Hemizygous Deficiency of Kruppel-Like Factor 2 Augments Experimental Atherosclerosis

ISSN: 1524-4571 Copyright © 2008 American Heart Association. All rights reserved. Print ISSN: 0009-7330. Online

TX 72514Circulation Research is published by the American Heart Association. 7272 Greenville Avenue, Dallas,

DOI: 10.1161/CIRCRESAHA.108.184663 published online Aug 28, 2008; Circ. Res.

K. Jain MukeshGao, Daiji Kawanami, Viswanath Natesan, Zhiyong Lin, Daniel I. Simon and

G. Brandon Atkins, Yunmei Wang, Ganapati H. Mahabeleshwar, Hong Shi, Huiyun Atherosclerosis

Hemizygous Deficiency of Krüppel- Like Factor 2 Augments Experimental

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Hemizygous Deficiency of Kruppel-Like Factor 2 AugmentsExperimental Atherosclerosis

G. Brandon Atkins, Yunmei Wang,Ganapati H. Mahabeleshwar, Hong Shi,Huiyun Gao, Daiji Kawanami,Viswanath Natesan, Zhiyong Lin,Daniel I. Simon, Mukesh K. Jain

Kruppel-like factor (KLF)2 is a central regulator ofendothelial and monocyte/macrophage gene expressionand function in vitro. Although the composite effects ofKLF2 in these 2 cell types predict that it likely inhibitsvascular inflammation, the role of KLF2 in this process invivo is uncharacterized. In this study, we provide evi-dence that hemizygous deficiency of KLF2 increaseddiet-induced atherosclerosis in apolipoprotein E–defi-cient mice. Our studies highlight an important role forKLF2 in primary macrophage foam cell formation viathe potential regulation of the key lipid binding proteinadipocyte protein 2/fatty acid–binding protein 4. Thesenovel observations establish that KLF2 is an atheropro-tective factor.

Kruppel-like factors (KLFs) are members of the zincfinger family of transcription factors that have been

implicated in the regulation of cellular growth and differen-tiation. 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 andinhibited by proinflammatory cytokines. Sustained overex-pression of KLF2 results in marked induction of endothelialnitric oxide synthase (eNOS) and thrombomodulin expres-sion while reducing cytokine-mediated activation of proin-flammatory genes such as vascular cell adhesion molecule(VCAM)-1. Similarly, previous studies indicate that KLF2inhibits monocyte proinflammatory gene expression andphagocytosis. Importantly, KLF2 expression in peripheralblood monocytes of patients with established atheroscleroticdisease is reduced by �30%.2 On the basis of these observa-tions, KLF2 has been viewed as a candidate atheroprotectivefactor. However, in vivo evidence in support of this hypoth-esis has been lacking.

Herein, we provide evidence that hemizygous deficiency ofKLF2 significantly increases diet-induced atherosclerotic le-sion formation in apolipoprotein E–deficient (ApoE�/�) mice.Our results also underscore the importance of KLF2 inmacrophage lipid uptake and foam cell formation via effectson the lipid binding protein adipocyte protein (aP)2/fattyacid–binding protein (FABP)4.

Materials and MethodsAnimals and DietsKLF2�/� mice (generously provided by J. Leiden) were generated aspreviously described.3 KLF2�/� mice of mixed background (calcu-lated to be �75% C57Bl/6) were mated to ApoE�/� mice4 on theC57Bl/6 background (The Jackson Laboratory) to generate KLF2�/�/ApoE�/� and KLF2�/�/ApoE�/� mice. All studies were performedwith strict age-matched, sex-matched littermate controls. Micewere weaned at 4 weeks, fed a normal rodent chow diet (4.5% fatby weight, 14% kilocalories; Laboratory Diet P3000), and, at 6 to8 weeks of age, were initiated on a high-fat (12.5% by weight, 40%kilocalories), high-cholesterol (1.25% by weight) diet (ResearchDiets D-12108) for a total of 20 weeks. Animal care and procedureswere performed according to NIH guidelines.

An expanded Materials and Methods section is available in theonline data supplement at http://circres.ahajournals.org.

ResultsEnhanced Atherosclerotic Lesion Formation inApoE-Deficient MicePrevious studies demonstrate that systemic knockout ofKLF2 is embryonic lethal.3,5 However, hemizygous deficient(KLF2�/�) mice are viable and fertile. KLF2�/� mice werecrossed to ApoE�/� mice to generate KLF2�/�/ApoE�/� andKLF2�/�/ApoE�/� mice. At 6 weeks of age, male mice (n�12to 14 per group) were initiated on a high-fat, high-cholesteroldiet for 20 weeks. Plasma lipid analysis reveals that there wasno significant difference in lipid profiles between mice atbaseline (data not shown) or after an atherogenic diet (TableI in the online data supplement).

After 20 weeks of an atherogenic diet, mice were eutha-nized and aortas were harvested for analysis of atheroscle-rotic lesion area by Sudan IV staining. KLF2�/�/ApoE�/�

mice exhibited a significant 31% increase in atheroscleroticlesion area when compared to KLF2�/�/ApoE�/� littermatecontrols (35.9�10.2% versus 27.3�9.9%; P�0.04) (Figure1A and 1B). Similarly, quantitative analysis using an en facepreparation revealed a 37% increase in atherosclerotic lesionarea (24.0�6.3% versus 17.6�5.4%; P�0.0099) (Figure 1Cand 1D).

Effect of KLF2 Deficiency on EndothelialGene ExpressionKLF2 is known to induce eNOS and inhibit VCAM-1expression in cultured endothelial cells.1 However, analysisof aortic sections demonstrated no appreciable difference inthe expression of endothelial eNOS or VCAM-1 (supplemen-tal Figure IA and IB). Consistent with this result, quantitativePCR analysis for eNOS, thrombomodulin, and VCAM-1revealed no significant change in KLF2�/� mice compared tolittermate controls (data not shown).

Original received August 4, 2008; revision received August 14, 2008;accepted August 18, 2008.

From the Case Cardiovascular Research Institute, Case WesternReserve University School of Medicine, Cleveland, Ohio.

Correspondence to Mukesh K. Jain MD, Wolstein Research Building,2103 Cornell Rd, Room 4-537, Cleveland, OH 44106-7290. [email protected]

(Circ Res. 2008;103:000-000.)© 2008 American Heart Association, Inc.

Circulation Research is available at http://circres.ahajournals.orgDOI: 10.1161/CIRCRESAHA.108.184663

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A recent study indicates that the Kruppel family memberKLF4 is capable of conferring gene regulatory effects similarto KLF2 in endothelial cells.6 Therefore, we reasoned that acompensatory increase in KLF4 in KLF2�/� mice mightaccount, in part, for the absence of any clear effect on theKLF2 endothelial targets assessed. Indeed, tissues fromKLF2�/� mice demonstrated a 39�4% increase in KLF4expression (P�0.0033) (Figure 2A).

Enhanced Lipid Uptake and aP2/FABP4Expression in KLF2 HemizygousDeficient MacrophagesWe next sought to assess the effect of KLF2 hemizygosity onmacrophage recruitment and function. Immunohistochemicalassessment using the macrophage specific antibody Mac-3revealed a nonsignificant trend toward increased macrophagearea staining in the aortic arch of KLF2�/�/ApoE�/� mice(3.13�1.70% versus 2.32�1.16%; P�0.176) (Figure 2B and2C). Thus, although KLF2 deficiency leads to increasedatherosclerotic burden (Figure 1), this cannot be explainedsimply by an increase in macrophage number.

Because previous studies indicated that KLF2 inhibitsphagocytosis,2 we reasoned that enhanced lipid uptake in

macrophages may account for the increase in atherosclerosis.Remarkably, peritoneal macrophages from KLF2�/� micetreated with oxidized LDL demonstrated a 40% increase inlipid uptake when compared to wild-type macrophages (Fig-ure 3A and supplemental Figure IIA). Conversely, adenoviraloverexpression 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 pro-cess, recent studies support a key role for the lipid chaperoneaP2/FABP4 in macrophage lipid accumulation and atherogene-sis.7,8 Indeed, KLF2�/� macrophages expressed higher proteinlevels of aP2 (Figure 3C). Conversely, overexpression ofKLF2 in RAW cells strongly inhibited aP2 expression(Figure 3D). These results are specific because the expres-sion of other factors involved in lipid accumulation such asCD36 were unaffected (data not shown). Taken together,these results indicate that reduced lipid uptake in KLF2�/� macro-phages is mediated, in part, by the regulation of aP2 by KLF2.

DiscussionAtherosclerosis is an extremely complex disease process witha number of important cellular contributors including endo-

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Figure 1. KLF2 heterozygous mice develop more atherosclerosis. Male littermate ApoE�/� and KLF2�/�/ApoE�/� mice at 6 weeks ofage were fed a high-fat, high-cholesterol diet for 20 weeks. Aortas were harvested and Sudan IV–stained for lipid. A, Two representa-tive pairs of fixed and stained whole aortas. B, Percentage lesion area of the entire isolated aorta area was calculated. ApoE�/�,27.3�9.9% (n�12); KLF2�/�/ApoE�/�, 35.9�10.2% (n�14). C, Two representative pairs of fixed and stained aortas en face. D, Percent-age lesion area of the entire aorta surface area was calculated. ApoE�/�, 17.6�5.4% (n�12); KLF2�/�/ApoE�/�, 24�6.3% (n�14). K2�/�

indicates KLF2�/�.

2 Circulation Research September 26, 2008



thelial cells, smooth muscle cells, and immune cells (mono-cyte and T cells). Because KLF2 has been shown to play aparticularly potent role in inhibiting endothelial cell andmonocyte/macrophage activation, we focused our mechanis-tic studies on these 2 cell types.

The key observation highlighted by our study relates to therole of KLF2 in monocyte/macrophage biology. Previously,Das et al reported that sustained expression of KLF2 inhibitedmonocyte activation and phagocytic capacity in vitro.2 Inaddition, Wu et al have shown that KLF2�/� mouse embry-onic fibroblasts exhibit accelerated lipid accumulation onadipocyte differentiation.9 Consistent with these observa-tions, KLF2�/� macrophages exhibit enhanced lipid accumu-lation (Figure 3) and increased proinflammatory genes(G.H.M. and M.K.J., unpublished observation). Thus, al-though absolute numbers of macrophages in atheroscleroticlesions of KLF2�/�/ApoE�/� and KLF2�/�/ApoE�/� micewere not significantly different, the increased lipid uptakecould account for the increase in lesion size. Mechanistically,our studies identify the lipid binding protein aP2 as a KLF2target. Work by Hotamisligil and colleagues has underscoredthe importance of aP2 in atherogenesis. Systemic and mac-rophage aP2 deficiency renders mice resistant to atheroscle-rosis.7,8 Furthermore, aP2-deficient macrophages exhibitedalterations in their ability to express inflammatory cytokines

and had reduced ability to uptake lipid when stimulated bymodified lipids.7 Given the results in KLF2�/� macrophages(Figure 3), it is likely that enhanced aP2 expression contrib-utes to the increase in lipid accumulation in KLF2�/� macro-phages. However, one cannot rule out the possibility thateffects on other aspects of lipid metabolism (eg, uptake orexport) in macrophages or alterations in aP2 expression inother tissues (eg, adipose) may also impact on the observedphenotype.

With respect to KLF2 effects in endothelial cells, ourstudies provide some important insights. Despite previous invitro observations, we did not observe any significant effectson the expression of KLF2 targets, including eNOS andVCAM-1. Importantly, however, our findings highlight a keycompensatory role for KLF4. Like KLF2, recent studiesreveal that KLF4 can also induce eNOS and inhibit VCAM-1.6

Thus, the increase in KLF4 observed may compensate forKLF2 deficiency. This is an interesting finding that raises theimportance of future studies to assess the effect of KLF4 ordual KLF2/KLF4 hemizygosity on atherosclerosis. Addition-ally, we note the possibility that other endothelial factor(s)not assessed in this study may be differentially regulated in anonredundant fashion by KLF2 and contribute to the pheno-type observed. These considerations, as well as generation of

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Figure 2. Effect of KLF2 deficiency on endothelial gene expression and macrophage recruit-ment. A, Quantitative PCR analysis was performed on RNA from isolated lung tissue harvestedfrom 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 malelittermate ApoE�/� and KLF2�/�/ApoE�/� mice fed an atherogenic diet for 20 weeks werestained for Mac-3 (n�8). Representative slides are shown. C, Mac-3 staining was quantified aspercentage staining area of the entire lesion area. KLF2�/�/ApoE�/�, 3.13�1.70%; ApoE�/�,2.32�1.16% (n�9).

A 1o Macrophages B RAW Cells AdGFP AdK2

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Figure 3. KLF2 regulates lipid uptake and macrophage aP2 levels. A and B, Peritoneal macrophages isolated from KLF2�/� and malewild-type littermate control mice (A) or RAW cells adenovirally infected with KLF2 (AdK2) or control virus (AdGFP) (B) were treated withoxidized LDL (oxLDL) (50 �g/mL) for 72 hours 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 levelsby Western blotting. A representative blot is shown.

Atkins et al KLF2 and Atherosclerosis 3



inducible tissue-specific KLF2 knockouts, are the subject ofongoing investigation.

In sum, these observations are the first in vivo evidence insupport of the hypothesis that KLF2 is atheroprotective.These findings are of particular interest as they suggest that areduction of KLF2 expression at levels that approximatethose observed in human subjects with coronary arterydisease is biologically relevant. Manipulation of KLF2 ex-pression may thus be beneficial in the treatment of vascularinflammatory disorders.

Sources of FundingThis 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.) and0725297B (to D.K.); an Alliance for Cancer Gene Therapy grant (toM.K.J.); a United Negro College Fund/Merck Science InitiativeFellowship grant (to G.B.A.); and a Robert Wood Johnson/HaroldAmos Medical Faculty Development grant (to G.B.A.).

DisclosuresNone.

References1. Atkins GB, Jain MK. Role of Kruppel-like transcription factors in endo-

thelial biology. Circ Res. 2007;100:1686–1695.

2. Das H, Kumar A, Lin Z, Patino WD, Hwang PM, Feinberg MW,Majumder PK, Jain MK. Kruppel-like factor 2 (KLF2) regulates proin-flammatory activation of monocytes. Proc Natl Acad Sci U S A. 2006;103:6653–6658.

3. Kuo CT, Veselits ML, Barton KP, Lu MM, Clendenin C, Leiden JM. TheLKLF transcription factor is required for normal tunica media formationand blood vessel stabilization during murine embryogenesis. Genes Dev.1997;11:2996–3006.

4. Zhang SH, Reddick RL, Piedrahita JA, Maeda N. Spontaneous hypercho-lesterolemia and arterial lesions in mice lacking apolipoprotein E. Science.1992;258:468–471.

5. Wani MA, Means RT Jr, Lingrel JB. Loss of LKLF function results inembryonic lethality in mice. Transgenic Res. 1998;7:229–238.

6. Hamik A, Lin Z, Kumar A, Balcells M, Sinha S, Katz J, Feinberg M,Gerzsten RE, Edelman ER, Jain MK. Kruppel-like factor 4 regulatesendothelial inflammation. J Biol Chem. 2007;282:13769–13779.

7. Makowski L, Boord JB, Maeda K, Babaev VR, Uysal KT, Morgan MA,Parker RA, Suttles J, Fazio S, Hotamisligil GS, Linton MF. Lack ofmacrophage fatty-acid-binding protein aP2 protects mice deficient in apo-lipoprotein E against atherosclerosis. Nat Med. 2001;7:699–705.

8. Boord JB, Maeda K, Makowski L, Babaev VR, Fazio S, Linton MF,Hotamisligil GS. Adipocyte fatty acid-binding protein, aP2, alters lateatherosclerotic lesion formation in severe hypercholesterolemia. ArteriosclerThromb Vasc Biol. 2002;22:1686–1691.

9. Wu J, Srinivasan SV, Neumann JC, Lingrel JB. The KLF2 transcriptionfactor does not affect the formation of preadipocytes but inhibits theirdifferentiation into adipocytes. Biochemistry. 2005;44:11098–11105.

KEY WORDS: atherosclerosis � Kruppel-like factor 2 � endothelium �macrophage � adipocyte Protein 2

4 Circulation Research September 26, 2008



Online Supplement Atkins et al., Hemizygous deficiency of Kruppel like Factor 2…

1

Supplementary Materials and Methods

Lipid analysis

Mice were fasted for 12-14 hours before blood was collected at the time of sacrifice.

Plasma was separated by centrifugation and stored at -80oC. Total cholesterol, high

density lipoprotein (HDL), and triglyceride concentrations were enzymatically measured

according to the outlined procedure in the corresponding Pointe Scientific Inc. kit.

Atherosclerotic lesion analysis

Mice were sacrificed and perfused with saline and formalin for 12 minutes. The heart

and aorta were harvested and fixed in 10% formalin. After careful removal of excess

adventitial tissue, the aorta was stained with Sudan IV (Sigma-Aldrich) for 15 minutes,

and differentiated in ethanol. For enface preparation, aortas were opened longitudinally

and pinned out on plain black wax. Images of Sudan IV stained aortas were taken with

a standard Nikon digital camera and analyzed with Zeiss image analysis software

(Axiovision Rel 4.5). Results were expressed as the percentage of the positive Sudan

IV color to the total aorta area.

Immunohistochemistry

Aortas were paraffin embedded and 5mm serial sections were obtained. Standard

immunohistochemistry procedures were used. Briefly, after peroxidase and serum

block, primary antibody was applied for 1-2 hours, followed by secondary antibody for

45 minutes, then peroxidase conjugated streptavidin applied for 18 minutes, and

developed using DAB substrate. Primary antibodies used were mouse macrophage-




2

specific marker Mac-3 (mAb M3/84) from BD Biosciences, VCAM antibody from Santa

Cruz (sc-8304), and eNOS from BD Transduction Laboratories. Immunostained

sections were quantified as the percent of positive staining area per total area using

Image Pro-Plus 6.0 program.

Quantitative PCR

Total RNA was isolated from lung tissue with Trizol (Invitrogen) as described by the

manufacturer. Quantitative PCR was performed as previously described 1. The

following primers were used: KLF2 (sense, 5’-ACCAAGAGCTCGCACCTAAA-3’;

antisense 5’-GTGGCACTGAAAGGGTCTGT-3’); KLF4 (sense, 5’-

CACAGGCGAGAAACCTTACCA-3’; antisense 5’-AATTTCCACCCACAGCCGT-3’);

36B4 (sense, 5’-GCTCCAAGCAGATGCAGCA-3’; antisense 5’-

CCGGATGTGAGGCAGCAG-3’). Fold induction was calculated after normalization to

36B4.

Macrophage isolation and culture

Peritoneal macrophages were isolated from 6-8 week old mice 4 days after an

intraperitoneal injection of 1 ml of 3% thioglycolate (Sigma-Aldrich). Isolated

macrophages were washed twice with 1XPBS. Cells were resuspended in DMEM

culture media (Cellgro) supplemented with 5%FBS (Atlanta Biologicals) and plated at a

concentration of 5 x 105 cells/ml on a 10cm culture plate for 18 hours. Cells were then

detached, counted, and re-plated at the required cell density for experimentation.




3

RAW264.7 were obtained from American Type Culture Collection and cultured following

the instructions as previously described 2.

Adenoviral infection of RAW264.7 cells

1 x 106 RAW264.7 cells were plated in a 60mm dish and infected with control (AdGFP)

or KLF2 (adKLF2) virus at a multiplicity of 100 in the presence of polybrene (final

concentration 10ug/ml). In general, ~80-90% infection was achieved within 48 hours of

incubation with adenovirus, at which time cells were used for experimentation.

Macrophage lipid uptake analysis

Mouse peritoneal macrophages were plated onto a 12 well tissue culture dish (5 x 104

cells/well) or adenovirally infected RAW cells (as described above) were incubated for

72 hours in the presence or absence of oxidized LDL (50µg/mL). Cells were then

washed once in ice-cold PBS followed by formaldehyde fixation (4% in PBS) for 30

minutes on ice. Neutral lipids were stained with 0.5% oil red O (Sigma-Aldrich) in

isopropanol (60%) for 60 minutes and photographs were taken. The oil red O stained

lipids were measured spectrophotometrically at 510nm after extraction with 100%

isopropanol, protocol adapted from 3.

Western blot analysis

Cellular protein was extracted in RIPA buffer (Sigma-Aldrich) supplemented with the

Complete protease inhibitor (Roche Applied Science) and western blot analysis using




4

aP2 antibody (Cell Signaling) and beta-actin antibody (Sigma-Aldrich) was performed as

previously described 4.

Statistical Analysis

Data are expressed as mean ± standard deviation (SD). For comparison between two

groups Student’s T-test was used. A value of p<0.05 was considered significant.

Online Table I

Genotype

Total Chol, mg/dL

Triglycerides, mg/dL

HDL, mg/dL

LDL, mg/dL

K2+/+/ApoE-/-

439±97

107±49

18±17

400±89

K2+/-/ApoE-/-

443±98

116±40

23±17

397±92

Plasmid lipid profile on high fat, high cholesterol diet. For all comparisons between

groups p was not significant. (K2+/+/ApoE-/- n=14, K2+/-/ApoE-/- n=16). Total Chol=total

cholesterol, HDL=high-density lipoprotein, LDL=low-density lipoprotein (calculated).




5

References

1. Hamik A, Lin Z, Kumar A, Balcells M, Sinha S, Katz J, Feinberg MW, Gerzsten

RE, Edelman ER, Jain MK. Kruppel-like factor 4 regulates endothelial

inflammation. J Biol Chem. 2007;282:13769-13779.

2. Feinberg MW, Jain MK, Werner F, Sibinga NE, Wiesel P, Wang H, Topper JN,

Perrella MA, Lee ME. Transforming growth factor-beta 1 inhibits cytokine-

mediated induction of human metalloelastase in macrophages. J Biol Chem.

2000;275:25766-25773.

3. Wu J, Srinivasan SV, Neumann JC, Lingrel JB. The KLF2 transcription factor

does not affect the formation of preadipocytes but inhibits their differentiation into

adipocytes. Biochemistry. 2005;44:11098-11105.

4. SenBanerjee S, Lin Z, Atkins GB, Greif DM, Rao RM, Kumar A, Feinberg MW,

Chen Z, Simon DI, Luscinskas FW, Michel TM, Gimbrone MA, Jr., Garcia-

Cardena G, Jain MK. KLF2 Is a novel transcriptional regulator of endothelial

proinflammatory activation. J Exp Med. 2004;199:1305-1315.




6

Figure Legends

Online Figure I. Effect of KLF2 deficiency on endothelial gene expression. A and B,

Descending aorta (A) and aortic arch (B) sections of male littermate wild-type and

KLF2+/- mice or male littermate ApoE-/- and KLF2+/-/ApoE-/- mice fed an atherogenic diet

for 20 weeks as indicated were stained for eNOS (n=3), VCAM-1 (n=8). Representative

slides are shown.

Online Figure II. Quantification of lipid uptake by macrophages. A and B, Peritoneal

macrophages isolated from KLF2+/- and male wild-type littermate control mice (A) or

RAW cells adenovirally infected with KLF2 (AdK2) or control virus (AdGFP) (B) were

treated with or without oxidized LDL (oxLDL) (50µg/mL) for 72hours as indicated and

stained for lipid uptake with oil red O. Quantification of oil red O staining was performed

by spectrophotometric analysis.



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Hemizygous Deficiency of Kruppel-Like Factor 2 Augments Experimental Atherosclerosis