EFFECT OF NEBIVOLOL TREATMENT ON ATHEROSCLEROTIC PLAQUE COMPONENTS IN APOE-KNOCKOUT MICE

Apolipoprotein E (ApoE)-knockout mice spontaneously develop atherosclerosis on a chow diet and display extensive atherosclerotic lesions in major arteries (1). This effect is much more pronounced in female ApoE-/- mice which develop markedly increased aortic lesions compared to males (2). Therefore, they are a useful animal model to study pathogenesis of atherosclerosis and to investigate the influence of potential anti-atherogenic drugs (3). The specific inflammatory process leading to the development of the atherosclerotic plaque involves multiple cell types, including monocyte-derived macrophages, vascular smooth muscle cells (VSMCs), T-lymphocytes, and endothelial cells, as well as profound remodeling of the extracellular matrix. The examination of the atherosclerotic lesion components provides the insight into mechanisms influencing plaque formation, development and stability.

Recent studies have demonstrated that nebivolol, a third generation beta1-selective blocker reduces the size of atherosclerotic lesions in cholesterol-fed rabbits and in apoE-deficient mice (4-6). The mechanisms of its antiatherogenic action are not clear and may include increase in the vascular nitric oxide release (7), improvement of endothelial function (4) and suppression of the infiltration and inflammatory activation of cells in the plaque (8-10). There is, however, very limited information concerning the effect of nebivolol on the composition of the atherosclerotic plaque in situ: the observations reported by other authors include only a decreased number of macrophages and smooth muscle cells (5, 9).

The aim of the present study was to broaden the research on the antiatherogenic effect of nebivolol in the apoE-deficient mouse model by analyzing its influence on the individual components of the atherosclerotic plaque: macrophages, smooth muscle cells,

T-lymphocytes, type I collagen and matrix metalloproteinases.

MATERIALS AND METHODS

Animals and treatment

Female 2-month-old apoE-knockout mice (n=12), background strain (C57BL/6J), were purchased from Taconic (Ejby, Denmark). The animals were fed regular chow diet (Ssniff, Soest, Germany) and given water ad libitum. They were kept under LD 12:12 h regime in air-conditioned rooms (22.5 ±0.5°C, 50±5% humidity). At the beginning of the experiment, one group of mice (n=6) was further fed normal chow diet and served as control, while the other group (n=6) was fed the same diet containing racemic mixture of D- and L-nebivolol (Janssen Pharmaceutica, Geel, Belgium) at a dose of 2.0 µmol per kg b.w. per day. This treatment was continued for 4 months.

The study was approved by the Jagiellonian University Ethical Committee on Animal Experiments and conforms to the guidelines for the care and use of laboratory animals published by the US National Institutes of Health.

Specimen preparation

Mice were injected with 1000 IU low molecular weight heparin (Fraxiparine, Sanofi-Synthelabo, France) to inhibit blood coagulation and they were sacrificed by carbon dioxide inhalation. The right atrium of the heart was incised and the heart was perfused with PBS through the apex of the left ventricle at a constant pressure of 100 mm Hg. Next, the heart with ascending aorta were dissected, embedded in OCT compound (CellPath, UK) and snap-frozen. Ten µm-thick serial cryosections were cut at the level of the aortic root using a standardized protocol (5), collected on poly-L-lysine coated slides and air dried. The sections were fixed with acetone (for immunohistochemistry), with 4% buffered formaldehyde (for other stainings) or left unfixed (for zymography).

Staining and microscopic examination

The sections were stained with Masson’s trichrome for general morphology, as well as with Sirius red for collagen. Individual components of the atherosclerotic plaque were visualized by immunostaining for collagen type 1, smooth muscle cells (α-smooth muscle actin, SMA), macrophages (CD68) and lymphocytes (CD3). In short, the sections were preincubated for 40 min in PBS containing 5% normal goat serum (Vector, Burlingame, CA), 0.01% sodium azide, 0.05% thimerosal, 0.1% bovine serum albumin, 0.5% Triton X-100, and 2% dry milk (11). They were next incubated overnight at room temperature with primary antibodies and after a rinse in PBS incubated for 30 min with the secondary antibodies. The used antibodies and their dilutions are listed in Table 1. Finally, sections were mounted in glycerol/PBS. To detect the gelatinase activity of matrix metalloproteinases (mainly MMP-2 and MMP-9), in situ gelatin zymography was employed: unfixed cryosections were incubated for 2 hours at 37°C in a dark humid chamber with 50 mg/ml FITC-labeled DQ-gelatin (Invitrogen, Eugene, OR). After a rinse in PBS, sections were fixed in 4% buffered formaldehyde and mounted in glycerol/PBS.

Table 1. Antibodies used in the study.

The sections were examined under Olympus BX50 brightfield/epifluorescence microscope (Olympus, Tokyo, Japan). Images of the aortic roots were recorded using Olympus DP71 digital CCD camera, stored as TIFF files and processed using LSM Image Browser 3 Software (Zeiss, Jena, Germany). Sections stained with Masson’s trichrome were used for lesion area and necrotic core measurements. The necrotic regions of the plaque were identified on the basis of their acellularity and the presence of crystalline clefts between collagen fibers (12, 13). In sections stained with Sirius red, total collagen content was assessed. The total plaque areas, necrotic core areas as well areas immunoreactive for macrophages, smooth-muscle cells, collagen type 1, and fluorescent areas showing MMP activity were measured semiautomatically in each image (the borders of the areas were hand-traced, the observer did not know whether the specimen comes from the control or treated mouse). Density of CD3-immunoreactive T lymphocytes was assessed using LSM Image Browser 3 software. At least eight consecutive sections encompassing the whole area occupied by the atherosclerotic plaque per animal per staining were used for the analysis.

Statistical analysis

The obtained results were analyzed using the Mann-Whitney U-test for nonparametric data. P<0.05 was regarded as statistically significant.

RESULTS

Histology of the plaque

Preliminary comparison of the cross-sectioned and Masson’s trichrome-stained aortic roots of the control and nebivolol-treated mice revealed in the control mice a thick vascular wall with atherosclerotic plaques containing prominent lipid/necrotic core areas characterized by the presence of collagen fibers interspersed with pale clefts corresponding to lipids (mostly cholesterol crystals). In nebivolol-treated mice, the total thickness of the vascular wall as well as the thickness of the plaques and their cores were reduced. The plaque caps separating the lipid cores from the lumen had similar thickness in the control and nebivolol-treated animals. In both cases the plaques occupied almost the entire circumference of the vessel (Fig. 1).

Fig. 1. Atherosclerotic lesions (A, B) and lesion area measurements (C) in the aortic root of apoE-knockout mice. Representative micrographs of Masson’s trichrome stained atherosclerotic plaques in control (A, top panel) and nebivolol-treated mouse (B, top panel). Atherosclerotic lesion area in control and nebivolol-treated mice (C, top panel). Close-up view of the atherosclerotic plaques (lower panels) in control (A, bottom panel) and nebivolol-treated mouse (B, bottom panel). Asterisks show necrotic regions. Necrotic core area in plaque in the control and drug-treated mice (C, bottom panel). A, B: original magnification – top panel ×40, bottom panel ×200. C: NTG – nebivolol-treated group. Mean ± S.E.M.; n=6 per group; *p<0.05.

Areas rich in CD68-positive macrophages were found in the lipid cores and in the shoulders of the atherosclerotic lesions. In the core, diffuse extracellular CD68 immunostaining indicative of macrophage debris was also observed. Immunoreactivity for SMA showed accumulation of smooth muscle cells in the fibrous cap of the plaque and in the aortic media. CD3-positive T lymphocytes were located in the outer region of the plaque and in the adventitia (Fig. 2). Collagen content and MMP activity were observed in the whole thickness of the neointima (Fig. 3). The location of the studied cells and ECM components was similar in both, control and nebivolol-treated mice.

Fig. 2. Cellular composition of the atherosclerosis lesions in the aortic root of control (A) and nebivolol-treated apoE-knockout mice (B). Immunohistochemical visualization (A, B) and quantitative analysis (C) of CD68-positive macrophages (top panel), SMA-positive smooth muscle cells (middle panel), and CD3-positive T lymphocytes (lower panels). A, B: L – lumen, M – media, arrows – fibromuscular cap; original magnification ×200. C: NTG – nebivolol-treated group. Mean ± S.E.M.; n=6 per group; *p<0.05.

Quantitative analysis of plaque components

The lesion area was significantly (by 25%) lower in nebivolol-treated mice as compared to untreated control animals (70173 ±9550 µm2 versus 93767 ±5651 µm2; p=0.03). Even more marked difference was observed in case of necrotic areas which were by 46% lower in the treated mice (13552 ±1088 µm2 versus 25289 ±940 µm2; p=0.03) (Fig. 1). In the control group, necrotic areas constituted 27% of the lesion, while in the nebivolol-treated group they were reduced to 19%.

The nebivolol treatment significantly (by 41%) decreased macrophage-occupied areas in the lesion (12981 ±1153 µm2 versus 22068 ±1777 µm2; p=0.008) (Fig. 2, CD68) and increased (by 46%) the content of smooth muscle cells in the fibromuscular cap (28380 ±2663 µm2 versus 15232 ±1159 µm2; p=0.008) (Fig. 2, α-SMA). The number of T lymphocytes in the plaques of nebivolol-treated mice was slightly (by 16%) but still significantly lower (17.2 versus 14.5 per mm2; p=0.03) (Fig. 2, CD3).

In the atheromatic plaques of nebivolol-treated mice, sirius red staining revealed a marked (by 49%) reduction of the total collagen content (22513 ±1979 µm2 versus 43995 ±4616 µm2 in the control; p=0.008). Likewise, immunostaining for type I collagen showed a 55% decrease in its content (18388 ±1091 µm2 versus 41024 ±4066 µm2; p=0.008) (Fig. 3). In the control and nebivolol-treated groups, type I collagen comprised 83% and 93% of total collagen content, respectively.

Fig. 3. Total collagen (top panel), type I collagen (middle panel) and MMP (gelatinase) activity (bottom panel) in atherosclerotic lesions of control (A) and nebivolol-treated apoE-knockout mice (B), as well as their quantitative measurements (C). A, B: Original magnification ×40. C: NTG – nebivolol-treated group. Mean ± S.E.M.; n=6 per group; *p<0.05.

In situ zymography showed that gelatinase activity area was by 48% lower in nebivolol-treated mice as compared with the control animals (25247 ±1776 µm2 versus 48458 ±3625 µm2; p=0.008) (Fig. 3).

DISCUSSION

Results of the present study demonstrate an inhibitory effect of nebivolol on several components of the atherosclerotic plaque which contribute to its progression. As compared to the control mice, the nebivolol-treated animals showed, along with significantly lower plaque size, a decrease in necrotic core size, collagen content, macrophage and T cell density, and activity of matrix metalloproteinases. In contrast, the drug increased the content of smooth muscle cells in the fibrous cap of the plaque. The differences showed statistical significance even though the mice groups were relatively small (n=6). However, according to Coleman et al. (14), in the histopathological studies of atherosclerotic lesions in apoE-deficient mice, a minimum of 5 animals per each control and experimental group provides sufficient data for statistical significance.

We found that nebivolol reduced atherosclerotic plaque size by about 25%. Comparable effects of nebivolol on plaque formation were demonstrated in apoE-knockout mice in other reports (4, 6).

The necrotic core is an area containing free cholesterol and apoptotic macrophage remnants. Its size directly correlates with the risk of plaque rupture (15). Formation of the necrotic core is driven by a combination of cholesterol-loaded macrophage apoptosis coupled with defective elimination of cell debris in advanced lesions (16). The decrease in macrophage density induced by nebivolol can therefore limit the size of the necrotic core and indeed in our study these two parameters were reduced in parallel (by 41% and 46%, respectively). A similar correspondence was found in apoE- and 11β-hydroxysteroid dehydrogenase type 1-deficient mice, in which the necrotic core was decreased by 30% and macrophage-specific CD68 immunostaining was reduced by 32% (13). CD68 is a marker of activated macrophages, mostly of the M1 type, that stimulate the inflammation by releasing proinflammatory cytokines and contribute to plaque vulnerability. Such macrophages are predominant in the symptomatic plaques (17). Hence, other effects of nebivolol-induced reduction of macrophage infiltration in the plaque observed in the present study can be a suppression of the inflammatory process and an increase in plaque stability.

The fibrous (fibromuscular) cap covering the plaque is formed relatively late in the course of the atherosclerotic lesion development. It is composed mostly of collagen fibers and smooth muscle cells (SMCs). The thickness of the cap and its composition is another important factor influencing plaque stability: plaques with thin caps, few smooth muscle cells and numerous macrophages are mostly prone to rupture (18). Our results indicate that in the apoE-deficient mouse model nebivolol enhances SMC content in the fibromuscular cap, increasing plaque stability. However, in other studies nebivolol was found to inhibit the proliferation of SMCs and to suppress neointima formation in human coronary arteries (19) and in rat carotid arteries (9). It seems possible that nebivolol induces proliferation of SMCs only in advanced atherosclerotic lesions, during the development of the fibromuscular cap. A reduction of plaque size accompanied by an increase in plaque smooth muscle cell content was also observed in apoE-deficient mice treated with angiotensin-(1-7) receptor agonist which shares some antiatherogenic mechanisms (e.g. increased endothelial NO production) with nebivolol (20).

The presence of T lymphocytes in the plaque reflects the involvement of the immune mechanisms modulating the inflammatory process driving the atherosclerosis. Depending on the subset, T cells can either promote or inhibit plaque development (21). In apoE-deficient mice, cytotoxic T cells were demonstrated to promote the formation of vulnerable plaque (22). Increased number of CD3+ cells was detected in complicated human carotid artery plaques as compared with uncomplicated ones (23). Our results show a relatively weak but still significant decrease in the number of CD3+ lymphocytes in nebivolol-treated mice, suggesting some suppression of the proinflammatory response of the immune system.

The development of the plaque is associated with an increase in collagen content, with type I collagen accounting for 70% of the total collagen (24, 25). A decrease in plaque collagen fibrillogenesis observed in the nebivolol-treated mice is another evidence of atherosclerosis-suppressing effect of that drug.

In advanced plaque, metalloproteinases (MMPs) initiate and execute proteolytic degradation of the fibrillar collagen types I and III, deposited by SMCs during plaque development. Enhancement of MMP-2 and MMP-9 activity in the extracellular matrix increases fragility of the plaque and the risk of its instability (26). Macrophages are the main source of MMPs in the plaque. The number of MMP-1, -2, -9-positive macrophages rises in parallel with plaque progression (27, 28). The release and extracellular activity of MMPs can affect stability of the plaque in several ways: it contributes to the formation of acellular necrotic core and can destroy the fibromuscular cap (29). In apoE-deficient mice, lesional over-expression of MMP-9 was found to increase plaque vulnerability and induce intra-plaque haemorrhage in advanced plaques (30), whilst doxycycline, a MMP inhibitor, ameliorated plaque development (31). The present study shows that inhibition of atherogenesis by nebivolol in apoE-knockout mice is associated with a decrease in MMP expression and thereby contributes to plaque stabilization. This effect can result not only from suppressed infiltration and activation of MMP-releasing macrophages, but also from a possible inhibitory influence of nebivolol on MMP gene expression, as demonstrated in human umbilical vein endothelial cells (32).

In conclusion, the present study has demonstrated that in the apoE-knockout mice nebivolol suppresses atherosclerotic plaque progression and enhances its stability by affecting several processes: macrophage and T cell infiltration, proliferation of smooth muscle cells in the fibromuscular cap, collagen fibrillogenesis and metalloproteinase activity. The mechanism(s) of antiatherogenic action of this drug is still unclear and it is difficult to conclude whether the observed effects are primary or secondary, but its beneficial properties demonstrated in the apoE-deficient mouse model justify further clinical studies on the possible application of nebivolol in the prevention and treatment of atherosclerosis.

Acknowledgements: This study was supported by statutory grant No K/ZDS/002851 from the Jagiellonian University Medical College to Grazyna Pyka-Fosciak and by the grant No 2012/05/B/NZ4/02743 from the Polish National Center of Science (NCN) to Jacek Jawien.

Conflict of interests: None declared.

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