Inhibition of macrophage proliferation dominates plaque regression in response to cholesterol lowering

Female APOE*3-Leiden.CETP mice were purchased from TNO (Leiden, Netherlands). We used female mice as they are more susceptible to cholesterol-containing diets by having higher plasma cholesterol levels relative to APOE*3-Leiden.CETP males, with established dose-dependent responses to statins [63, 70]. Correspondingly, we purchased female Apoe^−/− mice (B6.129P2-Apoe^tm1Unc) from The Jackson Laboratory (Bar Harbor, ME, USA). 8-week-old mice were fed a high-cholesterol diet (HCD, 1.25% w/w cholesterol, D12108 mod., Ssniff GmBH, Soest, Germany) ad libitum for 12 weeks to accelerate atherogenesis, followed by a low-cholesterol diet (LCD, 0.05% w/w cholesterol, semi-synthetic diet T, TNO, Leiden) for 4 weeks to lower proatherogenic plasma cholesterol to levels that allow for normalization through intervention. Next, mice were randomly assigned to up to three groups: 1. continued LCD (control group), 2. LCD supplemented with 0.01% w/w atorvastatin (Pfizer) corresponding to 10 mg/kg body weight per day (statin group), and 3. diet T without cholesterol (free group) for another 4 weeks. To translate the dosing used in our mouse studies (10 mg/kg/d) to human dosing, the following simplified calculation based on body surface area, as accepted by the FDA, can be used as a guide: mouse dose/12.3 × human body weight [36]. Accordingly, 10 mg/kg/day atorvastatin in mice correspond to 65 mg/day for an 80 kg human.

Female Ldlr^–/– mice (B6.129S7-Ldlr^tm1Her/J) were lethally irradiated (10 Gy) and reconstituted with a 1:1 mixture of bone marrow cells from CD45.1 C57Bl/6 (B6.SJL-Ptprc^a Pepc^b/BoyJ) and CD45.2 Msr1^–/– (B6.Cg-Msr1^tm1Csk/J) or CD45.2 CD36^–/– (B6.129S1-CD36^tm1Mfe/J) mice, as previously described [49]. Mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). Following 6 weeks of reconstitution consuming chow diet, mice were fed HCD (1.25% w/w cholesterol, D12108 mod., Ssniff GmBH, Soest, Germany) for 12 weeks to induce atherosclerosis.

Mice were housed under specific pathogen-free conditions

Twenty-three patients scheduled for elective carotid endarterectomy because of significant carotid artery stenosis gave written informed consent to having their blood and endarterectomy specimens analyzed as approved by the Institutional Review Board of the University Hospital of Freiburg. Blood and tissue samples were collected within 12 h after the last intake of statin drugs if applicable. Detailed patient characteristics are listed in supplemental table 1. Liver tissue biopsies were sampled from three patients during bariatric surgery that had been on oral atorvastatin 40–80 mg/day. Detailed patient characteristics are listed in supplemental table 2.

Four weeks into the high-cholesterol diet, 1-μm large yellow-green fluorescent beads (Fluoresbrite YG plain microspheres, Polysciences Inc., Eppelheim, Germany), diluted in 1:4 sterile PBS, were injected intravenously into APOE*3-Leiden.CETP and Apoe^−/− mice, respectively, for in vivo cell labeling and tracking of myeloid cells. Fluorescent bead accumulation in the plaque was quantified by immunofluorescence histology. 1 mg bromodeoxyuridine (BrdU) (BD Bioscience, San Jose, CA, USA) was injected intravenously 2 h prior to killing to label proliferating macrophages. BrdU incorporation into CD68⁺ macrophages in the plaque was quantified by immunofluorescence histology.

Female UBC-GFP mice (C57BL/6-Tg(UBC-GFP)30Scha/J) were purchased from The Jackson Laboratory (Bar Harbor, ME, USA) as donors for bone marrow cell transplantation. APOE*3Leiden.CETP mice with established atherosclerosis following 11 weeks of HCD were lethally irradiated with 10 Gy (RS-2000 Pro Biological System; Rad Source, Buford, GA, USA) while shielding the torso with a lead casing (Belly Shield, Braintree Scientific Inc, Braintree MA, USA, placed on a ¼-inch-thick lead plate) to protect the thoracoabdominal aorta from radiation. 1 × 10⁷ GFP⁺ bone marrow cells were intravenously transplanted into partially irradiated mice.

Modified LDL uptake by bone marrow-derived Mϕ 2 × 10⁵ bone marrow cells were isolated from CD45.1 C57Bl/6 wild-type (WT), CD45.2 Msr1^–/– and CD45.2 CD36^–/– mice which were used to generate mixed irradiation bone marrow chimeras in Ldlr^–/–, as described above. Bone marrow cells differentiating into bone marrow-derived Mϕ (BMDM) in the presence of 30 ng/ml colony stimulating factor 1 (Peprotech, Rocky Hill, NJ, USA) over 5 days were stimulated with DiI-medium oxidized LDL (10 μg/ml), DiI-acetylated LDL (1 μg/ml) (Kalen Biomedical, Montgomery Village, MD, USA) or PBS for 4 h before being detached from 24-well plates using trypsin/EDTA. Cells were stained with fixable viability dye (Thermofisher Scientific, Waltham, MA, USA) and anti-F4/80 (Biolegend, San Diego, CA, USA) for flow cytometric quantification of percent viable BMDM and mean fluorescence intensity (MFI) of DiI-labeled modified LDL into the respective cells.

Murine aortic roots and arches were embedded in Optimal cutting temperature (OCT) Tissue Tek (Sakura Finetek, Tokyo, Japan) and cut into serial cryostat sections (5 μm) starting at the level of the aortic valve (for root sections). Sections were stained with Oil-red O (Sigma-Aldrich, St. Louis, MO, USA), Masson Trichrome (Sigma-Aldrich, St. Louis, MO, USA), TUNEL (DeadEnd™ Fluorometric TUNEL System, Promega, Mannheim, Germany), anti-CD68 (clone FA-11, BioRad AbD Serotec, Puchheim, Germany), anti-BrdU (GTX128091, GeneTex, Irvine, CA, USA and ab1893, Abcam, Cambridge, UK), anti-GFP (ab290, Abcam, Cambridge, UK). Human carotid endarterectomy specimens were embedded in OCT, frozen, sectioned, permeabilized with 0.1% Triton X-100, and stained with with anti-Ki67 (ab15580, Abcam, Cambridge, UK), anti-CD68 (clone PG-M1, Agilent, Santa Clara, CA, USA), and Hoechst 33342 (Thermofisher Scientific, Waltham, MA, USA). Adjacent slides of the same plaques were stained for Oil-red O (Sigma-Aldrich, St. Louis, MO, USA). Secondary antibodies included rabbit–anti rat biotin conjugated (BA-4001) followed by ImmPACT AMEC Red Substrate (Vector Laboratories, Burlingame, CA, USA), rabbit–anti rat TRITC (PA1-28570, Thermofisher Scientific, Waltham, MA, USA), rabbit–anti rat AF647 (ab169349, Abcam, Cambridge, UK), goat–anti-rabbit (BA-1000) followed by fluorescein avidin DCS (A-2011, Vector Laboratories, Burlingame, CA, USA), donkey-anti-sheep TRITC (ab6897, Abcam, Cambridge, UK), alpaca-anti-rabbit AF488 (ChromoTek, Planegg-Martinsried, Germany), and DAPI Mounting Medium (Carl Roth, Karlsruhe, Germany) according to the manufacturers’ instructions. Images were recorded with the Axioplan 2 and Apotome 2 imaging light-/fluorescence microscope with an AxioCam camera (Carl Zeiss MicroImaging GmbH, Göttingen, Germany) and a confocal Leica TCS SP8 X microscope (Leica Microsystems, Wetzlar, Germany). Images were analyzed with Image Pro Premiere 9.2 (Media Cybernetics, Silver Springs, USA) and Zeiss Zen lite (Carl Zeiss MicroImaging GmbH, Göttingen, Germany).

Murine aortic cells were retrieved through enzymatic digestion with collagenase I, collagenase XI, hyaluronidase, DNAse I and HEPES solution (Sigma-Aldrich, St. Louis, MO, USA) in a thermocycler for 45 min at 750 rpm and 37 °C. Murine blood samples were lysed in RBC lysis buffer (Biolegend, San Diego, CA, USA). Isolated cells from the blood and aorta were counted using a Neubauer chamber (Marienfeld, Lauda-Königshofen, Germany). Cells were stained with specific fluorescent antibodies as indicated (Supplemental Table 3). Ly6C^high monocytes were identified as CD45⁺ CD11b⁺, Lin⁻ (Lin = CD3, CD19, NK1.1, Ly6G), Ly6C^high, CD115⁺, F4/80^low. Macrophages were identified as CD45⁺ CD11b⁺, Lin⁻, Ly6C^low, F4/80^high. Intracellular staining with anti-Ki67 and anti-active Caspase 3, BD Cytoxfix/Cytoperm (#554,722, BD Biosciences, San Diego, CA, USA), BD Perm/Wash (#554,723) and BD Permeabilization Buffer Plus (#561,651) was conducted according to the manufacturer’s instructions. Data were collected on a BD Facs Canto II (BD Bioscience, San Diego, CA, USA) and analyzed with FlowJo (Treestar, Ashland, OR, USA).

RNA was extracted from murine aortas using Qiazol and RNeasy Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. Quantitative TaqMan-PCR was performed using a Bio-Rad CFX96 Touch Real-Time PCR System and TaqMan probes Mm00443258_m1 (Tnfa), Mm01336189_m1 (IL1ß), Mm00446190_m1 (IL6), Mm00439614_m1 (IL10), Mm01178820_m1 (Tgfb1), Mm01320970_m1 (Vcam1), Mm00441242_m1 (Ccl2). Data were statistically analyzed using the 2^−▵Ct method.

Murine plasma cholesterol and triglyceride levels were measured using cholesterol and triglycerides FS 10′ Multi-purpose kits (DiaSys Diagnostic Systems GmbH, Holzheim, Germany) according to the manufacturer’s instructions. Lipoprotein profiling in murine plasma samples was conducted by LipoSEARCH services (Skylight Biotech Inc., Akita, Japan). Serum Amyloid A levels were measured using a SAA Mouse ELISA Kit (Thermo Fisher Scientific, Waltham, MA, USA), and ApoB levels were measured using the Mouse ApoB ELISA Kit (Abcam, Cambridge, UK) according to the manufacturers’ instructions.

Results are presented as mean ± SEM. Differences between two groups were analyzed with unpaired Student’s T test or Mann–Whitney test as indicated in the figure legend. To assess differences between more than two groups, one-way ANOVA with Holm–Sidak’s multiple comparisons testing or Kruskal–Wallis with Dunn’s multiple comparisons testing were applied. p values ≤ 0.05 denote significant changes. Pearson’s correlation coefficient was used to test for correlation.

To study mechanisms of statin-mediated plaque regression, we induced atherosclerosis in APOE*3-Leiden.CETP mice by feeding a 1.25% cholesterol diet (high-cholesterol diet, HCD) over 12 weeks, accelerating plaque formation, followed by 4 weeks of 0.05% cholesterol diet (low-cholesterol diet, LCD) to lower plasma cholesterol levels to about 10 mmol/L, that would allow for lipid normalization by subsequent therapeutic intervention. At 16 weeks, a baseline group was sacrificed and the remaining mice were randomized to continued LCD that allows for moderate plaque progression, LCD supplemented with 0.01% atorvastatin or a diet free of cholesterol. After 4 weeks, both atorvastatin and the cholesterol-free diet had cut plasma cholesterol levels by half, reaching normolipidemic starting levels. At this point, the three study groups were sacrificed for comparative analysis (Fig. 1a, b). Notably, the 10 mg/kg/d atorvastatin dose in APOE*3-Leiden.CETP mice decreased total and non-HDL cholesterol levels by about 50%, similar to the effects observed in humans taking atorvastatin 40–80 mg per day [53] (Fig. 1a, c). Moreover, atorvastatin or dietary cholesterol restriction significantly reduced circulating ApoB levels (Fig. 1d), a quantitative marker of atherogenic lipoprotein particles, in analogy to changes observed in patients treated with oral atorvastatin [27, 58]. Triglyceride levels fluctuated modestly over the course of diet changes and did not differ between the study groups, unlike cholesterol levels (Fig. 1b). Body weights were similar in all groups (Fig. 1e).

Atherosclerotic lesion size was quantified in the aortic root and arch. Adding atorvastatin to LCD, or feeding a diet free of cholesterol, prevented plaque growth and altered plaque composition. While continued LCD increased lipid and macrophage contents and decreased collagen deposition, atorvastatin and cholesterol-free diets reversed these changes. We refer to these compositional changes as phenotypic plaque regression (Fig. 2a; Supplemental Figs. 1 and 2). As reported previously [64], reducing cholesterol levels by atorvastatin or diet suppresses serum amyloid A levels in plasma, indicative of anti-inflammatory effects (Fig. 2b). Specifically, IL-1β and IL-6 expressions are reduced, and TGFβ is increased in atherosclerotic aortas (Fig. 2c).

Quantification of macrophage numbers in enzymatically digested atherosclerotic aortas confirmed a reduction by more than 42% in response to atorvastatin treatment or cholesterol-free diet compared to LCD controls (Supplemental Fig. 3a). Concomitantly, aortic Ly6C^high monocyte counts were reduced significantly in atorvastatin-treated mice, and tended to be lower in mice fed a diet free of cholesterol with aortic VCAM-1 and CCL2 expression levels being suppressed (Supplemental Fig. 3a–c). Since the numbers of circulating blood monocytes were similar in all groups we conclude that cholesterol lowering by atorvastatin or dietary restrictions impairs both monocyte infiltration and macrophage accumulation in atherosclerotic lesions (Supplemental Fig. 3a, b and Supplemental Fig. 7a).

Next, we asked whether these two processes were causally linked. To this end, we complemented the feeding and treatment scheme in mice by lethal irradiation before the end of the HCD feeding while shielding the torso to protect the thoracoabdominal aorta from irradiation (Fig. 3a, b). By doing so, Mϕ that had already accumulated in atherosclerotic plaques following 11 weeks of HCD feeding would not be affected by irradiation, preserving natural turnover kinetics. We then transplanted green fluorescent protein (GFP)⁺ bone marrow cells, which seeded in the irradiated, non-protected areas of the body, giving rise to GFP⁺ monocytes and their progeny as a tool to quantify recruitment-dependent and recruitment-independent contributions to Mϕ accumulation in established plaques. One week later, the diet was switched as before to LCD for 4 weeks (Fig. 3a). Meanwhile, a stable GFP cell chimerism of 17.3 ± 2.3% established in circulating Ly6C^high monocytes (Fig. 3d,e; Supplemental Fig. 4c). The baseline reference group was killed at week 16 and studied, while the remaining mice were randomized to continued LCD, LCD + 0.01% atorvastatin, or cholesterol-free diet. After 4 weeks of treatment, these three study groups were killed at week 20, and blood and aortas were analyzed with regard to lesion development and GFP cell chimerism. As in the original model (Fig. 1a), both atorvastatin treatment and cholesterol-free diet reduced plasma cholesterol levels by about 50% compared to continued LCD (control) in chimeric mice, while body weights and blood Ly6C^high monocyte numbers did not differ between the groups (Fig. 3a; Supplemental Fig. 4a, b). Likewise, histological evaluation of atherosclerotic lesions in the aortic roots and arches replicated the previously observed regression phenotype in irradiated, bone marrow transplanted mice, treated with atorvastatin or fed a diet free of cholesterol (Fig. 3c; Supplemental Figs. 4 and 5). The GFP cell chimerism in Ly6C^high monocytes was similar in the blood and aortic tissue of all four groups, indicating that circulating GFP⁺ monocytes infiltrated the atherosclerotic aortas, and that monocyte chimerism remained at equilibrium during the 4-week treatment period (Fig. 3d, e). A GFP chimerism of 15% in aortic Ly6C^high monocytes means that for every GFP⁺ monocyte recruited to the aorta, 6 to 7 GFP^– endogenous monocytes infiltrated as well (100%/15% = 6.7). If all macrophages in the plaque renewed through replacement by infiltrating monocytes during the 4 weeks of plaque progression (weeks 16 to 20), GFP chimerism among monocytes should translate 1:1 into GFP chimerism among macrophages. However, this was not the case. GFP chimerism in aortic macrophages was only 1/10 of the chimerism in aortic Ly6C^high monocytes at baseline (week 16) (Fig. 3d), and although it increased significantly, it did so only by an absolute 1.7 ± 0.6% following 4 weeks of plaque progression with continued LCD (week 20) (Fig. 3e). To estimate the relative contribution of all recruited monocytes to the aortic macrophage pool during plaque progression, accounting for both GFP⁺ and GFP^– Ly6C^high monocytes that infiltrated, the 1.7% absolute increase in GFP⁺ macrophages over 4 weeks of LCD needs to be multiplied by factor 6.7, resulting in about 11% (Fig. 3f). Although the increase in recruited monocyte-derived macrophages was blunted by atorvastatin treatment in our model of established atherosclerosis, a reduction in macrophage numbers exceeding 11%, such as the > 42% reduction observed with atorvastatin treatment compared to LCD controls (Supplemental Fig. 3a), will mainly depend on local processes of macrophage accumulation that are distinct from and additional to monocyte recruitment and differentiation. Low frequencies of recruited GFP⁺ CD68⁺ macrophages within established atherosclerotic lesions, and the decline in overall macrophage numbers in response to cholesterol lowering, were independently confirmed by histology (Fig. 3g; Supplemental Figs. 4d and 5).

We previously reported that local macrophage proliferation plays a dominant role during plaque progression [49], but its relevance for plaque regression has remained uncertain. Other local processes of cell turnover, such as cell death and egress, were described to influence macrophage content in atherosclerotic lesions [32]. Given that monocyte influx contributes minimally to plaque macrophage kinetics in our model of established atherosclerosis with plaque regression, we decided to compare the frequencies of macrophage proliferation, death and egress in the four study groups. Bromodeoxyuridine (BrdU), a thymidine analog that readily integrates into newly synthesized DNA during the cell cycle, was injected 2 h prior to killing. During this short time period, newly generated monocytes in the bone marrow will not have yet entered blood circulation, and they will, therefore, not contribute to the BrdU⁺ fraction of macrophages in the plaque. Under these conditions, BrdU incorporation into plaque macrophages indicates local proliferation [49]. The proportion of proliferating macrophages was quantified based on immunofluorescent co-staining of anti-BrdU with DAPI and anti-CD68 in aortic root and arch lesions. Macrophage proliferation was reduced to nearly half of time-matched control levels when adding atorvastatin to LCD or switching to cholesterol-free diet (Fig. 4a; Supplemental Figs. 2, 4d, 5, 6a). These data were independently confirmed by flow cytometric intracellular staining for Ki67 in aortic macrophages (Fig. 4b; Supplemental Fig. 7b). Macrophage death was quantified based on TUNEL co-staining with DAPI and anti-CD68 in aortic root and arch lesions, and flow cytometric intracellular staining for active caspase 3 (Fig. 4b, c; Supplemental Figs. 2 and 7b). We observed no difference between the groups in either test. Potential macrophage egress was determined using fluorescent bead labeling, as previously described [16, 29, 55, 67]. Fluorescent beads were injected early during atherogenesis after 4 weeks of HCD feeding (Supplemental Fig.6d). Within 7 days, these beads were cleared from the blood stream by circulating monocytes and neutrophils (Supplemental Fig. 6e) that infiltrate the nascent plaques. Since monocytes and neutrophils are short lived, beads that do not immediately leave the plaque within the cells that brought them in remain inside monocyte-derived macrophages. Alternatively, beads are disposed and trapped in the plaque when bead-laden cells die, until being taken up by other phagocytes. Only as bead-laden phagocytes emigrate from the plaque, the number of beads inside the plaque can decrease over time. Therefore, the total number of beads that are retained in the plaques is inversely proportional to the number of macrophages that might exit the plaque. We did not observe a significant decline in plaque bead content over the 4-week time period of plaque regression (Fig. 4d; Supplemental Figs. 2 and 6c), indicating that macrophage egress is unlikely to be a major contributor to the loss of macrophages with oral atorvastatin treatment. Taken together, our data show that suppression of local macrophage proliferation in the presence of unchanged cell death mediates atorvastatin induced plaque regression.

We wondered whether the observed effects of oral atorvastatin treatment were primarily mediated by cholesterol lowering or by so-called pleiotropic effects. Phenotypically, in the two APOE*3-Leiden.CETP mouse models, with and without partial body irradiation, both drug-induced and diet-mediated normalization of cholesterol levels resulted in similar plaque regression (Figs. 1a, 2a 3a–c Supplemental Figs. 2, 4d, 5). Mechanistically, both interventions suppressed local macrophage proliferation (Fig. 4a and Supplemental Figs. 2, 4d, 5 and 6a), suggesting that the cholesterol lowering effects matter. In addition, we quantified atorvastatin drug levels by mass spectrometry in livers, plasma and atherosclerotic aortas of APOE*3Leiden.CETP mice fed LCD + 0.01% atorvastatin. We detected high levels of atorvastatin in livers (59 ng/g) and lower levels in plasma (1.9 ng/ml), but virtually none in aortic tissues, arguing against atorvastatin affecting plaque macrophages directly when administered orally (Supplemental Fig. 8a, b).

Given that oral atorvastatin reduces the number of circulating ApoB-lipoprotein particles and their cholesterol contents (Fig. 1a, c, d), we asked instead, whether the uptake of cholesterol-rich modified LDL into plaque macrophages will influence proliferation, directly. To this end, we generated mixed-bone marrow chimeras in irradiated Ldlr^–/– mice reconstituted with a mixture of macrophage scavenger receptor 1 (Msr1) deficient and competent bone marrow (Fig. 5a). Msr1^+/+ bone marrow cells were isolated from CD45.1 C57Bl/6 mice to distinguish wild-type from knockout cells in the reconstituted recipients according to exclusive CD45.1 or CD45.2 expression. In analogy, we generated mixed Ldlr–/– bone marrow chimeras with CD45.1 CD36^+/+ and CD45.2 CD36^–/– cells (Fig. 5b). Msr1 preferentially mediates the uptake of LDL modified by acetylation into macrophages, whereas oxidized LDL is preferentially engulfed via CD36 (Supplemental Fig. 9a). Following 3 months of HCD feeding, atherosclerosis developed (Supplemental Fig. 9b, c), and blood and aortas were analyzed by flow cytometry for CD45.1/CD45.2 chimerism and differences in the expression of proliferation marker Ki67 in CD45.1 Msr1^+/+ versus CD45.2 Msr1^–/– macrophages, and in CD45.1 CD36^+/+ versus CD45.2 CD36^–/– macrophages, respectively. Proliferation of Msr1- or CD36-deficient macrophages was reduced by about 1/3 compared to wild-type (WT) macrophages in the same plaque exposed to the same lipids and external stimuli (Fig. 5a, b). The proliferative advantage of WT macrophages over knockout macrophages may have contributed to the shift in chimerism towards CD45.1 WT macrophages observed in atherosclerotic aortas.

Finally, we treated Apoe^–/– mice with established atherosclerosis in analogy to the feeding study in APOE*3-Leiden.CETP mice comparing LCD with and without supplementation of 0.01% atorvastatin (Fig. 6a). As previously reported [6, 34, 35, 39], oral atorvastatin at this dose range did not reduce systemic cholesterol levels, weight gain or monocytosis in Apoe^–/– mice (Fig. 6a; Supplemental Fig. 8c, d). Consequently, plaques continued to grow instead of regressing under atorvastatin treatment (Fig. 6b–d; Supplemental Fig. 8e). In the absence of a cholesterol lowering effect in Apoe^–/– mice, atorvastatin did not alter macrophage accumulation, proliferation, death and retention in atherosclerotic lesions (Fig. 6e, f; Supplemental Fig. 8f, g). Taken together, these data suggest that atorvastatin inhibits plaque macrophage proliferation indirectly via lowering of cholesterol and ApoB-lipoprotein levels.

Next, we asked whether the link between cholesterol levels and macrophage proliferation in atherosclerotic lesions also applied to human plaques. To this end, we collected blood and plaque tissue samples from 23 patients undergoing carotid endarterectomy (Supplemental Table 1). All but three patients were taking statins in line with guideline recommendations. Still, serum LDL-cholesterol levels ranged from 32 mg/dl to 161 mg/dl. Of note, atorvastatin plasma levels in patients corresponded to those measured in our APOE*3-Leiden.CETP mouse model, and, unlike the liver, atorvastatin was hardly detectable in human plaque tissues (Supplemental Fig. 8b, Supplemental Table 2). Macrophage proliferation was assessed by co-staining DAPI, anti-CD68 and anti-Ki67 (Fig. 7b). In adjacent slides, plaque lipid content was determined based on Oil-red O staining (Fig. 7a). Macrophage proliferation correlated positively with both serum LDL-cholesterol levels (r = 0.69) and plaque lipid contents (r = 0.63) (Fig. 7c).