Effect And Mechanism Of Pyruvate Dehydrogenase Kinase 4 In The Pathogenesis Of Vascular Calcification

Vascular calcification is a common pathological feature of patients with atherosclerosis,diabetic vascular complications,hypertension,end-stage renal disease,and ageing.It is an independent risk factor for adverse cardiovascular events and increased mortality.Vascular smooth muscle cell(VSMC)is the main components of vascular wall,and the phenotype transformation of VSMC from contractile to osteogenic/chondrogenic phenotype is the core of vascular calcification.Furthermore,VSMC phenotypic changes are tightly integrated with the overall energetic state of the cells.Pyruvate dehydrogenase kinase 4(PDK4)is an important regulator linking oxidative phosphorylation to glycolysis,and plays a critical role in glycolipid metabolism and ATP formation.Previous study revealed that PDK4 is an important pharmacological target for some metabolic diseases,such as cancer,diabetes and insulin resistance,and obesity.Furthermore,it is tightly associated with cardiovascular disease and participates in the pathological process of ischemia-reperfusion injury and myocardial remodeling.PDK4 is upregulated in the calcified vessels of patients with atherosclerosis,suggesting that PDK4 may be involved in the pathogenesis of vascular calcification.The purpose of this study is to explore the role and molecular mechanism of PDK4 and its related signaling pathway in vascular calcification.Part ? PDK4 in phenotype transformation of VSMC and vascular calcificationObjective: To investigate the role of PDK4 in phenotype transformation of VSMC and vascular calcification.Methods: We established an animal model of arterial calcification induced by vitamin D3 plus nicotine(VDN)in SD rats.In vivo,32 SD rats were randomly divided into four groups(eight rats for each group): 1)NC group(injected with normal saline and intragastric administrated with peanut oil);2)VDN group(injected with 3×105 IU/kg of vitamin D3 simultaneously intragastric administrated with nicotine and peanut oil);3)DCA + VDN group(intragastric administrated with 50 mg/kg of DCA simultaneously induced aortic calcification),and 4)DCA group(intragastric administrated with 50 mg/kg of DCA).After eight weeks of intervention,all rats were sacrificed.H&E staining and Von Kossa staining were performed to investigate the morphological changes and calcified nodules of the aorta,respectively.The expression of PDK4 and the phosphorylation level of PDHA1(S293)were analyzed by Western blotting.In vitro,cells were subjected to calcifying conditions by supplementing the medium of ?-glycerolphosphate(?-GP)to induce calcification.VSMC was transfected with lentivirus-mediated short hairpin RNA(shRNA)targeting PDK4 and divided into four group: Lv-shRNA-NC,Lv-shRNA-PDK4,Lv-shRNA-NC+?-GP,and Lv-shRNA-PDK4+?-GP.Calcium deposition in VSMC was determined by Alizarin Red S staining and quantified by the o-cresolphthalein calcium(OCPC)method.The expression of PDK4 and RUNX2 were analyzed by Western blotting.The m RNA levels of osteogenesis related genes were analyzed by RT-q PCR.Results:(1)Compared with that of the NC group,the morphology and arrangement of elastic fibres in the aortic wall were anomalous,accompanied by the mass black granules deposited in calcified aortic tissues of the VDN group.However,in DCA + VDN group,the structure of aortic wall was relatively integrated and a small number of granules was found.Compared with NC group,the protein expression of PDK4 and the phosphorylation level of PDHA1(S293)were increased in VDN group,while DCA treatment abolished this effect.(2)Compared with Lv-shRNA-NC+?-GP group,the number of calcified nodules and the m RNA levels of BMP2 and RUNX2 were decreased in Lv-shRNA-PDK4+?-GP group.Calcium quantification analysis showed that DCA could reduce the calcification of VSMC induced by ?-GP.Conclusions: PDK4 expression is upregulated under calcification conditions,knockdown of PDK4 or inhibition of its activity attenuates vascular calcification.Part ? Effect of PDK4 on mitochondrial structure and glucose metabolismObjective: To investigate the effect of PDK4 on mitochondrial structure and glucose metabolism.Methods: VSMC was transfected with lentivirus-mediated shRNA targeting PDK4 and divided into four group as before.Transmission electron microscopy was performed to investigate the role of PDK4 on mitochondrial structure.The protein expression of glycolysis-related genes expression was analyzed by Western blotting.The intracellular lactate content was detected by lactate kit.VSMC glucose uptake was detected by using the fluorescent glucose analog 2-NBDG.Results:(1)The results of transmission electron microscopy showed that compared with the untreated cells,the membrane structure of mitochondria was unclear in ?-GP-treated VSMC,along with the disordered mitochondria crista.However,inhibition of PDK4 activity by DCA improved mitochondria structural integrity.(2)Compared with the untreated cells,the expression levels of glycolytic related genes GLUT1,PKM2,LDHA,and MCT4 were significantly increased in ?-GP-treated VSMC,while DCA co-incubated with ?-GP or knockdown of PDK4 inhibited the protein expression of glycolytic related genes,along with the decrease of intracellular lactate content.The baseline and maximum oxygen consumption rate were significantly decreased in the empty vector-transduced cells under calcification conditions,whereas knockdown of PDK4 restored this effect.Conclusions: inhibition of PDK4 resulted in the impairment of mitochondrial structure and improved the oxidative phosphorylation of glucose in VSMC.Part ? Mechanisms of PDK4 via controlling VSMC autophagy in vascular calcificationObjective: To investigate the role of PDK4-mediated autophagy in vascular calcification and relevant mechanisms.Methods: VSMC was transfected with lentivirus-mediated shRNA targeting PDK4 and divided into four group as mentioned before.The protein expression of LC3,p62,LAMP1,LAMP2,and CTSD was detected using Western blotting.The RFP-GFP-LC3 double-labeled lentivirus was perform to detect autophagic flux.Lysosomal activity was detected by Lyso-Tracker Red staining.Lysosomal degradation activity in VSMC was measured using self-quenched BODIPY FL Conjugate of BSA.Results:(1)Compared with the untreated cells,the ratio of LC3?/LC3? and the protein expression of p62 were significantly increased in ?-GP-treated VSMC.(2)Compared with the untreated cells,the lysosomal activity was decreased in ?-GP-treated VSMC.Furthermore,the protein expression of LAMP1,LAMP2,and CTSD was also decreased.(3)Compared with Lv-shRNA-NC,the protein expression of LC3? and p62 was significantly decreased in Lv-shRNA-PDK4 group,suggesting that knockdown of PDK4 promotes autophagosomes degradation.Furthermore,under calcification conditions,compared with the NC-shRNA transfected cells,knockdown of PDK4 increased the RFP-LC3 dots.(4)Either 3-methyladenine(3-mA)or chloroquine(CQ)accelerated calcium deposition in VSMC triggered by ?-GP.Furthermore,stimulated autophagy activity by rapamycin resulted in a significant decrease in calcium deposition in the VSMC.(5)Under calcification conditions,compared with the VSMC transfected with NC-shRNA,the protein expression of RUNX2 was decreased in cells transfected with PDK4-shRNA.This was further identified by using calcium assay kit.However,inhibition of autophagy by CQ abolished the protective effect of PDK4 inhibition on calcium deposition in VSMC.Conclusions: Inhibition of PDK4 attenuates VSMC calcification via restoring autophagic flux.Part ? Effects of metformin on VSMC calcification by promoting mitochondrial biogenesisObjective: To investigate the effect of metformin(Met)on mitochondrial biogenesis during VSMC calcification,and the role of PDK4 involved in it.Methods: VSMC was transfected with lentivirus-mediated shRNA targeting PDK4 and divided into four group as mentioned before.Alizarin Red S staining was performed to assess mineral formation.The effect of Met on protein expression of PDK4 was performed by Western blotting.RT-q PCR was performed to detect the effect of Met on the m RNA levels of osteogenesis-and mitochondrial biogenesis-related genes.The ultrastructural changes of mitochondria were observed by using transmission electron microscopy.Mitochondrial membrane potential was analyzed using JC-1 dye staining.Cell apoptosis was measured by Annexin V-FITC/PI staining using flow cytometry.Results:(1)Compared with the untreated cells,the m RNA levels of RUNX2,BMP2,and OCN were increased upon ?-GP treatment.Alizarin red S staining analysis showed an increase in calcium mineralization in the ?-GP-treated VSMC compared with the mineralization shown in the normal controls.However,addition of Met gradually inhibited this effect.(2)Compared with the ?-GP-treated VSMC,the protein expression of PDK4 was significantly decreased in ?-GP-treated cells after supplement of Met.(3)Transmission electron microscopy analysis showed that incubation of VSMC by ?-GP resulted in destruction of the cell ultrastructure and a significant decrease in the mitochondrial density(compared with that of the control cells),which were restored after Met exposure.Furthermore,compared with the untreated cells,ATP content,the mitochondrial DNA copy number,and mitochondrial membrane potential were decreased in ?-GP-treated VSMC,whereas addition of Met restored these effects.RT-q PCR results revealed that the gene expression of PGC-1?,NRF1,and TFAM was decreased in ?-GP-treated cells,whereas supplementation with Met partially restored the m RNA levels.(4)Compared with the untreated cells,the protein expression of Bax and cleaved-caspase 3 was increased in cells treated with ?-GP,while the protein expression of Bcl-2 was decreased.This trend was further enhanced in ?-GP-treated VSMC suppled with AZT.(5)Compared with that in the ?-GP-treated group,the ratio of apoptotic cells was decreased in VSMC following treatment with DCA.Under calcification conditions,compared with NC-OE group,the decreased protein expression of Bax and Cleaved-caspase 3 and increased protein expression of Bcl-2 were observed in Met+NC-OE group.However,the anti-apoptotic effect was abolished upon PDK4 overexpression.Conclusions: Restoring mitochondrial biogenesis with Met attenuates VSMC calcification via inhibition of the PDK4-mediated apoptosis.