reduced AngII-induced H2O2 release; these effects were prevented by the use of the P2Y11R antagonist NF340. Human vascular SMCs and ECs were subjected to AngII or H/R simulation in vitro. P2Y11R agonist modulated vasoactive factors in human ECs, that is, endothelial nitric oxide synthase (eNOS) and endothelin-1, reduced SMC proliferation and prevented the switch towards a synthetic phenotype. H/R and AngII increased ECs secretome-induced SMC proliferation, an effect prevented by P2Y11R activation. Thus, our data suggest that P2Y11R activation may protect blood vessels from HR-/AngII-induced injury and reduce vascular dysfunctions. These results open the way for new vasculoprotective interventions.

Stimulation of P2Y11 receptor modulates cardiac fibroblasts secretome toward immunomodulatory and protective roles after Hypoxia/Reoxygenation injury.

Cardiac fibroblasts are important regulators of myocardial structure and function. Their implications in pathological processes such as Ischemia/Reperfusion are well characterized. Cardiac fibroblasts respond to stress by excessive proliferation and secretion of pro-inflammatory cytokines and other factors, e.g. ATP, leading to purinergic receptors activation. P2Y11 receptor (P2Y11R) is an ATP-sensitive GPCR playing an immunomodulatory role in human dendritic cells (DC). We hypothesized that P2Y11R stimulation modulated the pro-inflammatory responses of human cardiac fibroblasts (HCF) to Hypoxia/Reoxygenation (H/R) mainly by acting on their secretome. P2Y11R stimulation in HCF at the onset of reoxygenation significantly limited H/R-induced proliferation (-19%) and pro-inflammatory cytokines and ATP secretion (-44% and -83% respectively). Exposure of DC to HCF secretome increased their expression of CD83, CD25 and CD86, suggesting a switch from immature to mature phenotype. Under LPS stimulation, DC had a pro-inflammatory profile (high IL-12/IL-10 ratio) that was decreased by HCF secretome (-3,8-fold), indicating induction of a tolerogenic profile. Moreover, P2Y11R inhibition in HCF led to high IL-12 secretion in DC, suggesting that the immunomodulatory effect of HCF secretome is P2Y11R-dependant. HCF secretome reduced H/R-induced cardiomyocytes death (-23%) through RISK pathway activation. P2Y11R inhibition in HCF induced a complete loss of HCF secretome protective effect, highlighting the cardioprotective role of P2Y11R. Our data demonstrated paracrine interactions between HCF, cardiomyocytes and DC following H/R, suggesting a key role of HCF in the cellular responses to reperfusion. These results also demonstrated a beneficial role of P2Y11R in HCF during H/R and strongly support the hypothesis that P2Y11R is a modulator of I/R injury.

Hypoxia/Reoxygenation Inhibits P2Y11 Receptor Expression and Its Immunosuppressive Activity in Human Dendritic Cells.

High concentrations of extracellular ATP (eATP) resulting from cell damage may be found during an ischemia/reperfusion (I/R) episode at the site of injury. eATP activates purinergic receptors in dendritic cells (DCs) and may inhibit inflammation. This immunosuppressive activity could be of interest in the field of I/R, which is an inflammatory condition involved in myocardial infarction, stroke, and solid organ transplantation. However, the specific purinergic receptor responsible for this effect remains to be identified. In this study, we report that eATP induced maturation of human monocyte-derived DCs. Additionally, eATP inhibited IL-12 production whereas IL-10 levels remained unchanged in activated DCs. These effects were prevented by the P2Y11R antagonist NF340. Interestingly, a 5-h hypoxia prevented the effects of eATP on cytokine production whereas a 1-h hypoxia did not affect the eATP-mediated decrease of IL-12 and IL-6. We showed a time-dependent downregulation of P2Y11R at both mRNA and protein levels that was prevented by knocking down hypoxia-inducible factor-1α. In this study, we showed an immunosuppressive role of P2Y11R in human DCs. Additionally, we demonstrated that the time-dependent downregulation of P2Y11R by hypoxia orientates DCs toward a proinflammatory phenotype that may be involved in post-I/R injuries as observed after organ transplantation.

NaV1.5 Na⁺ channels allosterically regulate the NHE-1 exchanger and promote the activity of breast cancer cell invadopodia.

The degradation of the extracellular matrix by cancer cells represents an essential step in metastatic progression and this is performed by cancer cell structures called invadopodia. NaV1.5 (also known as SCN5A) Na(+) channels are overexpressed in breast cancer tumours and are associated with metastatic occurrence. It has been previously shown that NaV1.5 activity enhances breast cancer cell invasiveness through perimembrane acidification and subsequent degradation of the extracellular matrix by cysteine cathepsins. Here, we show that NaV1.5 colocalises with Na(+)/H(+) exchanger type 1 (NHE-1) and caveolin-1 at the sites of matrix remodelling in invadopodia of MDA-MB-231 breast cancer cells. NHE-1, NaV1.5 and caveolin-1 co-immunoprecipitated, which indicates a close association between these proteins. We found that the expression of NaV1.5 was responsible for the allosteric modulation of NHE-1, rendering it more active at the intracellular pH range of 6.4-7; thus, it potentially extrudes more protons into the extracellular space. Furthermore, NaV1.5 expression increased Src kinase activity and the phosphorylation (Y421) of the actin-nucleation-promoting factor cortactin, modified F-actin polymerisation and promoted the acquisition of an invasive morphology in these cells. Taken together, our study suggests that NaV1.5 is a central regulator of invadopodia formation and activity in breast cancer cells.

Stimulation of murine P2Y11-like purinoreceptor protects against hypoxia/reoxygenation injury and decreases heart graft rejection lesions.

OBJECTIVE: Myocardial ischemia reperfusion is a major cause of cell injury during cardiac transplantation and is responsible for increased graft rejection. Several in vitro studies demonstrated the protective effect of P2Y11-like purinoreceptor stimulation in the context of myocardial ischemia/reperfusion. In this study, we hypothesized a possible cardioprotective role of P2Y11R stimulation against ischemia/reperfusion lesions and validated its clinical effect in vivo in a heart transplantation model. METHODS: We subjected H9c2 rat cardiomyocyte-derived cell line to 5 hours of hypoxia and 1 hour of reoxygenation. P2Y11R selective agonist NF546 and antagonist NF340 were added at the onset of reoxygenation. Cell injuries were assessed by microculture tetrazolium reduction and intracellular adenosine triphosphate level. Clinical effect of P2Y11R stimulation was further investigated in vivo. Hearts from BALB/c mice were transplanted intra-abdominally into allogenic C57BL/6 mice (n = 104). Recipient mice were injected with P2Y11R agonist. Mice in the sham group were injected with saline solution. In the control group, hearts from C57BL/6 were transplanted into syngeneic C57BL/6 mice. Rejection lesions were investigated using histology and immunohistochemistry at days 3, 5, and 7 after transplantation. We measured caspase activities to quantify apoptosis. Production of proinflammatory and anti-inflammatory cytokines was investigated. RESULTS: P2Y11R stimulation at the onset of reoxygenation significantly reduced in vitro hypoxia/reoxygenation injuries. This protection was suppressed with P2Y11R antagonist. In vivo, cardiac allograft survival was significantly prolonged after P2Y11R stimulation. Rejection lesions, classified according to the International Society of Heart Lung Transplantation guidelines and quantified using the mean number of inflammatory cells per field, were significantly reduced in the treated group. At day 5 after transplantation, P2Y11R agonist pretreated allografts also demonstrated less apoptotic lesions. CONCLUSIONS: Our data suggest a novel cardioprotective role of P2Y11R at the onset of reoxygenation/reperfusion against reperfusion injuries. Pharmacologic conditioning using P2Y11 agonist may be beneficial after cardiac transplantation in improving myocardial ischemia/reperfusion outcomes and decreasing graft rejection lesions.

Stimulation of P2Y11 receptor protects human cardiomyocytes against Hypoxia/Reoxygenation injury and involves PKCε signaling pathway.

Sterile inflammation is a key determinant of myocardial reperfusion injuries. It participates in infarct size determination in acute myocardial infarction and graft rejection following heart transplantation. We previously showed that P2Y11 exerted an immunosuppressive role in human dendritic cells, modulated cardiofibroblasts' response to ischemia/reperfusion in vitro and delayed graft rejection in an allogeneic heterotopic heart transplantation model. We sought to investigate a possible role of P2Y11 in the cellular response of cardiomyocytes to ischemia/reperfusion. We subjected human AC16 cardiomyocytes to 5 h hypoxia/1 h reoxygenation (H/R). P2Y11R (P2Y11 receptor) selective agonist NF546 and/or antagonist NF340 were added at the onset of reoxygenation. Cellular damages were assessed by LDH release, MTT assay and intracellular ATP level; intracellular signaling pathways were explored. The role of P2Y11R in mitochondria-derived ROS production and mitochondrial respiration was investigated. In vitro H/R injuries were significantly reduced by P2Y11R stimulation at reoxygenation. This protection was suppressed with P2Y11R antagonism. P2Y11R stimulation following H2O2-induced oxidative stress reduced mitochondria-derived ROS production and damages through PKCε signaling pathway activation. Our results suggest a novel protective role of P2Y11 in cardiomyocytes against reperfusion injuries. Pharmacological post-conditioning targeting P2Y11R could therefore contribute to improve myocardial ischemia/reperfusion outcomes in acute myocardial infarction and cardiac transplantation.