The most significant and novel of the findings discussed in this manuscript are that 1) simulated microgravity promoted angiogenesis and migration behaviors through the increased eNOS expression and that 2) the up-regulation of eNOS induced by simulated microgravity is mediated by means of PI3K-Akt pathway. In this way, our studies reveal a previously unrecognized role of simulated microgravity in endothelial cell angiogenesis. We suggest underlying mechanisms for its generation and function in this process and novel complementary explanations to microgravity-induced vascular remodeling.
Angiogenesis, a process by which new blood vessels are formed, plays an important role in physiological and pathological processes in humans. ECs located in the entire inner surfaces of the blood vessels are the main players in angiogenesis. It has been reported that ECs are highly sensitive to microgravity, including the alterations in morphology and gene expression. However, effects of microgravity on ECs angiogenesis are largely unknown. Recently, Infanger et al. demonstrated that human endothelial EA.hy926 cells that were exposed to simulated microgravity formed tube-like structures (TSs) within 48 h of detaching from the bottom of a culture flask 
. Recently, they further described that these TSs consisted of several cell layers surrounding a central lumen. At the inner surface of the TSs, ECs were lined up and incorporated into abundant extracellular matrix so that the innermost layer of the TSs resembled the intima 
. The findings led to the idea that culturing ECs under conditions of modeled gravitational unloading could become a new strategy for engineering small blood vessels. In addition, it could become a new method of studying neovascularization, because angiogenesis in vivo
starts with the outgrowth of ECs, which assemble into tubes with tight cell-cell connections. Nevertheless, the angiogenic response of ECs to microgravity is still debatable. Morbidelli et al. showed that hypogravity conditions cause a marked impairment of ECs responsiveness to angiogenic factors and reduced their ability to migrate 
. Due to heterogeneity in experimental approaches, such as variations in the duration of exposure, levels of the gravity and cell types tested, the inconsistency of these results was understandable.
There are great differences between in vivo condition and in vitro model with clinorotation to study the vascular endothelium changes induced by microgravity. Recent researches showed that an exposure to microgravity shifts the mean arterial pressure of the head from 70 mmHg in the upright posture on Earth to 100 mmHg in space, and the opposite alterations occurred in lower body vascular systems. The redistribution of blood flows induced by microgravity included changes of transmural pressures and shear stress within the arterial vasculature. Therefore, in microgravity environment, the alterations of endothelium in human were not only related to microgravity, but also to the hemodynamic changes in the cardiovascular and the imbalance of nervous-humoral regulation. To investigate the effects of simulated microgravity as a single stimulation on ECs function, we performed a series of experiments with clinorotation in order to eliminate hemodynamic influences. In this study, we carried out an in vitro tube formation and wound healing experiment to evaluate the effects of simulated microgravity on endothelial cell function. Our results showed that 24 h of clinorotation enhanced angiogenesis and migration in endothelium. Our data obtained here are in agreement with previous reports 
. The cellular mechanisms involved in the alteration of endothelial functions induced by simulated microgravity are still not clearly understood.
NO, which is manufactured locally in the endothelium, is an important mediator of blood flow control, vascular permeability, and angiogenesis 
. eNOS-derived NO is a crucial contributor to the maintenance of cardiovascular homeostasis. The eNOS/NO pathway has been shown to exert a permissive role for angiogenesis in adult organisms 
. Inhibition of endogenously produced NO or disruption of the eNOS gene has been found to reduce new blood vessel formation in an animal model 
. Both in vivo
and in vitro
studies have found that suppression of eNOS activation can inhibit angiogenesis 
. In the present study, we evaluated the expression of the eNOS gene at the transcription and translation levels using RT-PCR, immunofluorescence staining, and Western blot analysis to determine whether eNOS activation is involved in the mechanisms underlying the potentiation of angiogenic responses. After 24 h of clinorotation, eNOS mRNA and protein expression were both significantly up-regulated. We then examined the angiogenesis after co-culturing with L-NAME, a specific inhibitor of eNOS. As expected, the tube formation and migration induced by simulated microgravity were dramatically suppressed in HUVEC-C.
Caveolae are specialized microdomains on the plasma membrane. They are involved in transcytosis and endocytosis, and a good body of evidence has shown that caveolae compartmentalize and integrate signaling events at the cell surface 
. A variety of protein signaling molecules involved eNOS are concentrated in caveolae. Caveolae membranes are characterized by a group of structural proteins called caveolins. Caveolin-1, a widely expressed isoform in ECs, has attracted much attention due to its ability to anchor eNOS. The binding of eNOS to caveolin-1 inhibits eNOS activity 
. Many studies have documented caveolae as endogenous negative regulator of eNOS function 
. Enzo and collaborators found that endothelial cell caveolae could constitute a mechanosensing system involved in hypergravity adaptation of human endothelial cells 
. They also indicated that short microgravity exposure strongly affected eNOS activity associated with caveolin-1 (Tyr14
) phosphorylation 
. Therefore, they have proposed that one of the early molecular mechanisms responsible for gravity sensing of endothelium involves endothelial cell caveolae and caveolin-1 phosphorylation. In this study, we observed the ultrastructure of caveolae by transmission electron microscopy and found that 24 h of exposure to simulated microgravity not only impaired the structural integrity of caveolae but also dramatically decreased the number of them in the membrane of HUVEC-C. Decreases in caveolae density in the plasma membrane are correlated with enhanced activity of eNOS.
All of these results confirmed that up-regulation of eNOS was correlated with the stimulated angiogenesis of HUVEC-C under simulated microgravity conditions. The mechanisms underlying the up-regulation of eNOS were then investigated. It has been reported that in ECs, eNOS is phosphorylated by the Akt protein kinase, resulting in an increase in eNOS activity. It plays a crucial role in the regulation of vascular tone, vascular remodeling, angiogenesis, and NO production 
. Early studies have suggested that the VEGF-induced increases in the release of NO release from ECsare attenuated by PI3K inhibitors and that VEGF stimulates Akt-mediated eNOS phosphorylation, leading to an increase in eNOS activity 
. It also has been shown to protect ECs from serum-deprivation-induced apoptosis and to promote the formation of capillary-like structures on Matrigel in an Akt-dependent manner 
. Dimmeler et al. reported that NO production in response to shear stress is controlled by the Akt-dependent phosphorylation of eNOS in cultured ECs 
. Taken together, these findings suggest that PI3K activates Akt, which in turn is responsible for regulating the phosphorylation and activation of eNOS 
. Therefore, to examine the functional involvement of Akt and eNOS in simulated microgravity-induced angiogenesis, we determined the effect of the chemical inhibitors of PI3K (LY294002) on simulated microgravity-induced signaling events. After exposure to clinorotation for 24 h, the phosphorylation of both Akt and eNOS was increased by approximately 2.9-fold and 5.4-fold, respectively, over control values. Co-culture with LY294002 not only downregulated the expression of phosphorylated Akt, but also downregulated eNOS phosphorylation, which suggested that the PI3K/Akt signal pathway participated in modulating the activity of eNOS. These experiments provide the first evidence that activation of PI3K-Akt-eNOS is a crucial event in the simulated microgravity-mediated signal transduction that leads to angiogenesis.
The present study involves examination of the underlying mechanisms of angiogenesis in HUVEC-C after exposure to simulated microgravity via clinostat device. Our results suggest that 24 h of simulated microgravity can promote angiogenesis, migration, and eNOS expression in HUVEC-C. Further investigations have confirmed that the increased levels angiogenesis after simulated microgravity in HUVEC-C are mediated via PI3K-Akt-eNOS signal pathway (). We plan a more detailed analysis of how microgravity affects the angiogenic process in ECs and we are currently investigating the precise molecular mechanisms responsible for the effects of simulated microgravity on the PI3K signaling pathway.
Role of PI3K-Akt-eNOS signaling cascade in simulated microgravity induced angiogenesis in HUVEC-C.