The Effects of Simulated and Real Microgravity on Vascular Smooth Muscle Cells

Under the various extraterrestrial phenomena present in spaceflight, astronauts undergo alterations to the vascular system and health in general (Blaber et al., 2010; Vernice et al., 2020). Spaceflight introduces cardiovascular deconditioning and physiological effects similar to aging (Demontis et al., 2017). As the vascular system is critical to healthy homeostasis across various major organ systems in the body, understanding the effects of the spaceflight environment on the various cells that comprise the vasculature is critical when considering the future of humans in space. Microgravity is one such component, which has been demonstrated to have effects across a number of cell populations via in vivo and in vitro experiments. While previous reviews have provided a summary of various cell types critical to human health, including endothelial cells (Maier et al., 2015) and macrophages (Ludtka, Silberman, et al., 2021; Sun et al., 2021), as well as the vasculature in general, there has been no previous review of the effects of microgravity on smooth muscle cells specifically to our knowledge. In particular, we focus on vascular smooth muscle cells since they are the focal point of existing experimental literature as well as being a key component of the overarching cardiovascular system, which is notably impacted by microgravity on an organismal level.

This review evaluates the literature covering vascular smooth muscle cells in microgravity over the last several decades, with a focus on recent developments. With the heightened public and research interest in spaceflight, as well as the further cross-validation and availability of various microgravity models, it is timely to collate and assess the major findings in the field regarding smooth muscle cells as a critical player in human health in space. Thus, we provide an organized compilation of the relevant research, as well as contextualizing it regarding existing microgravity models and experimental design considerations. As such, we aim to highlight similar findings and provide comparisons across the numerous reports using various cell types, microgravity models, and time points in order to provide an overview of the extent of existing literature in this research space.

Microgravity has been used as an experimental condition for numerous in vivo and in vitro studies, including for human, animal, plant, and bacterial responses. While some gravisensitive mechanistic insights have been gained for various cell populations, a critical limitation to investigations remains the limited access to spaceflight experiments. As a result, methods to simulate or achieve microgravity and its effects outside of actual spaceflight have become an alternative. As such, a key distinction across the microgravity models commonly used in research is simulated versus true microgravity systems. To validate these various models against one another, as well as against the gold standard of true spaceflight, numerous reviews and studies have been previously published in the literature (Clary et al., 2022; Herranz et al., 2013; Wuest et al., 2015). NASA recently had a comprehensive overview of microgravity methods and their predictive utility prepared (Williams, 2023). Simulated microgravity methods are useful analogs for a number of reasons, including their relative accessibility, cost, and ease of logistics compared to true microgravity approaches. Additionally, they simplify the isolation of the variables affecting the experimental population, as certain extraterrestrial factors cannot be removed for actual microgravity systems (e.g., hypergravity from launch/reentry and cosmic radiation). For benchtop in vitro experiments, the primary methods for microgravity simulation are 2D or 3D clinorotation and random position machines (RPM). 2D clinorotation is achieved via rotation along a singular axis. For instance, the rotating wall vessel (RWV) system originally developed by NASA rotates a fluid column with the rotational axis parallel to the ground (Figure 1A); this is also described as rotary cell culture. The rotational speed is optimized based on particle size to keep free-floating cells, cell aggregates, or cells on a scaffold such as microcarrier beads (Figure 1B) at a terminal (sedimentation) velocity, with gravity balanced by shear, centrifugal, and Coriolis forces (Hammond & Hammond, 2001). This system has previously been used in experiments for a number of cell types relevant to the vasculature, including macrophages (Ludtka, Moore, et al., 2021) and vascular stem cells (Ramaswamy, Dirr, et al., 2016; Ramaswamy, Goins, et al., 2016). In contrast, systems that operate on two rotational axes such as the Gravite 3D clinostat and Airbus/Yuri RPM allow for microgravity simulation using conventional adherent cell culture (e.g., flasks) (Figure 1C, 1D). The differentiating aspect of the RPM is that in addition to a conventional 3D clinostat mode, it can also operate in a random mode with varying angular velocities for each axis. However, gas exchange can prove a difficulty as plug caps are needed to prevent media leakage while samples are rotating on the device. For in vivo experiments, the hindlimb unloading model is common for rodents (Figure 1E) (Globus & Morey-Holton, 2016; Hawliczek et al., 2022), and some studies use the head-down tilt method as a microgravity analog for humans (Watenpaugh, 2016).

Figure 1.

True microgravity systems include parabolic flight and suborbital/sounding rockets, which provide weightlessness on the order of seconds to minutes. A limitation of these methods (or potentially, a benefit depending on the intended assessment being made) is the period(s) of hypergravity experienced with launch and reentry. Notably, the sinusoidal path of parabolic flights results in cycles of hypergravity and microgravity. In contrast, suborbital/sounding rocket trajectories offer a longer, continuous period of microgravity and experience hypergravity only at initial launch and reentry. Orbital spaceflight, such as experimentation conducted on the International Space Station and Space Shuttle prior to its retirement, is the gold standard for microgravity experiments. Naturally, this method also includes the consideration of hypergravity from launch as well as exposure to radiation, including galactic cosmic rays and solar radiation. However, these methods can prove more difficult to implement compared to simulation options on the ground. This is due to the costs, logistics, weight and volumetric limitations, and the limited availability of opportunities to have proposed payloads accepted for spaceflight launches.

Vascular smooth muscle cells (VSMCs) are stromal cells present in the tunica media of vasculature. The tunica media in the middle layer of blood vessels is flanked by the tunica intima toward the interior lumen of the vessel and the tunica adventitia that comprises the exterior of the vessel (Figure 2). Arteries are more elastic and have a thicker tunica media, internal elastic lamina, and external elastic lamina to accommodate the higher blood pressures they experience relative to veins.

Figure 2.

VSMCs' environment is comprised of both mechanical cues innate to the vascular system (e.g., cyclic stretch, pressure) as well as biochemical markers and signals present in the bloodstream. A major function of VSMCs in this space is the control of vascular tone, a large part of which is determined via interactions with endothelial cells (ECs) that compose the tunica intima of vessels and experience shear stress from direct contact with blood flow. This cellular interplay helps maintain hemodynamic homeostasis by reacting to mechanical and chemical cues within the vessel. VSMC tone is affected by mechanostimulation, Ca²⁺ signaling, angiotensin II, and nitric oxide, among others (Zieman et al., 2005). For instance, shear stress is a major driver of nitric oxide release by ECs, which then triggers relaxation in SMCs and corresponding vasodilation of the blood vessel. Meanwhile, angiotensin II is converted from angiotensin I in ECs and triggers SMC contraction, leading to vasoconstriction. As noted, the mechanical forces driving many of these reactions differ based on the cell types' location within the vessels. ECs experience shear in the tunica intima, which forms the hemocompatible lining of vessels. Meanwhile, VSMCs experience stretch within the tunica media, which serves as the elastic, resilient banding that helps maintain vessel shape and integrity. As such, it is important to be aware of and intentional about which stimuli are being included or excluded in your experimental design. For instance, the term ‘low-shear modeled microgravity’ (LSMMG) is often used in the literature for RWV systems, and Leguy et al. (2017) recently reported lower calculated shear values from RPM-simulated microgravity compared to previous reports for the RWV.

VSMCs are also a major contributor to vascular pathological developments such as arterial stenosis, sclerosis, and intimal hyperplasia. As such, understanding the underlying mechanisms of VSMC response to various signals, such as the microgravity environment, is critical for human health in general, as well as the development of interventions to mediate these pathologies. Indeed, many of these pathologies result, at least in part, from irregular VSMC proliferation, migration, or phenotypic changes.(Bennett et al., 2016; Lacolley et al., 2012) Additionally, under the pressure of pathological influences like stenosis, sclerosis, and intimal hyperplasia, VSMCs can undergo a phenotypic shift. They dedifferentiate from their contractile, quiescent phenotype and then enter a synthetic, pro-migratory, and pro-proliferative phenotype. This new phenotype also demonstrates increased extracellular matrix deposition. This phenotypic shift can be encouraged by both mechanostimulatory and chemostimulatory factors, as well as cell-cell and cell-matrix interactions in the vasculature (Kang, Fan, et al., 2013). As such, the effects of microgravity, the models used to achieve or simulate it, as well as the experimental design considerations (e.g., in vivo vs. in vitro) can have significant impacts on VSMC phenotype, expression, and behavior.

Vascular smooth muscle cells, as key cellular constituents of the vascular system, are responsive to changes in gravity. Depending on the type of experimental conditions (e.g., in vivo vs. in vitro), this may not only result from gravisensitive pathways and molecular mechanisms but also due to changes at the tissue and organ system level. For example, when subjected to microgravity, humans experience a cephalic fluid shift, as the typical vascular mechanisms for maintaining orthostatic blood pressure homeostasis have evolved around the existence of gravity acting on the human body (Tanaka et al., 2017). This fluid shift can lead to intracranial hypertension and correspondingly to spaceflight-associated neuro-ocular syndrome (SANS) due in part to intracranial and intraocular pressure mismatch (Zhang et al., 2018). Furthermore, central venous pressure is reduced in microgravity, with fluid redistributed from the legs to the head (Lawley et al., 2017). Tanaka et al. (2017) provide a more in-depth review of the impacts of microgravity on the cardiovascular system, such as cardiac atrophy. Based on these systemic effects, it is necessary to consider investigations of VSMC responses both on a cellular and organism level. For example, Hughson et al. noted the thickening of the carotid artery in astronauts, as well as arterial stiffening, cardiovascular deconditioning, and insulin resistance (Hughson et al., 2018). In their recent review, Gao and Chilibeck noted the various deleterious cardiovascular effects seen from spaceflight exposure, including resting and orthostatic tachycardia, impaired cardiac relaxation, reduced upright stroke volume, and microvascular dysfunction (Gao & Chilibeck, 2020).

As microgravity causes these systemic effects that can impact hemodynamics and present pathological symptoms, in vitro cellular studies can also be impacted by these indirect effects. It is also important to highlight the potential interaction that other aspects of spaceflight, like radiation, may have on human biology, confounding attempts to isolate the direct effects of microgravity specifically (Yatagai et al., 2019). In their discussion on the topic, Pietsch et al. (2011) note various microgravity-induced disease states (e.g., osteoporosis and muscle atrophy) on the organism level, while also noting more narrow effects on the cellular level. Numerous mammalian cell types have shown microgravity impacting apoptosis, cytoskeletal rearrangement, differentiation, proliferation, migration, and adhesion (Pietsch et al., 2011). Specifically, VSMCs are responsive to shear stress, stretch, and pressure, so it is important to consider these mechanical forces in the design of microgravity experiments (Haga et al., 2007; Shi & Tarbell, 2011). Additionally, changes to localized tissue function and structure are important to consider, given VSMCs' role in regulating vascular tone. For example, Prisby et al. (2015) report that increased vascular resistance seen in HU rodents results from reduced endothelium-dependent vasodilation, as opposed to increased vasoconstriction response. Similarly, Tarasova et al. (2020) noted that HU impairs sympathetic vasoconstriction, which is consistent with humans post-spaceflight. As vasoconstriction can be impacted by the presence or absence of the endothelium in an exercise and age-dependent manner, it is also important to note the differences in response that might be seen for in vivo vs. in vitro samples when considering microgravity effects (Ghosh et al., 2015).

Therefore, in vivo experiments in microgravity must consider the potential effects of fluid shift and altered localized vascular responses that result from microgravity, should the objective be to isolate microgravity's direct impacts. Similarly, certain simulated microgravity methodologies may introduce shear stress and introduce fluid convention that impacts the intercellular signaling gradient (Poon, 2020). As such, intentionality in experimental design is critical for utilizing appropriate controls and isolating the effects of variables (Figure 3).

Figure 3.

Additionally, making direct comparisons between the existing literature is complicated by the myriad of microgravity methods, cell types, and study durations used. This also can make it difficult to establish the direct and indirect effects of microgravity across studies, given the impacts on the organism, system, and cellular level previously noted. Table 1 summarizes these aspects of the VSMC microgravity literature: the experimental platform used for simulating or achieving microgravity, cell type(s), microgravity duration(s), primary results, in vivo vs. in vitro, and sex reporting. The effects demonstrated range from proliferation to gene expression and phenotype to RAAS, NOS, and Ca²⁺ signaling.

Coculture and Tissue Engineering

Microgravity, particularly that achieved via benchtop simulated microgravity systems, has been proposed as a tissue engineering methodology (Aleshcheva et al., 2016). Indeed, Grimm et al. (2014) have noted the formation of tubule-like multicellular constructs from RPM-simulated microgravity via the coculture of HUVEC-like EA.hy926 cells, SMCs, and fibroblasts (Figure 4). Similarly, Low and Giulianotti (2020) reviewed the potential for modeling human disease in microgravity via microphysiological systems. Scott et al. (2022) have recently described the potential research and tissue engineering opportunities that the spaceflight environment may offer for cardiovascular applications, such as for induced pluripotent stem cell–derived cardiomyocytes and organoids. Ren et al. (2023) have likewise recently highlighted in a comprehensive review the potential for microgravity in both tissue construct biomanufacturing as well as disease modeling across a number of tissues, including cardiac muscle. Zhang et al. (2014) conducted indirect coculture experiments with primary bovine aortic VSMCs and ECs using 2D clinostat simulated microgravity. Thus far, the assessment of VSMCs for these purposes has been relatively minimal, especially compared to the amount of literature regarding ECs.

Figure 4.

The proliferation of VSMCs is a significant concern for human health, as the pathological proliferation of the VSMCs can result in various cardiovascular conditions (e.g., stenosis and intimal hyperplasia). As such, considering the effects of microgravity on VSMC proliferation is prudent for assessing the potential harmful effects of long-term spaceflight on human health. Correspondingly, a number of studies have reported on VSMC proliferation rates.

Su et al. reported an increase in proliferation and decreased apoptosis in cerebral artery VSMCs in a 4-week rat HU study. (Su et al., 2020) Meanwhile, the opposite effects were seen in small mesenteric artery VSMCs. Zhang et al. (2020) found that simulated microgravity in a 28-day murine HU model resulted in increased proliferation of vascular VSMCs, as well as evidence of dedifferentiation. They also noted that this effect was mediated by the T-type CaV 3.1 channel through the downstream calcineurin/NAFTc3 pathway. In a similar murine HU study, Jiang et al. (2022) documented increased VSMC proliferation and phenotype switching, which could be attenuated via activation of the Erα-NRF1-OMI pathway to restore the disrupted mitophagy homeostasis. Indeed, hormones and their receptors like the estrogen receptor α (Erα) investigated by Jiang may have significant effects on the impacts of the microgravity environment on VSMCs, as Rabineau et al. (2022) noted in their review how the menstrual cycle can impact smooth muscle sensitivity to NO. Another research group reported that clinostat simulated microgravity in combination with EC-conditioned media helped mitigate the anti-proliferative effects of the media on bovine aortic VSMCs. Additionally, microgravity appeared to also increase VSMC migration compared to equivalent normogravity controls. The authors suggested these effects might be modulated through P2 receptor expression, which can also affect NO production and apoptosis signaling (Figure 5) (Y. Zhang et al., 2014).

Figure 5.

In contrast, Kang et al. (2013) reported a significant decrease in murine aortic VSMC proliferation and migration using an in vitro clinostat analog. Likewise, Coinu et al. (2006) reported decreased murine aortic vascular VSMC proliferation (represented by thymidine incorporation) after 24 h in RPM-simulated microgravity in vitro culture. Lv and Ai (2022) note the importance of the YAP/TAZ pathway for VSMC proliferation and apoptosis, as well as its mechanosensitivity. They also note reports of microgravity-induced inhibition of TAZ nuclear translocation, though demonstrated in mesenchymal stem cells (Chen et al., 2016). In their review of the cardiovascular effects of the space environment, Hughson et al. (2018) discuss the reported increase in renin-angiotensin-aldosterone hormones seen in spaceflight, highlighting that two of these – aldosterone and angiotensin II – are known to affect VSMC proliferation as well as resulting arterial stiffness. They further note reports of impacts on VSMC proliferation and migration following radiation exposure, another factor to consider when attempting to understand the effects of spaceflight on biological systems.

Microgravity has demonstrated a number of significant effects on VSMC expression and function. In particular, existing literature provides data supporting the impacts of microgravity on VSMC phenotype switching as well as the modulation of several major signaling pathways relevant to VSMCs (e.g., RAAS, NOS, and Ca²⁺ channels). These previous reports on VSMCs in microgravity utilize a number of different methods and experimental designs. As seen from Table 1, the most common among these appears to be the rodent HU model. For the purpose of assessing VSMCs in simulated microgravity, this model has the benefit of including EC support and intercellular interactions with the VSMCs being evaluated. However, there is a relative dearth of spaceflight and monoculture VSMC research to isolate cell-specific responses to microgravity and validate existing data against true spaceflight as the gold standard.

Furthermore, despite existing literature on the sex-based differences in several of the key aspects of VSMC function, there is very little reporting of sample population sex. Specifically, of the VSMC microgravity literature summarized in Table 1, only 2 (10%) use female-only samples, while 14 (70%) use male-only samples, and 4 (20%) are Not Specified. This is particularly important given the established differences in the vasculature, vascular cells, and overall cardiovascular health outside the context of microgravity.

Additional study of true spaceflight effects and the consideration of sex-based differences would help shed additional light on key aspects of VSMC responses to microgravity and the spaceflight environment. Isolating cell types for characterizations (i.e. separation of VSMCs and ECs together prior to collection of mRNA) could help elucidate cell-specific responses to microgravity. Furthermore, it would be valuable to determine the specific impact of other aspects of spaceflight (e.g., various types of cosmic radiation, long-term isolation) and their interaction with the effects of microgravity. This, in turn, would provide valuable information for human health considerations and potential interventions or mediators of pathological developments from exposure to long-term spaceflight.