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Attenuation of atherogenesis by systemic and local adenovirus-mediated gene transfer of interleukin-10 in LDLr^-/- mice ¹

JAN H. VON DER THÜSEN^*,, JOHAN KUIPER^*, MADELON L. FEKKES^*, PAULA DE VOS^*, THEO J. C. VAN BERKEL^* and ERIK A. L. BIESSEN^*2

^* Division of Biopharmaceutics, Leiden/Amsterdam Center for Drug Research, 2300 RA Leiden; and
Department of Cardiology, Leiden University Medical Center, 2300 RC Leiden, The Netherlands

²Correspondence: Division of Biopharmaceutics, Leiden/Amsterdam Center for Drug Research, Sylvius Laboratories, Leiden University, Wassenaarseweg 72, P.O. Box 9503, 2300 RA Leiden, The Netherlands. E-mail: biessen{at}lacdr.leidenuniv.nl

SPECIFIC AIM

In view of its multifaceted anti-inflammatory properties, interleukin 10 (IL-10) has been deemed to be potentially anti-atherogenic. We have evaluated the capacity of adenoviral gene transfer of IL-10 for the modulation of de novo atherosclerotic lesion formation by systemic and local overexpression of IL-10 in a model of collar-induced carotid atherosclerosis in low density lipoprotein receptor knockout (LDLr^-/-) mice.

PRINCIPAL FINDINGS

1. Systemic IL-10 overexpression leads to prolonged elevation of IL-10 levels, to systemic immunomodulation, and to lowering of plasma cholesterol levels
After intravenous administration of 1*10⁹ pfu of adenovirus bearing human IL-10 (Ad.IL-10) to female LDLr^-/- mice, plasma IL-10 levels peaked 12 days after injection at 3.4 ± 1.0 ng/ml and declined thereafter (Fig. 1 A). Significant expression was maintained for the duration of the experiment, as at the time of death (5 wk after injection) IL-10 remained raised at 1.2 ± 0.7 ng/ml. Indeed, in a separate experiment, IL-10 was found to be elevated for up to 217 days after inoculation, when the plasma level amounted to 0.32 ± 0.12 ng/ml. To establish whether overexpression of IL-10 had affected endogenous IL-10 production, we used quantitative RT-PCR to determine hepatic mRNA levels for IL-10 and IL-10 receptor (IL-10r). These were not found to differ significantly between treatment groups. Hepatic mRNA levels for tumor necrosis factor (TNF-) were markedly lowered by IL-10, as determined by quantitative RT-PCR 12 days after adenovirus inoculation (P<0.05, Fig. 1B ). In an ex vivo whole blood assay, TNF- production by monocytes in response to incubation with lipopolysaccharide (LPS) was used as a measure for the activation status of circulating monocytes. Ad.IL-10 was found to significantly reduce TNF- elaboration at all concentrations examined (ranging from 0.5 to 75 ng/ml (Fig. 1C ). Overexpression of IL-10 was also seen to result in a significant decrease in total plasma cholesterol by 12 days after injection (P<0.05), with a nadir at 21 days (810.4±148.2 mg/dl vs. 1841.9±155.2 mg/dl (-56.0%), P <0.001). Nonlinear regression fitting to a sigmoidal dose response curve revealed a high correlation between plasma total cholesterol levels and plasma IL-10 concentration (R²=0.80), with an estimated EC₅₀ of 3.4 ng/ml (i.e., 0.2 nM). The relative hypocholesterolemia was related to a lowered cholesterol content of the very low density lipoprotein (VLDL) and low density lipoprotein (LDL) fractions. The rate of VLDL synthesis, however, was not found to be altered 12 days after Ad.IL-10 administration, as determined by plasma triglyceride accumulation after Triton WR1339 injection.

View larger version (17K): [in this window] [in a new window]   Figure 1. A) Plasma levels of human IL-10 (ng/ml), as determined by ELISA, up to 35 days after systemic transfection with Ad.empty () or Ad.IL-10 () virus (n=7). Quantitative RT-PCR of hepatic mRNA (B) for endogenous IL-10, IL-10 receptor (IL-10r), and TNF- 12 days after systemic transfection. Levels of TNF- mRNA were significantly decreased by Ad.IL-10 (*=P<0.05, Student’s t test). C) Elaboration of TNF- by whole blood in response to lipopolysaccharide (LPS) (n=3). Whole blood was diluted 25x in DMEM and incubated overnight with varying concentrations of lipopolysaccharide. Across the entire concentration range, TNF- levels were lower in the Ad.IL-10-treated animals (P<0.05, ANOVA).

2. Systemic overexpression of IL-10 attenuates de novo atherogenesis
Systemic overexpression of IL-10 resulted in a 55.8% reduction of the plaque surface area (7852±2,302 vs. 17,768±4,673 (µm²), which translated to a marked (73.9%) decrease in the intima/media ratio (0.21±0.07 vs. 0.79±0.25, P<0.05) and a diminished degree of lumen stenosis (Fig. 2 A, B; intima/lumen ratio; 13.4±4.5% vs. 35.5±6.8%, P<0.02). The decrease in plaque size was found to be largely attributable to a decrease in the macrophage content of the lesion, as determined by MOMA-2 staining.

View larger version (132K): [in this window] [in a new window]   Figure 2. Representative cross sections of collar-induced plaques (hematoxylin-eosin staining) in carotid arteries of LDLr-/- mice after transfection with Ad.empty or Ad.IL-10 by either systemic (A, B, resp.) or local administration (C, D, resp.). Both methods of IL-10 transduction resulted in significant decreases in plaque size and complexity.

3. Transluminal IL-10 transfection inhibits atherogenesis virtually as effectively as systemic IL-10
Endothelial Ad.IL-10 incubation was not seen to lead to systemic overspill of IL-10, with levels averaging 0.03 ± 0.02 ng/ml 12 days after transfection, which did not differ from controls (P=0.27). Similarly, plasma cholesterol levels did not differ between groups after local transfection with Ad.empty or Ad.IL-10. The atheroprotective effects of local endothelial transduction with Ad.IL-10, however, were found to be only slightly less pronounced than those achieved with systemic transfer, as evidenced by a 49.5% reduction in plaque size (16,503±3907 vs. 32,659±4968 (µm², P<0.03) and a concomitant decrease in I/M ratio (0.65±0.12 vs. 0.98±0.15) and lumen stenosis (Fig. 2C , D ; 28.2±2.8% vs. 51.2±7.5%, P<0.05).

4. Systemic and local IL-10 limit the histological complexity of atherosclerotic plaques
Both methods of IL-10 administration were also found to lead to a similar decrease in the degree of lesion complexity. Whereas Ad.empty-treated arteries contained lesions consisting of a well-demarcated fibrous cap overlying a necrotic core, Ad.IL-10-treated animals had considerably less advanced plaques, consisting primarily of macrophages (Fig. 2) .

CONCLUSIONS

Systemic overexpression of IL-10 by adenoviral transfer was found to be highly efficacious in preventing de novo atherosclerosis in LDLr^-/- mice, as reflected by a threefold reduction in the intima/lumen ratio. The underlying anti-atherogenic mechanism may be bipartite, as systemic IL-10 overexpression was found to have both immunological and metabolic effects. Hepatic expression of Ad.IL-10 led to local and systemic immunosuppression, as evidenced by a marked decrease in hepatic mRNA levels for TNF- and by relative anergy of circulating monocytes with respect to LPS-induced TNF- production, respectively.

In addition to its immunomodulatory activity, we have shown systemic Ad.IL-10 administration to result in lowering of VLDL and LDL cholesterol levels in LDLr^-/- mice. Nonlinear regression analysis of plasma cholesterol levels relative to IL-10 levels returned a close correlation to a dose-effect model, with an EC₅₀ (3.4 ng/ml) that is of the same order of magnitude as the K_d found for the binding of human IL-10 to the murine IL-10 receptor (1.3 ng/ml). This led us to hypothesize that a direct receptor-mediated mechanism could be responsible for the observed hypocholesterolemia. Screening for differential expression of hepatic enzymes involved in lipid metabolism by quantitative RT-PCR, however, did not yield any significant differences, and neither fasting glucose nor insulin levels were affected by Ad.IL-10 treatment. Its precise mechanism of action thus remains to be determined, but the metabolic consequences of systemic IL-10 administration may contribute significantly to its therapeutic efficacy (Fig. 3 ). Local IL-10 treatment is virtually equipotent in comparison with systemic IL-10 overexpression (49.5% vs. 55.8% reduction in plaque size, respectively). Its anti-inflammatory properties are likely to have been instrumental in this respect, including down-regulation of NF-B activity, inhibition of cytokine production, and a reduction in endothelial adhesion molecule expression (Fig. 3) . This in turn this may have resulted in decreased monocyte extravasation and deactivation of intimal macrophages, and thus diminished accumulation of foam cells in the atherosclerotic plaque. We are currently performing additional studies to quantify the immunosuppressive effects of IL-10 in the vessel wall.

View larger version (44K): [in this window] [in a new window]   Figure 3. Putative mechanisms of the anti-atherogenic activity of IL-10 in the prevention of de novo atherosclerosis. Monocyte extravasation is attenuated by adenovirus-mediated hepatic overexpression of human IL-10 (blue) through the deactivation of endothelial cells and circulating monocytes and by lowering of serum cholesterol levels (particularly in the VLDL and LDL fractions). The former can lead to direct inhibition of monocyte extravasation at predisposed arterial sites, whereas the latter may exert indirect anti-inflammatory effects on endothelial cells and resident macrophages mediated by, inter alia, down-regulation of NF-B activity. In addition, IL-10 has been shown to shift T helper cells from a type I to a type II cytokine expression pattern, which may limit the amount of T cell-mediated inflammation in the developing plaque. The observed inhibition of atherosclerosis after local transfection is mediated by autocrine and paracrine effects of IL-10 expressed in endothelial cells (red), which result in suppression of vessel wall inflammation. Th = T helper cell; EC = endothelial cell; MO = monocyte; M = macrophage; ICAM-1 = intercellular adhesion molecule 1; VCAM-1 = vascular cell adhesion molecule 1; MCP-1 = monocyte chemoattractant protein 1; ROS = reactive oxygen species; iNOS = inducible nitric oxide synthase.

In conclusion, the marked efficacy of local IL-10 therapy obviates the need for systemic administration and yields potential benefits when considering the therapeutic applicability of IL-10 transfer for the prevention of human atherosclerosis. Local instillation of IL-10 may result in considerable amelioration of the side effect profile compared with systemic IL-10 therapy, as it does not lead to extended immunosuppression and modulation of lipid metabolism.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0483fje; to cite this article, use FASEB J. (October 29, 2001) 10.1096/fj.01-0483fje

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