Effects And Mechanisms Of Mitochondrial Fission And Mitophagy In Lactate Enhancing Vascular Calcification

PART Ⅰ.Correlation between plasma lactate levels and coronary artery calcificationObjective: To study the correlation between human plasma lactate levels and coronary artery calcification score(Agatston score).Methods: A total of 300 patients with angina or suspected coronary artery disease in the Zhongda Hospital of Southeast University or the Affiliated Hospital of Jiangsu University between November 2018 and May 2020 were enrolled.After being excluded by the exclusion criteria,274 patients were finally included.The Agatston score was calculated based on coronary CT angiography.The fasting peripheral blood of patients was collected,and the plasma lactate,CML and MGP levels were detected by ELISA kits.Results:(1)Human plasma lactate and CML levels were elevated with the increase of Agatston score,while the MGP levels were the opposite.(2)Human plasma lactate levels were independently correlated with Agatston score,and high lactate levels could predict the presence of coronary artery calcification(CAC).(3)Plasma lactate levels in CAC patients were correlated with MGP and CML levels.ROC curve analysis suggested that human plasma lactate levels(> 1.47 mmol/L)had diagnostic value for CAC,and combined analysis with MGP could further improve the diagnostic value of lactate for CAC.Conclusion: Human plasma lactate level was a biomarker for predicting and diagnosing CAC.PART Ⅱ.The mechanism of lactate regulating mitochondrial fission and mitophagy to promote calcification of rat thoracic aortaObjective: To study the effect of exogenous lactate on the calcification of the thoracic aorta in rats and its related mechanisms.Methods: A vascular calcification model was constructed on the basis of normal SD rats or type 2 diabetic SD rats.Vitamin D3 intramuscular injection and nicotine gavage were used to construct a model of vascular calcification.Lactate levels in plasma or tissues were detected.The thoracic aorta was taken for exogenous lactate intervention ex vivo.Alizarin Red S and Von Kossa staining were used to observe the formation of calcium nodules in the thoracic aorta;salting-out method was to detect the calcium content of the thoracic aorta;immunohistochemical staining and Western blotting were performed to detect the related molecules of osteogenesis,apoptosis,mitochondrial fission and mitophagy-related molecules;In addition,the enzyme activities of mitochondrial respiratory complexes and ATP content were measured.Results:(1)Compared with normal rats,lactate levels in plasma and tissues of calcified rats were significantly increased.(2)Compared with the control group,exogenous lactate promoted the osteogenesis phenotype transition(increased expression of RUNX2 and BMP2)and calcium salt deposition in the thoracic aorta.(3)Exogenous lactate promoted the translocation of Drp1 from cytoplasm to mitochondria,activated mitochondrial fission(decreased expression of OPA1 and increased expression of Mff and Drp1),inhibited autophagy(decreased expression of LC3-II and increased expression of p62)and BNIP3 mediated mitophagy(decreased mitochondrial membrane protein levels and increased BNIP3 expression),induced mitochondrialrelated apoptosis(increased expression of Bax and Cleaved-Caspase-3 along with decreased expression of Bcl-2).Compared with the control group,the mitochondrial respiratory chain complex enzyme activities in the exogenous lactate intervention group were impaired and the ATP production was decreased.Conclusion: Exogenous lactate induced mitochondrial-related apoptosis by interfering with the dynamic changes of mitochondria and mitophagy,and eventually promoted the calcification of the rat’s thoracic aorta.PART Ⅲ.The mechanism of lactate regulating mitochondrial fission and mitophagy to promote calcification of rat vascular smooth muscle cellsObjective: To study the effects of exogenous lactate on the calcification of rat vascular smooth muscle cells(VSMCs)and related mechanisms.Methods: The primary rat VSMCs were cultured,and β-glycerophosphate(β-GP)was used to construct a VSMC calcification model.Intracellular and extracellular lactate levels were detected.Glucose uptake rate of cells was determined by the 2-NBDG probe.Alizarin Red S staining method was used to observe the formation of calcium nodules in VSMCs;salting-out method was to detect calcium content in VSMCs;CCK-8 assay was performed to detect cell viability;DHE probe was used to determine the oxidative stress state in VSMCs;TUNEL staining was for the detection of VSMCs apoptosis;Mito-tracker red was used to observe the morphological changes of mitochondria under a confocal microscope;VSMCs were transfected with GFP-m RFPLC3 double-labeled adenovirus to observe intracellular autophagic flux;Mito-tracker red and Lyso-tracker green were used to label mitochondria and lysosome,and the colocalization of the two was observed;JC-1 probe was to detect mitochondrial membrane potential;transmission electron microscope was used to directly observe the changes in mitochondrial morphology and structure;Western blot analysis was used to detect osteogenic,apoptosis,mitochondrial fission and mitophagy related molecules in VSMCs.The NR4A1/DNA-PKcs/p53 signaling pathway was also verified;The enzyme activities of the mitochondrial respiratory complexes and ATP content were determined.Results:(1)Calcification induction promoted glucose uptake and lactate production in VSMCs.(2)Exogenous lactate activated Drp1 mediated mitochondrial fission(Drp1 translocated from cytoplasm to mitochondria;the morphology of mitochondria was changed towards punctate;expression of mitochondrial fission related molecules Drp1 and Mff was decreased),decreased intracellular autophagic flux(LC3-II expression was decreased,p62 expression was increased;after transfection with GFP-m RFP-LC3 double labeled adenovirus,the number of autophagosomes and autophagosomes was decreased),inhibited BNIP3-mediated mitophagy(BNIP3 expression was decreased and mitochondrial membrane protein levels were increased;mitochondrial and lysosomal co-localization was decreased),promoted the opening of mitochondrial permeability transition pore,interfered with mitochondrial membrane potential,damaged mitochondrial structure,and eventually led to impaired mitochondrial respiratory chain complex enzyme activity,decreased ATP generation,increased intracellular reactive oxygen species,and initiated endogenous mitochondria related apoptosis pathway(TUNEL positive cells increased;increased Bax and Cleaved caspase-3 expression,decreased Bcl-2 expression),promoted osteogenic phenotypic transformation and calcium salt deposition in VSMCs through the NR4A1/DNAPKcs/p53 signaling pathway.(3)Overexpression of BNIP3 alleviated calcification of VSMCs.Conclusion: Calcification induced lactate accumulation in VSMCs.Exogenous lactate activated Drp1-mediated mitochondrial fission and inhibited BNIP3-related mitophagy through the NR4A1/DNA-PKcs/p53 signaling pathway,activating the endogenous mitochondria related apoptosis pathway,and finally accelerating VSMCs calcification.