Genistein suppresses smooth muscle cell-derived foam cell formation through tyrosine kinase pathway

Accepted Manuscript Genistein suppresses smooth muscle cell-derived foam cell formation through tyrosine kinase pathway Jinghan Lin, Yi Xu, Tingting Zhao, Lina Sun, Meimei Yang, Tingjiao Liu, Hui Sun, Liming Zhang PII:

S0006-291X(15)30159-5

DOI:

10.1016/j.bbrc.2015.04.155

Reference:

YBBRC 34140

To appear in:

Biochemical and Biophysical Research Communications

Received Date: 11 April 2015 Accepted Date: 20 April 2015

Please cite this article as: J. Lin, Y. Xu, T. Zhao, L. Sun, M. Yang, T. Liu, H. Sun, L. Zhang, Genistein suppresses smooth muscle cell-derived foam cell formation through tyrosine kinase pathway, Biochemical and Biophysical Research Communications (2015), doi: 10.1016/j.bbrc.2015.04.155. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Genistein suppresses smooth muscle cell-derived foam cell formation through tyrosine kinase pathway JINGHAN LIN, YI XU, TINGTING ZHAO, LINA SUN, MEIMEI YANG,

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TINGJIAO LIU, HUI SUN, LIMING ZHANG

Liming Zhang, Department of Neurology, The First Affiliated Hospital of Harbin

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Fax: +86 451 53661120, E-mail: [email protected]

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Medical University, 23 Post Street, Harbin 150001, PR China, Tel: +86 451 85555026,

Abstract:

Purpose: Genistein, as a protein tyrosine kinase inhibitor, has been shown to possess anti-atherosclerotic effects. Since the smooth muscle cell-derived foam cells are key

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components of atherosclerotic plaques. The aim of this study is to investigate the influence of genistein on foam cell transformation from vascular smooth muscle cells

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and possible mechanisms contributing to these effects.

Methods and Results: Vascular smooth muscle cells exposed to ox-LDL developed

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into foam cell, as demonstrated by Oil Red O staining and cholesterol content analysis. Ox-LDL induced phenotype transformation of smooth muscle cells, decreased expression of α-actin and increased expression of CD68 (a specific marker for monocytes, can also function as a subtype of scavenger receptors). The expression of scavenger receptors CD36 and LOX-1 was measured, and their role in foam cell formation in the presence of genistein, daidzein (a structurally similar analogue of genistein) and herbimycin A (a commonly tyrosine kinase inhibitor). The results

ACCEPTED MANUSCRIPT showed that foam cell formation was markedly reduced by genistein and herbimycin A, as well as the expression of CD68, CD36 and LOX-1.However, daidzein had no such effect. In addition, genistein-induced down-regulation of CD68, CD36 and

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LOX-1 could be reversed by sodium orthovanadate (a membrane-permeable protein tyrosine phosphatase inhibitor).

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Conclusion: The results showed that ox-LDL induce smooth muscle cell-derived foam cell formation and transform the phenotype of smooth muscle cell. While

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tyrosine kinase inhibitor, genistein could suppress smooth muscle cell-derived foam cell formation through inhibiting the protein expressions of CD68, CD36 and LOX-1.

Keywords: genistein, smooth muscle cell-derived foam cells, CD36, LOX-1, CD68,

Introduction

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tyrosine kinase

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With aging, Atherosclerosis-related cardiovascular and cerebrovascular diseases represent the main conditions associated with mortality. Oxidized low-density

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lipoprotein (ox-LDL) is considered to play a major role in the progression of atherosclerosis [1]. Binding of extracellular ox-LDL to scavenger receptors (SRs) on membrane of macrophages, such as class A scavenger receptor (SR-A), CD36 and lectin-like oxidized-LDL receptor-1 (LOX-1) and its internalization leading to macrophages transformation into foam cells, is believed to be an early and significant process in atherosclerosis [2, 3]. Like macrophages, smooth muscle cells (SMCs) also express SRs and could convert to foam cells on the exposure to lipoproteins [4-7].

ACCEPTED MANUSCRIPT Thus, the SRs are considered as key regulators during foam cell formation.

In the early stage of progression of atherosclerosis, the presence of SMC-derived foam cells in the atherosclerotic plaque is play a critical role [8]. Recently, it has been

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reported that a large proportion (> 50%) of total foam cells in human coronary intima are derived from SMCs rather than from monocytes [9]. Rong et al. [10] reported that

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lipid loading of mouse SMCs in vitro led to transdifferentiation of SMCs toward a macrophage-like state, and the expression of SMC-related genes had decreased,

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whereas expression of macrophage-related genes CD68 had increased. CD68, a scavenger receptor D subtype, represents a specific marker of monocytes [11, 12]. It widely distributes on the surface of cell membranes and organelles and is involved in

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the deposition of intracellular cholesterol ester (CE).

Studies from epidemiological investigations have indicated that soybean isoflavones has a protective effect on cardiovascular and cerebrovascular diseases [13,

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14]. Genistein [15] is the most abundant phytoestrogen present in soybean isoflavones.

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It is used widely as a protein tyrosine kinase (PTK) inhibitor [16]. Results from in vivo experiments have revealed that genistein significantly inhibits LDL peroxidation [17]. It is beneficial in delaying the progression of atherosclerosis due to its capacity to diminish pathophysiologic vascular processes such as lipid profiles, angiogenesis, inflammation and tissue damage by reactive oxygen species [18-21]. Results from studies using in vitro models suggested that, as a tyrosine kinase inhibitors, genistein can inhibit the proliferation of vascular smooth muscle cells [22], regulate the vascular tone [23] and protect vascular endothelial functions [24]. In previous study,

ACCEPTED MANUSCRIPT genistein inhibited effect of Endothelin-1 on CD36 protein expression via the tyrosine kinase-dependent pathway [25]. Genistein can also inhibit the expression of macrophage SR-A and effect of the formation of macrophage derived foam cells [26].

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In addition, genistein can lower protein levels of CD36 and improve blood lipid levels and the degree of non-alcoholic fatty liver in ApoE-/- rats [27]. However, limited research of genistein has been directed at examining its role in inhibiting the

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formation of SMC-derived foam cells. Therefore, we hypothesized that genistein may

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affect the formation of SMC-derived foam cells by inhibiting SRs through its capacity to function as a tyrosine kinase inhibitor. These study might provide a new molecular mechanism by which gensitein potentially prevent atherosclerosis.

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Materials and methods

Vascular smooth muscle cell cultures

VSMCs were obtained from thoracic aortas of male Wistar mice and cultured by the

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tissue explant method. VSMCs were maintained in Dulbecco’s modified Eagle’s

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medium (Hyclone) supplemented with 10% fetal calf serum (GIBCO) containing 100mg/mL penicillin and 100mg/mL streptomycin (GIBCO). Cultured VSMCs were plated and grown at 37

in a humidified atmosphere of 95% air/5% CO2.They were

identified as VSMCs by their characteristic hill-and-valley growth patterns and immunofluorescence for SMC-specific α-actin. VSMC between the third and eighth passages were used in these experiments.

Foam cell preparation

ACCEPTED MANUSCRIPT Cells were grown to 80% confluence and then switched to serum-free media. After culture in serum-free media for 24h, VSMCs in the experiment were incubated in 0.2% bovine serum albumin (BSA)/DMEM medium containing ox-LDL100µg⁄mL

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(Yiyuan Biotechnologies, Guangzhou, China) for 72h, as well as pre-treated genistein (10, 50µmol/L), daidzein 50µmol/L or herbimycin A 2µmol/L in DMSO (<0.1%) for

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2h. Then cells were stained with Oil red O staining.

Determination of foam cell formation (Oil red O staining)

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Foam cell formation was evaluated by Oil red O staining. Oil red O powder (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in 99% isopropanol and diluted to 60% with ddH2O (working solution) 2 h before staining. Cells were fixed with 10%

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formalin for 30 min after being gently washed with ice-cold PBS. After rinsing with 60% isopropanol for 5 min, cells were stained with filtered oil red O working solution for 5 min followed by another rinse with distilled water to reduce the background and

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were imaged using an inverted microscope. The percent area of red color per cell was

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used for statistical analysis by Image-Pro Plus.

Quantitative measurement of intracellular cholesterol content The total cholesterol (TC) and free cholesterol (FC) were analyzed by the enzymatic fluorometric method. Briefly, VSMCs were harvested and washed twice with PBS. Lipids were extracted with the addition of 500 mL isopropanol to the cell pellet. After sonification, the samples were centrifuged for 15 min at 800g and the clear supernatant collected for TC and FC analysis (Cayman CHEMICAL, Ann Arbor, MI)

ACCEPTED MANUSCRIPT as measured in TECAN GENios Pro (excitation, 325 nm; emission,415nm). The concentrations of TC and FC were calculated using a standard curve, normalized by measuring the concentration of total cell protein using the BCA protein assay. The

FC from that of the TC.

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Immunofluorescence detection of α-actin and CD68

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concentration of cholesterol ester (CE) was calculated by subtracting the amount of

VSMCs were seeded upon cover slips at the bottom of culture dishes until

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subconfluence and were then fixed with 4% paraformaldehyde for 30 minutes. To block nonspecific reactions, 5% BSA was added for 30 minutes followed by anti-α -actin monoclonal antibody (1:100,Santa Cruz Biotechnology, USA) andanti-CD68

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polyclonal antibody (1:50,Santa Cruz Biotechnology, USA) administered for 18 hour while cultures were maintained at 4

. After washing, anti-rabbit IgG TRITC

fluorescein (1:800,Jackson) and anti-mouse IgG FITC fluorescein (1:800, Jackson)

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were added for 2 hours while cultures were maintained at room temperature. DAPI

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(1:2000, Sigma) was used to stain for total nuclei and imaging was performed using laser scanning confocal microscopy.

Western-blot

Cells cultured on 25 mm dishes or 6-well plates were lysed in lysis buffer (protease and phosphatase inhibitors). After centrifugation at 13500g for 15 min at 4

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lysates were collected. Protein concentration was assessed using the BCA protein assay. The aliquots were then mixed with Laemmli sample buffer and boiled at 99

ACCEPTED MANUSCRIPT for 5 min. The samples were resolved by 10% SDS–PAGE, followed by electrotransfer to polyvinylidene fluoride (PVDF) membranes. For visualization, blots were probed with antibodies against α-actin(1:800), CD68(1:500), LOX-1(1:500),

using

horseradish

peroxidase-conjugated

secondary

antibodies

(1:10000,Cell

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Signaling Technology).

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CD36(1:500)or GAPDH (1:1000)(Santa Cruz Biotechnology, USA) and detected

Statistical analysis

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Each experiment was replicated at least three times and all values presented represent the mean±SEM. Data were analysed by one-way ANOVA with the Bonferroni correction for multiple comparisons between groups. P<0.05 was considered

Results

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statistically significant.

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Ox-LDL Induces SMCs into SMCs-Derived Foam Cells

Following treatment with ox-LDL, VSMCs were transformed into foam cells with

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transformation rates of approximately 80% as indicated by oil red O staining (Figure 1Ab, B; p<0.001). To confirm the lipid accumulation seen by Oil Red O staining, we measured the amount of cholesterol content extractable from ox-LDL-treated VSMCs. The percentage of CE/TC in the foam cells was significantly increased by 40% following ox-LDL treatment (Figure 1C, p<0.001).

Ox-LDL Induces Phenotype Transformation of SMCs

ACCEPTED MANUSCRIPT Compared with the control group, the analysis of Immunofluorescence staining under laser confocal microscope shows that expression of α-actin is decreased and expression of CD68 is increased (Figure 2A). Western Blot analysis shows the

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ox-LDL could time-dependently decrease α-actin protein content (p<0.01) and increases the content of CD68 protein (p<0.05) (Figure 2B). The results show that, with ox-LDL loading in vitro, SMCs rapidly present a foam-cell appearance (Figure 1)

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and lose the expression of commonly accepted markers of the SMC phenotype

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(Figure 2) .

Genistein and Herbimycin A, but not Daidzein, Inhibits Foam Cell Formation

Results obtained from oil red O staining show that genistein at either concentrations

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of 10µmol/L (Figure1Ad and B; p<0.05) or 50µmol/L (Figure1Ae and B; p<0.01) and herbimycin A 2µmol/L (Figure1Af and B; p<0.01) could reduce the transformation rate of foam cells when co-incubated 100µg/ml ox-LDL. However, no effects were

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observed in response to daidzein (Figure 1Ac and B). In addition, percent of CE/TC

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(Figure1C) were also reduced by genistein and herbimycin A, but not daidzein (Figure 1).

Genistein and Herbimycin A, but not Daidzein, Inhibit CD68, LOX-1 and CD36 Expression

Protein expression of α-actin CD68, CD36 and LOX-1 were assessed after be pre-treated by genistein50µmol/L, daidzein 50µmol/L or herbimycin A 2µmol/L and without or with 100µg/mL ox-LDL co-cultures. Compared with the control group,

ACCEPTED MANUSCRIPT Western blot analysis demonstrated that ox-LDL could increase the protein expression of CD68 (Figure3C; p<0.05), CD36 (Figure3B; p<0.01) and LOX-1 (Figure 3A; p<0.01).Compared with the ox-LDL group, genistein inhibited the increase of CD68

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(Figure 3C; p<0.05), CD36 (Figure 3B; p<0.01) and LOX-1 (Figure 3A; p<0.01). Herbimycin A also could inhibited the increase expression of these proteins but there is less obvious than genistein. No effects were observed in response to

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daidzein(Figure 3).

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PTK-dependent effects of genistein on CD68, LOX-1 and CD36 Expressions in VSMCs

The protein tyrosine phosphatase inhibitor sodium orthovanadate (SOV) (50 µmol/L)

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was added to ascertain if genistein-induced effects on SRs are related to protein tyrosine kinase (PTK) inhibition, in which case SOV should antagonize these PTK-dependent effects [28]. Western blot analysis (figure 4) demonstrated that

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Genistein inhibit expression of LOX-1 (A), CD36 (B) and CD68 (C) with ox-LDL

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incubation, and after pre-incubation with SOV, this inhibitory effect was reversed, suggesting that the inhibition by genistein was PTK-dependent.

Discussion

Foam cells play an important role in the development of atherosclerotic lesions [29]. SMCs are the main cell type in intimal thickenings and some stages of atherosclerosis. Like macrophages, SMCs accumulate excess lipids and contribute to the total intimal foam cell population [30]. In this study, we demonstrated that

ACCEPTED MANUSCRIPT incubate rat primary thoracic aorta VSMCs treated with 100µg/mL ox-LDL for 72h could be successfully induced to SMC-derived foam cells (Figure 1). Such findings are consistent with a previous report [6]. And we first report that genistein could

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suppress smooth muscle cell-derived foam cell formation. It is valuable study that comprehensively investigated the regulating mechanisms of genistein in preventing

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arteriosclerosis process.

Intimal smooth muscle cells undergo phenotypic switching is also involved in

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the development of atherosclerosis, we demonstrated that (Figure 2) ox-LDL induces the decreasing expression of α-actin and increasing expression of CD68 as a result of loss of contractility phenotype of SMCs which are consistent with a previous report[10]. CD68, a specific marker for monocytes, can also function as a type D SR

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[11,12]. CD36 is a type B SR[3]. LOX-1 is a newly discovered SR[3]. They are distributed on the surface of SMCs, these receptors can recognize and engulf ox-LDL

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for decomposition into CEs within the cells. Excessive CE result in accumulation of CE stored as cytoplasmic lipid droplets and subsequently trigger the formation of

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foam cells. In this study, we demonstrated that the CE content was increased when cultured VSMCs were exposed to ox-LDL (Figure 1). ox-LDL also enhance CD36 and LOX-1 protein expression in SMCs (Figure 3). Therefore, we speculated that genistein represses smooth muscle cell-derived foam cell formation by preventing the protein expression of CD36 LOX-1 and CD68.

Genistein is the most abundant content of soybean isoflavones and it exerts effects like estrogen and tyrosine kinase inhibitors. The preventive effects of soybean

ACCEPTED MANUSCRIPT isoflavones on cardiovascular diseases and hormone-related cancers had been widely recognized for nearly a decade [31]. Findings from epidemiological investigations have indicated that long-term oral administration of the soybean isoflavones has a

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protective effect on cardiovascular and cerebrovascular diseases [13] and genistein exerts an anti-atherogenic effect that is particularly effective in postmenopausal women [32]. Results from in vivo experiments, have revealed that genistein

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significantly inhibits LDL peroxidation [17], and prevents the development of early

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atherosclerosis [33]. Experiments focusing upon the capacity for genistein to function as an anti-arteriosclerotic agent have mainly been directed at examining the protection of endothelial function [34] or inhibition of VSMC proliferation [35]. Only a very limited amount of genistein research has been directed at examining its role in

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inhibiting the formation of SMC-derived foam cells.

Results from studies using in vitro models, have shown that genistein can also

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inhibit the expression of macrophage SR-A and the formation from macrophage to foam cells [26]. In addition, when administered within the diet, genistein can lower

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protein levels of CD36 and improve blood lipid levels and the degree of nonalcoholic fatty liver in ApoE-/- rats [27]. As expected, by using oil red O staining and cholesterol ester determinations, we demonstrated (Figure 1) that genistein (10 or 50µmol/L) and herbimycin A suppressed intracellular lipid accumulation in SMCs, while daidzein had no effect. Then, in a follow-up experiment (Figure 3), we found that both genistein and herbimycin A were shown to decrease the protein expressions of CD68, CD36 and LOX-1, but effect of herbimycin A is less obvious than genistein.

ACCEPTED MANUSCRIPT However, daidzein has almost no effect on protein expressions. In addition, genistein daidzein and herbimycin A had no effect on α-actin expression.

In this study, daidzein was used as a structurally similar analogue of genistein to

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exert stimulatory effects. Although genistein and daidzein share a common benzene-ring structure, daidzein differs from genistein in that one hydroxyl group is

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absent and therefore it lacks tyrosine kinase inhibitory abilities [16]. Moreover, like genistein, daidzein also has antioxidant and estrogenic effects. Herbimycin A [36], a

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commonly used tyrosine kinase inhibitor, lacks antioxidant and estrogenic effects. Accordingly, with the use of these drugs, it is possible to identify some of the mechanism involved with genistein’s capacity to inhibit the formation of foam cells.

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Our results suggest two points. First, genistein could inhibit lipid deposition in SMCs through the inhibition of scavenger receptor (CD68, CD36 and LOX-1), but genistein could not reverse the transformation phenotype of SMCs. Second, the effect

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of gensitein on CD68, CD36 and LOX-1 protein expression are not attributable to its

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benzene ring and hydroxyl structure, but rather by its capacity to function as a tyrosine kinase inhibitor. However, as genistein is also a phytoestrogen [15], the potential for estrogen receptor action may also contribute to this effect.

In order to further investigate whether genistein is exerted through inhibition of

tyrosine kinase pathway, we use SOV (a membrane-permeable protein tyrosine phosphatase inhibitor) to antagonize genistein PTK-dependent effects [37, 38]. SOV was applied for 24h before genistein incubation. After pre-application of SOV (i.e.,

ACCEPTED MANUSCRIPT when the protein tyrosine substrates were phosphorylated and could not be dephosphorylated by PTK inhibitors), the inhibitory effect of genistein was reversed. In this study (Figure 4), enhanced expression of SRs (CD68, CD36 and LOX-1) was

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inhibited by genistein when its PTK inhibitory ability was antagonized by SOV in different extent. Based on these evidences, we concluded that CD68, CD36 and LOX-1 protein expression were PTK-dependently inhibited by genistein. The results

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from the present study provide evidence that genistein with its capacity to function as

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a tyrosine kinase inhibitors, can suppresses smooth muscle cell-derived foam cell formation through inhibiting CD68, CD36 and LOX-1.

Kwok et al [25]reported that endothelin-1 can inhibit CD36 expression in SMCs through tyrosine kinase-ERK pathway. As tyrosine kinases plays an important role in

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cell signal transduction, their inactivation may affect MAPK-ERK signal pathways downstream thus affecting protein synthesis of CD68, CD36 and LOX-1. This latter

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possibility still requires further study.

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In conclusion, our findings indicate that genistein can suppresses smooth muscle cell-derived foam cell formation through inhibiting. The mechanism of the modulation of SRs by genistein involves PTK-dependent inhibition. These findings might provide a new molecular mechanism by which gensitein potentially prevent atherosclerosis.

Acknowledgments This work was supported by the National Natural Science Foundation of China

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7-Difluoromethyl-5,4'-dimethoxygenistein, a novel genistein derivative, has therapeutic effects on atherosclerosis in a rabbit model, Journal of cardiovascular pharmacology, 54 (2009) 412-420. [34] L. Yi, C.Y. Chen, X. Jin, T. Zhang, Y. Zhou, Q.Y. Zhang, J.D. Zhu, M.T. Mi, Differential suppression of intracellular reactive oxygen species-mediated signaling pathway in vascular endothelial cells by

ACCEPTED MANUSCRIPT several subclasses of flavonoids, Biochimie, 94 (2012) 2035-2044. [35] W. Pan, K. Ikeda, M. Takebe, Y. Yamori, Genistein, daidzein and glycitein inhibit growth and DNA synthesis of aortic smooth muscle cells from stroke-prone spontaneously hypertensive rats, The Journal of nutrition, 131 (2001) 1154-1158.

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[36] Y. Uehara, H. Fukazawa, Use and selectivity of herbimycin A as inhibitor of protein-tyrosine

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[38] L. Zhao, Z. Wang, Y.C. Ruan, W.L. Zhou, Cellular mechanism underlying the facilitation of contractile response of vas deferens smooth muscle by sodium orthovanadate, Molecular and cellular

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biochemistry, 366 (2012) 149-157.

Figure Legend Figure 1

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The effect of genistein, daidzein and herbimycin A on intracellular lipid accumulation in VSMCs. (A) Representative pictures of oil red O staining of intracellular lipid

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droplets in cultured VSMCs that were pre-incubated with genistein (d, e), daidzein (c) or herbimycin A (f) for 2h then co-incubated without (control) (a) and with 100

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µg/mL ox-LDL(b) for 72h. Images are representative of three separate experiments (magnification ×400). (B and C) Bar graphs showing the quantification of lipid accumulation based on percent of cells positive for Oil Red O staining (B) and percent of CE/TC (C). Genistein suppressed intracellular lipid accumulation in VSMC. Data are mean+SEM (n=3 per group). ***p<0.001 vs control; ##p<0.01 #p<0.05 vs ox-LDL alone.

ACCEPTED MANUSCRIPT Figure 2 Ox-LDL Induces Phenotype Transformation of VSMCs. (A) VSMCs were treated in 0 or 100 µg/mL ox-LDL for 72h. Immunofluorescence showing the colocalization of

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α-actin and CD68 on the surface of VSMCs after treated with ox-LDL. α-actin and CD68 were identified using specific primary antibodies and fluorescence-labelled

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secondary antibodies. 4,6-Diamidino-2-phenylindol (DAPI) was used for nuclear counterstaining (blue fluorescence). Negative controls (Control) were performed with

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phosphate-buffered saline (PBS) instead of primary antibodies. Images are representative of three separate experiments (magnification × 200). (B and C) VSMCs were cultured in the absence (control) or presence of 100 µg/mL ox-LDL for 24-72h. Representative western-blot and summary data showing the effect of ox-LDL

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on the expression of α-actin (B) and CD68 (C) in VSMCs. Data represent mean+SEM( n=3 per group)*p<0.05 **p<0.01 vs controls.

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Figure 3

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Effects of genistein, daidzein and herbimycin A on expression of α-actin, CD68, CD36 and LOX-1 in VMSCs. VSMCs were pre-incubated with genistein daidzein or herbimycin A for 2h and co-incubate with ox-LDL for 72 h. Representative western blots showed that expression of CD68 (D), CD36 (B) and LOX-1 (A) protein all were inhibited by genistein and herbimycin A, while daidzein had no effect. However, α-actin expression did not been effected by genistein, daidzein and herbimycin A (C). Data represent mean+SEM (n=3 per group) **p<0.01 vs controls;

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p<0.01 #p<0.05

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Figure 4

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Effects of genistein on SRs after pre-incubation with SOV in VMSCs. Representative western blots showed that expression of CD68(C), CD36(B) and LOX-1(A) protein were enhanced in varying degrees after pre-incubation with SOV 50µM for 24 h. Data ##

p<0.01 #p<0.05 vs

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are mean+SEM (n=3 per group). **p<0.01 *p<0.05 vs control;

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ox-LDL+genistein.

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1,We incubated rat primary thoracic aorta vascular smooth muscle cells treated with ox-LDL, and successfully induced to smooth muscle cell-derived foam cells as indicated by oil red O staining and cholesterol content measuring.

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2,We demonstrated that ox-LDL induces the decreasing expression of α-actin and increasing expression of CD68, which means loss of contractility phenotype of smooth muscle cells which transformed to synthetic phenotype.

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3,We first reported that genistein could suppress smooth muscle cell-derived foam cell formation. Scavenger receptors can recognized and

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engulfed ox-LDL for decomposition into cholesterol esters within the cells. Therefore, we speculated that genistein represses smooth muscle cell-derived foam cell formation by preventing the protein expression of Scavenger receptors (CD36 LOX-1 and CD68).

4,Genistein inhibit lipid deposition in smooth muscle cells through the

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inhibition of scavenger receptor (CD68, CD36 and LOX-1), but genistein could not reverse the transformation phenotype of smooth muscle cells. 5,Foam cell formation was markedly reduced by genistein and

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herbimycin A (a commonly tyrosine kinase inhibitor), as well as the expression of CD68, CD36 and LOX-1. However, daidzein (a structurally

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similar analogue of genistein) had no such effect. In addition, genistein induced down-regulation of CD68, CD36 and LOX-1 could be reversed by sodium orthovanadate (a membrane-permeable protein tyrosine phosphatase inhibitor).It means that as tyrosine kinase inhibitor, genistein could suppress smooth muscle cell-derived foam cell formation through inhibiting the protein expressions of CD68, CD36 and LOX-1.