Caveolae Regulated Lysosomal Status Mediates Structural Remodeling Of Carotid Artery In Simulated Weightless Rats

BackgroundMedical monitoring in manned spaceflights and ground-based studies of simulated weightlessness show the remodeling of carotid artery(CA)like increased artery stiffness and intima-media thickness(IMT).CA is the major blood supply to the brain.Studies have shown that increased artery stiffness and IMT of CA are independent risk factors for cardiovascular diseases such as arteriosclerosis,stroke and myocardial infarction.Therefore,the mechanisms and protective measures of CA remodeling after weightlessness or simulated weightlessness are hot spots in space medicine research.At present,it is believed that the initial cause of CA remodeling after weightlessness or simulated weightlessness is the alteration of transmural pressure and yet the specific mechanism has not been illustrated clearly.Lysosomes,long regarded as the end point of metabolism,are important organelles in autophagy processes.Lysosomes are also critical intracellular signaling centers.They are involved in regulating vascular endothelial physiology and barrier function,and influencing the phenotype switch of vascular smooth muscle cells(VSMCs)and vascular vasomotion.Improving lysosomal functioning plays a protective role in the course of hypertension,arteriosclerosis,and vascular aging.Caveolae are flask-like invaginated structures in plasma membranes.Cholesterol is requisite to the assembly and functioning of Caveolae.Caveolae act as membrane mechanoreceptors.Studies have shown regulatory functions of caveolae on lysosomes in the course of some diseases.Previous researches have also confirmed decreased number of caveolae and downregulated autophagy in CA after simulated weightlessness.In conclusion,present aims of this study were to illustrate lysosomal modification,to explore the regulatory functions of caveolae on lysosomes and to elucidate the role of lysosomes in CA remodeling of simulated weightless rats.This work will elucidate possible mechanisms of CA remodeling after weightlessness or simulated weightlessness and its protective measures.AIM1.To elucidate lysosomal modification and its relation to the transmural pressure in CA of simulated weightless rats.2.To explore the regulatory functions of caveolae on lysosomes in CA of simulated weightless rats.3.To explore the role of lysosomes in CA remodeling of simulated weightlessness rats.Methods1.Rats model by hindlimb unloading tail suspension(HU)was used to simulate the effects of weightlessness on cardiovascular system.2.The number and distribution of lysosomes in CA were observed by transmission electron microscopy.Western blot(WB),immunohistochemistry(IHC),real-time polymerase chain reaction(q RT-PCR)were applied to examine the protein expression,distribution and m RNA levels of lysosome associated membrane protein 1(LAMP1),kinesin-1,transcription factor EB(TFEB),transcription factor E3(TFE3)and lysosome related genes(LRGs).3.CA of control(CTR)rats was incubated in vitro with methyl-cyclodextrin(MCD),a cholesterol depletion reagent.The expression and distribution of LAMP1 were detected by WB and IHC.4.A7R5 cell lines of vascular smooth muscle cells(VSMCs)were incubated with MCD.Nocodazole was used to interrupt polymerization of microtubule.The protein expression,distribution and m RNA levels of LAMP1,TFEB and LRGs were detected by WB,immunofluorescence and q RT-PCR.5.Intermittent fasting(IF)was applied in vivo to stimulate lysosomes.Lysosome related proteins were observed by WB and IHC in CA.The IMT of CA were detected by hematoxylin-eosin(HE)staining,and collagen content were observed by Masson staining.Terminal deoxynucleotidyl transferase-d UTP nick end labeling(TUNEL)was used to detect CA apoptosis.Results1.Lysosomal modification and its relation to the transmural pressure in CA of HU ratsIn CA of HU rats,the number of lysosomes decreased;the expression of LAMP1 decreased,which mainly happened in the media(P<0.05);the expression of kinesin-1increased(P<0.05);the transcription level of LRGs increased(P<0.05);the protein levels of TFEB and TFE3 decreased,but the transcriptional levels increased(P<0.05).In order to determine whether lysosomal modification in CA of HU rats is related to the change of transmural pressure,we also measured lysosomal changes in TA which are above the cardiac level and AA which are below the cardiac level.In TA of HU rats,the protein expression and distribution of LAMP1 decreased(P<0.05),which mainly happened in the media;the transcription level of LRGs increased(P<0.05);the protein levels of TFEB and TFE3 decreased and the transcription level increased(P<0.05);the changes of lysosomes in TA of HU rats were consistent with that of CA.In AA of HU rats,the expression of LAMP1 decreased,which mainly occurred in the media(P<0.05);the transcriptional level of LRGs decreased,which was opposite to that of CA(P<0.05);and the protein level and transcriptional level of TFEB and TFE3 decreased,which was opposite to that of CA(P<0.05).2.The regulation of lysosomes by caveolae in CA of HU rats.The expression of LAMP1 decreased in CA after incubation with MCD(P<0.05).In VSMCs,after MCD incubation,the expression of LAMP1 decreased,but lysosomal fusion to cell membrane increased(P<0.05);the transcription level of LRGs increased(P<0.05);the protein expression of TFEB decreased,which mainly happened in cytoplasm,but the transcription level increased(P<0.05).3.The role of lysosomes in CA structural remodeling of HU rats.Compared with ad libitum(AL)CTR,the expression of LAMP1,TFEB,TFE3 and IMT of CA in IFHU group remained unchanged,while the content of collagen of CA in IFHU group increased(P<0.05),and apoptosis level decreased in CA of IFHU group(P<0.05).Compared with ALHU,the expression of LAMP1,TFEB and TFE3 of CA in IFHU group increased(P<0.05),the IMT and the content of collagen of CA in IFHU group decreased(P<0.05),and apoptosis level increased in CA of IFHU group(P<0.05).Conclusions1.The number of lysosomes decreases and lysosomal moving to plasma membrane increases in CA of HU rats,which may be related to the changes of transmural pressure.2.Caveolae mediate lysosomal modification in CA of HU rats.3.Lysosomal modification is involved in the structural remodeling of CA in HU rats.