Microgravity simulation models

The term ‘microgravity’ is defined as the condition in which the sum of all forces producing accelerations does not exceed 10⁻⁶ g although it is usually mistaken as a synonym to the absence of gravity. Microgravity is actually achieved about 300–400 km over Earth, where accelerations on Moon and Mars have been referred to be 0.17 g and 0.38 g, respectively (Herranz et al., Reference Herranz, Anken, Boonstra, Braun, Christianen, De Geest, Hauslage, Hilbig, Hill, Lebert, Medina, Vagt, Ullrich, Van Loon and Hemmersbach2013).

Research under microgravity conditions is necessary in order to discover the effect of microgravity on biological processes. However, research under such circumstances is constrained by the high cost of space flights. Microgravity simulators on Earth are valuable tools which help scientists to reproduce conditions that resemble microgravity for their experiments (Herranz et al., Reference Herranz, Anken, Boonstra, Braun, Christianen, De Geest, Hauslage, Hilbig, Hill, Lebert, Medina, Vagt, Ullrich, Van Loon and Hemmersbach2013).

Long duration bed rest is a human microgravity analog used for the disclosure of the impact of microgravity on the human body. Especially, 6° head down tilt bed rest has been proposed by Russian investigators as the most suitable analog for microgravity simulation to study the impact of microgravity on bone metabolism and muscles, as well as the development of countermeasures used to eliminate the adverse effects (Hargens and Vico, Reference Hargens and Vico2016).

The rodent hindlimb unloading model by tail suspension was developed in the 1980s in order to study the effects of space flight on human organ systems, the underlying mechanisms and the countermeasures developed to deal with the detrimental effects on human health (Globus and Morey-Holton, Reference Globus and Morey-Holton2016).

The National Aeronautics and Space Administration (NASA) has developed a RWV (Rotating Wall Vessel) in order to study the effects of reduced mechanical forces on cell cultures, as a cellular analog to microgravity. The rotating wall vessel is a suspension culture vessel with a diffusion membrane for gas exchange without bubbles, where the cells are placed on micro-carrier beads and the culture medium rotates in a horizontal axis, which results in reduced shear stress and turbulence. The constant rotation of the culture results in a constant change of gravitational vector, which resembles free fall and some aspects of microgravity (Hammond and Hammond, Reference Hammond and Hammond2001).

The effect of microgravity on bone

Exposure to real and simulated microgravity results in alterations in bone metabolism (Table 1). A significant problem related to exposure to microgravity is bone loss. Members of space missions in RSS (Russian Space Station) and ISS (International Space Station) have experienced 1% reduction in lumbar spine bone mineral density and 1–1.6% reduction in hip bone mineral density during a month's time, where an equal percent of bone loss is experienced by menopause women in a year (Vico et al., Reference Vico, Collet, Guignandon, Lafage-Proust, Thomas, Rehaillia and Alexandre2000). Significant aspects of gravity influence cellular functions. For example, cells rearrange their cytoskeleton depending on the forces exerted on them and the cytoskeleton is less prominent under microgravity conditions (Arfat et al., Reference Arfat, Xiao, Iftikhar, Zhao, Li, Sun, Zhang, Shang and Qian2014). Reduced mechanical forces under microgravity result in increased bone resorption which cannot be compensated by bone formation (Hughes-Fulford, Reference Hughes-Fulford2003).

Table 1. Influence of microgravity on bone cells

There are four main types of bone cells: mesenchymal stem cells, osteoblasts, osteocytes and osteoclasts, which are influenced by microgravity. Microgravity results in maintenance of an undifferentiated state of mesenchymal stem cells induce their proliferation and lead to differentiation into adipocytes instead of osteoblasts (Yuge et al., Reference Yuge, Sasaki, Kawahara, Wu, Matsumoto, Manabe, Kajiume, Takeda, Magaki, Takahashi, Kurisu and Matsumoto2011). ERK kinases play a significant role in the signalling pathway which controls the differentiation of mesenchymal stem cells to adipocytes (Feve, Reference Feve2005). Modelled microgravity-induced alterations in the cytoskeleton of mesenchymal stem cells, such as thinner F-actin fibres and change of the shape of the cells (Buravkova et al., Reference Buravkova, Romanov, Konstantinova, Buravkov, Gershovich and Grivennikov2008). Some researchers support that alterations of the cytoskeleton contribute to restrictions in the differentiation of mesenchymal stem cells to osteoblasts (Zayzafoon et al., Reference Zayzafoon, Gathings and Mcdonald2004).

Real and modeled microgravity reduces osteoblastic differentiation, proliferation and activity, as well as their responses to environmental stimuli (Arfat et al., Reference Arfat, Xiao, Iftikhar, Zhao, Li, Sun, Zhang, Shang and Qian2014). MC3T3 cell line cultured under modeled microgravity developed less complex cytoskeleton, which is related to the nucleus activity. Besides this, microgravity disturbs microfibres of osteoblasts, which results in less bone formation (Arfat et al., Reference Arfat, Xiao, Iftikhar, Zhao, Li, Sun, Zhang, Shang and Qian2014). Carmeliet et al. (Reference Carmeliet, Nys and Bouillon1997) have proved that alkaline phosphatase expression and activity is significantly reduced under microgravity conditions (Carmeliet et al., Reference Carmeliet, Nys and Bouillon1997).

Osteocytes act as mechanical sensors through their dendritic axons (Bonewald and Johnson, Reference Bonewald and Johnson2008). The sensitivity of osteocytes to mechanical stimuli is reduced under modelled microgravity conditions and F-actin fibres are deranged (Di et al., Reference Di, Qian, Qu, Zhang, Wang, Ding, Li, Ren and Shang2011). Apoptosis of osteocytes is essential in order for bone remodelling to begin (Kennedy et al., Reference Kennedy, Herman, Laudier, Majeska, Sun and Schaffler2012). Besides, osteocytes produce sclerostin, which is an inhibitor of Wnt-β catenin signalling pathway, which is crucial in osteoblastogenesis (Lin et al., Reference Lin, Jiang, Dai, Guo, Weng, Wang, Li, Feng, Gao and He2009).

Osteoclast differentiation is induced under microgravity conditions and an increased amount of collagen telopeptides has been found in osteoclast cultures, indicating increased osteoclast activity (Tamma et al., Reference Tamma, Colaianni, Camerino, Di Benedetto, Greco, Strippoli, Vergari, Grano, Mancini, Mori, Colucci, Grano and Zallone2009). Sambandam et al. (Reference Sambandam, Blanchard, Daughtridge, Kolb, Shanmugarajan, Pandruvada, Bateman and Reddy2010) have proved a significant increase in expression of genes involved in osteoclast differentiation using microarray analysis (Sambandam et al., Reference Sambandam, Blanchard, Daughtridge, Kolb, Shanmugarajan, Pandruvada, Bateman and Reddy2010). Nabavi et al. (Reference Nabavi, Khandani, Camirand and Harrison2011) observed an increase in resorption pits in bones under microgravity conditions and a significant increase in osteoclast resorption activity (Nabavi et al., Reference Nabavi, Khandani, Camirand and Harrison2011). Modelled microgravity-induced the production of radical oxygen species in preosteoclast cell line RAW264.7, which have a crucial role in bone resorption and osteoclastogenesis induced by RANKL (Lee et al., Reference Lee, Choi, Baik, Han, Jeong, Bae, Kim and Lee2005).

Autophagy

Autophagy is a recycling cellular process of eukaryotic cells, which cells use to decompose damaged organelles and other macromolecules into amino acids, sugars, nucleonic acids and then use the ‘raw material’ to compose new macromolecules or produce the energy they need (Mizushima, Reference Mizushima2007). All cells show a basic level of autophagy, which serves as a quality control mechanism for cellular components. The level of autophagy increases under stress conditions, such as fasting periods, oxidative stress, hypoxia or infection, in order to protect the cell from potential damage (Kroemer et al., Reference Kroemer, Marino and Levine2010). It has recently been proved that autophagy is related to cellular life expectancy. Ageing reduces the level of autophagy in cells, whereas inducing autophagy pharmacologically or genetically results in longevity (Carmona-Gutierrez et al., Reference Carmona-Gutierrez, Hughes, Madeo and Ruckenstuhl2016). Defective autophagy has been related to diseases, such as infections, cancer and neurodegenerative diseases (Mizushima et al., Reference Mizushima, Levine, Cuervo and Klionsky2008).

Impact of autophagy on bone cells

Autophagy disorders have been recently related to bone cell dysfunction (Table 2). Induction of autophagy contributes to the survival of mesenchymal stem cells under oxidative stress circumstances (Song et al., Reference Song, Song and Tong2014). Autophagy is involved in the preservation of stemness, which is the ability of mesenchymal stem cells to renew themselves and differentiate into different cell types, as it has been observed in human cells from the umbilical cord (Hou et al., Reference Hou, Han, Jing, Yang, Zhang, Sun, Hao, Meng, Yu, Liu, Shi, Wu, Zhang and Wei2013). Nuschke et al. (Reference Nuschke, Rodrigues, Stolz, Chu, Griffith and Wells2014) have observed aggregated autophagosomes in early stages of differentiation of mesenchymal stem cells, which they consume as energy fuel in order to differentiate into osteoblasts (Nuschke et al., Reference Nuschke, Rodrigues, Stolz, Chu, Griffith and Wells2014).

Table 2. Impact of autophagy on bone cells

Experiments with osteoblast cell lines have proved that autophagy increased the differentiation of osteoblasts and bone mineralization, which is the main activity of osteoblasts. Liu et al. have observed that autophagy is important in terminal stages of osteoblastogenesis, without being implicated in proliferation or early stages of osteoblast differentiation (Liu et al., Reference Liu, Fang, Yuan, Yang, Chen, Williams, Goldstein, Krebsbach and Guan2013). Nollet et al. (Reference Nollet, Santucci-Darmanin, Breuil, Al-Sahlanee, Cros, Topi, Momier, Samson, Pagnotta, Cailleteau, Battaglia, Farlay, Dacquin, Barois, Jurdic, Boivin, Heymann, Lafont, Lu, Dempster, Carle and Pierrefite-Carle2014) have observed autophagosomes containing hydroxyapatite in osteoblasts. Osteoblasts use autophagy as a mechanism for exocytosis of hydroxyapatite, indicating the important role autophagy plays in bone mineralization (Nollet et al., Reference Nollet, Santucci-Darmanin, Breuil, Al-Sahlanee, Cros, Topi, Momier, Samson, Pagnotta, Cailleteau, Battaglia, Farlay, Dacquin, Barois, Jurdic, Boivin, Heymann, Lafont, Lu, Dempster, Carle and Pierrefite-Carle2014).

Osteocytes, because of their long life and their location being embedded in the mineralized bone matrix, face a constant state of stress, which results in a high level of basic autophagy. Zahm et al. (Reference Zahm, Bohensky, Adams, Shapiro and Srinivas2011) have observed a punctuate distribution of LC3, which is an autophagy marker, indicating a high level of basic autophagy in murine and human cortical bone osteocytes. They also observed induction of autophagy under starvation and hypoxia, conditions that are common in the environment of osteocytes (Zahm et al., Reference Zahm, Bohensky, Adams, Shapiro and Srinivas2011). Yang et al. (Reference Yang, Zheng, Li, Jiang and Jiang2014) proved that ovariectomy in murines resulted in increased oxidative stress and level of autophagy in osteocytes, as a result of estrogen deprivation (Yang et al., Reference Yang, Zheng, Li, Jiang and Jiang2014). Low doses of corticosteroids stimulate autophagy in osteocytes, as a mechanism to survive the stress caused by steroids (Jia et al., Reference Jia, Yao, Guan, Dai, Shahnazari, Kar, Bonewald, Jiang and Lane2011).

Autophagy and proteins taking part in the signalling pathway of autophagy have a significant role in differentiation and function of osteoclasts (Pierrefite-Carle et al., Reference Pierrefite-Carle, Santucci-Darmanin, Breuil, Camuzard and Carle2015). Wang et al. (Reference Wang, Niu, Kim and Kolattukudy2011) proved that upregulation of Beclin-1 and autophagy play an important role in osteoclastogenesis induced by MCP-1. Knocking down Atg7 suppressed the expression of TRAP and cathepsin K, which are osteoclast markers, indicating that autophagy is involved in osteoclastogenesis (Wang et al., Reference Wang, Niu, Kim and Kolattukudy2011). Chung et al. (Reference Chung, Jang, Choi, Song, Lee, Kim, Song, Kang, Yoon and Chang2014) showed that Beclin-1, which is an autophagy marker, induces reactive oxygen species formation and NFATc1 expression, which is an important transcription factor in the signalling pathway of osteoclastogenesis (Chung et al., Reference Chung, Jang, Choi, Song, Lee, Kim, Song, Kang, Yoon and Chang2014). TRAF3, which suppresses osteoclastogenesis induced by RANKL, is degraded by autophagy in pre-osteoclasts (Xiu et al., Reference Xiu, Xu, Zhao, Li, Morita, Yao, Xing and Boyce2014).

In addition, proteins taking part in the signalling pathway of autophagy, such as Atg5, Atg7, Atg4B, LC3, are necessary for the formation of the ruffled border of mature osteoclasts and their resorption activity. Atg5 is required for the transport of LC3II to the ruffled border, where LC3II promotes the fusion of lysosomes, which is necessary for the resorption activity of osteoclasts (DeSelm et al., Reference DeSelm, Miller, Zou, Beatty, Van Meel, Takahata, Klumperman, Tooze, Teitelbaum and Virgin2011). Chung et al. proved that osteoclast activity is related to the conversion of LC3I to LC3II, but not to the level of autophagy. Suppression of LC3 did not interfere with the formation of multinucleated cells expressing TRAP, which is an osteoclast marker. Instead, suppression of LC3 repressed the F-actin ring formation and exocytosis of cathepsin K, resulting in a reduction of osteoclast bone resorption activity (Chung et al., Reference Chung, Yoon, Choi, Sohn, Yoon, Kim, Kim and Chang2012).

Autophagy and microgravity

It has been noted since 1980s that microgravity modifies organ function of organisms subjected to microgravity. Research on the underlying mechanisms has started since then (Rokhlenko and Mul'diiarov, Reference Rokhlenko and Mul'diiarov1981). Rokhlenko and Mul'diiarov (Reference Rokhlenko and Mul'diiarov1981) first described the changes in myocardial cells of rats exposed to microgravity and noted the presence of more autophagosomes in animals exposed to microgravity than in those on earth (Rokhlenko and Mul'diiarov, Reference Rokhlenko and Mul'diiarov1981). Riley et al. (Reference Riley, Ellis, Slocum, Satyanarayana, Bain and Sedlak1987) supported that autophagy does not play an important role in microgravity because they found no significant increase in lysosomes in skeletal muscle cells of rats exposed to microgravity (Riley et al., Reference Riley, Ellis, Slocum, Satyanarayana, Bain and Sedlak1987). Thirty years later, autophagy is reevaluated as a mechanism responsible for adaptation of cells to exposure to microgravity.

Risks posed by microgravity to the cardiovascular system have been a challenging subject for researchers to study. Wang et al. (Reference Wang, Lu, Shi, Zhang, Yang, Wang, Cao, Du, Gao, Zhao and Sun2013) observed that autophagy is upregulated in endothelial cells of the human umbilical vein under exposure to modelled microgravity for 48 h, as indicated by increased autophagosomes and expression of beclin-1 and LC3, which are autophagy markers (Wang et al., Reference Wang, Lu, Shi, Zhang, Yang, Wang, Cao, Du, Gao, Zhao and Sun2013). Zhong et al. (Reference Zhong, Ling and Li2016) included autophagy in a review describing pathophysiological mechanisms responsible for cardiac atrophy under microgravity conditions (Zhong et al., Reference Zhong, Ling and Li2016).

Influence of microgravity on the musculoskeletal system has been extensively studied in order to develop compensatory measures. Sandona et al. (Reference Sandona, Desaphy, Camerino, Bianchini, Ciciliot, Danieli-Betto, Dobrowolny, Furlan, Germinario, Goto, Gutsmann, Kawano, Nakai, Ohira, Ohno, Picard, Salanova, Schiffl, Blottner, Musaro, Ohira, Betto, Conte and Schiaffino2012) investigated skeletal mouse muscles which were exposed to microgravity during a 90-days space mission. They observed that muscle atrophy was more obvious in muscles that support weight-bearing bones than muscles which are responsible for other movements, but there was not a statistically significant difference in expression of genes (LC3b and cathepsin L) which are considered autophagy markers (Sandona et al., Reference Sandona, Desaphy, Camerino, Bianchini, Ciciliot, Danieli-Betto, Dobrowolny, Furlan, Germinario, Goto, Gutsmann, Kawano, Nakai, Ohira, Ohno, Picard, Salanova, Schiffl, Blottner, Musaro, Ohira, Betto, Conte and Schiaffino2012). Ryu et al. (Reference Ryu, Choi, Namkoong, Jang, Seo, Choi, Kim and Park2014) observed an increased number of autophagosomes and expression of LC3II using Western blot analysis in myoblast cell cultures exposed to modeled microgravity for 72 h. They reached the conclusion that autophagy is induced under microgravity conditions and is probably involved in the pathogenesis of muscle atrophy under such circumstances (Ryu et al., Reference Ryu, Choi, Namkoong, Jang, Seo, Choi, Kim and Park2014).

Yoo et al. (Reference Yoo, Han and Kim2016) proved that levels of LC3II are time-dependently increased in preosteoblast cells MC3T3-E1 during their exposure to modeled microgravity for 72 h, which was attributed to increased phosphorylation of proteins ERK/Akt/mTOR. They also studied the effect of melatonin on the expression of autophagy markers and reached the conclusion that melatonin counteracts the effect of microgravity on preosteoblast autophagy markers’ expression (Yoo et al., Reference Yoo, Han and Kim2016).

Sambandam et al. (Reference Sambandam, Townsend, Pierce, Lipman, Haque, Bateman and Reddy2014) studied the impact of microgravity on the osteoclasts and proved that exposure in modelled microgravity for 24 h resulted in increased osteoclastogenesis and expression of autophagy markers Atg5 and LC3II in RAW264.7 cell culture as well as in mouse bone marrow cells (Sambandam et al., Reference Sambandam, Townsend, Pierce, Lipman, Haque, Bateman and Reddy2014). Exposure of seminoma TCam-2 cells to modeled microgravity resulted in temporary changes of cytoskeleton and autophagy induction. More specifically, expression of LC3II, as estimated with Western blot analysis, was increased after 24 h of exposure to modeled microgravity and then was restored to the basic level after 48 h, indicating a temporary and adaptive role of autophagy in seminoma cells under microgravity conditions (Ferranti et al., Reference Ferranti, Caruso, Cammarota, Masiello, Corano Scheri, Fabrizi, Fumagalli, Schiraldi, Cucina, Catizone and Ricci2014). Indo et al. (Reference Indo, Tomiyoshi, Suenaga, Tomita, Suzuki, Masuda, Terada, Ishioka, Gusev, Cornette, Okuda, Mukai and Majima2015) found no significant difference in BECN1 expression, which is an autophagy marker, among neuroblastoma cell line NB-1 cultured under modeled microgravity conditions for 24 h and the same cell culture under earth gravity conditions (Indo et al., Reference Indo, Tomiyoshi, Suenaga, Tomita, Suzuki, Masuda, Terada, Ishioka, Gusev, Cornette, Okuda, Mukai and Majima2015).

Human embryonic renal cell line HEK293, which expresses GFP-LC3 constantly, had increased numbers of autophagosomes after 72 h, but not after 24 h, of exposure to modeled microgravity and elevated levels of phosphorylated protein kinase dependent on AMP (AMPK). Suppression of AMPK expression resulted in the restoration of autophagy markers to comparable levels to the reference cell culture, indicating the involvement of AMPK in the induction of autophagy pathway, but not as a unique regulator (Ryu et al., Reference Ryu, Choi, Namkoong, Jang, Seo, Choi, Kim and Park2014). The effects of autophagy and microgravity are summarized in Table 3.

Table 3. Microgravity and autophagy

Microgravity, autophagy and osteoclastogenesis

Microgravity has detrimental effects on bone metabolism. Weight-bearing bones experience a significant decrease in bone density. Prolonged exposure to microgravity conditions results in increased bone resorption in the early stages and decreased bone formation later. Osteoclasts are responsible for bone resorption and play an important role under microgravity conditions. Autophagy is a mechanism which is activated under stress. Microgravity produces a stressful environment for the bone. Researchers, such as Sambandam et al. (Reference Sambandam, Baird, Stroebel, Kowal, Balasubramanian and Reddy2016), have studied the possible role of autophagy as the mediator of osteoclastogenesis under microgravity conditions (Sambandam et al., Reference Sambandam, Townsend, Pierce, Lipman, Haque, Bateman and Reddy2014).

Preosteoclasts cultured under microgravity conditions expressed increased levels of Atg5, LC3 and Atg16L, which are autophagy markers. The researchers also observed abundant autophagosomes in RAW264.7 cells cultured under modelled microgravity conditions using confocal microscopy. Sambandam et al. observed increased expression of genes taking part in signalling pathways associated with autophagy, such as GAA, Trp53, Prkaa1, Rps6kb1, or being components of autophagosomes, such as Atg16L, Atg9b, Irgm1, using RT2 profiler polymerase chain reaction array method. They also noted a significant increase in expression of phospholipase PLCγ2 under microgravity conditions, whereas the signalling pathway of PLCγ2/calcium regulates autophagy in preosteoclasts (Sambandam et al., Reference Sambandam, Townsend, Pierce, Lipman, Haque, Bateman and Reddy2014).

Administration of 3-methyladenine, which is an autophagy inhibitor, to mouse bone marrow cells suppressed the expression of Atg5 and LC3, which are autophagy markers, as well as cathepsin K, which is an osteoclast marker, indicating that autophagy probably plays a role in osteoclastogenesis under microgravity conditions (Sambandam et al., Reference Sambandam, Townsend, Pierce, Lipman, Haque, Bateman and Reddy2014).

Although autophagy is a significant player in bone metabolism under microgravity conditions, one possible treatment would be the attempt to inhibit the autophagic machinery and thus alleviate relative outcomes. Yet, although this entails a large ‘temptation’, autophagy inhibition can have several effects on cellular physiology. As aforementioned, autophagy consists of a defensive mechanism of a dual nature. Autophagy is relatively at low levels, in mammalian cells and it is triggered by starvation and oxidative stress (Guo and White, Reference Guo and White2016; Zhao et al., Reference Zhao, Qu, Wang, Li, Qiang, Xia, Duan, Huang and Zhu2016; Xu et al., Reference Xu, Wise, Wang, Schumann, Zhang and Shi2017), while it can function in a protective manner in the tumour microenvironment and promote tumour cell proliferation (Liu et al., Reference Liu, Mo, Yang, Du, Wang, Zhang, Kurihara, Xu and Cai2016a). Further on, it consists of a defense mechanism in DNA damage, which leads to controlled cell death (Roos et al., Reference Roos, Thomas and Kaina2016). On the other hand, numerous reports indicated the role of autophagy in malignant cell survival and proliferation. For example, it has been reported that autophagy inhibition has potential antitumor effects in glioblastoma (Zhang et al., Reference Zhang, Chen, Fan, Wang, Xian, Luan, Li, Wang, Nan, Luo, Li, Tian and Ju2018), pancreatic adenocarcinoma (Boone et al., Reference Boone, Zeh and Bahary2018), bladder urothelial carcinoma (Zeng et al., Reference Zeng, Liu, Cao, Li, Liu, Fan, Liu, Cheng, Xiong, Li, Bo, Zhu, Yang, Hu, Zhou, Zhou, Zou, Zhou and Cao2018), gastric cancer (Sun et al., Reference Sun, Jiang, Huang, Chen, Liao and Yang2017), acute lymphoblastic leukemia (Lambrou et al., Reference Lambrou, Papadimitriou, Chrousos and Vlahopoulos2012) and many others. It is evident that autophagy plays a significant role in cellular physiology as it consists of a protective mechanism under normal conditions and it is a facilitator of carcinogenesis and chemotherapy resistance under pathophysiological conditions. Thus, the mechanisms underlying those phenomena still remain to be elucidated and we still know very few for the complete role of autophagy in cellular pathophysiology. This leads us to the conclusion that the regulation of bone metabolism through autophagy inhibition could prove a risky alternative.