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KR-31378 ameliorates atherosclerosis by blocking monocyte recruitment in hypercholestrolemic mice¹

JIYUN KIM^*, KI-HOAN NAM^*, SUN-OK KIM, JAE-HOON CHOI^*, HYOUNG-CHIN KIM, SUNG-DON YANG, JOO-HYOUNG KANG^*, YOUNG-HAN RYU^*, GOO TAEG OH^*,2 and SUNG-EUN YOO

^* Laboratory of Development and Differentiation, Korea Research Institute of Bioscience and Biotechnology (KRIBB);
Laboratory of Experimental Animal Resources, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Yusong, Daejon, Korea;
AgroPharma Research Institute, Dongbu Hannong Chemical Company, Limited, Daeduck Science Town, Daejon, Korea; and
Center for Biological Modulators of the 21^st Century Frontier Program, Korea Research Institute of Chemical Technology, Yusong, Daejon, Korea

² Correspondence: Laboratory of Development and Differentiation, Korea Research Institute of Bioscience and Biotechnology, 52 Oun-dong, Yusong, Daejon 305-333, Korea. E-mail: gootaeg{at}kribb.re.kr

SPECIFIC AIMS

We sought to determine whether a recently characterized compound, KR-31378, could inhibit atherogenesis and monocyte accumulation in Ldlr knockout mice fed with a high-fat, high-cholesterol (HFHC) diet. Upon finding that this was the case, we then investigated the mechanisms underlying these effects by assessing cell adhesion molecule and chemokine production, and nuclear factor kappa B (NF-B) activation.

PRINCIPAL FINDINGS

1. KR-31378 reduced lesion formation and macrophage accumulation in Ldlr –/– mice
Ldlr knockout mice were divided into two groups and fed an HFHC diet. After 10 weeks of the high cholesterol diet, the control group showed a significant amount of lipid-enriched foam cells in the vessel walls. However, the mean lesion areas in the 0.1% KR-31378-supplemented group were reduced to 50.4% compared with the control group (Fig. 1 ). Upon hypothesizing that the mechanism of the anti-atherogenic effect of KR-31378 could involve decreased recruitment of monocytes into the arterial wall, we examined macrophage accumulation in the aorta by immunohistochemical staining with a macrophage-specific (MOMA-2) antibody. A marked accumulation of macrophages was observed in the subendothelial surface of the control group, but aortic arches from KR-31378-treated mice showed 63% less MOMA-2 staining than did arches from the control group. The proportional area of MOMA-2 staining to fatty streak lesion reduced 30% in the KR-31378-treated group (Fig. 2 ). We investigated possible mechanisms responsible for the lesion area reduction by assessing plasma lipid levels of mice in each group. The atherogenic diet-induced high level of total cholesterol was similar in the KR-31378 test group and controls, and LDL, HDL, and triglyceride levels showed no significant change. This indicated that KR-31378 ameliorated atherosclerosis via mechanisms other than improving lipid profiles.

View larger version (45K): [in this window] [in a new window]   Figure 1. Effect of KR-31378 on atherosclerotic lesion formation in Ldlr knockout mice fed a high-cholesterol diet for 10 weeks. Representative photograph of an aortic valve cross section (X 40) from a member of the control group (A), and the 0.1% (w/w) KR-31378-treated group (B). Quantification of Oil red O-stained aortic valve lesion areas from each group was performed by computer-associated morphometry and is presented in graph (C). Results are shown as mean ± SD, n = 8 (each group). *, P < 0.0001.

View larger version (54K): [in this window] [in a new window]   Figure 2. Reduction of macrophage accumulation in the lesions by KR-31378 treatment. A representative photograph of an aortic valve cross section (X 40) from a member of the control group (A), and the 0.1% (w/w) KR31378-treated group (B) which were stained with a rat monoclonal antibody to mouse macrophages (MOMA-2). The proportional ratio of MOMA-2 staining area to Oil red O staining area was shown in graph (C). Results are shown as mean ± SD, n =8 (each group). *, P < 0.0005.

2. KR-31378 inhibits monocyte attachment and expression of VCAM-1
Inflammatory responses such as monocyte or leukocyte recruitment to the artery wall are crucial to the early stages of atherosclerosis. We examined ability of KR-31378 to block THP-1 monocyte cell adhesion to HUVEC monolayers. Incubation with TNF- caused 3-fold increase in adhesion of THP-1 monocytes compared with adhesion to unstimulated HUVEC cells, but pretreatment of HUVECs with 10^–4 M of KR-31378 reduced the number of THP-1 cells attached to the basal level. Although HUVECs did not constitutively express VCAM-1 without TNF- treatment, the VCAM-1 expression on HUVECs was markedly induced by TNF- treatment for 12 h, and KR-31378 pretreatment reduced VCAM-1.

3. KR-31378 inhibits expression of chemoattractant molecules and monocyte migration
To investigate whether KR-31378 affects cell migration, we performed a ChemoTx assay. Conditioned medium taken from HUVECs stimulated with TNF- resulted in 2.5-fold increase of migrated THP-1 cells compared with medium taken from untreated HUVEC cultures; preincubation with KR-31378 showed attenuated migration of THP-1 cells in a dose-dependent manner. When the migration assay was performed with supernatant taken from THP-1 cells stimulated with LPS and PMA together, THP-1 migration was also reduced, indicating KR-31378 also suppresses monocyte-to-monocyte interaction. We used ELISA to examine the effects of KR-31378 on the release of chemotactic molecules. Incubation of HUVECs with KR-31378 for 2 h prior to TNF- stimulation resulted in a significant, dose-dependent decrease of secretion of IL-8, while there was no apparent change in MCP-1 secretion.

4. KR-31378 inhibits TNF--mediated NF-B activation
NF-B is a transcription factor that can regulate VCAM-1 and IL-8 expression. To determine whether KR-31378 affects NF-B-dependent transcription, we performed transcriptional activation assays using a reporter plasmid containing a minimal promoter with five-upstream B binding sites in the mouse macrophage cell line RAW 264.7. Treatment of RAW 264.7 cells with LPS enhanced the luciferase activity 6 fold, but pretreatment of the cells with KR-31378 diminished the activity in a dose-dependent manner. For relevant biological evidence that KR-31378 represses NF-B-dependent promoter, we performed transcription assay using pGLVCAM-1(-1.1), a reporter plasmid with mouse VCAM-1 promoter. As artificial 5xB-containing promoter, KR-31378 efficiently inhibited upregulation of transcription activity of pGLVCAM-1(-1.1). These results indicate that treatment with KR-31378 efficiently inhibited NF-B-dependent transcription. An electrophoretic mobility shift assay (EMSA) revealed that stimulation with TNF- (20 ng/mL, 15 min) resulted in a significant increase of NF-B DNA binding, while pretreatment with KR-31378 prevented the binding. These data suggest that KR-31378 inhibits TNF--mediated induction of NF-B-dependent gene transcription by blocking NF-B activation and its subsequent binding to DNA. We then performed Western blot assay to determine whether inhibition of activation and translocation of NF-B is due to the inhibition of IB degradation. TNF--induced IB degradation in HUVECs was apparent as early as 5 min after stimulation. However, when we treated the HUVECs with KR-31378 prior to TNF- stimulation, IB degradation was inhibited in a dose-dependent manner. We fractionated HUVECs, and resultant nuclear extracts were assayed for changes in relative abundance of p65 (RelA). In unstimulated cells, p65 was barely detected in nuclear extracts. By 10 min of TNF- treatment, nuclear p65 was abundantly detected, and KR-31378 diminished the protein level in a dose-dependent manner.

CONCLUSIONS AND SIGNIFICANCE

The early stages of atherosclerosis, characterized by recruitment and firm adhesion of leukocytes and monocytes to the arterial wall, closely resemble the inflammation response that follows injury to the endothelium. Inflammation is involved in the initiation, rupture, and thrombosis of atherosclerotic plaques, and recent data has suggested that inhibition of the inflammation response is beneficial to coronary heart disease, especially in the early stages of atherosclerosis. We demonstrated that in a mouse model of atherosclerosis, lesion formation was attenuated by diet with 0.1% KR-31378 without any indication of histological lesions in other tissues. However, an analysis of plasma lipid profiles and immunohistochemistry visualizing macrophage accumulation in the lesion suggest that KR-31378 exerts its anti-atherogenic effect through immune mechanisms rather than by improving lipid profiles. Our THP-1 cell adhesion assay demonstrated that KR-31378 efficiently inhibited monocyte attachment to endothelial cells stimulated by TNF- without attendant cytotoxicity in the tested range. Of the molecules secreted by the activated endothelium, VCAM-1 may preferentially contribute to monocyte adhesion and play a critical role in initiation of the inflammatory response in atherogenesis. In our experiment, pre-treatment with KR-31378 substantially decreased TNF--induced VCAM-1 expression (33%), which may explain the reduced monocytic attachment. Indeed, THP-1 migration and chemokine IL-8 secretion were also inhibited. We hypothesized that KR31378 may regulate endothelial cell and monocyte activity by down-regulating not only adhesion molecule expression but also monocyte chemoattractant production, resulting in a synergistic suppression monocyte recruitment in vivo.

VCAM-1 and IL-8 play roles in the recruitment of monocytes, and their expression is regulated via TNF--induced NF-B signaling. We showed that in RAW 264.7 monocytes, preincubation with KR-31378 repressed TNF--stimulated transcription from the NF-B-dependent promoter in a dose-dependent manner. KR-31378 apparently inhibited TNF--induced degradation of IB in the HUVECs not affecting IKK expression level. The data imply that KR-31378 might modulate IB degradation, which leads to down-regulation of NF-B. Consistent with results above, KR-31378 reduced translocation into the nucleus and DNA binding of NF-B. In the present study, it was not clarified how KR-31378 exerts an inhibitory effect on NF-B activation. Reportedly, hydrogen peroxide and oxygen radicals are produced during inflammatory processes and play second messengers, mediating activation of NF-B activation directly or indirectly. There is a great deal of evidence that inhibitors of reactive oxygen species (ROS), i.e., anti-oxidants, such as flavonoid, -tocopherol, ascorbate, troglitazone, aspirin, and gallate, have potential anti-atherogenic effects, suggesting that ROS inhibitors decrease NF-B activation induced by TNF- or IL-1, which then leads to decreased activation of adhesion molecules and chemoattractants needed for atherogenic migration and adhesion. In the previous study, KR-31378 showed the properties of scavenging the intracellular ROS and peroxyl radicals and KR-31378 effectively inhibited TNF- production induced by LPS in vitro. Thus, we speculate that the mechanism of inhibition of NF-B by KR-31378 involves anti-oxidant action.

Our results suggest that KR-31378 exerts its anti-atherogenic effects via suppressing cell adhesion molecule and chemokine expression by inhibiting NF-B activation (Fig. 3 ). This provides the possibility that in addition to its neuroprotective activity via its anti-inflammatory actions, which modulate multiple cellular activities and interactions, KR-31378 may be a valid therapeutic candidate for the treatment of vascular disease and atherosclerosis.

View larger version (19K): [in this window] [in a new window]   Figure 3. Scheme of the action mechanism of KR-31378. Stimulations outside the cell upregulate NF-B signal pathway, which regulates adhesion molecules and cytokines important to atherogenesis. By blocking activation of NF-kB activation, KR-31378 exerts anti-atherogenic effects.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0652fje; doi: 10.1096/fj.03-0652fje

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