RESUMO
Communication between endothelial cells and cardiomyocytes is critical for cardiac development and regeneration. However the mechanisms involved in these endothelial-cardiomyocyte interactions remain poorly understood. Nucleotides are released within the heart, especially under ischemia or pressure overload. The function of P2Y nucleotide receptors in cardiac development has never been investigated. Here we show that adult P2Y(4)-null mice display microcardia. P2Y(4) nucleotide receptor is expressed in cardiac endothelial cells but not in cardiomyocytes. Loss of P2Y(4) in cardiac endothelial cells strongly inhibits their growth, migration and PDGF-B secretion in response to UTP. Proliferation of microvessels and cardiomyocytes is reduced in P2Y(4)-null hearts early after birth, resulting in reduced heart growth. Our study uncovers mouse P2Y(4) receptor as an essential regulator of cardiac endothelial cell function, and illustrates the involvement of endothelial-cardiomyocyte interactions in post-natal heart development. We also detected P2Y(4) expression in human cardiac microvessels. P2Y(4) receptor could constitute a therapeutic target to regulate cardiac remodelling and post-ischemic revascularisation.
Assuntos
Coração/crescimento & desenvolvimento , Receptores Purinérgicos P2/fisiologia , Animais , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Neovascularização Fisiológica , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The effects of ADP on the biology of dendritic cells have been studied much less than those of ATP or adenosine. In this study, we showed that adenosine-5'-O-(2-thiodiphosphate) (ADPßS) induced intracellular Ca(2+) transients in murine dendritic cells (DCs). This effect was abolished by AR-C69931MX, a dual P2Y(12) and P2Y(13) receptor antagonist. RT-PCR experiments revealed the expression of both P2Y(12) and P2Y(13) mRNA in DCs. The Ca(2+) response to ADPßS was maintained in P2Y(13)-deficient DCs, whereas it was abolished completely in P2Y(12)(-/-) DCs. ADPßS stimulated FITC-dextran and OVA capture in murine DCs through macropinocytosis, and this effect was abolished in P2Y(12)(-/-) DCs. ADPßS had a similar effect on FITC-dextran uptake by human monocyte-derived DCs. OVA loading in the presence of ADPßS increased the capacity of DCs to stimulate OVA-specific T cells, whereas ADPßS had no effect on the ability of DCs to stimulate allogeneic T cells. Moreover, after immunization against OVA, the serum level of anti-OVA IgG1 was significantly lower in P2Y(12)(-/-) mice than that in wild-type controls. In conclusion, we have shown that the P2Y(12) receptor is expressed in murine DCs and that its activation increased Ag endocytosis by DCs with subsequent enhancement of specific T cell activation.
Assuntos
Difosfato de Adenosina/análogos & derivados , Células Dendríticas/imunologia , Receptores Purinérgicos P2Y12/imunologia , Tionucleotídeos/imunologia , Difosfato de Adenosina/imunologia , Difosfato de Adenosina/metabolismo , Animais , Apresentação de Antígeno/imunologia , Células Dendríticas/metabolismo , Endocitose/imunologia , Humanos , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores Purinérgicos P2Y12/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/imunologia , Tionucleotídeos/metabolismoRESUMO
P2Y receptors are G-protein-coupled receptors activated by extracellular nucleotides. The P2Y(6) receptor is selectively activated by UDP, and its transcript has been detected in numerous organs, including the spleen, thymus, intestine, blood leukocytes, and aorta. To investigate the biological functions of this receptor, we generated P2Y(6)-null mice by gene targeting. The P2Y(6) knockout (KO) mice are viable and are not distinguishable from the wild-type (WT) mice in terms of growth or fertility. In thioglycollate-elicited macrophages, the production of inositol phosphate in response to UDP stimulation was lost, indicating that P2Y(6) is the unique UDP-responsive receptor expressed by mouse macrophages. Furthermore, the amount of interleukin-6 and macrophage-inflammatory protein-2, but not tumor necrosis factor-alpha, released in response to lipopolysaccharide stimulation was significantly enhanced in the presence of UDP, and this effect was lost in the P2Y(6) KO macrophages. The endothelium-dependent relaxation of the aorta by UDP was abolished in KO P2Y(6) mice. The contractile effect of UDP on the aorta, observed when endothelial nitric-oxide synthase is blocked, was also abolished in P2Y(6)-null mice. In conclusion, we generated P2Y(6)-deficient mice and have shown that these mice have a defective response to UDP in macrophages, endothelial cells, and vascular smooth muscle cells. These observations might be relevant to several physiopathological conditions such as atherosclerosis or hypertension.