RESUMO
Nitric oxide (NO) acts as essential regulator of vasculogenesis and angiogenesis and is critical for arteriogenesis. Whether NO's effects in vivo are mediated through NO-sensitive guanylyl cyclase (NO-GC) and thus by cGMP-dependent mechanisms has been only poorly addressed. Mice lacking NO-GC globally or specifically in smooth muscle cells (SMC) or endothelial cells (EC) were subjected to two established models for arteriogenesis and angiogenesis, namely hindlimb ischemia and oxygen-induced retinopathy. Our data clearly show the involvement of NO-GC in the recovery of blood flow after hindlimb ischemia, and this effect could be attributed to NO-GC in SMC. In the retina, global deletion of NO-GC led to reduced oxygen-induced vessel loss and hypoxia-induced capillary regrowth, whereas pathological neovascularization was increased. These effects were also seen in mice with SMC-specific NO-GC deletion but not in animals lacking NO-GC in EC. Intriguingly, NO-GC was found to be strongly expressed in retinal pericytes. Our data prove the involvement of NO-GC in growth and plasticity of hindlimb and retinal vasculature after ischemic/hypoxic insult.
Assuntos
Guanilato Ciclase/metabolismo , Neovascularização Patológica , Óxido Nítrico/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , GMP Cíclico/metabolismo , Células Endoteliais/metabolismo , Éxons , Guanilato Ciclase/genética , Membro Posterior/irrigação sanguínea , Hipóxia/patologia , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Isquemia/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miócitos de Músculo Liso/metabolismo , Oxigênio/química , Pericitos/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Retina/metabolismo , Retina/patologia , Doenças Retinianas/patologia , Transdução de Sinais , Guanilil Ciclase Solúvel , Fatores de TempoRESUMO
Variants in genes encoding the soluble guanylyl cyclase (sGC) in platelets are associated with coronary artery disease (CAD) risk. Here, by using histology, flow cytometry and intravital microscopy, we show that functional loss of sGC in platelets of atherosclerosis-prone Ldlr-/- mice contributes to atherosclerotic plaque formation, particularly via increasing in vivo leukocyte adhesion to atherosclerotic lesions. In vitro experiments revealed that supernatant from activated platelets lacking sGC promotes leukocyte adhesion to endothelial cells (ECs) by activating ECs. Profiling of platelet-released cytokines indicated that reduced platelet angiopoietin-1 release by sGC-depleted platelets, which was validated in isolated human platelets from carriers of GUCY1A1 risk alleles, enhances leukocyte adhesion to ECs. I mp or ta ntly, p ha rm ac ol ogical sGC stimulation increased platelet angiopoietin-1 release in vitro and reduced leukocyte recruitment and atherosclerotic plaque formation in atherosclerosis-prone Ldlr-/- mice. Therefore, pharmacological sGC stimulation might represent a potential therapeutic strategy to prevent and treat CAD.
RESUMO
Radioimmunoassay is an established method to determine the amount of a specific substance in a given cell or tissue sample. Commercially available RIA or Elisa are very cost intensive. Here, we describe the generation of radioactive cGMP tracer and the quantification of cGMP. Although working with radioactive material requires experience and care, this method is very sensitive and rather cheap, once it is established.