The Mechanism Of Autophagy Activation And Its Effects On The Functional Changes Of Endothelial Cells Induced By Simulated Microgravity

Background The cardiovascular dysfunction induced by microgravity,which is manifested with orthostatic intolerance,disability on physical exercise and even fainting,is one of the health problems facing the astronaut during longterm spaceflight.Given that the countermeasures are limited,it is of significance to study the mechanism deeply in order to develop effective countermeasures.Forming a thin layer on the interior surface of vessels,endothelial cells play a pivotal role in coordinating vascular functions through autocrine,paracrine and participating in the regulation of smooth muscle contractions,vascular wall permeability,inflammatory cells adhesion as well as angiogenesis.Autophagy is important for maintaining normal cellular homeostasis in the cellular response to stress.Comprehensive investigations have demonstrated that autophagy in endothelial cells occurs in a number of cardiovascular diseases and involves in the endothelial dysfunction.In recent years,our study have found that the autophagy in endothelial cells both in vivo and in vitro is enhanced,which we assume plays an important role in the endothelial dysfunction after microgravity.Therefore,exploring the mechanism in depth is valuable to define the target of countermeasures.Objective This research aimed to explore the relationship between autophagy and functional changes of vascular endothelial cells and clarifying the mechanism of endothelial autophagy induced by simulated microgravity in vitro.Methods 1.HUVECs cultured in vitro were randomly divided into six groups: clinostat simulated microgravity for 24h(24h MG),48h(48h MG)and 72h(72h MG)and stationary control for 24h(24h Con),48h(48h Con)and 72h(72h Con).Western blot was performed to investigate the expression levels of LC3 and p62.LC3 puncta were explored by immunofluorescence assay in HUVECs and PMVECs after exposure to microgravity for 48 h.Transmission electron microscopy assay was used to observe autophagosome and autolysosome in HUVECs and PMVECs after clinorotation for 48 h.2.3-MA was used as the specific inhibitor of autophagy and HUVECs were divided into four groups randomly: stationary control(Con),stationary control with 3-MA(Con+3-MA),clinostat simulated microgravity for 48h(MG)and clinostat simulated microgravity for 48 h with 3-MA(MG+3-MA).The expressions of LC3 and p62 were explored by western blot the number of apoptotic cells was detected using Annexin V-FITC/PI staining by flow cytometry and the expressions of the apoptosis-related protein Bcl-2,Bax and cleaved caspase3 were detected by western blot,and cell migration was accessed using Transwell migration assay and scratch wound healing assay.3.The protein expressions of LC3,p62,p53,p-AMPK and p-p70S6 K in HUVECs which can reflect the activity of m TOR after microgravity for 48 h were tested byWestern blot.Then the si RNA and over-expression plasmid were used to knock down p53 under stationary condition and overexpress p53 under simulated microgravity condition respectively,and then the expression levels of LC3,p62 and p-p70S6 K were investigated by Western blot and LC3 puncta were explored by immunofluorescence assay.4.The m RNA level of p53 in HUVECs after microgravity for 48 h was tested by q RT-PCR and the expression of p53 in nucleus and cytoplasm respectively was tested by western blot.HDM2 was knocked down by si RNA and proteasome inhibitor MG132 was used to prohibit the degradation of protein in proteasome.Under the above two conditions,the expression levels of LC3,p62,p53 and p-p70S6 K were tested by Western blot and LC3 puncta were explored by immunofluorescence assay after exposure to microgravity for 48 h.Co-IP assay was performed to explore the interaction between HDM2 and p53.HUVECs were treated with LMB to block nuclear export while exposing to clinorotation and the expression levels of LC3,p62,p-p70S6 K as well as the expression of p53 in nucleus and cytoplasm were detected.LC3 puncta were examined by immunofluorescence assay.Results 1.After exposure to clinostat simulated microgravity for 24 h,48h and 72 h,the ratio of LC3II/LC3 I was increased and the expression of p62 were decreased in HUVECs.Simulated microgravity for 48 h increased the percentage of cells containing LC3 puncta(>5)in both HUVECs and PMVECs.TEM showed the typical autophagosome and autolysosome in HUVECs and PMVECs under clinorotation condition for 48 h.2.3-MA could reverse the increased ratio of LC3II/LC3 I and decreased p62 expression after simulated microgravity in HUVECs.Compared to MG group,the presence of 3-MA when exposure to simulated microgravity increased the the apoptosis rate,decreased the expression level of Bcl-2 and increased the expressions of Bax and cleaved caspase3.Exposure to microgravity enhanced the cellular migration but the presence of 3-MA weakened the effect according to Transwell migration assay and scratch wound healing assay.3.Compared to the control group,the ratio of LC3II/LC3 I was increased,the expressions of p62 and p-p70S6 K were decreased and the expression level of p-AMPK did not change after microgravity for 48 h.The knockdown of p53 under stationary condition increased the ratio of LC3II/LC3 I,decreased the expressions of p62 and p-p70S6 K and increased the percentage of cells containing LC3 puncta(>5)while the overexpression of p53 during simulated microgravity had the opposite results.4.Compared to control group,the m RNA level of p53 in HUVECs after microgravity for 48 h did not change while the expression levels of p53 in nucleus and in cytoplasm were both decreased.MG132 or knockdown of HDM2 weakened the effect of microgravity on the expression of LC3II/LC3 I,p53,p62,p-p70S6 K and LC3 puncta.The Co-IP assay showed increased binding of HDM2 to p53.The exposure to simulated microgravity with treatment of LMB had no effect on the expression of p53 in nucleus but decreased the expression level in cytoplasm,increased the ratio of LC3II/LC3 I,decreased the expression of p62 and p-p70S6 K and increased the percentage of cells containing LC3 puncta(>5)compared to control group.Conclusion 1.Simulated microgravity increases the autophagy in HUVECs and PMVECs.2.Autophagy can partly inhibit the apoptosis and enhance cell migration in HUVECs after clinorotation for 48 h.3.After clinostat simulated microgravity for 48 h,the decreased expression of p53 in HUVECs induces autophagy through decreasing the activity of m TOR and this process is independent of AMPK.4.p53 in cytoplasm is degraded at 26 S proteasome after binding to HDM2 during clinostat simulated microgravity for 48 h,thus decreasing the activity of m TOR and inducing autophagy in HUVECs.Taken together,this study found out the enhanced autophagy in endothelial cells after exposure to simulated microgravity for the first time.The induction of autophagy can inhibit the apoptosis and enhance cell migration.The mechanism is that simulatedmicrogravity induces the binding of HDM2 to p53 which results in the degradation of p53 in cytoplasm at 26 S proteasome,thus decreasing the activation of m TOR and inducing autophagy.This study offers new insights into the mechanism of cardiovascular dysfunction induced by microgravity and provides the theoretical basis for the development of methods to protect cardiovascular system.