Hemizygous deficiency of Krüppel-like factor 2 augments experimental atherosclerosis

Abstract

Krüppel-like factor (KLF)2 is a central regulator of endothelial and monocyte/macrophage gene expression and function in vitro. Although the composite effects of KLF2 in these 2 cell types predict that it likely inhibits vascular inflammation, the role of KLF2 in this process in vivo is uncharacterized. In this study, we provide evidence that hemizygous deficiency of KLF2 increased diet-induced atherosclerosis in apolipoprotein E-deficient mice. Our studies highlight an important role for KLF2 in primary macrophage foam cell formation via the potential regulation of the key lipid binding protein adipocyte protein 2/fatty acid-binding protein 4. These novel observations establish that KLF2 is an atheroprotective factor.

Figure 1

KLF2 heterozygous mice develop more atherosclerosis. Male littermate ApoE^−/− and KLF2^+/−/ApoE^−/− mice at 6 weeks of age were fed a high fat, high cholesterol diet for 20 weeks. Aortas were harvested and Sudan IV stained for lipid. A, Two representative pairs of fixed and stained whole aortas. B, Percent lesion area of the entire isolated aorta area was calculated. ApoE^−/− 27.3±9.9% (n=12), and KLF2^+/−/ApoE^−/− 35.9±10.2% (n=14). C, Two representative pairs of fixed and stained aortas en face. D, Percent lesion area of the entire aorta surface area was calculated. ApoE^−/− 17.6±5.4% (n=12), and KLF2^+/−/ApoE^−/− 24±6.3% (n=14). K2^+/− = KLF2^+/−.

Figure 2

Effect of KLF2 deficiency on endothelial gene expression and macrophage recruitment. A, QPCR analysis was performed on RNA from isolated lung tissue harvested from KLF2^+/− and wild-type littermate control mice, (n=3). Expression levels in KLF2^+/− mice (K2 0.44±0.08, K4 1.39±0.04) were normalized to wild-type. B, Aortic arch sections of male littermate ApoE^−/− and KLF2^+/−/ApoE^−/− mice fed an atherogenic diet for 20 weeks were stained for Mac-3 (n=8). Representative slides are shown. C, Mac-3 staining was quantified as percent staining area of the entire lesion area. KLF2^+/−/ApoE^−/− 3.13±1.70%, ApoE^−/− 2.32±1.16%, (n=9).

Figure 3

KLF2 regulates lipid uptake and macrophage aP2 levels. A and B, Peritoneal macrophages isolated from KLF^2+/− and male wild-type littermate control mice (A) or RAW cells adenovirally infected with KLF2 (AdK2) or control virus (AdGFP) (B) were treated with oxidized LDL (oxLDL) (50µg/mL) for 72hours and stained for lipid uptake with oil red O. Representative results are shown. C and D, Whole cell lysates were harvested from isolated peritoneal macrophages (n=4) (C) or RAW cells (n=12) (D) and assessed for aP2 levels by western blotting. A representative blot is shown.