Sphk1 Regulated Caveolae Mediates Remodeling Of Small Arteries In Simulated Weightless Rats

[Background]After medium-or long-term space flight,a series of adaptive change will happen in human body due to continuous exposure to weightlessness/microgravity environment,including disorder of water and electrolyte,cardiovascular dysfunction,loss of red blood cells,bearing skeletal muscles atrophy,bone loss.Among them,artery remodeling is one of the most important changes,which mainly manifests arterial wall thickening,compliance reduction,systolic function and innervation enhancement above the heart level,and artery wall thinning,systolic function and innervation reduction below the heart level^[1].Structural and functional changes in arteries are the main causes of postflight orthostatic intolerance,which may also affect working ability and even health status of astronauts.Therefore,research on relevant mechanism is an important topic in aerospace medicine.Small artery refers to artery with a diameter of 0.3～1mm.The vascular wall structure of small artery is like that of middle artery with all layers thinner.However,the muscle layer of the media vascular wall in small artery is relatively thicker than other arteries.Preliminary results showed that in weightlessness/simulated weightlessness environment,the IMT of CA thickened and the myogenic tone was enhanced,while the IMT of the mesenteric arteries became thinner and the MT was weakened,leading to significant changes in blood supply of the cerebral tissues and peripheral vascular resistance.Currently,it is believed that the causes of artery remodeling in weightlessness/simulated weightlessness involve contractile proteins,ion channels,kinases,etc.,but the detailed mechanism is still unclear.Caveolae is a flask-like structure with a diameter of 50～100 nm located on the cell membrane,which is rich in cholesterol and SLs.Caveolins are the main structural proteins of Caveolae.Plentiful important signaling molecules involved in cell growth,death and metabolism were accumulated in Caveolae.Therefore,it acts as a platform for regulating multiple cell functions,making the receptors,enzymes and regulatory molecules needed for signal transmission close to each other and accelerating the rate of signal transmission^[2].Caveolae/Caveolins are abundant in cardiovascular system and exist in cardiomyocytes,VSMCs,ECs,macrophages,and fibroblasts.It plays an important role in regulation of function of ECs,the contraction,proliferation and differentiation of smooth muscle cells,and the occurrence and development of hypertension and atherosclerosis.SLs are the structural basis of Caveolae,and their metabolic changes often lead to abnormal function of Caveolae.SLs are a batch of lipids with backbones of eighteen carbon amino alcohol as sphingosine,phytosphingosine or dihydrosphingosine.There are about dozens of types of SLs,among which the most well studied are Cer and S1P.S1P and S1PRs take part in proliferation and apoptosis of ECs,VSMCs,fibroblasts and macrophages,Ca²⁺signaling and inflammatory response,playing an important role in structure and function regulation of the arteries.Generation of S1P depends on Sphk1 and Sphk2,the former located in cytoplasm or near cell membrane,the latter located in the nucleus and organelles.It is reported that Sphk1 is closely related to Caveolae in cardiovascular system^[3,4].Our previous work confirmed that the number of Caveolae decreased in VSMCs of common carotid artery in simulated weightless rats,and Cav-1was involved in mediating the reduction of arterial contraction response to Ang II and the change of inhibition of AT₁R internalization^[5].The S1P/S1PRs of common carotid artery,thoracic aorta,abdominal aorta and femoral artery were significantly changed,and the expression and activity of Sphk1 underwent region-specific changes,taking part in the process of arterial structural remodeling,oxidative stress and inflammatory response^[6].Therefore,weightlessness/simulated weightlessness may result in structural and functional changes of small arteries and Caveolae is one of the important regulators.Caveolae is regulated by sphingolipid metabolism.Caveolae and Sphk1 have changed obviously after simulated weightlessness.According to the background and evidence,we proposed our speculation,that is,Sphk1 regulated Caveolae plays an important role in rat structural and functional remodeling of simulated weightless rats.[Aim]1.To elucidate the changes of Caveolae in small arteries of simulated weightless rats and its role in artery remodeling of structure and function2.To elucidate the changes in S1P/S1PRs pathway in small arteries of simulated weightless rats and the role of Sphk1 in artery remodeling of structure and function3.To elucidate the regulatory effect and mechanism of Sphk1 on Caveolae in small arteries of simulated weightless rats[Methods]1.The HU model was used to simulate the hemodynamic changes in weightlessness.After 4w suspension,CA and MA were carefully isolated from CON or HU rats.2.TEM and WB were used to observe the distribution of Caveolae,the main constituent protein Cav-1 and its phosphorylated expression p-Cav-1.3.After inoculation with MCD,the same sites of CA or MA without bifurcation were selected.MT was detected by Pressure Myograph System.HE staining and IHC were performed to observe the changes of structure or proliferation and apoptosis levels in CA and MA.Image J software was used to measure IMT and detect content of PCNA and Caspase 3.4.IHC and WB were used to observe the expression of enzymes involved in the generation and degradation of S1P and its receptors（S1PRs）in CA and MA of rats.5.MHP and SKI5C were incubated in CA and MA,respectively.The MT of CA and MA were detected by Pressure Myograph System.HE staining and IHC were performed to observe the changes of structure or proliferation and apoptosis levels in CA and MA.Image J software was used to measure in IMT and detect content of PCNA and Caspase 3.6.A7r5 VSMCs were cultured and incubated with agonist of ASM,TNF-α,and inhibitor Dpm,SKI5C or MHP to detect cholesterol content by commercial kits,changes in GM1 by CTB staining and Cav-1 expression by immunofluorescence staining.[Results]1.Distribution of Caveolae and Cav-1/p-Cav-1 protein expression in CA and MA of simulated weightless ratsThe results of TEM showed that compared with the CON group,the content of Caveolae in ECs and VSMCs of CA in HU group were significantly increased（P<0.05）,and they were all located on and near the cell membrane,expressed the flask structure.Changes in MA were contrary to those in CA.Caveolae contents on and around the cell membrane of the ECs in MA were significantly reduced（P<0.05）.In VSMCs,the number of Caveolae on the cell membrane was significantly reduced（P<0.05）,but the number of Caveolae around the cell membrane was significantly increased（P<0.05）.WB results in CA showed that compared with CON,the Cav-1 expression in HU group was significantly increased（P<0.05）,but there was no significant change in the content of p-Cav-1 protein.Both Cav-1 and p-Cav-1 protein expression of rats in MA of HU group were significantly increased（P<0.05）compared with CON.2.The role of Caveolae in structural and functional remodeling of small arteries in simulated weightless ratsThe MT results showed that after 4 wk simulated weightlessness,MT in CA of HU rats were significantly increased compared with CON under the pressures of 50 mm Hg（P<0.05）,75 mm Hg（P<0.01）,100 mm Hg（P<0.01）and 125 mm Hg（P<0.001）.The incubation of 1 m M MCD had no significant effect on the MT in CA of CON and HU rats,and the MT of the two groups was still significantly different under the pressure of 25mm Hg（P<0.05）,50 mm Hg（P<0.05）and 75 mm Hg（P<0.05）.3 m M MCD incubation had no significant effect on MT in CA of CON group,but the MT of HU rats under the pressures of 75 mm Hg（P<0.01）,100 mm Hg（P<0.0001）and 125 mm Hg（P<0.0001）was significantly reduced,and there was no significant difference in CA between the two groups after incubation.The MT of CA in CON group decreased significantly at 75 mm Hg（P<0.01）,100 mm Hg（P<0.0001）and 125 mm Hg（P<0.0001）after 10 m M MCD incubation.The MT of CA in HU group decreased significantly at 50 mm Hg（P<0.01）,75mm Hg（P<0.0001）,100 mm Hg（P<0.0001）and 125 mm Hg（P<0.0001）,and there was no significant difference in the MT of CA between the two groups.Different from CA,the MT of MA in 4wk HU rats was significantly reduced compared with CON group under pressures of 50 mm Hg（P<0.001）,75 mm Hg（P<0.01）,100 mm Hg（P<0.01）and 125 mm Hg（P<0.01）.After MCD incubation of 1 m M and 3 m M,there was no significant difference in MT of MA between the two groups.MT of MA in CON group under 4 mm Hg（P<0.05）,25 mm Hg（P<0.01）were significantly increased while were significantly reduced under 100 mm Hg（P<0.05）,125 mm Hg（P<0.01）after10 m M MCD incubation.The MT of MA in HU rats significantly decreased under the pressure of 100 mm Hg（P<0.05）and 125 mm Hg（P<0.01）.After 10 m M MCD incubation,the MT of MA in HU group was still significantly lower than that in CON group under the pressures of 25 mm Hg（P<0.001）and 50 mm Hg（P<0.05）.It was found that compared with CON,the content of eNOS in MA tissues of HU rats did not change significantly,but the content of p-eNOS increased significantly（P<0.05）by WB.WB and Co-IP detection found that the content of AT₁R in MA tissues of HU rats had no significant change compared with that of CON,but its combination with Cav-1was significantly increased（P<0.05）.In addition,IHC staining showed that the PCNA content of CA in the HU group was significantly higher than that in the CON group（P<0.05）and the content of Caspase3 was significantly lower（P<0.05）.The content of PCNA and Caspase3 of CA in CON group was not significantly affected by 3 m M MCD incubation for 1 h.The PCNA protein expression of CA in the HU group was significantly reduced（P<0.05）and the content of Caspase3 was significantly increased（P<0.05）after 3 m M MCD incubation.There was no significant difference in the content of PCNA and Caspase3 between the CON and the HU group after incubation.The change of MA was different from that of CA.Compared with CON group,PCNA protein expression in HU rats was significantly reduced（P<0.05）and Caspase3 was significantly increased（P<0.05）.After 10 m M MCD incubation for 1 h,PCNA of MA in the CON group was significantly reduced（P<0.05）and Caspase3 was significantly increased（P<0.05）,while the PCNA and Caspase3 contents of MA in the HU rats were not significantly changed,and the PCNA and Caspase3 contents of MA in the CON group and HU group were not significantly different.3.Changes of S1P/S1PRs in CA and MA of 4wk simulated weightless ratsWB and IHC were used to detect the expression and distribution of key enzymes for S1P synthesis and degradation and its receptors.It was found that Sphk1,Sphk2 and SGPL1 were expressed in both intima and media of CA.After 4 wk suspension,compared with CON group,Sphk1 protein expression of CA in HU rats was significantly reduced（P<0.05）,while Sphk2 was significantly increased（P<0.05）,and SGPL1 protein expression was significantly decreased（P<0.05）.At the same time,it was found that the Type 1-3 receptor of S1P,named S1PR_1-3,were also expressed in both intima and media of CA.Compared with CON,the protein expression of S1PR₁ in CA of HU rats was significantly decreased（P<0.05）,and the distribution and expression of S1PR₂ and S1PR₃were not significantly changed compared with CON group.Different from CA,IHC detection found that Sphk,SGPL1 and S1PR_1-3 were mainly distributed in media of MA.After 4 wk suspension,Sphk1 protein expression was significantly increased in HU group compared with CON group（P<0.05）,while Sphk2protein expression was significantly decreased（P<0.05）,and SGPL1 protein expression was significantly increased（P<0.05）.S1PR_1-3 was less expressed in both intima and media of MA,S1PR₁ of HU rats was significantly increased compared with that of CON group（P<0.05）,while S1PR₂and S1PR₃were not significantly changed.4.The role of Sphk1 in remodeling of small artery in simulated weightless ratsCA and MA were incubated with SKI5C and MHP to decrease or increase Sphk1activity respectively,and then MT was detected.The results showed that the MT of CA in the HU group was significantly higher than that in the CON group（P<0.01）.After the incubation of SKI5C,the MT of CA in the CON group showed a trend of strengthening but did not reach the significance level.The MT of CA in the HU group showed no significant change after incubation,and there was no significant difference between CON and HU group after the incubation of SKI5C.On the contrary,after MHP incubation,the pressure of CA in the CON group at 4 mm Hg,25mm Hg,50 mm Hg and 75 mm Hg showed an increasing trend of MT,while the pressure of CA at 100 mm Hg and 125 mm Hg showed a decreasing trend compared with that of CON,but none reached a significant level.After incubation,the MT of CA in HU group decreased significantly at 100 mm Hg（P<0.01）and 125 mm Hg（P<0.0001）,and there was no significant difference between CON and HU group after MHP incubation.The change of MA was more complicated.After SKI5C incubation,the MT of CON group MA increased slightly under the pressure of 4 mm Hg,25 mm Hg,50 mm Hg and 75mm Hg,while decreased slightly under the pressure of 125 mm Hg,and the change did not reach a significant degree.Incubation of SKI5C had no significant effect on MT in MA of HU rats.After incubation of SKI5C,there were still significant differences in MT between the CON and HU group under pressures of 50 mm Hg（P<0.01）and 75 mm Hg（P<0.05）.After MHP incubation,the MT of MA in CON group significantly decreased at125 mm Hg（P<0.05）,while that of MA in HU group decreased at 100 mm Hg and 125mm Hg,but did not reach a significant degree.After MHP incubation,there was no significant difference in MT between CON group and HU group.It was found by IHC staining that after incubation of SKI5C（10μM）or MHP（100μM）,there was no significant change in PCNA and Caspase3 protein expression of CA and MA in CON group and HU group.5.The regulatory effect of Sphk1 on Caveolae and its mechanism in small arteries of simulated weightless ratsIt was found that after SKI5C treatment,the number and distribution density of Caveolae on and near the VSMCs membrane of CA in CON rats increased significantly（P<0.05）,while in CA of HU rats,the number and distribution density had no significant change.After SKI5C incubation,there was no significant difference between CON and HU group.After MHP incubation,there was no significant change in Caveolae of CA in the CON group,but the number of Caveolae on and around the VSMCs membrane of CA in HU rats significantly decreased（P<0.01）,and there was no significant difference in the number and distribution of Caveolae between CON and HU after MHP incubation.After incubation of SKI5C and MHP,there was no significant change in the number of Caveolae on and below the VSMCs cell membrane in MA of CON group.However,for MA in HU group,Caveolae on the cell membrane did not change significantly after incubation of SKI5C or MHP but the number of Caveolae under the cell membrane significantly decreased after incubation of MHP（P<0.05）.After incubation of the two drugs,the number of Caveolae on MA cell membrane was still significantly different between CON and HU groups（P<0.05）.After incubation of SKI5C,Caveolae under the cell membrane still showed significant difference between the CON and HU groups（P<0.05）,but there was no significant difference between the CON and HU groups after incubation of MHP.There was no significant effect on protein content of Sphk1 after 10m M MCD incubation,and the difference in Sphk1 protein expression between CON and HU groups was still significant（P<0.05）.In order to further investigate the mechanism of Sphk1 regulating Caveolae,the A7r5VSMCs cells were incubated with SKI5C,MHP,TNF-αand Dpm,and the contents of three major structural components of Caveolae including sphingolipid ganglioside GM1,cholesterol and Cav-1 were detected.The results showed that when SKI5C or MHP were given alone,the contents of GM1 in the cell membrane did not change significantly,but after Dpm+SKI5C treatment,GM1 increased significantly（P<0.05）.However,after the incubation of TNF-αplus MHP,the cell membrane GM1 were significantly reduced（P<0.05）.Cholesterol content and Cav-1 protein expression were not significantly changed after single incubation of SKI5C or MHP,Dpm+SKI5C and TNF-α+MHP.[Conclusions]1.The Caveolae of CA and MA in rats shows obvious region-specific changes after simulated weightlessness.2.The changes of Caveolae are involved in the structural and functional remodeling of small arteries in simulated weightless rats.3.The S1P/S1PRs of CA and MA undergoes region-specific changes in simulated weightless rats and participates in the regulation of the structural and functional remodeling of arterioles small arteries.4.Sphk1 mediates the changes of Caveolae in the small arteries of in simulated weightless rats by regulating sphingolipid content.