With the SPHINX experiment, we aimed to investigate how microgravity on board the ISS modulates HUVEC behavior (4, 5), considering that the impaired EC functions could be due to changes in mechanotransduction responses. This challenging topic is important not only in the field of astrobiology but also for understanding common diseases, because microgravity rapidly generates alterations that are similar to those involved in age‐related diseases (5, 34–36). Within the evident limits of a spaceflight experiment, we chose the microarray approach together with the functional annotation tools provided by DAVID and the Bio‐Plex system to evaluate HUVEC global response to microgravity. Our data demonstrate that HUVECs are affected by spaceflight and respond using the mechanosensors responsible for sensing alterations in flow patterns (8), even though they are in a condition of quiescent fluid environment.

In space research, the mechanotransduction process has been mainly addressed in bone loss studies performed in simulated microgravity, while in the majority of the flown cell types ( 43 ) as well as in yeast ( 44 ) the cytoskeleton was considered as the main mechano‐sensor. Recently we demonstrated in our Saccharomyces cerevisiae Oxidative Stress Response Evaluation (SCORE; ref. 45 ) spaceflight experiment that cytoskeleton alterations and activation of ion channels are the main effects of microgravity on yeast, depending on changes in cell volume and metabolism, activation of the high osmolarity glycerol and cell integrity pathways, both of which respond to osmotic volume perturbations.

The cytoskeleton, the extracellular matrix and adhesion complexes, and the membranes are the first and most common cell mechanosensors. Considering that all proteins are deformable and therefore subjected to mechanical modulation, many enzymes, such as kinases, phosphatases, GTPases, cyclases, and G‐protein‐coupled receptors that change conformation in response to force, create transduction pathways that report mechanical stress. Force transduction can also involve changes in the kinetic rate constant of a mechanosensitive enzyme or, more qualitatively, expose cryptic binding sites on a molecule ( 42 ). In the specific case of ECs, several mechanosensors have been proposed, from cell‐cell adhesion and glycocalyx molecules, to ion channels, integrins, G‐protein‐coupled receptors, and the cytoskeleton ( 8 ).

All cells can respond to applied or cell‐generated mechanical forces by activating mechanosensors that mediate the complex process of mechanotransduction ( 37 – 39 ). Mechanotransduction is a rapidly expanding area of research ( 38 ) because the conversion of mechanical forces into biochemical signals governs many physiological processes. Accordingly, it is widely recognized that defects in mechanotransduction signaling can contribute to human diseases such as osteopenia, lung dysfunction, immune system disorders, muscular dystrophies, and cardiomyopathies. Also, it is well known that atypical mechanical stresses through normal mechanotransduction signaling can modulate cell processes and cause tissue function impairment or failure ( 39 , 40 ) as the disturbed fluid shear stress that triggers vascular remodeling and eventually atherosclerosis ( 8 , 12 ), or microgravity that induces the loss of bone mass ( 41 ).

Effects of spaceflight on HUVECs: alterations in focal adhesion, TXNIP expression, and oxidative phosphorylation lead to a condition of oxidative stress that promotes DNA damage and inflammation

The results of the GO analysis indicate that 15 of the 44 significantly modulated genes belonging to the cell adhesion biological process (Table 2) are associated with the focal adhesion pathway, which consists of integrins and many adaptor and signaling proteins (details of the genes are available in Supplemental Table S1). Focal adhesions link the actin cytoskeleton to the extracellular matrix through a cluster of actin‐associated proteins that modulate cell survival, migration, proliferation, and other important cell processes. Interestingly, integrins are rapidly activated after cell volume perturbations in many cell types and have been proposed as volume sensors after both swelling and shrinkage (46). Activated integrins transmit mechanical signals to ion channels, which adjust ion flux to restore cell volume, and the actin cytoskeleton, which transmits mechanical forces to the nucleus that modulates transcription processes.

Among the 1023 modulated genes (585 up‐regulated, 438 down‐regulated), the most up‐regulated is TXNIP, a stress‐responsive gene encoding a protein that inhibits the antioxidative action of thioredoxin (TRX) by interacting with its catalytic domain. TRX is an oxidoreductase that is ubiquitously expressed in ECs; it controls cell redox status and regulates cell growth, migration, angiogenesis, and apoptosis (47). It is increasingly recognized that TXNIP plays a crucial role in age‐related diseases (48) and cardiovascular disorders (49–53) as it is a critical sensor of biomechanical stress and a potent repressor of glucose uptake and glycolysis (54). Pressure overload decreases TXNIP expression in cardiomyocytes, and physiological fluid shear stress (which is atheroprotective) down‐regulates TXNIP. Also, it is worth noting that it inhibits endothelial migration, another important event in the healing of damaged vessel walls (55). Therefore, we conclude that an overexpression of TXNIP might contribute to the modulation of endothelial function in space.

In our experimental model, microgravity down‐regulated many of the genes involved in oxidative phosphorylation (Table 3), thus triggering mitochondrial dysfunction and possibly leading to bioenergetic incompetence and the overproduction of reactive oxygen species.

Mitochondrial dysfunction and TXNIP overexpression both contribute to the generation of a prooxidative environment. Oxidative stress not only causes lipid peroxidation and extensive DNA damage, but also contributes to the acquisition of an inflammatory phenotype and the onset of senescence in ECs (56–58). DNA damage activates the p53 signaling pathway (59), which leads to cell survival if the damage can be repaired, or apoptosis if the damage is too severe. In space‐flown HUVECs, TP53INP1 (60, 61) was up‐regulated (Table 1), and a number of miRNAs and genes involved in cell cycle arrest in response to DNA damage (CDK6, GADD45GIP1 and CCNF), DNA repair and damage prevention (GADD45GIP1, CCNF, SESN3, PTEN, and TSC2), and the initiation of apoptosis (CASP8) were modulated. TP53INP1 is a proapoptotic stress‐induced gene whose transcription is activated by p53. When overexpressed, it induces cell cycle arrest and enhances p53‐mediated apoptosis. Also, it interacts with the p53 gene and regulates p53 transcriptional activity (62). Recent studies indicate that p53 regulates the expression of various miRNAs (60, 61), a class of endogenously small, noncoding RNAs that play a key role in regulating gene expression by post‐transcriptionally silencing target genes through the RNA interference pathway (63). Space‐flown HUVECs showed increased levels of miR‐15a (64–66), which may contribute to cell survival or apoptosis: when energy is limited and under other stressful conditions, it induces cell cycle arrest and quiescence (64), but it also contributes to the induction of apoptosis by targeting the antiapoptotic factor BCL2 (67). A number of genes belonging to the BCL family (MCL1, BCL2L13, BAK, BNIP3; ref. 68) were modulated in our space‐flown ECs, some toward a proaptotic and others toward an antiapoptotic response. In line with cell cycle arrest and/or proapoptotic responses, we found the up‐regulation of CASP8 (which participates in apoptosis as well as cell proliferation and inflammation; refs. 69, 70), the 2 identified KEGG pathways (Alzheimer's and Parkinson's diseases), and the marked down‐regulation of HSPA1A, HSPA1B, and HSP90AA2. It is known that HSP70 and HSP90 protect cells against various stresses by regulating the cell cycle and interfering with key apoptotic proteins. Their down‐regulation suggests that HUVECs have adapted to the stress generated by exposure to microgravity. In our previous experiments in simulated microgravity we observed that HUVECs rapidly up‐regulated HSP70 as a mechanism of protection against stress‐induced apoptosis (18), which seems in contrast with spaceflight results. In SPHINX, due to spaceflight restraints, we could evaluate changes in gene expression after only 10 d in microgravity. The different timeframe explains why there is no contradiction in our results: once cells have adapted to the stress, HSP70 levels decrease. Analogously, we might interpret the controversial results on the modulation of NO synthases (Table 4) and the not significant differences found in spaceflight vs.1‐gNO releases as due to adaptation of cells after 10 d in space. Nevertheless, it is worth noting that HUVECs, although adapted after the 10‐d spaceflight, show 1023 modulated genes, even though their variation in expression is lower than expected on the basis of previous on‐earth laboratory experiments (8).

In summary, with SPHINX we took advantage of the extreme space environment to investigate the mechanisms responsible for HUVEC alterations in space. We are aware that the experiment has a number of limitations due to the well‐known inconveniences of space biology, including the strictly limited number of samples and possible fixatives, and the timing of the experiment dictated by the size of the bioreactors and the operational constrains. Nevertheless, SPHINX demonstrates that HUVECs are affected by microgravity and respond using the molecular machinery responsible for sensing alterations in flow patterns, even though they are in a condition of no flow. The use of a wide DNA microarray (a total of 28,869 genes) allowed us to identify the metabolic and structural most important pathways responsible for impaired EC function in space. Like disturbed flow, microgravity triggers TXNIP up‐regulation, which, together with mitochondrial dysfunction, generates a prooxidative environment that activates inflammatory responses, alters endothelial behavior, and promotes senescence. It can therefore be concluded that microgravity weakens and jeopardizes HUVEC function. Taken together, our findings may open up new avenues for the treatment of endothelial dysfunction, the common denominator of a number of space‐ and age‐related pathologies ranging from vascular diseases to bone loss.