| Fractalkine（FKN）and its specific receptor CX3CR1 play a critical role in the pathogenesis of atherosclerosis including recruitment of vascular cells and the development of inflammation.However,its contribution to regulating the development of atherosclerotic calcification has not been well documented.Osteogenic transformation of vascular smooth muscle cells（VSMCs）is critical in the development of calcification in atherosclerotic lesions.In this study,for the first time,we evaluated the effect of FKN/CX3CR1 on the progression of VSMCs calcification and defined molecular signaling that is operative in the FKN/CX3CR1-induced osteogenic transformation of VSMCs.We found that high-fat diet induced atherosclerotic calcification in vivo was markedly inhibited in the Apolipoprotein E（Apo E）and CX3CR1 deficient(Apo E-/-/CX3CR1-/-)mice compared with their control littermates.FKN and CX3CR1 were both expressed in VSMCs and up-regulated by oxidized low-density lipoprotein（ox-LDL）.FKN/CX3CR1 promoted the expression of osteogenic markers,including osteopontin（OPN）,bone morphogenetic protein（BMP）-2 and alkaline phosphatase（ALP）and decreased VSMCs markers,including smooth muscle（SM）α-actin and SM22-α in a dose-dependent manner.The essential role of FKN/CX3CR1 in VSMCs calcification was further confirmed by lentivirus-mediated knockdown or overexpression of CX3CR1 blocked or accelerated osteogenic transformation of VSMCs.This response was associated with reciprocal up-and down-regulation of osteogenic factor,runt-related transcription factor 2（RUNX2）,transcription factors in osteoclast differentiation,receptor activator of nuclear factor-κB（RANK）,RANK ligand（RNAKL）and osteoprotegerin（OPG）,respectively.Inhibition of FKN/CX3CR1-activated Jak2/Stat3 signaling by the Jak/Stat inhibitor AG490 blocked osteogenic transformation of VSMCs and RUNX2 induction concurrently.Taken together,our data uncovered novel roles of FKN/CX3CR1 in promoting VSMC osteogenic transformation and atherosclerotic calcification by activating RUNX2 through Jak2/Stat3 signaling pathway and suppressing OPG.Our findings suggest that targeting FKN/CX3CR1 may provide new strategies for the prevention and treatment of atherosclerotic calcification.Introduction Atherosclerosis is the main cause of cardiovascular and cerebrovascular diseases.Calcification is a common pathological process of atherosclerosis and is an independent risk factor for predicting acute cardiovascular and cerebrovascular events[1,2].In-depth investigations of the influencing factors and molecular mechanisms of atherosclerotic calcification play important roles in the prevention and treatment of atherosclerosis.Vascular smooth muscle cells（VSMCs）are involved in the process of atherosclerosis development and progression.Studies have shown that the transformation of VSMCs into an osteogenic phenotype is crucial for the calcification process in atherosclerosis[3].Under the action of inflammatory factors（such as tumor necrosis factor-α（TNF-α）and matrix metalloproteinases（MMPs））and oxidative stress,VSMCs in atherosclerosis lesions are transformed from a contractile phenotype to an osteogenic phenotype[4].Osteogenic VSMCs are able to secrete and mediate bone matrix deposition in blood vessels,eventually promoting the formation of calcification in atherosclerosis.The transformation of VSMCs into an osteogenic phenotype is a key step in the promotion of atherosclerotic calcification.However,the mechanism has not been elucidated thus far.In-depth studies of the mechanism of the osteogenic transformation of VSMCs and the identification of key targets for early intervention are important means of atherosclerosis prevention and treatment.Fractalkine（FKN,also known as CX3CL1）is the only member of the chemokine CX3 C subgroup.Through specific binding with CX3 C chemokine receptor 1（CX3CR1）,FKN recruits inflammatory cells and mediates their interaction with vascular wall cells,playing important roles in the process of atherosclerosis development and progression[5-8].Current studies suggest that FKN/CX3CR1 can induce VSMCs switching to the synthetic state and proliferation[8,9].The interaction between FKN and CX3CR1 can promote the expression of inflammatory cytokines such as TNF-α,MMP2 and MMP9 in VSMCs and macrophages in atherosclerosis lesions[4].The aforementioned inflammatory cytokines have been shown to promote the osteogenic transformation of VSMCs[10,11].FKN/CX3CR1 also plays an important role in the maturation and differentiation of osteogenic precursor cells and osteoblasts[12,13].Based on these findings,we speculated that FKN/CX3CR1 might be involved in promoting the osteogenic transformation of VSMCs in atherosclerosis lesions.The specific steps and possible molecular mechanisms involved have not been reported.Increasing evidence suggest that vascular calcification is an active regulation process similar to the formation of bone and cartilage.The imbalance between pro-calcification factors and anti-calcification factors promotes the osteogenic transformation of VSMCs[14].Therefore,whether FKN/CX3CR1 co-determines the osteogenic transformation of VSMCs through a similar pathway remains to be studied in the calcification process of atherosclerosis.The aim of this study was to investigate the effect of FKN/CX3CR1 on the osteogenic transformation of VSMCs in vitro and observe the intracellular changes in janus kinase/signal transducer and activator of transcription（Jak/Stat）-RUNX2 and OPG expression under the action of FKN/CX3CR1.We also investigated the relationship between the above signaling molecules and FKN/CX3CR1-induced osteogenic transformation of VSMCs.Based on these investigations,Apolipoprotein E（Apo E）and CX3CR1 deficient(Apo E-/-/CX3CR1-/-)mice were used to observe the effect of CX3CR1 gene knockout on the formation of atherosclerotic calcification and the osteogenic transformation of VSMCs induced by a high-fat diet.The purpose of this study was to investigate the regulatory mechanism of the osteogenic transformation of VSMCs in atherosclerosis and to provide experimental bases for finding new intervention targets for atherosclerosis and developing prevention and treatment measures.Materials and methods Experimental animals.Mice lacking Apolipoprotein E(Apo E-/-)have been widely used as a mice model to investigate the genetic and environmental factors that regulate pathogenesis of atherosclerosis as well as atherosclerotic calcification[15,16].CX3CR1 deficient mice in the Apo E-/-background were generated by crossing the Apo E-/-mice（Huafukang,CHN）with the CX3CR1 knocked out mice（Stock No.005582,Jackson Laboratory,US）,for receptor activator of nuclear factor-κB（RANK）and RANK ligand（RNAKL）are under the tight control of the female sex hormones estradiol and progesteron[17],the male ones were used.Characterization of atherosclerotic calcification in vivo was performed with mice crossed into Apo E-/-background.Apo E-/-/CX3CR1-/-mice were verified by PCR analysis and fed a high-fat diet（78% of general materials,16.4% of lard,3.6% of vegetable oil,2% of cholesterol）for 30 weeks,which were supplied by the Animal Research Committee of the Third Military Medical University.Cell culture and calcification model.VSMCs were harvested from C57BL/6 J genetic background mice,verified Apo E-/-and Apo E-/-/CX3CR1-/-mice at 6-8 weeks of age,3-8 passages were used.Cultures were incubated at 37 °C in a humidified chamber with 5% CO2.Cells（as above）were plated at 5í105 cells/ml and cultured in 6-well plate in DMEM medium supplemented with 10% FBS,penicillin（100 U/ml）,and streptomycin（100 μg/ml）for 24 h.The medium was removed and replaced with osteogenic medium to establish the calcification model,which is consist of 10 m M β-glycerophosphate,50 μg/ml ascorbic acid,10-8mol/l dexamethasone,100 U/ml penicillin,and 100μg/ml streptomycin in the presence of high glucose medium and 10% serum in the absence or presence of 10,30 or 50 ng/ml of recombinant mouse FKN chemokine domain（R&D Systems,258-MF）.The medium was changed every 3 days.Immunofluorescence and Confocal Laser Scanning Microscopy（CLSM）.At the end of the incubation period（7 or 14 days）the medium was removed and cells were rinsed in phosphatebuffered saline（PBS）,fixed with 4% paraformaldehyde for 15 min at room temperature,the aortas and aortic sections were embedded in OCT（Tissue-Tek,Alle-giance）,cut into 7-μm-thick sections and permeabilized with 0.1% Triton X-100 for 10 min,further PBS washes were followed by blocking with 5% normal donkey serum for 1 hour at room temperature.Primary antibodies were added and incubated overnight at 4℃.Unbound primary antibody was removed by washing in PBS prior to addition of a secondary antibody for 1 hour at room temperature,Alexa Fluor 488 donkey anti-rabbit and Alexa Fluor 594 donkey anti-mouse were used as secondary antibodies（Invitrogen,USA）,after washing by PBS for 3 times,cells were then incubated with 4′-6-diamidino-2-phenylindole（DAPI）for staining nuclei and then protected by anti-fademounting medium.Cells or were photographed using a confocal microscope（Leica,German）.Calcium assessment in VSMCs,aortas and aortic sections.Cells were cultured in osteogenic medium with or without gradient FKN for 7 or 14 days.All plates were washed in PBS and fixed with 4% paraformaldehyde for 15 minutes.Each plates were washed 3 times with PBS and stained with 1% alizarin red stain（p H 4.2,Sigma-Aldrich）in dd H2 O for 5 minutes at room temperature.The plates were then rinsed 3 times with dd H2 O and observed under a light microscope for calcified nodules.To quantify alizarin red staining（ARS）contents,ARS nodules were decalcified by 10% cetylaminopyridine（Sigma-Aldrich）and measured their absorbance at 450 nm using an ELISA reader.Aortas and aortic sections of Apo E-/-and Apo E-/-/CX3CR1-/-mice were perfused with saline and immersed 4% paraformaldehyde for 24 hours at room temperature and then directly stained with 1% alizarin red stain as described above.Aorta tissue blocks were dehydrated in a graded series of ethanol,embedded in paraffin,cut into 3μm-thick serial sections,and stained with ARS for detecting atherosclerotic calcification,hematoxylin and eosin（H&E）staining has also been used to see the section pattern.Staining was performed with 1% ARS in dd H2 O for 15 minutes at room temperature.After staining,the aortas and aortic sections were washed 3 times with dd H2 O,and images were acquired a Zeiss microscope equipped with a color camera.The calcium concentration was detected colorimetrically by the o-cresolphthalein complexone method（Calcium Kit,Abcam）.All sections of arteries were harvested from the aortic arches.Transfection of lentivirus.The lentivirus vector with CX3CR1 knockdown and overexpression were constructed by Han Heng（Shang Hai,China）.Scrambled sh RNA unrelated to mouse gene sequences was used as a negative control（sh Control）.In vitro,the chondrocytes were seeded before transfection at 5×104 cells/well in 24-well plates and incubated in serum-free growth medium.When cells reached 50% confluency,they were transfected with the specific lentivirus at a multiplicity of infection of 30（MOI=30）.Three days later,the transfection efficiency was measured by the percentage of GFP-positive cells/total cells,and the knocking-down efficiency was analyzed by western blot and RT-PCR.The transfection of CX3CR1 overexpression vectors were the same as described before.Western Blotting.Protein samples were obtained from cell lysates of homogenized cultured cells,and the protein concentration was determined by BCA protein assay kit（Thermo Scientific,USA）.Protein samples were separated by SDS-PAGE and transferred to a NC membrane（Bio-Rad,Hercules,USA）.The membranes were blocked in TBS containing 0.05 % Tween-20（TBST）and 5 % non-fat milk powder for 1 hour at room temperature before overnight incubation with primary antibodies at 4 °C.After washing with TBST,the membranes were then incubated with secondary antibodies for 2 hours at room temperature. Protein was scanned using the Odyssey scanner software（Li-COR Bioscience）and quantified using Image J 6.0.The expression of the various proteins was normalized to control groups.Real-Time Reverse Transcription–Polymerase Chain Reaction.Total RNA was isolated from VSMCs using the RNAeasy Isolation kit（Sangon Biotech,Shanghai,China）;1μg RNA was used to produce c DNA using i Script reverse transcription reagents（Bio Rad,US）,2íSYBR green PCR master mix was used（Bimake,China）,followed by quantitative real-time polymerase chain reaction.Primer sequences were listed in this article.Samples were heated to 95 for 3 min,followed by 39 cycles of: 95 for 10 s,57 for 30 s,72 for ℃ ℃ ℃ ℃10s,then72 for 10 s,65 for 5s,and 95 for 5s.Gene expression relative to ℃ ℃ ℃ β-actin was calculated using the-??CT formula method.Statistical Analysis.All experiments were performed at least three independent times,unless otherwise stated.Values are presented as the mean ± SEM.Unpaired Student’s t-test,one-way ANOVA,two-factor repeated measures ANOVA were used for two or multiple groups comparisons followed by Tukey’s multiple comparisons test.Data were plotted using Graph Pad Prism 7.0 software,P≤0.05 was considered significant.Results CX3CR1-/-impedes high-fat diet induced aortic atherosclerotic calcification in mice.Apo E-/-mice and Apo E-/-/CX3CR1-/-mice fed with high-fat diet（HFD）for 30 weeks were sacrificed for the detection of aorta.Apo E-/-mice showed extensive atherosclerotic calcification,visualized as red staining under intima and in media of aorta stained by Alizarin red.In contrast,Apo E-/-/CX3CR1-/-mice showed no significant calcium deposition in aorta.Furthermore,quantitative analysis showed markedly decreased calcium content in aorta from Apo E-/-/CX3CR1-/-mice than that from Apo E-/-mice.Consistently,osteogenic markers,including OPN,BMP2 and ALP,were expressed abundantly in aorta from Apo E-/-mice but not in that from Apo E-/-/CX3CR1-/-mice.Cultured VSMCs harvested from Apo E-/-mice showed significantly increased protein levels of OPN,BMP2 and ALP in response to FKN（50 ng/ml）stimulation for 7 days,while no significant enhancement was observed in VSMCs from Apo E-/-/CX3CR1-/-mice.These data suggest that FKN/CX3CR1 may be involved in atherosclerotic calcification induced by HFD,and exert important role in regulating the expression of osteogenic markers.FKN and CX3CR1 are expressed in VSMCs and up-regulated by oxidized low-density lipoprotein（ox-LDL）.To investigate the potential role of FKN and its specific receptor CX3CR1 in VSMC osteogenic transformation,the expression of FKN and CX3CR1 in cultured VSMCs were detected.It was shown that FKN and CX3CR1 expressed in membrane and cytoplasm of VSMCs cultured in DMEM medium.As a widely accepted atherogenic factor,ox-LDL（40 μg/ml for 72h）was found to up-regulate the m RNA and protein levels of FKN and CX3CR1 in cultured VSMCs,indicating that FKN/CX3CR1 may contribute to the high-fat diet induced VSMC osteogenic transformation.FKN/CX3CR1 plays important role in osteogenic transformation and calcification of VSMCs.Gradient FKN（0,10,30,50ng/ml）was added to the VSMCs cultured in osteogenic medium for 7 and 14 days,respectively.The osteogenic transformation in FKN-challenged VSMCs was subsequently detected.It was shown that FKN induced the osteogenic transformation of VMSCs in a dose dependent manner（reach peak at 50 ng/ml for 14 days’ treatment）,verified by red calcium node in Alizarin red staining,elevated OD value after decalcification and up-regulated protein and m RNA levels of osteogenic markers including OPN,BMP2 and ALP.In addition,the characters of VSMCs were simultaneously diminished,identified by decreased levels of α-SMA and SM22-α.Next,FKN induced osteogenic markers were further detected in VSMCs with CX3CR1 knockdown and overexpression,respectively.It was found that,compared with WT VSMCs,VSMCs with CX3CR1 knockdown displayed lower protein and m RNA levels of osteogenic markers（OPN,BMP2 and ALP）in response to FKN（50 ng/ml）stimulation.Whereas VSMCs with CX3CR1 overexpression showed enhanced levels of osteogenic markers on FKN（50 ng/ml）loading.These results provide supportive evidence that FKN/CX3CR1 exerts critical role in promoting VSMC osteogenic transformation.Activated RUNX2 and inhibited OPG contribute to the FKN/CX3CR1-regulated VSMC osteogenic differentiation and atherosclerotic calcification.We next sought to investigate whether osteogenic transcription factor,RUNX2,accounts for the osteogenic differentiation of VSMCs induced by FKN/CX3CR1.Expectedly,FKN increased RUNX2 protein and m RNA levels in VMSCs in a dose dependent manner（reach peak at 50 ng/ml for 14 days’ treatment）.Consistent with the increased RUNX2,RANK and RANKL displayed elevated protein and m RNA levels by FKN（50 ng/ml）（Fig.4B）.On the contrary,OPG,a well-established inhibitor of calcification,was down-regulated by FKN（50 ng/ml）. CX3CR1 knock-down VSMCs expressed less RUNX2 but more OPG than WT VSMCs in response to FKN（50 ng/ml）stimulation.Whereas CX3CR1 over-expression VSMCs expressed more RUNX2 and less OPG than WT VSMCs in response to FKN（50ng/ml）stimulation.Besides,consistent evidence was found in HFD-treated mice,which showed abundant expression of RUNX2,RANK and RANKL in aorta derived from Apo E-/-mice but not in that from Apo E-/-/CX3CR1-/-mice.RUNX2,RANK and RANKL were co-localized with α-SMA,suggesting an active osteogenesis in VSMCs.On the contrary,OPG showed an opposite expression in Apo E-/-and Apo E-/-/CX3CR1-/-mice.These results provide evidence that activated RUNX2,RANK/RANKL and inhibited OPG contribute to the FKN/CX3CR1-regulated VSMC osteogenic transformation and atherosclerotic calcification.Jak2/Stat3 signaling pathway mediates the FKN-induced osteogenic transformation of VSMCs.Jak/Stat is a well-known important signaling pathway mediating anabolic signals in osteoblasts and regulates osteogenic transformation.Our data showed that in cultured VSMCs,FKN（50 ng/ml）promoted the phosphorylation of Jak2 and Stat3,manifested by increased phosphorylated Jak2 and Stat3,which was further verified in VSMCs derived from Apo E-/-mice.However,in VSMCs from Apo E-/-/CX3CR1-/-mice,FKN failed to induce the phosphorylation of Jak2 and Stat3.These data suggest that FKN/CX3CR1 can activate Jak2/Stat3 signaling through promoting the phosphorylation of Jak2 and Stat3.In addition,inhibiting Jak2 using specific inhibitor AG490 largely impeded the FKN-induced RUNX2,OPN,BMP2 and ALP.These results indicate that Jak2/Stat3 signaling mediates the effect of FKN/CX3CR1 on promoting VSMC osteogenic transformation.Conclusions In summary,the present study found that FKN in atherosclerosis lesions binds to the membrane-specific receptor CX3CR1 on VSMCs and promotes the osteogenic transformation of VSMCs via two pathways.One is the activation of the Jak2/Stat3 signaling pathway,up-regulation of RUNX2（an initiation factor of the calcification）,and promotion of RANKL and other osteogenic marker protein expression.The second pathway is the inhibition of the anti-calcification protein OPG,thereby promoting the transformation of VSMCs into an osteogenic phenotype to participate in the formation of atherosclerotic calcification.Intervention measures targeting FKN/CX3CR1 may simultaneously inhibit inflammatory reactions and atherosclerotic calcification,thus providing new strategies for the prevention and treatment of atherosclerosis,atherosclerosis plaque stability,atherosclerotic calcification,and other cerebral or cardiovascular diseases. |
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