| Background:As the pathological basis of cardiovascular diseases, atherosclerosis (AS) has now become a threat to human health, especially in the elderly as the most important diseases, leading to heart cerebrovascular accident morbidity and high mortality rate, and also high morbidity in survivors. Risk factors of atherosclerosis involve in many areas, and the pathological process is complex. Although there are many theories attempting to explain its pathogenesis, the specific mechanism has not yet been defined.Numerous studies have demonstrated that atherosclerosis-induced acute clinical ischemic events associated more with the stability of the plaque, rather than the arterial lumen stenosis. Unstable plaque rupture and thrombosis has become a major clinical and pathological mechanism of acute cardiovascular events. The vulnerable plaque has a strong tendency to rupture of the plaque characterized by a fibrous cap becoming thinner, necrotic lipid core increasing macrophage infiltration within the plaques, smooth muscle cells and collagen content gradually reducing. And on this basis, the vulnerability index is introduced to evaluate quantitatively plaque ulnerability, defined as:vulnerability index=(lipid plaque area+macrophages area)/(collagen area+smooth muscle cells area). Therefore, exploring the impact of various risk factors on AS plaque stability, seeking effective intervention targeting on vulnerable plaque stability, and actively avoiding plaque rupture and secondary cardiovascular events in recent years have become a research hotspot.Serum amyloid A (serum amyloid A, SAA) family consists of SAA1, SAA2, SAA3and SAA4four family members. SAA1, as the main component, is the most widely expressed, the most active and the most sensitive response subtype in SAA family. Although under normal conditions, expression level of SAA1is low, but in the acute phase state its serum concentration increases1000times in a very short time. Serum SAA1is mainly synthesized and secreted by the liver cell, but also can be secreted by other cells. In AS plaque, SAA1can be synthesized and secreted by macrophages. In addition, SAA receptors on the cell membrane may include these types:type B Scavenger receptor1(the SR-B I), formyl peptide receptor1(FPRL1), toll-like receptors (TLR) and so on. According to the type of receptor binding, SAA1 may result in different biological effects on different cells.In recent years, with the in-depth study of SAA1, people found that there is a close link between SAA1and AS. SAA1can appear in the whole process of development in the AS plaque, and the concentration of serum SAA1positively correlated with coronary atherosclerotic heart disease. New clinical studies found that levels of serum SAA1may serve as an independent predictor of acute cardiovascular events. This suggests that, SAA1may not only affect the formation of AS plaque, but also further affect AS plaque stability through some mechanism and ultimately affect the occurrence of acute cardiovascular events. There are many factors affecting the stability of the AS plaque, involves the macrophage infiltration, lipid and migration of smooth muscle proliferation and extracellular matrix degradation, and endothelial dysfunction. Previous researches displayed that on the one hand, SAA1increased lipid deposition and accelerated the formation of lipid core, and increased inflammatory cell infiltration through the promotion of monocyte-macrophage chemotaxis and adhesion, which suggested that SAA1might diminish the plaque stability; while on the other hand, other studies showed that SAA could increase smooth muscle cell migration and proliferation, and could stimulate increased expression of extracellular matrix, which further showed that SAA1might increase the plaque stability. Thus, the impact of SAA on AS plaque stability is multifaceted, and may even be bidirectional. Based on the previous researches we can not simply infer the specific impact of SAA1on AS plaque stability. Direct evidences of SAA1influences the stability of the plaques have not been reported so far, therefore it is of great importance to discuss and solve this problem. Currently there are the following problems needed to be discussed and solved:the effects of SAA1on the stability of atherosclerotic plaque; effect of SAA1on atherosclerotic plaque composition; the signaling pathway involved the reaction.Objective:1. To certify the effects of SAA1overexpression on atherosclerotic plaque stability, through transfecting lentivirus carrying SAA1gene to ApoE-/-mice;2. To certify the effects of SAA1lentiviral transfection on lipids, macrophages, smooth muscle cells and collagen in the internal carotid artery plaque;3. To investigate the specific effect of SAA1on the expression of collagen and its associated signaling pathways. Methods:1. SAA1lentivirus constructionAfter obtaining the target gene by RT-PCR, we amplified gene fragments and connect the fragments with lentiviral vector plenti6.3-MSC-IRES-EGFP to construct plenti6.3-SAA1-IRES-EGFP. After transformation, packaging and determination of lentivirus titer, the lentiviral was used in the experiments.2. Animals breeding and ModelingThe100male ApoE-/-mice of6-8week-old fed for one week, and accepted the carotid artery cannula operation after that. Continued high fat diet for8weeks, the remaining95ApoE-/-mice were randomly divided into four groups:Control group (n=23), lenti-null group (n=25), low-lenti-SAA1group (n=23) and high-lenti-SAA1group (n=24). Lenti-null group were given1×107TU empty lentiviral vector, and low-lenti-SAA1group were given1×107TU SAAl lentivirus, and high-lenti-SAA1group were given1×109TU SAA1lentivirus and control group was given the same volume of saline. After4weeks high fat fed, mice were weighed and anesthetized with0.8%pentobarbital. The thorax was opened, and the heart bloods were collected, stored in-80℃freezer for subsequent detection. Saline was used to irrigate the hearts and aortas to flush out the residual blood. Following, some animals were irrigated with4%paraformaldehyde until the liver hard and then collect the mouse carotid arteries, with4%paraformaldehyde for another24hours. The remaining part of the animals’carotid arteries were directly collected and stored at-80℃refrigerator.3. ELISA (enzyme-linked immunosorbent assay, ELISA)Total cholesterol, LDL cholesterol, HDL cholesterol, triglycerides, and TNF-a, IL-6concentration in serum were detected by ELISA.4. Carotid oil red O staining5mm serial sections of mouse carotid artery, each of the10and1for oil red O staining, the severity of the lesions represented by the ratio of red stained lipid to carotid plaque area.5. Immunohistochemical staining tissueOn frozen sections, expression of SAA1, a-SMC actin, MOMA-2, collagen I, collagenⅢ, TGF-β1, MMP1, MMP8were evaluated by immunohistochemical staining.6. Western blot analysis Frozen tissue was quantitatively weighed, and cells were collected and extracted proteins. Western blot method was used to detect the expression of SAA1, collagen I, collagen Ⅲ, TGF-β1, MMP1, MMP8, MMP2, MMP9, p-p38, ERK1/2, JNK, Smad2and Smad3.7. Real-time quantitative PCR analysisFrozen tissue was quantitatively weighed, and Trizol extraction of total RNA. After reverse transcription, express of SAA1was determined with real-time quantitative PCR analysis.8. Immunofluorescence stainingAfter the SAA1protein stimulates smooth muscle cells and macrophages, the expression of collagen I, collagenⅢ, MMP1and MMP8were detected with immunofluorescence.9. Statistical analysisAll values are expressed as mean±standard deviation. Test for normally distributed data, the normal distribution of count data Student t test, groups using analysis of variance (ANOVA), a non-normal distribution of data rows Mann-Whitney test. Statistical significance was confirmed as P<0.05.Results:1. The general conditions of groups of miceThe weight serum TC, TG, LDL-C and HDL-C concentration levels did not differ (P>0.05) in control group(n=23), lenti-null group(n=25), low-lenti-SAAl group(n=23) and high-lenti-SAA1group(n=24) ApoE7-/-mice. The results showed that high expression of SAA1fragmentary lentivirus transfection did not affect the overall lipid metabolism in mice.2. Plenti6.3-SAA1-IRES-EGFP lentivirus transfection effect on serum SAA1and other inflammatory factor expressionSerum SAA1concentration has changed little in the control group, lenti-null, low-lenti-SAA1group. High-lenti-SAA1group is slightly higher than the previous three groups of serum SAA1expression but has yet to reach statistical difference (P>0.05). Serum TNF-a and IL-6concentrations of the four groups did not reach statistical difference (P>0.05), which indicateed that the local transfection of slow virus, did not cause significant inflammation in mice.3. Plenti6.3-SAA1-IRES-EGFP lentivirus transfection effect on SAA1expression in carotid plaquesSAA1immunohistochemical results showed that there was no difference between the control group and lenti-null group(P>0.05). SAA1positive staining area of low-lenti-SAA1group and high-lenti-SAA1were both significantly higher than lenti-null group and control group (P<0.05). Western blot and real-time PCR detection showed a consistent result with immunohistochemical results. The results indicate that SAA1lentivirus successfully achieved the purpose of the SAA1high expression within plaques.4. SAA1lentiviral transfection effect on the carotid artery plaque lipid accumulationOil red O staining of frozen sections of mouse carotid artery plaque showed that lenti-null group and the low-lenti-SAA1group changeed in lipid content compared with the control group; high-lenti-SAAl group increased significantly compared with the control group and low-lenti-SAA1group (P<0.05). This result suggested that, low concentration of SAA1did not significantly alter the plaque lipid deposition, while high concentration significantly promoted lipid accumulation in the plaque.5. SAA1lentiviral transfection effect on the carotid artery plaque macrophages accumulationMacrophages staining of frozen sections of mouse carotid artery plaque showed that compared with the control group, the proportion of macrophages in lenti-null group did not change significantly (P>0.05); low-lenti-SAA1group (P<0.05) and high-lenti-SAA1group (P<0.01) increased significantly; compared with low-lenti-SAA1group, the increase of macrophages in high-lenti-SAA1group did not reach statistical significance (P>0.05). This result suggests that both the concentration gradient SAA1overexpression lentivirus could significantly increase the accumulation of macrophages in the carotid artery plaque.6. SAA1lentiviral transfection effect on the carotid artery plaque smooth muscle cells accumulationSmooth muscle cells staining of frozen sections of mouse carotid artery plaque showed that accumulation had no significant change of smooth muscle cells among the four groups. This result suggests that, SAA1overexpression lentivirus can not change the aggregation of smooth muscle cells in the plaque.7. SAA1lentiviral transfection effect on the carotid artery plaque collagen accumulation Masson staining of frozen sections showed that lenti-null collagen group had no significant change compared with the control group (P>0.05); collagen in low-lenti-SAA1group increased significantly (P<0.01); no significant increase in high-lenti-SAAl group compared with the control group (P>0.05) and a significant reduction compared with low-lenti-SAA1group (P<0.05). The results showed that low concentration of SAA1overexpression lenti virus can significantly increase the accumulation of collagen, but high concentration of SAA1lenti virus make this effect disappeared.8. SAA1lenti viral transfection effect on vulnerability indexThe results showed that, compared with the control group, vulnerability index of lenti-null group had no significant change (P>0.05); low-lenti-SAA1group was significantly lower (P<0.05); high-lenti-SAA1group increased significantly (P<0.05) compared with control and low-lenti-SAA1groups reaching statistical significance (P>0.05). The results showed that low concentration of SAAl could significantly reduce plaque vulnerability, but high concentration of SAA1significantly increased plaque vulnerability.9. SAA1induced expression of collagen I and collagen IIICollagen I immunohistochemical staining showed that, compared with the control group, the percentage of collagen I in lenti-null group had no significant change (P>0.05); low-lenti-SAA1group was significantly improved (P<0.05); high-lenti-SAA1group did not change significantly compared with the control group (P>0.05), but reduced significantly compared with low-lenti-SAA1group (P<0.05). CollagenⅢ immunohistochemical staining showed that, compared with the control group, lenti-null group had no significant change (P>0.05); low-lenti-SAA1group III collagen was significantly increased (P<0.05); high-lenti-SAA1group significantly decreased (P<0.05) compared with control and low-lenti-SAA1group (P<0.05). Western blot results were consistent with the results of immunohistochemistry. The results showed that low concentrations of SAA1overexpression lenti virus could significantly increase the accumulation of collagen I and collagenⅢ, but high concentration of SAA1reversed this effect. Cultured smooth muscle cells in mice and humans showed that after SAA1stimulation, collagen I and collagenⅢ expression increase in dose-and time-dependent manners. Immunofluorescence results also showed that SAA1stimulation significantly increased the expression of collagen I and collagenⅢ.lO.MAPKs pathways didn’t mediate SAA1-induced collagens productionAfter different time of SAA1stimulation, the expression of p-JNK and p-p38MAPK did not change significantly (P>0.05); the expression of p-ERK1/2increased along with the time change. Then, JNK, ERK1/2and p38MAPK specific inhibitors pretreated smooth muscle cell, and the three inhibitors were not significantly inhibited SAA1induced collagen I and collagen III expression. The results show that the JNK, p38MAPK and ERK1/2signal pathways did not participate in the regulation of SAA1on the expression of collagen I and collagen III.11. Smad2/3pathway mediated SAA1-induced collagens productionAfter different time of SAA1stimulation, the expression of p-Smad2and p-Smad3changed significantly (P<0.05). Then Smad2/3siRNA pretreated smooth muscle cells, and the two siRNA can significantly inhibit SAA1induced collagen I and collagen III expression. The Smad2/3signaling pathways involved in the regulation of the collagen I and collagen III expression in smooth muscle eells.12. SAA1-induced collagens production was independent of TGF-βTGF-β1immunohistochemical staining showed that four groups have no statistical difference, indicating that SAA1lentivirus transfection caused no change in expression of TGF-β1in mouse carotid artery plaques. After recombinant SAA1stimulated smooth muscle cells for different times, expression of TGF-β1has not changed significantly (P>0.05); and TGF-β1siRNA and its specific inhibitor SB431542still has not changed SAA1induced collagenⅠ and collagenⅢ expression. These results demonstrate the process of collagenl and collagenⅢ expression induced by SAA1does not depend on the TGF-β1.13. SAA1increased MMP-8, MMP-2and MMP-9expression, but not MMP-1MMP1immunohistochemical staining showed that there was no statistical difference in the four groups (P>0.05). MMP8immunohistochemical staining showed that, compared with control group, lenti-null group and low-lenti-SAAl group had no significant change (P>0.05); high-lenti-SAA1group significantly increased (P<0.01). In vitro cultured mouse RAW264.7macrophages were stimulated with1μg/ml and10μg/ml concentrations of SAA1protein, and immunofluorescence detection showed that SAA1was unable to significantly increase MMP1expression (P>0.05);1μg/ml SAA1could not significantly increase the expression of MMP8, but10μg/ml SAA1 significantly enhanced MMP8expression. Western blot results showed that, after SAA1stimulation, no significant change was found in the expression of MMP1(P>0.05). The expression of MMP8was dose-dependently increased under the stimulation of SAA1. When the concentration reached more than5μg/ml, the change of MMP8expression reached statistical significance (P<0.01). In addition, concentration of1μg/ml SAA1recombinant protein could significantly increase the expression of MMP2and MMP9(P<0.05). With increasing concentration of SAAl, the expression of MMP2and MMP9showed a dose-dependent manner. These results indicated that SAA1could dose-dependently stimulate the expression of MMP8, MMP2, and MMP9.Conclusion:1. SAA1is closely related to the the stability of experimental atherosclerosis plaque;2. Low concentration of SAA1lentivirus transfection could significantly increase the stability of AS plaque, while high concentration SAA1of lentivirus transfection could significantly reduced the stability of AS plaque;3. Low SAA1concentration can increase the expression of collagen Ⅰ, Ⅲ, and0.1μ g/ml concentration of SAA1reach the maximum effect;4. SAA1increased the expression of collagen I, III through Smad2/3pathway independent MAPKs and TGF-β1;5. High concentrations of SAA1,5μg/ml and above, promotes collagen degradation and thus lower the stability of AS plaque through activation of MMP-8. |
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