Granulocyte-Macrophage Colony–Stimulating Factor Prevents the Progression of Atherosclerosis via Changes in the Cellular and Extracellular Composition of Atherosclerotic Lesions in Watanabe Heritable Hyperlipidemic Rabbits
Background—A cytokine network is involved in atherogenesis. This study was conducted to investigate the effects of granulocyte-macrophage colony–stimulating factor (GM-CSF) on the development and composition of atherosclerotic lesions in Watanabe heritable hyperlipidemic (WHHL) rabbits.
Methods and Results—GM-CSF (10 μg · kg−1 · d−1) was administered to 4-month-old WHHL rabbits (n=9) 5 days a week for 7.5 months, whereas an equal dose of human serum albumin was administered to controls (n=9). The cholesterol levels were not changed significantly by the treatment. Age-matched 4-month-old rabbits (n=7) had atheromatous plaques over 30.7±5.7% of the inner surface area of the aortic arch. After treatment, the percentages of surface atheromatous plaques to total aortic arch area were 45.0±12.6% in the GM-CSF group and 74.3±11.0% in controls (P<0.0001). Histological examination demonstrated that GM-CSF reduced the ratio of intima to media (P<0.01) and cross-sectional areas of atherosclerotic lesions (P<0.0001). Quantitative analysis indicated a marked decrease in the areas of smooth muscle cells (P=0.0001), collagen (P=0.0001), and extracellular lipid deposits (P<0.05) of atheromatous plaques in GM-CSF–treated rabbits compared with controls. The terminal deoxynucleotidyltransferase–mediated dUTP-digoxigenin nick end-labeling (TUNEL) method and immunohistochemistry were performed to examine the relationship between decreased atherosclerotic lesions and apoptosis. The percentage of TUNEL-positive cells increased in the GM-CSF group (GM-CSF, 24.1±4.4% versus control, 11.6±3.2%; P<0.0001). GM-CSF enhanced the apoptosis of smooth muscle cells in the shoulder region and the fibrous cap (P<0.0001), suggesting one of the mechanisms for the antiatherogenic effect.
Conclusions—GM-CSF altered the composition of atherosclerotic lesions and reduced the atherosclerosis in WHHL rabbits.
Macrophages and T lymphocytes possess the interleukin-2 receptor and major histocompatibility complex class II antigen in the early stage of atherosclerotic lesions, indicating that they are activated and can produce various cytokines.1 2 3 Stimulated endothelial cells and smooth muscle cells also are capable of producing a variety of cytokines and growth factors in the vascular wall.3 4 A large number of recent studies suggest that a cytokine network in the arterial wall is a critical process in atherogenesis.3 4
In 1988, Nimer et al5 reported a cholesterol-lowering effect of granulocyte-macrophage colony–stimulating factor (GM-CSF) in patients with aplastic anemia, suggesting that GM-CSF may play a role in lipid metabolism. We have shown that GM-CSF lowers cholesterol concentrations in normal and hypercholesterolemic rabbits and that the activity persists even after the termination of GM-CSF treatment.6 We found that this cholesterol-lowering effect was mediated in part through both the upregulation of VLDL receptor mRNA levels and the enhancement of macrophage functions.6 In addition, a variety of cells release GM-CSF, and it is also produced by activated cells in the arterial wall.7 8 However, the physiological role of GM-CSF in atherogenesis remains unclear.
In the present study, we investigated the effects of GM-CSF on the development of atherosclerosis and the composition of atheromatous plaques in Watanabe heritable hyperlipidemic (WHHL) rabbits with a dose of GM-CSF that did not significantly induce an overall reduction in plasma cholesterol levels. The cellular and extracellular compositions of atherosclerotic lesions were immunohistochemically and conventionally examined, and we attempted to clarify its associated mechanism of the antiatherogenic activity of GM-CSF in the arterial wall.
Female WHHL rabbits weighing ≈2.5 kg were bred at the Institute for Experimental Animals, Kobe University School of Medicine.9 The animals were kept in rooms equipped with laminar-flow filters at a temperature of ≈22°C and were fed a standard rabbit chow in the Experimental Animal Laboratory of Fukushima Medical University.
Recombinant human GM-CSF (GM-CSF) synthesized by Escherichia coli was a gift from Hoechst Japan Co Ltd (Tokyo, Japan). Its specific activity was 5×107 U/mg protein as determined with a bioassay of human granulocyte-macrophage colony formation.10
In Vivo Study Protocol
At 4 months of age, 20 rabbits were divided into 2 groups. A 100-μL aliquot of PBS (pH 7.4) containing 10 μg · kg−1 · d−1 of GM-CSF was administered to each WHHL rabbit (n=10) as a single subcutaneous injection from Monday through Friday for 7.5 months. An equal dose of human serum albumin (HSA) was given to control animals (n=10) because heterogenic serum develops an immune response and induces an antiatherogenic effect compared with saline-treated animals, as described previously.11 A preliminary study demonstrated that this dose did not induce a significant reduction in plasma cholesterol levels. Blood was drawn monthly to measure plasma lipid concentrations. One rabbit was lost in each group because of accidental lumbar dislocation at bleeding. After GM-CSF or HSA treatment, the aortas of rabbits were removed after injection of sodium pentobarbital (25 mg/kg IV). Aortas were also removed from 7 age-matched 4-month-old WHHL rabbits. As described below, the aortas were used for analyses of extent of inner surface areas with atheromatous plaques, histology, cellular and extracellular composition, cell density, and apoptotic cells. This study was carried out in conformance with the Guidelines on Animal Experiments in Fukushima Medical University and the Law Concerning the Protection and Control of Animals (Law No. 105) and Standards Relating to the Care and Management, etc, of Experimental Animals (Notification No. 6).
Evaluation of Aortic Inner Surface Area of Atheromatous Plaques
The aortas were cut longitudinally and fixed with 10% formaldehyde for 24 hours. After fixation, photographs of the inner surface were taken. The aortas were then divided into 3 parts (aortic arch, descending thoracic aorta, and abdominal aorta) at the levels of the first intercostal artery and celiac artery. The areas corresponding to atheromatous plaques, as judged visually, were delineated and their areas estimated by use of a color image analyzer (SP-500, Olympus Co). The extent of atheromatous plaques was calculated as the percent area of lesions to the total surface area of each segment.
Three atherosclerotic lesions were individually removed from each aortic arch and descending thoracic and abdominal aorta in rabbits treated with GM-CSF (n=9) or HSA (n=9) for conventional histology. A total of 9 specimens from 1 aorta were subjected to the following procedure. Sections were embedded in paraffin, and 4-μm-thick cross sections from each paraffin block were cut and stained with hematoxylin and eosin. The ratio of intima to media (I/M ratio) was determined in aortic arch and thoracic and abdominal aorta. To avoid error due to the pathological heterogeneity of atherosclerosis, the cross sections containing the thickest atherosclerotic lesions were selected individually from aortic arch and used for the analyses of cross-sectional absolute areas of atherosclerotic lesions, cellular and extracellular components, and cell density. Serial sections were cut and used for immunohistochemistry and elastic–van Gieson and Azan-Mallory’s stainings.
Analysis of Cellular and Extracellular Composition
To examine the cellular composition of atherosclerotic lesions, the serial cross sections of aortic arch of maximal thickness were stained immunohistochemically with monoclonal antibodies against smooth muscle α-actin (1A4, Dako) and rabbit macrophages (RAM11, Dako), reacted with an avidin-conjugated peroxidase (Dako), and visualized with 3,3′-diaminobenzidine (Dako) as previously described.12 Numbers of smooth muscle cells and macrophages were enumerated by light microscopy. We defined extracellular vacuoles and lacunae with Azan-Mallory’s staining as extracellular lipid deposits and fibers bearing only the blue element as collagen. The absolute area and proportion of macrophages and smooth muscle cells and extracellular components, including collagen and lipid deposits, were quantitatively estimated with a color image analyzer.9 The immunohistochemical analyses were performed in a blind fashion by 2 researchers.
DNA Nick End-Labeling of Tissue Sections
To determine the presence of apoptotic cells with DNA breaks in nuclei in situ, we performed terminal deoxynucleotidyltransferase (TdT)–mediated dUTP-digoxigenin nick end-labeling (TUNEL) on tissue sections as previously described by Gavrieli et al.13 Briefly, 4-μm cross sections of the ascending, arch, and descending regions in each aortic arch were deparaffinized and rehydrated by passing the slides through the following solutions: xylene, graded ethanol, and finally PBS. Nuclei were stripped of proteins by incubation with 20 μg/mL proteinase K (Promega Co) for 10 minutes. After washing, the slides were immersed in TdT buffer (30 mmol/L Trizma base, pH 7.2; 140 mmol/L sodium cacodylate; 1 mmol/L cobalt chloride). TdT (0.3 EU/μL, Takara Shuzo Co) and digoxigenin-labeled dUTP (digoxigenin 11-dUTP, Boehringer Mannheim) in TdT buffer were added to cover the tissue sections, which were then incubated at 37°C for 1 hour. One negative control slide per tissue was incubated in the absence of the TdT enzyme. The slides were washed in a buffer (300 mmol/L sodium chloride, 30 mmol/L sodium citrate). Sections were then covered with 2% aqueous BSA (Sigma Chemical Co) and immersed in PBS. Subsequently, the sections were incubated with a blocking solution and then alkaline phosphatase–labeled anti-digoxigenin antibody (Boehringer Mannheim). The sections were visualized with nitro blue tetrazolium (Boehringer Mannheim) and 5-bromo-4-chloro-3-indolyl phosphate (Boehringer Mannheim). One positive section was treated with DNase I (10 ng/mL; Boehringer Mannheim).
To identify the origin of TUNEL-positive cells, TUNEL and immunohistochemical staining were performed on the same sections. First, the sections were stained with the TUNEL method. Subsequently, immunohistochemistry was performed as described above. Evaluation of the TUNEL analysis was made in a blind fashion by 2 researchers.
Serological and Hematological Analyses
Blood was drawn from the central ear artery, and serological and hematological analyses were performed once a month. Total plasma cholesterol and plasma triglycerides were measured enzymatically. Complete blood cell counts were obtained with an automated hematological analyzer. Differential counts of leukocytes were carried out on slides stained with May-Grünwald-Giemsa.
Statistical analyses were performed with Student’s paired or unpaired t test or ANOVA with the Scheffé F test, as appropriate. A level of P<0.05 was considered statistically significant. Data are expressed as mean±SD.
Effect of GM-CSF on Plasma Lipid Content
Although there was a trend toward lower levels of total cholesterol in GM-CSF–treated rabbits, no significant differences in total cholesterol levels at each time point (Table 1⇓) and cumulative cholesterol exposure (cholesterol levels×duration of exposure, data not shown) were observed between the GM-CSF and the HSA groups. Neither HDL cholesterol nor triglycerides were altered by treatment with GM-CSF (Table 1⇓).
No significant difference in the number of granulocytes, monocytes, red blood cells, or platelets was observed in the 2 groups (data not shown).
Effect of GM-CSF on Extent of Inner Surfaces of Atherosclerotic Lesions
Atheromatous lesions were already found in the aortas of age-matched (4-month-old) WHHL rabbits before the initiation of treatment. The mean percentage of the extent of aortic arch atheromatous plaques was 30.7±5.7% in the 4-month-old intact rabbits (n=7, Figure 1⇓). After 7.5 months of treatment, the atherosclerotic lesions progressed with aging. However, the percentages of atherosclerotic lesions of the aortic arch in the GM-CSF group (45.0±12.6%) were significantly less than those in the HSA group (74.3±11.0%, P<0.0001). There was also a significant decrease in the percentages of atherosclerotic lesions in total aortic area between the GM-CSF (20.0±3.9%) and HSA (38.5±20.0%) groups (P<0.05). There was no significant difference between the GM-CSF and HSA groups in the extent of atheromatous plaques in the descending thoracic or abdominal aorta. Figure 2⇓ shows 2 representative aortas in the rabbits treated with GM-CSF (right 2 aortas) or HSA (left 2 aortas).
Estimation of Intimal Thickening
Examination by light microscopy showed that GM-CSF decreased the intimal thickness of atherosclerotic lesions in the aortic arch compared with HSA. As shown in Figure 3A⇓, the I/M ratio in the aortic arch was decreased by GM-CSF treatment compared with HSA (GM-CSF, 0.91±0.41 versus HSA, 1.78±0.72; each group, n=9; P<0.01). The I/M ratio of the thoracic and abdominal aorta showed no significant difference between the GM-CSF and HSA groups. Figure 3B⇓ shows a significant decrease in the cross-sectional area of atherosclerotic lesions in the thickest cross sections of aortic arch from GM-CSF–treated rabbits compared with HSA (GM-CSF, 1.05±0.51 mm2 versus HSA, 3.51±1.29 mm2; P<0.0001).
Quantitative Analysis of Cellular and Extracellular Composition
Figure 4⇓ shows the absolute area of each cellular and extracellular component of atherosclerotic lesions. The absolute area of smooth muscle cells decreased significantly in rabbits treated with GM-CSF compared with HSA (GM-CSF, 0.12±0.08 mm2 versus HSA, 0.79±0.38 mm2; P=0.0001). In contrast, no significant difference in the macrophage area was observed between the GM-CSF and HSA groups (GM-CSF, 0.33±0.22 mm2 versus HSA, 0.51±0.20 mm2; P=NS). Treatment with GM-CSF also reduced the absolute area of collagen compared with HSA (GM-CSF, 0.33±0.18 mm2 versus HSA, 1.15±0.28 mm2; P=0.0001). The area of extracellular lipid deposits in rabbits treated with GM-CSF was significantly smaller than that in the control rabbits (GM-CSF, 0.25±0.21 mm2 versus HSA, 0.62±0.29 mm2; P<0.05).
Table 2⇓ shows the ratio of the area of each lesional component to the area of the total lesion. The proportion of smooth muscle cells was decreased by GM-CSF treatment compared with HSA (P<0.005), whereas GM-CSF increased the ratio of macrophage area (P<0.05). A significant decrease was observed in the area ratio of extracellular lipid deposits in the GM-CSF group (P<0.05). There was no significant difference in the area ratio of collagen between the 2 groups.
The density of smooth muscle cells in the GM-CSF group (586±267/mm2) was significantly decreased compared with HSA (1012±275/mm2, P<0.02). Conversely, the macrophage density of atherosclerotic lesions in the GM-CSF group (876±402/mm2) tended to increase compared with that of the HSA group (573±239/mm2), but not significantly.
Sections were obtained from the ascending, arch, and descending regions of the aortic arch in rabbits treated with GM-CSF (n=25) or HSA (n=26). We examined 52 830 and 68 920 cells in each group, respectively, for apoptosis by use of the TUNEL method. TUNEL-positive cells were located primarily in the fibrous cap and the shoulder region of atheromatous plaques. Figure 5A⇓ and 5B⇓ shows TUNEL-positive cells of atherosclerotic lesions in rabbits treated with GM-CSF. The cellular origin was identified by immunohistochemistry as being predominantly smooth muscle cells in the fibrous cap, whereas TUNEL-positive cells in the shoulder appeared to be macrophages and smooth muscle cells. Figure 5C⇓ presents the result of double staining showing that the origin of TUNEL-positive cells is smooth muscle cells in the fibrous cap. A TUNEL-positive macrophage in the shoulder region is shown in Figure 5D⇓. A significant increase in TUNEL-positive cells was observed in rabbits treated with GM-CSF compared with controls (GM-CSF, 24.1±4.4% versus HSA, 11.6±3.2%; each group, n=9; P<0.0001), as shown in Figure 6⇓. Double staining by TUNEL and immunohistochemistry revealed that the frequency of TUNEL-positive smooth muscle cells in the GM-CSF group was significantly higher than in the control group (GM-CSF, 15.0±2.5% versus HSA, 6.2±2.7%; each group, n=6; P<0.0001). Conversely, there was no significant difference between the percentages of TUNEL-positive macrophages in the GM-CSF and HSA groups (GM-CSF, 25.8±6.9% versus HSA, 20.4±4.8%; each group, n=6). These findings suggest that the increase in apoptosis of smooth muscle cells may be one of the mechanisms associated with the decrease in intimal smooth muscle cells induced by GM-CSF.
In this study, GM-CSF was administered to 4-month-old WHHL rabbits for 7.5 months to examine its effect on the development of atherosclerosis. After 7.5 months of treatment, the percentages of atheromatous plaques in aortic arch in the GM-CSF group were markedly smaller than those in the control group (Figure 1⇑). Histological studies of cross sections indicated that GM-CSF reduced both intimal thickening and the absolute areas of atherosclerotic lesions in the aortic arch (Figure 3⇑). Moreover, the reduction of atherosclerosis after GM-CSF treatment was accompanied by alterations in the cellular and extracellular composition of atherosclerotic lesions. These data demonstrate that GM-CSF prevented the progression of atherosclerosis in this atherogenic animal model.
We used a dose of GM-CSF that tended to decrease plasma cholesterol levels, but not significantly (Table 1⇑). Statistical analysis also revealed no correlation of cumulative cholesterol exposure and final lesion size (inner surface area and cross-sectional area) between GM-CSF–treated animals and controls (data not shown). Thus, the antiatherogenic activity of GM-CSF was not associated with cholesterol-lowering. Probucol and macrophage colony–stimulating factor (M-CSF) also induce an antiatherogenic effect with negligible cholesterol-lowering.11 14 15 The histological changes in composition of atherosclerotic lesions have been shown after probucol treatment but not after M-CSF treatment.11 16 The present study is the first to document that a macrophage-stimulating factor, GM-CSF, induces an alteration in the cellular and extracellular composition of atherosclerotic lesions without significant cholesterol lowering, resulting in an antiatherogenic effect in WHHL rabbits. Our quantitative analysis revealed the following changes in cellular and extracellular composition in atherosclerotic lesions of the GM-CSF group: (1) a marked decrease in smooth muscle cells, (2) no significant difference in the macrophage area (a significant increase in the ratio of macrophage area to total area), (3) a reduction in extracellular matrix such as collagen, and (4) a significant decrease in extracellular lipid deposits.
Recent studies established that vascular smooth muscle cells and macrophages exhibit apoptosis in vitro and in vivo.17 18 19 20 To determine the mechanism associated with the decrease in smooth muscle cells in the GM-CSF group, TUNEL was performed in vascular walls in situ. We found that the percentage of apoptotic smooth muscle cells defined by the TUNEL method was increased in the atherosclerotic lesions by treatment with GM-CSF. The enhancement of apoptosis was observed in the cells of the fibrous cap and the shoulder region. The double staining by TUNEL and immunohistochemistry revealed that the percentage of apoptotic smooth muscle cells in the GM-CSF group was ≈2.4 times higher than in the HSA group. Our results raise the possibility that enhancement of apoptosis of intimal smooth muscle cells by GM-CSF may contribute to the reduction of atherosclerosis.
A major question is how GM-CSF treatment enhances apoptosis in vascular smooth muscle cells. It has been shown that macrophage-derived molecules, such as interferon-γ, tumor necrosis factor-α, and Fas ligand, can induce apoptosis of these cells.19 20 Therefore, one possibility is that GM-CSF can promote the production by macrophages of apoptosis-inducing factors for smooth muscle cells.2 19 20
In addition to enhanced apoptosis of smooth muscle cells, GM-CSF treatment may have affected the migration and proliferation of smooth muscle cells. However, we observed that GM-CSF had no effect on the proliferation and migration of smooth muscle cells in vitro in rabbits and humans (unpublished data). It is conceivable that GM-CSF can promote macrophage functions and modulate interactions of macrophages with other vascular cells, such as smooth muscle cells and endothelial cells.2 7 Because GM-CSF stimulates endothelial cells,21 stimulated endothelial cells may also affect the progression and composition of atheromatous plaques. Further studies are needed to elucidate the cellular interactions of the vascular wall using in vitro and in vivo systems.
The present study also demonstrated that GM-CSF treatment reduced the area of collagen in atheromatous plaques. One explanation for this is the decrease in intimal smooth muscle cells responsible for collagen production.22 A recent study using an in vitro system showed increased production of matrix metalloproteinases by macrophages.23 Further studies are needed to clarify the relationship between macrophage stimulation with GM-CSF and the production of matrix metalloproteinases by macrophages.
GM-CSF treatment also induced a profound reduction in the absolute area and the ratio of extracellular lipid deposits in atheromatous plaques. This suggests that GM-CSF may affect the reverse cholesterol transport system in addition to enhancing the uptake of modified LDLs into macrophages and the efflux of cholesterol from macrophages.24 25 It will be of interest to investigate this issue.
We found a marked decrease not only in smooth muscle cells and collagen but also in extracellular lipid deposits in atherosclerotic lesions of rabbits treated with GM-CSF. In this sense, our findings may be different from the characteristics of plaques in acute coronary syndromes in which lipids are abundant.26 However, the possibility cannot be excluded that GM-CSF may promote plaque instability because of the decrease in smooth muscle cells and collagen.
The ratio of macrophage area to total atherosclerotic area in the GM-CSF group was significantly greater than in the HSA group (Table 2⇑). The density of macrophages in atherosclerotic lesions in GM-CSF–treated rabbits tended to increase compared with control, but not significantly. Moreover, there was no significant difference in macrophage apoptosis in atherosclerotic lesions between the GM-CSF and HSA groups. These findings suggest the possibility of increased entry of monocytes from the circulation to the vascular wall and increased macrophage replication in the plaques. To understand the density of macrophages in atheromatous plaques, we should consider at least 4 parameters, including monocyte entry, replication, death, and life span. In the present study, however, we clarified only that macrophage apoptosis was not significantly affected by long-term treatment with GM-CSF compared with HSA. It will also be necessary to investigate the effect of GM-CSF on macrophage life span in the vascular wall in a short-term experimental model.
Recently, Rajavashisth et al27 showed the protective effect of M-CSF deficiency using a gene knockout mouse model. Although the cholesterol-lowering effect of M-CSF has been reported, as well as that of GM-CSF,5 6 28 29 we have clearly shown the difference in their associated mechanisms.6 In addition to differences between GM-CSF and M-CSF in targeting cells in the circulation and arterial walls, their receptor systems and signal transduction pathways are distinct.7 21 30 Therefore, GM-CSF and M-CSF appear to be distinct molecules in atherogenesis and lipid metabolism despite their sharing macrophage functions. To clarify the roles of GM-CSF and M-CSF in atherogenesis, it will be useful to generate GM-CSF gene knockouts in an atherogenic mouse model.
This study was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan (04770383).
- Received October 9, 1998.
- Revision received December 7, 1998.
- Accepted December 18, 1998.
- Copyright © 1999 by American Heart Association
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