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(Circulation. 2003;107:679.)
© 2003 American Heart Association, Inc.

Brief Rapid Communications

Osteopontin Transgenic Mice Fed a High-Cholesterol Diet Develop Early Fatty-Streak Lesions

Kikuo Isoda, MD, PhD; Yashuhiro Kamezawa, MD; Makoto Ayaori, MD; Masatoshi Kusuhara, MD, PhD; Norihiro Tada, PhD; Fumitaka Ohsuzu, MD, PhD

From Internal Medicine I, National Defense Medical College (K.I., Y.K., M.A., M.K., F.O.), Tokorozawa, Japan, and the Division of Biomedical Research Resources, Juntendo University School of Medicine (N.T.), Tokyo, Japan.

Correspondence to Kikuo Isoda, MD, PhD, Internal Medicine I, National Defense Medical College, 3-2, Namiki, Tokorozawa, Saitama, 359-8513, Japan. E-mail isoda{at}me.ndmc.ac.jp

	Abstract

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Background— Osteopontin (OPN) is a noncollagenous adhesion protein found at the site of atherosclerotic lesions. However, it has not yet been clarified whether or not OPN can promote atherosclerotic lesions.

Methods and Results— We investigated the contribution of OPN to atherosclerosis by evaluating aortic sinus lesions of both OPN transgenic (Tg) and non-Tg mice fed an atherogenic diet (1.25% cholesterol) for 16 weeks. The atherosclerotic lesions were found to be significantly larger in OPN-Tg compared with those in non-Tg (17 859±2010 versus 6469±485 µm², P<0.01). The lesions in both mice were fatty-streak lesions with an accumulation of mononuclear cells and lipids. We next investigated the production of interleukin (IL)-10 by macrophages from both mice. Compared with the non-Tg mice, a 42% (P<0.01) and 73% (P<0.001) decrease in the IL-10 production was identified in the OPN-Tg mice either without or with lipopolysaccharide.

Conclusions— The expression of OPN induces fatty-streak lesion formation in mice fed an atherogenic diet and inhibits IL-10 production by macrophages, thus suggesting that OPN plays an important role in the development of fatty-streak lesions in vivo.

Key Words: atherosclerosis • genes • interleukins

	Introduction

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Atherosclerosis is initiated by the infiltration of monocytes and T lymphocytes into an activated endothelium, and the expression of osteopontin (OPN) protein was detected in both macrophages and vascular smooth muscle cells (SMCs) within atherosclerotic lesions.^1–4 We recently reported that OPN transgenic (Tg) mice showed an increase in the medial thickness of artery without injury and an increase in the neointimal formation after injury.⁵ Taken together, these results suggest that OPN may play an important role in the initial step of atherosclerosis, including the proliferation and migration of smooth muscle cells. However, whether or not OPN can promote atherosclerotic lesions remains to be elucidated. The purpose of the present study was to determine whether OPN has a promoting or inhibitory effect on atherosclerosis in OPN-Tg mice.

	Methods

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Animals and Diet
Previously characterized OPN-Tg mice⁵ bred in our laboratory were used under protocols approved by the National Defense Medical College Board for Studies in Experimental Animals. In the present study, we compared heterozygous OPN-Tg to non-Tg littermates (non-Tg). Beginning at 8 weeks after birth and continuing for 16 weeks, both OPN-Tg and non-Tg mice were fed a high-fat/cholesterol diet containing 15% fat, 1.25% cholesterol, and 0.5% sodium cholate, hereafter referred to as the atherogenic diet. These mice were killed at 24 weeks of age to undergo a plasma lipoprotein analysis and lesion assay.

Plasma Lipid Measurements
On the day of analysis, food was removed from the cages in the morning, and the mice were fasted for 7 hours. The plasma total cholesterol, high-density lipoprotein (HDL) cholesterol, and triglyceride levels were then measured by enzymatic assays as previously described.⁶

Quantification of Aortic Sinus Lesions
The sections containing aortic sinus were prepared as previously reported,⁷ with modifications. The area of the lesion was measured with the National Institutes of Health Image 1.55 (public domain software). The values reported represented the mean lesion area from 5 sections for each animal. The extent of atherosclerosis in mouse aorta was also determined with an "en face" method.⁸

Immunohistochemical Studies
Frozen sections were incubated with either a primary rat monoclonal antibody against mouse macrophage, clone MOMA-2 (BioSource International), or a primary goat polyclonal antibody against mouse Interleukin (IL)-10 (Pharmingen). Immunostaining was visualized as previously described.⁵

Analysis of the Ability of IL-10 Production by Macrophages
Resident peritoneal macrophages from both non-Tg and OPN-Tg mice obtained by peritoneal lavage with phosphate-buffered saline were treated with red cell lysis buffer and were incubated (10⁵ macrophages per 100 µL) for 2 hours. The adherent fraction was incubated with lipopolysaccharide (LPS) (30 ng/mL) for 48 hours. Supernatant IL-10 was assayed with commercial ELISA kits (R&D Systems).

Statistical Analysis
The results are shown as the mean±SE. Two groups were compared with either Student’s t test or Student-Newman Keuls’s test with a one-way analysis of variance. P<0.05 was regarded as a significant difference.

	Results

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Development of Fatty-Streak Lesions
Our previous study demonstrated an increase in the medial thickness of the artery, but no evidence of the formation of early form cells in the OPN-Tg mice fed normal chow.⁵ To investigate the impact of OPN on the development of atherosclerotic lesions in vivo, studies were conducted with murine models on an atherogenic diet. After 16 weeks on the atherogenic diet, the total cholesterol (non-Tg: 361.2±15.1 versus OPN-Tg: 325.7±11.3 mg/dL), triglyceride (21.3±2.7 versus 29.2±2.9 mg/dL), and HDL cholesterol levels (58.0±3.8 versus 44.1±3.2 mg/dL), were comparable between the non-Tg (n=12) and OPN-Tg mice (n=12). Figure 1 shows the aortas of both a non-Tg and an OPN-Tg mouse on the atherogenic diet for 16 weeks. There was a marked difference in the lesion size between the OPN-Tg and non-Tg mouse, with the OPN-Tg mouse displaying a significantly larger fatty-streak lesion formation than the non-Tg mouse (Figure 1, upper panels). Furthermore, the thickness of the medial layer of the aorta was greater in the OPN-Tg mouse (Figure 1, middle panels). MOMA-2 staining revealed that the lesions in both mice consisted mainly of foam cells of macrophage (Figure 1, bottom panels), but more lipids were contained in the medial layer, which was not stained by MOMA-2 in the OPN-Tg mouse (Figure 1, arrow, right middle panel). After the mice were fed an atherogenic diet for 16 weeks, the aortic sinus lesions were increased in the OPN-Tg mice to a mean of 17 859±2010 µm² (n=12) versus the non-Tg to a mean of 6469±485 µm² (n=12, P<0.001). Analysis of the extent of atherosclerosis in the aortas en face yielded similar results with a 187% increase in the lesion aorta of OPN-Tg mice compared with non-Tg mice (0.89±0.23% and 0.31±0.01%, P<0.001). These results suggest that an overexpression of OPN may promote early fatty-streak lesions formation in OPN-Tg mice on an atherogenic diet.

View larger version (82K): [in this window] [in a new window]   Figure 1. Representative light photomicrographs of aortic atherosclerotic lesions of both non-Tg (left) and OPN-Tg (right). Sections were stained with oil red O and hematoxylin (upper and middle panels). Boxed areas are shown in middle of panels. Arrow shows lipids were contained in the medial layer. The bottom panels show MOMA-2 staining of both mice. Original magnification x25 (top panels), x50 (bottom panels), and x100 (bottom panels).

OPN Modulation of Macrophage IL-10 Production
The recent mice studies showed that mice deficient in OPN gene expression demonstrated an increase in production of IL-10,⁹ and showed that IL-10 overexpression can inhibit fatty-streak formation in the that were mice fed an atherogenic diet.^10,11 Based on the findings of these reports, we performed immunohistochemical analysis. As shown in Figure 2A, non-Tg mice expressed detectable levels of IL-10 compared with OPN-Tg mice. To clarify the contribution of IL-10, we next examined the production of IL-10 in mice. Figure 2B shows the production of IL-10 by peritoneal macrophages from ONP-Tg and non-Tg mice. Figure 2B (left) shows the production of IL-10 without stimulation, in which both peritoneal macrophage groups released little IL-10. However, the IL-10 levels were less in the OPN-Tg mice than in the non-Tg mice (19.8±1.7 versus 34.0±2.9 pg/mL, P<0.01). Next, the induction of IL-10 production by LPS was performed. These results are shown in Figure 2B (right). The LPS-dependent IL-10 production of macrophages was significantly less in the OPN-Tg mice than in the non-Tg mice (81.8±7.4 versus 304.8±15.0 pg/mL, P<0.0001). These results show that OPN-Tg may decrease in the production of IL-10, especially under the inflammatory conditions, and a decrease of IL-10 may contribute to the susceptibility to atherosclerosis in OPN-Tg mice.

View larger version (59K): [in this window] [in a new window]   Figure 2. A, Representative photomicrographs showing the immunohistochemical staining for IL-10 in atherosclerotic lesions of non-Tg (left) and OPN-Tg (right). IL-10 expression was detected in the lesion of non-Tg (brown staining). Original magnification x150. B, Production of IL-10 by peritoneal macrophages from non-Tg and OPN-Tg mice. The left panel shows the production of IL-10 without LPS and the right panel shows production of IL-10 by macrophages stimulated with LPS. Values represent the mean±SE. *P<0.01; **P<0.001.

	Discussion

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We recently reported that there was no neointimal formation in our OPN-Tg mice fed normal chow, although OPN-Tg mice showed increased medial thickening dependent on aging. However, in the response to arterial injury, more neointima formed in OPN-Tg than in non-Tg mice.⁵ These results suggest that the migration of SMCs and neointima formation by OPN are induced when the intima is impaired. Endothelial dysfunction has been reported to be induced by hypercholesterolemia.¹² As a result, we think hypercholesterolemia induced more atherosclerotic lesions in OPN-Tg than in non-Tg.

A previous study showed the expression of OPN in peritoneal cells of OPN-Tg to significantly increase,¹³ and the present study demonstrated the production of IL-10 by macrophages from OPN-Tg to decrease. These results are compatible with the results of the previous study, in which OPN suppressed the LPS-dependent IL-10 response of peritoneal macrophages.⁹ We think the decrease in IL-10 must contribute to the development of fatty-streak lesions. Indeed, previous studies showed that IL-10 overexpression can inhibit fatty-streak formation in mice fed an atherogenic diet,^10,11 and that IL-10 can induce endothelial cells to inhibit oxidized phospholipids-induced monocyte-endothelial interactions in vitro.¹⁰ These reports suggested that IL-10 might inhibit atherosclerosis. Although our findings suggest that the inhibition of IL-10 in OPN-Tg may play one of the most important roles in the development of atherosclerosis, we also believe that other factors most likely play a role in this phenomenon. For example, OPN has recently been classified as a T-helper 1 cytokine because of its ability to enhance interferon- and IL-12 production and to diminish IL-10.⁹ The strong influence toward a T-helper 1 phenotype has previously been demonstrated in an atherosclerotic model,^14,15 and therefore, we think that these factors may cause an increase of fatty-streak lesions in OPN-Tg mice. Further research is needed to clarify the mechanisms why OPN promotes atherogenesis.

Although the HDL levels decreased in the OPN-Tg, no significant difference was seen between OPN-Tg and non-Tg, and the total cholesterol levels tended to decrease in non-Tg. We thus believe that the HDL levels only slightly contributed to the increased fatty-streak lesions in OPN-Tg. The present study shows that early fatty-streak formation was promoted by OPN but the findings didn’t show whether or not OPN has any effect on more mature and advanced lesions. We thus believe that the OPN overexpression must be bred onto the apoE-deficient background to determine whether or not OPN has any effect on advanced lesions. However, it remains important to determine whether OPN can be modulated by treatment, and whether or not such an approach can be used to modulate the immune responses and reduce atherosclerosis.

	Acknowledgments

 
These studies were supported in part by New Energy and Industrial Technology Development Organization Grants.

Received November 7, 2002; revision received December 18, 2002; accepted December 19, 2002.

	References

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This Article Abstract Full Text (PDF) All Versions of this Article: 107/5/679    most recent 01.CIR.0000055739.13639.D7v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Isoda, K. Articles by Ohsuzu, F. Search for Related Content PubMed PubMed Citation Articles by Isoda, K. Articles by Ohsuzu, F. Related Collections Ion channels/membrane transport