Mechanism Of Serum Amyloid A Accelerates The Progression Of Atherosclerosis And Its Pro-inflammatory Role

BackgroundAtherosclerosis is an important underlying pathologic condition of many cardio-cerebro-vascular diseases, the leading cause of morbidity and mortality worldwide. Basing on the long-term experimental and clinical research, the theory that atherosclerosis is a chronic inflammatory disease has been proposed by Ross explicitly. The occurrence and development of atherosclerosis is a complex pathological process involving a series of influencing factors such as lipid metabolism, immuno-inflammatory response, macrophages infiltration and so on. Therefore, seeking the effective targets to avoid the formation of atherosclerosis plaque has already become a hot topic in the cardiovascular field.Serum amyloid A (SAA) is one of the most sensitive acute phase proteins in vertebrates. It is produced principally by the liver in response to acute inflammatory stimuli and its plasma concentration can increase by up to100-to1000-fold over the basal level. Meanwhile, accumulating evidences also support that under the chronic inflammatory status such as obesity and atherosclerosis, the secretion of SAA by human adipocytes, vascular endothelial cells and macrophages can cause a modest increase in the plasma and reflect the inflammatory level exactly.SAA is a family of homologous proteins including4isoforms which are encoded by the same gene. SAA1and SAA2are the major acute phase reactants, with primary structures that are93%identical. The human SAA3gene is a pseudogene. SAA4 which is constitutively expressed in humans but not in mice do not participate in the acute inflammatory reaction. Many studies have already demonstrated that SAA1predominates in plasma, where it functions as a major isotype.So far, many large clinical trials have demonstrated that increased plasma SAA level can be regarded as a useful indicator for identifying individuals at high risk of cardiovascular disease (CVD), suggesting that there may be a link between the SAA and prevalent CVD. With the further study, more and more biological function of SAA has been known by us. Firstly, SAA exhibits considerable chemoattractant activity for human monocytes and macrophages. Secondly, as the apolipoprotein of high-density lipoprotein (HDL) and low-density lipoprotein (LDL), SAA increases the binding affinity of cholesterol for macrophages and endothelial cells. In addition, a series of studies in vitro show that SAA can up-regulate the expression of many inflammatory factors such as monocyte chemotactic protein-1(MCP-1), tumor necrosis factor-α(TNF-α), interleukin (IL)-6and IL-8and so on. All the results mentioned above suggest that SAA should play an effective role in the progression of atherosclerosis.Despite the numerous reported proatherogenic properties of SAA, we lack direct proof that SAA is an active participant in the atherosclerosis process in vivo. To investigate whether SAA is purely a risk marker for atherosclerosis or is also an active participant in vivo, we examine the effect of high-level expression of SAA on atherosclerosis development by using apolipoprotein E-deficient (ApoE-/-) mice transfected with lentivirus to induce SAA overexpression. Findings from this study can provide the first in vivo evidence that an elevated plasma level of SAA accelerates the progression of atherosclerosis directly and independently, and then may be helpful in designing novel therapeutic strategies against atherosclerosis.Objectives1. To investigate the role of increased SAA plasma level on the formation of atherosclerosis plaque in ApoE-/-mice.2. To observe the deposition and the influence of SAA on the distribution of macrophages in atherosclerosis plaque.3. To elucidate the role of increased SAA plasma level on the expression of proatherogenic factors.Methods1. Establishment of animal model90male ApoE-/-mice (8weeks age) were randomly divided into3groups, and then were injected intravenously with lentivirus-expressing mouse SAA1(lenti-SAA group, n=30) at a total lentivirus dose of1×107TU/mouse, a null lentivirus (lenti-null group, n=30) or saline (saline control group, n=30). To avoid the influence of high plasma lipids on atherogenesis, all the animals were fed a chow diet (5%fat and no added cholesterol) throughout the entire experiment. At14weeks after lentivirus injection, mice were anesthetized with pentobarbital injected intraperitoneally, and then blood samples were taken. Serum was separated by centrifugation at4℃. Meanwhile, the hearts and aortas were removed and perfusion-fixed with4%paraform aldehyde for histological and morphological staining or with PBS for real-time polymerase chain reaction (real-time PCR).2. Biological measurementsThe levels of triglycerides (TG), total cholesterol (TC), HDL-C and LDL-C were measured by use of an automatic biochemistry analyzer. And serum levels of SAA, IL-6and TNF-α were detected by enzyme-linked immuno sorbent assay (ELISA).3. En face analysis of the aortaThe aorta was stripped of adventitia and dissected longitudinally from the iliac arteries to the aortic root, then the branching vessels were removed. The paraformaldehyde-fixed aorta was pinned flat on a black surface, and the atherosclerotic lesion area was readily visualized with Oil-Red-O staining. Average lesion area was quantified by use of ImagePro-Plus soft ware. The ratio of total atherosclerotic lesion area to aorta intimal surface area was calculated as an indicator to evaluate the level of atherogenesis.4. H&E and Oil-Red-O staining of aortic sinus Murine hearts were fixed in4%paraformaldehyde overnight and then embedded in optimal cutting temperature compound. At least50serial cryosections6-μm thick were cut, beginning at the junction of the left ventricle and the aorta. Sections were stained with hematoxylin and eosin (H&E). The lipid core was identified by Oil-Red-O staining. The atherogenesis level at the aortic sinus was evaluated by Oil-Red-O and H&E staining according to the ratio of total atherosclerotic lesion area to aortic lumen area.5. Immunohistochemical analysis Immunohistochemical analysis was used to detect the expression of macrophages and MCP-1in lesions. Data were analyzed by use of ImagePro-Plus software.6. ImmunofluorescenceCryosections of the aortic sinus were chosen to observe the expression of SAA, macrophages and vascular cell adhesion molecule-1(VCAM-1) through immunofluorescence analysis. The colocalization of SAA with macrophages was also examined in atherosclerosis plaque. Data were analyzed by use of ImagePro-Plus software.7. Quantitative real-time PCRTotal RNA was extracted from murine frozen aortic specimens by use of trizol, and then reverse transcribed. Real-time PCR was used to detect the mRNA levels of MCP-1and VCAM-1.Results1. Body weight and measurement of plasma variables ApoE-/-mice in3groups fed a chow diet did not differ in body weight, TG, TC, HDL-C or LDL-C (P>0.05). Therefore, we excluded the influence of lipid levels on atherosclerosis in this study. The lenti-null and saline control groups did not differ in plasma levels of IL-6or TNF-α, so the injection of the SAA1lentivirus vector was safe and did not induce inflammatory responses (P>0.05). The plasma levels of SAA were higher for the lenti-SAA group than lenti-null and saline control groups, so the SAA1lentivirus was efficiently transfected in vivo (P<0.01). Most importantly, with elevated SAA level, the plasma levels of IL-6and TNF-a were significantly higher in the lenti-SAA than lenti-null group, suggesting that the increased SAA level may aggravate the inflammatory reflect (P<0.01).2. Atherogenesis level analysisBy using en face analysis of the aorta, lesion area was significantly larger for the lenti-SAA than lenti-null group (P<0.01). Atherogenesis level at the aortic sinus was evaluated by Oil-Red-O and H&E staining by ratio of total atherosclerotic lesion area to aortic lumen area. The mean lesion size at the aortic sinus were greater for the lenti-SAA than lenti-null group (P<0.01). All the results demonstrated that increased plasma SAA level directly promotes atherosclerotic lesions.3. Accumulation of SAA and macrophages in atherosclerosis plaqueImmunohistochemistry analysis showed a greater increase in accumulation of macrophages for the lenti-SAA than lenti-null group (P<0.01). Because SAA is a classic chemoattractant to peripheral blood leukocytes, we observed the colocalization of SAA with macrophages in lesions on aortic cryosections through immunofluorescence. The distribution of macrophages was consistent with SAA protein localization.4. Expression of MCP-1in atherosclerosis plaqueBecause MCP-1is a key molecule regulating chemotactic migration of macrophages, we observed the expression of MCP-1in vivo by immunohistochemistry. MCP-1secretion was increased with elevated level of plasma SAA, which was also confirmed by real-time PCR (P<0.01). Our data suggested that SAA could enhance the chemotaxis of macrophages through up-regulating the expression of MCP-1in lesions.5. Expression of VCAM-lin atherosclerosis plaqueImmunofluorescence analysis revealed upregulated VCAM-1expression in vivo in the lenti-SAA group as compared with the lenti-null group (P<0.01). VCAM-1mRNA expression results agreed with protein level results. Our research suggested that SAA could promote the adhesion of macrophages in lesions through up-regulating the expression of VCAM-1. Conclusions1. In the present study, we achieved the persistent high expression of SAA protein by applying SAA1lentivirus in ApoE-/-mice effectively.2. Throughout the entire experiment, all the animals were fed a chow diet, so that the influence of lipid levels on atherosclerosis formation was excluded in our study.3. SAA accelerated the progression of atherosclerosis in ApoE-/-mice through aggravate the inflammatory level.4. SAA enhanced the chemotaxis and adhesion of macrophages through up-regulating the expression of MCP-1and VCAM-1, thus promoting the formation of atherosclerosis plaque BackgroundAtherosclerosis is the primary pathophysiological basis leading to the many cardio-cerebro-vascular diseases. A series of risk factors represented by obesity, hyperlipemia, hypertension and smoking participate in the occurrence of atherosclerosis. On the basis of the response-to-injury hypothesis, the theory that the chain reaction induced by inflammatory factors play an important role in the process of atherogenesis has been proposed by Ross explicitly. This chronic inflammation is characterized by the disfunction of vascular endothelial cell and the accumulation of macrophages and T lymphocytes in the arterial vascular walls.Serum amyloid A (SAA) is one of the most sensitive acute phase proteins in vertebrates. And it can reflect the inflammatory levels exactly under the status of the acute and chronic inflammations. Recently, a series of studies in vitro have demonstrated that SAA can up-regulate the expression of many inflammatory factors such as tumor necrosis factor-α (TNF-α), interleukin (IL)-6and IL-12and so on. And these findings suggest that SAA may be not only a prognostic indicator but also a pro-inflammatory mediator by inducing the expression of the inflammatory factors in mononuclear phagocytes and aortic endothelial cells.Pentraxins is an acute immunological response family of proteins that consists of three members including C reactive protein (CRP), serum amyloid P (SAP), and pentraxin3(PTX3). PTX3is one of the conserved proteins in evolution and produced by mononuclear phagocytes, dendritic cells (DCs), adipocytes and so on under the stimulation of IL-1and TNF-α. For one thing, just like CRP and SAA, PTX3belongs to classic acute phase reactants that can reflect the levels of inflammation exactly. Many studies have already proved that it is profound to estimate the levels of serum PTX3in the aspect of predicting the prognosis of acute cardiovascular events and hear failure. PTX3, as a cytokine-inducible factor, is mainly produced in vascular endothelial cells, fibroblasts and some other extrahepatic tissues and can reflect the inflammatory levels of local impaired tissues which make it distinguished from CRP and SAA that are synthesized majorly in the liver. For another, PTX3can activate the classical complement pathway through specific recognition and interaction with the complement component Clq and regulate the clearance of apoptotic cells, thus providing a link between the inflammation and innate immunity.However, the experimental evidence about the pro-inflammatory role of SAA is still inadequate. And the signaling pathway involved in this process is still poor understanding. Therefore, we investigate the capacity of human aortic endothelial cells (HAECs) to express PTX3, another sensitive inflammatory biomarker, after pro-incubation with SAA. Then the signaling pathway involved in this process has also been further discussed. Our research may be helpful in understanding the pro-inflammatory role of SAA and will provide a new theoretical support on elucidating the relative mechanism.Objectives1. To investigate the effect of SAA on up-regulating the expression of PTX3in HAECs and further discuss the pro-inflammatory role of SAA.2. To elucidate the relative signaling pathway involved in this process.Methods1. Experiment designHuman aortic endothelial cell line was obtained from American type culture collection (ATCC) and was cultured in endothelial culture medium (ECM) supplemented with5%fetal bovine serum. These cells were used for experiments between3rd and5th passage. 1) HAECs were treated with different concentration gradients (0,0.1,1,10,20μg/ml) of recombinant human SAA for24h or different checkpoints (0,3,6,9,12,15and24h) with10μg/ml of SAA respectively. The capacity of SAA on PTX3secretion in HAECs was observed through detecting the levels of PTX3secretion in cell-free supernatants by using Enzyme-linked immunosorbent assay (ELISA).2) HAECs were pretreated by the specific siRNA sequences of formyl peptide receptor like-1(FPRL1), or WRW4, an effective inhibitor of FPRL1, or pertussis toxin, an antagonist of G protein-coupled receptor. And then the cells were challenged by SAA (10mg/ml) for another24h. ELISA was used to investigate the role of FPRL1on SAA-induced expression of PTX3by detecting the levels of PTX3secretion in cell-free supernatants.3) HAECs were pretreated by the specific siRNA sequences c-Jun NH2-terminal kinase (JNK)1and JNK2for48h respectively, and then were challenged by SAA (10mg/ml) for another24h. ELISA was used to investigate the role of JNK1and JNK2on SAA-induced expression of PTX3by detecting the levels of PTX3secretion in cell-free supernatants.4) HAECs were pretreated by Tanshinone Ⅱ A, the specific inhibitor of activator protein (AP-1), or two effective nuclear factor-kappa B (NF-κB) inhibitors (BAY11-7082and SC-514) for1h respectively, and then were stimulated by SAA (10mg/ml) for another24h. ELISA was used to evaluate the role of AP-1and NF-κB activation in this process by detecting the levels of PTX3secretion in cell-free supernatants.2. ELISAHAECs were placed in ECM containing5%FBS in96-well plates and kept in a5%CO2incubator at37℃. After stimulation, cell-free supernatants were collected, centrifuged, and assayed for PTX3by ELISA according to the manufacturer's instructions.3. ImmunofluorescenceHAECs cultured on glass cover slips were pretreated by BAY11-7082and SC-514, two effective NF-κB inhibitors for1h respectively, and then were stimulated by SAA (10mg/ml) for another24h. After that, cells were fixed in2%paraformaldehyde and blocked in3%BSA, then incubated with anti-phospho-NF-kBp65antibody overnight. After being incubated with FITC conjugated secondary antibody, immunolabeled cells were counterstained with DAPI to detect cell nuclei. Then inhibitory role of BAY11-7082and SC-514on SAA-mediated NF-κB activation was observed by visualizing with a fluorescence microscope equipped with a digital camera.4. Western blotAfter being stimulated respectively, HAECs were harvested for protein extraction. Western blot was used to detect the protein expression levels of FPRL1, JNK1and JNK2.5. Quantitative real-time PCRTotal RNA was extracted from the harvested HAECs by the use of Trizol, and then reverse transcribed. Real-time PCR was used to detect the mRNA levels of PTX3, FPRL1,JNK1and JNK2.Results1. SAA up-regulates the PTX3expression in a time-and dose-dependent manner in HAECsHAECs were treated with different concentration gradients (0,0.1,1,10,20μg/ml) of SAA for24h and the supernatant concentration of PTX3was detected by ELISA. As a result, we observed that SAA could induce PTX3secretion in a concentration-dependent manner. Then cells were stimulated with SAA (10μg/ml) at various time points (0,3,6,9,12,15, and24h). The results demonstrated that SAA could induce PTX3accumulation in a time-dependent manner and reached its maximal activity at24h after stimulation. These results altogether indicate that the induction of PTX3by SAA is time-and dose-dependent. At the same time, the effect of SAA on PTX3at mRNA transcription level was examined via real-time PCR. Our data demonstrated a similar expression in compliance with its protein level, as the mRNA expression of PTX3increased by the time of stimulation after3-9h treatment with SAA which suggested that SAA-induced PTX3protein synthesis required transcriptional activation (P<0.05).2. SAA induces PTX3production via FPRL1Human FPRL1siRNA (Santa Cruz, sc-40123) was a convenient tool designed for FPRL1gene silencing and its effect had been examined carefully. Then, human FPRL1siRNA was used to confirm whether SAA-induced PTX3production in HAECs was mediated by FPRL1. After being preincubated with the siRNA sequences of FPRL1for48h and stimulated with10μg/ml SAA for another24h, both HAECs and supernatants were harvested for real-time PCR and Western blot to check the effect of the siRNA sequences. The data from real-time PCR and Western blot indicated that the SAA-induced PTX3secretion could be inhibited significantly by siRNA-mediated FPRL1gene silencing (P<0.05). All these results strongly support that FPRL1is likely to be one of the receptors through which SAA mediates its effects.3. SAA-induced PTX3production in HAECs is mediated through JNK pathwayAfter being preincubated with the siRNA sequences of JNK1and JNK2for48h, HAECs were stimulated with10μg/ml SAA for another24h. SAA-induced PTX3production levels were detected by ELISA. Our data showed that SAA-induced PTX3expression could be partially but significantly reduced (P<0.05). These results strongly suggest that JNK pathway is crucial for SAA-induced PTX3expression in HAECs, and activations of both JNK1and JNK2are involved in mediating PTX3production.4. SAA stimulates PTX3production in HAECs via AP-1activationAfter being incubated with Tanshinone Ⅱ A, an AP-1inhibitor, at various dose levels (0,5,10and20μg/ml) for24h, HAECs was exposed to SAA(10μg/ml) for additional24h. We found that Tanshinone Ⅱ A could inhibit SAA-induced PTX3expression dramatically in a dose-dependent manner (P<0.05), which suggested that the up-regulation of PTX3need the activation of AP-1.5. SAA induces PTX3production in HAECs via NF-κB activationWe examined the effect of SAA on NF-κB activity in HAECs by using two NF-κB inhibitors, BAY11-7082and SC-514. The results from immunofluorescence showed that both BAY11-7082and SC-514are effective in inhibiting the activation of NF-κB. After being incubated with BAY11-7082and SC-514at various dose levels (0,1,5,10and20μM) for1h, HAECs was exposed to SAA (10μg/ml) for additional24h. And both of them can block SAA-induced PTX3production significantly in dose-dependent manners (P<0.05). These results indicate that NF-κB activation is important for the SAA-induced PTX3production in HAECs.Conclusions1. In the present study, SAA can up-regulate the expression of PTX3in HAECs.2. SAA induces PTX3production via FPRL1.3. SAA-induced PTX3production in HAECs is mediated through JNK pathway4. SAA stimulates PTX3production in HAECs via AP-1and NF-κB activation...