| Atherosclerosis is the most important pathological process of the coronary artery and cerebrovascular diseases, which is the major cause of death. Angiogenesis is one of the key risk factors for atherosclerosis. Angiogenesis is a process that involves endothelial cell proliferation, migration and the formation of new vessels from pre-existing capillaries. The role of angiogenesis has two sides. On the one hand, angiogenesis can be used to treat ischemic heart disease, which needs to be promoted in treatment. On the other hand, angiogenesis can accelerate the progress of atherosclerosis and lead to plaque rupture, which needs to be inhibited in treatment. According to its two sides, angiogenesis can be divided into physiological angiogenesis and pathological angiogenesis. To explore the mechanism of pathological angiogenesis could provide new targets for the control of atherosclerosis.Serum amyloid A (SAA) is a family of highly conserved acute-phase proteins and consists of SAA1, SAA2, SAA3 and SAA4. SAA1 is the most important component. SAA is synthesized primarily by hepatocytes and its plasma concentration can increase up to 1000-fold over the basal level in response to inflammatory stimulation. Therefore, a high concentration of circulating SAA is a biomarker for acute and chronic inflammatory diseases. As an acute-phase protein, SAA can induce pathological angiogenesis in a variety of diseases, such as in giant cell arteritis and rheumatoid arthritis. Therefore, it is important to explore the mechanism of SAA to angiogenesis.Vascular endothelial growth factor (VEGF) is one of the most important growth factors of endothelial cells and the key regulator of angiogenesis. Vascular endothelial growth factor receptor 2 (VEGFR2) is one of the receptors of VEGF, which is the portal of VEGF signaling pathways to promote angiogenesis. The activation of VEGF/VEGFR2 signaling pathway promotes vascular endothelial cell survival, migration, proliferation and tube formation, which plays a critical role in the development of many diseases.VEGFR2 is mainly released from vascular endothelial cells, but the mechanism of its regulation remains to be explored. Previous studies have shown that SAA can promote the expression of VEGF in human carotid artery endothelial cells (HCtAE), and SAA can activate the p38 MAPK signaling pathways to induce angiogenesis. Based on the previous researches, we hypothesized that SAA may influence the expression of VEGFR2 and angiogenesis. Currently there are some problems needed to be solved:the effects of SAA on the expression of VEGFR2 in umbilical vein endothelial cells; the signaling pathway involved in SAA induced VEGFR2 expression; the effects of SAA induced VEGFR2 expression in the regulation of angiogenesis.Objective1. To clarify the effects of SAA on the expression of VEGFR2;2. To explore the signaling pathways involved in SAA induced VEGFR2 expression;3. To investigate the effects of SAA induced VEGFR2 expression in the regulation of angiogenesis.Methods1. Human umbilical vein endothelial cells culture Human umbilical vein endothelial cells (HUVECs) were cultured in endothelial complete medium (ECM). The cells were maintained at 37℃ in a humidified incubator under 5% CO2, with the medium replaced every 2-3 days until the cells reached proper density and then incubated with rSAA or with various antagonists and agonists for different concentrations and times.2. Quantitative real-time PCRThe mRNA was isolated from the HUVECs using TRIzol reagent, a reverse transcriptase reagent kit was used to obtain cDNA, and the expression of VEGFR2 mRNA was measured by quantitative real-time PCR.3. Western blot analysisCells were collected and extracted proteins. Western blot was used to examine the expression of VEGFR2, ERK1/2, JNK and p38.4. Tube formation assaySamples of matrigel were loaded into 96-well plates. After the gel had solidified, suspensions of HUVEC cells were added onto the matrigel. The final cell density was 2×104 cells/well. Take the groups without any stimulation as references, tube formation was evaluated under a microscope.5. Statistical analysisAll data were analyzed using Prism version 5 and SPSS 18.0. One-way analysis of variance was performed to determine the statistical significance. Results were expressed as mean ±SD. P<0.05 was confirmed as statistically significant.Results1. SAA up-regulates VEGFR2 expression in HUVECsCells were stimulated with different concentrations (0,5,10 and 50 μg/ml) of SAA for 24 h, and the protein and mRNA were extracted from the cells. Western blot and real-time PCR revealed that SAA dose-dependently up-regulated the expression of VEGFR2 (P<0.01). Then, cells were treated with SAA (10μg/ml) at different time points (0,2,3,6,12 and 24 h), and the protein and mRNA were extracted from the cells. Western blot and real-time PCR revealed that SAA time-dependently stimulated VEGFR2 expression (P<0.05).2. SAA induces VEGFR2 expression via FPRL1We pretreated HUVECs with different concentrations of WRW4 (a FPRL1 antagonist). Western blot revealed that SAA-induced VEGFR2 expression could be blocked inordinately by the pre-treatment with WRW4 compared with the SAA group (P<0.01). Then we stimulated the cells with WKYMVm (a FPRL1 agonist), and the expression of VEGFR2 increased evidently compared with the control group (P<0.01). These results revealed that FPRL1 can mediate SAA-induced VEGFR2 expression.3. SAA-induced VEGFR2 expression is mediated by the MAPK signaling pathwayWhen HUVECs were pre-treated with SAA, the relative expression of p-ERK1/2, p-JNK and p-p38 all increased and reached its peak production at 1 h compared with the control group (P<0.01). Then, we pre-incubated HUVECs with either PD98059, SP600125 or SB203580 for 1 h prior to SAA treatment for another 6,12 and 24 h. Western blot revealed that SAA-induced VEGFR2 expression was inhibited by the 3 inhibitors dramatically compared with the SAA groups (P<0.05). These results revealed that MAPK signaling pathway plays important roles in the process of SAA-induced VEGFR2 expression.4. The activation of the MAPK signaling pathway is mediated by FPRL1We pre-incubated HUVECs with the FPRL1 inhibitor WRW4. Western blot revealed that WRW4 inhibited the phosphorylation of MAPKs compared with the control group (P<0.05). In addition, HUVECs were also treated with the FPRL1 agonist WKYMVm. Western blot revealed that the phosphorylation of MAPKs increased in the WKYMVm-treated groups compared with the control group (P<0.01). In conclusion, these results strongly suggested that MAPKs were the downstream signaling molecules of FPRL1 in the process of SAA-induced VEGFR2 expression. 5. SAA-induced VEGFR2 expression increases angiogenesisWe pre-incubated HUVECs with the VEGFR2 inhibitor BIBF1120 and agonists and antagonists of FPRL1/MAPKs. We found that BIBF1120, WRW4, PD98059, SP600125 and SB20358 can inhibit tube formation compared with the SAA-treated groups (P<0.01). However, WKYMVm can induce tube formation compared with the control groups (P<0.01). These results indicated that SAA-mediated enhancement of tube formation might be dependent on the SAA-induced expression of VEGFR2.Conclusion1. SAA can induce the expression of VEGFR2 in HUVECs;2. FPRL1/MAPKs signaling pathways mediate SAA-induced VEGFR2 expression;3. SAA-induced VEGFR2 expression can enhance tube formation in endothelial cells. |
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