Effect of miR-27b-5p on apoptosis of human vascular endothelial cells induced by simulated microgravity.

Weightlessness-induced cardiovascular dysfunction can lead to physiological and pathological consequences. It has been shown that spaceflight or simulated microgravity can alter expression profiles of some microRNAs (miRNAs). Here, we attempt to identify the role of miRNAs in human umbilical vein endothelial cells (HUVECs) apoptosis under simulated microgravity. RNA-sequencing and quantitative real-time PCR (qRT-PCR) assays were used to identify differentially expressed miRNAs in HUVECs under simulated microgravity. Then we obtained the target genes of these miRNAs through target analysis software. Moreover, GO and KEGG enrichment analysis were performed. The effects of these miRNAs on HUVECs apoptosis were evaluated by flow cytometry, Western blot and Hoechst staining. Furthermore, we obtained the target gene of miR-27b-5p by luciferase assay, qRT-PCR and Western blot. Finally, we investigated the relationship between this target gene and miR-27b-5p in HUVECs apoptosis under normal gravity or simulated microgravity. We found 29 differentially expressed miRNAs in HUVECs under simulated microgravity. Of them, the expressions of 3 miRNAs were validated by qRT-PCR. We demonstrated that miR-27b-5p affected HUVECs apoptosis by inhibiting zinc fingers and homeoboxes 1 (ZHX1). Our results reported here demonstrate for the first time that simulated microgravity can alter the expression of some miRNAs in HUVECs and miR-27b-5p may protect HUVECs from apoptosis under simulated microgravity by targeting ZHX1.

Autophagy protects HUVECs against ER stress-mediated apoptosis under simulated microgravity.

Astronauts exposed to a gravity-free environment experience cardiovascular deconditioning that causes post-spaceflight orthostatic intolerance and other pathological conditions. Endothelial dysfunction is an important factor responsible for this alteration. Our previous study showed enhanced autophagy in endothelial cells under simulated microgravity. The present study explored the cytoprotective role of autophagy under microgravity in human umbilical vein endothelial cells (HUVECs). We found that clinorotation for 48 h induced apoptosis and endoplasmic reticulum (ER) stress in HUVECs. ER stress and the unfolded protein response (UPR) partially contributed to apoptosis under clinorotation. Autophagy partially reduced ER stress and restored UPR signaling by autophagic clearance of ubiquitin-protein aggregates, thereby reducing apoptosis. In addition, the ER stress antagonist 4-phenylbutyric acid upregulated autophagy in HUVECs. Taken together, these findings indicate that autophagy plays a protective role against apoptosis under clinorotation by clearing protein aggregates and partially restoring the UPR.

Clinorotation-induced autophagy via HDM2-p53-mTOR pathway enhances cell migration in vascular endothelial cells.

Individuals exposed to long-term spaceflight often experience cardiovascular dysfunctions characterized by orthostatic intolerance, disability on physical exercise, and even frank syncope. Recent studies have showed that the alterations of cardiovascular system are closely related to the functional changes of endothelial cells. We have shown previously that autophagy can be induced by simulated microgravity in human umbilical vein endothelial cells (HUVECs). However, the mechanism of enhanced autophagy induced by simulated microgravity and its role in the regulation of endothelial function still remain unclear. We report here that 48 h clinorotation promoted cell migration in HUVECs by induction of autophagy. Furthermore, clinorotation enhanced autophagy by the mechanism of human murine double minute 2 (HDM2)-dependent degradation of cytoplasmic p53 at 26S proteasome, which results in the suppression of mechanistic target of rapamycin (mTOR), but not via activation of AMPK in HUVECs. These results support the key role of HDM2-p53 in direct downregulation of mTOR, but not through AMPK in microgravity-induced autophagy in HUVECs.

Moderate exercise based on artificial gravity preserves orthostatic tolerance and exercise capacity during short-term head-down bed rest.

Numerous countermeasures have been proposed to minimize microgravity-induced physical deconditioning, but their benefits are limited. The present study aimed to investigate whether personalized aerobic exercise based on artificial gravity (AG) mitigates multisystem physical deconditioning. Fourteen men were assigned to the control group (n=6) and the countermeasure group (CM, n=8). Subjects in the CM group were exposed to AG (2 Gz at foot level) for 30 min twice daily, during which time cycling exercise of 80-95 % anaerobic threshold (AT) intensity was undertaken. Orthostatic tolerance (OT), exercise tests, and blood assays were determined before and after 4 days head-down bed rest (HDBR). Cardiac systolic function was measured every day. After HDBR, OT decreased to 50.9 % and 77.5 % of pre-HDBR values in control and CM groups, respectively. Exercise endurance, maximal oxygen consumption, and AT decreased to 96.5 %, 91.5 % and 91.8 % of pre-HDBR values, respectively, in the control group. Nevertheless, there were slight changes in the CM group. HDBR increased heart rate, sympathetic activity, and the pre-ejection period, but decreased plasma volume, parasympathetic activity and left-ventricular ejection time in the control group, whereas these effects were eliminated in the CM group. Aldosterone had no change in the control group but increased significantly in the CM group. Our study shows that 80-95 % AT aerobic exercise based on 2 Gz of AG preserves OT and exercise endurance, and affects body fluid regulation during short-term HDBR. The underlying mechanisms might involve maintained cardiac systolic function, preserved plasma volume, and improved sympathetic responses to orthostatic stress.

Simulated Microgravity Promotes Angiogenesis through RhoA-Dependent Rearrangement of the Actin Cytoskeleton.

BACKGROUND/AIMS: Microgravity leads to hydrodynamic alterations in the cardiovascular system and is associated with increased angiogenesis, an important aspect of endothelial cell behavior to initiate new vessel growth. Given the critical role of Rho GTPase-dependent cytoskeleton rearrangement in cell migration, small GTPase RhoA might play a potential role in microgravity-induced angiogenesis. METHODS: We examined the organization of actin filaments by FITC-conjugated phalloidin staining, as well as the expression and activity of RhoA by quantitative PCR and Western blot, in human umbilical vein endothelial cells (HUVECs) under normal gravity and simulated microgravity. Effect of simulated microgravity on the wound closure and tube formation in HUVECs, and their dependence on RhoA, were also analyzed by cell migration and tube formation assays. RESULTS: We show that in HUVECs actin filaments are disorganized and RhoA activity is reduced by simulated microgravity. Blocking RhoA activity either by C3 transferase Rho inhibitor or siRNA knockdown mimicked the effect of simulated microgravity on inducing actin filament disassembly, followed by enhanced wound closure and tube formation in HUVECs, which closely resembled effects seen on microgravity-treated cells. In contrast, overexpressing RhoA in microgravity-treated HUVECs restored the actin filaments, and decreased wound closure and tube formation abilities. CONCLUSION: These results suggest that RhoA inactivation is involved in the actin rearrangement-associated angiogenic responses in HUVECs during simulated microgravity.

The Impact of Simulated Weightlessness on Endothelium-Dependent Angiogenesis and the Role of Caveolae/Caveolin-1.

BACKGROUND/AIMS: The potential role of caveolin-1 in modulating angiogenesis in microgravity environment is unexplored. METHODS: Using simulated microgravity by clinostat, we measured the expressions and interactions of caveolin-1 and eNOS in human umbilical vein endothelial cells. RESULTS: We found that decreased caveolin-1 expression is associated with increased expression and phosphorylation levels of eNOS in endothelial cells stimulated by microgravity, which causes a dissociation of eNOS from caveolin-1 complexes. As a result, microgravity induces cell migration and tube formation in endothelial cell in vitro that depends on the regulations of caveolin-1. CONCLUSION: Our study provides insight for the important endothelial functions in altered gravitational environments.

Clinorotation enhances autophagy in vascular endothelial cells.

Individuals exposed to extended periods of spaceflight or prolonged 6° head-down-tilt bed rest often suffer from health hazards represented by cardiovascular deconditioning. Many studies have reported that alterations in vascular endothelial cells contribute to cardiovascular dysfunction induced by microgravity. Autophagy, a lysosomal degradation pathway, serves an adaptive role for survival, differentiation, and development in cellular homeostasis, and can be triggered by various environmental stimuli. However, whether autophagy can be induced in endothelial cells by real or simulated microgravity remains to be determined. This study was designed to investigate the effects of simulated microgravity on the activation of autophagy in human umbilical vein endothelial cells (HUVECs). We report here that clinorotation, a simulated model of microgravity, enhances autophagosome formation, increases LC3 and beclin-1 expression, and promotes the conversion of LC3-I to LC3-II in HUVECs. These results demonstrate that simulated microgravity for 48 h activates autophagy of vascular endothelial cells.

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.

Visual symptoms and G-induced loss of consciousness in 594 Chinese Air Force aircrew--a questionnaire survey.

A questionnaire survey was performed for the first time to assess the prevalence of visual symptoms and G-induced loss of consciousness (G-LOC) due to +Gz exposure in the Chinese Air Force (CAF) to determine the effectiveness of current G tolerance training. Responses were received from 594 individuals. Among them, 302 reported at least one episode of some sort of symptoms related to +Gz, including 110 (18.5%) with visual blurring, 231 (38.9%) with greyout, 111 (18.7%) with blackout, and 49 (8.2%) with G-LOC. Incidences were most common in aircrew with 250-1,000 flying hours (53.6%) and were more prevalent in those with fewer on type flying hours (p < 0.001). The most common situation was reported between +5 and 5.9 Gz. The results indicate a fairly high prevalence of visual symptoms and G-LOC among Chinese Air Force aircrew. There remains considerable scope for +Gz education, particularly in the early centrifuge training and selection of rational physical exercises.

Artificial gravity with ergometric exercise can prevent enhancement of popliteal vein compliance due to 4-day head-down bed rest.

Changes of venous compliance may contribute in part to postflight orthostatic intolerance. The purpose of the present study was to determine whether intermittent artificial gravity exposure with ergometric exercise could prevent venous compliance changes in the lower limbs due to simulated weightlessness. Twelve healthy male volunteers were exposed to simulated microgravity for 4 days of head-down bed rest (HDBR). Six subjects were randomly loaded 1.0-2.0 Gz intermittent artificial gravity (at foot level) with 40 W of ergometric workload every day (countermeasure group, CM). The six others served as the control (CON group). Venous compliance was estimated by measuring the corresponding change of cross-sectional area (CSA) of popliteal vein at each minute of various venous occlusion pressure stages. Basal CSA was significantly lower after bed rest in the control group, and preserved in the countermeasure group. The percent increase in the CSA of CON group was significantly greater almost at each minute of various venous cuff pressures after bed rest than before. Compliance of popliteal vein of CON group was significant greater when 40, 60 and 80 mmHg cuff pressure applied after bed rest than before of CON group. In conclusions, a 4-day simulated weightlessness leads to increase of popliteal venous compliance; centrifuge-induced artificial gravity with ergometric exercise can prevent enhancement of popliteal venous compliance due to 4-day head-down tilt bed rest, the effect of the countermeasure on compliance might involve changes in venous filling and changes in venous structure.

Microgravity-induced cardiovascular deconditioning: mechanisms and countermeasures / 中国应用生理学杂志

It has been demonstrated that individuals exposed to actual or simulated microgravity often experience cardiovascular dysfunctions when returning to Earth. The underlying mechanisms of orthostatic intolerance and countermeasure treatment are still poorly understood. In this paper, the progress in the mechanism of cardiovascular deconditioning from the view of vascular remodeling, increased venous compliance in the lower limbs, cellular proliferation and differentiation, and cell signaling pathway was reviewed. Meanwhile, an overview of the countermeasures including exercise, lower body negative pressure, thigh cuffs, traditional Chinese herb medicine and artificial gravity was presented.

Artificial gravity with ergometric exercise preserves the cardiac, but not cerebrovascular, functions during 4 days of head-down bed rest.

Cardiovascular and musculoskeletal deconditioning occurring in long-term spaceflight requires new strategies to counteract these adverse effects. We previously reported that a short-arm centrifuge produced artificial gravity (AG), together with ergometer, has an approving effect on promoting cardiovascular function. The current study sought to investigate whether the cardiac and cerebrovascular functions were maintained and improved using a strategy of AG combined with exercise training on cardiovascular function during 4-day head-down bed rest (HDBR). Twelve healthy male subjects were assigned to a control group (CONT, n=6) and an AG combined with ergometric exercise training group (CM, n=6). Simultaneously, cardiac pumping and systolic functions, cerebral blood flow were measured before, during, and after HDBR. The results showed that AG combined with ergometric exercise caused an increase trend of number of tolerance, however, there was no significant difference between the two groups. After 4-day HDBR in the CONT group, heart rate increased significantly (59±6 vs 66±7 beats/min), while stroke volume (98±12 vs 68±13 mL) and cardiac output (6±1 vs 4±1 L/min) decreased significantly (p<0.05). All subjects had similar drops on cerebral vascular function. Volume regulating hormone aldosterone increased in both groups (by 119.9% in CONT group and 112.8% in the CM group), but only in the CONT group there were a significant changes (p<0.05). Angiotensin II was significantly increased by 140.5% after 4-day HDBR in the CONT group (p<0.05), while no significant changes were observed in the CM group. These results indicated that artificial gravity with ergometric exercise successfully eliminated changes induced by simulated weightlessness in heart rate, volume regulating hormones, and cardiac pumping function and partially maintained cardiac systolic function. Hence, a daily 1h alternating +1.0 and +2.0 Gz with 40 W exercise training appear to be an effective countermeasure against cardiac deconditioning.

Horizontal canal benign paroxysmal positional vertigo in a fighter pilot.

Benign paroxysmal positional vertigo (BPPV) is the most common disorder of the peripheral vestibular system, characterized by intense, positional provoked vertigo. BPPV is thought to occur due to canalithiasis of the posterior semicircular canal. Recently, a new entity of BPPV, known as horizontal canal (HC)-BPPV, has been recognized. Although only 3 to 8% of BPPV is due to horizontal canal involvement, HC-BPPV is not rare. We present a case of a naval fighter pilot who had an incident of HC-BPPV on the ground. The pilot aeromedical evaluation and considerations are discussed.

Artificial gravity with ergometric exercise as a countermeasure against cardiovascular deconditioning during 4 days of head-down bed rest in humans.

We have shown previously that combined short-arm centrifuge and aerobic exercise training preserved several physiologically important cardiovascular functions in humans. We hypothesized that artificial gravity (AG) and exercise is effective to prevent changes of physical problems during head-down bed rest (HDBR). To test this hypothesis, 12 healthy male subjects had undergone 4 days of 6° HDBR. Six of them were exposed to AG of an alternating 2-min intervals of +1.0 and +2.0 Gz at foot level for 30 min twice per day with ergometric exercise of 40 W as a countermeasure during bed rest (CM group), while the remaining six served as untreated controls (no-CM group). Before and after 4 days of bed rest, leg venous hemodynamics was assessed by venous occlusion plethysmography and autonomic cardiovascular control estimated by power spectral analysis of blood pressure and heart rate. Further, orthostatic tolerance was evaluated by a 75° head-up tilt test and physical working capacity was surveyed by near maximal physical working capacity test before and after bed rest. The data showed that combined centrifuge and exercise applied twice daily for a total of 60 min during 4 days of HDBR prevented (a) a decrease in working capacity, (b) autonomic dysfunction (a decrease in the activity of parasympathetic cardiac innervation) and (c) an increase in leg venous flow resistance. The combination of a 30 min alternating of +1.0 and +2.0 Gz for twice per day of AG with 40 W ergometric exercise may offer a promising countermeasure to short duration simulated microgravity.

Combined short-arm centrifuge and aerobic exercise training improves cardiovascular function and physical working capacity in humans.

BACKGROUND: Musculoskeletal and cardiovascular deconditioning occurring in long-term spaceflight gives rise to the needs to develop new strategies to counteract these adverse effects. Short-arm centrifuge combined with ergometer has been proposed as a strategy to counteract adverse effects of microgravity. This study sought to investigate whether the combination of short-arm centrifuge and aerobic exercise training have advantages over short-arm centrifuge or aerobic exercise training alone. MATERIAL/METHODS: One week training was conducted by 24 healthy men. They were randomly divided into 3 groups: (1) short-arm centrifuge training, (2) aerobic exercise training, 40 W, and (3) combined short-arm centrifuge and aerobic exercise training. Before and after training, the cardiac pump function represented by stroke volume, cardiac output, left ventricular ejection time, and total peripheral resistance was evaluated. Variability of heart rate and systolic blood pressure were determined by spectral analysis. Physical working capacity was surveyed by near maximal physical working capacity test. RESULTS: The 1-week combined short-arm centrifuge and aerobic exercise training remarkably ameliorated the cardiac pump function and enhanced vasomotor sympathetic nerve modulation and improved physical working capacity by 10.9% (P<.05, n=8). In contrast, neither the short-arm centrifuge nor the aerobic exercise group showed improvements in these functions. CONCLUSIONS: These results demonstrate that combined short-arm centrifuge and aerobic exercise training has advantages over short-arm centrifuge or aerobic exercise training alone in influencing several physiologically important cardiovascular functions in humans. The combination of short-arm centrifuge and aerobic exercise offers a promising countermeasure to microgravity.

[Apoptosis in myocyte after repeated + Gz exposures in rats].

OBJECTIVE: To observe the effects of repeated + Gz exposures on the apoptosis of myocyte in rats. METHODS: Twelve male Sprague-Dawley rats were randomly divided into three groups: Control group, + 6Gz group and + 10Gz group. The rats of + Gz groups were exposed to + 6Gz for 3 min, + 10Gz for 3 min respectively, 1 b/d, 1 week. Four control rats were kept at the Earth gravity (1G) in the room with the centrifuge. All animals were anaesthetized and anatomies 1 day after the last exposure. Ventricular myocardium was studied by electron microscopy and terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL) method. RESULTS: The apoptosis of myocyte in control group and + 6Gz group were scarcely observed by electron microscopy, while heterochromatin concentration and margination were observed in + 10Gz group. The apoptotic index of myocardium increased significantly in + 6Gz and + 10Gz group compared with that of the control group (P < 0.05) and showed the largest value in the + 10Gz group (P < 0.05). CONCLUSION: Repeated + Gz exposures may induce apoptosis in myocyte, and the number of apoptosis in myocyte increases gradually with the increase of G value.

Hyperbaric oxygen preconditioning reduces ischemia-reperfusion injury by stimulating autophagy in neurocyte.

Cerebral ischemia-reperfusion injury (IRI) is a complex process resulting in cellular damage and death. Many studies have reported that an ischemic preconditioning could induce protection against ischemic insult. However, the safety concerns and practical feasibility have limited the application of ischemia preconditioning in practice. Subsequently, a number of substances including endotoxin and cytokines etc. have proven effective in inducing ischemic tolerance in the neurocyte. Unfortunately, the application of these substances to the clinical practice of neurosurgery still remains questionable for their toxicity or side effects. Therefore, a novel therapy to protect against cerebral IRI requires further study. Several recent studies confirmed that repeated hyperbaric oxygen preconditioning (HBO-PC) prior to cerebral ischemia or spinal cord ischemia can provide neuroprotection. HBO as a therapeutic measure has been widely accepted for its convenience and safety. However, information about the mechanism of how this neuroprotection works is still very limited. We hypothesize that autophagy induction is involved in HBO-PC induced neuroprotection on IRI in neurocyte. The hypothesis reveals that autophagy may be a new therapeutic target for cerebral IRI.

Clinorotation upregulates inducible nitric oxide synthase by inhibiting AP-1 activation in human umbilical vein endothelial cells.

Alterations of nitric oxide contribute to post-flight orthostatic intolerance. The aim of this study was to investigate the changes of inducible nitric oxide synthase (iNOS) and the mechanisms underlying regulation of iNOS by simulated microgravity in human umbilical vein endothelial cells (HUVECs). Clinorotation, a simulated-model of microgravity, increased iNOS expression and promoter activity in HUVECs. The transactivations of NF-kappaB and AP-1 were suppressed by 24 h clinorotation. A key role for AP-1, but not NF-kappaB in the regulation of iNOS was shown. (1) PDTC, a NF-kappaB inhibitor, had no effect on clinorotation upregulation of iNOS. (2) SP600125, a JNK-specific inhibitor, which resulted in inhibition of AP-1 activity, enhanced the iNOS expression and promoter activity in clinorotation. (3) Overexpression of AP-1 remarkably attenuated the upregulation effect of clinorotation. These findings indicate that clinorotation upregulates iNOS in HUVECs by a mechanism dependent on suppression of AP-1, but not NF-kappaB. These results support a key role for AP-1 in the signaling of postflight orthostatic intolerance.

Simulated weightlessness aggravates hypergravity-induced impairment of learning and memory and neuronal apoptosis in rats.

It has been demonstrated that altered gravity may lead to impairments in cognitive functions. However, the effect of a combination of hypergravity and weightlessness on cognitive functions is not well understood. In the present study, we report the effects of high sustained hypergravity after 7 days' simulated weightlessness on learning and memory abilities and neuronal apoptosis in rats. In the Y-maze tests, hypergravity (HG) or simulated weightlessness (SW) significantly decreases accuracy, and increases reaction time of rats compared to that of controls. On the contrary, in the passive avoidance test, HG or SW treatment significantly shortens latency and prolongs total time compared to those of controls. In addition, TUNEL staining shows a few apoptotic cells in cortex and hippocampus in the HG, SW and HG+SW groups, and the number of TUNEL positive cells was found to be the most in the HG+SW group. Furthermore, rats with combined HG and SW treatment reveal a synergistic effect in both the Y-maze and the passive avoidance tests, as well as increased neuronal cell death. These findings suggest that simulated weightlessness may exacerbate hypergravity-induced impairment of learning and memory, likely caused by neuronal cell death in rats.

Heart rate and respiration responses to real traffic pattern flight.

The purpose of this study was to observe heart rate and respiration responses to real traffic pattern flight. Nine experienced and nine less-experienced military pilots on active flying status participated in four uninterrupted traffic patterns flight missions with F-7 jet trainer. The heart rates and respiration waves were continuously recorded using a small recording device strapped around the chest. As compared with baseline values, significant increases in heart rates of the two groups (for experienced pilots group, F (11, 88) = 4.636, p = 0.000; for less-experienced, F (11, 88) = 4.437, p = 0.000) and mean respiration rates of less-experienced group (F (11, 88) = 4.488, p = 0.000) were obtained during the phases of take-off, final approach and landing. Heart rates of less-experienced pilots were significant higher than those of experienced pilots during the take-off phase (p < 0.05). There were no significant differences in respiration rates between the two groups at each phase of the whole flight. The results show that take-off, final approach and landing are the most mental workload phases in-flight, and less-experienced pilots show more mental workload than experienced pilots in take-off phase in-flight.