Simulated Microgravity Induces Human Umbilical Vein Endothelial Cells Autophagy

Mechanism of cardiovascular function changes under microgravity is always an important research topic of weightlessness, which plays a vital role on the possibility of human's long-term space activities. Cardiovascular dysfunction caused by weightlessness leads to a decrease in exercise tolerance and reduced orthostatic tolerance after astronauts'return to 1 G condition. They both will endanger the safety of astronauts and affect space exploration. Researchers found that after exposure to microgravity, hypovolemia, venous compliance changes and muscle atrophy may contribute to cardiovascular dysfunction. But none of them can exactly explain this problem.Some supposed that that changes in cardiovascular function under weightlessness conditions may involve multiple and complex mechanisms.In recent years, vascular remodeling after microgravity had draw a lot of attention from reseachers. Endothelial cells which were considered as the lining of blood vessels, play an vital role in the regulation of cardiovascular function and maintaining cardiovascular system homeostasis. Autophagy, distinct from apoptosis, is the second kind of programmed cell death. By lysosomal pathway, autophagy involves in the degradation process of the majority of all long-acting proteins and most of the macromolecules, which plays an important role in cells growth, development, environmental stimulation, and diseases. In addition, a large number of studies have shown that apoptosis and autophagy could be interchangeable or antagonistic in particular circumstance, or even exist in the same cell. What's more, there are crosses between the two signal regulatory pathway. Some studies had already confirmed that simulated weightlessness could lead vascular endothelial cell to apoptosis, few researches had been reported that microgravity or simulated microgravity could induce endothelial cells autophagy. In this study, rotating clinostat was used to simulated microgravity. We observed the autophagic activity of human umbilical vein endothelial cells (HUVEC) after simulated microgravity. The main results and findings of this research were as follows.1. 48h simulated microgravity with clinotate induced HUVEC autophagy. In this study, the expressions of autophagy-related gene MAP1-LC3-II and Beclin1 were observed after 48h simulated microgravity with clinostat. Meanwhile, transmission electron microscopy was used to investigate the ultrastructure change after simulated microgravity. It is found that the mRNA level of Beclin1 and MAP1-LC3 II after 48h clinorotation was significantly higher than that of 1G control group. Similar result was found on protein expression of Beclin1 with Western Blot, indicating that the 48h weightlessness can lead to autophagy-related genes'abnormal expression. The pEGFP-LC3 plasmid transfection experiment showed more transfected cells in 48h simulated microgravity group than that of 1G control group, indicating an increase in autophagy in HUVEC after simulated microgravity. The electron microscope found that HUVEC cells after clinotation showed the formation of autophagy and autophagosome membrane. That is another evidence of HUVEC'autophagic changes after simulated microgravity.2. Beclin1 and PI3k might the key signals in microgravity induced autophagy. In the following study, wortmannin, a specific inhibitor of phosphatidylinositol 3 kinase (PI3K) which is widely used to inhibit autophagy, was used to observe the autophagic change after simulated microgravity. We found that wortmannin treatment could partly atteduate the amount of Beclin1 and LC3 mRNA after clinotation but could not reverse it. This result indicated that simulated microgravity induced autophagy via PI3k and Beclin1, which might be intial signals of this kind of change. HUVEC Matrigel assay also confirmed the above results. The network of tube-like appeared no difference after simulated microgravity only or under the treatment of Wortmannin only, and meanwhile wortmannin could increase the formation of the tubes in the microgravity group.In summary, this study demonstrated that simulated microgravity of 48h could induce human umbilical vein endothelial cells autophagy. Wortmannin treatment could partly atteduate this kind of change but could not reverse it. Our findings indecated that Beclin1 and PI3k might the key signals in microgravity induced autophagy. Autophagy of HUVEC after microgravity might lead to the vascular remodeling. This information may help to ?nd the roots of the negative physiological changes which humans and animals would face during a longer stay in the orbit.