RESUMEN
ADAMTS1 inhibits capillary sprouting, and since capillary sprouts do not experience the shear stress caused by blood flow, this study undertook to clarify the relationship between shear stress and ADAMTS1. It was found that endothelial cells exposed to shear stress displayed a strong upregulation of ADAMTS1, dependent upon both the magnitude and duration of their exposure. Investigation of the underlying pathways demonstrated involvement of phospholipase C, phosphoinositide 3-kinase, and nitric oxide. Forkhead box protein O1 was identified as a likely inhibitor of the system, as its knockdown was followed by a slight increase in ADAMTS1 expression. In silico prediction displayed a transcriptional binding site for Forkhead box protein O1 in the promotor region of the ADAMTS1 gene, as well as sites for nuclear factor 1, SP1, and AP-1. The anti-angiogenic effects of ADAMTS1 were attributed to its cleavage of thrombospondin 1 into a 70-kDa fragment, and a significant enhancement of this fragment was indeed demonstrated by immunoblotting shear stress-treated cells. Accordingly, scratch wound closure displayed a slowdown in conditioned medium from shear stress-treated endothelial cells, an effect that could be completely blocked by a knockdown of thrombospondin 1 and partially blocked by a knockdown of ADAMTS1. Non-perfused capillary sprouts in rat mesenteries stained negative for ADAMTS1, while vessels in the microcirculation that had already experienced blood flow yielded the opposite results. The shear stress-dependent expression of ADAMTS1 in vitro was therefore also demonstrated in vivo and thereby confirmed as a mechanism connecting blood flow with the regulation of angiogenesis.
Asunto(s)
Proteínas ADAM/metabolismo , Capilares/fisiología , Células Endoteliales/metabolismo , Neovascularización Fisiológica , Estrés Mecánico , Proteínas ADAM/genética , Proteína ADAMTS1 , Animales , Capilares/ultraestructura , Línea Celular , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Humanos , Óxido Nítrico/metabolismo , Oxígeno/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Regiones Promotoras Genéticas , Ratas , Ratas Sprague-Dawley , Resistencia al Corte , Trombospondina 1/genética , Trombospondina 1/metabolismo , Fosfolipasas de Tipo C/genética , Fosfolipasas de Tipo C/metabolismoRESUMEN
Transcription factor Foxo-1 can be inactivated via Akt-mediated phosphorylation. Since shear stress activates Akt, we determined whether Foxo-1 and the Foxo-1-dependent, angiogenesis-related Ang-2/Tie2-system are influenced by shear stress in endothelial cells. Expression of Foxo-1 and its target genes p27Kip1 and Ang-2 was decreased under shear stress (6dyn/cm(2), 24h), nuclear exclusion of Foxo-1 by phosphorylation increased. eNOS and Tie2 were upregulated. No effects on Ang-1 expression were detected. In conclusion, Foxo-1 and Ang-2/Tie2 are part of the molecular response to shear stress, which may regulate angiogenesis.
Asunto(s)
Angiopoyetina 2/genética , Angiopoyetina 2/metabolismo , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Regulación de la Expresión Génica , Receptor TIE-2/genética , Receptor TIE-2/metabolismo , Angiopoyetina 1/genética , Angiopoyetina 1/metabolismo , Cromonas/farmacología , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/enzimología , Proteína Forkhead Box O1 , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Morfolinas/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Estrés Mecánico , Factores de TiempoRESUMEN
OBJECTIVE: The Interleukin 8 (IL-8) response of endothelial cells to lipoproteins has well known implications for the development and progression of atherosclerosis. In this study we sought for the role of zinc finger protein 580 (ZNF580) in the endothelial IL-8 response to lipoproteins. METHODS: In human umbilical vein endothelial cells (HUVEC) ZNF580 and IL-8 levels were examined by real-time-RT-PCR, immunoblotting and immunostaining or ELISA, respectively. RESULTS: ZNF580 is located in the nucleus and regulated by LDL and HDL depending on the oxLDL/LDL-ratio but not by TNFα. IL-8 expression profiles are inversely influenced by the oxLDL/LDL-ratio, both in vitro and in vivo. Knock down of ZNF580 enhances the expression and release of IL-8 and increases monocyte arrest under flow conditions in vitro. CONCLUSIONS: ZNF580 is a novel factor in the lipoprotein-dependent regulation of IL-8 and monocyte arrest. Therefore it may be a new potential target for intervention in atherosclerosis.