Acid Sphingomyelinase/ceramide Regulates Vascular Structural Remodeling In Simulated Weightless Rats

Weightlessness or simulated weightlessness will lead to a redistribution of blood and fluid,giving rise to an increase of transmural pressure in arteries upper than the heart,while a decrease lower than the heart.Longer exposure entails more variations like bone loss,load-bearing skeletal muscle atrophy as well as modification of immunology and the endocrine system,which exert an effect on vascular structural and functional remodeling.In decades,abundant researches performed on humans and animals under real weightless environment or simulated microgravity have confirmed a regionally specific artery remodeling.Specifically,arteries located upper than the heart endure a media thickness of vascular wall.However,arteries located lower than the heart experience a media attenuation of vascular wall.Structural remodeling of arteries may jeopardize physical health and working ability of astronauts.The prevailing fluid therapy and exercise are not very effective precautions.Although it’s been studied for years,the mechanism of vascular structural remodeling remains unclear.Sphingolipids is a group of compounds containing a long-chain fatty alcohol amine called sphingosine which is an essential constituent of membranes in eukaryotic cells.And it carries important roles in signal transduction.Ceramide(Cer)lies in the center of sphingolipids metabolism which is generated through multiple pathways.In vascular cells,the acid sphingomyelinase(ASM)catalyzing hydrolysis of sphingomyelin to produce Cer is associated with the regulation of apoptosis,proliferation and inflammatory response which underlies the vascular remodeling in different situations.However,the function of ASM/Cer pathway in vascular structural remodeling under simulated weightlessness is still unknown.According to the backgrounds,we proposed the following questions: 1.How would the ASM/Cer pathway change under simulated weightlessness? 2.What kind of role would ASM/Cer play in vascular remodeling of simulated weightless rats? And what is the downstream?To find out the possible answers to the above questions,we performed the following experiments in common carotid artery(CA),basilar artery(BA),mesenteric artery(MA)and femoral artery(FA): 1)Observe morphology change of arteries in simulated weightless rats.Hematoxylin-eosin(HE)staining is used to test morphology change in CA,BA,MA and FA.Immunohistochemistry,western blotting and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling(TUNEL)assay were conducted to test the level of proliferation,apoptosis or cellular phenotype in arteries.2)Observe the change of ASM/Cer in simulated weightless rats.Immunohistochemistry and western blotting were performed to test the content and distribution of ASM and Cer in CA,BA,MA and FA.3)Observe the impact of ASM/Cer on apoptosis,proliferation or cellular phenotype in arteries.HE staining,immunohistochemistry,western blotting and TUNEL assay were conducted to test morphology,proliferation,apoptosis or cellular phenotype in arteries.We also further proofed the impact of ASM/Cer in vascular smooth muscle cells.Afterwards,we tested the connection between lysosomal cathepsin D(CTSD)and ASM/Cer pathway preliminarily.The main findings of our study are as follows: 1)Structural remodeling in arteries after simulated weightlessness.After simulated weightlessness,CA and BA show a significant intima-media hypertrophy,with up regulated proliferation and down regulated apoptosis.MA shows a significant atrophy,with down regulated proliferation and up regulated apoptosis.FA shows a phenotypic transformation in vascular smooth muscle cells,with up regulated proliferation and apoptosis.2)ASM/Cer change in arteries of simulated weightless rats.After simulated weightlessness,CA and BA show a downregulation of ASM/Cer while MA and FA show an upregulation of ASM/Cer.3)The function of ASM/Cer in vascular remodeling of simulated weightless rats.Regulation of apoptosis,proliferation and phenotypic transformation of vascular smooth muscle cells following the changes of ASM/Cer:1)CA & BA:(1)Vascular incubation: C6-Ceramide(C6-Cer)incubation increases apoptosis level while decreases proliferation in arteries of hindlimb-unloaded tail-suspended(HU)rats.Desipramine(Dpm)incubation decreases apoptosis level while increases proliferation in arteries of control(CON)rats.(2)Intragastric administration: Doxepin(DOX),a specific ASM inhibitor,administration decreases apoptosis level while increases proliferation in arteries of CON rats.2)MA:(1)Vascular incubation: Dpm incubation decreases apoptosis level while increases proliferation in MA of HU rats.(2)Intragastric administration: DOX administration decreases apoptosis level while increases proliferation in MA of HU rats.3)FA:(1)Vascular incubation: Dpm incubation decreases apoptosis level and the ratio of synthetic/contractile phenotype marker,while increases proliferation in FA of HU rats.(2)Intragastric administration: DOX administration decreases apoptosis level and the ratio of synthetic/contractile phenotype marker,while increases proliferation in FA of HU rats.4)VSMCs:(1)C6-Cer incubation increases apoptosis level,the ratio of synthetic/contractile phenotype marker and expression of CTSD,while decreases proliferation level.Maturity and release of CTSD from lysosome are also promoted.(2)Dpm incubation decreases apoptosis level,the ratio of synthetic/contractile phenotype marker and expression of CTSD,while increases proliferation level.Maturity and release of CTSD from lysosome are also inhibited.In summary,the results intend to tell that simulated weightlessness could induce a significant decrease of ASM/Cer level in CA and BA,while an increase of ASM/Cer in MA and FA.The change of ASM/Cer level may regulate the expression,activity and distribution in cell of CTSD and then lead to region-specific vascular structural remodeling.