UTP is a regulator of in vitro and in vivo angiogenic properties of cardiac adipose-derived stem cells.

The ability of cardiac adipose-derived stem cells (cADSC) to differentiate into multiple cell types has opened new perspectives in cardiac cell-based regenerative therapies. P2Y nucleotide receptors have already been described as regulators of adipogenic differentiation of cADSC and bone marrow-derived stem cells. In this study, we defined UTP as a regulator of cADSC endothelial differentiation. A daily UTP stimulation of cADSC during endothelial predifferentiation increased their capacity to form an endothelial network in matrigel. Additionally, pro-angiogenic UTP target genes such as epiregulin and hyaluronan synthase-1 were identified in predifferentiated cADSC by RNA sequencing experiments. Their regulation by UTP was confirmed by qPCR and ELISA experiments. We then evaluated the capacity of UTP-treated predifferentiated cADSC to increase post-ischemic revascularization in mice subjected to left anterior descending artery ligation. Predifferentiated cADSC treated or not with UTP were injected in the periphery of the infarcted zone, 3 days after ligation. We observed a significant increase of capillary density 14 and 30 days after UTP-treated predifferentiated cADSC injection, correlated with a reduction of cardiac fibrosis. This revascularization increase was not observed after injection of UTP-treated cADSC deficient for UTP and ATP nucleotide receptor P2Y2. The present study highlights the P2Y2 receptor as a regulator of cADSC endothelial differentiation and as a potential target for the therapeutic use of cADSC in post-ischemic heart revascularization.

Loss of Mouse P2Y6 Nucleotide Receptor Is Associated with Physiological Macrocardia and Amplified Pathological Cardiac Hypertrophy.

The study of the mechanisms leading to cardiac hypertrophy is essential to better understand cardiac development and regeneration. Pathological conditions such as ischemia or pressure overload can induce a release of extracellular nucleotides within the heart. We recently investigated the potential role of nucleotide P2Y receptors in cardiac development. We showed that adult P2Y4-null mice displayed microcardia resulting from defective cardiac angiogenesis. Here we show that loss of another P2Y subtype called P2Y6, a UDP receptor, was associated with a macrocardia phenotype and amplified pathological cardiac hypertrophy. Cardiomyocyte proliferation and size were increased in vivo in hearts of P2Y6-null neonates, resulting in enhanced postnatal heart growth. We then observed that loss of P2Y6 receptor enhanced pathological cardiac hypertrophy induced after isoproterenol injection. We identified an inhibitory effect of UDP on in vitro isoproterenol-induced cardiomyocyte hyperplasia and hypertrophy. The present study identifies mouse P2Y6 receptor as a regulator of cardiac development and cardiomyocyte function. P2Y6 receptor could constitute a therapeutic target to regulate cardiac hypertrophy.

Loss of mouse P2Y4 nucleotide receptor protects against myocardial infarction through endothelin-1 downregulation.

Nucleotides are released in the heart under pathological conditions, but little is known about their contribution to cardiac inflammation. The present study defines the P2Y4 nucleotide receptor, expressed on cardiac microvascular endothelial cells and involved in postnatal heart development, as an important regulator of the inflammatory response to cardiac ischemia. P2Y4-null mice displayed smaller infarcts in the left descending artery ligation model, as well as reduced neutrophil infiltration and fibrosis. Gene profiling identified inter alia endothelin-1 (ET-1) as one of the target genes of P2Y4 in ischemic heart. The reduced level of ET-1 was correlated with reduction of microvascular hyperpermeability, neutrophil infiltration, and endothelial adhesion molecule expression, and it could be explained by the decreased number of endothelial cells in P2Y4-null mice. Expression analysis of metalloproteinases and their tissue inhibitors in ischemic heart revealed reduced expression of matrix metalloproteinase (MMP)-9, reported to be potentially regulated by ET-1, and MMP-8, considered as neutrophil collagenase, as well as reduction of tissue inhibitor of MMP-1 and tissue inhibitor of MMP-4 in P2Y4-null mice. Reduction of cardiac permeability and neutrophil infiltration was also observed in P2Y4-null mice in LPS-induced inflammation model. Protection against infarction resulting from loss of P2Y4 brings new therapeutic perspectives for cardiac ischemia and remodeling.

ATP confers tumorigenic properties to dendritic cells by inducing amphiregulin secretion.

ATP, which has an important proinflammatory action as danger signal, induces the semimaturation of dendritic cells (DCs) that can be associated with immune tolerance. We identified epidermal growth factor receptor ligands as target genes of ATPγS, a slowly hydrolyzed ATP derivative, by a gene profiling approach in DCs. Amphiregulin was the most highly up-regulated gene in response to ATPγS. Human monocyte-derived DCs and mouse bone marrow-derived DCs released amphiregulin (AREG) after purinergic receptor activation, with a contribution of P2Y(11) and A(2B) receptor, respectively. Supernatants of LPS+ATPγS-stimulated DCs induced smooth muscle cell and Lewis Lung Carcinoma (LLC) cell growth in vitro. The coinjection of LPS+ATPγS-stimulated DCs or their supernatants with LLC cells increased tumor weight in mice compared with LPS-treated DCs. The preincubation of LPS+ATPγS-treated DC supernatants with an anti-AREG blocking antibody inhibited their positive effect on smooth muscle cell density and tumor growth. The present study demonstrates for the first time that DCs can be a source of AREG. ATP released from tumor cells might exert a tumorigenic action by stimulating the secretion of AREG from DCs. Antagonists of purinergic receptors expressed on DCs and anti-AREG blocking antibodies could have a therapeutic potential as antitumor agents.

P2Y2 receptor regulates VCAM-1 membrane and soluble forms and eosinophil accumulation during lung inflammation.

ATP has been defined as a key mediator of asthma. In this study, we evaluated lung inflammation in mice deficient for the P2Y(2) purinergic receptor. We observed that eosinophil accumulation, a distinctive feature of lung allergic inflammation, was defective in OVA-treated P2Y(2)-deficient mice compared with OVA-treated wild type animals. Interestingly, the upregulation of VCAM-1 was lower on lung endothelial cells of OVA-treated P2Y(2)(-/-) mice compared with OVA-treated wild type animals. Adhesion assays demonstrated that the action of UTP on leukocyte adhesion through the regulation of endothelial VCAM-1 was abolished in P2Y(2)-deficient lung endothelial cells. Additionally, the level of soluble VCAM-1, reported as an inducer of eosinophil chemotaxis, was strongly reduced in the bronchoalveolar lavage fluid (BALF) of P2Y(2)-deficient mice. In contrast, we observed comparable infiltration of macrophages and neutrophils in the BALF of LPS-aerosolized P2Y(2)(+/+) and P2Y(2)(-/-) mice. This difference could be related to the much lower level of ATP in the BALF of LPS-treated mice compared with OVA-treated mice. Our data define P2Y(2) as a regulator of membrane and soluble forms of VCAM-1 and eosinophil accumulation during lung inflammation.

Mouse P2Y4 Nucleotide Receptor Is a Negative Regulator of Cardiac Adipose-Derived Stem Cell Differentiation and Cardiac Fat Formation.

Cardiac adipose tissue-derived stem cells (cASCs) have the ability to differentiate into multiple cell lineages giving them a high potential for use in regenerative medicine. Cardiac fat tissue still raises many unsolved questions related to its formation and features. P2Y nucleotide receptors have already been described as regulators of differentiation of bone-marrow derived stem cells, but remain poorly investigated in cASCs. We defined, in this study, the P2Y4 nucleotide receptor as a negative regulator of cardiac fat formation and cASC adipogenic differentiation. Higher expression of P2Y4 receptor in cardiac fat tissue was observed compared to other adipose tissues. P2Y4-null mice displayed a higher mass of cardiac adipose tissue specifically. We therefore examined the role of P2Y4 receptor in cASC adipogenic differentiation. An inhibitory effect of uridine 5'-triphosphate (UTP), ligand of P2Y4, was observed on the maturation state of differentiated cASCs, and on the expression of adipogenesis-linked genes and adiponectin, a cardioprotective adipokine. Higher adiponectin secretion by P2Y4-null adipocytes could be linked with cardioprotection previously observed in the heart of P2Y4-null ischemic mice. We realized here left anterior descending artery ligation on simple and double-knockout mice for P2Y4 and adiponectin. No cardioprotective effect of P2Y4 loss was observed in the absence of adiponectin secretion. In addition, P2Y4 loss was correlated with higher expression of UCP-1 (uncoupling protein-1) and CD137, two markers of brown/beige cardiac adipocytes. Our data highlight the P2Y4 receptor as an inhibitor of cardiac fat formation and cASC adipogenic differentiation, and as a potential therapeutic target in the regulation of cardioprotective function of cardiac fat.

Protective role of P2Y2 receptor against lung infection induced by pneumonia virus of mice.

ATP released in the early inflammatory processes acts as a danger signal by binding to purinergic receptors expressed on immune cells. A major contribution of the P2Y(2) receptor of ATP/UTP to dendritic cell function and Th2 lymphocyte recruitment during asthmatic airway inflammation was previously reported. We investigated here the involvement of P2Y(2) receptor in lung inflammation initiated by pneumonia virus of mice infection. We demonstrated that P2Y(2) (-/-) mice display a severe increase in morbidity and mortality rate in response to the virus. Lower survival of P2Y(2) (-/-) mice was not significantly correlated with excessive inflammation despite the higher level of neutrophil recruiters in their bronchoalveolar fluids. Interestingly, we observed reduced ATP level and lower numbers of dendritic cells, CD4(+) T cells and CD8(+) T cells in P2Y(2) (-/-) compared to P2Y(2) (+/+) infected lungs. Lower level of IL-12 and higher level of IL-6 in bronchoalveolar fluid support an inhibition of Th1 response in P2Y(2) (-/-) infected mice. Quantification of DC recruiter expression revealed comparable IP-10 and MIP-3α levels but a reduced BRAK level in P2Y(2) (-/-) compared to P2Y(2) (+/+) bronchoalveolar fluids. The increased morbidity and mortality of P2Y(2) (-/-) mice could be the consequence of a lower viral clearance leading to a more persistent viral load correlated with the observed higher viral titer. The decreased viral clearance could result from the defective Th1 response to PVM with a lack of DC and T cell infiltration. In conclusion, P2Y(2) receptor, previously described as a target in cystic fibrosis therapy and as a mediator of Th2 response in asthma, may also regulate Th1 response protecting mice against lung viral infection.