Flow-dependent expression of ectonucleotide tri(di)phosphohydrolase-1 and suppression of atherosclerosis
Yogendra Kanthi, … , Hanjoong Jo, David J. Pinsky
Yogendra Kanthi, … , Hanjoong Jo, David J. Pinsky
Published June 29, 2015
Citation Information: J Clin Invest. 2015;125(8):3027-3036. https://doi.org/10.1172/JCI79514.
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Research Article Vascular biology

Flow-dependent expression of ectonucleotide tri(di)phosphohydrolase-1 and suppression of atherosclerosis

Abstract

The ability of cells to detect and respond to nucleotide signals in the local microenvironment is essential for vascular homeostasis. The enzyme ectonucleotide tri(di)phosphohydrolase-1 (ENTPD1, also known as CD39) on the surface of leukocytes and endothelial cells metabolizes locally released, intravascular ATP and ADP, thereby eliminating these prothrombotic and proinflammatory stimuli. Here, we evaluated the contribution of CD39 to atherogenesis in the apolipoprotein E–deficient (ApoE-deficient) mouse model of atherosclerosis. Compared with control ApoE-deficient animals, plaque burden was markedly increased along with circulating markers of platelet activation in Cd39+/–Apoe–/– mice fed a high-fat diet. Plaque analysis revealed stark regionalization of endothelial CD39 expression and function in Apoe–/– mice, with CD39 prominently expressed in atheroprotective, stable flow regions and diminished in atheroprone areas subject to disturbed flow. In mice, disturbed flow as the result of partial carotid artery ligation rapidly suppressed endothelial CD39 expression. Moreover, unidirectional laminar shear stress induced atheroprotective CD39 expression in human endothelial cells. CD39 induction was dependent upon the vascular transcription factor Krüppel-like factor 2 (KLF2) binding near the transcriptional start site of CD39. Together, these data establish CD39 as a regionalized regulator of atherogenesis that is driven by shear stress.

Authors

Yogendra Kanthi, Matthew C. Hyman, Hui Liao, Amy E. Baek, Scott H. Visovatti, Nadia R. Sutton, Sascha N. Goonewardena, Mithun K. Neral, Hanjoong Jo, David J. Pinsky

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Figure 7

Cd39 expression is regulated by KLF2 via direct interaction with the CD39 promoter.

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Cd39 expression is regulated by KLF2 via direct interaction with the CD...
(A) Cd39 expression is decreased by silencing Klf2 in WT bone marrow–derived macrophages using siRNA (siKLF2) versus controls (siScramble) (n = 3 per group). *P < 0.05; **P < 0.01. (B–E) Silencing KLF2 with shRNA targeting KLF2 (shKLF2) decreased KLF2 gene transcript by more than 65% in static conditions and more than 65% under laminar shear stress versus controls (shScramble) in HUVECs (B) and HAECs (D) (n = 3–4 per group). *P < 0.05; **P < 0.005. KLF2 silencing abrogated laminar shear stress–induced expression of CD39 versus nonsilenced controls in HUVECs (C) and HAECs (E) (n = 3–4 per group). *P < 0.05. (F) CD39 induction by KLF2 overexpression. HUVECs transduced with a recombinant lentivirus (rLV-KLF2) to overexpress human KLF2 or vector alone (rLV-Control). Induction of KLF2 (left) and CD39 (right) transcripts were measured with quantitative RT-PCR. (n = 3 per group). *P < 0.05; **P < 0.001. (G) ChIP of HUVECs treated with static or laminar shear stress using anti-KLF2 antibody or nonspecific IgG revealed KLF2 binding to the CD39 promoter in static conditions, which was significantly enhanced under laminar shear stress conditions (n = 3 per group) *P < 0.05; **P < 0.01. Data are expressed as mean ± SEM. Student’s t test was used when comparing 2 variables. One-way ANOVA and Tukey’s multiple comparison tests were used when comparing more than 2 variables.