Endothelial Kruppel-like factor 4 protects against atherothrombosis in mice

Abstract

The endothelium regulates vascular homeostasis, and endothelial dysfunction is a proximate event in the pathogenesis of atherothrombosis. Stimulation of the endothelium with proinflammatory cytokines or exposure to hemodynamic-induced disturbed flow leads to a proadhesive and prothrombotic phenotype that promotes atherothrombosis. In contrast, exposure to arterial laminar flow induces a gene program that confers a largely antiadhesive, antithrombotic effect. The molecular basis for this differential effect on endothelial function remains poorly understood. While recent insights implicate Kruppel-like factors (KLFs) as important regulators of vascular homeostasis, the in vivo role of these factors in endothelial biology remains unproven. Here, we show that endothelial KLF4 is an essential determinant of atherogenesis and thrombosis. Using in vivo EC-specific KLF4 overexpression and knockdown murine models, we found that KLF4 induced an antiadhesive, antithrombotic state. Mechanistically, we demonstrated that KLF4 differentially regulated pertinent endothelial targets via competition for the coactivator p300. These observations provide cogent evidence implicating endothelial KLFs as essential in vivo regulators of vascular function in the adult animal.

Figure 1. Endothelial KLF4 protects against atherosclerosis.

(A–D) BM transplantation was performed on Cre and Klf4^fl-Cre mice, giving rise to EC-Klf4^+/+ and EC-Klf4^–/– mice, respectively. (E–H) EC-KLF4 Tg mice and nontransgenic WT controls were also generated, using the human KLF4 coding sequence under control of the VE-cadherin promoter. All mice were on an Apoe^–/– background. (A and E) Sudan IV staining of the entire aorta. (B and F) Atherosclerotic lesion area. (C and G) H&E and CD45 staining at the aortic root. Original magnification, ×100 (H&E); ×200 (CD45). See Supplemental Figure 1 for enlarged views of boxed regions. (D and H) Infiltration of inflammatory cells. *P ≤ 0.05; **P ≤ 0.01.

Figure 2. Endothelial KLF4 protects against thrombosis.

(A) Ex vivo assays demonstrated early fibrin clot formation using cells isolated from EC-Klf4^–/– mice in the absence (Con) and presence of TNF-α pretreatment (n = 10–12 replicates from ECs pooled from 5 mice). t_1/2max, half-time to complete clot formation. (B) EC-Klf4^–/– mice were predisposed to early thrombosis, as assessed by in vivo carotid injury assay (n = 9 pairs). (C) Primary ECs from EC-KLF4 Tg mice demonstrated a delayed time to fibrin clot formation in ex vivo assays (n = 13–17 replicates from ECs pooled from 5 mice). (D) EC-KLF4 Tg mice showed delayed time to formation of occlusive thrombus (n = 7 pairs). *P ≤ 0.05; **P ≤ 0.01.

Figure 3. KLF4 regulates endothelial gene expression.

(A) KLF4 expression was upregulated by an atheroprotective hemodynamic shear stress profile (versus atheroprone). (B and C) Adenoviral overexpression of KLF4 maintained an atheroprotective gene program in HUVECs exposed to (B) atheroprone flow and (C) cytokine (n = 6 experiments). EV, empty virus; AdK4, KLF4-expressing adenovirus. (D) siRNA-mediated KLF4 knockdown led to a proatherogenic gene program even in the presence of atheroprotective flow (n = 8 experiments). siK4, KLF4 siRNA; NS, nonspecific siRNA. (E) KLF4 binding to p300 was mediated via the P3ID. Immunoprecipitation experiments were performed 3 times in 293 cells. A representative experiment is shown. (F) KLF4 worked synergistically with p300 in activation of the TM promoter. COS-7 cells were transfected with vector plasmid, expression plasmids, and a TM promoter reporter plasmid. Reporter activity is expressed as relative luciferase units (RLU). (G) Inhibition by KLF4 of p65-mediated VCAM1 promoter activity required the KLF4 P3ID (n = 3 experiments; 6 replicates per experiment). *P ≤ 0.05; **P ≤ 0.01.