Hyperglycemia Drives Atherosclerotic Calcification Through PARP-1/Statl Axis

1.BackgroundAtherosclerotic calcification occurs in atherosclerotic plaques and is the most common calcific vascular disease.Under pathological stimuli such as inflammatory factors and oxidized lipids,the vascular cell subpopulations in atherosclerotic plaques undergo osteogenic differentiation.Atherosclerotic calcification is closely associated with plaque instability and is an independent risk factor for cardiovascular disease.In the diabetic microenvironment,there are general pro-calcific stimuli,such as inflammation,oxidative stress damage,high glucose and inorganic phosphate,which could promote the conversion of vascular smooth muscle cells and other vascular cells into osteoblast-like cells.Although in vitro studies have confirmed that high glucose could promote osteoblast differentiation of vascular cells,the molecular mechanism of accelerated atherosclerotic calcification in diabetes is still poorly understood,and there is still lack of corresponding in vivo studies.Runt-related transcription factors(Runx2/Cbfa1)plays a central role in the osteogenic differentiation of cells.Runx2 regulates the expression of osteocalcin and alkaline phosphatase by binding to osteoblast-specific element 2(OSE2)to regulate osteogenic differentiation.The expression of Runx2 is low in normal blood vessels,which is elevated in atherosclerotic calcific lesions both from human and mouse samples.In addition,studies have shown that diabetes increased Runx2 activity and accelerated aortic stiffness.In contrast,intervention against Runx2 has been shown to be beneficial in reducing the development of atherosclerotic calcification.PARP-1 is the most abundant subtype of PARP(poly ADP ribose polymerase)family and can participate in various pathophysiological processes such as DNA repair,gene transcription,and cell death.It plays an important role in various diabetic complications.Recent studies have reported that PARP-1 can negatively regulate bone resorption,and that inhibition of PARP can inhibit osteogenic differentiation of osteosarcoma cells and mouse mesenchymal stem cells.However,the relationship between PARP-1 and Runx2 expression remains unclear,and it is still unclear whether diabetes can accelerate atherosclerotic calcification by activating PARP-1.To explore the above issues,we bred ApoE-/-PARP-1-/-double knockout mice,established an animal model of diabetes and given high-fat diet to observe the effect of PARP-1 knockout on atherosclerotic calcification in diabetic mice and explore the potential mechanisms.2.Objectives(1)To observe the effect of diabetes on atherosclerotic calcification in high-fat diet-fed ApoE-/-mice;(2)To explore whether PARP-1 knockout can reduce atherosclerotic calcification in diabetes;(3)To explore the mechanisms by which PARP-1 knockout improves atherosclerotic calcification in diabetic mice through gene microarray analysis.3.Method(1)Establishment of animal modelsPARP-1-/-mice were crossed with ApoE-/-mice to obtain experimental ApoE-/-PARP-1-/-double knockout mice and littermates ApoE-/-mice.They were divided into high-fat group and diabetic high-fat group.The mice were rendered diabetic by daily intraperitoneal injections of STZ(Sigma)at a dose of 50 mg/kg for five consecutive days.We fed 12-week-old male ApoE-/-background mice high fat diet(HFD)for 12 weeks.Spontaneous atherosclerotic calcification can occur after 12 weeks of continuous high fat feeding.(2)Measurement of PWVPWV was measured via a modification of the transit time method using echocardiography with Vevo770 imaging system(Visual Sonics).Mice were anesthetized with continuous inhalation of 1-2%isoflurane.The curvilinear distance from the aortic arch to the abdominal aorta was measured(D2-D1;in mm)by the exact coordinates of the Doppler sample volumes.The time delay(T2-T1;in msec)was measured relative to the simultaneously recorded electrocardiogram signal.PWV was calculated by the formula(D2-D1/T2-T1),expressed as m/sec.(3)Alizarin Red stainingThe mouse aortic roots were fixed in 4%paraformaldehyde for 48 hours,then embedded in OCT and serially sectioned at 5μm.Briefly,frozen sections were incubated in the alizarin red staining solution at room temperature in the dark for 15 minutes.The sections were further washed in PBS for 3 times × 5 minutes,then dehydrated and observed under a microscope.Positive areas of alizarin red staining were observed and photographed under a microscope.The percentage of positively stained area for each aortic section was quantified using Image J software.(4)Determination of aortic calcium contentDissected aortas were briefly incubated at 70℃ for 30 min to obtain their dry weight and decalcified with 0.6 mM HC1 at 37℃ for 48 hr.The vascular calcium concentration was quantified using the Calcium Colorimetric Assay Kit(Sigma)and normalized by the dry weight of the aortas,which was expressed as fold change compared to control.(5)Detection of serum lipids,calcium,and phosphorusWe detect serum lipids,serum calcium and phosphorus in the laboratory of Qilu Hospital,Shandong University.(6)Immunohistochemical stainingFrozen sections were incubated with primary antibodies(rabbit anti-PARP-1 and anti-Runx2)overnight at 4℃.Sections were further washed and exposed to a second-ary antibody at 37℃ for 30 min.The antibody binding signals were visualized with diaminobenzidine.(7)Microarray AnalysisTotal RNA was extracted from the aortic tissues of diabetic ApoE-/-and diabetic ApoE-/-PARP-1-/-mice for microarray analysis.Microarray expression profiles were detected using the Affymetrix Gene Chip Mouse Genome 430 2.0 Array.Heatmap generation and Ingenuity Pathway Analysis were performed by Genechem Co.,Ltd.(8)Electrophoretic Mobility Shift Assay(EMSA)We performed EMSA to detect DNA-protein binding with a Light shift chemiluminescence EMSA kit(Thermo Fisher Scientific).The biotin-labeled Stat1 probe was added to different groups of nucleoproteins,and the loading buffer was added for further electrophoresis.The membrane was transferred to a nylon membrane,and then cross-linked for 15 minutes with a ultraviolet lamp.The ECL luminescent solution was used to detect the signal.4.Results(1)PARP-1 deletion had no significant effect on blood glucose,serum lipids,calcium,and serum phosphorus in ApoE-/-mice.After 12 weeks of diabetes,diabetic mice showed higher levels of blood glucose,total plasma cholesterol,and triglyceride,whereas PARP-1 deletion had no obvious effect on mice body weight,blood glucose,total cholesterol,or triglyceride levels.Diabetes and PARP-1 deficiency had no significant effect on serum calcium and serum phosphorus.(2)PARP-1 deletion reduced the calcification area of aortic root in the diabetic ApoE-/-miceWe found that HFD feeding for 12 weeks was sufficient to induce apparent arteriosclerotic calcification in the aortic roots of diabetic ApoE-/-mice.Compared with the HFD-fed mice,the proportion of calcium nodules in atherosclerotic lesions significantly increased in diabetic HFD-fed mice.However,the proportion of calcium nodules in the aortic roots of diabetic ApoE-*-PARP-1-/-mice was significantly reduced.In addition,the ratio of aortic root calcification in ApoE-/-PARP-1-/-mice was also significantly reduced compared to high-fat fed ApoE-/-mice.(3)PARP-1 deletion reduced aortic calcium content and vascular stiffness in the diabetic ApoE-/-miceWe found that the calcium content of aorta as well as PWV determined by echocardiography in the diabetic mice fed with high-fat feeding were significantly higher than that in the high-fat fed group.Increased aortic calcium content and aortic stiffness in diabetic ApoE-/-mice suggested that diabetes significantly accelerated vascular calcification in ApoE-/-mice.Compared with the diabetic ApoE-/-mice,PARP-1 deletion significantly reduced aortic calcium content and vascular stiffness in diabetic ApoE-/-mice.(4)PARP-1 deletion could reduce Runx2 expression in the aorta of diabetic ApoE-/-miceThe results of immunohistochemistry showed that the area of PARP-1 and Runx2 positive areas in the atherosclerotic lesions of diabetic ApoE-/-mice with high-fat diet was significantly higher than that of the high-fat diet group.The area of Runx2 positive aortic root was significantly reduced in diabetic ApoE-/-mice with PARP-1 deletion.Further RT-PCR results showed that the mRNA level of the aortic Runx2 was significantly increased in the diabetic high-fat fed group compared with the high-fat fed group,and PARP-1 deletion significantly inhibited Runx2 mRNA levels.(5)PARP-1 deletion attenuated diabetic atherosclerotic calcification by targeting osteogenic differentiation-associated transcription factorsA genome-wide microarray assay of the aorta was performed to profile differentially expressed genes that might be responsive to PARP-1 deletion.A heatmap was used to illustrate the expression profile of genes that were differentially expressed in diabetic ApoE-/-mice compared with ApoE-/-PARP-1-/-mice.Among these genes,TRAIL and ESRRG(encoding ERRy)were associated with arteriosclerotic calcification.In addition,several candidate genes including ATF3,TBX3 and ELOVL6 were associated with osteogenic differentiation.Notably,our results indicated that PARP-1 acted on the genes for transcription factors(TFs)including STAT1,ATF3,EGR1,EGR2,EGR3,ID4,NR4A1 and TBX3.Protein-protein interaction networks highlighted the osteogenic genes downstream of these targeted TFs,which may explain the underlying mechanism of PARP-1 deletion-attenuated atherosclerotic calcification.(6)PARP-1 deletion could inhibit transcriptional activity of StatlIt has been reported that transcription factor Statl can negatively regulate the expression of Runx2 in osteoblasts.Our results of EMSA showed that diabetes upregulated the transcriptional activity of Stat1 in the aortic tissues,while the elevated transcriptional activity of Statl in the diabetic aortic tissues were hindered by PARP-1 deletion.It is suggested that the mechanism of Statl regulating Runx2 in vascular tissues may be different from that of osteoblasts.Diabetes activates Statl/Runx2 expression in the aortic tissues of ApoE-/-mice,while PARP-1 deletion inhibits this activity.5.Conclusion(1)Increased Statl/Runx2 activity,aortic root calcification,aortic calcium content and aortic stiffness were found in the aorta of high-fat fed diabetic ApoE-/-mice,which suggested that diabetes accelerated vascular calcification in ApoE-/-mice.(2)PARP-1 deletion reduced the calcification area of aortic root,reduced the aortic calcium content and aortic stiffness in the diabetic ApoE-/-mice.(3)PARP-1 deletion inhibited Statl/Runx2 activity and reduced atherosclerotic calcification in diabetic ApoE-/-mice.1.BackgroundVascular calcification refers to the pathological deposition of calcium minerals in the extracellular matrix of the arterial wall and is closely related to cardiovascular morbidity and mortality.Vascular calcification is mainly divided into medial calcifycation and intimal calcification,and intimal calcification is usually associated with atherosclerosis,also known as atherosclerotic calcification.However,the transforma?tion of vascular smooth muscle cells(VSMCs)into osteoblast-like cells are the main reason for both intimal calcification and medial calcification.When VSMCs transfor?med into osteoblast-like cells,the activity of alkaline phosphatase(ALP),and the expression of Bone Morphogenetic Protein-2(BMP-2),Osteopontin(OPN)and osteo?calcin(OCA)increases.At the same time,VSMCs release matrix vesicles and become the place where calcium salts and phosphates accumulate,eventually leading to the occurrence of calcification.VSMCs in normal vascular tissues are in a contrac?tile phenotype,expressing a series of contractile proteins such as a-SMA,SM-22 a,calponin,and smoothoelin,to maintain the structure and function of the arterial wall.However,VSMCs are not terminally differentiated cells and can be converted from a contractile phenotype to a synthetic phenotype under stimulation of high glucose,high phosphorous,oxidized low-density lipoprotein,and glycosylation end products.Dur?ing the process,the contractility of VSMCs is decreased,accompanied by increased expression of osteogenic ALP and OPN,and finally become osteoblast-like cells with secretory function.High glucose,inflammation,oxidative stress and inorganic phosphate in the diabe?tic microenvironment can promote the conversion of VSMCs to osteoblast-like cells and thus accelerate vascular calcification.Although in vitro studies have shown that high concentrations of glucose can promote osteogenic differentiation of VSMCs? the exact molecular mechanisms are still not fully understood and there are no corresponding interventions yet.In the process of osteogenic differentiation,Runt-related trans?cription factor 2(Runx2)plays a central role.Runx2 can regulate the expression of OCA and ALP by binding to OSE2 to regulate osteogenic differentiation.The use of gene knockout and other measures to inhibit the expression of Runx2 in VSMCs has also been shown to reduce osteogenic differentiation and calcification of VSMCs.Recent studies have reported that PARP inhibitors can inhibit the expression of Runx2 and Bmp2,and thus inhibit osteogenic differentiation in both bone marrow mesenchy?mal stem cells and osteosarcoma cells.However,the intrinsic relationship between PARP-1 and Runx2 remains unclear,and whether inhibition of PARP-1 could reduce osteogenic differentiation of VSMCs needs to be clarified.We have also confirmed in the first part of the study that the upregulation of Statl transcriptional activity as well as Runx2 expression in the diabetic aortic tissues,which may be an important molecu?lar mechanism for the accelerated atherosclerotic calcification in diabetes.Knockout of PARP-1 inhibits transcriptional activity of Statl and Runx2 expression.Therefore产whether high glucose accelerates osteogenic differentiation of VSMCs through the Statl/Runx2 axis? Whether knockout of PARP-1 in VSMCs attenuate high glucoseinduced osteogenic differentiation by suppressing Statl/Runx2 axis? Based on the above problems,we extracted the VSMCs from mice in this study,and cultured with ’high-glucose osteogenic medium to observe the effect of PARP-1 knockout on the osteogenic differentiation of VSMCs.At the same time,we investigate whether high"glucose accelerates osteogenic differentiation of VSMCs through PARP-1/Statl axis.2.Objectives(1)To observe the effect of high glucose on osteogenic differentiation of VSMCs;(2)To investigate whether knockout of PARP-1 can alleviate high glucose-induced osteogenic differentiation by inhibiting Statl/Runx2;(3)To explore the molecular mechanism of Statl regulating Runx2 expression and osteogenic differentiation.3.Method(1)Extraction,culture and identification of mouse primary VSMCsSix-week-old mice were sacrificed.The aorta was excised to small pieces under a stereo microscope.A small amount of medium was added and gently applied to the culture flasks.Smooth muscle cells would be seen after 3 days of culture,climbing out from the vascular tissue mass.The extracted smooth muscle cells were seeded on cell slides.Then immunofluorescent a-SMA staining was performed to identily the extracted smooth muscle cells.(2)Establishment of calcification model in vitroAllocation of osteogenic medium: DMEM medium was supplemented with 10%fetal bovine serum and 10 mM P-phosphoglycerol.The above mentioned VSMCs(at passage 3-5)were seeded in 6-well plates,cultured with osteogenic medium contain?ing 5.5 mM glucose(NG)or osteogenic medium with 27.5 mM glucose(HG).And medium was changed every 3 days.(3)Alizarin Red staining,Von Kossa staining,and calcium determinationAfter the VSMCs were cultured for a predetermined time,the medium was discard?ed,washed with PBS and fixed with 4% paraformaldehyde.For Alizarin Red staining,add Alizarin Red dye to the culture plate,incubate at room temperature in the dark for15 minutes,rinse with PBS,and take a photograph with the camera.When staining with Von Kossa,5% silver nitrate solution was added to the culture plate and exposed to ultraviolet light for 15 minutes.Then add 5% sodium sulfite solution,wash with PBS,and take a picture.When measuring the calcium content of the cells,an equa]amount of lysate was added to each well to lyse the cells,and the protein concentra?tion of each group was measured according to the Sigma calcium assay kit(colorime?tric method).Subsequent operations were performed and the calcium concentration was corrected for protein concentration.Results were expressed in ^ig/mg protein.(4)Real-time quantitative RT-PCRThe Trizol method was used to extract RNA from each group.Reverse transcript-tion and RT-PCR were performed to determine the mRNA levels of Runx2,OCA and ALP.(5)Western BlotCells were harvested,and RIPA lysates were used to extract total cellular protein.Western Blot was used to detect the expression of PARP-1,Statl,Runx2,a-SMA,OPN and Vimentin in each group.(6)TransfectionThe cells were evenly plated and transfected when the cell density grew to about40%.Adenovirus encoding murine wild-type Statl(Ad-Statl)or control virus(AdNC)was transduced into VSMCs at a multiplicity of infection(MOI)of 10.After 24 hr,virus-containing medium was replaced with fresh complete medium.For siRNA transfection,take two sterile EP tubes and add 250 Opti MEM+5 \iL Lipo3000 and 250 μL Opti MEM+100 pmol siRNA respectively,mix and add it to the culture well.After 6 h,Opti MEM was replaced with fresh complete medium.(7)Luciferase Activity AssayThe constructed wild-type and mutant reporter plasmids were co-transfected with pRL-TK plasmid into 293 cells using Lipo3000 and P3000,respectively.The pGL3-Basic plasmid was used as a negative control.After 48 hours,cells were harvested and luciferase activity was detected with Luciferase Activity Assay kit.(8)Chromatin Immunoprecipitation Assay(ChlP)The cells were fixed with 4% paraformaldehyde,the cells were collected and disrupted by sonication.The Input group,Statl antibody group,and IgG antibody group were incubated overnight.Then the beads were added and the precipitated complexes were washed and cross-linked overnight at 65°C.The DNA fragments were subjected to PCR amplification.The products were subjected to agarose gel electrophoresis and the results were analyzed.4.Results(1)Knockout of PARP-1 inhibited high glucose-induced VSMCs calcificationWe found that VSMCs showed obvious calcification after cultured with osteogenic medium for 3 weeks,especially in osteogenic medium with high glucose.Deletion of PARP-1 inhibits high glucose-induced VSMCs calcification.At the same time,the calcium content of VSMCs cultured in high glucose osteogenic medium increased significantly,but the knockout of PARP-1 inhibited the increment of calcium content.(2)Knockout of PARP-1 inhibited the expression of key genes involved in osteogenic differentiation of VSMCs induced by high glucoseWe found the mRNA levels of the key genes for osteogenic differentiation such as Runx2,ALP,and OCA were significantly higher in hyperglycemia-stimulated VSMCs.The absence of PARP-1 inhibited the expression of these genes induced by high glucose.Western blot showed that high glucose stimulation significantly up-regulated Statl/Runx2 protein expression,whereas knockout of PARP-1 inhibited Statl/Runx2 expression.This is consistent with the first part of the study.(3)Knockout of PARP-1 inhibited the high glucose-induced VSMCs transformation from a contractile phenotype to a synthetic phenotypeWestern blot results showed that high glucose stimulation can inhibit the express?ion of contractile protein a-SMA,and promote the expression of synthetic-related proteins OPN and Vimentin,indicating that high glucose drives the transformation of VSMCs into osteoblast-like cells with secretory function.However,in the VSMCs with PARP-1 knockout,the contractile protein a-SMA did not show a significant decrease even when stimulated by high glucose,but there was no significant increase in the synthetic protein OPN or Vimentin.(4)Knockout of Statl reduced high glucose-induced VSMCs calcificationWe used Statl-flox mice and Acta2-Cre mice to obtain smooth muscle cell-specific Statl deletion mice,and then cultured primary mouse smooth muscle cells with high glucose osteogenic medium.After 3 weeks,it was found that the wild type VSMCs showed significant calcification,whereas the Statl knockout VSMCs didn’t show significant calcification.At the same time,the calcium content of VSMCs with Statl knockout was significantly reduced.(5)Statl knockout inhibited high-glucose induced expression of osteogenic differentiation related genes in VSMCsKnockout of Statl significantly inhibited high glucose-induced expression of osteogenic differentiation related genes such as Runx2,ALP and OCA.Western blot results also showed that Statl knockout significantly inhibited Runx2 protein express?ion.Statl knockout did not significantly affect PARP-1 protein expression,indicating that Statl is downstream of PARP-1,and PARP-1 could inhibit Runx2 expression through Statl.These results also confirmed the hypothesis of Statl/Runx2 axis in the first part of this study.Statl positively regulates the expression of Runx2 in vascular tissues,which is different from the negative regulation in osteoblasts.(6)Knockout of Statl inhibited the high glucose-induced VSMCs transformation from a contractile phenotype to a synthetic phenotypeUnder high glucose osteogenic medium stimulation,compared with the wild-type VSMCs,the Stat 1-knockout VSMCs had higher contractile protein a-SMA,while the synthetic proteins OPN and Vimentin decreased significantly.(7)Overexpression of Statl upregulated Runx2 and aggravated VSMCs calcificationOverexpression of Statl significantly up-regulated the expression of Runx2.Two weeks after cultured in high glucose osteogenic medium,both Alizarin Red and Von Kossa staining showed that Statl overexpression significantly aggravated the calcifi?cation of wild-type VSMC,and the calcium content also increased significantly.(8)Statl directly bound to the Runx2 promoter and regulated its expressionWe found that Statl siRNA treatment significantly inhibited the activity of the wild-type Runx2 luciferase reporter plasmid.However,mutation of 5’-TCTCCAG-TAAT-3’ significantly decreased Runx2 promoter activity,indicating that this she is the core region of Runx2 prcmioter activity.However,Statl siRNA treatment didn5 t affect the luciferase activity of the mutant plasmid.These data suggested that Statl may transcriptionaUy regulate Runx2 expression through this binding site.ChIP assay was performed with specific primers overlaying the Runx2 promoter region5f-TCTCCAGTAAT-3’ and the results confirmed that Statl specifically binds to the5’-TCTCCAGTATAAT-3f region of the Runx2 promoter.5.Conclusion(1)High glucose stimulation can accelerate the calcification of VSMCs and increase the calcium content,which were reversed by PARP-1 knockout.(2)Knockout of PARP-1 attenuates high glucose-induced VSMCs calcification by inhibiting Statl/Runx2 axis.(3)Knockout of PARP-1 inhibits high glucose-induced VSMCs transformation from a contractile phenotype to a synthetic phenotype by targeting Statl.(4)Statl can bind directly to the core region of the Runx2 promoter and transcription?ally regulates its expression.1.BackgroundWhen atherosclerotic lesions progress to a certain extent,calcification can occur within the plaque,known as atherosclerotic calcification.Atherosclerotic calcification is an important independent risk factor for cardiovascular morbidity and mortality.Especially in patients with diabetes,the risk of atherosclerotic calcification is greatly increased.Atherosclerotic calcification is formed by the osteoblast-like differentiation of vascular cells under pathological stimuli.However,the origin of atherosclerotic calcification is not clear,and the role of macrophages in atherosclerotic calcification remains controversial.Previous studies using in vitro co-culture methods found that macrophages enhanced the calcification of vascular smooth muscle cells(VSMCs)by releasing pro-inflammatory cytokines.Although most of the calcified cells in atheros?clerotic plaques originate from VSMCs,genetic lineage tracing techniques have found that nearly 20% of Runx2 positive cells in atherosclerotic plaques are derived from bone marrow derived cells.Recent studies have shown that in addition to VSMCs,macrophages can also release matrix vesicles(MVs)for the accumulation of calcium salt and phosphate,leading to the occurrence of calcification.These studies indicated that macrophages may be directly involved in the occurrence of atherosclerotic calcification.Phenotype switch of macrophages may also influence the progression of atheroscle?rotic calcification.Studies have reported that gene expression profiles of macrophages that contribute to osteogenic differentiation are significantly positively correlated with Ml macrophage,but negatively correlated with M2 macrophage.When stimulated by high glucose,CD1 lc,iNOS and TNF-a expression in macrophages were up-regulated,while the expression of Arg-1 was down-regulated,which promoted the conversion to Ml phenotype.However,whether high glucose can promote macrophage differentia?tion into osteoblast-like cells has not been reported.Runt-related transcription factor 2(Runx2)plays a central role in osteogenic differentiation.Runx2 inhibition in VSMCs has been shown to reduce osteogenic differentiation and calcification.However,it is unclear whether high glucose can aggravate osteogenic differentiation and calcifica?tion by upregulating Runx2.Recent studies have reported that PARP-1 can bind to the promoter of TRAP and thus regulate bone resorption and osteoclast activity.PARP inhibition can prevent osteogenic differentiation of mouse mesenchymal stem cells and osteosarcoma cells.We have also confirmed in the second part of the study that PARP-1 deficiency in VSMCs can reduce high glucose-induced osteogenic different?iation and calcification by inhibiting Statl/Runx2 axis.However,it is still unclear whether similar mechanisms exist in macrophages.To answer these questions,we used primary macrophages and RAW264.7 macro?phages in this study,cultured in osteogenic medium and stimulated with normal or high glucose to observe the effect of PARP-1 inhibition.We also investigated whether high glucose accelerated the osteogenic differentiation of macrophages through the PARP-l/Statl axis.2.Objectives(1)To observe whether macrophages could undergo osteogenic differentiation and calcification in osteogenic medium;(2)To observe the effect of high glucose on the osteogenic differentiation of macrophages;(3)To investigate whether PARP-1 deficiency can reduce the high glucose-induced macrophage osteogenic differentiation by inhibiting Statl/Runx2;(4)To investigate whether PARP-1 regulates macrophage polarization through Stat]signaling.3.Method(1)Extraction,culture and identification of mouse primary peritoneal macrophages8-week-old mice were injected intraperitoneally with 3% thioglycollate.After 72 hours,the mice were sacrificed.The abdomen was cleaned and the abdomen skin was disinfected to expose the abdominal muscles.Inhale the DMEM medium into the abdominal cavity with a sterile pipette.After gently sucked the suspension into the centrifuge tube,centrifuged at lOOOrpm for 5 minutes,and then resuspended it in 10%DMEM and cultured in dishes.Extracted macrophages were labeled with APC-labeled anti-mouse F4/80 antibody and the positive rate was measured by flow cytometry.(2)Establishment of calcification model in vitroAllocation of osteogenic medium: DMEM medium was supplemented with 10%fetal bovine serum and 10 mM P-phosphoglycerol.The above mentioned VSMCs(at passage 3-5)were seeded in 6-well plates,cultured with osteogenic medium contain?ing 5.5 mM glucose(NG)or 27.5 mM glucose(HG).And medium was changed every 3 days.(3)Alizarin Red staining and calcium determination(4)Real-time quantitative RT-PCRThe Trizol method was used to extract RNA from each group.Reverse transcript-tion and RT-PCR were performed to determine the mRNA expression of Runx2,OCA and ALP.(5)Western blotCells were harvested and RIPA lysates were used to extract total cellular protein.Western Blot was used to detect the expression of PARP-1,Statl,p-Statl,Runx2,Argl,and iNOS in each group.(6)Measurement of Dil-ox-LDL UptakePeritoneal macrophages were plated in dishes and incubated with Dil-ox-LDL for 4hours.Then,fluorescence imaging was performed by laser-scanning confocal micro?scopy.(7)Flow cytometryAfter exposure to osteogenic medium,macrophages were co-labeled with PE antiCD 1 lc and APC anti-CD206 antibodies(eBioscience).Then the cells were examined using a FACS Calibur system(BD Biosciences)and data analyzed using Flow Jo software.4.Results(1)Knockout of PARP-1 inhibited high glucose-induced macrophages calcificationWe found that calcification was evident in macrophages cultured in osteogenic medium,whereas calcification in high-glucose osteogenic medium was more pronounced.Knockout of PARP-1 inhibits high glucose-induced macrophages calcification.At the same time,calcium content in macrophages cultured in highglucose osteogenic medium was significantly increased compared to normal osteogenic medium,and knockout of PARP-1 inhibited the increment in calcium content A similar phenomenon was also found in RAW264.7 macrophages(2)Knockout of PARP-1 can inhibit the expression of key genes involved in macrophages osteogenic differentiation induced by high glucoseThe mRNA levels of Runx2,alkaline phosphatase and osteocalcin in macrophages were significantly increased under high glucose stimulation,and knockout of PARP-1inhibited the high glucose-induced expression of these key genes for osteogenic differentiation.Simultaneous,Western blot results also showed that PARP-1 knock?out inhibited high glucose-induced Runx2 expression.The PARP-1 inhibitor PJ34 was also found to inhibit high glucose-induced Runx2 expression in RAW264.7macrophages.(3)Knockout of PARP-1 can inhibit high glucose-induced macrophages polarizationThe expression of CDllc and iNOS in macrophages of wild-type mice was increased under high glucose stimulation,while the expression of anti-inflammatory phenotype markers CD206 and Arg-1 was significantly decreased,indicating that high glucose stimulation can promote macrophages to Ml type transformation.Knockout of PARP-1 can inhibit this transformation.At the same time,high glucose can up-regulate phosphorylated Statl and total Stall expression,but PARP-1 knockout can reverse this activity.In RAW264.7 macrophages,it was also found that the CD206 expression of macrophages cultured in osteogenic medium was reduced,indicat-ing that macrophages transformed to M2 type were decreased during osteogenic differentiation.And high glucose stimulation could promote this conversion,while this process was reversed by treatment of PARP inhibitor PJ34.(4)PARP-1 knockout can inhibit endocytosis of Dil-labeled ox-LDL in macrophagesCompared to wild-type macrophages,PARP-1 knockout significantly attenuated the endocytosis of Dil-labeled ox-LDL.(5)Conditioned medium from PARP-1 knockout macrophages didn’t exacerbate VSMCs calcificationThe VSMCs treated with wild-type macrophage medium showed obvious calcifica?tion,but the VSMCs treated with PARP-1 knockout macrophage culture medium had lower Runx2 expression and decreased calcification.(6)Knockout of Statl inhibited high glucose-induced macrophages calcificationStatl knockout macrophages cultured in high-glucose osteogenic medium didn5 t show apparent calcification.At the same time,the calcium content of Statl knockout macrophages was significantly reduced.It indicated that knockout of Statl can inhibit high glucose-induced calcification of macrophages,whereas PARP-1 knockout can inhibit macrophage calcification through the Statl/Runx2 axis.(7)Knockout of Statl can inhibit the expression of key genes involved in high-glucose-induced macrophage osteogenic differentiationKnockout of Statl significantly inhibited high glucose-induced Runx2,alkaline phosphatase,and osteocalcin mRNA levels.Further western blot also showed that knockout of Statl significantly inhibited Runx2 protein expression in macrophages.It also showed that knockout of PARP-1 in macrophages inhibited the expression of key genes involved in osteogenic differentiation through Statl.(8)Knockout of Statl can inhibit high glucose-induced macrophage polarizationKnockout of Statl can inhibit the expression of CD11 c and iNOS,and p...