Chronic unpredictable stress accelerates atherosclerosis through promoting inflammation in apolipoprotein E knockout mice

Thrombosis Research 126 (2010) 386–392

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Thrombosis Research j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / t h r o m r e s

Regular Article

Chronic unpredictable stress accelerates atherosclerosis through promoting inflammation in apolipoprotein E knockout mice Tao Zhang a, Yundai Chen a, Hongbin Liu a,⁎, Zhenhong Zhou b, Yongzhi Zhai c, Junjie Yang a a b c

Department of Cardiology, PLA General Hospital, Beijing 100853, China Department of Pathology, PLA General Hospital, Beijing 100853, China Department of Emergency, PLA General Hospital, Beijing 100853, China

a r t i c l e

i n f o

Article history: Received 16 May 2010 Received in revised form 5 July 2010 Accepted 30 July 2010 Available online 25 August 2010 Keywords: Chronic unpredictable stress Atherosclerosis Inflammation Vascular cell adhesion molecule-1 Intercellular adhesion molecule-1 C-reactive protein Interleukin-6

a b s t r a c t Introduction: Chronic unpredictable stress (CUS) has been suggested to accelerate atherosclerosis. However, the underlying mechanism of this adverse effect is not fully understood. Since chronic stress can promote or even initiate inflammation response, which is thought to be a major contributor to atherogenesis, we postulated that stress-induced inflammatory response might be one important reason for CUS-promoted atherosclerotic disease. Materials and methods: We used the CUS treated apolipoprotein E (ApoE)-deficient mice, which have been shown to spontaneously develop atherosclerosis with features similar to those seen in humans, as an animal model. Haematoxylin and eosin staining and immunohistostaining were used to analyze the plaque formation and composition. Results: Histological analysis clearly demonstrated that CUS treatment promoted the development of atherosclerotic lesions, such as triggering plaque rupture, increasing plaque size and plaque-to-surface ratio, and also led to profound changes in plaque composition, as evidenced by increased macrophage and T cell infiltration and decreased smooth muscle cell mass, all reflecting an unstable plaque phenotype. Moreover, adhesion molecular vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1), acute phase reactant C-reactive protein (CRP), and proinflammatory cytokine interleukin-6 (IL-6) -/were significantly enhanced in CUS treated ApoE mice compared with untreated control animals (P b 0.01). Conclusion: The involvement of CUS in the pathogenesis of atherosclerosis is at least partially attributable to its acceleration of inflammation. © 2010 Elsevier Ltd. All rights reserved.

Introduction Atherosclerosis and related cardiovascular diseases represent one of the greatest threats to human health worldwide. Despite important progress in prevention and treatment, these conditions still account for one third of all deaths annually [1]. Atherosclerosis is not only merely a lipid disorder, but also a chronic inflammatory disease [2,3]. Inflammation is thought to be a major contributor to atherogenesis through adverse effects on lipoprotein metabolism and arterial wall biology, which is mediated by components of the innate immune system, including macrophages and dendritic cells (DCs) [4,5] and by components of the adaptive immune system, including T lymphocytes [6,7]. Monocytes and T-cells migrate from the circulation into the intima of the arterial wall where monocytes differentiate into macrophages, which then take up modified lipoproteins thereby transforming into foam cells. Both monocyte-derived macrophages and T lymphocytes are abundantly present at all stages of atheroscle-

⁎ Corresponding author. E-mail address: [email protected] (H. Liu). 0049-3848/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2010.07.022

rotic process and the lesion development [8–10]. Their pivotal role in atherogenesis has been demonstrated by the attenuation of lesion formation in monocyte-deficiency in apolipoprotein E (ApoE)-/- mice and Low-density lipoprotein receptor (LDLR)-/- mice [11,12]. Many factors contribute to atherosclerosis, including high blood pressure, tobacco smoke, diabetes, high levels of cholesterol in the blood [13]. However, the absence of such "traditional" risk factors does not completely protect from the disease, indicating additional factors involved in the development of atherosclerosis [13,14]. Psychosocial stress, especially chronic stress is one kind of these nontraditional risk factors for atherosclerosis. Potential toxic elements in the personality construct such as hostility, anger, cynicism, mistrust, and unhealthy lifestyle [15–17], as well as social isolation [16], lack of social support [17], and work-related stress [18], increase the risk for cardiovascular disease, suggesting a strong causal relationship between chronic stress and the development of atherosclerosis [15]. Moreover, experimental studies also demonstrated that chronic stress accelerates atherosclerosis in the ApoE deficient mice through increasing atheroma and corticosterone concentrations [19]. Another study also showed that social isolation increases atherosclerosis and plasma lipids in ApoE-/- mice [20].

T. Zhang et al. / Thrombosis Research 126 (2010) 386–392

Despite the threat of psychosocial stress to public health, not much is known about the mechanisms linking psychosocial health and cardiovascular disease. Goran Bergstrom's research group hypothesizes that repeated and/or exaggerated episodes of activation of stress-related neurohormonal systems promote atherosclerosis and thereby cardiovascular disease [21]. Bierhaus and his colleges has shown that noradrenaline-dependent adrenergic stimulation results in activation of NF-κB in vitro and in vivo, which represents a downstream effector for the neuroendocrine response to stressful psychosocial events and links changes in the activity of the neuroendocrine axis to the cellular response [22]. Stress-induced activation of mononuclear interleukin-1β (IL-1β) is a novel mechanism potentially linking stress and heart disease [23]. Gu et al showed that chronic mild stress (CMS) mice increased aortic atherosclerosis, which were associated with significant increases in levels of expression of Toll-like receptor (TLR4), myeloid differentiation primary response gene 88 (MyD88), nuclear factor kappa-lightchain-enhancer of activated B cells (NF-κB), monocyte chemotactic protein-1(MCP-1), IL-1β, tumor necrosis factor-α (TNF-α), and soluble intercellular adhesion molecule-1 (sICAM-1). They concluded that TLR4 signaling pathway plays an important role in atherosclerosis in their CMS mouse model [24]. Considering that chronic stress can promote or even initiate inflammation reactions [25], we postulate the hypothesis that stress-induced inflammatory responses are an important reason for promoting atherosclerotic disease. To test this hypothesis, we used a chronic unpredictable stress (CUS) model in ApoE-/- mice to investigate the effect of CUS on inflammatory cell components, adhesion molecular vascular cell adhesion molecule-1 (VCAM-1) and ICAM-1, acute phase reactant C-reactive protein (CRP), and proinflammatory cytokine interleukin-6 (IL-6). Material and methods Animals Male ApoE-/- mice on a Balb/c background (n = 20) were purchased from Peking University (Beijing, China). Mice were maintained in a specific pathogen-free environment on a 12-hour dark/light cycle. They were provided with standard rodent chow and water ad libitum and housed at 23 ± 2 °C. At 20 weeks of age the ApoE-/- mice were assigned to two groups. One group of ApoE-/- mice received CUS treatment (n = 10). The other group of ApoE-/- mice (n = 10) remained untreated and were used as a control group for the comparison with the CUS treated mice. Before and during CUS treatment, mice were weighted at Week 0 (initial week) and Week 4 of the procedure. All the procedures with animal were approved by Institutional Animal Care and Use Committee of PLA General Hospital, Beijing, China. CUS treatment Chronic unpredictable stress mouse model was induced as described by Willner and his colleagues [26]. Briefly, ApoE-/- mice assigned to the chronic stress group were exposed to the following CUS protocol: Day 1: 8:00 a.m. heat stimulation at 45 °C for 5 min (animals were forced to swim in a plastic cylinder with 40 cm in diameter and 60 cm in height, which were filled with a half-volume of water at 45 °C); Day 2: 10:00 p.m. cage tilting for 12 h (animals were placed in a polycarbonate cage and tilted at an angel of 45°); Day 3: 10:00 a.m. wet bedding for 12 h (400 ml tap water in home cage); Day 4: 10:00 p.m. lights on overnight; Day 5: 8:00 a.m. tail pinch (2 cm apart from the end of the tail) for 1 min; Day 6: 3:00 p.m. high-speed agitation for 1 min; Day 7: 8:00 a.m. cold stimulation at 4 °C for 5 min (animals were forced to swim in water at 4 °C); Day 8: 12:00 p.m. overhang for 5 min; Day 9: 8:00 a.m. water deprivation for 24 h; Day 10: 8:00 a.m. food deprivation for 48 h; Day 12: 10:00 p.m. lights on

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overnight; Day 13: 10:00 a.m. wet bedding for 12 h; Day14: 10:00 p.m. cage tilting for 12 h. The stress procedure was repeated once during Day 15 to Day 28. Open field test Open field test was done as described by Dunn and his colleges [27]. The Spontaneous locomotor activity was quantified for 5 min in an open field, a wooden box (59 × 59 cm) with its floor divided into 16 squares illuminated by white light. Four squares were defined as the center and the 12 squares along the walls as the periphery. Each mouse was gently place in the very center of the box and anxiety-like behavior was scored by the time spent in the central squares, the numbers of line crossing (defined as numbers of with which the mice crossed one of the grid lines with all four paws ), and the numbers of grooming. Quantitation of atherosclerosis, plaque composition and immunohistochemisty Atherosclerotic sections were prepared as described by Desai et al [28]. Briefly, the thorax and abdomen were opened, and blood samples were collected. After perfusion with phosphate-buffered saline (PBS), the brachiocephalic artery was removed, fixed in buffered formalin, embedded in paraffin, and serially sectioned at 5 μM. For quantification of atherosclerotic lesions, 45 serial sections from the brachiocephalic artery root were collected. Ten sections sampled from every four consecutive sections were stained with hematoxylin and eosin. Lesion areas were quantitated by light microscopy (Olympus BX40). Lesion surface area and total aortic surface area were measured using Image Pro Plus version 6 (Media Cybernetics, Bethesda, MD). Three to four sections from eight animals per group were analyzed by immunohistochemical staining. Paraffin-embedded brachiocephalic artery sections were deparaffinized by immersion in xylene, followed by a series of alcohol treatments. Endogenous peroxidase activity was quenched by immersing the slides in 0.3% hydrogen peroxide in methanol for 15 minutes. The sections were rinsed in PBS three times for 5 minutes each and were blocked with 5% normal goat serum for 30 min. Primary antibodies were incubated for 90 minutes (rabbit anti-mouse CD68, BOISYNTHESIS, CHINA, 1:200 dilution; rabbit anti-mouse CD3, BOISYNTHESIS, CHINA, 1:200 dilution; rabbit anti-mouse α-actin, Chemicon, USA, 1:200 dilution; rabbit antimouse VCAM-1, Santa Cruz, USA, 1:200 dilution; rabbit anti-mouse ICAM-1, Santa Cruz, USA, 1:200 dilution). Sections were washed and incubated with HRP-conjugated secondary antibodies for 30 minutes. Staining was detected using the Alkaline Phosphatase Standard ABC Kit (Vector Labs, Burlingame, CA). The slides were counterstained with hematoxylin (Sigma, St. Louis, MO, USA). Positive staining areas were automatically traced using the Image Pro Plus Version 6.0. The total optical density (OD) was calculated using the following formula: OD = (1/red intensity + 1/blue intensity + 1/green intensity) × area for positive staining [29]. The OD was measured at 2 levels in the aortic root and an average OD was calculated for each mouse. Lipid analysis and plasma VCAM-1, ICAM-1, high sensitivity CRP (hsCRP) and IL-6 Blood samples were taken for measurement of lipids from control and CUS-treated ApoE-/- mice. The levels of triglycerides (TG), total cholesterol (TC) and cholesterol linked to high density lipoproteins (HDL-C) were measured using colorimetric enzymatic methods using a Roche/Hitachi analyzer (Roche Diagnostics, Indianapolis, IN, USA). The fraction of cholesterol linked to low density lipoproteins (LDL-C) was calculated using the Friedewald formula [30].

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Venous blood sampling from ApoE-/- mice was collected in plain tubes, and was centrifuged. Serum was subsequently removed, aliquoted and stored at -80 °C for use in biochemical analysis. Plasma levels of sVCAM-1, sICAM-1 and IL-6 were measured by ELISA kits (R&D Systems Inc., USA) according to the manufacturer's instructions. Plasma hsCRP level was measured by mouse high sensitivity C-Reactive Protein,hs-CRP ELISA Kit (Wuhan EIAab Science Co.,Ltd., Wuhan, China) according to the manufacturer's instructions.

Statistical analysis Data are expressed as mean ± SD. All statistical analyses were performed with SPSS.11 software for windows (SPSS Inc., Chicago, IL, USA). Significance between controls and treatment samples was calculated by Student's t test. P-values less than 0.05 were considered as significant.

The cellular components of atherosclerotic plaque in CUS-treated ApoE-/mice As changes in plaque composition can have profound influences on plaque instability and thus the risk to patient [31,32], we analyzed the plaque composition by measuring the relative content of inflammatory cells, macrophages and T cells and smooth muscle cells in the atherosclerotic lesions of the brachiocephalic artery in both CUS treated and untreated ApoE-/- mice by immunohistochemical staining of the cell surface molecules: CD68, CD3 and α-SM actin, which are the cell markers of macrophages, T cells, and smooth muscle cells, respectively. The lesions in CUS-treated ApoE-/- mice revealed a marked increase in macrophage and T cell content in all sectors compared with untreated ApoE-/- mice (P b 0.01). Whereas smooth muscle cell content in the lesions of CUS treated mice was markedly lower than that in control mice (7.76 ± 0.44% vs. 4.87 ± 0.61%, P b 0.01; Fig. 2G-I). Taken together, all reflected an unstable atherosclerotic plaque phenotype. CUS treatment elevates the concentrations of VCAM-1 and ICAM-1 in plaques and in plasma

Results The effect of CUS treatment on the locomotor activity of ApoE-/- mice To evaluate the behavioral responses of ApoE-/- mice to CUS treatment, open field test was done during the CUS treatment. The locomotor activity was not shown any significant difference between CUS treated and control ApoE-/- mice at week 0, 1, 2, and 3 (data not shown). But at week 4, CUS treatment markedly increased the time spent in the central squares and decreased the numbers of line crossing and grooming in ApoE-/- mice compared with control ApoE-/mice (P b 0.01) (Table 1). All of these results indicated that CUS treatment started to decrease the locomotor activity and induce anxiety-like behavior in ApoE-/- mice at week 4.

Exposure to CUS increases aortic atherosclerotic development in ApoE-/mice To investigate the effect of CUS treatment on atherosclerotic lesion formation in ApoE-/- mice, H&E staining was used to analyze atherosclerotic lesion size (Fig. 1A and B). Advanced atherosclerotic plaques were observed in CUS treated and untreated ApoE-/- mice. Exposure to CUS for 4 weeks promoted the development of atherosclerotic lesions, such as triggering plaque rupture, and increasing plaque sizes, in brachiocephalic artery of ApoE-/- mice (Fig. 1B). CUS treatment significantly increased both atherosclerotic plaque size and plaque-to-surface ratio in ApoE-/- mice (P b 0.05 relative to control ApoE-/- mice) (Fig. 1C and D). Prior to CUS treatment, the body weight and the baseline of serum lipid levels (TC, TG, LDL-C and HDL-C) were comparable between CUS treated and untreated ApoE-/- mice (Table 2). However, 4 weeks after exposure to CUS, both the body weight and serum lipid levels of ApoE-/- mice, in terms of TC, TG and LDL-C levels, were significantly higher than that of untreated ApoE-/- mice (P b 0.05). Whereas, the HDL-C levels in CUS treated ApoE-/- mice was markedly lower than that in control ApoE-/- mice (P b 0.05).

Table 1 Effect of CUS treatment on the locomotor activity of ApoE-/- mice at week 4. Group

Time spent in the central squares (s)

Numbers of line crossing

Numbers of grooming

Control CUS

9.1 ± 2.0 13.5 ± 1.6⁎⁎

25.6 ± 3.5 19.2 ± 2.4⁎⁎

9.6 ± 2.5 6.0 ± 2.3⁎⁎

⁎⁎ P b 0.01 compared to the control mice; n = 10.

Elevated levels of adhesion molecules on the endothelial cells facilitate the infiltration of the entry of monocytes, T cells and atherogenic lipoproteins into the sub-endothelial space, during the earliest fatty streak lesion formation [33]. We tested the effect of CUS treatment on the levels of endothelial cell adhesion molecules ICAM-1 and VCAM-1 by immunohistochemical staining (Fig. 3A-F). Fig. 3C and F showed that CUS treatment significantly (P b 0.01) enhanced the expression levels of both VCAM-1 and ICAM-1 in the atherosclerotic lesions of ApoE-/- mice. Soluble adhesion molecules can promote plaque instability and weaken the plaque fibrous cap and accelerate plaque rupture and thrombosis [34–36]. We also tested the soluble VCAM-1 (sVACM-1) and ICAM-1 (sICAM-1) in serum (Fig. 3G and H). Exposure to CUS for 4 weeks also significantly increased the sVACM-1 and sICAM-1 in serum compared with untreated control ApoE-/- mice (P b 0.01). CUS treatment enhanced the production of hsCRP and IL-6 in plasma in ApoE-/- mice Acute phase reactant hsCRP and its regulator proinflammatory cytokine IL-6 have been shown to contribute to atherosclerotic plaque development and plaque destabilization via a variety of mechanisms [37,38]. Therefore, we investigated the effect of CUS treatment on the production of hsCRP and IL-6 in the serum of ApoE-/- mice. ELISA analysis showed that CUS treatment significantly increased the production of both hsCRP (Fig. 4A) and IL-6 (Fig. 4B) in the serum of ApoE-/- mice compared with untreated control mice (P b 0.01). Discussion Although the impact of psychosocial stress on atherosclerosis have frequently been described in clinical and experimental research [15–20], much less is known about the molecular mechanisms converting psychosocial stress into biochemical changes and cellular dysfunction. In present study, we present that CUS treatment increases the atherosclerotic plaque size and promotes the formation of unstable atherosclerotic lesions through modulating the serum lipid levels (increasing TC, TG and LDL-C levels and decreasing HDL-C levels), enhancing macrophage and T cell accumulation, decreasing smooth muscle cell mass on the atherosclerotic plaques and elevating the production of adhesion molecular VCAM-1 and ICAM-1, acute phase reactant CRP and proinflammatory cytokine IL-6. Consistent with the published data [19,20], we also showed that CUS treatment accelerates atherosclerosis. We found that CUS

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Fig. 1. Photographs of atherosclerotic lesions in the brachiocephalic artery of ApoE-/- mice with or without CUS treatment. H&E staining were used to analyze the development of atherosclerotic lesions in untreated control ApoE-/- mice (A) and CUS treated ApoE-/- mice (B). CUS treatment promoted the formation of unstable plaque formation (B, asterisk: plaque rupture) and significantly enhanced the plaque size (C) and plaque-to-surface ratio (D). The data shown are the mean ± SD of three independent experiments. ⁎P b 0.05 compared to untreated control ApoE-/- mice. Original magnification, × 100.

treatment not only increases atherosclerotic lesion size, but also increases plaque-to-surface ratio that exhibited severe endothelial dysfunction. Interestingly, we also found that CUS treatment triggers plaque rupture, the predominant pathological substrate of acute cerebrovascular and cardiovascular events such as stroke and myocardial infarction (MI) [39], which indicates that CUS-promoted atherosclerosis is associated with plaque progression and instability. Atherosclerotic plaques consist of an accumulation of vascular smooth muscle cells (VSMCs), inflammatory cells (macrophages, T lymphocytes, dendritic cells, and mast cells) underlying a dysfunctional endothelium, together with extracellular lipid, collagen and matrix [28]. The changes in plaque composition can have profound influences on plaque instability [28,31]. For example, the continued recruitment of T-cells and macrophages at sites of ‘dysfunctional endothelium’ appears to be a constant feature of lesion initiation and progression as it accentuates the chronic inflammatory nature of atherosclerosis [2–12]. In contrast, loss of VSMCs from the fibrous cap, for example via the process of apoptosis, would be predicted to promote plaque instability, and both human and animal data support this assertion [32,40]. Immunohistochemical staining showed that CUS treatment significantly increases the accumulation of macrophages and T cells on the atherosclerotic lesions and

Table 2 Effect of CUS on body weight and serum lipid composition in ApoE–/– mice at week 4. Parameter

Body weight (g) TC (mg/dl) TG (mg/dl) LDL-C (mg/dl) HDL-C (mg/dl)

Week 0

Week 4

Control

CUS

Control

CUS

18.1 ± 0.1 209.3 ± 13.2 578.5 ± 84.7 313.6 ± 34.2 169.3 ± 32.6

18.1 ± 0.2 210.6 ± 5.2 580.3 ± 73.6 322.6 ± 30.7 171.8 ± 24.3

30.2 ± 0.9 381.8 ± 92.4 1025.2 ± 96.7 750.4 ± 81.2 186.4 ± 30.1

25.3 ± 0.8⁎ 571.4 ± 163.2⁎ 1411.6 ± 101.2⁎ 1024.8 ± 142.5⁎ 235.1 ± 22.7⁎

TC: total cholesterol; TG: triglyceride; LDL-C: low-density lipoprotein cholesterol; HDLC: high-density lipoprotein cholesterol. ⁎ P b 0.05 compared to the control mice; n = 10.

decreases smooth muscle cell mass, which indicates CUS treatment mediated progression of atherosclerosis. CUS-mediated changes in plaque composition also predict to promote plaque instability, which is consistent with triggering plaque rupture in CUS-treated ApoE-/mice. Endothelial cell adhesion molecules, such as VCAM-1 and ICAM-1, play an important role on the infiltration of the entry of macrophages and T cells into the sub-endothelial space during the lesion formation [32]. We tested the effect of CUS treatment on the expression of VCAM-1 and ICAM-1. Consistently, CUS treatment also significantly increased the expression of VCAM-1 and ICAM-1 in atherosclerotic plaques. It seems that CUS treatment increases the expression of adhesion molecular VCAM-1 and ICAM-1, which in turn initiates the formation of atherosclerotic lesions and promotes the accumulation of macrophages and T cells in the atherosclerotic lesions. We also tested the soluble VCAM-1 and ICAM-1 in the serum, which have been documented to promote plaque instability, weaken the plaque fibrous cap, and accelerate plaque rupture and thrombosis [32–35]. Interestingly, CUS treatment increased the VCAM-1 and ICAM-1 in serum, which could be used to explain the formation of unstable plaques in the CUS treated ApoE-/- mice. However, it is still unclear to what extent the increase of endothelial cell and soluble VCAM-1 and ICAM-1 expression contributes to the accumulation macrophages and T cells in atherosclerotic lesions and the formation of unstable atherosclerotic plaques. Blockade of VCAM-1 and ICAM-1 using their specific antibodies or knockdown their expression using RNA interference would be helpful to resolve this issue. CRP, an important downstream marker of inflammation, was shown to have proinflammatory and proatherogenic effects during atherosclerosis development in numerous studies, such as decreasing nitric oxide and prostacyclin, increasing endothelin-1, cell adhesion molecules, monocyte chemoattractant protein-1 (MCP-1) interleukin-8 (IL-8), and plasminogen activator inhibitor-1 (PAI-1) in endothelial cells; inducing tissue factor secretion, increasing reactive oxygen species and proinflammatory cytokine release, promoting monocyte chemotaxis and adhesion, and increasing oxidized low-density lipoprotein uptake

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Fig. 2. Exposure to CUS for 4 weeks enhanced the accumulation of macrophages and T cells and decreased smooth muscle cells in the atherosclerotic plaques in brachiocephalic artery of ApoE-/- mice. Representative sections were immunochemical staining of CD68 (A and B), CD3 (D and E) and α-SM actin (G and H), which are the cell surface markers of macrophages, T cells and smooth muscle cells, respectively. There were much more macrophages (B) and T cells (E) accumulation in CUS treated group than untreated group (A and D). However, smooth muscle cells in CUS treated group (H) were significantly lower than untreated control group (G). C, F and I showed the quantitative immunochemical staining of CD68, CD3 and α-SM actin. Dark brown color represents positive staining. The data shown are the mean ± SD of three independent experiments. ⁎⁎P b 0.01 compared to untreated control ApoE-/- mice. Original magnification, × 100. The total optical density (OD) was calculated using the following formula: OD = (1/red intensity + 1/blue intensity + 1/green intensity) × area for positive staining [27]. The OD was measured at 2 levels in the aortic root and an average OD was calculated for each mouse.

Fig. 3. CUS treatment enhanced the expression of adhesion molecular VCAM-1 and ICAM-1 in the atherosclerotic plaques in brachiocephalic artery and in the serum of ApoE-/- mice. Immunochemical staining of VCAM-1 (A and B) and ICAM-1 (D and E) showed that CUS treatment (B and E) significantly increased the expression of VCAM-1 and ICAM-1 compared with untreated control group (A and D). C and F showed the quantitative immunochemical staining of VCAM-1 and ICAM-1. Dark brown color represents positive staining. CUS treatment elevated soluble VCAM-1 (G) and ICAM-1 (H) in the serum of ApoE-/- mice was measured by ELISA. The data shown are the mean ± SD of three independent experiments. ⁎⁎P b 0.01 compared to untreated control ApoE-/- mice. Original magnification, × 100.

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Fig. 4. CUS treatment enhanced the secretion of CRP and IL-6 in the serum of ApoE-/- mice. The production of acute phase reactant CRP and proinflammatory cytokine IL-6 in the serum of ApoE-/- mice were measured by ELISA. CUS treatment markedly increased the secretion of CRP (A) and IL-6 (B) production. The data shown are the mean ± SD of three independent experiments. ⁎⁎P b 0.01 compared to untreated control ApoE-/- mice.

in monocyte-macrophages; and increasing inducible nitric oxide production, increasing NF-κB and mitogen-activated protein kinase (MAPK) activities, upregulating angiotensin type-1 receptor (AT1R) resulting in increased reactive oxygen species and cell proliferation in vascular smooth muscle, and hence was thought as one important risk marker for atherosclerosis [37]. Therefore, we investigated the effect of CUS treatment on the production of CRP in the serum of ApoE-/- mice. Our results demonstrated that CUS treatment markedly enhanced the secretion of CRP in the serum of ApoE-/- mice. Proinflammatory cytokine IL-6 and its signaling events have been shown to contribute to both, atherosclerotic plaque development and plaque destabilization via a variety of mechanisms, one of which is through regulation of CRP [38]. ELISA analysis showed that CUS treatment also significantly increased the production of IL-6 in the serum of ApoE-/- mice. The enhancement of the production of CRP and IL-6 might be another mechanism to explain the unstable plaque phenotype, the changes of plaque components and the increase of adhesion molecular VCAM-1 and ICAM-1 in CUS treated ApoE-/- mice. However, similarly, blockade of CRP and IL-6 would be also needed to address the question of how important both factors are in CUS-promoted atherosclerosis. High levels of cholesterol in the blood is one important risk factors for atherosclerosis and published data showed that CUS can enhance TG and LDL-C levels in serum in Wistar rats [37]. We measured the serum lipid concentrations in CUS treated ApoE-/- mice. Similarly, we also found that CUS treatment significantly increases the serum lipid levels in term of TG and LDL-C levels. Moreover, our results also showed that TC levels of stressed ApoE-/- mice were also markedly upregulated compared with nonstressed ApoE-/- mice. Instead of no obvious changes in HDL-C levels in Wistar rats [41], significantly lowered HDL-C levels were found in our CUS treated ApoE-/mice. Thus, the enhancement of TC, TG, LDL-C level and lessening of HDL-C levels by CUS treatment may be one reason to explain CUS treatment-promoted atherosclerosis in ApoE-/- mice. In conclusion, our data show the accelerating effect of CUS on atherosclerosis in ApoE-/- mice, which is coupled with the modulation of serum lipid contents (i.e., increasing TC, TG and LDL-C levels and decreasing HDL-C levels), the accumulation of macrophages and T cells in atherosclerotic plaques, and the elevation of CRP, IL-6, VCAM-1 and ICAM-1 production. These observations reveal a close link between the CUS-induced inflammation and the development of atherosclerosis, and may motivate future efforts to explore the possibility to interfere with the CUS-mediated atherosclerosis through anti-inflammation treatments. Conflict of interest statement The authors of the manuscript, “Chronic Unpredictable Stress Accelerates Atherosclerosis through Promoting Inflammation in Apolipoprotein E Knockout Mice”, declare that we have no proprietary, financial, professional or other personal interest of any nature or kind in any product, service and/or company that could be construed

as influencing the position presented in, or the review of, the manuscript entitled.

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