4 research outputs found
haemoreologic aspects of regulation of expression of angiogenesis modulating factors ADAMTS1, Angiopoietin-2 and Interleukin-8
Ziel dieser Arbeit war die Untersuchung der strömungsabhängigen
Expressionsregulation des antiangiogenen Faktors ADAMTS1 und des angiogenen
Faktors Angiopoietin-2 in Endothelzellen in vitro und in vivo. ADAMTS1 wurde
durch Strömung Zeit- und Kraft-abhängig induziert, Angiopoietin-2 dagegen
supprimiert. Phospholipase C, Phosphoinositol-3-Kinase, eNOS und FoxO1 waren
an der Expressionsregulation von ADAMTS1 beteiligt. Phosphoinositol-3-Kinase,
Akt und FoxO1 waren an der Expressionsregulation von Angiopoietin-2 beteiligt.
Im Bereich von 20-100 mmHg war die Expression von ADAMTS1 darüber hinaus
direkt mit dem Sauerstoffpartialdruck korreliert. Der antiangiogene Effekt von
ADAMTS1 wurde durch ein 70 kDa Fragment von Thrombospondin-1 vermittelt. Im
scratch wound assay war der Verschluß des Zelldefektes unter Medium von
strömungsbehandelten Zellen verzögert. Dieser Effekt wurde durch Blockung
(siRNA) von ADAMTS1 teilweise und Blockung von TSP1 vollständig aufgehoben.
Sprossende Kapillaren in Totalpräparaten des Rattenmesenteriums ließen sich
für Angiopoietin-2, nicht jedoch für ADAMTS1 anfärben. Dagegen waren reife,
perfundierte Blutgefäße positiv für ADAMTS1 dessen Expression darüber hinaus
mit der intravitalmikrokopisch gemessenen Wandschubspannung dieser Gefäße eng
korreliert war. LDL mit niedrigem oxLDL/LDL Quotienten induzierte die
Expression von Zinkfingerprotein 580, welches seinerseits die Expression von
IL-8 supprimierte. Einer Hemmung von ZNF580 (siRNA) folgte ein Anstieg der
Adhäsion von Monozyten am Endothel. Zusammenfassend sprechen die Ergebnisse
dieser Arbeit für eine strömungsregulierte Synthese von ADAMTS1 und
Angiopoietin-2 in Endothelzellen und den Einfluß des Transkriptionsfaktors
ZNF580 auf die Expressionsregulation von IL-8. Die hier untersuchten
Mechanismen werden daher eine Rolle spielen für die Steuerung des Wachstums
von Blutgefäßen in vivo und die Initiierung der Atherosklerose.This study aimed at analyzing blood flow-dependent effects on the expression
regulation of ADAMTS1 and angiopoietin-2 in endothelial cells. ADAMTS1 is a
putative inhibitor of angiogenesis while angiopoietin-2 promotes angiogenesis.
Expression of ADAMTS1 was strongly induced by flow-elicited shear stress
acting on endothelial cells both on the mRNA- and the protein level. This
induction was time- and shear stress-dependent. Activation of phospholipase C,
phosphoinositol-3-kinase, and generation of nitric oxide were all likewise
necessary to induce the flow-dependent increase in ADAMTS1 with a small
contribution of down-regulation of FoxO1. By contrast to ADAMTS1,
angiopoietin-2 was reduced by shear stress. This reduction was transmitted via
the phosphoinositol-3-kinase-Akt-FoxO1-pathway. In addition to shear stress,
increasing oxygen pressure between 20-100 mmHg similarly increased ADAMTS1.
Looking for anti-angiogenic mechanisms of ADAMTS1 revealed an increase of a
thrombospondin-1 fragment (70 kDa) in shear stress-treated endothelial cells
in an ADAMTS1-dependent manner. According to this, scratch wound closure
(using the scratch wound assay) was delayed with shear stress-treated cells
conditioned medium. This delay was partly abolished if ADAMTS1 was knocked
down with specific siRNA and it was completely absent if TSP1 was knocked
down. In vivo, sprouting capillaries stained positive for angiopoietin-2 and
negative for ADAMTS1. Moreover, ADAMTS1 staining of mature blood vessels
correlated with the amount of shear stress measured by means of intravital
microscopy. LDL with only minute parts of oxLDL (low oxLDL/LDL ratio) induced
the expression of a new zink finger protein (ZNF580) which in turn suppressed
the expression of interleukin 8. Consequently, MonoMac6 adhesion to
endothelial cells was enhanced if ZNF580 was knocked down (siRNA). Taken
togehter, the data presented here demonstrate shear stress-dependent
endothelial cell mechanisms for the regulation of angiogenesis and the control
of atherosclerosis
Regulation of Foxo-1 and the angiopoietin-2/Tie2 system by shear stress
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
Expression of ADAMTS1 in endothelial cells is induced by shear stress and suppressed in sprouting capillaries
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