Effects Of Simulated Microgravity On Pulmonary Microvascular Endothelial Cells

ObjectiveMicrogravity or simulated microgravity can induce dysfunction of pulmonary circulation, in which the changes of vascular structure and function have important role. The function of pulmonary microvascular endothelial cells play important role in maintaining the normal functions of pulmonary circulation. Moreover, pulmonary microvascular endothelial cells compose the main physical barrier for microvascular permeability, which can not only guarantee the normal substance exchange in microvascular, but also excrete all kinds of factors regulating the contractile and relaxing function of microvascular. However, there is not any report concerning the effects of microgravity or simulated microgravity on the function of pulmonary microvascular endothelial cells until now, the mechanisms of changes for pulmonary microvascular endothelial cells structure and function are not clear. So, the purpose of this study is following::(1) Observe effects of simulated microgravity induced by clinostat on ultrastructure of pulmonary microvascular endothelial cells.(2) Study the effects of simulated microgravity induced by clinostat on NO production and protein expression of eNOS in pulmonary microvascular endothelial cells.(3) Study the effects of simulated microgravity on cytoskeleton of pulmonary microvascular endothelial cells.(4) Study the effects of simulated microgravity on apoptosis in pulmonary microvasuclar endothelial cells and its molecular mechanisms.MethodsPulmonary microvascular endothelial cells were cultured by implanting tissue pieces and were identified by cell morphology under inverted phase contrast microscope,â…§factor immunofluorescence staining, scanning electron microscopy and transmission electron microscopy. Simulated microgravity was induced by clinostat, scanning and transmission electron microscopy were used to observe the ultrastructure of pulmonary microvascular endothelial cells. NO concentration released by pulmonary microvascular endothelial cells was measured by Griess method, eNOS protein expression was measured by western blot and immunostaining methods. Annexinâ…¤-FITC and PI were used to label cells, apoptosis of pulmonary microvascular endothelial cells was detected by nucleus staining with Hoechst 33258 and flow cytometry. By immunofluorescence technique, phalloidin labeled by Texas Red-X was used to incubate with pulmonary microvascular endothelial cells to detect the microfilament cytoskeleton. Radioimmunoassay technique was used to detect the ET-1 concentration released by pulmonary microvascular endothelial cells, western blot and immunofluorescence methods were used to measure the changes of Akt phosphorylation and P21Cip1 protein expression. Protein expression of apoptosis regulating factors Bcl-2 and Bax were measured by western blot.ResultsThe results from sanning electron microscopy showed that the contacts of pulmonary microvascular endothelial cells became incompact and obvious crack appeared after 48h clinostat, which were not observed in control pulmonary microvascular endothelial cells. These results illuminated that simulated microgravity can destroy the cell monolayer of pulmonary microvascular endothelial cells and induced the increased permeability of cell monolayer. The results from transmission electron microscopy showed that the mitochondrion became hyperplasia and swelling. The results of NO measurement, western blot and immunostaining showed that simulated microgravity induced the increased NO production and eNOS protein expression which were dependent with time of clinostat. The results from Hoechst33258 and flow cytometry showed that 48h clinostat induced the increased apoptosis rate of pulmonary microvascular endothelial cells. Microfilament staining by phalloidin showed that simulated microgravity induced microfilament depolymerization which was progressive with time of clinostat. After 48h clinostat, the results from radioimmunoassay showed that ET-1 released by pulmonary microvascular endothelial cells was lower than that of control. The results of western blot and immunofluorescence showed that the phosphorylation level of Akt was decreased, the protein expression of P21Cip1 was increased. The measurements of apoptosis related proteins by western blot showed that simulated microgravity significantly decreased the protein expression of Bcl-2 but increased the expression of Bax protein.Conclusion(1) Simulated microgravity induces mitochondrion hyperplasia and swelling in pulmonary microvascular endothelial cells, destroys the cell monolayer of pulmonary microvascular endothelial cells and induced the increased permeability of cell monolayer.(2) Simulated microgravity stimulates NO synthesis and releaseing, increases the protein expression of eNOS, which are dependent with time of clinostat.(3) Simulated microgravity induces early apoptosis of pulmonary microvascular endothelial cells.(4) Simulated microgravity induces microfilament cytoskeleton depolymerization in pulmonary microvascular endothelial cells.(5) Simulated microgravity decreases the secretion of ET-1, increases the opening of mPTP, inhibits the phosphorylation level of Akt, increases the protein expression of P21Cip1 in pulmonary microvascular endothelial cells. Moreover, the protein expression of Bcl-2 was decreased and Bax was increased by simulated microgravity induced 48h clinostat.