| Background: Atherosclerosis (AS) is a chronic inflammatory disease of the arterial wall characterized by the progressive accumulation of lipids, extracellular matrix, and cells. Studies have revealed that many different cell types, including macrophages, lympho-cytes, endothelial cells, and vascular smooth muscle cell (VSMC), are involved in atherosclerotic lesion formation. Understanding the molecular and cellular mechanisms that lead to the development of atherosclerosis is critical for identifying strategies to limit the disease progression before it leads to clinical consequences.Human cellular repressor of E1A-stimulated genes (CREG) is a 220-amino acid secreted glycoprotein (human isoform). It can inhibit proliferation and promote differentiations of human embryonic carcinoma cells (NTERA-2) and cultured VSMC via E1A-induced transcriptional activation and cellular transformation. Overexpression of CREG inhibits cell cycle progression and induces differentiation of NTERA-2 cells, even in the absence of an inducer such as retinoic acid . CREG knockdown prevents the in vitro maturation of VSMC induced by serum starvation. Restenosis and AS are two disorders characterized by abundant proliferation and migration of VSMC. Previous studies have demonstrated a protective effect of the CREG against restenosis. However, the role of CREG in AS is undetermined.Objective: The aim of the present study is to examine the impact of CREG on the VSMC of AS.Methods:1. Speciman (1) Human atherosclerotic tissue specimens were obtained from peripheral arterial occlusion patients who under went amputated surgery. Healthy aortas were obtained from three organ donors. (2) Ten Albino New Zealand rabbits were maintained on high fat diet (1% cholesterol, 10% fat, 10% dried egg yolk) for about 1 or 2 months. The root of aorta was extracted after the rabbit was anaesthetized with sodium phenobarbial. (3) ApoE-/- mice on the C57BL/6 background were obtained from Department of Laboratory Animal Science, Peking University, China. Mice were weaned at 8 weeks of age and fed an atherogenic diet ad libitum (containing 21% fat and 0.25% cholesterol).2. Immunohistochemistry and immunofluorescence(1) Tissue specimens were dissected in phosphate-buffered saline (PBS) and fixed overnight in 4% paraformaldehyde at 4°C. Fixed specimens were dehydrated in graded ethanols and embedded in paraffin. Serial 5μm sections were cut and dewaxed in xylene and subsequently rehydrated in graded ethanol and water. The sections were incubated in methanol containing 3%(v/v) hydrogen peroxide for 30 minutes to quench endogenous peroxidase activity, then blocked by incubation in 4% BSA (Sigma) for 60 minutes at room temperature to prevent nonspecific binding of antibodies to the tissue. Thereafter, the sections were incubated with monoclonal antibody against CREG, SMαactin, SM MHC and CD31 (1:100 dilution in PBS, Sigma, USA) overnight at 4°C. After this incubation period, the slides were rinsed with three changes of PBS for 5 minutes each, then exposed to a 1:150 dilution of peroxidase-conjugated goat anti-mouse IgG (Sigma, USA) for 90 minutes at room temperature. After rinsing as before, the peroxidase label was developed by exposure to 0.05% (w/v) diaminobenzidine in phosphate buffer containing 0.01% hydrogen peroxide for 2 minutes. The slides were washed in PBS, counterstained with hematoxylin (Sigma), dehydrated in an ethanol/xylene series and mounted under coverslips. The specimens were viewed using a standard light microscope. (2) The brachiocephalic artery of apoE(-/-) were dissected and immersed in 4% paraform for 30 min, then in 7.5% sucrose overnight. After that the specimens were embeded in optimal cutting temperature (OCT). Frozen serial sections (5μm) were treated with 0.3% H2O2 in PBS to block endogenous peroxidase activity, followed by blocking in 4% BSA (Sigma). Slides were incubated (2 hours at 20℃) with purified rat monoclonal antibody against mouse VSMC (CREG, SMα-actin) (Sigma) at 1:100 dilution (20 mg/mL). A second biotinylated antibody was added, anti-rat at 1:50 dilution. After incubation with the appropriate TRITC-conjugated secondary antibodies (Zhongshan Biological Technology), the sections were observed using confocal laser scanning microscopy (CLSM).3.VSMCs CulturesVSMCs were obtained from human nonatherosclerotic arteries of hearts removed in transplant operations by using a modification of the explant technique, and those used in the experiments were between the third and fifth passage. Briefly, VSMCs were cultured in DMEM supplemented with 10%FCS. Cell culture media and reagents were from GIBCO/BRL (Invitrogen). Cells were seeded in plates, and were arrested at subconfluency with medium containing 0.4% FCS for 48 hours. Arrested cells were stimulated with 100μg/mL oxLDL for different times. When inhibitors were used, VSMCs were preincubated with them for 30 minutes before stimulus. The inhibitor used was PD98059 (an ERK kinase inhibitor, Sigma).4. Protein purification and western blot analysisFor Western analysis, cell lysates or homogenates of human, rabbits or mice artery were prepared in the lysis buffer. After centrifugation at 15 000 g for 10 minutes, the supernatant was used for western blotting. Total protein concentration was determined by using the DC Protein Assay Kit (BIO-RAD). Proteins were resolved in SDS-polyacrylamide gels and transferred to PVDF membranes. The membranes were incubated with appropriate primary antibodies. The specific binding was detected with HRP-conjugated secondary antibodies and an ECL Western Blotting Kit. The blots were quantified on a Bio-Rad Gel Documentation System.5. Quantification of AtherosclerosisAll images were captured and analyzed by National Institutes of Health Image software. Lipid lesion formation was analyzed by determining the percent area of oil red O stained to the total cross-sectional vessel wall area. The average value for the 5 locations for each mouse was used for analysis.6. TNF-αand IL-1βELISAThe amount of TNF-αand IL-1βsecreted into the cell culture supernatant was determined by ELISA using the human TNF-αand IL-1βkit (R&D Systems). The levels of TNF-αand IL-1βwere quantified by comparison with standard curves concurrently by using recombinant TNF-αand IL-1βaccording to the manufacturer’s protocol.Results:1. Downregulation of CREG in VSMC in Atherosclerotic Artery(1) The expression of CREG protein was downregulated in AS artery of high-fat diet rabbit compared with normal artery of normal diet rabbit. Simultaneously, the VSMC markers of SMα-actin and SM MHC are also decreased in media of arteriosclerotic artery. Western blot was also displayed CREG was decreased in AS artery compared with nomal artery and downregulation of CREG was time-dependent. (2) In Human AS Artery, CREG was detected in endothelial cells and media VSMC. We discovered that CREG was expressed at lower levels in AS aorta sections accompany with SMα-actin and SM MHC expressions compared with normal artery. (3) The change of expression CREG in arterial wall of apoE(-/-) mice: From 1 to 8 weeks of atherogenic diet, we discoved that CREG did not decrease at the early stage of AS. However, its expression obviously increased within two weeks of atherogenic diet. At the fourth weeks of atherogenic diet, CREG expression droped down rapidly and later it rose up gradually till the 8th week. But CREG did not resumethe the level of normal. At the same time we observed the change of nuclear factor-kappa B (NF-κB) expression, an inflammatory marker, in a time-dependent fashion. Within 8 weeks of atherogenic diet, NF-κB incresed gradually, which means that inflamation continued and became more serious throughout atherosclerosis.2.OxLDL downregulated CREG level and overexpression of CREG inhibite prolifertion in cultured human VSMCHuman VSMC were serum-depleted for 48 hours and then exposed to oxLDL for 24 hours. We observed a significant reduction in CREG protein level within 12 to 24 hours of exposure to oxLDL. We obsevered that SMα-actin and SM MHC decreased and phosphorylation of ERK1/2 (P-ERK1/2) increased in a time-dependent fashion. CREG-DW cells were pretreated with PD98059 (a ERK inhibitor, tested at concentrations 50μmol/L) for 30 minutes before they were stimulated with oxLDL for 12 hours. The results showed that pretreatment with PD98059 (50μmol/L) significantly reduced the number of oxLDL-induced proliferation cells ( 29.62±2.1% vs. 42.23±1.8%, P < 0.05 ). Western Blot also displayed that P-ERK1/2 was inhibited by PD98059. The number of cells stimulated with 100μg/mL oxLDL after 24 hours in CREG-UP group was significantly lesser than CREG-NR group, and in CREG-DOWN group the number of cells was much more compared with CREG-NR group. The number of Brdu positive cells stimulated with 100μg/mL oxLDL 24 hours later was the least in CREG-UP group among the three groups. While the number was much more in CREG-DOWN group than the other two groups. These deminstrated that CREG-UP significantly inhibited VSMC proliferation induced by oxLDL stimulation (P<0.05) . 3.CREG overexpression inhibited NF-κB p65, TNF-αand IL-1βin human VSMCSerum-starved, human VSMCs were incubated with 100μg/ml oxLDL for 12~24 hours. CREG-NR exposing to oxLDL (100μg/ml), the NF-κB protein expression in nuclear extracts was markedly up-regulated. However, if CRGE-UP stimulated with the same concentration of oxLDL (100μg/ml), the expression of NF-κB protein was up-regulated slightly. In CREG-DOWN, NF-κB was more abound than that in CREG-NR group. The supernatant were collected at indicated time and TNF-αand IL-1βwere detected by ELISA. We surprisingly discovered that the levels of TNF-αand IL-1βat 24h was also markedly decreased in CREG-UP cells compared with that in CREG-DOWN or CREG-NR group.4.Adnovirus-mediated overexpression of CREG reduce atherosclerotic in vivoTo further elucidate the involvement of CREG in atherosclerosis, AD-GFP-CREG or control (AD-GFP) was injected into the tail vein of apoE(-/-)mice fed an atherogenic diet. After 6 weeks on a atherogenic diet, apoE(-/-) mice received a single systemic administration of GFP-CREG adenovirus (AD-GFP-CREG) (4.75×1010pfu) or control adenovirus (AD-GFP) and euthanized 6 weeks later. After injection of AD- GFP - CREG or AD-GFP via tail vein, Western blot analysis of CREG and GFP indicated higher CREG levels as well as expression of GFP levels in the AD-GFP-CREG group compared with controls between 1 and 5 weeks. In the AD-GFP-CREG group, CREG and GFP protein levels rose progressively between 1 and 2 weeks after adenoviral delivery, after which CREG and GFP levels gradually declined and tapered off by 4 weeks. The AS lesion area in the control group was obviously larger than that of the AD-GFP-CREG group, as indicated by a significantly increased lipid-positive area (oil red O) in the aortae (P<0.05). In addition brachiocephalic plaque volume was determined as a measure of the effect of AD-GFP-CREG on atherogenesis in apoE-/- mice. AD-GFP-CREG treated mice showed a highly significant reduction in AS plaque lesion size compared with control mice (0.257±0.03 mm2 versus 0.462±0.04 mm2; n=10/group; P < 0.01). Lesion size in a separate group of uninfected animals (0.521±0.05 mm2; n=4) was similar to AD-GFP treated mice, indicating that adenoviral infection per se did not increase AS. AD-GFP-CREG treated mice showed a highly significant reduction in NF-κB expression compared with control mice receiving AD-GFP after two weeks of injection. And TNF-αlevel of artery was significantly decreased in AD-CREG-GFP group compared with control. IL-1βwas also decreased, but had not significant difference between the two groups.Conclusions: CREG has a protective effect against atherosclerosis, which is related to inhibition of proliferation and inflammatory response of VSMCs. |
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