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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.

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AQP1 is preferentially expressed in endothelium overlying plaque-free intimae and is induced by KLF2.(A, B) Heatmaps of the top 25 ranking genes as determined by GSEA. (A) Gene expression in endothelium overlying plaque-free (PF) intimae from human large arteries is compared to gene expression in endothelium from early- and advanced (ADV) lesions. Data set from Volger et al. [2]. Aquaporin 1 (AQP1) is identified as the highest ranking membrane-expressed gene in plaque-free lesions (green rectangle). (B) Time courses (24, 48 and 72 hours) of gene expression in mock-transduced HUVEC (control, c) and KLF2-transduced HUVEC are compared. Data set from Boon et al. [7]. The position of AQP1 is indicated by a green rectangle. (C) Correlation of KLF2—and AQP1 transcript levels during time courses (24, 48 and 72 hours) of gene expression in mock- and KLF2- transduced HUVECs (same data set as used for Fig 1B).
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pone.0145777.g001: AQP1 is preferentially expressed in endothelium overlying plaque-free intimae and is induced by KLF2.(A, B) Heatmaps of the top 25 ranking genes as determined by GSEA. (A) Gene expression in endothelium overlying plaque-free (PF) intimae from human large arteries is compared to gene expression in endothelium from early- and advanced (ADV) lesions. Data set from Volger et al. [2]. Aquaporin 1 (AQP1) is identified as the highest ranking membrane-expressed gene in plaque-free lesions (green rectangle). (B) Time courses (24, 48 and 72 hours) of gene expression in mock-transduced HUVEC (control, c) and KLF2-transduced HUVEC are compared. Data set from Boon et al. [7]. The position of AQP1 is indicated by a green rectangle. (C) Correlation of KLF2—and AQP1 transcript levels during time courses (24, 48 and 72 hours) of gene expression in mock- and KLF2- transduced HUVECs (same data set as used for Fig 1B).

Mentions: In a dataset comprising transcriptome data from endothelium overlying normal intimae and intimae containing early- and advanced lesions in human large arteries [2], we determined the top 25 ranking genes in a comparison between the plaque-free situation and early- and advanced lesions using Gene Set Enrichment Analysis. AQP1 was identified as the highest ranking endothelial cell-surface exposed signature gene in the plaque-free situation (Fig 1A). Likewise, we identified AQP1 as the fourth ranking gene in a comparison between transcriptomes of endothelial cells that were lentivirally transduced with either a mock- or KLF2 encoding construct and subsequently cultured for different periods of time [7] (Fig 1B).A further analysis of transcript levels during this time course revealed a significant (p = 0.0007) positive correlation between KLF2- and AQP1 mRNA (Fig 1C), suggesting that AQP1 might be part of the repertoire of KLF2-downstream atheroprotective genes. This idea is consistent with the observed preferential expression of AQP1 in endothelium overlying healthy intimae. Together, these in silico analyses qualify AQP1 as a potential cell-surface marker for healthy, non-dysfunctional endothelium.


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)

AQP1 is preferentially expressed in endothelium overlying plaque-free intimae and is induced by KLF2.(A, B) Heatmaps of the top 25 ranking genes as determined by GSEA. (A) Gene expression in endothelium overlying plaque-free (PF) intimae from human large arteries is compared to gene expression in endothelium from early- and advanced (ADV) lesions. Data set from Volger et al. [2]. Aquaporin 1 (AQP1) is identified as the highest ranking membrane-expressed gene in plaque-free lesions (green rectangle). (B) Time courses (24, 48 and 72 hours) of gene expression in mock-transduced HUVEC (control, c) and KLF2-transduced HUVEC are compared. Data set from Boon et al. [7]. The position of AQP1 is indicated by a green rectangle. (C) Correlation of KLF2—and AQP1 transcript levels during time courses (24, 48 and 72 hours) of gene expression in mock- and KLF2- transduced HUVECs (same data set as used for Fig 1B).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0145777.g001: AQP1 is preferentially expressed in endothelium overlying plaque-free intimae and is induced by KLF2.(A, B) Heatmaps of the top 25 ranking genes as determined by GSEA. (A) Gene expression in endothelium overlying plaque-free (PF) intimae from human large arteries is compared to gene expression in endothelium from early- and advanced (ADV) lesions. Data set from Volger et al. [2]. Aquaporin 1 (AQP1) is identified as the highest ranking membrane-expressed gene in plaque-free lesions (green rectangle). (B) Time courses (24, 48 and 72 hours) of gene expression in mock-transduced HUVEC (control, c) and KLF2-transduced HUVEC are compared. Data set from Boon et al. [7]. The position of AQP1 is indicated by a green rectangle. (C) Correlation of KLF2—and AQP1 transcript levels during time courses (24, 48 and 72 hours) of gene expression in mock- and KLF2- transduced HUVECs (same data set as used for Fig 1B).
Mentions: In a dataset comprising transcriptome data from endothelium overlying normal intimae and intimae containing early- and advanced lesions in human large arteries [2], we determined the top 25 ranking genes in a comparison between the plaque-free situation and early- and advanced lesions using Gene Set Enrichment Analysis. AQP1 was identified as the highest ranking endothelial cell-surface exposed signature gene in the plaque-free situation (Fig 1A). Likewise, we identified AQP1 as the fourth ranking gene in a comparison between transcriptomes of endothelial cells that were lentivirally transduced with either a mock- or KLF2 encoding construct and subsequently cultured for different periods of time [7] (Fig 1B).A further analysis of transcript levels during this time course revealed a significant (p = 0.0007) positive correlation between KLF2- and AQP1 mRNA (Fig 1C), suggesting that AQP1 might be part of the repertoire of KLF2-downstream atheroprotective genes. This idea is consistent with the observed preferential expression of AQP1 in endothelium overlying healthy intimae. Together, these in silico analyses qualify AQP1 as a potential cell-surface marker for healthy, non-dysfunctional endothelium.

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