STEM Today

STEM TODAY July 2018, No. 34

STEM TODAY July 2018, No. 34

CONTENTS CV8: Can manifestations of sub足clinical or environmentally induced cardiovascular diseases during spaceflight be predicted?

Limited data are available to definitively establish the individual roles of spaceflight stressors (i.e. exposure to microgravity, radiation, oxidative and mental stress, or lifestyle alterations in diet and exercise) on short足term and long足term cardiovascular health outcomes. Existing evidence suggest increased vascular stiffness and carotid intimal media thickness immediately post足flight, but it is unclear if these effects persist or resolve over time.

Editorial Editor: Mr. Abhishek Kumar Sinha Editor / Technical Advisor: Mr. Martin Cabaniss

STEM Today, July 2018, No. 34

Disclaimer ( Non-Commercial Research and Educational Use ) STEM Today is dedicated to STEM Education and Human Spaceflight. This newsletter is designed for Teachers and Students with interests in Human Spaceflight and learning about NASA’s Human Research Roadmap. The opinion expressed in this newsletter is the opinion based on fact or knowledge gathered from various research articles. The results or information included in this newsletter are from various research articles and appropriate credits are added. The citation of articles is included in Reference Section. The newsletter is not sold for a profit or included in another media or publication that is sold for a profit.

Cover Page A Closer View of the Moon Posted to Twitter by Astro_Alex, European Space Agency astronaut Alexander Gerst, this image shows our planet’s Moon as seen from the International Space Station. As he said in the tweet, "By orbiting the Earth almost 16 times per day, the ISS crew travel the distance to the Moon and back - every day. Horizons" Image Credit: NASA

Back Cover The Amazon River and Its Surrounding Lakes iss055e086505 (May 25, 2018) – The Amazon River and its surrounding lakes cut through the South American country of Brazil in this photo taken by the Expedition 55 astronauts on board the International Space Station.

Image Credit: NASA

STEM Today , July 2018

Editorial Dear Reader All young people should be prepared to think deeply and to think well so that they have the chance to become the innovators, educators, researchers, and leaders who can solve the most pressing challenges facing our world, both today and tomorrow. But, right now, not enough of our youth have access to quality STEM learning opportunities and too few students see these disciplines as springboards for their careers.

According to Marillyn Hewson, "Our children - the elementary, middle and high school students of today - make up a generation that will change our universe forever. This is the generation that will walk on Mars, explore deep space and unlock mysteries that we can’t yet imagine". "They won’t get there alone. It is our job to prepare, inspire and equip them to build the future."

STEM Today will inspire and educate people about Spaceflight and effects of Spaceflight on Astronauts.

STEM Today, July 2018, No. 34

Editor Mr. Abhishek Kumar Sinha

Editorial Dear Reader The Science, Technology, Engineering and Math (STEM) program is designed to inspire the next generation of innovators, explorers, inventors and pioneers to pursue STEM careers.

According to former President Barack Obama, " Science is more than a school subject, or the periodic table, or the properties of waves. It is an approach to the world, a critical way to understand and explore and engage with the world, and then have the capacity to change that world..."

STEM Today addresses the inadequate number of teachers skilled to educate in Human Spaceflight. It will prepare , inspire and educate teachers about Spaceflight. STEM Today will focus on NASA’S Human Research Roadmap. It will research on long duration spaceflight and put together latest research in Human Spaceflight in its monthly newsletter.

Editor / Technical Advisor Mr. Martin Cabaniss

STEM Today, July 2018, No. 34

Human Health Countermeasures (HHC) CV8: Can manifestations of sub-clinical or environmentally induced cardiovascular diseases during space ight be predicted?

Limited data are available to de nitively establish the individual roles of space ight stressors (i.e. exposure to microgravity, radiation, oxidative and mental stress, or lifestyle alterations in diet and exercise) on short-term and long-term cardiovascular health outcomes. Existing evidence suggest increased vascular sti ness and carotid intimal media thickness immediately post- ight, but it is unclear if these e ects persist or resolve over time.

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SA 3. To determine how alterations in vascular function and structure correlate with changes in circulating biomarkers of oxidative and in ammatory stress.

Changes in arterial structure and function are associated with risk of Cardiovascular disease (CVD) and cerebrovascular disease in the general population. Spaceflight can affect arterial structure and function through changes in the balance of mechanical stimuli, neural and hormonal activities, and radiation. Mechanical stimuli, including circumferential stress related to blood pressure and shear stress related to blood flow, are markedly altered over a 24-h period in space as a consequence of both removal of the effect of gravity and the overall reduction in physical activity.

Animal models have shown that arterial walls adapt to the distending pressures, so hypertrophy of arteries above the heart and atrophy of vessels below the heart might be expected. The carotid arteries of astronauts on the ISS develop thicker walls as measured by the intima-media thickness (FIG. 1), and the carotid arteries become stiffer. Stiffer central and peripheral arteries have also been observed in astronauts from the ISS, as assessed by shorter transit times for the arterial pulse wave from the heart to the finger, and from the heart to the ankle of male, but not female, astronauts. Reduced transit time to the ankle is consistent with the finding of increased intima-media thickness in the femoral arteries of astronauts, but this observation contrasts with the anticipated effect of reduced distending arterial pressure.

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However, increased intima-media thickness of carotid and femoral arteries was also observed in the groundbased confinement simulation of a Mars mission, suggesting that factors other than distending pressure have a role in determining arterial structure and function during spaceflight. In the progression of atherosclerosis, differentiation of stem cells to vascular smooth muscle cells contributes to the increase in intima-media thickness, but the mechanisms are not known, and potential links to spaceflight are speculative.

Neural and hormonal factors change with spaceflight, potentially affecting artery structure and function. Although total peripheral vascular resistance is reduced during spaceflight, evidence for increased sympathetic nervous activity exists, including elevated circulating catecholamine levels and directly measured peroneal nerve sympathetic activity, which might affect arterial stiffness.

As noted above, concentrations of hormones of the renin-angiotensin-aldosterone system increase during spaceflight. Angiotensin II and aldosterone are intimately involved in processes causing increased arterial stiffness through endothelial dysfunction and stimulation of collagen formation, matrix remodelling and hypertrophy, and proliferation of vascular smooth muscle cells. Furthermore, development of insulin resistance could result in increased formation of glycation end products, including crossbridge formation in the vascular extracellular matrix Evidence of changes in arterial structure might also come from animal investigations in spaceflight and groundbased analogues. However, data to date from space-flown mice point to the need for caution in the interpretation of vascular responses. Ground-based models with hindlimb suspension of male rats induced cerebral vessel hypertrophy and improved vasoconstrictor responses, which were attributed to the elevated arterial pressure in this head-down position and to activation of the intrinsic renin-angiotensin-aldosterone system of the vascular wall.

By contrast, female mice flown in space for 2 weeks had reduced myogenic vasoconstrictor responses, and the vessels were more distensible than those of control mice; a follow-up hindlimb-suspension study in male mice found no significant change in cerebral artery structure or function, in contrast to studies with rats. Subsequent 30-day spaceflight studies of male mice revealed attenuated vasoconstrictor and vasodilator properties, along with less-distensible cerebral arteries.

These results raise issues of potential species-specific differences that could relate to body size and pressure gradients. The results also suggest potential sex-specific differences with an increase in arterial stiffness in male mice, but reduced stiffness in female mice - observations that are consistent with the changes in pulse wave transit time, but not carotid artery distensibility, observed in astronauts.

The vascular endothelium is an important regulatory organ responsible for the release of vasoactive factors, preventing platelet aggregation and leukocyte adhesion, and regulating vascular smooth muscle cell proliferation.

Nitric oxide (NO), prostacyclin, and endothelium-dependent hyperpolarization factor have multiple beneficial effects on vascular health, with counter mechanisms resulting from reduced bioavailability of NO in oxidative stress, and from release of vasoconstrictor prostaglandins and endothelin 1. A consequence of endothelial dysfunction can be increased expression of adhesion proteins that facilitate leukocyte adhesion and penetration as precursors to vascular disease.

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The effect of spaceflight on endothelial function is not known, although bed-rest studies unexpectedly reported improvements in function (as assessed by flow-mediated dilatation) in men who performed no exercise countermeasures, and maintenance of function when exercise countermeasures were applied. These data are in contrast to reports of impaired microvascular endothelial function in women after bed rest, and to data from the rat hindlimb-suspension model (with endothelial dysfunction and increased expression of vascular cell adhesion protein 1 in cerebral and carotid arteries).

Endothelial dysfunction occurs as a result of the imbalance of pro-oxidant and antioxidant levels, as is often observed with physical inactivity, and might, therefore, be anticipated with spaceflight. Reduced daily activity levels in space are also associated with risk of insulin resistance, which is strongly associated with endothelial dysfunction in patients with diabetes mellitus. Endothelial dysfunction has been clearly demonstrated in animal models of exposure to high-energy ionizing radiation, with the dysfunction persisting for 2 weeks to 6 months after exposure to radiation. Whether endothelial dysfunction occurs with radiation exposure in spaceflight is not known, but future studies can assess one index of endothelial dysfunction by focusing on flow-mediated dilatation in astronauts in space and on return to Earth (FIG. 2).

SPHINX - SPaceflight of Huvec: an Integrated eXperiment

Exposure to microgravity generates alterations that are similar to those involved in age-related diseases, such as cardiovascular deconditioning, bone loss, muscle atrophy, and immune response impairment. Endothelial dysfunction is the common denominator. The objective of this study is to determine how HUVECs (Human Umbilical Vein Endothelial Cell - cells that line the interior of blood vessel) modify their behaviour when exposed to real microgravity.

It has been previously demonstrated that simulated microgravity reversibly stimulates the growth of macrovascular ECs (Endothelial Cell). In order to investigate the effects of microgravity on HUVEC genomics, proteomics and Nitrogen monoxide synthesis, cell cultures will be flown and incubated in microgravity for several days.

At the end of this period, the culture medium will be separated from the cells and both will be fixed for postflight analysis. The specific benefits of the proposed space experiment range from a better understanding of the molecular mechanisms influencing endothelial behaviour to the possibility of outlining new countermeasures against the astronaut cardiovascular deconditioning and bone demineralisation.

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Endothelial dysfunction is common to age-related diseases and some pathological conditions observed in space, such as cardiovascular deconditioning, bone loss, muscle atrophy, and impaired immune responses. Its exact mechanism is unknown under both conditions, but it is reasonable that alterations in mechanotransduction are implicated , because endothelial cells (ECs) are very sensitive to mechanical forces. Indeed, ECs, which cover the entire inner surface of blood vessels and play a crucial role in maintaining the functional integrity of the vascular wall, are continuously exposed to various hemodynamic forces to which they respond with significant changes in gene expression and in the protein network.

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SPHINX, which formed part of the Progress 40P space mission, was designed to obtain insights into the mechanism responsible for endothelial dysfunction implicated in the abovementioned pathological conditions by taking advantage of the microgravity environment on board the ISS. HUVECs were selected as a consolidated in vitro model of macrovascular ECs.

The SPHINX flight set (Fig. 1) consisted of 12 experiment hardware (EH) modules developed by Kayser Italia (Livorno, Italy; http://www.kayser.it/), each of which had one experiment unit (EU) integrated into the KUBIC interface container single level (KIC-SL). Each EU consisted of a brick made of biologically compatible plastic [polyetheretherketone (PEEK)] containing 5 cylinders (for the medium and chemicals), a cell culture chamber (CC), and connecting channels.

Five small valves were placed to separate the different fluids and the CC. Each cylinder had a piston to inject a new fluid into the CC; the waste medium was collected in the previously emptied cylinder and suitably preserved. During the experiment (Fig. 2), all the medium exchanges and fixation operations were automated

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on the basis of a predefined timeline: T0: installation in KUBIK; T0 + 7 h: first medium exchange (medium A); T0 + 91 h: second medium exchange (medium B); T0 + 167 h: third medium exchange with PBS (medium C); the experiment was stopped by means of 2 subsequent RNAlater fixations (Sigma-Aldrich) separated by an interval of 6 min.

After each exchange, the exhausted culture medium was fixed using a protease inhibitor cocktail (SigmaAldrich). After the experiment was completed, the EH modules were kept at 6◦ C inside the KUBIK incubator before being returned to Earth on Soyuz 23S. Twelve identical EH control modules were prepared in Baikonur and run in parallel using the same experimental protocol. At the end, each module provided a 230-mm2 cell sample and the related preserved media (media A-C, 1.8 ml each).

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Results To evaluate whether microgravity affects EC function, author cultured HUVECs in space and on Earth using suitable bioreactors with automatic fluid exchanges (Fig. 1). Given the temperature and preservation limits of the spaceflight experiment, the cells were cultured for 4 d at 27◦ C, followed by approximately 7 d (160 h) at 36.5◦ C using suitable medium exchanges and the final addition of RNAlater (Fig.2); 27◦ C was the temperature in the Progress vehicle before docking (i.e., from launch to installation in the KUBIK incubator on board the ISS), and, before the flight, authors verified in the laboratory that this temperature would not jeopardize the success of the experiment by culturing HUVECs in the EH modules at 27◦ C for selected time intervals and determining that they were vital (data not shown).

There were some restrictions in selecting the postflight analyses, i.e., authors could not determine cell numbers due to the use of RNAlater. At the end of the in-flight and on-Earth experiments, the cells were confluent with the characteristic static distribution and well attached to the culture support as determined by visual inspection using contrast phase microscopy, thus indicating that they were still alive when fixed with RNAlater.

The long storage time before landing had no influence on the conditions of the fixed flown samples, in accord with the manufacturer’s indications (4 wk) and the preflight experiments. Nevertheless, since the principal aim was to study gene expression in space-flown HUVECs, authors decided to proceed with the RNA analyses, as indicated in the original project.

All of the SPHINX EH modules (in flight and on Earth) were correctly activated, and cells and culture media fixed according to the timeline. The extracted RNA passed the quality check for microarray analysis. The culture media were clear and immediately frozen for further analyses. Out of the 12 spaceflight samples and 12 groundbased controls, authors selected the best ones in terms of RNA quality for microarray analysis.

Microarray analysis of HUVECs The effects of microgravity on HUVEC gene expression were investigated by means of cDNA microarray analyses of 6 randomly chosen samples (3 for each of the 2 conditions of spaceflight and 1 g) using Affymetrix Gene Human 1.0 ST arrays. A separate array was prepared for each of the samples. Since the results were highly reproducible, authors decided to use the remaining samples to perform experiments confirming the results obtained by cDNA arrays. The raw data were imported into Partek Genomic Suite 6.5software.

Principal component analysis (PCA), which allows a visual estimate (3D plot) of the number of clusters rep-

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resented by the data, indicated that the spaceflight samples partitioned away from the 1 g controls, thus confirming the existence of 2 separate clusters (Fig. 3A). Hierarchical clustering showed patterns consistent with PCA (Fig.3B). The microarray hybridization signal intensities of the spaceflight and 1-g control samples were averaged, and a list of the differentially expressed genes was obtained using ANOVA. A 1.3-FC cutoff value coupled with P ≤ 0.01 was used to distinguish statistically higher or lower gene expression from random variation.

We found 1023 genes (585 up-regulated, 438 down-regulated) with a statistically different expression pattern in spaceflight vs. 1-g control samples.

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Inspection of the gene list showed that thioredoxininteracting protein (TXNIP) was the most up-regulated gene, and that heat-shock 70-kDa proteins (HSPA1A and HSPA1B) were the most down-regulated. Most of the top modulated genes were involved in membrane trafficking, cell adhesion/communication/migration, response to stress/oxidative stress, proliferation/angiogenesis, inflammation, DNA damage responses, and cell cycle arrest/apoptosis, which is in line with the knowledge that microgravity affects the cytoskeleton organization, cell adhesion, and migration and may have controversial effects on proliferation and apoptosis. Accordingly, Table 1 shows a selection of these genes.

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GO classification To provide a global functional view of the effects of microgravity, the filtered gene list was classified in terms of GO using DAVID Bioinformatics Resources 6.7 software, which recognized 815 IDs among the 1023 genes modulated by spaceflight. The classification is shown in Table 2.

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A further submission of the gene list to the Kyoto Encyclopedia of Genes and Genomes (KEGG; Kyoto University, Kyoto, Japan) pathway database indicated a statistically significant over representation (corrected P < 0.05) of genes associated with 1) key cellular pathways as oxidative phosphorylation, focal adhesion, spliceosome, and glycophosphatidylinositol (GPI)-anchor biosynthesis; and 2) neurodegenerative and immune diseases (Fig. 4).

Quantitative real-time PCR analysis of selected genes To validate the microarray results, the mRNA expression of 6 top modulated genes (TXNIP, MIR15A, TP53INP1, HSPA1B, HSPA1A, CLCA2) was analyzed by means of RT-PCR. This confirmed the modulation of the 6 tested genes and showed that mRNA expression was higher/lower, thus indicating that the microarray tends to underestimate the FC.

Authors also evaluated the mRNA expression of 6 genes (EDN1, IL1A, IL6, NOS2, NOS3, TNF) known to be involved in inflammation and angiogenesis, and possibly altered in space (Table 3). RT-PCR indicated the up-regulation of IL1A, NOS2, and NOS3 in spaceflight vs. 1-g control samples.

Secretome analysis of HUVEC and NO release HUVEC secretome in the spaceflight and 1-g control conditioned media was first analyzed using the RayBio Human Antibody L-series 507 Array (RayBiotech, Norcross, GA, USA), which simultaneously detects 507 proteins.

This approach did not allow us to reach any substantial conclusion because of the small amount of protein in the medium and the insufficient sensitivity of the analytical approach when applied to this case.

For this reason, author switched to the Bio-Plex multiplex suspension array technique and limited the selection to a panel of 13 proteins involved in inflammation and angiogenesis. Authors did not analyze the A and B media because what was released in A media was due to the combined effects of the initial hypergravity, low temperature gradient (27◦ C), and microgravity; and what was released in B media may have been due to differences in cell number, which could not be assessed under the experimental conditions.

Nevertheless, as both the spaceflight and 1-g control cells were confluent at T0 + 167 h (as revealed by visual inspection), authors assumed that what was released in C media was due to a similar cell number. The results (Table 4) indicate that the secretions of IL-1α and IL-1β were significantly increased in the spaceflight samples vs. 1-g control samples, and the secretion of IP-10 and VEGF was slightly decreased. Authors also measured NO release in the spaceflight and 1-g control C media. Unexpectedly, the results indicated no significant difference between the spaceflight and 1-g controls: 3.15 ± 0.43 vs. 2.86 ± 1.02 µM.

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All cells can respond to applied or cell-generated mechanical forces by activating mechanosensors that mediate the complex process of mechanotransduction. Mechanotransduction is a rapidly expanding area of research 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 as the disturbed fluid shear stress that triggers vascular remodeling and eventually atherosclerosis, or microgravity that induces the loss of bone mass.

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 proteincoupled 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.

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.

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 as well as in yeast the cytoskeleton was considered as the main mechanosensor.

Recently authors demonstrated in our Saccharomyces cerevisiae Oxidative Stress Response Evaluation (SCORE) 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 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. Focal adhesions link the actin cytoskeleton to the extracellular matrix through a cluster of actinassociated proteins that modulate cell survival, migration, proliferation, and other important cell processes.

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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. 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.

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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. It is increasingly recognized that TXNIP plays a crucial role in agerelated diseases and cardiovascular disorders as it is a critical sensor of biomechanical stress and a potent repressor of glucose uptake and glycolysis.

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. Therefore, authors conclude that an overexpression of TXNIP might contribute to the modulation of endothelial function in space.

In this 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. DNA damage activates the p53 signaling pathway, which leads to cell survival if the damage can be repaired, or apoptosis if the damage is too severe. In space-flown HUVECs, TP53INP1 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.

Recent studies indicate that p53 regulates the expression of various miRNAs, 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. Space-flown HUVECs showed increased levels of miR-15a, which may contribute to cell survival or apoptosis: when energy is limited and under other stressful conditions, it induces cell cycle arrest and quiescence, but it also contributes to the induction of apoptosis by targeting the antiapoptotic factor BCL2.

A number of genes belonging to the BCL family (MCL1, BCL2L13, BAK, BNIP3) were modulated in the spaceflown ECs, some toward a proaptotic and others toward an antiapoptotic response. In line with cell cycle arrest and/or proapoptotic responses, authors found the up-regulation of CASP8 (which participates in apoptosis as well as cell proliferation and inflammation), the 2 identified KEGG pathways (Alzheimerâ&#x20AC;&#x2122;s and Parkinsonâ&#x20AC;&#x2122;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 in-

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terfering with key apoptotic proteins. Their down-regulation suggests that HUVECs have adapted to the stress generated by exposure to microgravity. In authorâ&#x20AC;&#x2122;s previous experiments in simulated microgravity authors observed that HUVECs rapidly up-regulated HSP70 as a mechanism of protection against stress-induced apoptosis, which seems in contrast with spaceflight results.

In SPHINX, due to spaceflight restraints, authors 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-g NO releases as due to adaptation of cells after 10d 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.

Effects of short-term microgravity (22 s) on the gene expression and morphology of endothelial cells (ECs) and evaluated gravisensitive signaling elements It has been shown that astronauts experience postflight orthostatic intolerance and a variety of other health problems. Among them are cardiovascular problems, which appear to be due to the dysfunction of the endothelium. Spaceflight is associated with vascular impairment and alterations in the cytokine network. Several groups have cultured ECs under simulated and real microgravity conditions to test the effect on ECs.

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Romanov et al. demonstrated that clinorotation leads to cytoskeletal remodeling in cultured ECs. Long-term gravity vector changes also modulate the expression of surface adhesion molecules, such as intercellular adhesion molecule-1 (ICAM-1), E-selectin, and vascular cell adhesion molecule-1 (VCAM-1), on cultured ECs. Furthermore, RPM exposure remodeled the actin cytoskeleton and reduced the total amount of actin, whereas hypergravity (3 g) did not affect EC growth, markedly stimulated migration, and enhanced NO synthesis. In ECs cultured in a rotating wall vessel (RWV) device, Carlsson et al. detected an overexpression of heat-shock protein (HSP) 70 and a down-regulation of IL-1Îą, which is a potent inhibitor of EC growth and also plays a role in promoting senescence. In addition, these gravitationally unloaded ECs rapidly remodeled their cytoskeleton and, after a few days, markedly down-regulated actin through a transcriptional mechanism. The reduction in the amounts of actin in response to simulated microgravity appears to represent an adaptive mechanism to avoid the accumulation of redundant actin fibers. After exposure to microgravity on a RWV for 6 d, Cotrupi et al. measured low levels of IL-6, which may contribute to EC growth retardation as well as to the enhancement of NO synthesis.

Authors investigated the gravisensitive molecular mechanisms of ECs to identify the biological interface between gravity force and EC function. Long-term simulated microgravity on a RPM increased extracellular matrix proteins and cell adhesion molecules and induced an altered release of cytokines and apoptosis in ECs in vitro.

Several of these changes are also found in ECs in cardiovascular disease and play a role in the development of cancer and neoangiogenesis.

However, the underlying molecular mechanisms are not known. Recently, authors discovered altered signaling pathways in ECs after RPM exposure. The RPM is used to simulate microgravity, allowing the characterization of the cellular behavior and EC function under such conditions, and to prepare future spaceflights and experiments on the International Space Station (ISS). It is important to investigate the first shortterm effects occurring within the first seconds of exposure to microgravity in the rocket. These changes are practically undetectable directly, as the spacecraft is still flying, but a parabolic flight campaign with its 22-s phases of real microgravity provides an ideal environment to analyze them.

Therefore, the aim of this study was to investigate the early effects of microgravity and hypergravity occurring during a parabolic flight on human ECs. Ultrashort, initial, and primary effects and mechanisms were studied during and after the first (P1) and the 31st parabola (P31) on gene expression as a response to altered gravity conditions. In addition, the possible influence of vibrations was tested. The hypergravity and vibration experiments on ground will help to separate their effects on gene and protein expression from those caused by microgravity alone.

Human endothelial EA.hy926 cells were grown in RPMI 1640 medium (Invitrogen, Eggenstein, Germany) supplemented with 10% FBS (Biochrom, Berlin, Germany), 100 U of penicillin/ml, and 100 Âľg of streptomycin/ml.

The cell culture procedure for the parabolic flight campaigns was published recently. In brief, for the flight experiments we used both T75 cell culture flasks (75 cm2 ; Sarstedt, Numbrecht, Germany) and slide flasks (Nunc, Langenselbold, Germany) with subconfluent layers of cells (106 and 105 cells, respectively) and filled them with

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10 ml (T75) and 2 ml (slide flask) of medium. Syringes containing the appropriate fixative were connected to both types of flasks via a flexible tube. In addition, the T75 flasks were also equipped with a 3-way valve (Fig. 1D, E).

RNAlater (Applied Biosystems, Darmstadt, Germany) at a ratio of 4:1 was used for the fixation of the gene array and quantitative real-time PCR samples, whereas the cells designated for Western blot analysis were fixed by addition of ethanol up to a final concentration of 70%. Cells grown in slide flasks were fixed using paraformaldehyde (Carl Roth, Karlsruhe, Germany) at a final concentration of 3%. ECs on slide flasks for acridine orange/ethidium bromide and Hoechst 33342 staining remained unfixed and were stained directly after the flight (Fig. 1E).

One hour before each flight, the cell culture flasks were transported to the aircraft in transportable Cell Trans 4016 incubators (Labotect, Gottingen, Germany) and placed into similar devices that were installed on an experimental rack and preheated to 37◦ C (Fig. 1C). Furthermore, in-flight 1-g control samples were incubated in a centrifuge that was also mounted on the rack. This centrifuge was controlled by a gravity sensor and began operation on reaching microgravity. In addition, corresponding static 1-g samples were cultured in the laboratory (1-g controls).

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Cells for the gene array analyses and quantitative real-time PCR were fixed after P1 and P31, whereas fixation for Western blot samples was done after P31. Moreover, fixation for the slide flasks was done after the first microgravity phase. All cell samples were transported back to the laboratory for further investigations immediately after landing of the aircraft. For each application [Western blot (P31), quantitative real-time PCR (P1 and P31), and gene arrays (P1 and P31)], authors collected n=6 T75 cell culture flasks from both parabolic flight samples (microgravity) and the 1-g control group, as well as n=6 slide flasks for histology (Fig. 1E).

All parabolic flight experiments were conducted aboard the Airbus A300 ZERO-G, which is operated by Novespace and is based in Bordeaux, France (Fig. 1A). On each of the 3 days of the campaign, a parabolic flight, which lasts ∼ 3 h, including takeoff and landing, and encompasses 31 parabolas, takes place. Every parabola started from a steady normal horizontal flight and typically included 2 hypergravity (1.8 g) periods of 20 s, separated on average by a 22-s microgravity period (Fig.1B). The first test parabola was followed by 6 series of 5 parabolas, separated by breaks of 4 and 8 min, respectively. The microgravity level achieved by parabolic flights is 0 ± 0.05 g. The data presented emerged from the 12th, 13th,14th, and 16th parabolic flight campaigns of the German Space Agency, representing a total of 12 parabolic flights or 372 parabolas.

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Results Viability tests of ECs cultured under short-term microgravity To verify whether parabolic flight maneuvers have a damaging effect on cells cultured under these conditions, authors performed both Hoechst 33342 and acridine orange/ethidium bromide staining (Fig. 2). For either staining method, no differences were found between cells grown under 1 g and cells exposed to 31 consecutive parabolas. Apoptotic cells, usually characterized by increased chromatin condensation and formation of apoptotic bodies, could not be detected by Hoechst 33342 in any groups (Fig. 2A,C). Moreover, dead or necrotic cells were also absent, as shown by acridine orange/ethidium bromide staining (Fig. 2B, D).

Short-term microgravity changes the microtubule network of ECs Western blot analyses showed that both β-tubulin and vimentin were significantly down-regulated after 31 parabolas of microgravity, whereas no significant change in cytokeratin-8 protein levels could be observed (Fig. 3A). In addition, immunofluorescence showed that 22 s of microgravity led to a rearrangement of β-tubulin. Instead of a more even distribution as seen in the 1-g samples, cells exposed to microgravity showed accumulations of β-tubulin around the nucleus (Fig. 3B).

Microarray analysis of ECs PCA mapping over 12,321 expressed probes showed that the first and second principal components accounted for 41.7 and 21% of the data set variance, respectively (Fig. 4A). A total of 3605 probes were detected as differentially regulated under the experimental conditions. To further narrow the genes of interest, only those 320 transcripts with a fold change of 2.5 between 1 g vs. P1 were selected. A subsequent filtering by Gene Ontology biological processes resulted in a list of 192 gene symbols and 196 GenBank accession numbers significantly

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regulated with assignable biological function (Table 2). Of those genes, 61 were up-regulated and 131 were downregulated. The corresponding heat map is displayed in Fig. 4B.

To examine the major physical and functional interactions between proteins related to the 320 2.5-fold and significant differentially expressed probes, string network analysis was performed using a stringent confidence score of 0.7. Around the signal transducer and transcription activators STAT1 and STAT2, a subnetwork of proteins related to immune response and type I interferon-mediated signaling could be separated (Fig. 4C, blue cluster). In addition, authors identified an integrin cell surface interaction and hemostasis-related subnetwork (Fig. 4C, green cluster) and a set of cell cycle-related genes connected to mitotic spindle assembly checkpoint protein MAD2A (Fig. 4C, yellow cluster).

Quantitative real-time PCR analysis of selected genes involved in EC signaling Authors selected a total of 23 genes of interest, which were either detected as differentially expressed by the microarray analysis or which have been analyzed by quantitative real-time PCR. The list included mostly genes that were involved in the cytoskeleton, extracellular matrix/cell-cell contact, and endothelial function and comprised ABL2, ACTA2, ADAM19, CARD8, CAV2, CD40, CEACAM1, COL4A5, COL8A1, EDN1, GSN, ITGA6, ITGA10, ITGB3, NOS3, PRKAA1, PRKCA, SERPINH1, TNFRSF12A, TUBB, VASH1, VIM, and VWF. Already after one parabola CAV2, ABL2, EDN1, ABL2, PRKAA, and TNFRSF12A were significantly up-regulated compared with the 1-g control. Of these genes, only ABL2 and TNFRSF12A remained overexpressed after 31 parabolas; all other transcripts returned to 1-g levels. ADAM19, CARD8, CD40, CEACAM1, COL4A5, COL8A1, GSN, ITGA10, ITGB3, NOS3, VASH1, and VWF, on the other hand, were found to be down-regulated already after one parabola.

The expression of COL8A1, ITGA10, NOS3, and VASH1 remained significantly reduced after 31 parabolas, whereas all other transcripts did not differ from the 1-g control. ITGA6, SERPINH1, TUBB, PRKCA, ACTA2, and VIM showed some tendencies toward regulation, but the differences in transcript levels did not prove to be mathematically significant. This result seems to be due to a high variability in the corresponding quantitative real-time PCR measurements, most certainly caused by a generally low expression of these genes (Fig. 5).

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Vibration and hypergravity in general had only a minor effect on the expression of the genes analyzed. No significant changes at all could be observed under the simulated vibration of a parabolic flight. The genes affected by hypergravity according to the acceleration profile during a parabolic flight all answered with decreased expression. NOS3, VASH1, SERPINH1, and CARD8 were significantly down-regulated after one cycle of hypergravity but did not show any change after 31 cycles. In contrast to this, we observed a significant reduction after the 31 cycles of hypergravity for ADAM19, CAV2, CD40, ITGA6, and TNFRSF12A (Fig. 6).

When cells are grown on ground-based devices that simulate microgravity on Earth, they undergo a number of changes. Thyroid cancer cells, lymphocytes, adherently growing ECs, and others develop apoptosis and show changes in differentiation and growth behavior. Genes involved in proliferation, apoptosis, biosynthesis, and secretion are repressed in the absence of gravity in primary human T cells.

Moreover, it was found that altered gravity as achieved by an RWV or an RPM induced cytoskeletal remodeling in ECs, stimulated EC growth, enhanced NO production, and reduced the total amount of actin. Hypergravity, on the other hand, did not affect EC growth, also enhanced NO synthesis, and altered the actin distribution but not its amount. Simulation, especially created by different experimental approaches, needs verification under real conditions. Thus, experiments in real microgravity are absolutely necessary for the interpretation of the ground-based simulation approaches.

So far, the short-term effects of microgravity on ECs are not fully understood. From the technical point of view, it is also difficult to simulate short periods of microgravity, such as 22 s, on machines such as the RPM, because after starting up they all need some time to reach a steady state.

Therefore, authors studied the early changes in ECs induced by shortterm microgravity during a parabolic flight.

To authorâ&#x20AC;&#x2122;s knowledge, this is the first microarray analysis of human ECs exposed to 22 s of real microgravity with the corresponding control experiments (vibration and hypergravity), which allowed for a clear separation of the microgravity effects.

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By analyzing the protein distribution and content in the cells, authors observed a rearrangement of the cytoskeletal protein β-tubulin (Fig. 3B). Similar changes were found for the actin cytoskeleton. F-actin was detectable at the cellular membrane and around the nucleus. Furthermore, Western blot analyses revealed a significant decrease in vimentin and β-tubulin in samples exposed to real microgravity, whereas cytokeratin-8 remained unchanged. Although the rearrangement of β-tubulin was comparable to author’s earlier findings, authors did not observe an up-regulation of cytoskeletal proteins, which authors detected in long-term experiments. This might therefore represent the initial cellular response to microgravity, which is later compensated for by other mechanisms, leading to an increase in cytoskeletal components.

Short-term microgravity-induced changes in gene expression Cytoskeleton ABL2 (also known as ARG) was significantly upregulated after 1 and 31 parabolas of real microgravity, whereas

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both vibration and hypergravity did not change its expression (Figs. 5B and 6B). ABL2 belongs to a family of tyrosine kinases that regulate cellular morphogenesis and motility through functional interactions with the actin cytoskeleton. It possesses an internal F-actin-binding site and ABL2- F-actin interaction leads to F-actin bundling.

Therefore, it was proposed that ABL2 uses its F-actinbundling activity to directly regulate the cytoskeletal structure. In parallel, the ACTA-2 gene expression as measured by quantitative real-time PCR was down-regulated (Fig. 5B), which is in accordance with the corresponding gene array result (Table 2). In addition, although not mathematically significant, VIM gene expression was slightly down-regulated, and TUBB mRNA was elevated after P1 and P31 (Fig. 5B).

Cell viability GSN mRNA was significantly down-regulated under short-term microgravity (P1), but it was increased again

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after P31. Vibration and hypergravity did not change its expression pattern (Figs. 5E and 6E). GSN protein mediates cellular motility, morphogenesis, and actin cytoskeleton remodeling. The best-known functions of GSN are its actin filament severing, capping, uncapping, and nucleating activities.

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Moreover, GSN also mediates apoptosis. Full-length GSN, as well as its C-terminal half, have been found to be mostly antiapoptotic, whereas the N-terminal half is apoptotic. When intact, GSN forms a stable complex with actin and DNase I, inhibiting DNase I activity. In contrast to this, the N terminus alone disrupts the actinDNase I interaction and releases DNase I, which is a potent effector of apoptosis. GSN is a substrate of caspase-3, -7, and -9 and has been shown to be down-regulated in different human cancers, which usually can evade apoptosis.

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CARD8 (also known as CARDINAL) gene expression was also found to be decreased after 22 s of real microgravity (Fig. 5E). The CARD family proteins play a central role in the activation of caspases, the regulation of apoptosis, and the mediation of NF-ÎşB activation. CARD-containing proteins occur repeatedly in stress response pathways that lead to activation of either caspases or NF-ÎşB. Activation of the latter molecules results in either apoptosis or transcription of proinflammatory genes, responses that are consistent with the preservation of the

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integrity of multicellular organisms. CARD8 down-regulation may be an early adaptation of the ECs toward conditions of microgravity.

In addition, the ECs exhibited an increase in TNFRSF12A (TNF-related weak inducer of apoptosis receptor, TWEAKR or FN14) transcript levels (Fig.5E). TWEAKR is a member of the TNF receptor family, binds the TWEAK cytokine with high affinity, and is mainly expressed in vascular smooth muscle cells and ECs. The TWEAK/TWEAKR signaling cascade mediates NF-κB-activity via different TRAF (TNF receptor-associated factor) signal transducers, and it has been shown that it can promote cell proliferation, migration, and angiogenesis in human vascular ECs. CD40 (TNFRSF5), also belonging to the TNF receptor family, is a 48-kDa transmembrane glycoprotein cell surface receptor. It was first identified and characterized on B cells but it is also expressed, among others, in ECs. Activation of CD40 by its ligand CD40L leads to inflammatory responses such as secretion of leukocyte adhesion molecules, cytokines, or matrix metalloproteinases.

CD40 and cell adhesion molecules such as ICAM-1 and VCAM-1 are involved in angiogenesis, because CD40/CD40L interaction up-regulates these cell adhesion molecules, which in turn bind immune cells, supporting vessel formation. After the first parabola, CD40 mRNA levels were reduced in comparison with the 1-g controls but were elevated after 31 parabolas (Fig. 5E). This is in accordance with earlier long-term experiments showing that the relative quantities of TNFRSF5 mRNA expressed in ECs incubated for 7 d either under normal gravity or under simulated microgravity on a RPM were not changed.

ADAM19 exhibits proteolytic activity and is able to cleave the membrane-anchored precursor of the potent proinflammatory cytokine TNF-α. Over expression of ADAM19 in mouse embryonic fibroblasts showed that it can contribute to the constitutive release of TNF-α, thus exerting an increased inflammatory effect. In ECs, on the other hand, we measured reduced ADAM19 gene expression in cells exposed to 22 s of microgravity compared with the 1-g control (Fig. 5E).

AMPK (PRKAA1) is able to mediate cell proliferation and regulates the antioxidant status of ECs. Colombo and Moncada have shown that PRKAA1 silencing leads to reduced NOS3 levels, reduced cell proliferation, increased ROS accumulation, and apoptosis. The PRKAA1 expression in our cells was significantly elevated after 1 parabola and remained up-regulated after 31 parabolas (Fig. 5E). It is very likely that this finding has a bene-

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ficial effect for the cells.

Taken together, the expression patterns of GSN, CARD8, TNFRSF12A, CD40, ADAM19, and PRKAA1 seem to hint at a very early protective mechanism of cell survival/proliferation induced by short-term microgravity, which may later be replaced by inflammatory or apoptotic processes after the environmental stimulus of altered gravity affected the cells for a longer period of time.

Angiogenesis Two genes that were detected as differentially regulated by the microarray and quantitative real-time PCR method are involved in the regulation of angiogenesis: CEACAM1 and VASH1. Both genes have opposite functions. CEACAM1 is expressed in microvessels of proliferating tissues, in wounded tissues, and in solid human tumors.

Moreover, purified CEACAM1 exhibits angiogenic properties in in vitro and in vivo angiogenesis assays by stimulating cell proliferation, chemotaxis, and tube formation. CEACAM1 expression is regulated by VEGF. A monoclonal anti-CEACAM1 antibody completely blocked VEGFmediated tube formation in vitro, showing that CEACAM1 is a potent angiogenic factor and a strong effector of VEGF.

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VASH1 is a VEGF-inducible protein in human cultured ECs and has antiangiogenic properties. VASH1 differs from other angiogenesis inhibitors by being selectively induced in ECs by proangiogenic factors such as VEGF and basic fibroblast growth factor.

Thus, VASH1 is considered to be an intrinsic and highly specific negative feedback regulator of activated ECs engaged in angiogenesis. The protein inhibits migration, proliferation, and EC network formation in vivo. VASH1negative feedback action is EC-exclusive, as transfection into human lung carcinoma cells did not alter their proliferation but affected tumor angiogenesis.

Authors found both CEACAM1 and VASH1 transcripts to be down-regulated in ECs exposed to 22 s of real microgravity (P1 and P31; Fig. 5C). In long-term experiments (7 d) authors have measured an increase in the VEGF gene expression and the VEGF protein content in ECs cultured under simulated microgravity on a RPM compared with 1-g controls, but the corresponding VEGF level in the supernatant was reduced in these cultures.

This may explain the reduction in CEACAM1 and VASH1 mRNAs. Gravisensing/mechanosensing and regulation of vasotonus Caveolae are found in different cell types but are most abundant in vascular ECs, adipocytes, fibroblasts, and epithelial cells. The proteins CAV-1, -2, and -3 are the main structural components of caveolae.

CAV-1 and CAV-2 are expressed in most cardiovascular cell types, whereas CAV-3 is mostly present in vascular, cardiac, and skeletal muscle. CAV-1 and CAV-3 expression seems to be necessary for the formation of caveolae, whereas the role of CAV-2 is less clear.

It has been shown, however, that CAV-2 is almost always coexpressed with CAV-1 and supports caveolae assembly by forming heterooligomers with CAV-1. It has also been shown that NOS3 (eNOS) is localized inside the caveolae. By binding to it, CAV-1 can inhibit NOS3 activity and therefore reduce NO production. NO, on the other hand, is able to directly downregulate EDN1 gene expression.

It has been reported that caveolae can also act as mechanosensors by converting mechanical stimuli caused by the blood flow into chemical signals, helping the endothelium to adapt to different conditions inside the vessel. Likewise, caveolae also react to changes in gravity. Spisni et al. demonstrated that hypergravity (3 g) changes CAV-1 expression, leading to altered NOS3 expression and NO production.

Thus, authors speculate that authors observed a similar effect in our cells. Authors detected increased CAV2 gene expression, which might lead to the formation of new caveolae or to a higher CAV protein density inside them. The elevated CAV-2 levels could affect NOS3 on both transcriptional and protein levels by down-regulating NOS3 gene expression and inhibiting NOS3 activity by means of direct interaction. These processes would lead to reduced NO production, followed by induction of EDN1 gene expression by the lower NO levels. In summary, the observations suggest vasoconstriction as the fast initial endothelial reaction to microgravity (Fig. 5A).

Extracellular matrix ECs express collagens I, III, IV, VIII, and XIII. Different collagen subtypes are increased in cardiovascular disease, diabetes, and ECs grown under microgravity conditions. Authors found COL4A5 and COL8A1 gene expression

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to be downregulated after 22 s of real microgravity (Fig. 5D).

Both collagens belong to the group of nonfibrillar collagens and they are involved in the formation of vascular basement membranes. The function of collagen VIII is still unclear, but it is believed to play a central role in maintaining vascular integrity and structure. SerpinH1 (HSP47) was not significantly down-regulated (Fig. 5D). HSP47 is known to be involved in the synthesis and/or intracellular transport of type I collagen in ECs. VWF is known to bind to collagen IV, and it can promote EC attachment via the so-called vitronectin receptor (αv β3 ), a member of the β3 integrin family. Interestingly, authors found COL4A5, COL8A1, VWF, ITGB3, and ITGA10 gene expression to be reduced under conditions of real microgravity (Fig. 5D).

This finding seems to hint at a tendency for a less stable extracellular matrix and a weaker cell-cell contact.

In previous long-term RPM studies, authors observed the detachment of ECs and the formation of tube-like structures in the cell culture medium. The finding may therefore represent the first steps toward the rearrangement of the extracellular matrix favoring the development of endothelial tubes. The results show that ECs are very sensitive to real microgravity and that a relatively short period of only 22 s of gravitational unloading is sufficient to induce a multitude of adaptive mechanisms inside the cells, ranging from alterations in the cytoskeleton to regulation of cell survival. Vibrations similar to those occurring during parabolic flights had no effects, and the effect of hypergravity (1.8 g) was very small and did not interfere with the microgravity findings.

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The string network analysis indicated a network of proteins around STAT1 and STAT2, which is related to immune responses. In addition, integrin signaling and a set of cell cycle-related genes could be defined. As measured by quantitative real-time PCR, the integrins ITGA10 and ITGB3 were significantly downregulated by microgravity. These signaling pathways will be investigated in more detail in future parabolic flight campaigns.

SPHEROIDS

The science team will investigate the effects of microgravity on endothelial cell function, their program of differentiation and apoptosis. The goal of the study is to investigate the three-dimensional cell assembly under real microgravity, while emphasizing proliferation, differentiation and induction of apoptosis (programmed cell death). Extensive studies have been performed on cultured endothelial cells over the last few years using the Random Positioning Machine (RPM).

Expected results: • Induced formation of multicellular tubular structures • Induced apoptosis in human endothelial cells • VEGF inhibited apoptosis • Over-production of extracellular matrix • Re-arrangement of cytoskeletal proteins • Biomarkers released into the culture supernatants • Changes in gene expression (RNA formation) Based on the recent RPM experiments, it is suggested that the space flight experiments will show these results: Exposure of the endothelial cells to real microgravity should trigger the endothelial cells to detach from the bottom of the culture dish and to reassemble to tubular structures, which could comprise a wall of one layer of endothelial cells and thus be similar to natural blood vessel intimas. The tube formation may go off, while a rate of apoptosis varying between 20 % to 30 % is initiated by the microgravity. This rate could be lower, when VEGF is added in the beginning but should remain stable until the end of the experiment. The formation of the three dimensional cellular structures is expected to be accompanied by an overproduction of extracellular matrix components, which stabilize the structures. The biomarkers released into the culture supernatants and the changes in gene expression (RNA formation) will help us to understand, what is happening at a molecular level, when endothelial cells form blood vessel intimas in vitro.

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