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

KLF2 dependency of AQP1 expression.(A, B) Expression of KLF2 was induced in HUVEC by laminar flow (shear), lentiviral transduction (KLF2) or incubation with atorvastatin (statin). mRNA levels were determined by semi-quantitative RT-PCR for AQP1 (A) and eNOS (B), normalized to P0 and shown as mean and SEM of the fold induction (grey) relative to the corresponding control (white). The following conditions are depicted: shear; exposure to laminar shear stress for ≥ 4 days at an average of 18 dyne/cm2, induction relative to static control (N = 6), KLF2; transduction with a lentiviral vector carrying KLF2 under control of the PGK promoter and subsequent growth for ≥ 4 days, induction relative to mock transduced cells (N = 7), statin; incubation with atorvastatin at a final concentration of 10 μM during 24 hours, induction relative to vehicle control (N = 5). *P<0.05, **P<0.01. (C) HUVECs were transduced with a lentiviral vector encoding an shRNA targeting KLF2. 24 hours later, expression of KLF2 was induced by incubation with atorvastatin at a final concentration of 10 μM during 24 hours. mRNA levels were determined for KLF2, AQP1 and eNOS, normalized to P0 and shown as mean and SEM mRNA level (grey) relative to control cells transduced with a non-targeting construct (white)(N = 3). *P<0.05, **P<0.01.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0145777.g002: KLF2 dependency of AQP1 expression.(A, B) Expression of KLF2 was induced in HUVEC by laminar flow (shear), lentiviral transduction (KLF2) or incubation with atorvastatin (statin). mRNA levels were determined by semi-quantitative RT-PCR for AQP1 (A) and eNOS (B), normalized to P0 and shown as mean and SEM of the fold induction (grey) relative to the corresponding control (white). The following conditions are depicted: shear; exposure to laminar shear stress for ≥ 4 days at an average of 18 dyne/cm2, induction relative to static control (N = 6), KLF2; transduction with a lentiviral vector carrying KLF2 under control of the PGK promoter and subsequent growth for ≥ 4 days, induction relative to mock transduced cells (N = 7), statin; incubation with atorvastatin at a final concentration of 10 μM during 24 hours, induction relative to vehicle control (N = 5). *P<0.05, **P<0.01. (C) HUVECs were transduced with a lentiviral vector encoding an shRNA targeting KLF2. 24 hours later, expression of KLF2 was induced by incubation with atorvastatin at a final concentration of 10 μM during 24 hours. mRNA levels were determined for KLF2, AQP1 and eNOS, normalized to P0 and shown as mean and SEM mRNA level (grey) relative to control cells transduced with a non-targeting construct (white)(N = 3). *P<0.05, **P<0.01.

Mentions: We studied the effect of KLF2 expression on AQP1 mRNA levels. Either KLF2 was constitutively overexpressed from a lentiviral vector, or KLF2 was induced by mechanical or pharmaceutical stimuli. The following conditions were applied: 1. Cells were exposed to prolonged laminar shear stress (≥ 4 days at an average of 18 dyne/cm2) [4]. 2. Cells were transduced with a lentiviral vector expressing KLF2 from the phosphoglycerate kinase-1 promoter and subsequently grown for ≥ 4 days [5]. 3. Cells were incubated with atorvastatin at a final concentration of 10 μM during 24 hours [9,10]. Total RNA was harvested and used to determine induction of AQP1 by means of semi-quantitative RT-PCR. As a control, we determined induction levels of an established KLF2 direct target gene; eNOS. Compared to the other conditions applied, duration of statin incubation was limited to 24 hours due to adverse effects of prolonged cholesterol-deprived growth that results from HMG-CoA inhibition. Despite differences in duration of stimulation, AQP1 showed a significant upregulation of between three- and four-fold with no significant differences between applied stimuli (Fig 2A). eNOS showed a comparable, significant five-fold upregulation during 24 hours culture in the presence of statins but tended to be higher upregulated under conditions of prolonged shear (fourteen-fold) or ≥4 days of KLF2 overexpression (twelve-fold), which is in full agreement with earlier studies of our group [4,5] (Fig 2B). These results reveal that AQP1 mRNA is induced within 24 hours of KLF2 induction by statin, suggesting a direct role of KLF2 in AQP1 transcription. Furthermore, the data suggest that AQP1 mRNA steady state levels are reached within a shorter time-span as compared to eNOS mRNA.


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)

KLF2 dependency of AQP1 expression.(A, B) Expression of KLF2 was induced in HUVEC by laminar flow (shear), lentiviral transduction (KLF2) or incubation with atorvastatin (statin). mRNA levels were determined by semi-quantitative RT-PCR for AQP1 (A) and eNOS (B), normalized to P0 and shown as mean and SEM of the fold induction (grey) relative to the corresponding control (white). The following conditions are depicted: shear; exposure to laminar shear stress for ≥ 4 days at an average of 18 dyne/cm2, induction relative to static control (N = 6), KLF2; transduction with a lentiviral vector carrying KLF2 under control of the PGK promoter and subsequent growth for ≥ 4 days, induction relative to mock transduced cells (N = 7), statin; incubation with atorvastatin at a final concentration of 10 μM during 24 hours, induction relative to vehicle control (N = 5). *P<0.05, **P<0.01. (C) HUVECs were transduced with a lentiviral vector encoding an shRNA targeting KLF2. 24 hours later, expression of KLF2 was induced by incubation with atorvastatin at a final concentration of 10 μM during 24 hours. mRNA levels were determined for KLF2, AQP1 and eNOS, normalized to P0 and shown as mean and SEM mRNA level (grey) relative to control cells transduced with a non-targeting construct (white)(N = 3). *P<0.05, **P<0.01.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0145777.g002: KLF2 dependency of AQP1 expression.(A, B) Expression of KLF2 was induced in HUVEC by laminar flow (shear), lentiviral transduction (KLF2) or incubation with atorvastatin (statin). mRNA levels were determined by semi-quantitative RT-PCR for AQP1 (A) and eNOS (B), normalized to P0 and shown as mean and SEM of the fold induction (grey) relative to the corresponding control (white). The following conditions are depicted: shear; exposure to laminar shear stress for ≥ 4 days at an average of 18 dyne/cm2, induction relative to static control (N = 6), KLF2; transduction with a lentiviral vector carrying KLF2 under control of the PGK promoter and subsequent growth for ≥ 4 days, induction relative to mock transduced cells (N = 7), statin; incubation with atorvastatin at a final concentration of 10 μM during 24 hours, induction relative to vehicle control (N = 5). *P<0.05, **P<0.01. (C) HUVECs were transduced with a lentiviral vector encoding an shRNA targeting KLF2. 24 hours later, expression of KLF2 was induced by incubation with atorvastatin at a final concentration of 10 μM during 24 hours. mRNA levels were determined for KLF2, AQP1 and eNOS, normalized to P0 and shown as mean and SEM mRNA level (grey) relative to control cells transduced with a non-targeting construct (white)(N = 3). *P<0.05, **P<0.01.
Mentions: We studied the effect of KLF2 expression on AQP1 mRNA levels. Either KLF2 was constitutively overexpressed from a lentiviral vector, or KLF2 was induced by mechanical or pharmaceutical stimuli. The following conditions were applied: 1. Cells were exposed to prolonged laminar shear stress (≥ 4 days at an average of 18 dyne/cm2) [4]. 2. Cells were transduced with a lentiviral vector expressing KLF2 from the phosphoglycerate kinase-1 promoter and subsequently grown for ≥ 4 days [5]. 3. Cells were incubated with atorvastatin at a final concentration of 10 μM during 24 hours [9,10]. Total RNA was harvested and used to determine induction of AQP1 by means of semi-quantitative RT-PCR. As a control, we determined induction levels of an established KLF2 direct target gene; eNOS. Compared to the other conditions applied, duration of statin incubation was limited to 24 hours due to adverse effects of prolonged cholesterol-deprived growth that results from HMG-CoA inhibition. Despite differences in duration of stimulation, AQP1 showed a significant upregulation of between three- and four-fold with no significant differences between applied stimuli (Fig 2A). eNOS showed a comparable, significant five-fold upregulation during 24 hours culture in the presence of statins but tended to be higher upregulated under conditions of prolonged shear (fourteen-fold) or ≥4 days of KLF2 overexpression (twelve-fold), which is in full agreement with earlier studies of our group [4,5] (Fig 2B). These results reveal that AQP1 mRNA is induced within 24 hours of KLF2 induction by statin, suggesting a direct role of KLF2 in AQP1 transcription. Furthermore, the data suggest that AQP1 mRNA steady state levels are reached within a shorter time-span as compared to eNOS mRNA.

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