| Background:Cardiovascular disease(CVD)is a major threat to human health and survival worldwide.The "China Cardiovascular Disease Report 2018" showed that the mortality rate of cardiovascular sufferers whether it is urban or rural residents is the top cause of death in 2018,and it is far higher than tumors and other sufferers.Atherosclerosis(AS)has been accounted as the main potential cause of CVD.Oxidized low-density lipoprotein(ox-LDL)is considered to involve in the development of atherosclerotic lesions.Hypertension,smoking and diabetes are risk factors for atherosclerosis and thrombosis.There are many factors that induce the development of AS such as foaming of macrophages.The accumulation of macrophages in the blood vessel wall is a sign of atherosclerosis.In atherosclerotic lesions,various environmental stimuli can change functional phenotypes of macrophages,such as modified lipids,cytokines,and senescent red blood cells.Understanding the function of macrophages on the formation and development of atherosclerotic plaques will help to delay or prevent the development of diseases and also provide new theoretical supports and treatment strategies to atherosclerosis-related diseases.In addition,studies have shown that chemokines secreted by macrophages also play an important role in the development of atherosclerosis.Chemokine CXCL12 also known as stromal cell-derived factor 1(SDF-1),chemokine-like functional chemokine or macrophage migration inhibitory factor(MIF).CXCL12 is a widely expressed and highly conserved protein.CXCL12 plays an important role in cell homeostasis,recruitment and arrest by binding to its corresponding chemokine receptors CXCR4 and ACCR3.In addition,many studies have confirmed that CXCL12 plays a key role in the homing of hematopoietic progenitor cells to the bone marrow and migration to peripheral tissue.In addition to these effects on progenitor cells,CXCL12/CXCR4 may also affect the development of atherosclerosis-related diseases by affecting various atherosclerosis-related cells.However,the role of CXCL12 secreted by macrophages in atherosclerosis is unclear.In this study,we used a series of experimental designs to explore the role of macrophage CXCL12 in atherosclerosis and its related mechanisms.To solve this scientific problem will provide a new theoretical basis for the treatment of atherosclerotic diseases and may create new treatment strategies.Objective1.The study will clarify the role of CXCL12 secreted by macrophages in the development of atherosclerosis.2.The study will determine whether CXCL12 secreted by macrophages affects development of atherosclerosis through vascular smooth muscle cells(VSMCs).Methods1.Establish cell modelMononuclear macrophages(THP-1)were induced with myristoyl phorbol ethyl ester(PMA)for 48 hours for transform into macrophages.Then,ox-LDL was used to induce the macrophages to foam cells as a cell model of atherosclerosis.2.Immunofluorescence(IF)assayVSMCs were inoculated on the coverslips of 12-well plates overnight.After paraformaldehyde fixation,cells were permeabilized with Triton X-100 and then sealed with BSA.These cells were incubated with a primary antibody against ?-SMA at 4? overnight,and a secondary antibody labeled with Alexa Fluor 488 for 1 hour at room temperature.The cell nucleus was stained with DAPI.Finally,the laser scanning confocal microscope is used for inspection.3.HE,IF or immunohistochemistry(IHC)detectionParaffin block was made from pulmonary artery or thoracic aorta of rats or mice in control group and model group,and stained after sectioning4.Transwell co-cultivationVSMCs were inoculated into the lower chamber of the Transwell plate,and THP-1 cells were inoculated into the upper chamber.THP-1 cells were then treated with ox-LDL.Following,VSMCs and THP-1 cells were co-cultured for 24 or 48 hours for subsequent experiments.5.Cell proliferation detectionVSMCs were seeded in the lower chamber of the Transwell plate,and THP-1 cells were seeded in the upper chamber.Then,MTT was added to VSMCs for 2 hours,and DMSO was added finally.The OD490 was measured with a microplate reader.6.Enzyme-linked immunosorbent assay(ELISA)detectionAfter incubation for 48 hours,the cell culture supernatants(upper and lower chambers of transwell co-culture system)were collected and tested with ELISA kit for CXCL12.7.Western blot detectionTotal protein extracted from THP-1 cells or VSMCs were separated by 10%SDS-PAGE.Then,the protein was transferred to the PVDF membrane and sealed with 5%skim milk.The membrane was incubated with the primary antibody of CXCL12 or GAPDH at 4? overnight.The next day,it wes washed 3 times with 1×TBST and incubate with HRP-labeled goat anti-mouse or anti-rabbit IgG for 1 hour at room temperature.The protein bands were detected with ECL substrate.8.Oil Red O stainingCells were cultured on slides overnight,and then treated with ox-LDL.After fixing with 4%paraformaldehyde,the cells were stained with 0.3%oil red O for 20 minutes,and the images were collected with a microscope.9.RNA extraction and qPCR detectionTRIZOL were used to extract total RNA from cells.The total RNA sample was reverse transcribed into cDNA using a reverse transcriptase kit.Real-time quantitative PCR was performed with SYBR Green qPCR kit to determine the relative levels of GAPDH and CXCL12 expression.10.siRNA and shRNA were transfected into cellsCells were plated in a six-well plate and surface to 50-80%.Following the instruction manual,siRNA was transfected into cells with Lipofectamine 2000 for subsequent experiments.We design gene shRNA,construct vectors(provided by Anhui General Biotechnology Co.,Ltd.),extract high-purity plasmids,transfect cells,and screen for lentivirus stable cell lines?11.Flow cytometry was used to detect cell cycleThe cell culture supernatant was discarded,and then the cells were washed with cold PBS.Cells were rinsed carefully.We took 100 ?L of the cell suspension into a Eppendorf tube,and add PI(final concentration 50 ?g/mL)and RNase A(The final concentration is 20 ?g/mL)into tube.After incubating on ice for 30 minutes in the dark,we filtered the cells with a 200-mesh strainer.Finally,we used a flow cytometer to detect the cell cycle12.Atherosclerosis animal model1)Establishment of atherosclerosis model in ratsTen male Sprague-Dawley rats(12 weeks old)were randomly divided into a control group(n=5 rats)and a model group(n=5 rats).According to the reference,an animal model of atherosclerosis was established by feeding rats a high-fat diet plus vitamin D2 for 3 weeks.2)lEstablishment of atherosclerosis model in mice14 female ApoE-/-mice(8 weeks old)were randomly divided into a control group(n=7 mice)and a model group(n=7 mice).According to the reference,an atherosclerosis model was established by feeding mice high-fat diet for 24 weeks.13.Statistical analysisGraphPad Prism 7.0 software was used to analyze all data.The data between the two groups was compared using an independent sample t test.P value<0.05 had statistical differenceResults1.Atherosclerosis cell model were successfully constructed and validatedAfter 24 hours of inducing macrophages by ox-LDL,the results of Oil Red O staining showed that lipid accumulated in macrophages.After 48 hours,the results of Oil Red O staining showed that lipid accumulation and foaming of macrophages increased significantly.After treated with ox-LDL,the secreting of CXCL12 in the culture supernatant and transcriptional level of CXCL12 in macrophages was significantly increased.2.The stimulation of ox-LDL can also promote the foaming of VSMCsIF was carried out for testing expression of a-SMA in VSMCs.The results showed that all cells were positive for a-SMA.Using oil red O staining to evaluate the level of foam cell formation,the results showed that ox-LDL treatment can induce lipid droplet accumulation after 24 hours,and significantly increased after 48 hours.The transcript levels of CXCL12 in VSMCs were tested by ELISA and qPCR,the results showed that the concentration of CXCL12 in VSMCs culture supernatant and the transcription level of CXCL12 in VSMCs after ox-LDL treatment was significantly increased.3.THP-1 macrophages treated with ox-LDL promote the proliferation of VSMCs and the formation of foam cellsThe proliferation of VSMCs co-cultured with THP-1 macrophages pre-treated with ox-LDL was significantly improved.Compared with the control group without THP-1 macrophage in the upper chamber and only ox-LDL,the proliferation of VSMCs was not significantly improved.In addition,the results also showed that in VSMCs co-cultured with THP-1 macrophages pretreated with ox-LDL,foam cell formation was significantly increased.The concentration of CXCL12 in the lower chamber culture after ox-LDL treatment was significantly increased,and the expression of CXCL12 in THP-1 macrophages in the upper chamber was also significantly increased used ELISA and Western blot.4.CXCL12 secreted by ox-LDL-treated THP-1 macrophages promotes VSMCs proliferation and foam cell formationIf CXCL12 expression ability is inhibited by siRNA in THP-1 macrophages,the ability of ox-LDL stimulates THP-1 macrophages to secrete CXCL12 was significantly reduced,and its ability to promote VMSCs proliferation was also significantly decreased.On the basis of this condition,if exogenous CXCL12 could reverse the effects brought about by siRNA inhibition of THP-1 macrophages,and the cell proliferation ability of VSMCs was again significantly improved.Consistent with the proliferative outcome of VSMCs foam cell formation detected by red oil 0 staining,THP-1 macrophages had a significantly decreased ability of ox-LDL to stimulate THP-1 macrophages to promote VSMCs foaming if CXCL12 expression was inhibited by siRNA.Exogenous CXCL12 also could significantly improve the foaming of VSMCs.In addition,changes in the concentration of CXCL12 in the lower chamber medium and CXCL12 expression in THP-1 macrophages were detected using ELISA and Western blot,results showed it has the same trend with proliferation of VSMCs.We designed and cloned the shRNA of CXCL12 into PLKO.1 vector,and established THP-1 cell line which can stably interfere with CXCL12 through retroviral vector system.The stable and low expression of CXCL12 was confirmed by qPCR and Western blot,and the THP-1 cell line which stably interfered with CXCL12 was successfully constructed.In this system,the results showed that the proliferation trend of VSMCs cells was consistent with results of siRNA interference,and it could promote VSMCs cells to enter the S phase of cell cycle.5.The secretion level of CXCL12 in the serum of rats in AS rat model wassignificantly increased.The results of HE staining showed that no obvious atherosclerotic lesions were found in the control group,but the lesions in the main pulmonary artery region of model rats were significantly enlarged;the results of immunohistochemical staining(IHC)showed that a-SMA and IBA-1 were significantly increased in the successfully induced atherosclerotic rat model.The concentration of CXCL12 in the serum of atherosclerotic rat models was also significantly enhanced.6.In the AS mouse model,CXCL12 secreted by macrophages and the proliferation of VSMCs was significantly increased.The results of HE stains showed that no obvious atherosclerotic lesions were found in the control group,but there were significant plaques in the thoracic aorta region of the model mice.CXCL12 secretion by macrophages was significantly elevated after atherosclerosis.Meanwhile,the proliferation of VSMCs in the model group was significantly increased compared with the control group.Macrophage apoptosis in plaques was examined by caspase3,and the results showed that macrophage apoptosis was significantly increased in plaques.Conclusions:1.The secretion levels of CXCL12 in THP-1 and VSMCs were significantly increased and foaming of THP-1 and VSMCs were also significantly increased after treated with ox-LDL.2.THP-1 treated with ox-LDL can promote the proliferation of VSMCs3.The proliferation of VSMCs was promoted by THP-1 macrophages treated with ox-LDL through the secretion of CXCL12.4.In vivo models,the secretion of CXCL12 by macrophages and the proliferation of VSMCs were significantly increased.Background:Many key signaling pathways are highly correlated with atherogenesis.These pathways include:insulin receptor(and other receptor tyrosine kinases);ras and MAPK activation;TNF-? and related family members that cause NF-?B activation;effect of reactive oxygen species(ROS)on signal;the activation of endothelial cells and other cells by modified lipoproteins;purine signaling;control of leukocyte adhesion to endothelial cells,migration and further activation;the formation of foam cell;and macrophages and vascular smooth muscle cell(VSMC)signals associated with proliferation,exocytosis and apoptosis.They have become the focus of modern atherosclerosis research and will undoubtedly provide rich resources for innovative intervention and prevention of the leading cause of death in the modern world.With the new understanding of the interaction between macrophages and vascular smooth muscle cells in atherosclerotic plaques and the influence of macrophages secretory profile on VSMCs phenotype,the communication between macrophage derived foam cells and VSMCs has become more and more clear.However,it is not clear what kind of signal pathway ox LDL stimulates macrophages to secrete CXCL12 and how CXCL12 promotes VSMCs proliferation.Therefore,exploring the role of the above signals in this process may help us to further clarify the molecular mechanism of CXCL12 secretion by macrophages in promoting atherosclerosis.After the initial identification of activated nuclear factor ?B(NF-?B)in human atherosclerotic lesions,more attention has been paid to the involvement of this transcription factor family in the formation of atherosclerosis.It is now clear that the activation of the NF-?B signaling pathway and the role of NF-?B constitute major participants in all phases of the atherosclerotic process.NF-?B has long been considered a pro-atherogenic factor,and recent research suggests that its actual role may be more complex.In addition to activating many of the pro-inflammatory genes associated with atherosclerosis,NF-?B also regulates cellular processes such as cell survival and proliferation.In addition,its important role in inflammatory regression and anti-inflammatory gene transcription suggests that its activation in different cell types or stages of the atherogenic process may have different and opposite results.At the same time,NF-?B signaling pathway plays an important role in the transformation of macrophages into foam cells.However,whether it is involved in the secretion of CXCL12 by macrophages is not clear and further research is needed.Intracellular mitogen-activated protein kinase(MAPK)signaling cascade plays an important role in the pathogenesis of cardiovascular diseases.Numerous basic scientific studies have identified many details of MAPK pathway organization and activation,but the role of individual signaling proteins in the pathogenesis of various cardiovascular diseases is still being elucidated.In this study,we will investigate whether ERK(extracellular signaling kinase),JNK(c-jun amino-terminal kinase)and p38 signaling pathways activate the MAPK signaling pathway of VSMCs when macrophages secreted CXCL12 to promote VSMCs proliferation in an ox-LDL-induced atherosclerosis model.This study is help to the exploration of new targets for drug treatment of atherosclerosis.The phosphatidylinositol 3 kinase(PI3K)/AKT/mammalian target of rapamycin(mTOR)pathway is one of the key signaling pathways induced by various receptor-tyrosine kinases.More and more evidences show that this pathway is an important promoter of cell growth,metabolism,survival,proliferation and differentiation,and is closely related to the regulation of autophagy.There has been a great deal of research on this pathway,but its exact role in atherosclerosis is still unknown.Study found that selective inhibition of the AKT/mTOR signaling pathway can reduce macrophages by promoting autophagy of macrophages and stabilize vulnerable atherosclerotic plaques.Studies have also shown that the PI3K/AKT signaling pathway is an important signal transduction pathway that mediates VSMC proliferation.Therefore,it is necessary to further explore whether CXCL12 functions through this signaling pathway.In conclusion,the solution of these key scientific problems will provide new theoretical basis and new therapeutic targets for the treatment of atherosclerosis.Objective1.The study will determine whether ox-LDL induces THP-1 macrophages through the NF-?B signaling pathway.2.The study will determine whether CXCL12 secreted by ox-LDL induced THP-1 macrophages is completed by activating the MAPK signaling pathway of VSMCs.3.The study will determine whether CXCL12 secreted by ox-LDL induced THP-1 macrophages is completed by activating the PI3K/AKT/mTOR signaling pathway of VSMCs.Methods:1.Culture human mononuclear cell line THP-1.THP-1 cells were incubated in a T75 cell culture bottle with 1640 medium(containing 10%FBS and FBS inactivated at 55? for 30min)at 37? and incubated in a 5%C02 incubator.The cells grew in suspension,THP-1 cell suspension was collected and resuspended with 1640 medium(containing 10%inactivated FBS,foboester PMA10ng/mL,and PMA for stimulating cell adherence),and spread in 24 well plates for further culture after counting.The next experiment was carried out.2.BMDM induction experiment.The hind legs of C57 mice were taken aseptically,the muscle was removed.The bone marrow was blown with a 2ml syringe for several times.Tris-NH4CL lysate lyses red blood cells.The cells were resuspended and counted.The concentration was adjusted to 2×106 cells/ml.In a 10cm culture dish,7-8ml cells could be added and cultured in BMDM induction medium.The suspension cells could be removed by changing the fluid every 2-3 days.Mature BMDM could be harvested in 7 days.3.Transwell co culture.VSMCs were inoculated into the lower chamber of the Transwell plate(3422,Corning,NY,U.S.A.)and THP-1 cells were inoculated into the upper chamber.THP-1 cells were then treated with ox-LDL.Then,VSMCs and THP-1 cells were cultured for 24 or 48 hours.4.Total protein extraction and Western Blot.The supernatant of cell culture in each well was sucked away,and an appropriate amount of cell lysate containing protease inhibitor was added into each well to fully lyse cells,and the protein concentration was determined with the BCA protein concentration detection kit.The corresponding volume of each pore was added into 6xprotein loading buffer,fully mixed,heated for 5 min at 98? after cooling,the sample could be directly used for SDS-PAGE,or stored temporarily at-80? or-20?.According to the needs of the experiment,the corresponding volume of protein samples and samples of predye protein marker were taken for electrophoresis.ECL was Shadowed with a hypersensitive chromogenic solution(Thermo scientific,34095).5.siRNA transfection.Cells were seeded in a six well plate and cultured to 30-50%fusion.Then,according to the manufacturer's instructions,siRNA was transfected with Lipofectamine 2000(Invitrogen,Carlsbad,CA,U.S.A.6.Mouse model of atherosclerosis.Female ApoE-/-mice(8 weeks old,purchased from Beijing Charles River)were randomly assigned to the control group(n=7 mice)and the model group(n=7 mice).The atherosclerosis model was established by feeding the mice with a high fat diet for 24 weeks,as described in the literature.7.Statistical analysis.The GraphPad Prism7.0 software was used to analyze all the data.The data between the two groups were compared by independent sample t test,and P<0.05 showed a statistical difference.Results:1.ox-LDL induces the expression and secretion of CXCL12 in macrophages through the NF-kB signaling pathway.The expression of I?B?(p-I?B?)phosphorylated in both THP-1-derived macrophages and BMDM was significantly increased after induction of ox-LDL for 24h.Furthermore,the expression of p65(p-p65)phosphorylated after ox-LDL stimulation was consistent with that of I?B? for 24 h.However,there was no significant change in the expression of p52(p-p52)phosphorylated by the two types of macrophages after ox-LDL stimulation for 24h.After blocking the NF-?B signaling pathway by resveratrol,the levels of CXCL12 secretion or expression in the cultured supernatant or THP-1 macrophages were significantly decreased under ox-LDL induction by ELISA and Western blot.2.CXCL12 promotes the proliferation of VSMCs by activating the ERK or JNK signaling pathways.In the presence of THP-1,the phosphorylation of ERK of VSMCs was significantly increased after ox-LDL induction.On this basis,the phosphorylation level of ERK in VSMCs can be significantly increased by adding exogenous CXCL12.The trend of JNK was consistent with ERK,but there was no significant difference in p38 between groups3.The PI3K/AKT/mTOR pathway is not involved in the abnormal proliferation of VSMCs stimulated by CXCL12 in the Transwell system.There was no significant difference in the phosphorylation of AKT in VSMCs and GSK-3? in VSMCs under different conditions.The phosphorylation level of mTORCl in VSMCs did not change significantly.4.Resveratrol significantly reduced the atherosclerotic plaque area in ApoE-/-mice on a high-fat diet.The atherosclerotic plaque area in ApoE-/-mice on a high-fat diet was significantly reduced if they were given resveratrol water at the same time.Conclusions1.ox-LDL promotes CXCL12 expression and secretion by activating p65 phosphorylation in the NF-?B signaling pathway of THP-1 or BMDM.2.The possible effector molecules involved in CXCL12 stimulation of VSMCs proliferation are MAPK signaling pathway downstream regulators of ERK or JNK,rather than p3 8.3.AKT,Raptor or GSK-3 in the PI3K signaling pathway did not involve in the proliferation of VSMCs stimulated by CXCL12. |
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