The Role Of StarD7 And Mechanism Of StarD7 Action In Ang Ii-induced Mitophagy Of Vascular Smooth Muscle Cells

Angiotensin ??Ang??is essential for the regulation of blood pressure in mammals,but long-term stimulation of higher concentration of Ang? can cause damage to vascular smooth muscle cells in the media wall of blood vessels.It has been proved that Ang? can cause inflammation,oxidative stress and organelle damage of VSMCs.How to provent cell injuries caused by Ang? and how to remove damaged organelles have become the focus of recent research.In recent years,cell autophagy has been extensively studied.Mitophagy is an important way to remove the damaged mitochondria and maintain homeostasis.In addition to the classical PINK1/Parkin/LC3pathway,more and more regulatory networks have been found,such as NIX/BNIP3 pathway and FUNDC1 pathway.In addition,it has been demonstrated that during the occurrence of mitophagy,the interaction of mitochondria with endoplasmic reticulum?ER?,occurs through mitochondria-associated ER membrane?MAM?,suggesting that the contact and communication between these two organelles might be related to the occurrence of organelle autophagy.Increasing evidence suggests that MAM serves as an important cellular signaling platform associated with several important functions,including Ca²⁺storage,lipid biogenesis,mitochondrial division and induction of autophagy.In recent years,the some MAM-related proteins are identified in the MAM.It has been reported that some lipid transfer proteins located on MAM are involved in the formation of autophagosome.St AR-related lipid transfer protein 7?starD7?is an important lipid transfer protein participated in the non-vesicular lipid transport in cells,but whether the starD7 locating in MAM structure is involved in mitophagy is still unclear.Part? Ang? induces mitophagy in VSMCsObjective:To test whether Ang? induces the mitophagy of VSMCs.Methods:1.Mito SOX?red fluorescent probe was used to detect the changes in mitochondrial ROS levels in Ang?-treated VSMC.2.Luciferase-based luminescence-detected ATP level was used to evaluate the effect of Ang? on VSMC mitochondrial functions.3.The effect of Ang? on VSMC mitophagy was detected by using Western blot and immunofluorescence staining to examine mitophagy-related markers.Results:1. Ang? significantly elevates the levels of mitochondrial ROS in VSMCMito SOX?red fluorescent probe staining showed that the red fluorescence intensity of Mito SOX?in Ang?-treated VSMC was significantly higher than that in the control group,suggesting that Ang? stimulation facilitates the production of mitochondrial ROS in VSMCs,which is a prerequisite for the oxidative damage of mitochondria.2.Ang? can induce mitochondrial dysfunction in VSMCsCompared with the control group,ATP content in VSMCs was significantly decreased after Ang? treatment,suggesting that Ang? induces mitochondrial dysfunction in VSMCs.3.Ang? promotes the mitophagy in VSMCsAfter treating VSMCs with Ang?,the mitophagy marker PINK1was significantly upregulated compared with the control group.The LC3-?/LC3-I ratio was also significantly increased.The immunofluorescence staining showed that,in Ang?-treated VSMCs,PINK1 was mainly localiz ed on the mitochondria.Summary:Ang? causes oxidative damage to VSMC mitochondria,thus leading to mitochondrial dysfunction and mitophagy.Part ? starD7 participates in Ang?-induced VSMC mitophagythrough regulating mitochondria-ER contact sitesObjective:To elucidate whether and how starD7 participates in mitophagy of VSMCs induced by Ang?.Method:1.The subcellular localization of starD7 was observed by triple immunofluorescence staining in Ang?-treated VSMCs.2.Immunofluorescence staining was used to observe the effect of Ang? on mitochondria-ER contact sites.3.The fractionation by ultra centrifugation and immunofluorescence staining were used to detect whether starD7 is localized at the mitochondria-ER contact sites.4.The starD7 expression and subcellular localization in Ang?-treated VSMCs were analyzed by triple immunofluorescence,western blot analysis and RT-PCR.5.Western blot and immunofluorescence staining were used to detect Ang?-induced mitophagy in starD7-knocked down VSMCs.Results:1. Ang? stimulation facilitates the co-localization of starD7 with mitochondria and autophagosome in VSMCsImmunofluorescence staining showed that the co-localization of starD7 with mitochondria and LC3 was increased significantly in Ang?–treated VSMCs.2. Ang? treatment increased in mitochondria-ER contact sites in VSMCsMito-Tracker red fluorescent probe was used to label the mitochondria,and KDEL was used to label the endoplasmic reticulum.Confocal microscopy results showed that the mitochondria-ER contact sites were significantly increased after Ang? treatment.3. starD7 is localized on the mitochondria-associated ER membraneThe rsubcellular localization by ultra centrifugation showed that starD7 was present in the isolated mitochondria-associated ER membrane.Immunofluorescence staining showed that starD7 co-locatlized with mitochondria-associated ER membrane marker GRP78.4. Ang? promotes the accumulation of starD7 at mitochondria-ER contact sitesTriple immunofluorescence staining showed that Ang? significantly promoted the accumulation of starD7 at mitochondria-ER contact sites.Western blot analysis and RT-PCR showed that Ang? promoted starD7 expression at both m RNA and protein levels.5. starD7 is involved in Ang?-induced VSMC mitophagyAfter starD7 was knocked down with small interfering RNA,western blot showed that the expression of PINK1 and LC3 was significantly decreased.Immunofluorescence staining showed that the level of mitophagy significantly decreased in starD7-knocked down VSMCs.Summary:starD7 is localized to mitochondria-associated ER membrane.Ang? promotes the accumulation of starD7 at the mitochondria-ER contact sites.After starD7 was knocked down in VSMCs,the level of mitophagy induced by Ang? was significantly reduced.Conclusion:1.Ang? induces mitophagy in VSMCs.2.starD7 participates in the mitophagy of VSMCs induced by through regulating mitochondria-ER contact sites.