Limits...
Expression of Nitric Oxide-Transporting Aquaporin-1 Is Controlled by KLF2 and Marks Non-Activated Endothelium In Vivo.

Fontijn RD, Volger OL, van der Pouw-Kraan TC, Doddaballapur A, Leyen T, Baggen JM, Boon RA, Horrevoets AJ - PLoS ONE (2015)

Bottom Line: Chromosome immunoprecipitation (CHIP) confirms binding of KLF2 to the AQP1 promoter.We conclude that AQP1 expression is subject to KLF2-mediated positive regulation by atheroprotective shear stress and is downregulated under inflammatory conditions both in vitro and in vivo.Thus, endothelial expression of AQP1 characterizes the atheroprotected, non-inflamed vessel wall.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Cell Biology and Immunology, VU Medical Center, Amsterdam, the Netherlands.

ABSTRACT
The flow-responsive transcription factor Krüppel-like factor 2 (KLF2) maintains an anti-coagulant, anti-inflammatory endothelium with sufficient nitric oxide (NO)-bioavailability. In this study, we aimed to explore, both in vitro and in human vascular tissue, expression of the NO-transporting transmembrane pore aquaporin-1 (AQP1) and its regulation by atheroprotective KLF2 and atherogenic inflammatory stimuli. In silico analysis of gene expression profiles from studies that assessed the effects of KLF2 overexpression in vitro and atherosclerosis in vivo on endothelial cells, identifies AQP1 as KLF2 downstream gene with elevated expression in the plaque-free vessel wall. Biomechanical and pharmaceutical induction of KLF2 in vitro is accompanied by induction of AQP1. Chromosome immunoprecipitation (CHIP) confirms binding of KLF2 to the AQP1 promoter. Inflammatory stimulation of endothelial cells leads to repression of AQP1 transcription, which is restrained by KLF2 overexpression. Immunohistochemistry reveals expression of aquaporin-1 in non-activated endothelium overlying macrophage-poor intimae, irrespective whether these intimae are characterized as being plaque-free or as containing advanced plaque. We conclude that AQP1 expression is subject to KLF2-mediated positive regulation by atheroprotective shear stress and is downregulated under inflammatory conditions both in vitro and in vivo. Thus, endothelial expression of AQP1 characterizes the atheroprotected, non-inflamed vessel wall. Our data provide support for a continuous role of KLF2 in stabilizing the vessel wall via co-temporal expression of eNOS and AQP1 both preceding and during the pathogenesis of atherosclerosis.

Show MeSH

Related in: MedlinePlus

Suppression of AQP1 transcription by TNF-α is restrained by KLF2.HUVECs were transfected with either empty lentiviral vector (mock) or with a lentiviral vector carrying KLF2 under control of the PGK promoter (KLF2) and two days after transfection incubated with vehicle (ctr) or TNF-α at a concentration of 20 ng/ml during 24 hours (TNF). mRNA levels were determined for AQP1, normalized to P0 and expressed as mean and SEM (N = 4). *P<0.05, **P<0.01.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4696733&req=5

pone.0145777.g004: Suppression of AQP1 transcription by TNF-α is restrained by KLF2.HUVECs were transfected with either empty lentiviral vector (mock) or with a lentiviral vector carrying KLF2 under control of the PGK promoter (KLF2) and two days after transfection incubated with vehicle (ctr) or TNF-α at a concentration of 20 ng/ml during 24 hours (TNF). mRNA levels were determined for AQP1, normalized to P0 and expressed as mean and SEM (N = 4). *P<0.05, **P<0.01.

Mentions: In contrast to the atheroprotective effects of shear stress and KLF2 expression, intimal inflammation is a driving force in the pathology of atherosclerosis. We examined the response of AQP1 transcription to the inflammatory mediator TNF-α. HUVECs, either mock-transduced or transduced with an KLF2 encoding lentiviral vector, were incubated with 20 ng/ml TNF-α during 24h. As expected, levels of overexpressed KLF2 mRNA did not significantly change upon TNF-α incubation (S2A Fig). Basal expression levels of both AQP1 and eNOS were significantly reduced under TNF-α-induced inflammatory conditions. Constitutive overexpression of KLF2 completely preserved eNOS mRNA expression upon TNF-α incubation, (S2B Fig), whereas AQP1 mRNA expression was partially maintained (Fig 4). These results indicate that AQP1 transcription is reduced under TNF-α mediated inflammatory conditions and that KLF2 is able to significantly counteract this reduction.


Expression of Nitric Oxide-Transporting Aquaporin-1 Is Controlled by KLF2 and Marks Non-Activated Endothelium In Vivo.

Fontijn RD, Volger OL, van der Pouw-Kraan TC, Doddaballapur A, Leyen T, Baggen JM, Boon RA, Horrevoets AJ - PLoS ONE (2015)

Suppression of AQP1 transcription by TNF-α is restrained by KLF2.HUVECs were transfected with either empty lentiviral vector (mock) or with a lentiviral vector carrying KLF2 under control of the PGK promoter (KLF2) and two days after transfection incubated with vehicle (ctr) or TNF-α at a concentration of 20 ng/ml during 24 hours (TNF). mRNA levels were determined for AQP1, normalized to P0 and expressed as mean and SEM (N = 4). *P<0.05, **P<0.01.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4696733&req=5

pone.0145777.g004: Suppression of AQP1 transcription by TNF-α is restrained by KLF2.HUVECs were transfected with either empty lentiviral vector (mock) or with a lentiviral vector carrying KLF2 under control of the PGK promoter (KLF2) and two days after transfection incubated with vehicle (ctr) or TNF-α at a concentration of 20 ng/ml during 24 hours (TNF). mRNA levels were determined for AQP1, normalized to P0 and expressed as mean and SEM (N = 4). *P<0.05, **P<0.01.
Mentions: In contrast to the atheroprotective effects of shear stress and KLF2 expression, intimal inflammation is a driving force in the pathology of atherosclerosis. We examined the response of AQP1 transcription to the inflammatory mediator TNF-α. HUVECs, either mock-transduced or transduced with an KLF2 encoding lentiviral vector, were incubated with 20 ng/ml TNF-α during 24h. As expected, levels of overexpressed KLF2 mRNA did not significantly change upon TNF-α incubation (S2A Fig). Basal expression levels of both AQP1 and eNOS were significantly reduced under TNF-α-induced inflammatory conditions. Constitutive overexpression of KLF2 completely preserved eNOS mRNA expression upon TNF-α incubation, (S2B Fig), whereas AQP1 mRNA expression was partially maintained (Fig 4). These results indicate that AQP1 transcription is reduced under TNF-α mediated inflammatory conditions and that KLF2 is able to significantly counteract this reduction.

Bottom Line: Chromosome immunoprecipitation (CHIP) confirms binding of KLF2 to the AQP1 promoter.We conclude that AQP1 expression is subject to KLF2-mediated positive regulation by atheroprotective shear stress and is downregulated under inflammatory conditions both in vitro and in vivo.Thus, endothelial expression of AQP1 characterizes the atheroprotected, non-inflamed vessel wall.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Cell Biology and Immunology, VU Medical Center, Amsterdam, the Netherlands.

ABSTRACT
The flow-responsive transcription factor Krüppel-like factor 2 (KLF2) maintains an anti-coagulant, anti-inflammatory endothelium with sufficient nitric oxide (NO)-bioavailability. In this study, we aimed to explore, both in vitro and in human vascular tissue, expression of the NO-transporting transmembrane pore aquaporin-1 (AQP1) and its regulation by atheroprotective KLF2 and atherogenic inflammatory stimuli. In silico analysis of gene expression profiles from studies that assessed the effects of KLF2 overexpression in vitro and atherosclerosis in vivo on endothelial cells, identifies AQP1 as KLF2 downstream gene with elevated expression in the plaque-free vessel wall. Biomechanical and pharmaceutical induction of KLF2 in vitro is accompanied by induction of AQP1. Chromosome immunoprecipitation (CHIP) confirms binding of KLF2 to the AQP1 promoter. Inflammatory stimulation of endothelial cells leads to repression of AQP1 transcription, which is restrained by KLF2 overexpression. Immunohistochemistry reveals expression of aquaporin-1 in non-activated endothelium overlying macrophage-poor intimae, irrespective whether these intimae are characterized as being plaque-free or as containing advanced plaque. We conclude that AQP1 expression is subject to KLF2-mediated positive regulation by atheroprotective shear stress and is downregulated under inflammatory conditions both in vitro and in vivo. Thus, endothelial expression of AQP1 characterizes the atheroprotected, non-inflamed vessel wall. Our data provide support for a continuous role of KLF2 in stabilizing the vessel wall via co-temporal expression of eNOS and AQP1 both preceding and during the pathogenesis of atherosclerosis.

Show MeSH
Related in: MedlinePlus