Suppressed monocyte recruitment drives macrophage removal from atherosclerotic plaques of Apoe–/– mice during disease regression (original) (raw)
Reduction in lesional macrophage content is preceded by de-esterification of cholesterol within plaques. Treatment of Apoe–/– mice with viral vectors encoding apoE (ad-hApoE3) causes lesion regression characterized by loss of oil red O (ORO) staining (to identify neutral lipids) and macrophages from plaques (28), but the kinetics by which changes occur in Apoe–/– plaques following apoE complementation have not been reported. Thus, we analyzed the course of changes observed from 2 days to 1, 2, 4, or 6 weeks after treatment of high-fat diet–fed (HFD-fed) Apoe–/– mice with ad-hApoE3, employing empty adenoviral vector (ad-Empty) as a control of plaque progression. Total cholesterol decreased from approximately 1,000 mg/dl to 100 mg/dl within 2 days following infection with ad-hApoE3 vector, and remained low for up to 6 weeks (Figure 1, A and B, and data not shown). Within 2 days, HDL was elevated more than 4-fold and continued to rise, reaching a plateau by day 7 (Figure 1B). Plaque area in the aortic sinus (Figure 1C) and aortic arch (data not shown) increased by 4 weeks in control ad-Empty vector–treated mice, whereas plaque area was stabilized in ad-hApoE3–treated animals, showing no changes in relation to a baseline cohort euthanized at the time when vector treatment began (Figure 1C and data not shown). In a separate experiment wherein Apoe–/– mice were euthanized 6 weeks after vector treatment, ad-hApoE3 vector reduced plaque area and stenosis relative to baseline (Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI43802DS1), consistent with past evidence indicating that over many weeks, regression of plaque area occurs (28).
Kinetics of changes within Apoe–/– plaques following apoE complementation. Apoe–/– mice were fed a HFD for 9 weeks before establishment of a baseline group or starting treatment with ad-hApoE3 or ad-Empty vector. The evolution of changes within plaque after 1, 2, or 4 weeks following adenoviral infection was characterized. (A) Total cholesterol measurements. Dashed line depicts the values obtained from age- and sex-matched C57BL/6 Apoe+/+ mice. (B) The plasma lipoprotein profile as a function of time following apoE complementation. (C) Lesion area was measured on 8-μm sections at 48-μm intervals, starting from the initiation of valves. (D) Macrophage area was quantified by CD68+ area and normalized to the average CD68+ area from the corresponding baseline group lesions. (E) Representative photomicrographs of CD68+ staining in sections of the aortic sinus of each experimental group. Red, CD68; blue, DAPI. Original magnification, ×50. (F) Neutral lipid area within lesions was quantified by ORO staining within each treatment group over time; the dashed line refers to baseline values obtained in Apoe–/– mice after 9 weeks of HFD feeding. (G and H) Total, free, and esterified cholesterol mass analysis at baseline or 14 days after apoE complementation normalized to wet weight of tissue (G) or tissue protein content (H) from 7–8 mice per group. Data represent mean ± SEM, except in A, which depicts mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. Results are compiled from 3 independent experiments with 5–8 animals per group per experiment.
We next quantified macrophage area in plaques as a function of treatment with ad-hApoE3 or ad-Empty vector. CD68+ macrophages disappeared in a linear fashion from plaques over the weeks following treatment with ad-hApoE3 vector (Figure 1D) in contrast to the ad-Empty group, where they increased substantially over time (Figure 1D). However, the reduction in macrophage content only became statistically significant 4 weeks after ad-hApoE3 treatment (Figure 1, D and E), when it was reduced by an average of 72%. The marked loss of macrophages in the ad-hApoE3–treated group was further confirmed by staining for other macrophage markers, F4/80 and MOMA-2 (Supplemental Figure 2, A and B). Accompanying the decrease in macrophage content in plaques of ad-hApoE3–infected mice, we observed increased collagen accumulation, as analyzed with Sirius red staining (Supplemental Figure 2C), and conserved smooth muscle cell content, as revealed by α-actin staining (Supplemental Figure 2D).
To document how changes in plaque cholesterol distribution and mass correlated with the decrease in circulating cholesterol or plaque macrophages observed in ad-hApoE3–treated mice, we first stained plaque sections with ORO and then quantified total, free, and esterified cholesterol using mass spectroscopy (29). ORO+ area did not change in ad-Empty vector–treated mice over time. By contrast, in ad-hApoE3 vector–treated mice, ORO+ area in the plaque was decreased by more than one-half (Figure 1F and Supplemental Figure 3A), and also showed substantial reduction in other tissues, such as the liver, 4 weeks after infection (Supplemental Figure 3B). We verified that the adenovirus infection had no detrimental effect on liver function by assessing alanine transaminase (ALT) activity in plasma at different time points (Supplemental Figure 3C). By 4 weeks after infection, ALT levels approached baseline in all groups (Supplemental Figure 3C). Nevertheless, we noted a transient effect of ad-hApoE3 on day 7 (Supplemental Figure 3C). The specific and prominent increase in ALT on day 7 after ad-hApoE3 infection was paralleled with a peak of large cholesterol-rich HDL particles (big HDL) accumulation in the plasma (Figure 1B), consistent with the possibility that mobilization of cholesterol to the liver peaks around day 7 and transiently affects liver metabolism. This finding is reminiscent of a previous study on virally induced DGAT1 overexpression, where a similar outcome was observed (30).
Given the impressive reduction in ORO+ area within plaques, we set out to quantify the changes in aortic cholesterol mass. Cholesterol ester mass dropped substantially, by one-third (Figure 1G), whereas the clear trend downward in total cholesterol mass did not reach statistical significance (Figure 1G). These data corresponded to an increased ratio of free cholesterol to total cholesterol within the aorta in response to apoE complementation (49% ± 3% to 61% ± 4%, before versus after apoE complementation), indicating that the proportion of cholesterol present in esterified form was decreased (51% ± 3% to 39% ± 4%, before versus after apoE complementation). These results are consistent with our ORO staining analyses (Figure 1F), as ORO staining detects only esterified cholesterol (31). Putting these observations together, we observe that an initial reduction in plasma cholesterol and increase in circulating HDL were rather rapidly followed by reduced cholesterol ester–laden macrophages in plaque. The reduction in cholesterol ester–laden foam cell content occurred prior to, rather than concomitantly with, the loss of plaque macrophages.
Reduction in plaque macrophages does not depend upon CCR7 or migratory egress of cells. We next set out to determine how the decrease in macrophage content within plaques was achieved. Given our past findings in the surgical model of regression, we hypothesized that monocyte-derived cell disappearance would be a consequence of their emigration out of plaque, through mobilization into adventitial lymphatics (24). Accordingly, we anticipated that deficiency in CCR7, a key mediator in leukocyte emigration through lymphatics (32–34) and a proposed modulator of regression in the surgical model (25), would prevent loss of plaque macrophages in response to ad-hApoE3 treatment. We thus crossed Apoe–/– mice with Ccr7–/– mice to generate Apoe–/–Ccr7–/– double-knockout mice and analyzed the evolution of macrophage area within plaques after ad-hApoE3 infection. The absence of CCR7 and ApoE led to increased levels of circulating leukocytes and decreased lymphocyte and dendritic cell accumulation in the lymph nodes similar to the CCR7 single knockout (32, 33) (data not shown). However, after 9 weeks of HFD feeding (14–15 weeks of age), atherosclerotic plaques of Apoe–/–Ccr7–/– mice showed no significant difference in size or in macrophage content compared with Apoe–/–Ccr7+/+ control littermates, although trends toward somewhat smaller lesions in the absence of CCR7 were observed (Figure 2, A and B). In contrast to expectations generated from observations in the surgical model of regression (25), lipid and macrophage loss from plaques of Apoe–/–Ccr7–/– mice was massive after treatment with the ad-hApoE3 vector (Figure 2, C and D), thus illustrating that CCR7 is not necessary for profound loss of lipid or macrophages from plaques.
CCR7-independent removal of Apoe–/– plaque macrophages after apoE complementation. Apoe–/–Ccr7–/– and Apoe–/–Ccr7+/+ (littermate control) mice were fed HFD for 9 weeks. A cohort of animals from each genotype was sacrificed to establish baseline (white and gray bars). A second cohort of Apoe–/–Ccr7–/– was then infected with ad-hApoE3 vector for 4 weeks (black bar) before sacrifice. (A) Plaque area was quantified in aortic sinus sections and (B) en face aortic arch preparation. (C) Lipid area was measured by ORO coloration and (D) macrophage content by CD68+staining. (E) Representative photomicrographs of macrophage (CD68+) area in sections of the aortic sinus at time of sacrifice in each group. Green, CD68; blue, DAPI. Original magnification, ×50. Data represent mean ± SEM from 2 independent experiments; n = 3 mice in Apoe–/–Ccr7+/+ baseline group, n = 5 in Apoe–/–Ccr7–/– baseline group, n = 6 in Apoe–/–Ccr7–/– ad-hApoE3 group; ***P < 0.001.
To more generally address the importance of migratory egress from plaques as a means of removing macrophages during plaque regression, which may occur in a CCR7-independent manner, we employed a modified version of the phagocyte-tracking approach previously developed in our laboratory (9, 35). This technique independently labels about 10%–20% of the two endogenous subsets of monocytes in the circulation and is useful in monitoring and quantifying monocyte subset entry into (9) and egress from plaques (36). Over time, if latex bead+ monocyte-derived cells die or exocytose beads, the beads will not disappear, as they are non-degradable and are too large to cross the endothelial barrier passively, but may instead label other phagocytes within the plaque. In past studies, we have documented that latex bead transfer from phagocyte to phagocyte can occur (37) and that emigration of bead-labeled monocyte-derived cells is not impeded (9, 38, 39). Once peak accumulation of latex bead+ monocytes occurs in plaques, if thereafter some monocyte-derived cells migrate out of lesions, then the total number of bead+ cells in the lesions will decrease. On the other hand, if there is no emigration of monocyte-derived cells out of lesions, the bead frequency within plaques will remain constant.
To verify that this method can track emigration from plaques, we first tested it using the surgical model of regression, wherein migratory egress from mouse plaques was initially described (24). We labeled blood monocytes with beads and then harvested aortic arches from these animals after 3 days. A subset of aortic arches was used to quantify baseline recruitment, while the others were transplanted into wild-type C57BL/6 mice (Apoe+/+) to allow plaque regression. Mice receiving these aortic transplants were sacrificed 5 days later (Figure 3A). We then quantified the number of beads per 6-μm section of the lesser curvature of the aortic arch, comparing the number of beads per section at baseline prior to surgery to the number of beads per section 5 days after surgery. A 63% decline in bead content was observed at the post-transplantation end point (Figure 3B). Thus, in the surgical model of regression, monocyte-derived cell emigration from the intima indeed occurred, and the process appeared quantitatively significant enough to mediate loss of macrophages that accompanies regression. Furthermore, these data serve as a proof of concept that beads within plaques can be removed under conditions of phagocyte mobilization.
Analysis of migratory egress from atherosclerotic plaques in Apoe–/– mice following apoE complementation. (A) Diagram depicts experimental design in which the Ly-6Clo monocytes of mice were labeled with beads in the blood before the mice served as transplant donors in a surgical model of regression. (B) Quantification of the number of beads per cross section of the aortic arch before (baseline) and after plaque regression in WT transplant recipients. Data were pooled from 3 independent experiments; n = 5 in the baseline group, n = 7 in the WT recipient group. Difference from baseline is significant, P < 0.01. (C) Experimental design to study migratory egress from plaques of Apoe–/– mice after apoE complementation. (D) Quantification of bead number in aortic sinus plaques (blue lines) or aortic arch (lesser curvature; red lines) after Ly-6Chi (upper graph; n = 5–7 mice per data point) or Ly-6Clo (lower graph; n = 10–16 mice per data point) monocytes were initially labeled in the blood. (E) Microphotograph of beads (green particles, some indicated by arrows) localized within an aortic arch plaque 4 weeks after labeling in the control group. Non-overlapping ORO (red) and DAPI (blue) staining reveals necrotic core. Data represent mean ± SEM.
To monitor migratory egress from Apoe–/– plaques after apoE complementation, we labeled circulating monocytes in Apoe–/– mice with latex beads and tracked the persistence of this label in plaques as a function of ad-hApoE3 or ad-Empty vector treatment (Figure 3C). We set up two parallel studies wherein we independently labeled the classical (Ly-6Chi) and nonclassical (Ly-6Clo) subsets of monocytes as the populations to carry latex beads into plaques (2, 9). Mice were housed without further manipulation for the next 8 days to permit accumulation of bead+ monocytes in the plaques. Within this period, 73% of bead+ monocytes were cleared from the blood due to recruitment into tissues and normal turnover (Supplemental Figure 4). At day 8, we sacrificed one-third of the mice for baseline analysis of plaque area, lipid and macrophage content, and number of beads per plaque section. Other similarly bead-labeled mice were infected with ad-hApoE3 or ad-Empty vector 8 days after monocyte labeling and sacrificed at 4 weeks after adenoviral infection. Bead number was quantified in the aortic sinus and the lesser curvature for comparison to the number of bead+ cells in analogous sections obtained at baseline.
From the baseline data obtained in aortic sinus, we observed that, in contrast to previous studies in the aortic arch (9), labeled Ly-6Chi monocytes did not outnumber labeled Ly-6Clo monocytes in entering aortic sinus plaque (Figure 3D), where robust recruitment of both monocytes was apparent. To analyze migratory egress from plaques, accumulation of bead+ monocytes at the experimental end point was compared with that in the baseline group. The number of beads per plaque section in Apoe–/– mice treated with ad-Empty for 4 weeks after baseline monocyte entry was the same as observed at baseline. This result was true whether circulating classical Ly-6Chi or nonclassical Ly-6Clo monocytes were initially labeled or whether the aortic sinus or lesser curvature of the aorta was examined (Figure 3D). In agreement with previous findings (24), this result suggests that migratory egress of plaque phagocytes is rare or does not occur in conditions of plaque progression. Unexpectedly, we did not observe any diminution in bead labeling in Apoe–/– mice treated with ad-hApoE3 (Figure 3, D and E), even though (as shown in Figure 1) macrophage loss from plaque was significant under these conditions. This lack of bead removal from plaques was not because the label was sequestered outside of macrophages, as even at late time points, very few beads were present in necrotic core or outside of macrophages (Figure 3E and data not shown). Overall, our analyses failed to support the hypothesis that migratory egress is responsible for the marked removal of macrophages that characterizes ad-hApoE3–treated Apoe–/– mice.
Markedly inhibited infiltration of both monocyte subsets into plaque mediates macrophage loss from plaques. Without experimental support for a role of migratory egress in mediating removal of macrophages from Apoe–/– plaques after apoE complementation, we initiated studies to determine whether monocyte recruitment was altered. Since hypercholesterolemia increases monocytes in the circulation, making more monocytes available for recruitment into plaques (9, 40), we first examined whether ad-hApoE3 or ad-Empty vector treatment influenced monocyte counts or subset frequency in the blood. Blood monocyte subsets were discriminated by flow cytometry, based on their expression of CD115 and Ly-6C (9). Neither ad-hApoE3 nor ad-Empty virus significantly influenced monocyte numbers of either circulating subset (Figure 4A), indicating that the loss of macrophages observed in plaques of ad-hApoE3–treated mice was not downstream of a notable decrease in blood monocyte counts. However, monocytes in the circulation altered their phenotype. Monocytes from ad-hApoE3 vector–treated mice had reduced surface CD11b, known to be elevated upon monocyte activation in general and following postprandial hyperlipidemia (ref. 41 and Figure 4B). Extending evidence that monocyte activation was reduced, surface CD62L, which is expressed only on the Ly-6Chi monocyte subset (42) and which is shed upon activation, was more abundant on the plasma membrane of Ly-6Chi monocytes after apoE complementation (Figure 4C). Lipid loading of monocytes in the plasma may also have been modulated, because the increased side scatter associated with lipid uptake into circulating Ly-6Clo CD11c+ monocytes (43), which preferentially express scavenger receptors and lipid response pathways (44), was reversed by apoE complementation (Figure 4, D and E). Ly-6Clo monocytes also expressed less CD115 (CSF-1 receptor), a growth factor receptor for CSF-1 known to promote atherosclerosis (45), after apoE complementation (Figure 4F).
Analysis of blood monocyte numbers and phenotype following apoE complementation. (A) Total monocytes and monocyte subsets were quantified by flow cytometry in blood of baseline mice or 4 weeks after infection with ad-Empty and ad-hApoE3 (n = 10–34 mice per bar). (B) CD11b and (C) CD62L (Ly-6Chi monocytes only) expression was analyzed with (black bars) or without apoE complementation (white bars) of Apoe–/– mice maintained on a HFD. n = 5–9 mice per bar; *P < 0.01. (D) Flow plot overlays show whole leukocyte fraction in blood (gray) overlaid with profiles of Ly-6Clo monocytes (black) from Apoe–/– mice fed a HFD followed by apoE complementation or no complementation. (E) Summary of relative changes in SSC-A shifts in Ly-6Clo monocytes from Apoe–/– mice on a HFD complemented (black bar) or not (white bars) with apoE-encoding vector. Data are compared with SSC-A of monocytes from Apoe+/+ control mice. (F) Effect of hApoE3 complementation on surface expression of CD115. n = 5 mice per bar; **P < 0.001.
For quantification of monocyte recruitment into plaques by labeling monocyte subsets in vivo using the bead-labeling approach, monocyte subsets were labeled 1, 2, or 4 weeks after adenoviral treatment, and plaques were harvested for quantification from 1 to 5 days later, during the major period of monocyte entry (9). This experimental design was such that the bead-labeling assay could be used to monitor monocyte entry into plaques rather than their persistence, as in the experiments presented in Figure 3. Importantly, we verified that adenoviral infection did not affect the bead-labeling efficiency of blood monocytes and that the same number of monocytes was labeled in each group (data not shown). Quantification of monocyte recruitment into plaques after infection with the control adenoviral ad-Empty vector revealed that it slightly but non-significantly increased monocyte recruitment into plaques 1 week after infection, and this effect did not persist up to 2 weeks following infection (Figure 5A). To compile results from independent experiments, we compared the relative magnitude of monocyte subset entry into plaques between ad-hApoE3–treated mice and ad-Empty vector–treated mice. By 2 weeks after ad-hApoE3 vector treatment and thereafter, we observed a dramatic inhibition of both nonclassical (Figure 5B) and classical monocyte subset entry (Figure 5C) into the plaque. Inhibition of monocyte recruitment into plaques appeared somewhat delayed relative to the kinetics of cholesterol lowering in plasma, since apoE complementation non-significantly reduced monocyte recruitment to plaques at 1 week after infection with ad-hApoE3 vector (Figure 5B).
Monocyte recruitment and apoptotic death during macrophage loss from plaques. (A) Effect of ad-Empty vector infection on Ly-6Clo monocyte recruitment into the plaque using the bead labeling assay. n = 5–7 mice/group. (B) Recruitment of Ly-6Clo monocytes was quantified 1, 2, and 4 weeks (10, 11, and 13 weeks of HFD) after ad-hApoE3 infection and ad-Empty infection. Monocytes were labeled 2–6 days before sacrifice, depending on the experiment. Relative bead number was obtained by dividing the number of beads for each mouse by the number of beads averaged from the corresponding control ad-Empty group. Data represent 4 experiments; n = 5–7 mice per group. (C) Recruitment of Ly-6Chi monocytes was quantified 2 weeks after ad-hApoE3 infection and ad-Empty infection. n = 5–7 mice/group. (D) Quantification of the number of CD68+ TUNEL+ DAPI+ cells per section in the aortic sinus, 2 weeks after ad-Empty or ad-hApoE3 infection (n = 3–7/group). (E) Lesion area of chow-fed Apoe–/– mice, with baseline group analysis conducted at 31 weeks of age and further analysis 2 or 4 weeks after ad-hApoE3 vector infection; n = 5 mice per group. (F) Total plasma cholesterol in 17-week-old Apoe–/– mice fed a HFD, 31-week-old Apoe–/– mice fed a chow diet, and WT mice fed a chow diet (white bars). Additional measurements were made 2 weeks after ah-hApoE3 infection (black bars); n = 5 per group. (G) Fraction of lesion area, from Apoe–/– animals in F, containing CD68+ cells. (H) Relative effect of ad-hApoE3 in altering monocyte recruitment in chow-fed Apoe–/– mice, using same approach as in B for HFD-fed Apoe–/– mice. (I) Correlation between total plasma cholesterol and monocyte recruitment measured after bead labeling of blood monocytes. Correlation is significant; P < 0.001. Data in all panels represent mean ± SEM; *P < 0.01; **P < 0.001.
In order for impaired monocyte recruitment to result in macrophage loss from plaques, there must also be a loss of macrophages due to turnover. We thus assessed whether apoE complementation altered apoptosis of plaque macrophages by quantifying the number of plaque cells that were CD68+, DAPI+, and TUNEL+ per section, 2 weeks after ad-Empty or ad-hApoE3 vector treatment (Figure 5D). The vast majority of the DAPI+ TUNEL+ cells costained for CD68+ (data not shown). Our analysis found a similar rate of macrophage apoptosis in plaques treated with ad-hApoE3 or ad-Empty (Figure 5D), indicating that an enhanced burst of apoptosis did not contribute to macrophage loss within Apoe–/– plaques after treatment of these mice with apoE-complementing virus. Instead, a stable rate of apoptosis coupled with a markedly reduced new supply of incoming monocytes served to reduce macrophage content in plaques over time. It is possible that macrophage content may also be influenced by changes in proliferation, but an analysis of Ki67 staining to assess this question revealed that the vast majority of proliferation in the plaques we examined was observed within α-actin+ smooth muscle cells, whose rate of proliferation nearly doubled in response to apoE complementation (data not shown).
To lend support for the importance of monocyte recruitment in maintaining macrophage content of established plaques, we compared macrophage area in Apoe–/– plaques treated for 3 weeks with biologically active pertussis toxin (PTX), an inhibitor of αi-type G protein–coupled receptors that regulate the responsiveness of most chemoattractant receptors, versus those treated with PTX denatured by boiling. While systemic treatment with PTX no doubt has multiple effects, including induction of leukocytosis, we reasoned that its use would allow us to confirm that blocking monocyte entry into plaques would be associated with a reduction in plaque macrophage accumulation, independently of aggressively lowering cholesterol. Using a model of skin contact sensitization (46), we first verified that a single i.p. injection of PTX (2.5 μg/mouse), but not boiled PTX or PBS, efficiently impaired skin dendritic cell migration to lymph nodes (data not shown). Just prior to the end of a 3-week period of PTX treatment in HFD-fed Apoe–/– mice, circulating monocytes were bead labeled. Treatment with PTX partially inhibited monocyte recruitment into plaque (Supplemental Figure 5A) and decreased CD68+ macrophage content by approximately one-half (Supplemental Figure 5B), with smaller changes in lesion area and ORO content (Supplemental Figure 5, C and D). PTX-treated mice showed a similar percentage of TUNEL+ cells per plaque section (data not shown). These data further support the concept that targeting monocyte recruitment in established advanced plaques is a viable strategy for lowering plaque macrophage burden.
Next, we carried out a series of experiments in Apoe–/– mice fed a standard chow diet to discern whether apoE complementation under conditions of less extreme hypercholesterolemia led to a similar reduction in monocyte recruitment. We studied 31-week-old Apoe–/– mice fed a standard chow diet, as these older-age chow-fed Apoe–/– mice had lesion size similar to that in the HFD-fed younger Apoe–/– mice we studied (Figure 5E), while baseline cholesterol levels were approximately one-half those of the HFD-fed cohort (Figure 5F). The rise in cholesterol following its nadir in HFD-fed Apoe–/– mice (Figure 1A) was less apparent in the chow-fed animals, wherein cholesterol levels still hovered near Apoe+/+ levels 2 weeks after apoE complementation (Figure 5F). Within 4 weeks following apoE complementation in the chow-fed Apoe–/– mice, lesion area was decreased (Figure 5E), in contrast to HFD-fed mice (Figure 1, B and C), consistent with earlier work (28). Macrophage area reductions in chow-fed Apoe–/– mice were significant 2 weeks after apoE complementation (Figure 5G). None of these differences were sufficient to lead to migratory egress from plaques, as assessed using the bead-labeling method (data not shown), but monocyte recruitment to plaques was dramatically reduced (Figure 5H) to an extent similar to that observed in the HFD-fed mice (Figure 5B). Indeed, these studies in chow-fed animals with or without apoE complementation extended the range of plasma cholesterol variations under which we quantified monocyte recruitment. Total plasma cholesterol and the magnitude of monocyte recruitment to plaque were positively correlated (Figure 5I), consistent with the concept that disease reversal characterized by aggressive cholesterol lowering is pivotally regulated by modulation of the recruitment of monocytes to plaque.
Understanding the complete mechanism behind suppression of monocyte recruitment in the context of apoE complementation is beyond the scope of this study. However, we did observe reduced expression of ICAM-1, VCAM-1, and osteopontin, molecules involved in the recruitment of monocytes across the arterial endothelium, in plaque sections 2 weeks after ad-hApoE3 vector treatment (Figure 6). Thus, apoE complementation leads to changes both within plaque (Figure 6) and within circulating monocytes (from Figure 5) that could, especially collectively, strongly suppress monocyte recruitment.
Effect of apoE complementation on the expression of adhesion molecules in atherosclerotic plaque. Immunofluorescent staining of ICAM-1, VCAM-1, and osteopontin (red) in aortic sinus lesions was carried out and quantified. Blue, nuclei. Original magnification, ×100 for ICAM-1 and VCAM-1, and ×50 for osteopontin. Quantifications of red area in a series of sections in 5 animals per stain are shown below each representative micrograph. All reductions in staining intensity of adhesion molecules were statistically significant, with a P value of 0.01 or less.