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
Wound healing and the grow-in of free tissue grafts critically depend on blood vessel growth, i.e., on the angiogenic invasion of endothelial cells, which is critically reduced in smokers, in patients suffering from microangiopathies (e.g., in diabetes), or in those who are treated with immunosuppressives. Although several angiogenic factors have been tested to accelerate wound healing in such critically patients, their combinations have not yet been systematically investigated. This study was done to reveal which combination of proangiogenic with promaturating factors is the most effective in an endothelial wound closure assay. Human umbilical vein endothelial cells were isolated, cultured to confluence, and subjected to a scratch wound assay with the addition of vascular endothelial growth factor (VEGF)-A(165), platelet-derived growth factor (PDGF)-AB, angiopoietin-1 (ANG1), or ANG2 and all of their 16 possible combinations. VEGF-A(165) plus ANG1 was most effective at accelerating endothelial scratch closure. Moreover, VEGF-A(165) stimulated wound closure in all combinations tested, while it was attenuated by PDGF-AB. Thus, with respect to their effects on endothelial cells, a combination of VEGF-A with ANG1 is the most promising and is superior to combinations with PDGF-AB.