| BackgroundVascular calcification,the ectopic deposition of calcium and phosphate crystals in the blood vessel wall,is an important complication of chronic kidney disease(CKD),which will increase the morbidity and mortality of cardiovascular diseases.Vascular calcification is mainly divided into intima calcification,media calcification and adventitia calcification,among which the media calcification is most likely to occur in patients with CKD,and the vascular calcification induced by CKD has become an independent risk factor for cardiovascular diseases.The mechanism of vascular calcification involves the absence of inhibitors of vascular calcification,the increase of marker molecules of osteoblast/osteoclast,the composition and content changes of extracellular matrix and matrix hydrolase,and autophagy.Vascular calcification is an active,cell-regulated osteogenic process,in which vascular smooth muscle cells(VSMCs)play a key role.Long-term exposure to high levels of mineral ions in pathological environment can lead to osteogenic differentiation of VSMCs,namely the contraction markers of VSMCs including alpha smooth muscle actin(α-SMA)and smooth muscle 22 alpha(SM22α)decrease,while the secretory markers of VSMCs including Runt related transcription factor 2(RUNX2),muscle segment homeobox(MSX2)and osteopontin(OPN)increase,which is the core link of vascular calcification.Currently,there are no drugs to inhibit osteogenic differentiation of VSMCs and delay vascular calcification.Therefore,it is urgent to further study the mechanism of vascular calcification and effective targets in its progression.Oxidative stress is common in patients with chronic kidney disease(CKD).There is an imbalance of oxidation-antioxidant system in the early stage of CKD,which can be aggravated with the progression of renal failure.Oxidative stress can promote the osteogenic differentiation of VSMCs.During oxidative stress,polyunsaturated fatty acid(PUFA)is attacked by reactive oxygen,resulting in lipid peroxidation,which produces large amounts of toxic reactive carbonyl compounds such as 4-hydroxynonenal(4-HNE),malondialdehyde(MDA)and acrolein.However,the effect of lipid peroxide derivatives on vascular calcification has been poorly studied.4-HNE is the main end product of lipid peroxidation and is the most toxic reactive carbonyl compound.Its role in cell proliferation,cell death and other cell behaviors has been fully studied,but the relationship between 4-HNE and vascular calcification remains unclear.Aldehyde dehydrogenase 2(ALDH2)is the key enzyme in alcohol metabolism,and is the main metabolic enzyme of 4-HNE.The mutation of ALDH2 gene at rs671 site(glutamate to lysine)leads to the decrease of its enzyme activity,causing the accumulation of toxic aldehydes,and producing facial flushing after drinking alcohol.Therefore,ALDH2 is also known as the "blush gene".It is reported that subjects who blushed after drinking alcohol had higher levels of coronary artery calcification than those who did not.In addition,ALDH2 has been confirmed to be closely related to a variety of cardiovascular diseases such as aortic dissection,atherosclerosis and pulmonary hypertension,and its mechanism involves phenotypic transformation and proliferation of VSMCs.However,it is unclear whether ALDH2 is involved in the development of vascular calcification.In order to clarify the effect of ALDH2 and 4-HNE on vascular calcification and explore the potential mechanisms,we have constructed ALDH2 knockout(ALDH2-KO),ALDH2 VSMCs conditioned knockout(ALDH2SMKO),and ALDH2 transgenic(ALDH2-Tg)mice,,established CKD induced vascular calcification model and vitamin D3(Vit D3)induced vascular calcification model in vivo and extracted mouse primary VSMCs.Objectives1.To clarify the role of ALDH2 in the occurrence and development of vascular calcification.2.To reveal that 4-HNE is involved in vascular calcification by regulating phenotypic transformation of VSMCs.3.To explore the specific molecular mechanism of ALDH2 regulating phenotypic transformation of VSMCs.Methods1.Patients and sample processingAll procedures involving human samples and information complied with the principles outlined in the Declaration of Helsinki and were approved by Qilu Hospital of Shandong University.Non-CKD and CKD patients.71 healthy persons(non-CKD)and 80 patients diagnosed with chronic kidney disease(CKD)admitted to Qilu Hospital of Shandong University.Clinical and biochemical parameters were collected from the electronic medical record in the hospital.Patient sera were collected for detection of 4-HNE and 8-iso-PGF2α.The diagnosis of CKD was based on eGFR decline present for 6 months or longer.CKD stage 3 to 5 was defined as an estimated GFR<60 mL/min per 1.73 m2 among study participants before receiving dialysis following the Kidney Disease Outcomes Quality Initiative(KDOQI)guidelines.Patients were excluded for the following:(a)age<18;(b)pregnant;(c)administration of any medication as phosphate binders or vitamin D analogs within 3 months before recruitment;(d)acute renal failure.Non-calcification and calcification patients.253 patients underwent coronary CT angiography in Qilu Hospital of Shandong University were recruited in our study to evaluate the association between coronary artery calcification and ALDH2 mutation.Patients were excluded for the following:(a)deletion of ALDH2 genotype results;(b)old myocardial infarction;(c)chronic kidney disease;(d)history of intracoronary stent implantation or coronary artery bypass grafting.Calcification scores of images,clinical and biochemical parameters were collected from the electronic medical record in the hospital.Patient sera were collected for detection of ALDH2 genotype.2.Animal Models2.1 Vitamin D3 induced vascular calcification model.Six-to seven-week-old male ALDH2-Tg,ALDH2-KO,ALDH2SMKO and corresponding WT mice were injected subcutaneously with 500,000 IU/kg body weight of vitamin D(Vit D3,Sigma-Aldrich)or vehicle for 3 days as previously described[5].For Alda-1 experiments,mice were injected intraperitoneally with 20 mg/kg body weight of ALDH2 specific activator Alda-1 or vehicle every two days.After 6 days of treatment,mice were sacrificed,blood was collected,and tissues were snap-frozen in liquid nitrogen.2.2 Chronic kidney disease induced vascular calcification model.Six-to seven-week-old male ALDH2-Tg,ALDH2-KO,ALDH2SMKO and corresponding WT mice were anesthetized by inhalation of sevoflurane.Then,the upper and lower poles of the left kidney were resected.One week later,the right kidney was then removed completely.Sham group mice underwent the same surgery over the same period as the 5/6 nephrectomy mice,but only the renal capsule was stripped after exposure of the kidney and then the abdomen was closed.To accelerate the process of aortic calcification,animals were fed with a high-phosphate diet(0.9%Pi)after completing renal ablation for the duration of the study.The uraemia mouse model was set up successfully after twelve weeks,which was confirmed by a dramatic increase level of serum urea and creatinine compared with the sham group.Finally,the mice were sacrificed,blood was collected,and tissues were snap-frozen in liquid nitrogen.3.Induction of vascular calcification in vitro.Primary aortic smooth muscle cells(VSMCs)were isolated from the aortas.10 mMβ-glycerophosphate and 1.5 mM CaCl2 were used as calcification media for von Kossa or for Alizarin red S staining,respectively.Fresh media with agents were added every 2-3 days for 14 days.4.Alizarin red or von Kossa StainingDetermination of calcium deposition by calcium staining.mVSMCs in 6-well plates were fixed in 4%formaldehyde for 15 min at room temperature.For Alizarin red staining,vascular tissues sections or VSMCs were exposed to 2%Alizarin red S for 10 min and washed with double distilled H2O.Positively stained cells showed a reddish/purple color.For von Kossa staining,vascular tissues sections or VSMCs were incubated with silver nitrate solution and exposed to ultraviolet light for 30 min,washed with double distilled H2O.Calcified nodules were stained brown to black.5.Western BlotTissue or cell lysates with the same protein content(determined by the BCA method)were prepared.Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)and transferred to 0.2μ m polyvinylidene difluoride(PVDF)membrane.The membranes were incubated with primary antibodies overnight at 4℃.After being washed,the membranes were incubated with corresponding horseradish peroxidase(HRP)-coupled secondary antibodies.Blots were visualized by chemiluminescence reagents and were analyzed with ImageJ software.6.Histological analysisThe entire aorta was fixed with 10%formalin for 24 hours at room temperature before being embedded in paraffin for sectioning.Tissues were sectioned at 5 μm and underwent HE staining and immunohistochemical staining.Immunohistochemical staining was carried out with the commercial kit and primary antibody was 4-HNE.All images were captured using a microscope.7.Quantitative real-time PCR(RT-qPCR)The total RNA was isolated using TRIzol,then cDNA was synthesized using the commercial kit following the instructional manual.RT-qPCR was performed using the Takara kit on the ABI7500 Real-time PCR System.The relative expression of each gene was normalized to the β-actin gene and analyzed with 2-ΔΔCT method.8.Immunoprecipitation AnalysisFor immunoprecipitation(IP),cells were lysed with IP lysis buffer.Primary antibody was coupled with protein A/G beads,and immune complex was added to the cell lysates and incubated at 4℃ overnight.After IP,the samples were washed with IP lysis buffer 3 times.Proteins were eluted with 2× SDS sample buffer.9.Luciferase AssayTo establish the p6xOSE2-luc-stably transfected cell line,C2C12 cells were cotransfected with p6xOSE2-luc reporter vector and pcDNA3.0 in a 5:1 ratio.Stably transfected clones were selected in media containing 200 μg/ml of Hygromycin B Gold.Individual Hygromycin B Gold-resistant clones were isolated by using separation rings,transferred,and subcultured.Stable cell clones were screened by luciferase assay.For transient transfection assays,1×105 cells were plated per well in 6 well plates and transfected with RUNX2 aa 1-521 plasmid 24 h after seeding with 0.5 μg of p6xOSE2-luc or 0.5 μg of other expression vectors.4-HNE was treated 12 h before luciferase assay.After 24-48 h of transfection,cells were harvested and assayed for luciferase activity using Luciferase Reporter Assay kit according to the manufacturer’s instructions.Results1.4-HNE is increased during vascular calcification.Vascular calcification is very common and accelerated in CKD patients.To explore the relationship between 4-HNE and vascular calcification,we first tested the level of 4-HNE in serum of CKD patients.Increased 4-HNE was detected in serum of CKD stage 3-5 patients compared with that of non-CKD subjects.8-epi-PGF2α,which has been viewed as the most reliable biomarker of lipid peroxidation,was also increased in serum of CKD patients.Moreover,more 4-HNE was immunostained in coronary calcified atherosclerotic plaques than that in noncalcified plaques.To further verify the change of 4-HNE during vascular calcification,a mouse vascular calcification model with CKD which induced by 5/6 nephrectomy and high-phosphorus diet was established.Obvious calcification in aortas were stained by alizarin red and von Kossa staining.Similar to CKD patients,elevated 4-HNE was produced in serum of CKD mouse.Also,4-HNE was significantly increased in calcified mouse aortas.2.ALDH2 deficiency accelerates the development of vascular calcification.As ALDH2 is the key enzyme of metabolizing 4-HNE,ALDH2 knockout(ALDH2-KO)mice were used to investigate the effect of 4-HNE on vascular calcification in vivo.After vitamin D3 treatment,calcium deposition was significantly increased in aortas of ALDH2-/mice compared with that of control mice.Meanwhile,more 4-HNE was immunostained in aortas of ALDH2-KO mice.Using the CKD mouse model,we confirmed that vascular calcification and 4-HNE was more stained in aortas of ALDH2-KO mice.To further elucidate the role of ALDH2 in vascular calcification,ALDH2SMKO mice was constructed and exposed to vitamin D3 for 3 days.Expectedly,ALDH2SMKO mice developed much more severe calcification in aortas than control mice.Also,4-HNE was increased in aortas of ALDH2SMKO mice.Similar results were found using the CKD mouse model.These results indicated that ALDH2 in VSMC played a critical role in vascular calcification by detoxifying 4-HNE.3.ALDH2 Glu504Lys mutation is positively associated with coronary calcification in patients.A G-to-A missense mutation at exon 12(rs671)in ALDH2 gene results in substitution of glutamate(Glu)by lysine(Lys)(Glu504Lys).In the carriers of the variant ALDH2 homozygous and heterozygous genotypes,the ALDH2 activity is significantly lost,and nearly 0%and 17-38%of the normal activity respectively22.To confirm the potential effect of 4-HNE on vascular calcification,we analyzed whether ALDH2 rs671 polymorphism has correlation with coronary calcification.A total of 253 consecutive CAD patients from Qilu Hospital of Shandong University were recruited from Nov.9th,2017 to Mar.31st,2019.To reduce the selection bias,a greedy propensity score matching strategy was used and 146 CAD patients were included in the final analysis.More ALDH2 rs671 polymorphism occurred in patients with calcified coronary(24/73,32.9%)than that in patients with noncalcified coronary(19/73,26.0%).4.ALDH2 attenuates the severity of vascular calcification.Regarding the critical role of ALDH2 in detoxifying 4-HNE,we next determined the effects of overexpressing or activating ALDH2 on vascular calcification.Using ALDH2transgenic(ALDH2-Tg)mice,we found that calcium deposition and 4-HNE was obviously reduced in ALDH2-overexpressed aortas.To confirm the effects of overexpressing ALDH2 on vascular calcification,the primary mouse aortic smooth muscle cells(mVSMCs)were isolated and culture from ALDH2-Tg and control mice.Overexpression of ALDH2 effectively suppressed mASMCs calcification and expression of osteochondrogenic markers(RUNX2,BMP2,OPN,and MSX2),accompanied by increased contractile marker(α-SMA).Moreover,less 4-HNE was produced in ALDH2-Tg mASMCs under β-GP and CaCl2 stimulation.Activating ALDH2 using intraperitoneally injecting Alda-1 also suppressed calcium deposition and 4-HNE accumulation in mouse aortas.In vitro experiments confirmed that ALDH2 activation prevented cell calcification,expression of osteochondrogenic markers and production of 4-HNE in β-GP+CaCl2 treated mVSMCs.5.4-HNE promotes osteochondrogenic differentiation of VSMCs.To confirm the in vivo results.mVSMCs and primary human aortic smooth muscle cells(hVSMCs)cells were treated with 4-HNE.mRNA sequencing was conducted with mVSMCs stimulated by 4-HNE,and GO analysis results showed that differential genes were enriched in osteogenic pathway.4-HNE treatment enhanced calcium deposition in mVSMCs under β-GP and CaCl2 stimulation.The smooth muscle cell contractile markers SM22α and aSMA were reduced while the osteochondrogenic markers BMP2,MSX2,OPN and RUNX2 were increased in 4-HNE treated mVSMCs.To further explore the effect of 4-HNE on the osteochondrogenic differentiation of VSMC.we interfered the activity of ALDH2 using Alda-1-an ALDH2 activator to eliminate 4-HNE and found that alda-1 reversed the osteochondrogenic effects of 4-HNE on mVSMCs and hVSMCs.6.RUNX2 mediates the osteochondrogenic effect of 4-HNE on VSMCs.To confirm the role of RUNX2 in osteoblast differentiation of vascular smooth muscle cells induced by 4-HNE,RUNX2 was knocked down using RUNX2 small interference,and results showed that knockdown RUNX2 significantly inhibited the increase of BMP2,MSX2,OPN.and RUNX2 proteins induced by 4-HNE stimulation.Alizarin red and von Kossa staining results showed that lentiviral knockdown RUNX2 significantly inhibited β-GP combined with CaCl2 and 4-HNE induced calcification.These results suggest that 4-HNE induced vascular smooth muscle cell calcification is mediated by RUNX2.7.4-HNE prevents ubiquitin-dependent degradation of RUNX2 by modifying RUNX2 K176.To explore the underlying mechanism how 4-HNE upregulated the protein level of RUNX2,both the gene and the protein changes of RUNX2 were detected.Under 4-HNE treatment,the mRNA level of RUNX2 was not changed compared with control.Under the condition of inhibiting protein synthesis using CHX,more 4-HNE protein was detected in 4-HNE treated mVSMCs,indicating that the protein degradation of RUNX2 was reduced by 4-HNE.In eukaryotic cells,protein is degraded depending on two major pathways-the ubiquitin-proteasome pathway and lysosomal proteolysis pathway23.RUNX2 was markedly increased by MG-132(a proteasome inhibitor)in 4-HNE treated mVSMCs.Moreover,the autophagy inhibitors CQ and 3-MA did not change the levels of RUNX2 in 4-HNE treated mVSMCs.These results demonstrate that the ubiquitin-proteasome pathway contributes to the upregulation of RUNX2 by 4-HNE.4-HNE stimulate RUNX2 in cells.To confirm the relationship between the carbonylation and ubiquitination of RUNX2,we overexpressed Flag-tagged RUNX2 in mVSMCs and hVSMCs and found that more 4-HNE and fewer ubiquitin was co-immunoprecipitated with Flag under 4-HNE stimulation.The human RUNX2 gene consists of 8 exons encoding a 521-amino acid protein.To figure out the carbonylation site of RUNX2 which reduces the binding of ubiquitin,we constructed three cDNA fragments that encoded Flag-full-length RUNX2 aa 1-521,Flag-RUNX2 aa 1-350,and Flag-RUNX2 aa 1-229 and then cloned them into pcDNA 3.1 plasmid.Co-immunoprecipitation tests indicated that the carbonylation of Flag-RUNX2 1-521,Flag-RUNX2 1-350,and Flag-RUNX2 1-229 was increased whereas the ubiquitination of them was simultaneously reduced under 4-HNE stimulation.It demonstrates that the key modified site should be within RUNX2 aa 1-229.Using the software tool-CarSPred,we found 7 potential carbonylation sites among RUNX2 aa 1-229 including K176,R94,R228,R229,P96,P227 and T22024.Then,pcDNA 3.1 plasmid which contains Flag-RUNX2 K176R or Flag-RUNX2 R94K R228K R229K or Flag-RUNX2 P94A P227A or Flag-RUNX2 T220S was constructed and transfected into HEK293T cells.Using co-immunoprecipitation,we found that the carbonylation and ubiquitination of Flag-RUNX2 K176R was not changed by 4-HNE compared with control.However,the carbonylation of Flag-RUNX2 R94K R228K R229K,Flag-RUNX2 P94A P227A,and Flag-RUNX2 T220S was all increased accompanied with less ubiquitination under 4-HNE stimulation.Altogether,these results suggest that the carbonylation of RUNX2 K176 should be the key reason for the downregulation of ubiquitination by 4-HNE.Conclusions1.ALDH2 plays a protective role in the process of vascular calcification.2.4-HNE promotes the carbonylation of RUNX2 K176,prevents the ubiquitination of RUNX2 and promotes phenotypic transformation of VSMCs.3.ALDH2 inhibits phenotypic transformation of VSMCs through metabolism of 4-HNE. |
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