Cholesteryl ester transfer protein (CETP) is a 74-kDa

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1 Importnt Role for Bone Mrrow Derived Cholesteryl Ester Trnsfer Protein in Lipoprotein Cholesterol Redistriution nd Atherosclerotic Lesion Development in LDL Receptor Knockout Mice Mirnd Vn Eck, Dn Ye, Reeni B. Hildernd, J. Kr Kruijt, Willeke de Hn, Menno Hoekstr, Ptrick C.N. Rensen, Christin Ehnholm, Mtti Juhiinen, Theo J.C. Vn Berkel Astrct Aundnt mounts of cholesteryl ester trnsfer protein (CETP) re found in mcrophge-derived fom cells in the rteril wll, ut its function in therogenesis is unknown. To investigte the role of mcrophge CETP in therosclerosis, LDL receptor knockout mice were trnsplnted with one mrrow from CETP trnsgenic mice, which express the humn CETP trnsgene under control of its nturl promoter nd mjor regultory elements. CETP production y one mrrow-derived cells induced 1.8-fold (P 0.01) increse in therosclerotic lesion development. The increse in lesion size coincided with n increse in VLDL/LDL cholesterol nd decrese in HDL cholesterol. The cholesterol redistriution in serum ws direct effect of the sustntil serum CETP ctivity nd mss (38 3 nmol/ml/h nd g/ml, respectively) induced y CETP production y one mrrow-derived cells. Conversely, specific disruption of CETP production y one mrrow-derived cells in CETP trnsgenic mice resulted in 2-fold (P ) reduction in serum CETP ctivity nd mss, demonstrting the quntittive relevnce of one mrrowderived CETP. Finlly, we show tht in liver Kupffer cells, heptic mcrophges, contriute 50% to the totl heptic CETP expression. In conclusion, one mrrow-derived CETP induces protherogenic lipoprotein profile nd promotes the development of therosclerotic lesions in LDL receptor knockout mice. Most importntly, we show for the first time tht one mrrow-derived CETP is n importnt contriutor to totl serum CETP ctivity nd mss. (Circ Res. 2007;100: ) Key Words: therosclerosis mcrophges lipoproteins one mrrow trnsplnttion mouse models Cholesteryl ester trnsfer protein (CETP) is 74-kD glycoprotein tht fcilittes the trnsfer of cholesteryl esters from ntitherogenic HDL to protherogenic pobcontining lipoproteins nd concomitnt equimolr trnsfer of triglycerides to HDL. 1 High HDL levels protect ginst the development of therosclerosis y virtue of its essentil role in the removl of excess cholesterol from peripherl cells nd its ntioxidtive, ntiinflmmtory, nd ntithromotic properties. 2,3 Severl lines of evidence indicte tht CETP is protherogenic. Introduction of the simin or the humn CETP gene in mice nturlly lcking CETP, results in dose-dependent reduction of HDL cholesterol nd n incresed susceptiility to therosclerosis. 4 6 Furthermore, humns possessing genetic deficiency for CETP hve higher HDL cholesterol levels nd reduced prevlence of coronry rtery disese (CAD). 7,8 However, in certin popultions CETP deficiency ppers to hve deleterious effects on CAD risk. 9,10 CETP mrna shows widespred tissue distriution, with the highest levels found in liver, spleen, nd dipose tissue Interestingly, CETP is lso expressed loclly in the rteril wll. 14,15 Aundnt mounts of CETP mss re found in mcrophge-derived fom cells in humn therosclerotic lesions, ut not in the helthy rteril wll. By promoting the trnsfer of cholesteryl esters from HDL to pob-contining lipoproteins CETP remodels the HDL prticle, which is ccompnied y reduction in size nd y the dissocition of pre -migrting, lipid poor, 16,17 which is n importnt cceptor of ABCA1-medited cholesterol efflux from mcrophges. 18,19 Loclly, in the rteril wll the ction of CETP might thus e implicted in the conversion of lrge cholesteryl ester enriched HDL into lipid-poor pre -HDL nd Originl received Septemer 7, 2006; revision received Jnury 12, 2007; ccepted Jnury 31, From the Division of Biophrmceutics, Leiden/Amsterdm Center for Drug Reserch, Gorleus Lortories (M.V.E., D.Y., R.B.H., J.K.K., M.H., T.J.C.V.B.), Leiden University, Leiden, The Netherlnds; Deprtment of Moleculr Medicine (C.E., M.J.), Ntionl Pulic Helth Institute, Biomedicum, Helsinki, Finlnd; Deprtment of Generl Internl Medicine, Endocrinology nd Metolic Diseses (W.d.H., P.C.N.R.), Leiden University Medicl Center, Leiden, The Netherlnds. These uthors contriuted eqully to this work. Correspondence to M. Vn Eck, Division of Biophrmceutics, Gorleus Lortories, Einsteinweg 55, 2333 CC Leiden, The Netherlnds. E-mil M.Eck@LACDR.LeidenUniv.nl 2007 Americn Hert Assocition, Inc. Circultion Reserch is ville t DOI: /01.RES f 678

2 Vn Eck et l Bone Mrrow Derived CETP nd Atherosclerosis 679 thus my lso hve n ntitherogenic function. Interestingly, mcrophge cholesterol loding results in dose-dependent increse in mcrophge secretion of CETP ctivity. 20 Furthermore, in vitro studies indicted direct role for CETP in cholesterol efflux from COS cells 14 nd J774 mcrophges. 21 The in vivo effects of mcrophge CETP production, however, re currently unknown. Mcrophges, present in therosclerotic lesions primrily depend on infiltrtion from one mrrow (BM)-derived monocytes into the rteril wll. Therefore, in the present study, we investigted the effects of selective introduction of CETP in BM-derived cells nd thus mcrophges on lipoprotein metolism nd therosclerotic lesion development y using one mrrow trnsplnttion. We show tht CETP production y mcrophges nd other BM-derived cells, under conditions of impired clernce of pob-contining lipoproteins, is highly therogenic ecuse of its significnt contriution to the serum cholesteryl ester trnsfer cpcity. Mterils nd Methods For detiled methodology, plese refer to the online dt supplement ville t Bone mrrow trnsplnttion (BMT) ws used to selectively introduce CETP in hemtopoietic cells, including mcrophges. Briefly, femle LDL receptor knockout (LDLr / ) mice (C57Bl/6J N5) were exposed to single dose of 9 grys (Gy) (0.19 Gy/min, 200 kv, 4 ma) X-ry totl ody irrdition, using n Andrex Smrt 225 Röntgen source (YXLON Interntionl, Copenhgen, Denmrk) 1 dy efore trnsplnttion. Irrdited recipients were trnsplnted y intrvenous injection of 0.5x10 7 one mrrow cells, isolted from mle CETP trnsgenic mice (; strin 5203; C57Bl/6J N10), overexpressing humn CETP under the control of its own promoter nd other mjor regultory elements, 22 or from wild-type littermtes. To induce the development of therosclerosis, the mice were fed Western-type diet (D), contining 15% (w/w) totl ft nd 0.25% (w/w) cholesterol (Diet W, Specil Diet Services, Withm, UK) for 9 weeks, strting t 8 weeks fter trnsplnttion fter which the mice were euthnized nd therosclerotic lesion development nd the composition of the lesions ws quntified. For determintion of serum lipid levels lood ws drwn fter n overnight fsting-period. CETP ctivity in serum ws mesured s the trnsfer/exchnge of 14 C-cholesterol olete etween exogenously dded humn LDL nd HDL, while CETP mss ws determined using CETP ELISA (Diichi). In ddition, CETP mrna expression ws determined in whole livers nd spleens of trnsplnted mice t 17 weeks posttrnsplnt nd in prenchyml, endothelil, nd Kupffer cells isolted from livers of mice using rel time-quntittive PCR. Results CETP Production y BM-Derived Cells Induces Atherosclerotic Lesion Development in LDLr / Mice To ssess the role of CETP production y BM-derived cells in therosclerotic lesion development, we used BMT to selectively introduce CETP in hemtopoietic cells. Hereto, one mrrow from CETP trnsgenic mice ws trnsplnted into LDLr / mice (3LDLr / ), which represents n estlished niml model for the development of therosclerosis. LDLr / mice trnsplnted with one mrrow from nontrnsgenic littermtes devoid of CETP expression were used s controls (3LDLr / ). For our experiments we used CETP trnsgenic mice expressing humn CETP under control of its own promoter nd nturl flnking regions. 22 These mice disply tissue distriution pttern of humn CETP mrna expression tht is similr to those oserved in humns (liver, spleen, smll intestine, kidney, nd dipose tissue) nd the expression is regulted y dietry cholesterol. Plsm CETP levels in the CETP trnsgenic mice re similr to the levels in normolipidemic humns. 23 Furthermore, plsm CETP ctivity in the CETP trnsgenic nimls (45 6 nmol/ml/h) is comprle to the ctivity tht we hve previously descried for helthy Finnish sujects (35 7 nmol/ml/h 24 ). To induce therosclerotic lesion development, the trnsplnted mice were fed Western-type diet, contining 0.25% cholesterol nd 15% ft, strting t 8 weeks fter trnsplnttion. After 9 weeks on Western-type diet therosclerotic lesion development ws nlyzed in the ortic root of 3LDLr / mice nd in 3LDLr / nimls. As shown in Figure 1, production of CETP y BM-derived cells induced 1.8-fold increse in lesion size (561 52x10 3 m 2 in 3LDLr / mice versus x10 3 m 2 in 3LDLr / mice, n 9, P 0.01). The mcrophge content of the lesions of 3LDLr / mice ws 64 6%, while the collgen content ws 4 1%. No significnt effect of CETP expression y BM-derived cells ws oserved on the reltive mcrophge nd collgen contents of the lesions (60 5% nd 7 2%, respectively). A predominnt prt of the lesions lso consisted of cellulr necrotic res which did not differ significntly etween the 2 groups (11 6% in 3LDLr / mice nd 17 4% in 3LDLr / nimls). Thus, CETP production y BM-derived cells induces the growth of the therosclerotic lesions without mrkedly ffecting lesion composition. Effect of Mcrophge CETP Production on Cholesterol Efflux To study the effect of endogenous CETP production on mcrophge cholesterol efflux, efflux of cholesterol from 3 H-cholesterol-loded peritonel mcrophges from humn CETP trnsgenic mice nd wild-type littermtes tht do not produce CETP ws compred (Figure 1). No effect of mcrophge CETP expression ws oserved on cholesterol efflux to lipid-free. After 24 hours, % of the cholesterol ws effluxed from wild-type mcrophges to s compred with % from CETP producing mcrophges. In ddition, no effect ws oserved on efflux to humn HDL ( % for wild-type nd % for CETP producing mcrophges fter 24 hours) or mouse HDL devoid of CETP ( % for wild-type nd % for CETP producing mcrophges fter 24 hours). It is thus unlikely tht the increse in lesion development is cused y effects of mcrophge CETP production on cholesterol efflux. CETP Production y BM-Derived Cells Induces Redistriution of Cholesteryl Esters From HDL to ApoB-Contining Lipoproteins The primry function in which CETP is implicted is the trnsfer of cholesteryl esters from HDL to protherogenic pob-contining lipoproteins. Therefore, next we nlyzed the effect of CETP production y BM-derived cells on serum

3 680 Circultion Reserch Mrch 16, 2007 Cholesterol efflux (%) ) LDLr-/- LDLr-/- 5x 5x 6h lipid levels. On regulr chow diet, no effect of CETP production y BM-derived cells on the concentrtion of free cholesterol nd cholesteryl esters in the circultion ws oserved (Tle). CETP, however, did induce prominent Lesion re (x10 4 µm 2 ) Donor one mrrow 24h BSA hhdl mhdl BSA hhdl mhdl Figure 1. CETP expression in BM-derived cells promotes therosclerotic lesion development in LDLr / mice, ut does not influence mcrophge cholesterol efflux., Formtion of therosclerotic lesions ws determined t the ortic root of LDLr / mice reconstituted with wild-type () or CETP trnsgenic () one mrrow tht were fed Western-type diet for 9 weeks. Representtive photogrphs of oil red O-stined cross sections of the ortic root t the level of tricuspid vlves (left). The lesion re ws clculted from 10 consecutive sections (right). P 0.01 vs mice trnsplnted with one mrrow., Effect of mcrophge CETP production on cholesterol efflux. Thioglycollte-elicited mcrophges derived from mice (open rs) nd from mice (filled rs) were loded with 3 H-cholesterol for 24 hour. 3 H-cholesterol loding ws nmol/mg cell protein nd nmol/mg for nd mcrophges, respectively. After wshing, cholesterol efflux ws susequently determined for the indicted times t 37 C in DMEM/0.2% BSA (BSA) or in the presence of 5 g/ml lipid-free () or 50 g/ml humn HDL (hhdl) nd mouse HDL devoid of CETP (mhdl). Vlues re mens SEM of 4 individul mice. redistriution of cholesteryl esters from HDL to pobcontining lipoproteins (Figure 2). As result, HDL cholesterol ws reduced from mg/dl in control 3LDLr / nimls to mg/dl (P 0.05) in CETP Tg3LDLr / mice (Tle). On chllenging the mice with the Western-type diet, the effect of CETP production y BM-derived cells ws even more pronounced. Under this condition serum free cholesterol nd cholesteryl esters were 2.4-fold (P 0.001) nd 1.7- fold (P 0.05), respectively, higher in 3LDLr / mice ecuse of n increse in VLDL nd LDL cholesterol levels (Tle, Figure 2). HDL cholesterol levels were reduced from 76 7 mg/dl in control 3LDLr / nimls to 53 7 mg/dl (P 0.05) in 3LDLr / mice. CETP production y BM-derived cells thus mrkedly influences serum cholesterol levels nd the distriution of cholesterol mong the different lipoproteins. To investigte chnges in HDL suclss distriution, serum of the trnsplnted mice ws lso nlyzed y crossed immunoelectrophoresis (Figure 2). On Western-type diet, the mount of pre -HDL prticles ws 3-fold (P 0.001) incresed in serum of CETP Tg3LDLr / mice (40 4%) s compred with control 3LDLr / nimls (14 2%). BM-derived CETP is thus cple of trnsferring cholesterol from HDL to pobcontining lipoproteins, therey trnsforming HDL nd generting metoliclly more pre -HDL. Despite the incresed ntitherogenic potentil of HDL s result of CETP production y BM-derived cells, the drmtic increse in protherogenic VLDL nd LDL levels still resulted in n incresed susceptiility to lesion development. BM-Derived Cells Significntly Contriute to Serum CETP Activity nd Mss As shown in Figure 3, CETP production y BM-derived cells resulted in sustntil levels of CETP ctivity in the serum of 3LDLr / mice (38 3 nmol/ml/h). This level of serum CETP ctivity is comprle to the ctivity in the donor mice, expressing CETP in ll endogenous tissues (45 6 nmol/ml/h) s well s the ctivity tht we hve previously descried for helthy Finnish sujects (35 7 nmol/ml/h 24 ). In ddition, CETP mss of g/ml ws determined in ser of the LDLr / nimls reconstituted with one mrrow. BM-derived cells overll re thus n importnt contriutor to serum totl CETP ctivity nd mss, therey explining the oserved redistriution of cholesteryl esters from HDL to protherogenic pob-contining lipoproteins nd the enhnced formtion of Effect of CETP Production y BM-Derived Cells on Serum Lipid Levels in LDLr / Mice Diet Western-Type Diet Mice Free Cholesterol Cholesteryl Esters HDL Cholesterol Mice Free Cholesterol Cholesteryl Esters HDL Cholesterol 3LDLr / LDLr / LDLr / LDLr / Blood smples were drwn fter n overnight fst t 8 weeks posttrnsplnt while feeding regulr chow diet nd t 17 weeks fter BMT fter 9 weeks feeding of high-cholesterol Western-type diet. Ser from individul mice were frctionted using superose 6 column to determine HDL cholesterol levels. The mount of free nd esterified cholesterol, nd HDL cholesterol in LDLr / mice trnsplnted with wild-type or one mrrow is shown. Vlues re the men SEM of t lest 9 mice. P 0.05, P vs control nimls trnsplnted with wild-type one mrrow.

4 Vn Eck et l Bone Mrrow Derived CETP nd Atherosclerosis 681 Lipids Free cholesterol VLDL LDL HDL Cholesterol esters LDL HDL VLDL LDLr-/- LDLr-/- -HDL stndrd D -HDL stndrd stndrd D -HDL preß-hdl -HDL stndrd Lipids D VLDL LDL HDL VLDL LDL Frction numer D HDL AUC (% totl HDL) preß-hdl D pre -HDL in 3LDLr / mice. No effect of introduction of CETP in BM-derived cells ws oserved on serum phospholipid trnsfer protein (PLTP) ctivity (23 1 mol/ ml/h nd 22 1 mol/ml/h for 3LDLr / nd CETP Tg3LDLr / mice, respectively). Circulting CETP levels re highly correlted with heptic CETP mrna levels nd heptic CETP output. 25 Therefore, CETP mrna expression ws determined in the trnsplnted mice oth in liver nd in spleen (Figure 3). As expected, reconstitution of LDLr / mice with one mrrow resulted in the ppernce of CETP mrna in spleens. Interestingly, CETP mrna levels in livers of the trnsplnted mice were 5.2-fold higher s compred with the levels in spleens ( in liver s compred with in spleen, P 0.05). Preß-HDL -HDL Preß-HDL -HDL Figure 2. CETP expression y BM-derived cells increses VLDL nd LDL cholesterol levels, reduces totl HDL cholesterol, nd increses pre -HDL in LDLr / mice. Blood ws collected fter n overnight fst t 8 weeks post trnsplnttion when the mice hd een on regulr chow diet (chow) nd t 17 weeks fter BMT when the mice hd een on Western-type diet (D) for 9 weeks., The mount of free cholesterol (left) nd esterified cholesterol (right) nd their distriution over the different lipoproteins in LDLr / mice trnsplnted with wild-type (open circles) or (closed circles) one mrrow is shown. Inserts show free cholesterol distriutions t smller scle. Frctions 3 to 7 represent VLDL; frction 8 to 14, LDL; nd frctions 15 to 19, HDL, respectively. Vlues re the men SEM of 9 mice., The mount of pre -HDL nd -HDL ws determined y crossed immunoelectrophoresis (top) in the ser of LDLr / mice trnsplnted with wild-type or one mrrow. The ottom pnel illustrtes the quntifiction of the chnges oserved. Open rs represent LDLr / mice trnsplnted with wild-type one mrrow nd filled rs mice trnsplnted with one mrrow. Vlues re the men SEM of 9 to 10 mice. P vs mice trnsplnted with wild-type one mrrow. CETP ctivity (nmol/ml/h) Serum Donor one mrrow CETP mrna expression (A.U.) Spleen The liver contins severl different cell types including prenchyml cells, endothelil, nd Kupffer cells. Kupffer cells re liver-specific mcrophges tht develop from BMderived monocytes nd ccount for 15% of ll liver cells nd 2.5% of liver protein. 26 To nlyze the replcement of Kupffer cells fter BMT, LDLr / mice were trnsplnted with one mrrow from mice, expressing enhnced green fluorescent protein (EGFP). As shown in Figure 4, lood cells re quntittively replced fter trnsplnttion y the donor EGFP-positive cells reching level of % t 8 weeks fter trnsplnttion. At this time point lredy 54 4% (n 5) of the F4/80-expressing Kupffer cells were EGFP-positive nd thus of donor-origin (Figure 4). Kupffer cells re thus suspected to e predominnt source of CETP in livers of LDLr / mice reconstituted with CETP- Liver Donor one mrrow Figure 3. CETP production y BM-derived cells significntly contriutes to serum CETP ctivity nd heptic nd splenic CETP expression., CETP ctivity in the ser of LDLr / mice trnsplnted with one mrrow from wild-type () or CETP trnsgenic () mice., CETP mrna expression in liver nd spleen of LDLr / mice following BMT (s ove), determined y rel-time PCR. CETP mrna expression is shown reltive to the verge expression of the housekeeping genes HPRT, -ctin, nd GAPDH. Open rs represent LDLr / mice trnsplnted with one mrrow nd filled rs mice trnsplnted with one mrrow. Vlues re the men SEM of 9 to 10 mice. P 0.05 nd P vs mice trnsplnted with wild-type one mrrow.

5 682 Circultion Reserch Mrch 16, 2007 Blood cell replcement (%) Time post-trnsplnt (dys) Liver Lung 8 wks posttrnsplnt Spleen Lesion 40x Figure 4. Reconstitution of lood cells nd mcrophges in liver, spleen, lung, nd lesions with donor-derived EGFPexpressing cells fter one mrrow trnsplnttion., The ppernce of EGFP-expressing lood cells in the circultion determined y FACS nlysis t different time points fter trnsplnttion of LDLr / mice (n 5) with EGFP-expressing one mrrow., Representtive merged photomicrogrphs of EGFP fluorescence (green), immunohistochemicl stining ginst mouse mcrophge F4/80 (red) nd Dpi for nuclei (lue) in liver, spleen, lung t 8 weeks fter trnsplnttion s well s therosclerotic lesions. Colocliztion of mcrophges with EGFP is yellow. expressing one mrrow. In ddition to the liver, EGFPpositive mcrophges were found in other orgns, including spleen nd lung. Interestingly, in spleen EGFP-positive cells were primrily found in the red pulp, which is rich in mcrophges s well s in the mrginl zone tht is rich in B-cells, while only limited replcement of T-cells in the white pulp ws oserved. In lesions EGFP ws expressed y mcrophge fom cells, ut not y endothelil cells overlying the lesion. To investigte the potentil contriution of Kupffer cells to totl heptic CETP production, the expression of CETP ws evluted in purified prenchyml cells, endothelil cells, nd Kupffer cells of livers from totl-ody mice tht express CETP in ll endogenous tissues (Figure 5). CETP mrna expression in prenchyml liver cells ws Expression in endothelil cells ws 10-fold higher ( ; P 0.01) s compred with prenchyml liver cells. However, 47-fold higher expression ws found in Kupffer cells ( ; P ). Thus, lthough Kupffer cells only contriute to 2.5% of the totl liver protein, they do contin t lest 48% of the totl liver CETP expression, s compred with 38% nd 14% for prenchyml cells nd endothelil cells, respectively. Immunohistochemicl stining of liver sections from mice lso indicted the predominnt Kupffer cell locliztion of CETP in the liver (Figure 5). To determine which types of BM-derived cells, in ddition to mcrophges contriute to the circulting CETP ctivity levels, CD4 T-helper cells, CD8 cytotoxic T-cells, B-cells, nd mcrophges/neutrophils were isolted from spleens of mice. As shown in Figure 5c, CETP mrna ws found in ll splenocytes nlyzed, with no significnt difference in the level of expression etween the different cell types. Immunohistochemicl stining of spleen sections for CETP showed CETP protein expression in mcrophges of the red pulp of the spleen nd in the mrginl zone surrounding the white pulp, which is rich in B-cells (Figure 5d). Thus, other BM-derived cells lso contriute to CETP production esides mcrophges. Reverse Bone Mrrow Trnsplnttion of Wild-Type Bone Mrrow to Mice To confirm the quntittive importnce of hemtopoieticllyderived CETP for determining plsm CETP levels, the effect of specific disruption of BM-derived CETP in mice ws determined. Hereto reverse BMT experiment ws performed, in which one mrrow from wild-type littermtes ws trnsplnted into mice hving functionl LDL receptor. At 8 weeks fter trnsplnttion, CETP ctivity in control trnsplnted 3 nimls ws nmol/ml/h, s compred with only nmol/ml/h in 3 mice (P ) (Figure 6). CETP mss ws reduced from g/ml to g/ml (P ). Specific disruption of CETP production y BM-derived cells thus resulted in 53.8% reduction in CETP ctivity nd 41.9% reduction in CETP mss, confirming our conclusions out the quntittive importnce of BM-derived CETP. The oserved reduction in plsm CETP ctivity nd mss levels lso coincided with 2-fold lower (P 0.029) CETP mrna expression levels in the liver ( for control 3 nimls nd for 3 mice). The therosclerosis susceptiilities of 3 CETP Tg nimls nd 3 mice were compred fter 8 weeks on high-cholesterol diet, contining 1% cholesterol, 15% ft, nd 0.5% cholte, strted t 8 weeks fter trnsplnttion. Both on chow diet nd fter feeding the high-cholesterol diet, no effect of disruption of CETP production y BM-derived cells ws oserved on serum cholesterol levels (dt not shown). In ddition, no differences were oserved in therosclerotic lesion size ( m 2 for CETP Tg3 nimls [n 9] nd m 2 for 3 mice [n 8]) (Figure 6). In oth groups of mice lesions were composed of multiple lyers of fom cells.

6 Vn Eck et l Bone Mrrow Derived CETP nd Atherosclerosis 683 Liver c CETP mrna expression (A.U.) CETP mrna expression (A.U.) Prenchyml cells Endothelil cells Kupffer cells Spleen CD8+ CD4+ CD11+ CD45R+ Thus, in greement with the in vitro studies which showed tht cholesterol efflux from mcrophges ws not ffected y CETP expression, lso no locl effects of disruption of mcrophge CETP expression on therosclerotic lesion development were found in mice with functionl LDL receptor. Discussion It hs lredy een known for lmost two decdes tht CETP is produced y mcrophges. 20 In ddition, undnt mounts CETP ctivity (nmol/ml/h) Serum Donor one mrrow d Red pulp White pulp 5x 5x Lesion re (x10 4 µm 2 ) Figure 5. CETP expression y different cell types of the liver nd spleen., Heptic CETP mrna expression ws determined in totl-ody CETP trnsgenics y rel-time PCR fter seprtion of the prenchyml cells, endothelil cells, nd Kupffer cells in the liver y collgense perfusion., For determintion of CETP mrna expression in spleen, splenocytes were hrvested using mgnetic nnoprticles conjugted with nti-mouse CD4, CD8, CD45R/B220, or CD11 monoclonl ntiodies for the isoltion of CD4 T-helper cells, CD8 cytotoxic T-cells, B-cells, nd mcrophges/ neutrophils. CETP mrna expression is shown reltive to the verge expression of the housekeeping genes HPRT, -ctin, nd GAPDH. Vlues re the men SEM of 6 femle mice. P 0.01, P vs prenchyml cells. nd d, For detection of CETP protein cryostt sections of liver () nd spleen (d) of mice were stined immunohistochemiclly for CETP (green), F4/80-positive mcrophges (red) nd nuclei (lue). Colocliztion of CETP nd mcrophge F4/80 is yellow. of CETP protein nd mrna re detected in mcrophge-derived fom cells in humn ortic nd coronry therosclerotic lesions, indicting tht CETP production y mcrophges might ply n importnt role in cholesterol homeostsis loclly in the rteril wll. 14,15 Until now, however, no in vivo studies were performed on the role of mcrophge-derived CETP in therosclerotic lesion development. To specificlly study the role of mcrophge CETP in therosclerosis, we ns Donor one mrrow Figure 6. Disruption of CETP production y BM-derived cells reduces serum CETP ctivity ut does not ffect therosclerotic lesion development in mice with functionl LDL receptor., CETP ctivity in the ser of humn CETP trnsgenic () mice trnsplnted with one mrrow from or wild-type () mice. Open rs represent mice trnsplnted with one mrrow (n 9) nd filled rs mice trnsplnted with one mrrow (n 9). P vs mice trnsplnted with one mrrow., Formtion of therosclerotic lesions ws determined t the ortic root of mice reconstituted with or one mrrow tht were fed high cholesterol/cholte diet for 8 weeks. Representtive photogrphs of oil red O-stined cross sections of the ortic root t the level of tricuspid vlves (left). The lesion re ws clculted from 10 consecutive sections (right).

7 684 Circultion Reserch Mrch 16, 2007 hve creted chimers tht express humn CETP in BMderived cells, including rteril intim mcrophges, y trnsplnttion of one mrrow from humn CETP trnsgenic mice into LDLr / mice. Interestingly, we found tht CETP production y BM-derived cells promoted the development of therosclerosis, wheres efflux of cholesterol from peritonel mcrophges to lipid-free or HDL ws not ffected. Thus, in contrst to previous in vitro studies suggesting n ntitherogenic function for mcrophge CETP 14,15,20,21 in the current study we provide strong in vivo evidence tht CETP production y BM-derived cells, including mcrophges is protherogenic in mice lcking the LDL receptor. The incresed therosclerosis in these mice expressing CETP in BM-derived cells coincided with significnt redistriution of cholesteryl esters from HDL to protherogenic pob-contining lipoproteins nd drmtic increse in the levels of protherogenic pob-contining lipoproteins s result of the ppernce of sustntil mounts of ctive CETP in the circultion. The lrge increse in plsm cholesteryl ester levels in the 3LDLr / mice is primrily the consequence of the circulting CETP ctivity levels on the cholesteryl ester trnsfer process in these mice. In sence of the LDL receptor, the removl of triglyceride-rich pob-contining lipoproteins from the circultion is severely impired, leding to their ccumultion. Similr effects of CETP expression on the ccumultion of pob-contining lipoproteins hve previously een descried in poe nd LDL receptor knockout mice. 5 By specific disruption of CETP production y BM-derived cells in CETP Tg mice we estlished tht BM-derived cells contriute for 50% to the totl circulting CETP ctivity levels. In these nimls with functionl LDL receptor, no effects on serum cholesterol levels or therosclerotic lesion development were oserved, indicting tht LDL receptor deficiency is mjor contriutor to the oserved effects of BM-derived CETP in LDL receptor knockout mice. In ddition, these studies implicte tht mcrophge CETP does not ffect cholesterol homeostsis loclly in the rteril wll. Mcrophges re n importnt source of BM-derived cells tht reside within lmost ll tissues. Importnt sources of serum CETP re liver nd spleen CETP production y the liver hs primrily een ttriuted to the prenchyml cells. However, the liver consists of 15% Kupffer cells, representing the heptic mcrophges. 26 In the current study we lso provide evidence tht Kupffer cells re n importnt source of CETP in livers of totl-ody CETP trnsgenic mice nd tht disruption of CETP expression in BM-derived cells results in 50% decrese in heptic CETP mrna expression ecuse of the sence of Kupffer cell CETP expression. In greement with this, nonprenchyml cells of the heptic sinusoids re the principl source of CETP in livers of cynomolgus monkeys tht like humns nturlly express CETP. 27 Thus, lthough it still remins to e determined whether nd how much mcrophges nd other BM-derived cells contriute to plsm CETP ctivity in humns, the comprle findings in humn CETP trnsgenic mice nd cynomolgus monkeys suggest tht our findings pply to species tht nturlly express CETP. In our BMT chimers lso significnt expression of CETP ws found in spleen. In greement, CETP expression ws previously shown in spleens of dult totl-ody CETP trnsgenic mice, 28 hmsters, 13 nd humns. 29 In the current study, we show tht B-cells, T-cells, nd mcrophges/neutrophils isolted from spleens of totl-ody mice ll express CETP mrna. CETP protein coloclized with mcrophges in the red pulp of the spleen. In ddition, sustntil CETP protein expression ws found in the mrginl zone, the interphse etween the nonlymphoid red pulp nd the lymphoid white pulp, n re rich in B-cells. In hmsters, CETP is primry loclized t the periphery of germinl follicles of the spleen. 13 Also in humn spleens CETP protein ws found in white pulp germinl centers nd coloclized with B-cells nd proportion of mrginl zone B-cells. 29 Thus, CETP is not only restricted to mcrophges, ut is lso produced y other cells from one mrrow origin. An importnt question remins out the function of CETP production y mcrophges. Mcrophge fom cells re primrily restricted to therosclerosis, wheres ctivted mcrophges re common feture of mny inflmmtory diseses. Interestingly, CETP elongs to the fmily of lipid trnsfer/lipopolyscchride-inding proteins. 30 Furthermore, dministrtion of lipopolyscchride to hmsters with endogenous CETP expression or to trnsgenic mice expressing humn CETP induces rpid decrese in serum CETP concentrtion, 31,32 suggesting tht CETP with its moleculr mimicry to lipopolyscchride-inding proteins might e le to modulte the lipopolyscchride response nd thus cn ply role in innte immunity in ddition to its role in cholesterol metolism. In conclusion, the current study strongly suggests tht CETP produced y BM-derived cells, including mcrophges, is n importnt contriutor to totl serum CETP ctivity. It modultes lipoprotein metolism y mediting the redistriution of cholesteryl esters from HDL to pobcontining lipoproteins, nd promotes the development of therosclerosis in LDL receptor knockout mice with impired clernce of pob-contining lipoproteins. The sustntil contriution of BM-derived cells to circulting totl CETP ctivity implictes n importnt role for CETP in mcrophge function. Acknowledgments The uthors thnk Jri Metso, M.Sc. for expert nlysis of pre -HDL prticles in mice ser nd determining CETP ctivities. Sources of Funding This work ws supported y The Netherlnds Orgniztion of Scientific Reserch (VIDI grnt to M.V.E., VIDI grnt to P.C.N.R.), the Netherlnds Hert Foundtion (grnt 2001T041 to M.V.E.), the Finnish Foundtion for Crdiovsculr Reserch, nd the Sigrid Juselius Foundtion. None. Disclosures References 1. Tll AR. Plsm lipid trnsfer proteins. Ann Rev Biochem. 1995;64:

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