Homocysteine promotes migration of adventitial broblasts via Angiotensin II type 1 receptor activation to aggravate atherosclerosis

Background: Hyperhomocysteinemia (HHcy) is an independent risk factor for atherosclerosis. However, the mechanisms of HHcy-induced arteriosclerosis are largely unknown. Objective: To clarify the effect of Hcy on adventitial broblasts (AFs) and its relation with angiotensin II type 1 receptor (AT1R). Method: The apolipoprotein E gene-decient mice (Apoe−/−) were used as the murine model for atherosclerosis. HHcy was induced and treated by feeding them 1.5% methionine and telmisartan (gavage 10 mg/kg/d) for 12 weeks, respectively. The AFs and HEK293A cells transfected with the AT1R plasmid were used to investigate the interaction between Hcy and AT1R. All data were expressed as mean ± SEM. The data were analyzed by one-way ANOVA or repeated measurement ANOVA. Results: HHcy aggravated the plaque area of the aortic root and increased the expression of IL-6, MCP-1, and the macrophage marker Mac3 in the plaque and adventitia of the aorta, whereas telmisartan improved this effect. Hyperhomocysteinemia induced the occurrence of the AFs marker protein ER-TR7 in the plaque and the entire layer of the aorta, whereas telmisartan also improved this effect, indicating that hyperhomocysteinemia induced AFs migration and that AT1R mediated this process. Hcy increased the production of AFs H 2 O 2 , ROS, and IL-6 of AFs, indicating that Hcy activated the oxidative stress and inammatory reactions, which may induce cell migration. The subsequent scratch and transwell experiments conrmed that Hcy induced the AFs migration and that telmisartan inhibited this effect. Hcy increased the expression of AFs AT1R and the phosphorylation levels of PKC and ERK1/2 in the AF and HEK293A cells transfected with the AT1R plasmid, whereas telmisartan inhibited this effect, indicating that Hcy activated AT1R intracellular

experiments con rmed that Hcy induced the AFs migration and that telmisartan inhibited this effect. Hcy increased the expression of AFs AT1R and the phosphorylation levels of PKC and ERK1/2 in the AF and HEK293A cells transfected with the AT1R plasmid, whereas telmisartan inhibited this effect, indicating that Hcy activated AT1R intracellular signaling pathway.

Background
Hcy is a nonessential sulfur-containing amino acid derived from the essential amino acid methionine and actively participates in various biochemical reactions. 1 HHcy (circulating Hcy ≥ 15 μ m) is an independent risk factor for many cardiovascular diseases, such as atherosclerosis, coronary heart disease, and abdominal aortic aneurysm. Although there is evidence that HHcy can cause vasodilation, damage endothelial cells, promote intravascular proliferation, promote outer membrane activation, and disrupt hemostasis/coagulation, 2-5 the mechanism by which Hcy aggravates atherosclerosis remains elusive.
AT1R is a key player in the renin-angiotensin system. AT1R activation leads to cardiac remodeling, ventricular hypertrophy, intimal formation, smooth muscle cell proliferation, and migration. 6,7 Genetic deletion of AT1R effectively prevents pathological vascular injuries in various animal models, such as models hypertension and atherosclerosis. 8 Although recent studies indicate Hcy directly interacts and activates AT1R to aggravate vascular injury 1 and Hcy accelerates collagen accumulation in the adventitia of balloon-injured rat carotid arteries via AT1R expression, 9 little is known about the association between Hcy and AT1R in atherosclerosis.

Migration and proliferation responses of cells in the vascular wall and deposition of extracellular matrix
(ECM) play a key role in restenosis and atherosclerosis. 10 Although medial smooth muscle cells have been regarded as the main source of cells, 11,12 there is now increasing evidence that the adventitial layer can also be a signi cant contributor to the arterial remodelling process through increased angiogenesis. 13 Findings by Richard C.M. et al. 10 provide direct evidence that adventitial fibroblasts migration contribute to neointima formation after balloon injury. Hcy induced AT1R expression that activates various signaling molecules extracellular signal-regulated protein kinases 1 and 2 (ERK-1/2) and protein kinase C (PKC). These signaling molecules activation can promote cell migration and invasion. [14][15][16] However, the precise role of Hcy in the migration and invasion of adventitia broblasts is unclear in the process of atherosclerosis formation. Therefore, we investigated whether HHcy stimulates AFs migration and invasion in mice via AT1R activation to accelerate the formation atherosclerosis.

Methods
Materials L-homocysteine (69453), telmisartan (T8949), was purchased from Sigma-Aldrich. Disposable consumables such as cell culture bottle and 6-well plates were purchased from Guangzhou Jet Bio-Filtration Co., Ltd.

Animals and Treatments
A total of 21 ApoE-/-mice (male, 8 weeks) were purchased from Beijing Vital River Laboratory Animal Technology Co., Beijing, China (Ltd. Number: SCXK2016-0006). The mice were randomly divided into three groups: a control group with a standard mouse diet (n = 7), an HHcy group fed with standard mouse diet plus 1.5% methionine (n = 7), 17 Tail-cuff measurement of systolic blood pressure Systolic blood pressure (SBP) was recorded by a computerized non-invasive tail-cuff system (Cat. NO.BP-300A, Chengdu Techman Software CO.,LTD). Measurements were performed in quiet environment to avoid causing mice anxiety. The mice underwent 7 consecutive days of training sessions to become accustomed to the tail-cuff procedure. Blood pressure of mice underwent 12 consecutive weeks was measured from 1 to 6 PM on weekends every week. Three measurements were weekly performed on each mouse, so that the average of total of 3 measurements was represented as the SBP of each mouse.

Measurement of Serum Homocysteine Level
The levels of TG, TC, HDL, and LDL were assessed with kits from Beijing Sino-UK Institute of Biological Technology. Hcy concentration was measured using enzyme-linked immunosorbent assay (HY-N0080) in Beijing Sino-UK Institute of Biological Technology.

Histology and Immunostaining
The heart and approximately 2 mm of proximal aortas were fresh frozen in OCT compound. 20 Tissue samples were cut into 7μm thin slices. Frozen tissue sections were stained with Oil Red O, H&E, Masson, and immunostaining. 21 A Masson's trichrome staining kit was used to detect collagen deposition in the atherosclerotic plaque. Analyzed with Image-Pro Plus 6.0 or ImageJ software. 20 Cryosections were xed in 4% paraformaldehyde and subsequently treated with H 2 O 2 and after the primary antibody was blocked in 5% goat serum (1:100) at 4°C overnight. The tissue sections were washed in PBS then incubated with secondary antibody (1:100). They were incubated in diaminobenzidine until a change in color was observed under the microscope and then the nucleus was stained with hematoxylin. 22 Immuno uorescence staining procedure was similar to immunostaining.

Cell Culture
Primary Rat AFs Cells were prepared according to common methods 19 and with some modi cations. SD rat were euthanized with sodium pentobarbital at 120 mg/kg. Thoracic aortas were removed and cleaned under sterile conditions. The adventitia was stripped from the aorta and cut into pieces about 2-3 mm3 in size. Then, the tissue pieces were attached to the 6-well plates and immersed in Dulbecco's modi ed Eagle's medium (DMEM, Gibco) containing 30% fetal bovine serum (FBS, Gibco) and maintained at 37°C, 5% CO 2 . Cells were collected, and when the cells grew to about 70%-80% fusion, the tissue pieces could be cultured again. AFs were veri ed by positive immuno uorescent staining of vimentin and ER-TR7. The AFs after passage were cultured in DMEM supplemented with 10% FBS at 37°C, 5% CO2. All AFs used in this study were from early passage with a maximum of four passages.
The human embryonic kidney cell line HEK293A was a generous gift from our laboratory and cultured in DMEM (high glucose) supplemented with 10% FBS at 37°C in a humidi ed atmosphere containing 5% CO 2 . The cells were con rmed of no mycoplasma contamination before application.

Cell Proliferation Assay
The proliferation of cells was examined with Cell Counting Kit-8 (Cat. NO. K1018, ApexBio Technology LLC). Brie y, cells were seeded in 96-well plates with 5 × 10 3 cells/well, cultured for 24 hours, and subjected to the indicated treatment. 23 The absorbance at 450 nm was measured using an automatic microplate reader (n = 3).

Wound-Healing Assay
AFs (5x10 5 cells/well) were inoculated into 12-well plates. After the cells completely covered the bottom wall of the 12-well plate, three vertical lines were drawn in the 12-well plate with a sterile 200-μl pipette tip, and the oating cells were removed by PBS washing. 24 DMEM 1000 μl containing 1% FBS and Hcy 200 μmol/L was added into the 6-well plate. The cells were cultured in an incubator at 37°C and 5% CO 2 for 48 hours. At the same location, the images of 0 and 48 hours were taken to observe the cell migration.
Cell migration analysis was processed by Image-Pro Plus 6.0 software 24 (n = 3).

Transwell Matrigel Invasion Assays
Transwell Matrigel invasion assays were performed using a transwell membrane (8-μm pore size, 6.5-mm diameter; Cat. NO. 35488, Corning Incorporated) in a 24-well plate. The Hcy-treated cells were digested with trypsin (0.25%) and then centrifuged and the supernatant was discarded. The cell suspension was prepared with DMEM without serum. The cells (1x10 5 cells/well) were loaded to the upper side of the chamber (500 μl/well), and the lower chambers contained 750 μl/ of DMEM with 10% FBS as chemoattractant. The cells were cultured in the cell incubator for 6 hours. The lter inserts were then removed from the wells. Cells on the upper surface of the lter were removed using cotton swabs and the membranes were xed and stained with crystal violet reagent. 21,25 Invasion was quanti ed by counting 5 random elds under a light microscope (Leica, Germany) at x40 magni cation.

Detection of Intracellular ROS
Intracellular ROS generation was monitored by using Reactive Oxygen Species Assay Kit (WLA070a, Wanleibio). Cells (1x10 6 cells/well) were seeded in 6-well plates. Cells were treated with Hcy for 48 hours and stained with 20 µmol/L DCF-DA in culture medium for 30 minutes in the dark. The cells were then harvested and washed in PBS three times and oxidation-induced DCF uorescence was assayed by uorescence microscopy. 26 Assay of Hydrogen Peroxide Level Hydrogen peroxide level was assayed using a Hydrogen Peroxide Assay Kit (Cat. NO. A064-1-1, Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer's instructions.

Statistical Analysis
All data were expressed as mean ± standard error of the mean (SEM), except Table 1 and table 2 (mean ± standard deviation). All experiments were repeated at least three times. The data were analyzed by GraphPad Prism 5.0 (GraphPad Software, San Diego, California, USA) and SPSS version 19.0 (SPSS Inc., Chicago, IL, USA). The data were analyzed by one-way ANOVA or repeated measurement ANOVA, without assuming sphericity. When multiple measurements were made in the same cell, Greenhouse-Geisser correction was performed. The data that failed to pass the normality test were analyzed by nonparametric test. The difference was signi cant (P < 0.05).

Vital parameters
HHcy was induced by feeding ApoE-/-mice a high-methionine diet for 12 weeks. The results showed that serum Hcy concentrations in the HHcy group were signi cantly higher (35.89 ± 3.26 μmol/L) than the control group (23.85 ± 2.09 μmol/L), (*P < 0.05). Therefore, the high-methionine diet signi cantly increased serum concentrations of Hcy, suggesting that the diet-induced HHcy model was successful (Table 1). Total cholesterol (TC), and high-density lipoprotein (HDL) in HHcy group were signi cantly difference except triglyceride (TG). TC level in HHcy group was higher than that in control group (P < 0.05). TC decreased signi cantly after telmisartan treatment (P < 0.05), Low-density lipoprotein (LDL) level in HHcy group was higher than that in control group, but there was no statistical signi cance. After telmisartan treatment, LDL decreased signi cantly (P < 0.05), as shown in Table 1.
Telmisartan treatment decreased systolic blood pressure; however, there was no statistical difference in three groups, showing no signi cant changes in body weight (Table 2).

HHcy accelerates atherosclerosis formation
A large amount of lipid deposition was found in the aortic root of mice in the HHcy group. The area of atherosclerotic plaque accounted for about( 23.1%) of the aortic root, which was larger than that in the control group (6.3%), as shown by the Oil Red O staining. The plaque area in the HHcy+Telmi group (9.1%) was signi cantly lower than in the HHcy group (Fig. 1). These results demonstrated that Hcy signi cantly induced atherosclerosis in ApoE-/-male mice, which may be related to AT1R activation.
The AT1R mediates HHcy-aggravated plaque instability Hematoxylin and eosin staining showed higher number of foam cells and a larger acellular necrotic core area in the HHcy group, which was improved in the HHcy+Telmi group (Fig. 2a). Moreover, Masson staining (Figs. 2b and 2c) showed that total collagen content, expressed as a percentage of the section area, was signi cantly reduced in HHcy group compared with that in control group ( P < 0.001), while it was signi cantly increased in the HHcy+Telmi group. These results suggested that Hcy increased plaque instability via AT1R.
The number of in ammatory cells such as MCP-1, IL-6, and Mac3 in aortic root plaques in HHcy group was signi cantly higher than that in control group (P < 0.05), while it was reduced in the HHcy+Telmi group (Figs. 3a and 3b). These results suggested that HHcy increased the in ltration of in ammatory cells in plaques and accelerated the progression of atherosclerosis via AT1R.

HHcy ampli es aortic adventitial in ammation in ApoE −/− male mice
Results showed that in ammatory cells MCP-1, IL-6, and Mac3 were preferentially produced in the adventitial layer. Quantitative analysis of aortic adventitia revealed higher expression of MCP1, IL-6, and Mac3 in the HHcy group than in control group. Furthermore, the AT1R blocker telmisartan markedly attenuated MCP1, IL-6, and Mac3 expression (Fig. 4), which is consistent with observations in a previous study. 2 These results demonstrated that Hcy signi cantly exaggerated vascular in ammation in ApoE -/male mice, which may be related to AT1R activation.

HHcy induces AFs migration in ApoE -/male mice
Immuno uorescence was used to detect AFs marker ER-TR7 (Fig. 5). In the control group, staining showed ER-TR7 expression in aortic adventitia. Moreover, deposition of ER-TR7 increased in the whole aortic wall and plaque in the HHcy group. This phenomenon was reduced by telmisartan, as shown in the HHcy+Telmi group. These results further demonstrated that Hcy induced AFs migration and invasion in ApoE-/-male mice, which may be related to AT1R activation.

Hcy upregulates proin ammatory effects in AFs of rat
Our results showed that Hcy of 200 μmol/L greatly enhanced hydrogen peroxide (H 2 O 2 ) secretion ( Fig.   6a) and increased intracellular ROS at 48 hours after stimulation (Fig. 6b). Hcy induced IL-6 expression in a time-dependent manner, and the most effective Hcy time was 36 hours as indicated by using western blot (Figs. 6c and 6d). These results demonstrated that Hcy could activate AFs oxidative stress and in ammatory response.

Hcy induces expression of MMP-9 in AFs
Hcy induced MMP-9 expression in a time-dependent manner, and the most effective Hcy time was 36 hours as indicated by using western blot (Fig. 7). Matrix remodeling is a critical step to initiate and support AFs motility.

HHcy induces AFs proliferation, migration, and invasion
AFs were treated with increasing concentrations of Hcy (0, 50, 100, 200, and 400 μmol/L) for 48 hours. Results showed that cell viability was increased signi cantly in a dose-dependent manner, indicating that Hcy promoted cell proliferation (Fig. 8a). Cells were treated with 200 μmol/L Hcy for 48 hours. The wound areas of the cells were observed via scratch assay under a microscope at 0 hour and 48 hours. Results showed that Hcy signi cantly increased cell migration (Figs. 8b and 8c). Cell invasion was detected by transwell Matrigel invasion assays. In AFs stimulated by 200 µmol/L Hcy for 48 hours, cell invasion was signi cantly increased (Figs. 8d and 8e). After telmisartan (1 mmol/L) pretreatment for 1 hour, this phenomenon was inhibited. Our results suggested that Hcy promoted migration and invasion via AT1R activation in AFs.

Hcy enhances AT1R expression in cultured AFs
Hcy induced AT1R expression in a time-dependent manner as indicated by using western blot (Fig. 9). There was a signi cant increase in AT1R expression at 36 hours (Figs. 9a and 9c). Moreover, Hcy of 50 to 300 μmol/L signi cantly facilitated AT1R expression, with a peak at 200 μmol/L (Figs. 9b and 9d). This phenomenon was further veri ed by immuno uorescence staining of AT1R protein (Fig. 9e).
To further verify the results, we constructed EGFP-AT1R fusion protein particles and transfected HEK293A cells to assess the effect of Hcy on PKC and ERK1/2 signaling pathways. We used agarose gel electrophoresis to detect the extracted plasmids and transfected into HEK293A cells (Figs. 11a and 11b). Western blot indicated AT1R levels in HEK293A cells (Figs. 11c and 11d). The results con rmed the successful construction of the plasmids by GeneChem Co. Ltd. (Shanghai, China) (Fig. 11a). Fluorescence microscopy showed that the green uorescence was much brighter in the HEK293A cells transfected with AT1R compared with the control group (Fig. 11b). Western blot indicated higher AT1R expression in plasmids transfected into HEK293A cells than in the control group (Figs. 11c and 11d).
These results indicated that the plasmid was successfully constructed and transfected into HEK293A cells.
In HEK293A cells transfected with AT1R, Hcy at 200 μmol/L resulted in delayed but sustained PKC and ERK1/2 phosphorylation, which was decreased by telmisartan pretreatment (1 mmol/L) (Figs. 12a to  12c). These results suggested that Hcy could activate the downstream PKC and ERK1/2 signaling pathway downstream of AT1R.

Discussion
Previous studies have demonstrated that AFs migrate from the adventitia to the neointima upon balloon injury in rats. 10 Recent data have demonstrated that Hcy directly interacted and activated with AT1R to aggravate adventitial in ammation, collagen accumulation, and vascular injury. 1,2,9 However, little is known about the possible mechanisms by which Hcy can promote AFs migration and invasion and accelerate atherosclerosis formation via AT1R activation. This study revealed that Hcy induced AFs migration and invasion, which was inhibited with telmisartan treatment, an AT1R inhibitor. Furthermore, Hcy increased the expression of AT1R and MMP9 in AFs and activated the ERK and PKC signaling pathway of AT1R.
A large body of evidence suggests the crucial role that dyslipidemia play in vascular pathology associated with HHcy. 27 Vascular mitochondrial oxidative stress increases under hyperlipidemic conditions, resulting in enhanced atherosclerotic plaque in ammation, necrotic core and brous cap remodeling, and susceptibility to rupture. 20 In this study, ApoE-/-mice receiving methionine diet displayed high levels of serum Hcy, TC (Table 1), and typical atherosclerotic plaques, thereby causing accumulated lipid, increased necrotic core, 17 decreased collagen ber content, and in ltrated in ammation cells in the aortic root. After telmisartan treatment, these effects of HHcy were inhibited 28 (Figs. 1-3). Consistent with the study by Schlimmer, our data revealed that telmisartan can effectively reduce oxidative stress in aortic tissue and subsequent peroxidation of membrane lipids, thereby preventing atherosclerosis formation. 29 Based on these results, we speculated that Hcy might promote atherosclerosis formation via AT1R activation. Furthermore, lipid loading increased adventitial metalloproteinase-9 levels to promote cell migration. 30 Intriguingly, we found that HHcy induced AFs migration and invasion in vitro and in vivo. Telmisartan also inhibited the migration and invasion of broblasts induced by HHcy (Figs. 5 and 8).
Consistent with Dan Yao, 9 Hcy promoted AT1R expression in AFs in a time-and concentration-dependent manner (Fig. 9). Hcy increased ROS production in AFs, increased H 2 O 2 in cell supernatant, and promoted IL-6 and MMP9 expression in AFs (Figs. 6-7). ROS produced by NADPH oxidase is an important mediator in signal transduction. 31 Cell migration in its essence is an invasive process that requires degradation of ECM through activation of matrix metalloproteinases (MMPs). 31 Compelling evidence suggests that AT1R leads to ROS production. 2 Moreover, ROS can directly or indirectly activate MMPs, hence inducing cell migration. These results suggest that Hcy may promote AFs migration and invasion by enhancing AT1R expression and then accelerate atherosclerosis formation. The development of atherosclerosis depends largely on local in ammation. A large number of macrophages in atherosclerotic lesions is an indicator of a more unstable and rupture-prone phenotype. 32,33 In fact, these cells are aggregated in plaques and swallow lipids, become foam cells, and release cytokines and growth factors, as well as MMPs and ROS that degrade the structures of plaques to provide conditions for cell migration. 34 Our study also showed that HHcy induced the in ltration of in ammatory cells in aortic root plaque and aortic adventitia in ammation in mice, which was consistent withLili et al. 2 (Figs. 3-4). The results of mice aortic immunohistochemistry showed that in ltrations of MCP-1, IL-6, and Mac3 were mainly observed in the tunica adventitia of mice aorta. MCP-1 is a potent chemokine that stimulates cells migration. 35 There is increasing evidence suggesting that Hcy has a possible role in triggering arterial in ammation. 2,36,37 ERK-1/2 activation has strongly been shown to be associated with in ammation 38,39 and atherosclerosis. 40 A large number of studies have shown that ERK and PKC activation can promote cell migration and invasion. 41,42 To further explore relevant mechanisms, AFs and HEK293A cells transfected with human AT1R were stimulated by Hcy, with or without telmisartan pretreatment for 1 hour, and the expression level of ERK-1/2 and PKC signaling pathway in AT1R was detected. Our results demonstrated that Hcy induced a robust and signi cant increase of ERK-1/2 and PKC phosphorylation, which was markedly attenuated by telmisartan (Figs. 11-12). These results clearly suggest the involvement of AT1R in mediating ERK-1/2 and PKC phosphorylation in Hcy.
Furthermore, our ndings suggest that Hcy induces AT1R and ERK-1/2 and PKC activation. Activated ERK-1/2 and PKC further contribute to AFs proliferation, migration, and invasion and participate in the process of atherosclerosis by modulating MMP-9.

Conclusion
In conclusion, our experiments con rm that HHcy aggravates atherosclerosis formation, at least in part, by activating AFs through AT1R, causing AFs proliferation, migration, and invasion. These ndings suggest that strategies designed to block AT1R may reduce HHcy-related adventitial migration and invasion and thus reduce HHcy-related vascular diseases. SBP and weight were measured in five to seven mice per group. Data represent mean ± SD.

Abbreviations
Control, standard mouse diet group; HHcy, 1.5% methionine group; HHcy+Telmi, HHcy + telmisartan treatment group; SBP, systolic blood pressure. SBP before treatment: the systolic blood pressure of mice without 1.5% methionine diet and telmisartan gavage. SBP after treatment: the systolic blood pressure of mice underwent 12 consecutive weeks after receiving 1.5% methionine diet and telmisartan gavage.