PCSK9 inhibition fails to alter hepatic LDLR, circulating cholesterol, and atherosclerosis in the absence of ApoE.

LDL cholesterol (LDL-C) contributes to coronary heart disease. Proprotein convertase subtilisin/kexin type 9 (PCSK9) increases LDL-C by inhibiting LDL-C clearance. The therapeutic potential for PCSK9 inhibitors is highlighted by the fact that PCSK9 loss-of-function carriers exhibit 15-30% lower circulating LDL-C and a disproportionately lower risk (47-88%) of experiencing a cardiovascular event. Here, we utilized pcsk9(-/-) mice and an anti-PCSK9 antibody to study the role of the LDL receptor (LDLR) and ApoE in PCSK9-mediated regulation of plasma cholesterol and atherosclerotic lesion development. We found that circulating cholesterol and atherosclerotic lesions were minimally modified in pcsk9(-/-) mice on either an LDLR- or ApoE-deficient background. Acute administration of an anti-PCSK9 antibody did not reduce circulating cholesterol in an ApoE-deficient background, but did reduce circulating cholesterol (-45%) and TGs (-36%) in APOE*3Leiden.cholesteryl ester transfer protein (CETP) mice, which contain mouse ApoE, human mutant APOE3*Leiden, and a functional LDLR. Chronic anti-PCSK9 antibody treatment in APOE*3Leiden.CETP mice resulted in a significant reduction in atherosclerotic lesion area (-91%) and reduced lesion complexity. Taken together, these results indicate that both LDLR and ApoE are required for PCSK9 inhibitor-mediated reductions in atherosclerosis, as both are needed to increase hepatic LDLR expression.

express human IgG antibodies were immunized with human PCSK9. Determination of binding affi nity, screening for cross reactivity to mouse PCSK9, and activity in a cell-based LDL uptake assay led to mAb1 selection.
cDNA sequences encoding the variable domains of heavy and light chains of mAb1 were fused to constant domains of mouse IgG1 heavy chain and mouse lambda light chain. The resulting cDNA sequences encoding the chimeric mAb1 (CmAb1) heavy chains and light chains were inserted into pTT5 expression plasmid separately. CmAb1 mouse IgG1 was expressed by cotransfecting 293 6E cells with pTT5 plasmids containing light chain and heavy chain sequences . Expressed chimeric antibody was purifi ed by capturing on a MabSelect SuRe column and polished on a SP-Sepharose column as previously described ( 25 ).
Binding of mAb1 and CmAb1 to mouse PCSK9 was measured in a kinetic binding assay by BIAcore. Mouse anti-His antibody (Qiagen, Valencia, CA) was immobilized on all four fl ow cells of a CM5 chip using amine coupling reagents (GE Healthcare, Piscataway, NJ) with an approximate density of 5,000-6,000 RU. Histagged PCSK9 was captured on the second and fourth fl ow cells at an approximate density of 130 RU for mouse PCSK9. Flow cells one and three were used as background controls. Anti-PCSK9 antibody at 100 nM was diluted in PBS plus 0.1 mg/ml BSA, 0.005% P20, and injected over the captured PCSK9 surface with a 50 ul/min fl ow rate (5 min association and 5 min dissociation). CmAb1 showed very similar binding activity compared with mAb1 ( 25 ).
Control mouse IgG1 was raised against a PeptiBody peptide AGP-3. The resulting antibody was produced in stably transfected Chinese hamster ovary cells and purifi ed using the same method as CmAb1 .

In vivo
All animal care and experimental procedures were approved by the Institutional Animal Care and Use Committee at Amgen for work performed at Amgen and by the Institutional Animal Care and Use Committee of the Netherlands Organization for Applied Research for work performed at TNO Metabolic Health Research. All mice were housed and maintained under standard environmental conditions with a 12 h light-dark cycle and had free access to food and water. All mice were in a C57Bl/6 background.
Ldlr Ϫ / Ϫ and apoE Ϫ / Ϫ mice were obtained from Jackson Labo- In pharmacologic inhibitory studies, antibodies were administered by sc injection (10 mg/kg) every 10 days for 14 weeks, to examine effects on atherosclerotic plaque development.
Whole blood was collected by tail nick, vena cava, or cardiac puncture. At study termination, animals were euthanized either and an increased risk of experiencing a cardiovascular event (20)(21)(22). Additionally, PCSK9 loss-of-function carriers have 15-30% lower circulating LDL-C and a disproportionately lower risk (47-88%) of experiencing a cardiovascular event ( 23 ). This disproportionate reduction in risk is in contrast to statins, where 5 year treatment reduced cardiovascular events by 40% even when LDL-C was reduced to 80 mg/dl ( 24 ). Whether this disproportionate reduction in risk is due to PCSK9 having a direct negative effect at the atherosclerotic lesion or if the additional benefi t is driven by a modest lifelong reduction in serum cholesterol is unclear. These observations have led to the development of PCSK9 inhibitors as a means to therapeutically reduce LDL-C and the associated CVD risk (25)(26)(27)(28)(29). Inhibition of PCSK9 by monoclonal antibodies, adnectins, or siRNAs reduces LDL-C levels in patients, and clinical trials designed to assess the effect of anti-PCSK9 therapies on cardiovascular outcomes are underway (30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42).
ApoE, like ApoB, is present in lipoproteins and functions as a ligand of the LDLR and is important for the clearance of TG-rich lipoproteins. The decrease in HDL cholesterol (HDL-C) in pcsk9 Ϫ / Ϫ mice has been attributed to the binding of ApoE containing HDL to the upregulated LDLR ( 11 ). Even with a functional LDLR and ApoB, mutations in APOE in humans can lead to hypercholesterolemia (43)(44)(45)(46). To date the role of ApoE in the lipid lowering and athero-protective effects of PCSK9 inhibition is unclear. PCSK9 overexpression in an apoe -defi cient background has been reported to be proatherogenic, while PCSK9 deletion in apoe -defi cient mice leads to a reduction in the amount of cholesterol ester found within the aorta, even though the plaque size and total plasma cholesterol levels remain unchanged ( 47 ). The contribution of cholesterol ester content to atherosclerotic lesion development in the absence of changes in lesion area are unknown, but these data hint that a functional ApoE-LDLR pathway is essential for PCSK9-mediated changes in atherosclerosis that are driven by decreases in plasma cholesterol. To investigate this, we utilized both genetically engineered knockout mice ( pcsk9 Ϫ / Ϫ ) and an anti-PCSK9 antibody to examine the effect of PCSK9 inhibition on plasma lipoproteins and atherosclerotic lesion development in mice lacking the LDLR or ApoE, as well as in APOE*3Leiden.cholesteryl ester transfer protein (CETP) mice ( 47 ), which have mouse ApoE and LDLR but hampered clearance of the ApoBcontaining lipoproteins due to the expression of human mutant APOE*3Leiden ( 48 ). The APOE*3Leiden.CETP mice are a well-established mouse model for familial dysbetalipoproteinemia with human-like lipoprotein metabolism and atherosclerosis development, which respond in a human-like manner to both lipid lowering as well as HDLraising drugs (like statins, fi brates, niacin, etc.) used in the treatment of CVD (49)(50)(51)(52).

Antibody generation and purifi cation
The fully human PCSK9-targeting antibody, mAb1, was generated as described previously ( 25 ). Briefl y, mice engineered to with either Verhoeff-Van Gieson (VVG) or hematoxylin-phloxinesaffron to measure lesion area. In some studies, histological analysis was performed by Charles River Discovery Research Services and sections were stained with Mac-2 to monitor macrophage content. For each mouse, three or four sections at intervals of 40 to 50 m were used for quantitative and qualitative assessment of the atherosclerotic lesions ( 54,55 ). To qualify lesion severity, the lesions were classifi ed into one of fi ve categories according to the American Heart Association classifi cation: early fatty streak (I), regular fatty streak (II), mild plaque (III), moderate plaque (IV), and severe plaque (V), as previously described ( 56 ). To assess lesion severity as a percentage of all lesions, type I through III lesions were classifi ed as mild lesions and type IV and V lesions were classifi ed as severe lesions. Images were acquired with an Olympus BX51 microscope. Atherosclerosis development was quantifi ed by measuring lesion areas using Cell D imaging software (Olympus Soft Imaging Solutions).
For en face analysis, aortas were soaked in PBS followed by 70% ethanol (5 min each). Aortas were subsequently soaked with Sudan IV stain for 6 min with occasional agitation. Aortas were then rinsed twice with 80% ethanol followed by PBS (3 min each). Aortas were mounted and photographed under a stereo microscope. Aortic plaque area was quantifi ed by Image-Pro.

LDLR is the predominant means for PCSK9-mediated regulation of circulating cholesterol and is required for PCSK9 inhibitor-mediated regulation of atherosclerosis
To investigate whether LDLR infl uences circulating PCSK9 levels, we measured plasma PCSK9 levels in ldlr Ϫ / Ϫ and WT mice and found a signifi cant elevation in plasma PCSK9 in ldlr Ϫ / Ϫ mice (2,083 ± 1,529 ng/ml and 98 ± 98 ng/ml) providing further confi rmation that PCSK9 and LDLR infl uence the clearance of one another ( Fig. 1A ) ( 57,58 ).
Pcsk9 Ϫ / Ϫ mice exhibit increased hepatic LDLR levels leading to lower circulating cholesterol levels ( 26 ). However, it is unclear if PCSK9 also infl uences the levels of circulating cholesterol independent of the LDLR ( 47 ). We investigated this possibility by comparing the levels of circulating cholesterol in 12-week-old ldlr Ϫ / Ϫ / pcsk9 Ϫ / Ϫ mice relative to ldlr Ϫ / Ϫ littermate controls. Mice were fed a WTD (40% kcal from fat and 1.25% cholesterol) for 12 weeks, and circulating cholesterol and TG levels were measured at 5 and 12 weeks from the initiation of WTD feeding. The ldlr Ϫ / Ϫ / pcsk9 Ϫ / Ϫ mice exhibited a slight but signifi cant To determine whether these reductions in TC levels translated into reduced atherosclerosis development, we measured the amount of atherosclerotic plaque by CO 2 asphyxiation or by exsanguination under anesthesia (100 mg/kg ketamine, 5 mg/kg diazepam). For liver collection, sections of the right medial or left lobe were excised, fl ash frozen, and stored until further use. For heart and aorta isolation, hearts were either isolated and placed directly in formalin or animals were perfused by gravity fl ow under anesthesia. Perfusion was performed by inserting a 25 gauge needle into the apex of the left ventricle and nicking the right atrium. Animals were perfused with saline for 10 min followed by 4% paraformaldehyde for 10 min for fi xation. Hearts and aortas were removed, immersed in 4% paraformaldehyde, and stored at 4°C.

Cholesterol, TG analysis, PCSK9 ELISA
Mouse serum or EDTA plasma was obtained from whole blood collected via centrifugation. Serum or plasma cholesterol and TGs were analyzed using either a Cobas Integra 400 chemistry analyzer or enzymatic kits according to the manufacturer's instructions (catalog numbers 1458216 and 1488872, respectively; Roche/Hitachi). In some instances, pooled serum from mice treated with either control or anti-PCSK9 monoclonal antibodies was fractionated by fast protein liquid chromatography (Superose 6 10/300 GL column). Cholesterol content of each fraction was measured using the HDL-C E kit omitting the phosphotungstatemagnesium salt precipitation step (Wako Pure Chemical Industries, Osaka, Japan). Mouse PCSK9 serum protein levels were measured by sandwich ELISA (R&D Systems; MPC900) according to the manufacturer's instructions.

Hepatic LDLR mRNA and protein expression
Total RNA was extracted from liver tissue samples using RNA-Bee (Amsbio, Oxon, UK) according to the manufacturer's instructions. Random primers were used to convert RNA to single stranded cDNA by reverse transcription (Promega, Fitchburg, WI) according to the manufacturer's protocol. Levels of cDNA were measured by real-time PCR using the 7500 Fast real-time PCR system (Applied Biosystems, Foster City, CA), according to the manufacturer's instructions. Assay-on-demand primers and probes were obtained from Applied Biosystems. The mRNA levels were normalized to mRNA levels of three housekeeping genes (i.e., cyclophilin, HPRT , and GAPDH). The level of mRNA expression for each gene of interest was calculated according to the manufacturer's instructions (Applied Biosystems).
For protein expression, liver tissues were homogenized in lysis buffer (Santa Cruz Biotechnology, Inc.) containing complete protease inhibitors (Roche Diagnostics) and incubated on ice for 30 min. The lysis buffer consisted of 50 mM Tris-HCL (pH 7.4), 150 mM NaCl, 0.25% deoxycholic acid, 1% NP-40 (Igepal), 1 mM EDTA, protease inhibitor cocktail (complete, Roche), 1 mM PMSF, and 1 mM Na3VO4. Samples were then centrifuged at 13,000 g at 4°C for 20 min. Protein concentration in cell lysates was determined by BioRad protein assay reagents (BioRad) according to the manufacturer's instructions. Fifty micrograms of protein lysates were separated by SDS-PAGE and then transferred to polyvinylidene fl uoride membranes (BioRad). Blots were subjected to goat anti-mouse LDLR from R&D Systems and rabbit anti-goat HRP from Santa Cruz. Mouse anti-␣ -actin from Cell Signaling Technologies was used to confi rm equal loading in conjunction with horse anti-mouse HRP from Santa Cruz Biotechnology, Inc., according to the manufacturer's instructions. Blots were developed with Bio-Rad Clarity Western ECL (BioRad) and subjected to ChemiDoc™ XRS+ imaging system. Intensities of protein bands were quantifi ed using Image Lab™ software.

Atherosclerosis measurements
Hearts embedded in paraffi n were cross-sectioned (5 m each) through the entire aortic root area. Sections were stained exhibit comparable PCSK9 plasma and hepatic LDLR protein levels relative to WT mice (67 ± 23 ng/ml and 98 ± 98 ng/ml, respectively; Fig. 2A , supplementary Fig. III). Comparing apoe Ϫ / Ϫ / pcsk9 Ϫ / Ϫ and apoe Ϫ / Ϫ littermate controls revealed that there was no difference in circulating TC, HDLs, LDLs, or TGs, and there was no signifi cant difference in hepatic LDLR protein levels ( Fig. 2B, C ; supplementary Figs. IV, V). In addition, there was no difference in either atherosclerotic plaque accumulation (0.18 ± 0.08 mm 2 and 0.14 ± 0.07 mm 2 ) or macrophage content (0.006 ± 0.005 mm 2 and 0.006 ± 0.003 mm 2 ) within the aortic root between apoe Ϫ / Ϫ / pcsk9 Ϫ / Ϫ and apoe Ϫ / Ϫ littermate controls at 32 weeks of age, respectively ( Fig. 2D-F ). Together these data demonstrate that in the absence of ApoE, expected lipid lowering and atheroprotective effects caused by the deletion of PCSK9 are not apparent.
PCSK9 inhibition is effective in APOE*3Leiden.CETP mice but not in apoe ؊ / ؊ mice Recently, several PCSK9 monoclonal antibodies have been developed as a therapy to reduce plasma lipids. To determine whether anti-PCSK9 antibody treatment can within the aorta and the aortic root after 12 weeks. Atherosclerotic lesions covered 11 ± 5% and 10 ± 3% of the aortic area in the ldlr Ϫ / Ϫ and ldlr Ϫ / Ϫ mice, respectively (supplementary Fig. II). Similarly, no signifi cant difference was observed in lesion area (0.26 ± 0.10 mm 2 and 0.28 ± 0.11 mm 2 ) or macrophage content (0.04 ± 0.02 mm 2 and 0.05 ± 0.02 mm 2 ) in the aortic sinus of ldlr Ϫ / Ϫ and ldlr Ϫ / Ϫ / pcsk9 Ϫ / Ϫ mice, respectively ( Fig. 1D-F ). Thus, deletion of PCSK9 in the absence of the LDLR reduces circulating cholesterol levels, suggesting that other receptors or mechanisms are involved in the PCSK9-mediated cholesterol clearance.

PCSK9 deletion in apoe ؊ / ؊ mice does not affect circulating cholesterol and atherosclerosis
Another key player and essential protein for normal particle uptake by the liver via the LDLR gene family is ApoE, which is present on chylomicrons, VLDLs, IDLs, and LDLs, and also on large HDL particles. Consequently, apoe Ϫ / Ϫ mice exhibit elevated circulating cholesterol levels leading to accelerated atherosclerotic plaque accumulation on a chow diet ( 59,60 ). In contrast to ldlr Ϫ / Ϫ mice, apoe Ϫ / Ϫ mice In contrast, in APOE*3Leiden.CETP mice the single dose sc injection of anti-PCSK9 antibody signifi cantly reduced both cholesterol (up to 69%) and TGs (up to 70%) during 14 days posttreatment ( Fig. 3C, D ) compared with control antibody. This corresponded to a signifi cant increase in hepatic LDLR mRNA and protein expression (supplementary Fig. IX). We next assessed the effect of anti-PCSK9 antibody (10 mg/kg, sc, every 10 days) on atherosclerosis in APOE*3Leiden.CETP mice on a WTD. As compared with a chow diet, the WTD, containing 0.15% cholesterol, increased PCSK9 levels by 51% (from 135.4 ± 14.2 ng/ml to 205.2 ± 41.9 ng/ml, P < 0.05; Fig. 4A ). Treatment with anti-PCSK9 antibody further increased the circulating PCSK9 levels by another 166% (to 545.8 ± 399.7 ng/ml, P < 0.01; Fig. 4A ), demonstrating circulating complexes of antibody bound to PCSK9. During the 14 week treatment, consistent and signifi cant reductions in TC and TG levels were observed as measured 3 and 10 days after the fi rst (week 1) and ninth (week 12) injection ( Fig. 4B, C ). On average, TC was reduced by 67% ( P < 0.001), which was driven by a decrease in nonHDL-C ( Fig. 4D ), and TGs were reduced by 61% ( P < 0.001), as compared with control. After 14 weeks of treatment, atherosclerosis development was reduced by 91% ( P < 0.001) in the mice treated lower circulating lipids in the absence of ApoE, we administered a single dose (10 mg/kg, sc) of either an anti-PCSK9 antibody (CmAb1) or control antibody to either apoe Ϫ / Ϫ or APOE*3Leiden.CETP mice, which express both mouse ApoE and the human mutant APOE3*Leiden, as well as a functional LDLR.
Consistent with our observations utilizing pcsk9 Ϫ / Ϫ mice, a single dose of anti-PCSK9 antibody did not signifi cantly lower circulating cholesterol levels at up to 14 days posttreatment or affect hepatic LDLR protein levels in apoe Ϫ / Ϫ mice ( Fig. 3A , supplementary Fig. VI). This is in contrast to C57Bl/6 mice, where a signifi cant increase in hepatic LDLR was observed following anti-PCSK9 antibody treatment (supplementary Fig. VII), which is consistent with our previous fi ndings for PCSK9 inhibition in WT mice ( 25 ). Circulating TGs were not signifi cantly different at day 3, 10, or 14, but did reach signifi cance at the day 5 time point ( P < 0.05, Fig. 3B ). Additionally, chronic administration of anti-PCSK9 antibody (10 mg/kg, sc, every 10 days) failed to reduce circulating lipid levels or atherosclerosis in apoe Ϫ / Ϫ mice (supplementary Fig. VIII). Together, these data suggest that ApoE is required for cholesterol and TG lowering, and atherosclerosis reduction, by anti-PCSK9 antibody. , and APOE*3Leiden.CETP mice. We demonstrate that circulating cholesterol and atherosclerotic lesions are minimally modifi ed in pcsk9 Ϫ / Ϫ mice on either an ldlr Ϫ / Ϫ or an apoe Ϫ / Ϫ background, strongly suggesting requirement of both proteins for robust atheroprotection mediated by PCSK9 inhibition. It is likely that the minor effects on plasma cholesterol lowering are the major reason for the lack of lesion reduction, as the key role of lipids in driving atherosclerotic lesion development in rodent models has been well-defi ned ( 62 ). We also demonstrate the ability of anti-PCSK9 monoclonal antibody to robustly reduce atherosclerosis in a mouse model with a functional ApoE-LDLR pathway, but with no effect when ApoE is absent. We observed small but signifi cant reductions in serum cholesterol levels after deletion of pcsk9 in ldlr Ϫ / Ϫ mice. These data are in contrast with previous studies showing no effect of pcsk9 deletion or PCSK9 inhibition by mAbs ( 25,47 ). However, these studies used low-cholesterol diets ( р 0.2% w/w cholesterol) in contrast to the current study (1.25% w/w), which might be the reason for the discrepancy in effect. The small but signifi cant reduction in serum cholesterol levels after deletion of pcsk9 in ldlr Ϫ / Ϫ mice might relate to potential effects of PCSK9 in enabling ApoB secretion in nascent VLDL, or perhaps in upregulation with anti-PCSK9 antibody as compared with control ( Fig. 4E-G ). Lesion severity was also reduced, with 8-fold more lesion-free segments in the animals treated with anti-PCSK9 antibody, as compared with control (7.8 ± 9.2% in control and 62.5 ± 31.0% in anti-PCSK9 antibody; P < 0.001), and a strong signifi cant reduction in the percentage of severe lesions (46.2 ± 23.9% in control and 7.8 ± 15.1% in anti-PCSK9 antibody; P < 0.001; Fig. 4H ). All together these data suggest that LDLR and ApoE are required for the atheroprotective effects of PCSK9 inhibition. Moreover we clearly demonstrate that an anti-PCSK9 antibody is highly effi cacious in reducing lipid levels and atherosclerosis development in diet-induced hyperlipidemic APOE*3Leiden.CETP mice (a translational model for dysbetalipoproteinemia), which have an intact ApoE-LDLR clearance pathway.

DISCUSSION
Classic work, such as that by Ishibashi et al. ( 61 ), has set the foundation of understanding of ApoE and LDLR in lipoprotein homeostasis. To study the role of LDLR and ApoE on PCSK9-mediated regulation of plasma cholesterol and atherosclerosis lesion development, we utilized explains the absence of lipid lowering effects. This phenomenon, however, was not previously explained. We hypothesize that in the absence of ApoE there is no uptake and intracellular traffi cking of LDLR (bound to the lipoprotein), and consequently there is no shuttling of the LDLR into the lysosomal degradation pathway. In this situation when the LDLR is not degraded, PCSK9 inhibition, rescuing the LDLR from degradation, is not effective. Supportive data was provided by Mortimer et al. ( 70 ), showing that under normal circumstances, chylomicron remnants are rapidly internalized by the LDLR and catabolized in hepatocytes, with a critical requirement for ApoE. Ishibashi  After deleting or inhibiting PCSK9 in apoe Ϫ / Ϫ mice we anticipated decreases in ApoB-containing lipoproteins (and hence some plasma cholesterol reduction) by the expected increase in liver LDLR expression, even though lipoproteins normally containing ApoE would be unaffected. However, our experiments clearly show that in absence of ApoE, PCSK9 deletion or inhibition does not reduce lipids, whereas in the presence of ApoE, using the APOE*3Leiden.CETP mice, PCSK9 inhibition is effective. Further, we show that in the absence of ApoE, PCSK9 inhibition does not lead to LDLR upregulation, which was also unexpected. Of course the lack of effect on LDLR expression after PCSK9 inhibition in apoe Ϫ / Ϫ mice most likely Fig. 4. Anti-PCSK9 antibody treatment reduces atherosclerosis in APOE*3Leiden.CETP mice. Plasma PCSK9 levels (A) in APOE*3Leiden. CETP mice were determined on chow diet and WTD, as well as two weeks after a single injection with anti-PSCK9 antibody (10 mg/kg, sc) in mice fed WTD. Data represented as the means (bars) ± SD (n = 8 per group). * P < 0.01 versus chow; # P < 0.05 versus WD, one-way ANOVA, Tukey posttest. To assess the effect on atherosclerosis, control or anti-PCSK9 antibody was injected sc every 10 days for 14 weeks in APOE*3Leiden.CETP mice. Plasma TC (B) and TGs (C) were measured at 3 and 10 days postinjection in the fi rst and twelfth week of treatment. Data represented as means (bars) ± SD (n = 15 per group). *** P < 0.001 versus control antibody. D: Fast protein liquid chromatography fractionation of pooled plasma samples are shown from week 8. Atherosclerosis development was determined in the aortic sinus of APOE*3Leiden.CETP mice. E-F: Representative pictures of control antibody-and anti-PCSK9 antibody-treated mice are shown. The total lesion area per cross-section (G) was measured and lesion severity (H) was determined. Data represented as means (bars) ± SD (n = 15 per group). *** P < 0.001 versus control antibody. trials. The lipid lowering effect of statins has been shown to reduce the risk of cardiovascular events and death in several outcome trials (30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)79 ). Determining whether anti-PCSK9 antibody therapies will be effi cacious in reducing the risk of cardiovascular events and death, as suggested by the current study using APOE*3Leiden.CETP mice, will be defi ned in the current outcome trials.
in apoe Ϫ / Ϫ / pcsk9 Ϫ / Ϫ , as compared with their respective apoe Ϫ / Ϫ controls. We did not measure aortic cholesterol ester levels in our studies.
Here we provide further evidence that ApoE is necessary for the atheroprotective effects of PCSK9 inhibition, as treating APOE*3Leiden.CETP mice with anti-PCSK9 antibodies resulted in signifi cant and sustained reductions in TC and TG levels, which translated to reduced atherosclerosis development in the aortic root. While normal WT mice have a very rapid clearance of apoB-containing lipoproteins, APOE*3Leiden mice have an impaired clearance and increased TG levels, and are thereby mimicking the slow clearance observed in humans, particularly in patients with familial dysbetalipoproteinemia ( 45 ). Upon feeding saturated fat and cholesterol, hyperlipidemia and atherosclerosis will develop. These animals also respond in a human-like manner to drugs used in the treatment of CVD (like statins, fi brates, antihypertensives, etc.) ( 49,54,(72)(73)(74). However, APOE*3Leiden mice (like WT mice) do not possess a CETP gene, and therefore these mice do not respond to HDL-modulating interventions. By crossbreeding the APOE*3Leiden mice to mice expressing the human CETP gene ( 75 ), APOE*3Leiden.CETP mice were obtained that respond to both lipid-lowering as well as HDL-raising interventions (50)(51)(52)(53)76 ). In the current study, we found signifi cant lowering effects of anti-PCSK9 antibodies on TC and TG levels in APOE*3Leiden.CETP mice, but HDL-C was not affected (data not shown).
Other than the plasma cholesterol modulating effect, other potential atherosclerosis-related effects of PCSK9 have been described or suggested. Previously, Ferri et al. ( 77 ) reported that PCSK9 is expressed in human vessel walls and produced locally by vessel smooth muscle cells causing a local effect, and it was suggested that PCSK9 could enter the subendothelial space from the circulation either by itself or in association with LDL. In addition, it has been hypothesized that PCSK9 could impact the expression of LDLR on lesion monocytes and macrophages modulating foam cell formation and/or promoting apoptosis ( 78 ). Although our analysis was quite limited, we conclude that PCSK9-mediated local effects at the lesion, refl ected by lesion area and macrophage number, is not signifi cant in the models utilized here. However, we cannot rule out the possibility that certain biochemical changes may have occurred in the lesion due to the absence or inhibition of PCSK9.
The diffi culty in examining the effect of human and humanized biologics in animal models, particularly when chronic dosing is required, is the potential appearance of neutralizing anti-drug antibodies reducing the effi cacy of the therapeutic. In the study described here, there was clear evidence that after 12 weeks of treatment (9 injections) this was the case for approximately 30% of the mice, as demonstrated by a reduced effi cacy in lipid lowering in those mice. Regardless, even with all study animals included in the analysis, the effect on atherosclerotic lesions was highly signifi cant .
The ability of anti-PCSK9 therapies to lower LDL-C in human subjects is evident from numerous late stage clinical