Myeloid Krüppel-Like Factor 4 Deficiency Augments Atherogenesis in ApoE−/− Mice—Brief Report
Objective—To investigate the role of Krüppel-like factor 4 (KLF4), an essential transcriptional regulator of macrophage polarization (M1/M2), in the pathogenesis of atherosclerosis.
Methods and Results—Despite the acknowledged importance of macrophages in atherosclerosis, the role of M1 (classically activated or proinflammatory) versus M2 (alternatively activated or anti-inflammatory) macrophages in this process remains incompletely understood. We recently identified KLF4 as a regulator of macrophage subset specification; that is, KLF4 promotes M2 and inhibits M1 phenotype. Here, we provide evidence that KLF4-deficient macrophages exhibit enhanced proinflammatory activation and foam cell formation in response to oxidized lipids. In vivo, myeloid KLF4-deficient mice (ApoE−/− background) develop significantly more vascular inflammation and atherosclerotic lesion formation.
Conclusion—Our findings identify myeloid KLF4 as an essential regulator of vascular inflammation and experimental atherogenesis.
Macrophages are central to the development of vascular inflammation and atherogenesis.1 Recent investigations highlight the importance of 2 subsets of macrophages termed M1 (classical) and M2 (alternative) in a broad array of biological processes.2 These 2 subsets have also been identified in atherosclerotic lesions, but their importance remains incompletely understood.3–5
Krüppel-like factor 4 (KLF4) has been shown to regulate cell growth, development, differentiation, and function of diverse cell types.6,7 Recently, we showed that KLF4 controls macrophage speciation by promoting the M2 phenotype and inhibiting M1 phenotype.8 However, the importance of myeloid KLF4 in atherogenesis remains unknown.
Myeloid KLF4 Regulates Inflammatory Targets in Response to Oxidized Lipids
We first determined whether proinflammatory cytokines and lipids modulate KLF4 expression. Peritoneal macrophages were treated with 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphatidylcholine (POV-PC), an oxidized low-density lipoprotein (LDL) species,9 interleukin-1β, and tumor necrosis factor-α. As shown in Figure 1A, KLF4 expression is significantly reduced by proinflammatory cytokines and modified lipids. Next, peritoneal macrophages from control (LysMCre/Cre:KLF4+/+ designated KLF4+/+) and myeloid KLF4-deficient mice (LysMCre/Cre:KLF4Flox/Flox; designated KLF4Δ/Δ) were treated with POV-PC, and inflammatory targets were analyzed by quantitative polymerase chain reaction (Figure 1B) and ELISA/enzymatic activity (Figure I in the online-only Data Supplement). KLF4-deficient macrophages expressed higher levels of classical M1 markers, such as inducible NO synthase, interleukin-6, and interleukin-1β. Next we assessed the role of KLF4 in modified lipid uptake. As shown in Figure 1C, the uptake of fluorescent labeled oxidized LDL (but not acetylated LDL, data not shown) was enhanced in the KLF4-deficient macrophages. Consistent with these observations, quantitative polymerase chain reaction analysis revealed enhanced expression of CD36 (receptor for oxidized LDL) but not MSR1 (receptor for acetylated LDL) in KLF4Δ/Δmacrophages (Figure 1D).
Myeloid KLF4 Deficiency Augments Experimental Atherosclerosis
To determine the role of myeloid KLF4 on atherogenesis, KLF4+/+ and KLF4Δ/Δ mice were crossed to ApoE−/− mice and fed a normal chow (n=9 per group) or high-fat diet (HFD, n=18 per group). After 20 weeks of feeding, mice were euthanized and aortas harvested for assessment of atherosclerotic lesion area by Sudan IV staining. Intriguingly, as shown in Figure 2, enhanced lesion area was observed on both a normal chow (22.84 ± 3.50 versus 9.15 ± 2.85; P<0.001) as well as HFD (35.50 ± 9.8 versus 24.38 ± 10.42; P=0.002). Quantitative polymerase chain reaction analysis of aortic tissues revealed increased expression of proinflammatory (M1) targets (Figure IIA and IIB in the online-only Data Supplement). In addition, a reduction was observed in some M2 markers, particularly after HFD (Figure IIA and IIB in the online-only Data Supplement). Mac-3 staining confirmed increased macrophage infiltration in aortic lesions (Figure III in the online-only Data Supplement). Finally, plasma lipid analyses revealed a modest increase in low-density lipoprotein, triglycerides, high-density lipoprotein, and total cholesterol in KLF4Δ/Δ mice ApoE−/− after HFD but not chow diet (Table in the online-only Data Supplement).
Macrophages are major cellular constituents of atherosclerotic lesions, but how different subsets impact atherogenesis remains poorly understood.3–5 We recently identified the transcription factor KLF4 as a key molecular determinant of macrophage subset specification.8 Myeloid deficiency of KLF4 was found to enhance M1 gene expression and reduce M2 gene expression.8 Furthermore, myeloid KLF4-deficient mice exhibited diet-induced obesity, insulin resistance, and glucose intolerance.8 The availability of this model provided an opportunity to determine the effect of altering macrophage polarization on atherogenesis. Here we show that myeloid deficiency of KLF4 leads to enhanced atherosclerotic lesion formation and macrophage accumulation in the atherosclerotic lesion, supporting a critical role of myeloid KLF4 in vascular inflammation and atherogenesis after an HFD. Intriguingly, a significant increase in lesion formation and vascular inflammation (Figure 2 and Figure II in the online-only Data Supplement) was also observed under normal chow conditions, observations that underscore the importance of KLF4 in disease pathogenesis. However, there are several caveats to our work. We note that, in response to HFD, myeloid KLF4-deficient mice exhibited altered lipid profiles that may contribute to the observed phenotype. Also, as there are differences in mouse versus human macrophage subsets, one must be cautious in directly extending these findings to the biology of human atherosclerosis.
Our study also expands our understanding of stimuli that regulate myeloid KLF4 expression. Previously, we showed that inflammatory cytokines reduce KLF4 expression in macrophages.8 Here we extend these observations and show that oxidized lipids also reduce macrophage KLF4 expression. The importance of KLF4 is highlighted by the fact that its absence results in enhanced proinflammatory gene expression in response to POV-PC. As inflammatory cytokines and certain lipid species are well known to contribute to obesity and atherosclerosis, this downregulation may be functionally quite important.10 Indeed, consistent with this idea, we recently found that KLF4 expression was reduced in macrophages derived from obese subjects.8
In summary, our observations implicate myeloid KLF4 as a key regulator of experimental atherogenesis in mice. The fact that KLF4 has been shown to confer favorable anti-inflammatory effects in both the endothelium11 and myeloid compartments8 suggest that targeting this factor could be exploited for therapeutic gain.
Sources of Funding
This work was supported by National Institutes of Health grants HL097593, HL086548, HL076754 HL084154, HL075427 (to M.K.J), and HL097023 (to G.H.M), and American Heart Association grants 09POST2060203 and 11POST7200004 (to N.S).
The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.112.300471/-/DC1.
- Received April 16, 2012.
- Accepted September 26, 2012.
- © 2012 American Heart Association, Inc.
- Shields JM,
- Christy RJ,
- Yang VW
- Podrez EA,
- Poliakov E,
- Shen Z,
- Zhang R,
- Deng Y,
- Sun M,
- Finton PJ,
- Shan L,
- Gugiu B,
- Fox PL,
- Hoff HF,
- Salomon RG,
- Hazen SL
- Hamik A,
- Lin Z,
- Kumar A,
- Balcells M,
- Sinha S,
- Katz J,
- Feinberg MW,
- Gerzsten RE,
- Edelman ER,
- Jain MK