Hemizygous Deficiency of Krüppel-Like Factor 2 Augments Experimental Atherosclerosis
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.
Krüppel-like factors (KLFs) are members of the zinc finger family of transcription factors that have been implicated in the regulation of cellular growth and differentiation. KLF2 is a key regulator of endothelial and monocyte/macrophage proinflammatory action.1,2 In endothelial cells, KLF2 expression is induced by laminar shear stress and inhibited by proinflammatory cytokines. Sustained overexpression of KLF2 results in marked induction of endothelial nitric oxide synthase (eNOS) and thrombomodulin expression while reducing cytokine-mediated activation of proinflammatory genes such as vascular cell adhesion molecule (VCAM)-1. Similarly, previous studies indicate that KLF2 inhibits monocyte proinflammatory gene expression and phagocytosis. Importantly, KLF2 expression in peripheral blood monocytes of patients with established atherosclerotic disease is reduced by ≈30%.2 On the basis of these observations, KLF2 has been viewed as a candidate atheroprotective factor. However, in vivo evidence in support of this hypothesis has been lacking.
Herein, we provide evidence that hemizygous deficiency of KLF2 significantly increases diet-induced atherosclerotic lesion formation in apolipoprotein E–deficient (ApoE−/−) mice. Our results also underscore the importance of KLF2 in macrophage lipid uptake and foam cell formation via effects on the lipid binding protein adipocyte protein (aP)2/fatty acid–binding protein (FABP)4.
Materials and Methods
Animals and Diets
KLF2+/− mice (generously provided by J. Leiden, Abbott Laboratories, Chicago, Ill.) were generated as previously described.3 KLF2+/− mice of mixed background (calculated to be ≈75% C57Bl/6) were mated to ApoE−/− mice4 on the C57Bl/6 background (The Jackson Laboratory) to generate KLF2+/−/ApoE−/− and KLF2+/+/ApoE−/− mice. All studies were performed with strict age-matched, sex-matched littermate controls. Mice were weaned at 4 weeks, fed a normal rodent chow diet (4.5% fat by weight, 14% kilocalories; Laboratory Diet P3000), and, at 6 to 8 weeks of age, were initiated on a high-fat (12.5% by weight, 40% kilocalories), high-cholesterol (1.25% by weight) diet (Research Diets D-12108) for a total of 20 weeks. Animal care and procedures were performed according to NIH guidelines.
An expanded Materials and Methods section is available in the online data supplement at http://circres.ahajournals.org.
Enhanced Atherosclerotic Lesion Formation in ApoE-Deficient Mice
Previous studies demonstrate that systemic knockout of KLF2 is embryonic lethal.3,5 However, hemizygous deficient (KLF2+/−) mice are viable and fertile. KLF2+/− mice were crossed to ApoE−/− mice to generate KLF2+/−/ApoE−/− and KLF2+/+/ApoE−/− mice. At 6 weeks of age, male mice (n=12 to 14 per group) were initiated on a high-fat, high-cholesterol diet for 20 weeks. Plasma lipid analysis reveals that there was no significant difference in lipid profiles between mice at baseline (data not shown) or after an atherogenic diet (Table I in the online data supplement).
After 20 weeks of an atherogenic diet, mice were euthanized and aortas were harvested for analysis of atherosclerotic lesion area by Sudan IV staining. KLF2+/−/ApoE−/− mice exhibited a significant 31% increase in atherosclerotic lesion area when compared to KLF2+/+/ApoE−/− littermate controls (35.9±10.2% versus 27.3±9.9%; P=0.04) (Figure 1A and 1B). Similarly, quantitative analysis using an en face preparation revealed a 37% increase in atherosclerotic lesion area (24.0±6.3% versus 17.6±5.4%; P=0.0099) (Figure 1C and 1D).
Effect of KLF2 Deficiency on Endothelial Gene Expression
KLF2 is known to induce eNOS and inhibit VCAM-1 expression in cultured endothelial cells.1 However, analysis of aortic sections demonstrated no appreciable difference in the expression of endothelial eNOS or VCAM-1 (supplemental Figure IA and IB). Consistent with this result, quantitative PCR analysis for eNOS, thrombomodulin, and VCAM-1 revealed no significant change in KLF2+/− mice compared to littermate controls (data not shown).
A recent study indicates that the Krüppel family member KLF4 is capable of conferring gene regulatory effects similar to KLF2 in endothelial cells.6 Therefore, we reasoned that a compensatory increase in KLF4 in KLF2+/− mice might account, in part, for the absence of any clear effect on the KLF2 endothelial targets assessed. Indeed, tissues from KLF2+/− mice demonstrated a 39±4% increase in KLF4 expression (P=0.0033) (Figure 2A).
Enhanced Lipid Uptake and aP2/FABP4 Expression in KLF2 Hemizygous Deficient Macrophages
We next sought to assess the effect of KLF2 hemizygosity on macrophage recruitment and function. Immunohistochemical assessment using the macrophage specific antibody Mac-3 revealed a nonsignificant trend toward increased macrophage area staining in the aortic arch of KLF2+/−/ApoE−/− mice (2.16±1.65% versus 1.30±0.86%; P=0.176) (Figure 2B and 2C). Thus, although KLF2 deficiency leads to increased atherosclerotic burden (Figure 1), this cannot be explained simply by an increase in macrophage number.
Because previous studies indicated that KLF2 inhibits phagocytosis,2 we reasoned that enhanced lipid uptake in macrophages may account for the increase in atherosclerosis. Remarkably, peritoneal macrophages from KLF2+/− mice treated with oxidized LDL demonstrated a 40% increase in lipid uptake when compared to wild-type macrophages (Figure 3A and supplemental Figure IIA). Conversely, adenoviral overexpression of KLF2 in a mouse macrophage cell line (RAW 264.7 cells) strongly inhibited oxidized LDL uptake (Figure 3B and supplemental Figure IIB).
Although lipid accumulation within cells is a complex process, recent studies support a key role for the lipid chaperone aP2/FABP4 in macrophage lipid accumulation and atherogenesis.7,8 Indeed, KLF2+/− macrophages expressed higher protein levels of aP2 (Figure 3C). Conversely, overexpression of KLF2 in RAW cells strongly inhibited aP2 expression (Figure 3D). These results are specific because the expression of other factors involved in lipid accumulation such as CD36 were unaffected (data not shown). Taken together, these results indicate that reduced lipid uptake in KLF2+/− macrophages is mediated, in part, by the regulation of aP2 by KLF2.
Atherosclerosis is an extremely complex disease process with a number of important cellular contributors including endothelial cells, smooth muscle cells, and immune cells (monocyte and T cells). Because KLF2 has been shown to play a particularly potent role in inhibiting endothelial cell and monocyte/macrophage activation, we focused our mechanistic studies on these 2 cell types.
The key observation highlighted by our study relates to the role of KLF2 in monocyte/macrophage biology. Previously, Das et al reported that sustained expression of KLF2 inhibited monocyte activation and phagocytic capacity in vitro.2 In addition, Wu et al have shown that KLF2−/− mouse embryonic fibroblasts exhibit accelerated lipid accumulation on adipocyte differentiation.9 Consistent with these observations, KLF2+/− macrophages exhibit enhanced lipid accumulation (Figure 3) and increased proinflammatory genes (G.H.M. and M.K.J., unpublished observation, 2008). Thus, although absolute numbers of macrophages in atherosclerotic lesions of KLF2+/+/ApoE−/− and KLF2+/−/ApoE−/− mice were not significantly different, the increased lipid uptake could account for the increase in lesion size. Mechanistically, our studies identify the lipid binding protein aP2 as a KLF2 target. Work by Hotamisligil and colleagues has underscored the importance of aP2 in atherogenesis. Systemic and macrophage aP2 deficiency renders mice resistant to atherosclerosis.7,8 Furthermore, aP2-deficient macrophages exhibited alterations in their ability to express inflammatory cytokines and had reduced ability to uptake lipid when stimulated by modified lipids.7 Given the results in KLF2+/− macrophages (Figure 3), it is likely that enhanced aP2 expression contributes to the increase in lipid accumulation in KLF2+/− macrophages. However, one cannot rule out the possibility that effects on other aspects of lipid metabolism (eg, uptake or export) in macrophages or alterations in aP2 expression in other tissues (eg, adipose) may also impact on the observed phenotype.
With respect to KLF2 effects in endothelial cells, our studies provide some important insights. Despite previous in vitro observations, we did not observe any significant effects on the expression of KLF2 targets, including eNOS and VCAM-1. Importantly, however, our findings highlight a key compensatory role for KLF4. Like KLF2, recent studies reveal that KLF4 can also induce eNOS and inhibit VCAM-1.6 Thus, the increase in KLF4 observed may compensate for KLF2 deficiency. This is an interesting finding that raises the importance of future studies to assess the effect of KLF4 or dual KLF2/KLF4 hemizygosity on atherosclerosis. Additionally, we note the possibility that other endothelial factor(s) not assessed in this study may be differentially regulated in a nonredundant fashion by KLF2 and contribute to the phenotype observed. These considerations, as well as generation of inducible tissue-specific KLF2 knockouts, are the subject of ongoing investigation.
In sum, these observations are the first in vivo evidence in support of the hypothesis that KLF2 is atheroprotective. These findings are of particular interest as they suggest that a reduction of KLF2 expression at levels that approximate those observed in human subjects with coronary artery disease is biologically relevant. Manipulation of KLF2 expression may thus be beneficial in the treatment of vascular inflammatory disorders.
Sources of Funding
This work was supported by NIH grants HL72952, HL75427, HL76754, HL086548, HL084154, and P01 HL48743 (to M.K.J.); HL085816 and HL057506 (to D.I.S.); and HL088740 (to G.B.A.); American Heart Association grants 0635579T (to Z.L.) and 0725297B (to D.K.); an Alliance for Cancer Gene Therapy grant (to M.K.J.); a United Negro College Fund/Merck Science Initiative Fellowship grant (to G.B.A.); and a Robert Wood Johnson/Harold Amos Medical Faculty Development grant (to G.B.A.).
Original received August 4, 2008; revision received August 14, 2008; accepted August 18, 2008.
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