| Background:As the chronic inflammatory pathological basis, atherosclerosis is mainly involved in large and medium arteries and leads to various cerebral-cardiovascular diseases such as coronary heart disease, ischemic necrosis, abdominal aortic aneurysm, heart failure and stroke. It has become the major risk factor that results in high mortality and high morbidity throughout the world. As a complex pathological progression, AS is associated with various components, metablosim and immune inflammation. Recently, the pathogenetic mechanism of AS has been studied intensively, however, the specific mechanism and risk factors leading to AS were not clearly clarified. Thus, it has been a hotspot to investigate the risk factors resulting in AS and explore the pathological mechanism of AS. The specific target may provide preventive and therapeutic strategies and favor the atherosclerotic events.The prolyl hydroxylase domain proteins (PHDs) belongs to the family of 2-oxoglutarate and iron-dependent dioxygenases, which could hydroxylate the proline residues of hypoxia inducible factor a (HIF-a) causing proteasomal degradation via combination with the von Hippel-Lindau protein (pVHL) ubiquitin ligase under normoxia. In hypoxia condition, inhibition of PHDs activity results in the stabilization of HIF-la. Aggregated HIF-la together with HIF-1 β forms dimers and then translocate into the nucleus where a series of genes transcription are activated. Three PHD isoforms (PHD1, PHD2 and PHD3) in mammalian cells have been identified to date. PHD3 is highest in enzymatic activity among the three isoforms and it has being a research focus of PHDs family. PHD3 is abundantly expressed in heart, muscle and great vessels, and is evenly distributed in the cytoplasm and nucleus.PHD3 is mainly regulated in two ways. The first way is HIF-dependent pathway. Under hypoxia, the hydroxylation and ubiquitin of HIF-1 a were decreased causing its elevation in cytoplasm. Increased HIF-1 a can directly combined with the promoter of PHD3 and promote its expression at the transcriptional level. The second way is HIF-independent pathway. In mice renal tubular juxtaglomerular cells, the expression of PHD3 in mRNA and protein level was elevated with Angiotensin II treatment.Besides, previous studies demonstrated reduced erythrocytes, platelet aggregation and vascular spasm with PHD3 kockout in microvascular thrombosis, which alleviated symptoms, in myocardial infarction and ischemia reperfusion animal models, knockout of PHD3 can decrease the apoptosis of erythrocyte and retard cardiac remodeling, which consequently improves cardiac function. Compared with neonatal pulmonary vascular smooth muscle cells, expression of PHD3 in adult pulmonary vascular smooth muscle cells is increased, and elevated PHD3 level is associated with apoptosis, migration and secretion of pulmonary vascular smooth muscle cells. In aortic balloon injury rat model, PHD3 increased in neovascularization indicating that PHD3 may participate in the process of angiogenesis. PHD3 also involves in regulating proliferation, differentiation and apoptosis of various inflammatory cells, and subsequently, regulating the systemic and local inflammation. Previous studies indicated that PHD3 expressed in diverse inflammatory cells including neutrophils, macrophages and mast cells. A study associated with neutrophils illustrated that PHD3 could mediate the adhesion, chemotaxis and secretion of inflammatory cells. Besides, PHD3 exerts high expression in macrophages with proinflammatory functions (M1). During AS progression, M1 and M2 subtype of macrophages can co-exist in the atherosclerotic plaque. At the early stage, a variety of inflammatory mediators can induce M1 macrophages which promote the generation of various proinflammatory factors such as tumor necrosis factor a (TNF-α), interleukin (IL) 6 and Monocyte chemotactic protein (Monocyte chemo-attractant protein, MCP) 1. As inflammation progression, M2 macrophages increase and ameliorate inflammation, and thus promote restoration of the damage tissues. Previous studies displayed that PHD3 expressed in M1 macrophages and M1 macrophages in local inflammation exerts high PHD3 expression, which reminds us that PHD3 might promote the migration and necrosis of M1 cell and induce the secretion of various inflammatory mediators.In general, PHD3 is commonly expressed in aortic endothelial cells, vascular smooth muscle cells and macrophages. With ischemic, hypoxic and inflammatory stimuli, PHD3 was activated and exerted its activity to hydroxylate substrate. Under pathological condition, PHD3 regulates function of a variety of cells including vascular smooth muscle cells, macrophages and neutrophils. In addition, PHD3 could affect the stability of the extracellular matrix via promoting the secretion of various inflammatory mediators and metal matrix protease. Thus, we speculate that PHD3 was likely to involve in the progression and stability of atherosclerotic plaque. However, it has not been reported that PHD3 influence atherosclerotic plaque progression at home and abroad. Thus, exploring the specific mechanism of PHD3 in AS is a great issue remaining to be solved.Objective:1. To investigate the effects of PHD3 on atherosclerosis progression through transfecting lentivirus carrying PHD3 gene to ApoE-/-mice;2. To investigate the effects of PHD3 on lipids, macrophages, smooth muscle cells and collagen in atherosclerotic plaques;3. To investigate the specific mechanism of PHD3 on endothelial cells and the associated signaling pathways.Methods:1. Atherosclerotic animals modelingMale ApoE-/-mice (n=100) of eight-week-old were randomly divided into the following 5 groups (n=20 per group):(1) control group (fed on chow diet), (2) high-fat diet(HFD) group (fed on high-fat diet, saline control at a dose of 0.2ml/mouse), (3) lentivirus-PHD3 group (fed on high-fat diet, lentivector at the dose of 2x107 TU/mouse), (4) shRNA-PHD3 group (fed on high-fat diet, lentivector at the dose of 2×107 TU/mouse), and (5) NC group(fed on high-fat diet, null lentivirus at the dose of 2×107 TU/mouse). The control group was fed a basal diet, and the other groups were fed a high-fat diet (HF diet containing 42% fat and 0.2% cholesterol).2 week after the transfection, three mice were sacrificed for detecting the transfection efficiency. After 12 week, mice were anaesthetized with an intraperitoneal injection of pentobarbital (30 mg/kg body weight). Then blood was collected and stored at -80℃. The hearts and aortas were perfused with saline in case of residual blood. Some animals were perfumed with 4% paraformaldehyde for following histologic expriment. The other animals were collected and stored at -80 ℃ for western blot analysis.2. En face analysis of the aortaOil Red O staining was performed to assess the size of the atherosclerotic lesion. The aortic trees and hearts were fixed in 4% paraformaldehyde, and washed by phosphate-buffered saline (PBS). Then the aorta was opened longitudinally and fixed flat on a black surface. After 30min, the atherosclerotic plaque was stained with red color. The degree of atherosclerosis was measured by the percentage of total plaque area upon aorta intimal surface.3. H&E and Oil red staining of aortic sinusThe aortic trees and hearts were fixed in 4% paraformaldehyde, and washed by phosphate-buffered saline (PBS). The hearts were embedded in OCT and aortic sinuses were cut into cross sections with the thickness of 6 μm. H&E and Oil red staining were used to detect the morphology and lipids in aortic sinus. The degree of AS was evaluated by the ratio of total plaque area to aortic lumen area.4. Immunohistochemical analysisImmunohistochemical analysis of PHD3, ICAM-1, VCAM-1, MOMA-2 and a-SMA were used to investigate the effects of PHD3 on ICAM-1,VCAM-1, macrophages and smooth muscle cells. Sirius red and Masson staining were used to clarify the effects of PHD3 on collagen fibers in AS plaque.5. TUNEL assayTUNEL assay was used to explore the effect of PHD3 on the apoptosis of AS plaque and HUVECs.6. Western blot analysisAortic tissues in -80℃ and cells were collected to extracted proteins. Western blot analysis was used for detecting the expression of PHD3, ICAM-1, VCAM-1, MCP-1, TNF-a, IL-1 β and the phosphorylation of ERK1/2, JNK and P38. 7. StatisticsAll study data were presented as the mean± standard deviation(SD). Comparisons among multiple groups were performed via one-way ANOVA, and differences between two groups were compared by Student’s t test. A P value<0.05 was considered as statistically significant. All statistical analyses were performed via SPSS 18.0 (SPSS Inc., Chicago, IL). All data were repeated at least 3 times.Results:1. The general conditions of miceThe body weight and serum level of TG、TC、HDL-C and LDL-C in each group were detected before mice were sacrificed. The data showed that there was no statistical difference among each group (P> 0.05), which leads to the confirmation that PHD3 lentivirus transfection did not influence the lipid metabolism.2. PHD3 expression in atherosclerotic plaques after lentivirus transfection in ApoE-/-miceImmunohistochemical analysis demonstrated that PHD3 expression was elevated in HFD group compared with control group (P<0.05). Compared with NC group, PHD3 lentivirus treatment increased its level in atherosclerotic plaques and inhibition of PHD3 decreased the level of PHD3 in atherosclerotic lesions (P<0.05). The results of western blot were in line with immunohistochemical analysis. The data indicated that PHD3 lentivirus is effective in mediating the expression of PHD3.3. Effects of PHD3 on the lipid accumulation in atherosclerotic plaques in ApoE-/-miceEn face analysis of aortic tissues stained with Oil Red O from ApoE-/-mice with injection of lentivirus-PHD3 revealed larger atherosclerotic lesions compared with NC group (P<0.05), while atherosclerotic lesion area decreased with in shRNA-PHD3 group compared with NC group (P<0.05). In addition, the atherosclerotic lesions in cross-sections of the aortic sinus stained with Oil Red O exhibited enhanced lesion areas in ApoE-/- mice fed on a high fat diet compared with mice fed on a chow diet. Lentivirus-PHD3 treatment demonstrated larger lesion areas than NC group (P<0.05), and inhibition of PHD3 showed less lesion area compared to NC group (P<0.05).4. Effect of PHD3 on the apoptosis in atherosclerotic plaques in ApoE-/-miceMacrophage apoptosis plays a pivotal role in the progression of atherosclerotic plaque. The proportion of TUNEL-positive apoptotic cells in atherosclerotic lesions was increased in mice fed on high-fat diet compared with control group. Overexpression of PHD3 exhibited significantly increased percentage of TUNEL-positive apoptotic cells in atherosclerotic lesions compared with NC group, and PHD3 inhibition effectively decreased the proportion relative to vehicle treatment. The results above demonstrated that PHD3 was associated with atherosclerotic apoptosis.5. Effect of PHD3 on macrophages in atherosclerotic plaques in ApoE-/-miceImmunohistochemical staining of Moma-2 was used to analyze the percentage of macrophages in atherosclerotic lesions. The percentage of Moma-2+macrophages in atherosclerotic lesions was higher in HFD group than the control group (P<0.05). Treatment with lentivirus-PHD3 displayed an increased percentage of Moma-2+ macrophages compared with NC group (P<0.05), and inhibition of PHD3 expression decreased its percentage (P<0.05). Our data suggested that PHD3 overexpression increased macrophages percentage, and inhibition of PHD3 decreased the number of macrophages in atherosclerotic plaques.6. Effect of PHD3 on smooth muscle cells in atherosclerotic plaques in ApoE-/-miceImmunohistochemical staining of α-SMA was used to analyze the percentage of smooth muscle cells in atherosclerotic lesions. The percentage of α-SMA+smooth muscle cells in atherosclerotic lesions was higher in HFD group than the control group(P<0.05). Treatment with lentivirus-PHD3 displayed an increased percentage of α-SMA+smooth muscle cells compared with NC group(P<0.05), and inhibition of PHD3 expression decreased its percentage(P<0.05). Our data suggested that PHD3 overexpression increased smooth muscle cells number, and inhibition of PHD3 decreased the percentage of smooth muscle cells in atherosclerotic plaques.7. Effect of PHD3 on collagen expression in atherosclerotic plaques in ApoE-/-miceCollagen deposition in atherosclerotic lesions is associated with the stability of the plaques. Masson’s trichrome and Picrosirius red staining were used to detect the collagen deposition in atherosclerotic plaques. The results demonstrated that collagen content exhibited larger percentage in mice fed high-fat diet than the control mice (P<0.05). PHD3 overexpression increased the percentage of collagen content compared with NC group (P<0.05), and PHD3 inhibition displayed reduced collagen deposition relative to vehicle treatment(P<0.05). The above results indicated that PHD3 overexpression increased the content of collagen fibers and inhibition of PHD3 decreased the production of collagen fibers.8. Effect of PHD3 on ICAM-1 in ApoE-/-miceImmunohistochemical staining of ICAM-1 was used to analyze the expression of ICAM-1 in atherosclerotic lesions. Immunohistochemical staining analysis illustrated an obviously increase in the expression of adhesion molecules ICAM-1 in the HFD group relative to control group. Over-expression of PHD3 diplayed elevated levels of ICAM-1 compared with NC group (P<0.05), and shRNA-PHD3 treatment attenuated their levels (P<0.05). The above results indicated that PHD3 overexpression increased the expression of ICAM-1 and inhibition of PHD3 decreased its production in atherosclerotic plaques in ApoE-/-mice.9. Effect of PHD3 on VCAM-1 in ApoE-/-miceImmunohistochemical staining of VCAM-1 was used to analyze the expression of VCAM-1 in atherosclerotic lesions. Immunohistochemical staining analysis illustrated an obviously increase in the expression of adhesion molecules VCAM-1 in the HFD group relative to control group. Over-expression of PHD3 diplayed elevated levels of VCAM-1 compared with NC group (P<0.05), and shRNA-PHD3 treatment attenuated their levels (P<0.05). The above results indicated that PHD3 overexpression increased the expression of VCAM-1 and inhibition of PHD3 decreased its production in atherosclerotic plaques in ApoE-/-mice.10. Effect of PHD3 on the expression of inflammatory mediators in ApoE-/-miceWestern blot analysis displayed that the protein levels of pro-inflammatory markers, such as MCP-1, IL-1β, and TNF-a were increased in atherosclerotic lesions in lentivirus-PHD3 group compared with NC group (P<0.05), and inhibition of PHD3 treatment revealed a decrease in their expression in contrast to NC group (P<0.05). The data led to the comfirmation that PHD3 is associated with the expression of inflammatory cytokines.Conclusion:1. Over-expression of PHD3 accelerates the formation of atherosclerosis, and inhibition of PHD3 ameliorates the progression of atherosclerosis.2. PHD3 accelerates the progression of atherosclerosis via increasing the percentage of macrophages and smooth muscle cells in atherosclerotic plaques.3. Over-expression of PHD3 increases the expression of ICAM-1 and VCAM-1 in atherosclerotic plaques.4. PHD3 affects the function of HUVECs via MAPK signaling pathway.Background:As the pathological foundation of chronic inflammatory diseases, atherosclerosis is considered as the major risk factor that leads to high mortality and high morbidity throughout the world. AS is a complex pathological process, which involves a variety of vascular components, metabolism and immune inflammatory response, etc. The rupture of AS plaque can result in a series of acute cardiovascular events such as acute coronary syndrome, and severe cases can be life-threatening. In recent years, mechanisms that influence the progression and stability of AS plaques have been carried on the exploration, however, the specific pathogenesis affecting AS plaque stability is not yet clear. Thus, looking for new target which causes plaque rupture and exploring the effective measures for intervention would provide effective prevention for patients and furthermore reduce the the mortality of acute cardiovascular events.Visfatin was also known as the former B cell colony enhancement factor (PBEF) and nicotinamide phosphoribosyltransferase (Nampt). Visfatin is an adipocytokines exerting the proinflammatory and insulin-like effects and is associated with blood lipid metabolism, insulin resistance and function of pancreatic B cells. In cytoplasm, the main function of visfatin is regulating the metabolism of intracellular NAD and participating in cell metabolism, survival and apoptosis. Extracellular visfatin can promote inflammation reaction by enhacing the release of inflammatory cytokines.Visfatin can regulate the expression of virious proinflammatory factors and anti-inflammatory factors including IL-6, IL-1β and TNF-α, etc., especially IL-6. Visfatin can also promote the release of TGF-β 1 and influence cell growth, differentiation and apoptosis. In HUVECs, visfatin can increase the expression of MMP-2 and MMP-9, and upregulate the expression of vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor 2 (VEGFR2) via ERK1/2 and PI3K/AKT signaling pathway. Visfatin may also promote the release of VCAM-1 and ICAM-1 in HUVECs through NF-κB pathway. In addition to the influence on inflammatory factors, visfatin was also reported to be associated with multiple inflammatory diseases. In Crohn’s disease and ulcerative colitis, serum visfatin level is higher than healthy people and is more obvious in mRNA level. In osteoarthritis, visfatin can promote excessive release of prostaglandin 2. Visfatin is also involved in rheumatoid arthritis (RA), and visfatin level in patients is regulated by toll-like receptor ligands.Previous studies showed that visfatin is closely associated with AS. In patients with metabolic syndrome or carotid atherosclerosis, their serum visfatin levels increase significantly. Visfatin gene expression is upregulated in carotid plaques of AS patients, and visfatin gathered mostly in macrophages rich in lipids. Serum visfatin level in patients with acute myocardial infarction increased significantly. Ox-LDL and TNF-α can induce the expression of visfatin in monocytes. In male patients with acute ST segment elevation myocardial infarction, visfatin was found to increase in neutrophils. Ruptured plaque and macrophages in the coronary arteries exerted high visfatin expression. In addition, visfatin can elevate the mRNA expression of CCR2 and MCP-1 in HUVECs.Visfatin may also increase the expression of CD36 and SRA and decrease the expression of ABCA1 and ABCG1, thereby increasing the intake of ox-LDL and reducing the outflow of cholesterol which is through PI3K and ERK dependent way. In THP1 cells, visfatin can increase the generation of EMMPRIN and MMP-9 in a dose-dependent way. The inflammation is through the NAMPT-MAPK (p38 lightning, ERKl/2)-the NF-κB signaling pathways.In general, visfatin is involved in a variety of inflammatory diseases and promotes the progression of the AS. Elevated serum visfatin levels in patients with acute myocardial infarction remind us that visfatin may be closely related to the AS plaque stability, but the specific mechanism leading to vulnerable plaques is not fully clarified. Thus, the following problems still need to be addressed:(1) the specific influence of visfatin on AS plaque stability; (2) influence of visfatin on AS plaque conternts; (3) signal mechanism through which visfatin exerts its effects.Objective:1. To clarify the effects of visfatin overexpression on the stability of AS plaques via visfatin lentivirus transfection.2. To investigate the influence of visfatin lentivirus transfection on macrophages, lipid accumulation, smooth muscle cells and collagen fibers;3. To investigate the expression of MMP-8 mediated by visfatin in AS plaque and the related signaling pathway.Methods:1. Animals modelingMale apolipoprotein E (ApoE-/-) mice(n=80,6 weeks of age) were given a constrictive silastic tube, which was placed around the left common carotid artery after 2 weeks of a chow diet (5% fat and no added cholesterol). They were then given a high-fat diet (16% fat and 0.25% cholesterol) for 8 weeks. Eight weeks after the collar placement, the mice were randomly divided into 2 groups, the lenti-null group (n=40, local injection of 1×107 TU null lentivirus in left carotid artery region) and the lenti-visfatin group (n=40, local injection of 1×107 TU lentivirus containing visfatin in the left carotid artery region). Finally, the mice were given a high-fat diet for another 4 weeks. Mice were anaesthetized with an intraperitoneal injection of pentobarbital (30 mg/kg body weight). Then blood was collected and stored at -80 ℃. The hearts and aortas were perfused with saline in case of residual blood. Some animals were perfumed with 4% paraformaldehyde for following histologic expriment. The other animals were collected and stored at -80℃ for western blot analysis.2. The serological detectionThe serological detection was used to detect the levels of serum total cholesterol, triglyceride, low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C).3. Oil red staining of aortic sinusThe carotid arteries were embedded in OCT and cut into cross sections with the thickness of 6 μm. Oil red staining was used to detect the morphology and lipids in carotid artery. The degree of leisions was evaluated by the ratio of red stained area to carotid plaque area.4. Immunohistochemical analysisImmunohistochemical analysis of visfatin、MOMA-2、α-SMA、MMP-8 were used to investigate the effects of visfatin on macrophages, smooth muscle cells and MMP-8. Sirius red staining was used to clarify the effects of visfatin on collagen fibers in carotid plaque.5. Western blot analysisTissues in -80℃ and cells were collected to extracted proteins. Western blot analysis was used for detecting the expression of visfatin, MMP-1, MMP-2, MMP-8, MMP-9 and the phosphorylation of P65'I κ B α6. Real-time quantitative PCR analysisTissues in -80 ℃ and cells were collected to extracted total RNA. Real-time quantative PCR analysis was used to detect the expression of visfatin and MMP-8.7. Immunofluorescence staining Immunofluorescence staining was used to detect the expression and location of visfatin in carotid plaques. After stimulating with visfatin, the expression of MMP-8 was detected with immunofluorescence.8. StatisticsAll study data were presented as the mean± standard deviation(SD). Comparisons among multiple groups were performed via one-way ANOVA, and differences between two groups were compared by Student’s t test. A P value <0.05 was considered as statistically significant. All statistical analyses were performed via SPSS 18.0 (SPSS Inc., Chicago, IL). All data were repeated at least 3 times.Results:1. Body weight and serum profilesWe found no significant difference in the body weight of the lenti-visfatin group compared with the lenti-null group (P>0.05). These results demonstrated that the transfection of lenti-visfatin was safe in these animals. There was still no significant difference in the serum TC, TG, LDL-C and HDL-C levels among the 2 groups of ApoE-/-mice (P>0.05), suggesting that the local transfection of lenti-visfatin did not affect the circulating lipid panel and that the effects of lenti-visfatin transfection were independent of the serum lipid profiles.2. Visfatin lentivirus effects the expression of visfatin in carotid plaquesThe results demonstrated that mRNA level (P<0.05), but not protein level (P>0.05), was significantly enhanced after 3 days post-transduction. However, both mRNA level and protein level were significantly enhanced after 7 days post-transduction (P<0.01). After 4 weeks, the transfection efficiency of visfatin in the carotid plaques was detected again, and the expression of visfatin was still significantly up-regulated in the lenti-visfatin group compared with the lenti-null The results above demonstrated that transfection of visfatin lentivirus successfully up-regulated the expression of visfatin in the murine carotid arteries.3. Visfatin is mainly expressed in macrophages in carotid plaquesIn the present study, to further confirm where visfatin is generated in the plaques, we colocalized visfatin and macrophages in carotid lesions. The immunofluorescence confirmed that although other cells, which cannot be labeled by MOMA-2 antibody could also express small amount of visfatin, the majority of visfatin originated from macrophages.4. Effects of visfatin overexpression on macrophages in carotid plaquesThe relative area of macrophages in the lenti-visfatin group was significantly elevated compared with the lenti-null group (P<0.01), indicating that visfatin overexpression can induce the infiltration of macrophages in carotid plaques.5. Effects of visfatin overexpression on lipids in carotid plaquesThe ratio of the lipids areas to the total lesion areas of the lenti-visfatin transfection group was significantly elevated compared with the lenti-null group, indicating that visfatin overexpression increased the accumulation of lipids in carotid plaques.6. Effects of visfatin overexpression on SMC in carotid plaquesThe relative area of SMCs in the lenti-visfatin group was also significantly decreased compared with the lenti-null group (P<0.05), indicating that visfatin overexpression decreased the accumulation of SMC in carotid plaques. 7. Effects of visfatin overexpression on collagen fibers in carotid plaquesThe relative collagen levels in the lenti-visfatin group were also greatly diminished compared with the lenti-null group (P<0.05), indicating that visfatin overexpression decreased the expression of collagen fibers in carotid plaques.8. Effects of visfatin overexpression on vulnerability indexThe vulnerability index of the lenti-visfatin group was much higher than that of the lenti-null control group (P<0.01). We also calculated the sizes of the lipid cores of the 2 groups, and the results demonstrated that the lipid core size was also larger in lenti-visfatin group than that in the lenti-null group (P<0.01). These results demonstrated that lentivirus transfection of visfatin changed the composition of carotid plaques and significantly increased the plaque fragility.9. Effects of visfatin overexpression on expression of MMP-8 in carotid plaquesBoth immunohistochemisty and western blot detections were performed and the results demonstrated that the expression of MMP-8 was significantly enhanced in the lenti-visfatin group compared with the lenti-null group (P<0.05). The results indicate that visfatin overexpression upregulated the expression of MMP-8 in carotid plaques.10. Visfatin upregulated the expression of MMP-8 in RAW264.7 cells in a concentration-and time-dependent wayRAW264.7 macrophages were cultured and treated with gradient concentrations of visfatin (0,0.5,5,50 and 500 ng/ml). After 24h of visfatin treatment, MMP-8 was detected with Western blot, and MMP-8 was up-regulated in a concentration-dependent manner.5 ng/ml of visfatin could significantly elevate the expression of MMP-8 (P<0.05), and the peak elevation was achieved at a concentration of 500 ng/ml (P<0.01). Then, RAW264.7 macrophages were cultured and treated with visfatin at a concentration of 500 ng/ml for different times (0,3,6,12,24 and 48 h).12 h of stimulation with visfatin could significantly elevate the expression of MMP-8 (P<0.01), which peaked at 48 h (P<0.01). Meanwhile, the results of real-time PCR showed a similar tendency. Subsequently, the MMP-8 expression was evaluated using immunofluorescence. After stimulation with visfatin (500ng/ml) for 24 h, RAW264.7 macrophages were examined using immunofluorescence, and there was a significantly elevated effect of visfatin on MMP-8. All of these data suggested that visfatin could dramatically improve the expression of MMP-8 in vivo and in vitro, promoting collagen degradation in the arteries and altering the plaque vulnerability index.11.Visfatin upregulated the expression of MMP-1, MMP-2 and MMP-9MMP-1, MMP-2 and MMP-9 in the carotid plaques were assessed with western blot. The expressions of MMP-1, MMP-2 and MMP-9 were all significantly increased in the lenti-visfatin group compared with the lenti-null group (P<0.05). Then, RAW264.7 macrophages were cultured and treated with visfatin (500ng/ml) for different time, and the results demonstrated that visfatin could not increase the expressions of MMP-1, MMP-2 and MMP-9 in vitro within 24h. But when we prolonged the stimulation time to 48h, the expressions of MMP-1, MMP-2 and MMP-9 were significantly increased. Compared with the effect on MMP-8, which was significantly increased after 12h-stimulation, the results of other MMPs reminded us that visfatin might induce MMP-1, MMP-2 and MMP-9 in an indirect way.12. Visfatin induced MMP-8 expression via the NF-κB pathwayVisfatin (500ng/ml) treatment of RAW264.7 macrophages significantly increased the expression of p65 in the nucleus and decreased its expression in the cytosol in a time-dependent manner. Meanwhile, the treatment also increased the phosphorylation of IxBa in the cytosol. Both of the effects on p65 and IκBα reached statistical significance after 8 h treatment, and they peaked after 12 h of treatment. Then, to further determine the effect of the NF-xB pathway on the reaction, we blocked the NF-xB pathway with 2 h of treatment with two inhibitors, BAY11-7082 (20 μM) and SC-514 (20 μM), before visfatin was added to the cells. Both of the inhibitors significantly suppressed visfatin-induced MMP-8 production. All of the results indicated that the activation and translocation of NF-κB is critical for visfatin-induced MMP-8 production.Conclusions:1.Visfatin is closely related to the stability of experimental atherosclerosis plaques;2. Visfatin lentivirus transfetion could significantly decrease the stability of AS plaques;3.Visfatin could increase the expression of MMP-1, MMP-2, MMP-8 and MMP-9 in a concertration and time dependent way;4. Visfatin induced MMP-8 expression via the NF-κ B pathway... |
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