Osteoprotegerin Promotes Fibrous Cap Formation in Atherosclerotic Lesions of ApoE-Deficient Mice—Brief Report
Objective— Osteoprotegerin (OPG) is a tumor necrosis factor receptor–related cytokine, initially found to inhibit osteoclastogenesis. In the present study we investigated the effect of OPG treatment on atherosclerosis.
Methods and Results— Hypercholesterolemic apoe−/− mice were treated with recombinant 15 mg/kg OPG or vehicle injections twice a week for 10 consecutive weeks. Mice treated with OPG showed increased amounts of smooth muscle cells and collagen within the atherosclerotic lesions. OPG treatment did not affect atherosclerotic lesion size (8.2% versus 7.6%) or total vessel area but led to a 250% increase in lesion collagen, formation of mature collagen fibers in subendothelial fibrous caps, and upregulated mRNA for lysyl oxidase that promotes collagen crosslinking. In cell culture studies, OPG promoted cell proliferation in rat aortic smooth muscle cells. In contrast, OPG treatment did not affect markers of vascular or systemic inflammation.
Conclusion— OPG treatment promotes smooth muscle accumulation, collagen fiber formation, and development of fibrous caps but does not affect inflammatory properties of atherosclerotic lesions. Its effects may contribute to plaque stabilization.
Osteoprotegrin (OPG), a cytokine of the TNFR superfamily, was initially recognized as an osteoprotector.1 It is a circulating decoy receptor for the receptor activator of nuclear factor κB ligand (RANKL), thereby inhibiting its binding to RANK.2 RANKL and RANK are overexpressed in T cells and monocytes of patients with acute coronary syndromes,3 and we have suggested that RANKL expression could be an important mediator of plaque destabilization.3 In contrast, OPG-deficient mice exhibit not only low bone density but also increased vascular calcification.4
These data points to a role for the OPG-RANKL-RANK system in the atherosclerotic process. Therefore, we have investigated the effect of OPG treatment on atherosclerosis in apoe−/− mice.
Materials and Methods
For comprehensive Material and Methods, please see supplemental materials (available online at http://atvb.ahajournals.org).
Eight-week-old female apoe−/− mice on a C57BL/6 background were assigned to treatment with recombinant murine OPG at 15 mg/kg SC twice per week, or injections with vehicle (NaCl), for 10 consecutive weeks, beginning at 9 weeks of age. Bone mineral density (mg/cm3) was measured repeatedly during treatment, using quantitative computed tomography of the left distal femur and proximal tibia. The heart and ascending aorta were cryosectioned, and 9 10-μm sections were collected at 100-μm intervals starting at a 100-μm distance from the appearance of the aortic valves. Formaldehyde-fixed sections were stained with hematoxylin and oil red-O, and lesion size was analyzed using Leica QWin image analysis software. Acetone-fixed sections of the ascending aorta were stained using rat anti-mouse CD68, rat anti-mouse VCAM-1, biotinylated mouse anti-mouse I-Ab, rat anti-mouse CD3, or mouse anti–α-smooth muscle actin. Picrosirius red staining was used for the assessment of collagen fibers in the lesions. The total collagen content of lesions in 4 sections was measured using the Leica QWin program and calculated as the ratio of thresholded chromogen area to total lesion area. Total aortic RNA was isolated, analyzed by capillary electrophoresis, and reverse-transcribed with Superscript-II and random hexamers. cDNA was amplified by real-time PCR in an ABI 7700 Sequence Detector by using TaqMan Universal Master Mix and premanufactured primers and probes sets (Assay on Demand, Applied Biosystems). Rat vascular smooth muscle cell (VSMC; between passages 4 and 6) were cultured in DMEM containing 10% FCS and penicillin/streptomycin. After 48 hours of serum starvation in DMEM/0.5%FCS, cells were treated with recombinant murine OPG (0.1 to 4.0 μg/mL), RANKL (1.0 μg/mL), or a mix of both cytokines. The Mann–Whitney U test was used to analyze data. A probability value less than 0.05 was considered significant. Results are shown as mean±SEM.
Ten weeks of OPG treatment did not affect atherosclerotic lesion size or lumen area in the aortic root of apoe−/− mice (Figure 1A through 1C), nor did it affect the proportions of CD68+ macrophages or CD3+ T cells (Figure 1D), or VCAM-1 and I-A protein compared to vehicle treated controls (Figure 1D).
Total collagen was increased throughout the lesions of OPG-treated mice, with prominent mature red collagen fibers in subendothelial parts, where cap-like structures were observed (Figure 2A). Lesions of control mice contained less total collagen and did not display any signs of cap formation at this age (Figure 2B). OPG treated mice displayed a significant 300% increase in the cross-section area stained with the SMC marker, α-SM-actin, in lesions of the aortic root (Figure 2F), and 250% increase in the collagen stained cross-section area of aortic lesions, as compared with vehicle treated controls (Figure 2F). OPG treatment also led to a significant increase in mRNA for lysyl oxidase (LOX), a collagen-crosslinking enzyme (Figure 2G), and a 50% reduction of MMP-12 mRNA (Figure 2G).
Because SMC content was increased in lesions of OPG-treated mice, we studied the effect of OPG on SMC proliferation in vitro. Addition of recombinant murine OPG at 1 μg/mL to cultured rat VSMCs increased DNA replication by 74% (Figure 2H). Addition of recombinant RANKL together with OPG did not affect the growth response, suggesting that it is an intrinsic property of the OPG molecule.
As expected, OPG treatment increased bone mineral density (supplemental Figure I) serum levels of OPG, RANKL and CTX in apoe−/− mice, whereas osteocalcin (supplemental Figure II) decreased. There was no significant difference in IL-10 or MCP-1 levels between OPG-and vehicle-treated mice (supplemental Figure III). Serum concentrations of IL-6, IL-12, IFN-γ, and TNF-α were below the detection level (data not shown). Plasma lipoproteins (supplemental Figure IV) or elastin content and elastolysis (supplemental Figure V) did not differ between OPG-treated and control mice.
The present data show that chronic OPG treatment increases the content of SMC and collagen in lesions and promotes the formation of fibrous caps with mature collagen fibers, whereas it does not affect SMC or elastin content of the media. There were no signs of altered systemic or local inflammation, and OPG-treated mice had similar atherosclerotic lesion area as controls. Therefore, a major effect of OPG may be to stabilize lesions by promoting cap formation.
The lack of effect on inflammation in this study concurs with the report that OPG reduces calcification in lesions of ldlr −/− mice without affecting lesion size.5 The increased SMC content of lesions observed in our study may reflect a survival-promoting activity of OPG,6,7 supported by the OPG stimulated entry of SMC into S phase. These findings prompt a reevaluation of OPG, which appears to act not only as a decoy receptor but also as a bona fide cytokine with intrinsic biological effects.
The observed increase in collagen content of lesions was likely attributable to enhanced collagen fiber maturation. This process depends on LOX-mediated crosslinking of lysyl residues.8 LOX mRNA was significantly upregulated in the aorta of OPG-treated mice, suggesting that OPG promotes collagen crosslinking and fibrous cap formation. Hyperinflamed lesions of apoe−/− mice with increased T cell activation contain reduced LOX mRNA and display reduced fibrous cap formation.9 These findings together with the present data support a key role for LOX-mediated collagen crosslinking in the development of the fibrous cap.
Bennett et al recently reported that lesions in the brachiocephalic artery of old apoe−/− × opg−/− mice display increased size, reduced cellularity, increased calcification, and increased proteoglycan accumulation in older mice.7 Only part of this picture was mirrored when we administered recombinant OPG. Of note, because congenital OPG deficiency may have a dramatic impact on hard-tissue metabolism, compensatory changes may take place in matrix composition, and age differences may also be important. In spite of this, both studies support the conclusion that OPG enhances plaque stability by promoting SMC and collagen accumulation.
Sources of Funding
This study was supported by grants from the Swedish Research Council, Heart-Lung Foundation, the Leducq Transatlantic Network of Excellence in Atherothrombosis and Västerbottens county council, Sweden.
O.O. and Å.G. and G.K.H. and P.N. contributed equally to this study.
Received March 17, 2009; revision accepted June 21, 2009.
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