Effects of simulated microgravity on human umbilical vein endothelial cell angiogenesis and role of the PI3K-Akt-eNOS signal pathway.
Endothelial cells are very sensitive to microgravity and the morphological and functional changes in endothelial cells are believed to be at the basis of weightlessness-induced cardiovascular deconditioning. It has been shown that the proliferation, migration, and morphological differentiation of endothelial cells play critical roles in angiogenesis. However, the influence of microgravity on the ability of endothelial cells to foster angiogenesis remains to be explored in detail. In the present study, we used a clinostat to simulate microgravity, and we observed tube formation, migration, and expression of endothelial nitric oxide synthase (eNOS) in human umbilical vein endothelial cells (HUVEC-C). Specific inhibitors of eNOS and phosphoinositide 3-kinase (PI3K) were added to the culture medium and gravity-induced changes in the pathways that mediate angiogenesis were investigated. After 24 h of exposure to simulated microgravity, HUVEC-C tube formation and migration were significantly promoted.This was reversed by co-incubation with the specific inhibitor of N-nitro-L-arginine methyl ester hydrochloride (eNOS). Immunofluorescence assay, RT-PCR, and Western blot analysis demonstrated that eNOS expression in the HUVEC-C was significantly elevated after simulated microgravity exhibition. Ultrastructure observation via transmission electron microscope showed the number of caveolae organelles in the membrane of HUVEC-C to be significantly reduced. This was correlated with enhanced eNOS activity. Western blot analysis then showed that phosphorylation of eNOS and serine/threonine kinase (Akt) were both up-regulated after exposure to simulated microgravity. However, the specific inhibitor of PI3K not only significantly downregulated the expression of phosphorylated Akt, but also downregulated the phosphorylation of eNOS. This suggested that the PI3K-Akt signal pathway might participate in modulating the activity of eNOS. In conclusion, the present study indicates that 24 h of exposure to simulated microgravity promote angiogenesis among HUVEC-C and that this process is mediated through the PI3K-Akt-eNOS signal pathway.
Department of Aerospace Biodynamics, Fourth Military Medical University, Xi'an, Shaanxi, China.
Fei
F
Shi
Yong-Chun
YC
Wang
Tian-Zhi
TZ
Zhao
Shu
S
Zhang
Ting-Yuan
TY
Du
Chang-Bin
CB
Yang
Ying-Hui
YH
Li
Xi-Qing
XQ
Sun
D021941:Caveolae / Q000187:drug effects / Q000378:metabolism; D002465:Cell Movement / Q000187:drug effects; D003094:Collagen / Q000494:pharmacology; D004338:Drug Combinations; D004789:Enzyme Activation / Q000187:drug effects; D061307:Human Umbilical Vein Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000201:enzymology* / Q000648:ultrastructure; D006801:Humans; D007797:Laminin / Q000494:pharmacology; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D018919:Neovascularization, Physiologic / Q000187:drug effects; D052250:Nitric Oxide Synthase Type III / Q000235:genetics / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D011509:Proteoglycans / Q000494:pharmacology; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D015854:Up-Regulation / Q000187:drug effects / Q000235:genetics; D018474:Weightlessness Simulation
D004338:Drug Combinations; D007797:Laminin; D011509:Proteoglycans; C059485:matrigel; D003094:Collagen; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D019331:NG-Nitroarginine Methyl Ester