Toll-Like Receptor 4 Mediated Oxidized Low-Density Lipoprotein-Induced Foam Cell Formation in Vascular Smooth Muscle Cells

Background: Oxidized low-density lipoprotein (oxLDL) induced a foam-cell like phenotype of the vascular smooth muscle cells (VSMCs), leading to the inammatory responses incorporating Toll-like receptors (Tlrs)-mediated cellular alterations. We previously found that Tlr4 participated in inammation response in VSMCs under oxLDL stimulation. However, the role of Tlr4 in foam-cell formation and underlying molecular pathways has not been comprehensively elucidated. This study aimed to investigate the role of Tlr4-mediated mechanisms in oxLDL induced foam-cell formation within VSMCs. Methods: After incubated with different dose of oxLDL, the lipid, reactive oxygen species (ROS) accumulation and foam-cell phenotype of the VSMCs were detected. The alteration of Tlr family, ROS and lipid accumulation regulators including the Src kinase, Nox2, Nox4, Mnsod and sirtuins were measured. Then the Tlr4 was knock down and underlying cellular change and altered molecules were detected. Results: We showed that oxLDL induced foam-cell like phenotype in VSMCs and led to lipid and ROS accumulation in a dose-dependent manner. OxLDL induced the strongest upregulation of Tlr4 in the Tlrs family and initiated change of Src activation, Nox2, Mnsod, sirt1 and sirt3 expression. The effect of oxLDL was abolished by Tlr4 knockdown. Furthermore, knocking down of Tlr4 reduced Src activation and led to restored Sirt1/Sirt3 expression. Moreover, inhibiting or knocking down the Src kinase diminished lipid accumulation in VSMCs under oxLDL treatment. And overexpression of Sirt1/3 relieved the oxLDL induced ROS accumulation and foam-cell phenotype in VSMCs. Conclusions: These results demonstrated that Tlr4 is a critical regulator in oxLDL induced foam cell formation of VSMCs via mediating Src kinase as well as Sirt1 and Sirt3. Beyond the role of Tlr4 in inammation response of VSMCs, we provide an integrated mechanism about TLR4 in VSMCs standard deviation (SD). Paired samples were compared using Student’s paired t-test. One-way ANOVA followed by Friedman’s post-test was used for multiple group comparisons. A two-sided p value less than 0.05 was considered statistically signicant. Data were analyzed and plotted using the Graphpad Prism Version 7.0.


Background
Coronary artery disease (CAD) is a leading health burden contributing to high morbidity and mortality worldwide [1]. And atherosclerosis serves as the major cause driving the occlusion of coronary arteries and cardiovascular events [2]. During the process of atherosclerosis, mounting foam cells formation and necrosis invoked the in ammation storm, which aggravated instability of plaque and led to acute myocardial infraction [3,4]. Previous studies showed that despite the well-established essential role of monocyte-derived macrophages, vascular smooth muscle cells (VSMCs) were equipped with macrophage features, constituting a substantial source of foam cells and in ammatory response in plaques [5,6]. Basically, low density lipoprotein underwent oxidative modi cation beneath the vascular intimal, and could be ingested by VSMCs [7], which, promoted transition of VSMCs from the mature contractile state to the macrophage-like phenotype [8]. As a result, lipid accumulated in the VSMCs, and such VSMC-derived foam cells accelerated the progression of the atherosclerosis [9,10]. Though a few scavenger receptors participated in lipid uptake during foam cells formation, the speci c mechanism contributing to lipid accumulation in VSMCs was still unclear. To acquire a better understanding of VSMCs alteration in atherosclerosis, it is necessary to clarify the mechanism underlying the lipid accumulation in VSMCs.
Along with the continuous formation and necrosis of the foam cells, regional in ammatory storm induced by excessive cytokines caused damage to the vessels [11]. OxLDL ingestion induced foam cells formation and soon afterwards, accelerated mitochondrial oxidative stress [12] , [13], which led to accumulating reactive oxygen species (ROS) production [13].Taken together, these factors evoked in ammatory response signaling pathway in foam cells [14,15]. In our previous studies, oxLDL activated pre-in ammatory signaling pathway and raised expression and secretion of in ammatory cytokines in VSMCs via Tlr4 [16]. Moreover, oxLDL promoted the bond of Tlr4 with Src kinase to induce lipid uptake and foam cell formation in macrophages [17]. Other studies also indicated that Tlr4 was implicated in foam cell formation in VSMCs. These results hint that Tlr4 might regulate lipid uptake process and subsequently contribute to foam cells formation in VSMCs. Nevertheless, such potential role of Tlr4 in the VSMCs has not been explored.
Additionally, excessive production of reactive oxygen species (ROS) was widely observed in atherosclerosis. Although it has been well-established that the broken oxidative homeostasis could promote the vascular in ammation response, the relationship between oxidative stress and foam cell formation in VSMCs has not been elucidated. Several previous studies showed that Tlr4 mediated ROS accumulation via regulating Nox2 [18]. The pivotal role of emerging sirtuins family in maintaining the balance of the ROS metabolism has also been increasingly reported [19]. However, whether they were involved in oxLDL-TLR4 induced VSMCs in ammation and ROS accumulation remain obscure.
In this study, we hypothesized that Tlr4 mediated oxLDL-induced foam cell formation via regulating lipid accumulation and ROS production in VSMCs. Based on cellular and molecular research, we aimed to clarify the mechanism underlying the ROS and lipid accumulation induced by oxLDL in VSMCs, thus deepening the insights about the formation of foam cell-like VSMCs during atherosclerosis.

Primary Smooth Muscle Cells Culture
Wild-type (C57BL/6) mice were purchased from Model Animal Research Center of Nanjing University (Nanjing, China) and were euthanized at 4 weeks old. To obtain the primary smooth muscle cells, the aortas of the mice were dissected and the adventitia was removed. The aortic explants were cultured after mechanical dissection and twice washes in PBS. The explant-derived VSMCs were cultured at 37℃,5% CO 2 in F12: DMEM (1:1) medium with 20% fetal bovine serum and 1% antibiotic-antimycotic.
The animal experiment protocol complied with the Animal Management Rules of the Chinese Ministry of Health (Document No. 55, 2001) and was approved by the Animal Care Committee of Shanghai Jiaotong University.

Assessment of Intracellular Lipids
VSMCs were cultured in 6-well plates and incubated with oxLDL for 72 hours. Afterward, the VSMCs were washed by PBS for twice followed by 15-minute 4% paraformaldehyde/PBS xation and then were stained by 100 ng/mL Nile Red for intracellular lipids detection [20]. All cell samples were observed and photographed microscopically (ZEISS LSM 800, Zeiss Microsystems). 5 elds of view were randomly acquired and representative images were shown. Intracellular lipids were quanti ed by HDL and LDL/VLDL Cholesterol Assay Kit. Quanti cations of total lipoprotein was conducted according to description in the manufacturer's protocols and demonstrated as relative values-to-total protein ratio (n=3).

Intracellular Reactive Oxygen Species Assay
Cellular ROS protection was detected by carboxy-H2DCFDA kit. Based on the kit instruction, 25 μM carboxy-H2DCFDA working solution was used to label VSMCs for 30 minutes at 37°C in dark. Afterward, cells were gently washed in warm HBSS/Ca/Mg and incubated with Hoechst 33342 for another 5 minutes. After 3 times washes, the ROS signals were observed via a uorescent microscope. 5 elds of view were randomly acquired and representative images were showed.

Assessment of intracellular Mitochondrial Superoxide
After incubation with oxLDL, VSMCs were gently washed by warm Hanks buffer. The mitochondrial superoxide was detected using the MitoSOX™ Red indicator. According to the manufacturer's recommendation, the MitoSOX™ reagent was diluted into a working concentration and added to the 6 well plate covering the VSMCs. After 10 minute-incubation at 37 ℃ in dark, the cells were washed by Hanks buffer for three times. Image were obtained by uorescence microscope using a green excitation light. 5 elds of view were randomly acquired and representative images were showed.
Quantitative Real-time RT-PCR Total RNA was extracted by Trizol reagent, and 5 ug of total RNA undergone the reverse-transcription. Polymerase chain reactions (PCR) were carried out using Power SYBR Green PCR Master Mix (Applied BioSystems, CA, USA) according to the manufacturer's recommendation in a StepOne System (Applied BioSystems, CA, USA). Primers for the promoter sequences were listed in (Table 1). Gene expression were normalized with β-actin as the reference gene. StepOne software v2.1 (Applied BioSystems) was used for data analysis.  VSMCs were lysed in Western & IP Cell lysate on ice for 15 min. The total protein was collected after centrifugation. Protein concentration was measured using a BCA-protein assay kit. Equal quanti cation of protein (20 μg) was applied in a 15% SDS-polyacrylamide gel and transferred to polyvinylidene uoride (PVDF) membranes. The membranes were blocked by 5% milk for 1 hours at room temperature and then incubated with the primary antibody overnight at 4°C. After 3 times washed in TBS buffer, the membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 2 hours. Finally, Images were captured in a Tanon-5500 chemiluminescent imaging system (Tanon Science and Technology Co., Ltd., Shanghai, China) and quanti ed by ImageJ software (Bio-Rad, Hercules, CA, USA).
Oligonucleotide Transfection RNA interference was conducted using the Oligo-fectamine reagent (Invitrogen). A non-targeting sequence was used as control siRNA. And cultured VSMCs were transfected with the siRNA and control according to the instruction. Cells had 60%-70% con uency at the day of transfection. After transfecting for 48 hours, the knockdown e ciency was tested by western-blot.
Pretreatment with PP2 and PP3 PP2 (10 μmol/L) and PP3 (10 μmol/L) were respectively added to VSMCs and incubated for 30 min before oxLDL stimulation. PP3 served as the negative control for PP2.

Sirt1 and Sirt3 Overexpression Lentivirus production and Transfection
The Sirt1 and Sirt3 overexpression was completed using recombinant lentivirus vectors containing the overexpression plasmid of the corresponding gene. Empty vector lentivirus was also transfected as control. Cells were infected with lentivirus for 72 hours followed by a RT-PCR for e ciency determination.

Statistical Analysis
Values were showed as mean with standard deviation (SD). Paired samples were compared using Student's paired t-test. One-way ANOVA followed by Friedman's post-test was used for multiple group comparisons. A two-sided p value less than 0.05 was considered statistically signi cant. Data were analyzed and plotted using the Graphpad Prism Version 7.0.

Results
OxLDL induced dose-dependent lipid accumulation, oxidative stress and foam cell formation in vascular smooth muscle cells.
After stimulated by gradient dose (12.5, 25 and 50 μg/mL) of oxLDL for 48 hours, lipid accumulation, cellular ROS accumulation, mitochondrial superoxide generation and foam cells formation was examined. The Nile Red staining and lipoprotein quanti cation showed that oxLDL induced lipid accumulation in VSMCs in a dose-dependent manner (Figure 1.A). The DCFH-DA and MitoSOX served as detectors for labeling cellular ROS and mitochondrial superoxide generation respectively. Similar to the oxLDL-induced lipid accumulation, higher concentration of oxLDL triggered severe oxidative stress within cell and mitochondrial (Figure 1.B and C).
Moreover, after oxLDL stimulation, the expression of αSma and Myh11, the contractile phenotype-speci c mRNA and protein, was down-regulated, while the foam cells' phenotype markers, Cd68 and Mac2, were signi cantly upregulated (Figure 1. D-F).
OxLDL mediated signi cant upregulation of Tlr4 along with expression/ activation of lipid metabolism and oxidative stress regulators.
To explore the overall change of the Tlrs family under the oxLDL stimulation in VSMCs, we further measured mRNA levels of the Tlrs in VSMCs after 48 hours incubation with 50 μg/mL of oxLDL to gure out which members of the Tlrs experienced drastic change. Remarkably, under oxLDL treatment, expression of Tlr4 increased more signi cant than any other Tlrs among Tlr1-Tlr13 ( Figure 2.A), with over 1.5-fold expression than the control. In our previous study had explored the protein level of Tlr4 increased with oxLDL stimulation in VSMCs [16].
Based on our previous observations in macrophages [17], activation of Src in VSMCs was measured after gradient dose (12.5, 25 and 50 μg/mL) of oxLDL stimulation for 1 hour. In line with the macrophage, we found that the phosphorylation site of Src (418-Tyr) was obviously activated in VSMCs. But different from the result of macrophages, Src did not show a dose-dependent effect in such activation and the extend of activation was comparable across different oxLDL concentration-treated group (upper of Figure   2.B). Meanwhile, the expression of the ROS elimination-relevant gene Mnsod as well as Nox2 and Nox4, which are responsible for ROS generation were examined. Only the highest dose (50 μg/mL) of oxLDL resulted in signi cant elevation of Nox2 and remarkable decreased Mnsod, whereas the signi cant change of Nox4 expression was not observed. (bottom of Figure 2.B). Finally, the alteration of the oxidative balance maintainer, sirtuins family, were also explored. It is interesting to note that only expression of Sirt1 and Sirt3 were remarkably downregulated while no signi cant effect was observed in terms of other members of sirtuins family (Figure 2.C).
Tlr4 mediated oxLDL-induced lipid accumulation, oxidative stress and foam cell formation in VSMCs To investigate the role of Tlr4 in oxLDL-induced pathophysiological change and its relation with those altered regulators in VSMCs, Tlr4 was knockdown to further elaborate subsequent cellular phenomenon and molecular pathway. Tlr4 were signi cantly knockdown by targeted siRNA and negative control siRNA (NC) was also transfected to serve as negative control (Figure 3.A). After 50μg/mL of oxLDL stimulation for 48 hours, lipid accumulation, ROS and mitochondrial superoxide were still sharply promoted in NC group. By contrast, these alterations were ameliorated in Tlr4-knockdown VSMCs (Figure 3.B-D). More importantly, although oxLDL still led to signi cant decrease of VSMCs contractile phenotype marker (Myh11 and αSma) and elevated foam cells marker (Mac2 and Cd68) in NC VSMCs, TLR4 knockdown had interrupted most of these alterations, indicating that TLR4, at least partly mediated the oxLDL induced lipid and ROS accumulation and contribute to foam cell formation (Figure 3.E and F).

Tlr4-Src kinase regulated lipid accumulation and cellular phenotype transition in VSMCs
Notably, after knocking down the Tlr4 in VSMCs, the activation in Tyr-418 phosphorylation site of Src kinase were deprived to a great extent, compared with the NC group, after 1-hour oxLDL treatment ( Figure  4.A and B). Such effect indicated that Tlr4 regulated Src kinase activation under oxLDL stimulation. And we further hypothesized that Src kinase might be downstream executor of TLR4 to impact lipid uptake in VSMCs. To illuminate this hypothesis, either the expression or the activation of Src were disturbed by siRNA or PP2 respectively, and then the change of intracellular lipid concentration and cell phenotype markers were determined following oxLDL treatment. The e ciency of siRNA knockdown Src was tested with western-blot (Figure 4.C and D). Compared with untreated group, higher intercellular lipid levels were observed in oxLDL-treated groups, but Src knockdown or activation-blocked groups showed relative less lipid accumulation than NC or PP3 groups (Figure 4.E). Furthermore, knocking down the expression or blocking the activation of Src signi cantly postponed the loss of VSMCs contractile markers loss and of foam cell phenotype gaining, which were induced by oxLDL (Figure 4.F and G).
Tlr4 mediated Sirt1/Sirt3 alteration which regulated oxLDL-induced oxidative stress and foam cell formation in VSMCs Interestingly, we also observed that when Tlr4 in VSMCs was knocked down, the expression of Sirt1 and Sirt3 restored compared with the NC group after 48-hours oxLDL treatment accompanied by reduced Additionally, though oxLDL led to signi cantly elevated Nox2 and decreased Mnsod, Sirt1 or Sirt3 overexpression almost reversed such impact on these genes (upper, Figure 5.G and H). Moreover, increased expression of Sirt1 or Sirt3 also relive the oxLDL-induced VSMCs contractile phenotype marker (Myh11 and αSma) loss and foam cells marker (Mac2 and Cd68) acquisition, though they were still signi cantly altered compared with the untreated group, which implied that Sirt1 and Sirt3 partly assist in VSMCs phenotype rebalancing under oxLDL stimulation (bottom, Figure 5.G and H).

Discussion
In this study, we demonstrated that oxLDL induced the transition of VSMCs to foam cells by promoting lipid accumulation and ROS production via raising the expression of a key linking molecular-Tlr4. Furthermore, oxLDL-induced lipid and ROS accumulation were at least partly attributed to Tlr4-regulated Src activation and Sirt1 and Sirt3 upregulation, which also contributed to foam cells formation. Our ndings unraveled a crucial role of Tlr4 in oxLDL-induced foam cell formation in VSMCs.
The toll-like receptors family constitute important members of pattern recognition receptors (PRRs), which identify special ligand of receptor to evoke pathogen-associated molecular patterns (PAMPs) [21][22][23]. These innate immune responses are commonly activated in innate immune cells. Likewise, as a chronic in ammation process, atherosclerosis was closely bound with the continuous innate immune response triggered by activation of PPRs [24]. Basically, oxLDL is a principle component of endogenous lipid ligand that causes endothelial cell injury, accumulates in macrophage and VSMCs and induces a cellular in ammation response [25,26]. Tlrs were reported to participate in oxLDL induced in ammation response [27][28][29], but an integrated expression feature of Tlrs family in VSMCs under oxLDL stimulation remained unelaborated. In our present study, we found that oxLDL raised the expression of Tlr4 more signi cantly, which was over 1.5-fold than other Tlrs in VSMCs. Such observations suggested that among the Tlrs family, Tlr4 might serve as major participant in oxLDL induced alterations of VSMCs.
Foam cell phenotype and in ammation response are two major interrelated alterations in VSMCs that exacerbated the progression of atherosclerosis [6]. Our work provided evidence for the role of Tlr4 throughout the oxLDL-induced change in VSMCs. Previously, we certi cated that Tlr4 was a crucial in ammation regulator in oxLDL-induced in ammatory cytokine expression/secretion as well as p38 and NFκB activation [16]. Herein, we also detected the role of Tlr4 in the prior foam cell formation in the VSMCs. In this study, besides gradient upregulation of Tlr4, we evidenced that knocking down Tlr4 could reverse oxLDL-induced phenotype change, which was characterized by emerging foam cells phenotype and weakened contractile phenotype in VSMCs. Taken together, we found that Tlr4 not only played a crucial role in oxLDL-induced in ammatory response, but also mediated oxLDL-induced foam cells formation in VSMCs (Fig. 6).
Foam cell formation, characterized by accumulation of intracellular lipids and occurrence of in ammatory phenotype, is a hallmark in progression of atherosclerosis [30]. Lipid loading in VSMCs activated multiple pro-in ammatory genes, suppressed expression of VSMC marker genes, and led to the phenotype switching as well as in ammatory cytokines secretion [31]. Herein, we found that oxLDL evoked lipid accumulation in VSMCs, and knocking down the Tlr4 inhibited such lipid uptake in VSMCs, implying that Tlr4 mediated oxLDL-induced lipid accumulation and promoted subsequent foam cells formation. In a previous studies we were able to demonstrate that Tlr4 directly participated in oxLDLinduced lipid uptake in macrophages by regulating Src kinase [17], indicating that Tlr4 was not only an innate immune receptor in in ammatory response, but also a mediator for lipid accumulation. Although little was known about the role of Tlr4 in lipid accumulation of VSMCs, herein we evidenced that Tlr4-Src signaling contributed to lipid accumulation of VSMCs and further demonstrated that the foam cell phenotype could be partly reversed after interfering Tlr4 or Src. These observations suggest that TLR4-Src pathway might be a common regulation mechanism of oxLDL-induced lipid accumulation and foam cell formation in atherosclerosis (Fig. 6). Actually, it might be reasonable that Src could serve as a downstream molecule contributing to the lipid loading in VSMCs. Firstly, activation of Src may mediate the rearrangement of the cytoskeleton, which is a major cellular alteration underling endocytosis [32]. Additionally, Src signaling activates c-Jun N-terminal kinase and enhances the trans-activity of c-Jun in response to LPS, thus triggering the expression of in ammation markers [33,34]. Therefore, Src is likely to alter the lipid accumulation and in ammation response in the VSMCs.
ROS was a well-known mediator that exerts severe intracellular oxidative stress and prompts in ammatory response, structural reorganization, and even cell phenotype transition [35,36]. In our present study, we found that oxLDL promoted intracellular ROS and mitochondrial superoxide accumulation. In terms of this remarkable cytological event, we explored the remarkable cytological event from dialectical aspects, including both the ROS generation through Nox2 and Noxr4 and the ROS elimination via Mnsod, which constitute the balance of "in" and "out" on a "tank". We found that oxLDL led to over ow of ROS production through accelerating speed of "in" (increasing Nox2 expression but not Nox4) and decelerating speed of "out" (decreasing Mnsod expression) in VSMCs. Along with the reversed phenotype alteration of VSMCs, knockdown of Tlr4 also inhibited ROS and mitochondrial superoxide production, reduced Nox2 expression and increased Mnsod expression, suggesting that the ROS production speed of "in" or "out" were regulated by Tlr4. These results indicated that Tlr4 mediated oxLDL-induced ROS accumulation through regulating the balance of ROS homeostasis, which might be another potential mechanism of Tlr4 during foam cells formation in VSMCs.
Since previous studies showed that Sirtuin family members were located in nuclear and mitochondrial organelles, maintaining redox homeostasis via regulating the oxidative stress-associate genes [19], we also examined whether the Sirtuin family participate in the Tlr4-mediated oxidative alteration during foam cell formation of VSMCs. We found that amid the Sirtuins family, Sirt1 and Sirt3 were downregulated most signi cantly after oxLDL stimulation. As previous publications revealed that Sirt1 and Sirt3 were capable of suppressing ROS accumulation through inhibiting the activities of Nox and activating the Mnsod in aging and carcinogenesis [37-39], we hypothesized that Sirt1 and Sirt3 may also serve as upstream molecules to regulate Nox2 and Mnsod expression in VSMCs during atherosclerosis. In line with these ndings, we observed that overexpression of Sirt1 and Sirt3 upregulated Mnsod but downregulated Nox2 under oxLDL treatment. Furthermore, raising the expression of Sirt1 and Sirt3 inhibited production of ROS and foam cells phenotype in VSMCs. These results illustrated that Sirt1 and Sirt3 participated in ROS accumulation and foam cells formation via regulating Nox2 and Mnsod expression and regulating Sirt1 and Sirt3 might alleviate the oxidative stress and foam-cell formation in VSMCs. More importantly, when considering reversed expression of Sirt1 and Sirt3 after knockdown of Tlr4, we concluded that oxLDL promoted ROS accumulation via Tlr4-Sirt1/3 signaling pathway, thus inducing the foam-cell phenotype of VSMCs (Fig. 6).

Conclusions
Our study demonstrated that Tlr4 is a critical regulator in oxLDL induced foam cell formation of VSMCs via mediating Src kinase as well as Sirt1 and Sirt3. Beyond the role of Tlr4 in the in ammation response of VSMCs, we provided an integrated mechanism about Tlr4 in VSMCs phenotype transition under oxLDL stimulation. Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests. Overall network of Tlr4 in regulating oxLDL induced foam cell formation in VSMCs.