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.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

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

info:eu-repo/semantics/publishe

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.JOURNAL ARTICLESCOPUS: ar.jinfo:eu-repo/semantics/publishe

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

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 post-natal 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

Higher mortality rate in PVM-infected P2Y₂-deficient mice.

<p>Following intranasal inoculation of PVM (1000 PFUs), P2Y₂^+/+ and P2Y₂^−/− mice were monitored daily for survival (<b>A</b>) and weight loss (<b>B</b>). Weight curves (mean ± SEM) are relative to initial body weight. The displayed data result from the pooling of four independent experiments.</p

Quantification of neutrophils and macrophages, and their recruiters in the lungs of PVM-infected P2Y₂^+/+ and P2Y₂^−/− mice.

<p><b>A–D.</b> The level of the chemokines KC/CXCL-1 (A), MIP-2/CXCL-2 (B), MIP-1α/CCL3 (C) and MCP-1/CCL2 (D) was determined by ELISA in the BALFs of PVM-infected P2Y₂^+/+ and P2Y₂^−/− mice. (N = 9) <b>E.</b> Flow cytometry quantification of neutrophils and macrophages in the BALFs of PVM-infected P2Y₂^+/+ and P2Y₂^−/− mice at day 8 and 10 post-inoculation (N = 12). <b>F.</b> Cytospin preparations were made from BALFs of P2Y₂^+/+ and P2Y₂^−/− mice at day 8 post-infection using a Shandon III cytocentrifuge and were stained using Diff-Quick staining. Magnification: ×400.</p

Cellular infiltration in the lungs of PVM-infected P2Y₂^+/+ and P2Y₂^−/− mice.

<p>50 µL of the viral suspension were instilled into the nostrils of the anesthetized mouse maintained in a vertical position as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050385#s2" target="_blank">Materials and Methods</a>. <b>A</b>, BALF was collected and the total number of cells was evaluated at days 8, 9, 10 and 12 (N = 7). <b>B.</b> Quantification of ATP level in the BALF of PVM-infected P2Y₂^+/+ and P2Y₂^−/− mice. ATP level was quantified in the BALF of P2Y₂^+/+ and P2Y₂^−/− mice using ATP detection assay system ATPlite at d8, d9 and d10 post-infection with PVM. <b>C</b>, Histological analysis of representative lungs of P2Y₂^+/+ and P2Y₂^−/− mice 10 days and 15 days after infection with PVM. Paraffin sections (7 µM) of lungs of P2Y₂^+/+ and P2Y₂^−/− mice infected by PVM were stained with haematoxylin-eosin (magnification: ×200).</p

Defective infiltration of DCs, CD4⁺ T cells and CD8⁺ T cells in the lungs of PVM-infected P2Y₂-deficient mice.

<p><b>A.</b> The percentage of DCs (MHC II⁺ CD11c⁺ CD11b⁺), CD4⁺ T cells and CD8⁺ T cells were determined by flow cytometry analysis in BALFs of P2Y₂^+/+ and P2Y₂^−/− mice at day 8 and days 10 (d8 and d10) post-infection (N = 5). <b>B.</b> Analysis of viral titer in P2Y₂^+/+ and P2Y₂^−/− lungs after infection with PVM. PVM viral titer was quantified by quantitative PCR in lung homogenates of P2Y₂^+/+ and P2Y₂^−/− mice 8 or 10 days post-infection. Data were normalized to the viral titer obtained for P2Y₂^+/+ lungs at d8. <b>C.</b> IL-12, IFN-γ, TNF-α and IL-6 levels were determined by ELISA in the BALFs of PVM-infected P2Y₂^+/+ and P2Y₂^−/− mice (N = 11). <b>D.</b> The level of DC recruiters was determined by ELISA (MIP-3α, IP-10) or qPCR (BRAK) in the BALFs of PVM-infected P2Y₂^+/+ and P2Y₂^−/− mice (N = 5).</p