Cholesterol-Lowering Independent Regression and Stabilization of Atherosclerotic Lesions by Pravastatin and by Antimonocyte Chemoattractant Protein-1 Therapy in Nonhuman Primates
Objective— Anti-atherosclerotic effects of statins might be mediated partly by pleiotropic cholesterol-lowering independent mechanisms. We used nonhuman primates and examined whether treatment with pravastatin or antimonocyte chemoattractant protein-1 (MCP-1) therapy can induce regression and stabilization of established atherosclerotic lesions through cholesterol-lowering independent mechanisms.
Methods and Results— Advanced atherosclerosis was induced in the abdominal aorta and the common iliac artery of cynomolgus monkeys by undergoing balloon injury and giving atherogenic diet for 6 months. At 6 months, the diet was changed to normal chow, and the animals were allocated to 4 treatment groups: control vehicle group and other groups treated with pravastatin (1 or 10 mg/kg) or with mutant MCP-1 gene transfection for additional 6 months. Each compound was treated instead of the atherogenic diet, and cholesterol contents in pravastatin-treated groups were adjusted to equalize plasma cholesterol level among groups. Pravastatin reduced neointimal formation in the aorta, but not in the common iliac artery. Pravastatin reduced intimal macrophage area and other markers of plaque destabilization in the common iliac artery. Equivalent inhibitory effects were observed in animals that received mutant MCP-1 gene transfection. No serious side effects were noted by 2 therapeutic modalities.
Conclusion— This study demonstrated cholesterol-lowering independent regression and stabilization of established atherosclerotic lesions by pravastatin and by anti-MCP-1 therapy in nonhuman primates. An anti-inflammatory mechanism may be involved in the beneficial effects of pravastatin.
- 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors
- nonhuman primates
There is clinical evidence that 3-hydroxyl-3-methylglutaryl-coenzyme A reductase inhibitors (statins) improve endothelial dysfunction and reduce the incidence of atherosclerotic events such as myocardial infarctions and ischemic strokes.1–4 Although anti-atherosclerotic effects of statins are attributed to their lipid-lowering effects, it is suggested that some of the beneficial effects of statins are mediated by pleiotropic effects independent of cholesterol-lowering.1–3,5–7 There is ample evidence that statins improve endothelial dysfunction8,9 and reduce vascular inflammation and oxidative stress10 by their cholesterol-lowering independent effects. These data suggest the possibility that treatment with statins has cholesterol-lowering independent anti-atherosclerotic effects in humans. It is practically impossible, however, to investigate such beneficial effects of statins in clinical settings, because clinical doses of statins inevitably lower serum cholesterol levels.
Nonhuman primate models may be useful to gain insight into cholesterol-lowering independent anti-atherosclerotic effects of statins. There are at least 2 studies that had examined cholesterol-lowering independent effects of statins on atherosclerosis in cynomolgus monkeys. Sukhova et al11 reported that statins reduced markers of plaque destabilization, and Williams et al12 reported that pravastatin improved endothelial dysfunction and reduced macrophage infiltration of coronary arteries. These studies, however, failed to demonstrate reduction or regression of atherosclerosis formation by statins. One notable caveat in interpreting these previous studies is that a high dose (20 or 40 mg/kg per day) of pravastatins was used, which might cause serious side effects.
In the present study, we aimed to investigate cholesterol-lowering independent effects of pravastatin on regression and stabilization of established atherosclerotic lesions in nonhuman primates (cynomolgus monkeys). For clinical implication, we selected at least a clinical dose of pravastatin (1 mg/kg per day). To obtain mechanistic data regarding anti-inflammatory effects, we examined whether anti-atherosclerotic actions afforded by pravastatin resemble those obtained by antimonocyte chemoattractant protein-1 (MCP-1) therapy. Because recruitment of monocyte/macrophage is a major histopathologic finding in atherosclerosis, an anti-inflammatory strategy targeting MCP-1 is considered to be a reasonable approach for vascular inflammation leading to atherosclerosis.13 We have recently devised a new strategy of anti-MCP-1 gene therapy by transfecting plasmid cDNA encoding a mutant MCP-1 gene into skeletal muscle.14 This mutant MCP-1, called 7ND, lacks the N-terminal amino acid 2 to 8 and has been shown to work as a dominant-negative inhibitor of MCP-1. With this strategy, we have demonstrated that blockade of MCP-1 signals reduces neointimal formation after arterial injury in animals, including monkeys.15–17 We report here that treatment with pravastatin or anti-MCP-1 therapy not only reduced plaque size but also changed characteristics of plaques to more stable phenotype in cynomolgus monkeys with established atherosclerotic lesion caused by arterial injury and hypercholesterolemia.
Forty-five 4-year-old male cynomolgus monkeys were used. The study protocol was reviewed and approved by the Committee on the Ethics of Animal Experiments, Kyushu University Graduate School of Medical Sciences. A part of this study was performed at the Station for Collaborative Research and the Morphology Core, Kyushu University Graduate School of Medical Sciences.
Treatment and Tissue Preparation
All animals were fed a high-cholesterol diet (0.5% cholesterol and 6% corn oil) for 6 months and received balloon injury of the descending aorta and the right common iliac artery at 1 month after the initiation of the diet. Nine animals were euthanized after 6 months of high-cholesterol diet and were considered the baseline group. Thereafter, the diet was changed to normal chow, and the animals were randomized to 4 groups as follows: (1) vehicle control group (0.5% carboxymethyl cellulose sodium salt); (2) low-dose pravastatin (1 mg/kg per day) group (low-P group); (3) high-dose pravastatin (10 mg/kg per day) group (high-P group); and (4) 7ND transfection group (n=9 each). In the animals treated with pravastatin, 0.4% to 4% cholesterol solution was orally administered and adjusted on a biweekly basis to make the serum cholesterol level equal to that in the vehicle group.
To transfect 7ND gene, 7ND plasmid (2.5 mg/500 μL PBS) was injected into the femoral muscle of the animals in the 7ND group biweekly. To enhance transgene expression, all monkeys were pretreated with intramuscular injection of bipuvacine (0.25 mg/kg) at the injected site.18 Human 7ND cDNA was constructed by recombinant polymerase chain reaction using a wild-type human MCP-1 cDNA as template and inserted into the BamHI (5′) and NotI (3′) sites of the pcDNA3 (Invitrogen) expression vector plasmid.14 We have reported biological efficacy of 7ND gene transfer by in vivo matrigel plug assay in monkeys.18 In brief, MCP-1-induced inflammatory angiogenesis in the plugs was suppressed until 14 days after 7ND gene transfer. All monkeys were euthanized with a lethal dose of anesthesia after 6 months of treatment for morphometric analysis (Figure 1A).
After the monkeys were euthanized, the abdominal aortas and the right iliac arteries were perfused with saline and fixed with the solution of 95% ethanol and 1% acetic acid and were used for histology and immunohistochemistry.
Histology and Immunohistochemistry
All tissues were dehydrated and embedded in paraffin, and serial sections (5 μm) of the abdominal aortas and the common iliac arteries were prepared and mounted on slides. Some of these sections were stained with hematoxylin-eosin, Elastica van Gieson, or Elastica Masson. Interstitial collagens were stained by Picrosirius red (Direct Red, Aldrich Chem) and photographed using a polarization microscopy. The remaining sections were used for immunohistochemical analysis. They were stained with an antibody for smooth muscle cell (mouse anti-human HHF-35 antibody, DAKO, Kyoto, Japan), macrophages/monocytes (mouse anti-human CD-68 antibody, DAKO), or nonimmune mouse IgG (DAKO). After incubation with appropriate biotinylated affinity-purified secondary antibodies (DAKO), the sections were incubated with alkaline phosphatase-labeled streptavidin solution (DAKO) and visualized using a fast red substrate kit (DAKO). The sections were then counterstained with Karachi-hematoxylin.
A single observer who was blinded to the experiment protocol performed morphometry and cell counting. All images were captured by an Olympus microscope equipped with a digital camera (HC-2500) and were analyzed using Adobe Photoshop 6.0 and National Institute of Health Image 1.62 Software. Elastica-van Gieson staining, which stains elastic fiber, was used to delineate internal elastic lamina for determination of the intimal area. The percent area of extracellular lipid deposition (area dropped off in Elastica Masson staining), macrophage accumulation (CD68-positive area), smooth muscle cell area (HHF-35–positive area), and collagen deposition (Picrosirius red staining area) were estimated.
Biochemical Analysis and Measurements of Cytokines
Biochemical parameters listed in Tables 1 and 2⇓ were measured by SRL Inc, Japan. Chemokines including MCP-1, IL-8, and transforming growth factor-β1 (TGF-β1) were measured using commercially available enzyme-linked immunosorbent assay (ELISA) kits (Biosource International Inc).
Measurement of Antibody Productions in 7ND-Transfected Animals
We examined whether anti-7ND or anti-MCP-1 IgG and IgM antibodies were produced in the 7ND transfected animals. Ninety-six well plates were coated with 7ND protein (0.1 μg/mL) or with human MCP-1 protein (0.1 μg/mL). Paired serum before and 7 or 28 days after 7ND transfection was incubated on each coated well for 90 minutes at 37°C, followed by incubation with HRP-conjugated goat antibodies against monkey IgG or IgM (Kirkegaard & Perry Laboratories) for 1 to 2 hours at 37°C. TMB one solution (Promega) was used, and the absorbencies of each well were detected by using an ELISA plate reader.
We also checked the presence of newly produced immunoglobulins in the 7ND-transfected animals 11 and 17 months after transfection. The control monkey serum and the 7ND-treated monkey serum were diluted 500-fold, and the diluted serum was mixed with an equivalent solution of 4% SDS in Tris buffer, and the solution was boiled for 5 minutes. The treated protein solution was then electrophoresed on a 5% to 20% polyacrylamide gradient gel according to the method of Laemmli. The developed protein bands on the gel were visualized by staining with SYPRO Ruby. Molecular weight was determined in comparison with the molecular weight markers.
Data were expressed as mean±SEM. Differences between groups were determined using 2-way analysis of variance and a multiple comparison test. P<0.05 was considered to be statistically significant.
Clinical Status and Biochemical Parameters
All animals had no clinical signs (decreased spontaneous motor action, decreased food consumption, diarrhea, limping, and prone position) during experimental period and survived. There was no significant treatment effect on body weight among 4 groups (Table 1). Pravastatin treatment and 7ND gene transfection had no adverse effects on biochemical parameters, including CPK and liver transaminases (Table 1). As designed, time courses of serum total cholesterol levels and low-density lipoprotein cholesterol levels were comparable among 4 groups (Figure 1B, P>0.10).
There were also no treatment effects on plasma renin activity or angiotensin II in the pravastatin groups and 7ND group (Table 2). Plasma MCP-1 levels in all groups decreased with time. However, pravastatin treatment and 7ND gene transfection had no treatment effect on cytokine concentrations (Table 2).
Pravastatin-Induced Regression of Atherosclerotic Lesions in Abdominal Aorta
There was no significant difference in the degrees of neointimal formation (intimal area and intima/media ratio) between the baseline group and the vehicle group. In contrast, pravastatin treatment and 7ND gene transfer significantly reduced neointimal formation of injured abdominal aorta compared with the vehicle group (Figure 2, Table 3). There was no difference in medial area among 4 treatment groups.
Pravastatin Changes Characteristics of Atheromatous Plaques Composition
In common iliac arteries, although significant reduction of intimal area was noted in the 7ND group, there were no treatment effects on the intima/media ratio in the pravastatin group and the 7ND group compared with the vehicle group (Figure 3, Table 3).
Because neither pravastatin nor 7ND reduced the neointimal formation in the common iliac arteries, we then investigated the characteristics of plaque composition (Figure 4). Macrophage infiltration into atherosclerotic lesion was markedly less in the pravastatin groups and the 7ND group compared with the vehicle group. Percent areas of lipid deposition were also reduced in the pravastatin groups and the 7ND group compared with the vehicle group. However, there was no significant difference in the percent area of collagen and smooth muscle cells among 4 treatment groups.
Antibody Production in 7ND-Transfected Animals
In ELISA assay, IgG and IgM antibodies against 7ND protein and wild-type MCP-1 protein were not detected after 7ND transfection (n=6 each). The electrophoresis pattern of the7ND-transfected monkey serum was almost consistent with that of the control monkey serum, and the abnormal bands of immunoglobulin heavy chain protein and light chain protein were not observed in the electrophoresis result of the 7ND-transfected monkey serum compared with that of the control monkey serum (Figure 5).
A novel finding of this study was that compared with dietary lipid-lowering therapy alone, additional treatment with pravastatin induced regression of established atherosclerotic lesions of the injured abdominal aorta in nonhuman primates. In contrast, pravastatin did not reduce the size but induced stabilization of atherosclerotic lesions of the common iliac arteries, suggesting that the effects of the statin on regression might differ according to the size/site of artery. Because of our experimental design, these beneficial effects on regression and stabilization were independent of the cholesterol-lowering effects of the statin. Overall, the present data provide the notion that pravastatin treatment in addition to strong dietary lipid-lowering can induce regression and plaque stabilization of atherosclerotic lesions by mechanisms beyond its cholesterol-lowering effects.
Previous studies in animals have not addressed cholesterol independent regression of atherosclerosis by statins. As mentioned, 2 recent studies that examined effects of statins on atherogenesis in cynomolgus monkeys11,12 showed no detectable reduction in plaque size after statin treatment. Our model differs from those models in several aspects that deserve discussion. First, in addition to high-cholesterol diet, balloon injury was performed in the present study. Balloon injury to hypercholesterolemic animals accelerates atheroma formation and makes lesions more uniform in size.19,20 Importantly such injured lesions resemble the so-called vulnerable plaque in humans more closely, compared with the foam cells and macrophage-rich lesions produced by hypercholesterolemia alone. Therefore, it is possible that such “vulnerable” plaque was more sensitive to pravastatin treatment. Second, we used relatively lower doses of pravastatin (1 mg/kg and 10 mg/kg per day) compared with the previous studies (20 mg/kg or 40 mg/kg per day). The low dose (1 mg/kg per day) used in the present study is the range of clinical doses. Therefore, the present data imply that a clinically relevant dose of pravastatin can induce regression and stabilization of atherosclerotic lesions in nonhuman primates. Reasons why the higher doses (10 mg/kg per day or more) of pravastatin could not show greater action on regression and stabilization of atherosclerosis in our present study and previous studies are not clear. In preliminary experiments, we administered higher doses (20 and 30 mg/kg per day) of pravastatin to hypercholesterolemic monkeys, which resulted in serious side effects such as body weight loss and labdomyolysis leading to death (data not shown). We speculate, therefore, that some toxic actions at high doses of pravastatin might detract from beneficial effects of the low dose of pravastatin in the present and those previous studies.
It has been shown that increased macrophage infiltration and lipid deposition enhance plaque destabilization and that increased interstitial collagens and smooth muscle cells increase plaque stability.21 We show here that pravastatin did not affect percent areas of collagen and smooth muscle cells but did reduce the degrees of macrophage infiltration and lipid deposition into atherosclerotic plaque of the common iliac arteries. These data support the previous reports that treatment with statins promotes transformation of destabilized plaque of atheroma to more stable phenotype through cholesterol-lowering independent actions.
We13,15,17,18,22,23 have previously shown that blockade of MCP-1 by 7ND gene transfer reduces neointimal formation after mechanical injury and initiation and progression of hypercholesterolemia-induced atherosclerosis by suppressing monocyte infiltration and activation. We therefore examined whether the observed effects of pravastatin were similar to those of 7ND gene transfer and found that the effects of pravastatin on plaque size and composition were comparable to those obtained by 7ND gene transfer. Of note is that striking decreases in macrophage infiltration and lipid deposition were achieved by pravastatin treatment and by 7ND gene transfer. Reduced local lipid deposition might be the result of reduced macrophage infiltration. These data imply that pravastatin might function as local anti-inflammatory or anti-MCP-1 therapy beyond its cholesterol-lowering effects. We further examined whether systemic inflammatory process was involved and showed that there were no treatment effects of pravastatin or 7ND gene transfer on plasma concentrations of chemokines and cytokines listed in Table 2. These data suggest that the observed beneficial effects of pravastatin in the present study may not be mediated by its inhibitory effects on plasma MCP-1, renin-angiotensin system, and other systemic factors.
No appreciable side effects were observed in animals treated with pravastatin or those that received 7ND gene transfer. Thus, 7ND gene transfer may be as safe as pravastatin, although caution should be used when 7ND is used clinically. 7ND gene transfer can be used clinically because the anti-MCP-1 gene therapy for patients with severe forms of atherosclerotic vascular disease and inflammatory disease cannot be treated successfully by conventional therapeutic regimens.
In conclusion, this study demonstrates that compared with strong dietary lipid lowering alone, additional treatment with pravastatin induces regression and stabilization of established atherosclerotic lesions in nonhuman primates through mechanisms beyond its cholesterol-lowering actions. The observed effects of pravastatin were identical to those of anti-MCP-1 therapy. Thus, an anti-inflammatory mechanism may be involved in the beneficial effects of pravastatin. This study implies that compared with modest to moderate lipid-lowering therapy with statins that have been conventionally introduced to patients with hypercholesterolemia, stronger dietary lipid lowering plus statin treatment may induce significant regression and destabilization of human atherosclerotic lesions resulting in stronger reduction in atherothrombotic events in the patient.
This study was supported by grants-in-aid for Scientific Research (14657172, 14207036) from the Ministry of Education, Culture, Sports, Science, and Technology, Tokyo, Japan, by Health Science Research Grants (Comprehensive Research on Aging and Health, and Research on Translational Research) from the Ministry of Health Labor and Welfare, Tokyo, Japan, and by the Program for Promotion of Fundamental Studies in Health Sciences of the Organization for Pharmaceutical Safety and Research, Tokyo, Japan.
- Received April 5, 2004.
- Accepted May 20, 2004.
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