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Shear stress modulates endothelial KLF2 through activation of P2X4

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Abstract

Vascular endothelial cells that are in direct contact with blood flow are exposed to fluid shear stress and regulate vascular homeostasis. Studies report endothelial cells to release ATP in response to shear stress that in turn modulates cellular functions via P2 receptors with P2X4 mediating shear stress-induced calcium signaling and vasodilation. A recent study shows that a loss-of-function polymorphism in the human P2X4 resulting in a Tyr315>Cys variant is associated with increased pulse pressure and impaired endothelial vasodilation. Although the importance of shear stress-induced Krüppel-like factor 2 (KLF2) expression in atheroprotection is well studied, whether ATP regulates KLF2 remains unanswered and is the objective of this study. Using an in vitro model, we show that in human umbilical vein endothelial cells (HUVECs), apyrase decreased shear stress-induced KLF2, KLF4, and NOS3 expression but not that of NFE2L2. Exposure of HUVECs either to shear stress or ATPγS under static conditions increased KLF2 in a P2X4-dependent manner as was evident with both the receptor antagonist and siRNA knockdown. Furthermore, transient transfection of static cultures of human endothelial cells with the Tyr315>Cys mutant P2X4 construct blocked ATP-induced KLF2 expression. Also, P2X4 mediated the shear stress-induced phosphorylation of extracellular regulated kinase-5, a known regulator of KLF2. This study demonstrates a major physiological finding that the shear-induced effects on endothelial KLF2 axis are in part dependent on ATP release and P2X4, a previously unidentified mechanism.

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Abbreviations

HUVECs:

Human umbilical vein endothelial cells

HAECs:

Human aortic endothelial cells

IVF:

In vitro fertilization

qRT-PCR:

Quantitative real-time polymerase chain reaction

HKG:

Housekeeping genes

KLF2:

Krüppel-like factor 2

KLF4:

Krüppel-like factor 4

NFE2L2:

Nuclear factor (erythroid derived-2) like-2

NOS3:

Nitric oxide synthase 3

THBD:

Thrombomodulin

SNP:

Single nucleotide polymorphism

Y315C:

Tyr315>Cys

WT:

Wild type

ERK5:

Extracellular regulated kinase-5

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Acknowledgments

We thank Siv Svensson, Hanna Gustafsson, and Joanna Daszkiewicz-Nilsson for their technical assistance and Dr. Aliaa Abdelrahman for performing calcium assays for PSB-12253 at P2X receptor subtypes. The Swedish Heart and Lung Foundation, Swedish Scientific Research Council, ALF, and Skåne University Hospital funds supported this work.

Conflict of interest

The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Department of Cardiology, Clinical Sciences, Lund University, Lund, Sweden

    R. Sathanoori, B. Olde & D. Erlinge

  2. PharmaCenter, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, Bonn, Germany

    F. Rosi & C. E. Müller

  3. Florey Neuroscience Institutes, University of Melbourne, Parkville, Victoria, Australia

    B. J. Gu & J. S. Wiley

  4. Department of Cardiology, Lund University, Tornavägen 10, BMC-D12, Room D1219b, 221 84, Lund, Sweden

    R. Sathanoori

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  1. R. Sathanoori
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Correspondence to R. Sathanoori.

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Sathanoori, R., Rosi, F., Gu, B.J. et al. Shear stress modulates endothelial KLF2 through activation of P2X4. Purinergic Signalling 11, 139–153 (2015). https://doi.org/10.1007/s11302-014-9442-3

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