RESUMO
OBJECTIVE: Angiotensin II (AngII) has been shown to regulate angiogenesis and at high pathophysiological doses to cause vasoconstriction through the AngII receptor type 1. Angiotensin 1 to 7 (Ang-(1-7)) acting through the Mas1 receptor can act antagonistically to high pathophysiological levels of AngII by inducing vasodilation, whereas the effects of Ang-(1-7) signaling on angiogenesis are less defined. To complicate the matter, there is growing evidence that a subpressor dose of AngII produces phenotypes similar to Ang-(1-7). APPROACH AND RESULTS: This study shows that low-dose Ang-(1-7), acting through the Mas1 receptor, promotes angiogenesis and vasodilation similar to a low, subpressor dose of AngII acting through AngII receptor type 1. In addition, we show through in vitro tube formation that Ang-(1-7) augments the angiogenic response in rat microvascular endothelial cells. Using proteomic and genomic analyses, downstream components of Mas1 receptor signaling were identified, including Rho family of GTPases, phosphatidylinositol 3-kinase, protein kinase D1, mitogen-activated protein kinase, and extracellular signal-related kinase signaling. Further experimental antagonism of extracellular signal-related kinases 1/2 and p38 mitogen-activated protein kinase signaling inhibited endothelial tube formation and vasodilation when stimulated with equimolar, low doses of either AngII or Ang-(1-7). CONCLUSIONS: These results significantly expand the known Ang-(1-7)/Mas1 receptor signaling pathway and demonstrate an important distinction between the pathological effects of elevated and suppressed AngII compared with the beneficial effects of AngII normalization and Ang-(1-7) administration. The observed convergence of Ang-(1-7)/Mas1 and AngII/AngII receptor type 1 signaling at low ligand concentrations suggests a nuanced regulation in vasculature. These data also reinforce the importance of mitogen-activated protein kinase/extracellular signal-related kinase signaling in maintaining vascular function.
Assuntos
Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Artéria Cerebral Média/metabolismo , Neovascularização Fisiológica , Proteínas Proto-Oncogênicas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Vasodilatação , Angiotensina I/farmacologia , Angiotensina II/farmacologia , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Animais , Relação Dose-Resposta a Droga , Estimulação Elétrica , Células Endoteliais/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/inervação , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação da Expressão Gênica , Masculino , Artéria Cerebral Média/efeitos dos fármacos , Artéria Cerebral Média/inervação , Neovascularização Fisiológica/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/agonistas , Ratos Sprague-Dawley , Receptor Tipo 1 de Angiotensina/efeitos dos fármacos , Receptor Tipo 1 de Angiotensina/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Vasodilatação/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Bone marrow-derived endothelial progenitor cells (BM-EPCs) are stimulated by vascular endothelial growth factor-A (VEGF-A) and other potent proangiogenic factors. During angiogenesis, an increase in VEGF-A expression stimulates BM-EPCs to enhance endothelial tube formation and contribute to an increase in microvessel density. Hypoxia is known to produce an enhanced angiogenic response and heightened levels of VEGF-A have been seen in oxygen deprived epithelial and endothelial cells, yet the pathways for VEGF-A signaling in BM-EPCs have not been described. This study explores the influence of hypoxia on VEGF-A signaling in rat BM-EPCs utilizing a novel proteomic strategy to directly identify interacting downstream components of the combined VEGF receptor(s) signaling pathways, gene expression analysis, and functional phenotyping. VEGF-A signaling network analysis following liquid chromatographic separation and tandem mass spectrometry revealed proteins related to inositol/calcium signaling, nitric oxide signaling, cell survival, cell migration, and inflammatory responses. Alterations in BM-EPC expression of common angiogenic genes and tube formation in response to VEGF-A during hypoxia were measured and combined with the proteomic analysis to enhance and support the signaling pathways detected. BM-EPC tube formation assays in response to VEGF-A exhibited little tube formation; however, a cell projection/migratory phenotype supported the signaling data. Additionally, a novel assay measuring BM-EPC incorporation into preformed endothelial cell tubes indicated a significant increase of incorporated BM-EPCs after pretreatment with VEGF-A during hypoxia. This study verifies known VEGF-A pathway components and reveals several unidentified mechanisms of VEGF-A signaling in BM-EPCs during hypoxia that may be important for migration to sites of vascular regeneration.