STEM Today by Abhishek Sinha

STEM TODAY August 2018, No. 35

CONTENTS CV8: Can manifestations of subÂclinical 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-term and long-term cardiovascular health outcomes. Existing evidence suggest increased vascular stiffness and carotid intimal media thickness immediately post-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

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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 Hurricane Hector just south of the Hawaiian island chain iss056e126638 (Aug. 7, 2018) — Hurricane Hector was pictured by an Expedition 56 crew member as the International Space Station orbited nearly 250 miles above the Pacific Ocean just south of the Hawaiian island chain. Image Credit: NASA

Back Cover Soaring Into an Orbital Sunrise The International Space Station soars into a sunrise every 90 minutes, each and every day. This image, taken on July 20, 2018, shows one of four basketball court-sized main solar arrays that power the space station, in contrast to the bright blue glow of Earth’s limb in the background as the orbital complex flew over eastern China. Image Credit: NASA

STEM Today , August 2018

Editorial Dear Reader

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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. 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

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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.

Effect of Microgravity and Hypergravity on Endothelial Cells

The endothelial cells (ECs), which line the inner surface of vessels, play a fundamental role in maintaining vascular integrity and tissue homeostasis, since they regulate local blood flow and other physiological processes. ECs are highly sensitive to mechanical stress, including hypergravity and microgravity. The endothelium is semipermeable and regulates the transport of various molecules between the blood and underlying interstitial space by expressing specific carriers. ECs also control vascular permeability, especially in microvascular districts. Moreover, ECs importantly contribute to maintaining a nonthrombogenic blood-tissue interface since they release various antithrombotic and fibrinolytic factors as well as molecules that impact on platelets.

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The endothelium is an immunocompetent organ because it exposes histocompatibility and blood group antigens can be induced to express adhesion molecules for leukocytes and produce cytokines. Finally, a functional relation exists between endothelial and smooth muscle cells, as a consequence of the presence of junctions allowing the passage of electric charges and metabolites, and the production and release of vasoactive mediators. Indeed, ECs finely control vasomotor responses through the production and metabolism of vasoactive molecules acting on smooth muscle cells, as endothelin-1 (ET-1), nitric oxide (NO), and angiotensin II (AngII). They also tightly control smooth muscle cells proliferation. ECs are protagonists in angiogenesis, that is, the formation of new blood vessels from preexisting ones. Angiogenesis involves the most dynamic functions of the endothelium, since it requires the migration of ECs, their ability to degrade the extracellular matrix, their proliferation and differentiation, ultimately leading to functional capillaries. This highly organised process is modulated by the balance between stimulators and inhibitors of angiogenesis. Vascular endothelium is structurally and functionally heterogeneous. This heterogeneity is detectable at different levels, that is, markers of cell activation, gene expression, responsiveness to growth factors, and antigen composition, and differentiates the behaviour between microand macrovascular ECs, as well as between cells isolated from different organs and from different vascular districts of the same organ. In fact, the arteriolar endothelium is different from the venous one, as well as fromthe micro- and macrovessel derived ECs. The endothelium of the cerebral circulation- which is the main component of the bloodbrain barrier to protect the brain from toxic substances- deserves special consideration. It is continuous, has tight junctions, and differs both from fenestrated endothelium, where cells have pores, and fromdiscontinuous endothelium, where cells have intracellular and transcellular discontinuities. ECs are normally quiescent in vivo with a turnover rate of approximately once every three years. Most of ECs in the adult have a cell cycle variable from months to years, unless injury to the vessel wall or angiogenesis occurs. Only endothelium from endometrium and corpus luteum has a doubling time of weeks. ECs act as mechanotransducers, whereby the transmission of external forces induces various cytoskeletal changes and activates second messenger cascades, which, in turn, may act on specific response elements of promoter genes. Therefore, it is not surprising that ECs are sensitive to variations of gravity.

Effects of Microgravity on Endothelial Cells

Endothelial Cells are highly sensitive to microgravity and undergo morphological, functional, and biochemical changes under these conditions. The studies have used a variety of in vitro cell models with divergent results. One of the reasons for these discrepancies can be EC heterogeneity or the isolation from different species. Indeed, human, bovine, murine, and porcine endothelial cells have been investigated under gravitational unloading.With concern to human cells, studies are available on human ECs from the umbilical vein (HUVEC),

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widely considered a model of macrovascular endothelial cells, as well as on human microvascular ECs (HMEC). Moreover, studies have been performed on EA.hy926 cells, a fusion of HUVEC with the lung carcinoma cell line A549. Although immortalized cell lines offer significant logistical advantages over primary cells in in vitro studies, they exhibit important differences when compared to their primary cell counterparts. Indeed, microarrays used for a genome-wide comparison between HUVEC and EA.hy926 in their baseline properties have shown that EA.hy926 cells are useful in studies on genes encodingmolecules involved in regulating thrombohemorrhagic features, while they appear to be less suited for studies on the regulation of cell proliferation and apoptosis. Moreover, immortalized endothelial cell lines show different expression pattern of biomarkers when compared to primary cells. The controversial results reported about the response of ECs to microgravity could be due also to the diverse experimental approaches utilized, such as the device simulating microgravity, the duration of exposure to simulated microgravity, and the degree of reduction of the gravity that can be reached operating these devices differently.

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Nevertheless, altered EC morphology, cell membrane permeability and senescence are documented by spaceflight experiments on cultured endothelium. Several aspects of endothelial behaviour have been studied in simulated and real microgravity. Table 1 summarizes the published findings.

No significant modulation of cell migration under basal condition and in response to the angiogenic factor hepatocyte growth factor (HGF) was observed in HUVEC as well as in HMEC cultured in the RPM [12, 46]. Shi et al. demonstrated that, after 24 h of exposure to simulated microgravity in a clinostat, HUVEC migration was significantly promoted through the eNOS pathway upregulation by means of PI3KAkt signalling. On the contrary, the endothelial cell line EA.hy926 in simulated microgravity migrated more than controls, while in a study on porcine aortic endothelial cells (PAEC), microgravity modelled by a RPM caused a marked impairment of cell migration induced by serum or the angiogenic factors vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2).

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Proliferation and Formation of 3D Structures Carlsson and Versari, using the RWV and the RPM, respectively, found that the proliferation rate of HUVECs was reversibly increased under simulatedmicrogravity. Also bovine aortic ECs (BAEC) grew faster in the RWV than controls. On the contrary, simulated microgravity inhibited the growth of HMEC and murine microvascular ECs. The

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results obtained using microvascular EC are reinforced by the in vivo finding showing an impairment of angiogenesis in space. Wound healing, in which neovascularization is an early and fundamental step, is retarded in space-flown animal models , and the development of vascular channels in a rat fibular osteotomy model is inhibited after flight, as shown by an experiment carried out during a shuttle mission. Also in PAECs a marked impairment of EC responsiveness to angiogenic factors and a reduced ability to proliferate were reported. Using the endothelial cell line EA.hy926, Grimm et al. showed the formation of 3D tubular structures in clinorotation. After two weeks, a subtype of 3D aggregates was observed with a central lumen surrounded by one layer of ECs. These single-layered tubular structures resembled the intimas of blood vessels. Characterization of these tubular structures revealed that they might originate fromdouble-rowcell assemblies formed between the fifth and seventh day of culture under simulated microgravity.

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Apoptosis Increased apoptosis after culture in the RPM has been observed in PAEC and the endothelial cell line EA.hy926 . In particular, following exposure to simulated hypogravity, PAEC change their morphology and gene expression pattern, triggering proapoptotic signals.The gene expression profile demonstrated the upregulation of p53, FAS-L, and BAX genes, and the concomitant downregulation of the antiapoptotic protein Bcl-2 and proliferation marker PCNA. The induction of apoptosis was accompanied by mitochondrial disassembly, thus suggesting the activation of the mitochondrial intrinsic pathways. In pulmonary HMEC simulated microgravity-induced apoptosis by downregulating the PI3K/Akt pathways and increasing the expression of NFκ B . On the contrary, no apoptosis was observed in HUVEC and dermal HMEC cultured for various times in the RWV or in the RPM, and this has been linked to the rapid induction of heat shock protein (hsp)-70. Indeed, hsp-70 protects endothelial cells from apoptotic stimuli acting downstream of cytochrome c release and upstream of caspase 3. Alterations of Cytoskeleton and Extracellular Matrix The cytoskeleton plays a key role in the adaptation to mechanical stress, including alterations of gravity. Therefore, the changes that cytoskeletal components, such as microtubules, undergo in microgravity can be a key to explaining the effects of weightlessness on cells. Carlsson et al. studied actin microfilaments in HUVEC exposed to microgravity simulated by the RWV. In comparison with controls, the cells showed elongated and extended podia, disorganization of actin microfilaments that clustered in the perinuclear area, and decrease in stress fibers. Moreover, after 96 h exposure, actin RNA levels were downregulated and total actin amounts were reduced. The cytoskeletal modifications were reversible upon return to normal growth conditions (1 X g). The authors speculated that the reduction in actin amount could be an adaptive mechanism to avoid the accumulation of redundant actin fibers. The same results were obtained when the experiment was replicated by using a RPM to model the microgravity conditions. More recently, in HUVEC exposed to mechanical unloading by RPM, Grenon et al. found disorganization of the actin network with clustering of the fibers around the nucleus. Moreover, they observed that caveolin-1 was less associated with the plasma membrane and adopted a perinuclear localization. Thus the authors advanced the hypothesis that disruption of the actin cytoskeleton organization could impair the translocation of caveolin-1 to the caveolae. After spaceflight (Soyuz TMA-11), readapted HUVEC cells with subsequent passages exhibited persisting changes in the organization of microtubules, with prominent bundles that occupied the peripheral cytoplasm. In a study carried out by Zhang et al. HUVEC activated with TNF-α and exposed to microgravity modeled by RWV demonstrated that, after 30 min, depolymerization of F-actin and clustering of ICAM-1 on cell membrane occurred. Moreover, ICAM-1 and VCAM-1 RNA were up regulated. After 24 h, actin fiber rearrangement was initiated, clustering of ICAM-1 became stable, and the mRNAs of ICAM-1 and VCAM-1 returned to levels comparable with the controls. The authors speculated that actin cytoskeleton rearrangement and changes in levels and distribution of surface adhesion molecules could significantly affect transendothelial migration processes. Grosse et al. studied the effect of parabolic flight on the cytoskeleton of the endothelial cell line EA.hy926. Every parabola (P) included two hypergravity (1.8 g) periods of 20 s, separated by a 22 s microgravity period. After P1, they observed a rearrangement of β-tubulin that accumulated around the nucleus. After P31, β-tubulin

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and vimentin were down regulated. Using the EA.hy926 cell line exposed to parabolic flight, Wehland et al. reported that the actin network underwent a drastic rearrangement, mostly affected by vibration. Grimmet al. studied the walls of tube-like structures spontaneously formed by the endothelial cell line EA.hy926 cultured in a RPM. They found that the walls consisted of single-layered endothelial-like cells which had produced significantly more Î˛1 -integrin, laminin (LM), fibronectin (FN), and Îą-tubulin than controls. Microgravityinduced up regulation of proteins involved in the extracellular matrix building was confirmed in studies carried out by Monici et al. on cultured bovine coronary venular endothelial cells (CVECs) exposed for 72 h to microgravity modelled by a RPM. The authors observed an increase in actin content and impressive production of actin stress fibers, accompanied by the overexpression and clustering of Î˛1 - integrin, 40% increase in LM, 111% increase in FN content, and formation of a tight and intricate network of FN fibrils. Since FN and LMare strongly involved in the regulation of cell adhesion/migration, their upregulation and altered networking, together with the changes in actin and integrin patterns induced the authors to hypothesize that the exposure to microgravity causes a dysregulation in cell motility and adhesion to the substrate.

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In summary, all of the studies carried out so far demonstrated that microgravity strongly affects cytoskeleton organization and induces a rearrangement of the actin network with clustering of the fibers in the perinuclear area. A similar behavior has been observed also analyzing the microtubule network. Moreover, clustering of adhesion molecules on the plasma membrane and over expression of proteins of the extracellular matrix have been reported by some authors. The results are less consistent when considering the expression of cytoskeleton proteins or their RNA. Probably the discrepancies are due to differences in experimental models (different cell populations), protocols, and analytical procedures. However, it is widely accepted that the microgravity induced changes in the cytoskeleton can strongly affect the behavior of endothelial cells in terms of adhesion, migration, and production of extracellular matrix and can interfere with other processes such as translocation of molecules inside the cells, transendothelial migration, and even inflammation and angiogenesis. Synthesis of Vasoactive Molecules The levels of vasoactive molecules, such as NO, and ET-1 are modified under microgravity conditions, which also indicates that microgravity may influence both hemodynamic changes and angiogenesis. In particular, HUVEC and HMEC exposed to simulated microgravity using RWV and RPM produce more NO than controls as the result of increased levels of endothelialnitric oxide synthase (e-NOS), which correlates with the increase of caveolins. In particular, Grenon et al. suggested that the alterations in NO production are mediated by changes in the cytoskeleton detected in all the endothelial types studied. Wang et al. explained the increased amounts of NO in HUVEC after 24 h in simulated microgravity as the results of the up regulation of inducible NOS through a mechanism dependent on the suppression of the activity of the transcription factor AP-1. Also in BAEC NO production was increased. In the endothelial cell line EA.hy926, a reduced release of ET-1 and VEGF was reported, while the production of NO was increased via the iNOS-cGMP-PKG pathway . If confirmed in vivo in space, these results might, in part, explain the hemodynamic changes and the redistribution of blood flows induced by microgravity. Genomic and Proteomic Analysis Microgravity affects several molecular features of ECs markedly modulating gene expression. In HUVEC cultured in the RPM, the secretome was evaluated by a 2D proteomic approach. The proangiogenic factor FGF-2 and the proinflammatory cytokines interleukin-1 (IL-1) and IL-8 were decreased in simulated microgravity, whereas two chemokines involved in leukocyte recruitment, Rantes and Eotaxin, were increased. The unprecedented gene profile analysis on HUVEC cultured on the ISS for 10 days was performed by Versari et al. . 1023 genes were significantly modulated, the majority of which are involved in cell adhesion, oxidative phosphorylation, stress responses, cell cycle, and apoptosis, thioredoxin-interacting protein being the most upregulated. Briefly, in cultured HUVEC, real microgravity affects the same molecular machinery which senses alterations of flow and generates a prooxidative environment that alters endothelial function and promotes senescence. Similar conclusions were reached by Kapitonova et al., who described premature senescence in space-flown

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HUVEC. By accelerating some aspects of senescence, microgravity offers a big challenge to study the mechanisms implicated in the onset of aging. Looking at the endothelial cell line EA.hy926, a short term lack of gravity (22 s) generated by parabolic flights significantly influences the signalling pathways. When these cells are cultured for various times from 4 to 72 h on the RPM, a number of proteins of the extracellular matrix implicated in apoptosis are modulated when compared to control cells. In the RPMsome EA.hy926 cells form tube like 3D aggregates, while others continue to grow adherently. 3D aggregates and adherent cells were analyzed by gene array and PCR techniques and compared to controls. 1625 differentially expressed genes were identified and, in particular, the levels of expression of 27 genes changed at least 4-fold in RPM-cultured cells when compared to controls.These genes code for angiogenic factors and proteins implicated in signal transduction, cell adhesion, membrane transport, or enzymes. Fifteen of them, with IL-8 and von Willebrand factor being the most affected, showed linkages to genes of 20 proteins that are important in the maintenance of cell structure and in angiogenesis.

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EA.hy926 cell line and human dermalmicrovascular ECs (HMVECs) were then compared after culture on the RPM for 5 and 7 days . A total of 1175 types of proteins were found in EA.hy926 cells and 846 in HMVECs, 584 of which were common and included metabolic enzymes, structure-related and stress proteins. This proteomic study also highlights that HMVECs develop tube-like 3D structures faster than EA.hy926 possibly through a transient augmentation of ribosomal proteins during the 3D assembling of ECs. Effects of Hypergravity on ECs A summary of published data on endothelial cell behaviour is reported in Table 2. HUVECs exposed to hypergravity (3 X g) for 24-48 h showed inhibition of cell growth but unaltered apoptosis, increased COX-2, eNOS, and Cav-1, suggesting a possible role of caveolae in mechanotransduction.

Also an increased synthesis of PGI2 and NO, which are also proangiogenic, was observed. However, surprisingly, the formation of capillary-like structure was inhibited. Versari et al., studying the same cells exposed to 3.5 X g, found increased NO production, enhanced cell migration, but no effects on proliferation.

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Moreover, altered distribution of actin fibers without modifications of the total amounts of actin was detected. In the same conditions, HUVEC showed a time-dependent decrease in occludin correlating with an increase in paracellular permeability and a decrease in transendothelial electrical resistance, indicating a decrease in EC barrier function, with exactly opposing results in BAEC cultured under hypogravity in RWV where increased barrier properties were detected. Koyama et al. reported that, after a few minutes of exposure to 3 X g in a centrifuge, BAECs showed actin reorganization via Rho activation and FAK phosphorylation, increased cell proliferation, and ATP release.A daily exposure of 1-2 h repeated for 5 consecutive days promoted cell migration. Wehland et al. investigated short term (s) effects of hypergravity (1.8 X g) on EA.hy926 cells and found that the cells were weakly affected by loading in the conditions used for the experiment. On the contrary, short term effects of microgravity were much more evident.

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Authors studied and compared the behavior of coronary venular endothelial cells (CVEC) and BAEC exposed to 10 X g for 5 periods of 10 minutes each spaced with four recovery periods of the same duration. Following exposure, both the cell types showed similar changes in cytoskeleton organization and αvβ3 integrin distribution.The peripheral ring of actin microfilaments was substituted by trans-cytoplasmic stress fibers, microtubules, and intermediate filaments gathered in the perinuclear area, focal contacts in the protruding lamellipodia disappeared, and αvβ3 integrin molecules clustered in the central body of the cells. Both in CVEC and in BAEC the expression of the cytoskeletal proteins β-actin and vimentin increased. In BAEC the transcripts for the matrix proteins LM and FN decreased. In both the cell types exposure to hypergravity decreased the transcription of genes encoding for the proapoptotic factors Fas and FasL, Bcl-XL. Cell energy metabolism, assessed by auto fluorescence spectroscopy and imaging, did not change significantly in BAECs. On the contrary, CVECs exposed to hypergravity showed an increase of the anaerobic metabolism, in comparison with 1 X g controls. The phenotypic expression of molecules involved in inflammation and angiogenesis such as eNOS, FGF-2, and COX-2, which is not expressed in basal conditions, did not significantly change as assessed by immunofluorescence microscopy in CVECs. Nevertheless, in BAECs the expression of COX-2 and other genes controlling the calibre of the vessels, that is, renin, ET processing enzyme, and inflammation, such as TNFα and its receptor CD40, P and E selectins, CD54, was downregulated. Briefly, hypergravity does not seem to affect significantly the survival of both macro- and microvascular ECs. However, significant changes have been observed in cytoskeleton and integrin distribution in all the ECs studied, and changes in cell energy metabolism have been observed only in CVECs, while the downregulation of some genes involved in inflammation and vasoconstriction has been found only in BAECs.

Considering the expression of growth modulators, hypergravity increased VEGF expression while it decreased a series of interleukins acting as inhibitors of EC proliferation. These results are consistent with the hypothesis that the EC response to gravitational alterations depends, at least in part, on the vascular district from which the cells are derived. Briefly, from studies on different types of ECs exposed to simulated microgravity :

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• Impact on cell proliferation and survival: all the studies indicate alterations of cell proliferation. Only HUVEC and BAEC have been reproducibly found to proliferate faster in microgravity than controls. Microvascular EC and other endothelial cells are growth inhibited or induced to apoptosis. • Impact on NO synthesis: most studies agree on the increased production of NO through the modulation of NOS isoforms. • Impact on cytoskeleton: all the studies described important cytoskeletal remodelling in all the different EC analyzed. • Impact on gene expression: no doubt exists about the profound modifications of gene expression by exposure to simulated or real microgravity.

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The impact of hypergravity on ECs is less defined. Due to the different experimental approaches adopted on various cell types the findings are not consistent and deserve further consideration. The effects of gravitational forces on mechanotransduction in ECs responses have been the matter of only a few investigations and remain largely unknown. The plausible mechanosensing targets for gravity changes appear to be the cytoskeletal structure and particularly caveolae.

Simulated microgravity promotes nitric oxide-supported angiogenesis via the iNOS-cGMP-PKG pathway in macrovascular endothelial cells

The effects of microgravity on angiogenesis are largely unknown. In this study, authors have used in vitro and in ovo models of angiogenesis to grow blood vessels and tubes from endothelial cells (EC) under microgravity. EAhy926, an immortalized macrovascular cell line was gifted by Dr. Cora-Jean Edgell, University of North Carolina, Chapel Hill, USA. Bovine lung microvascular endothelial cells were harvested from slaughtered animals as described previously. Bovine pulmonary aortic endothelial cells were isolated from the pulmonary artery. Porcine ventricular endocardial endothelial cells (PVEEC) was gifted by Dr. C.C. Kartha, Rajiv Gandhi Centre for Biotechnology, Trivandrum, India (Table 1).

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Three-dimensional clinostat was used to simulate microgravity as described previously. Cells grown in culture plates were subjected to microgravity for 2 h at 37 ◦ C. To cancel the dynamic simulation of gravity in any direction, cycles of rotation were electronically controlled at an outer frame rotation of 20 rpm and an inner frame rotation of 13 rpm.

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Results Microgravity stimulates endothelial cell functions Wound healing assay using EAhy926 monolayer showed 60% faster healing under microgravity for 2 h (Fig. 1A). Number of EAhy926 cells that migrated from upper to lower compartments of Boyden’s chamber was significantly more in microgravity treated group (Fig. 1A). Authors also investigated effects of microgravity on endothelial cell proliferation by incubating EAhy926 cells with MTT and CMFDA for 30 min and 2 h, respectively. Microgravity treatment for 2 h promoted EC proliferation (Fig. 1A and B). Microgravity treatment of 3 day incubated egg for 2 h also showed significant neovascularization (Fig. 1A inset).

Microgravity induces iNOS dependent NO generation Measurement of NO in macrovascular cells by three independent assays viz., by NO electrode, DAF imaging and Griess assays showed increased generation of NO under microgravity that was attenuated by 1400 W, a selective inhibitor of iNOS (Fig. 2A and B). In accordance, iNOS mRNA level was also higher in microgravity treated cells vis-à -vis gravity control (Fig. 2A).

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Microgravity induces iNOS in macrovascular EC EAhy926 cells transfected with iNOS siRNA generated almost half the amount of NO than their untransfected counterparts under microgravity (Fig. 3A). Further, immunofluroscence assay demonstrated no change in eNOS expression in 2 h microgravity treated macrovascular and microvascular EC (Fig. 3B). Also, estimation of iNOS levels in microvascular, macrovascular and PVEEC demonstrated that microgravity treatments elevates iNOS level only in EAhy926 cells while no changes were observed in microvascular and PVEEC (Figs. 3C and 4A). In corroboration, higher NO production as assayed by DAR-4AM fluorescence probe was seen only in EAhy926 cells but not in microvascular EC or PVEEC kept under microgravity (Fig. 4B). Finally, wound healing assays showed higher healing response in EAhy926 cells than in microvascular ECs under microgravity (Fig. 4C).

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Microgravity acts via cGMP/PKG pathway The role of cGMP/PKG pathway in iNOS dependant angiogenesis was assessed by enzyme measurement and tube formation assays. iNOS inhibitor showed marked inhibitory effects on microgravity induced cGMP levels in EAhy926 cells (Fig 5A). Tube formation assay was performed using pharmacological blockers for soluble guanylate cyclase (sGC) (ODQ) and PKG (KT5823). As shown in Fig 5B, a significant drop in number of tubes was observed upon treatment with ODQ and KT5823. Simultaneous treatment with 8Bromo-cGMP or Sildenafil citrate partially restored ODQ mediated inhibition of tube formation under microgravity (Fig. 5B).

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iNOS plays a key role in modulating endothelial functions under microgravity. Authors observed higher iNOS expression levels in macrovascular EC but not in endocardial and microvascular EC after microgravity treatment (Figs. 3C and 4A and B). Our observation is in agreement with others demonstrating that in rat cerebral artery; hind limb unloading reduces NO activity and increases myogenic tone of the blood vessels. This study also demonstrates that while simulated microgravity enhances endothelial migration in macrovascular endothelial cells, it inhibits migration in microvascular endothelial and endocardial vascular monolayers (Fig. 5A). Nevertheless, iNOS and NO are not the only determinates of endothelial functions under microgravity. Cortrupi et al. showed that microgravity reversibly inhibits endothelial growth that correlates with an upregulation of p21, a cyclindependent kinases inhibitor and down regulation of interleukin-6. Taken together, our work introduces a concept that in the EC, microgravity de-couples iNOS from caveolin-1 via a mechanosensing pathway, which in turn produces bulk NO stimulating angiogenesis. Authorâ&#x20AC;&#x2122;s unpublished observation that a 15 min treatment of EC with NO induces the formation of endothelial tubes supports this postulation. However, the present study used limited number of representative endothelial cell lines and cells of macrovascular, microvascular and endocardial origin. Further investigation warrants confirming differential effects of microgravity on primary endothleial cells of different origin. Targeting key steps of the downstream NO signaling with sGC modulators, authors also demonstrate that microgravity induced tube formation from endothelial monolayer is cGMP dependent (Fig. 4A).

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Cyclic GMP-dependent protein kinase, PKG is a key enzyme in NO-cGMP signaling cascade and sGC-PKG pathway has been implicated in angiogenesis under various physiological and pathological contexts. It has recently been demonstrated that in critical limb ischemia, sildenafil therapy results in increased angiogenic activity in a PKG-dependent manner. In summary, the experiments performed in this study established that simulated microgravity increases the number of EC tubes by activating endothelial iNOS in macrovascular EC. The work also established that iNOS is the key molecular switch in the differential effects of microgravity on macro and microvascular EC. Finally, the dissection of NO-downstream signaling brought to light the key role of cGMP-PKG pathway in microgravity induced angiogenesis.

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

Endothelial cells, as key players that maintain vessel integrity, immune function and metabolic homeostasis, have been extensively studied for the effects of simulated microgravity on vascular biology. Abnormalities in endothelial cells induced by microgravity may be implicated in orthostatic intolerance associated with cardiovascular dysfunction following space flights. Nevertheless, some important aspects of endothelial biology during simulated microgravity and underlying mechanisms still remain unclear. As a structural adjustment to increase vascular perfusion, a form of new vessel growth from the existing vasculature, termed angiogenesis, can be induced by simulated microgravity. For instance, microgravity treatment in human umbilical vein endothelial cells (HUVECs), a well-characterized cell model for studying angiogenesis in vitro, results in enhanced formation of capillary-like tubes in a 3D-matrix culture system.

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Furthermore, previous studies including authors have demonstrated that the increased angiogenic capacity of HUVECs induced by simulated microgravity depends on nitric oxide (NO) production. It was found that phosphoinositide 3-kinase (PI3K) pathway can stimulate endothelial nitric oxide synthase (eNOS) phosphorylation and activation in HUVECs exposed to microgravity, in which caveolin-1 (Cav-1), a scaffolding protein that functions as a potential mechanosensor for simulated microgravity, could be a critical mediator. The decreased Cav-1/eNOS interactions in HUVECs exposed to microgravity can lead to eNOSphosphorylationassociated enzyme activation, thus inducing endothelium-dependent angiogenesis during simulated microgravity. In addition, another possible player in mediating microgravity-stimulated angiogenesis is cytoskeleton dynamics. The coordinated remodeling of actin networks in cells is required by multiple steps of angiogenesis including sprouting, migration and adhesion. Cytoskeletal components such as microtubules and actin filaments are known to be affected by simulated microgravity. These rearrangements of cytoskeletons correlate with morphological alterations of cells under simulated microgravity conditions, thus influencing a variety of cellular processes such as transcriptional response, cell differentiation and oxidative stress. Notably, disorganization of actin induced by simulated microgravity was shown to be linked with eNOS activation in endothelial cells. It seems that cytoskeleton dynamics could cooperate with NO signaling during simulated microgravity. Importantly, several studies also demonstrated effects of real microgravity environment on the cytoskeleton. Both short-term (22 second) and long-term (10 days) weightlessness could induce changes in cytoskeleton of human cells. Using live-cell imaging during a rocket flight, Corydon et al. observed significant alterations in the organization of cytoskeleton. However, the role of actin cytoskeleton rearrangement in angiogenesis stimulated by microgravity is unknown. RhoA, a member of the Rho GTPase family of small G proteins, is a master regulator of actin dynamics. When activated, GTP-bound RhoA recruits and stimulates downstream effectors to facilitate polymerization of actin monomers. Although the mechanisms are illusive, a decreased activity of RhoA was observed in brain microvascular endothelial cells responding to simulated microgravity, which is also accompanied by a disorganization of actin cytoskeleton. A delicate balance in RhoA activity seems essential for cell-specific migration depending on signaling context. It is known that efficient RhoA activity is required for migration, but high RhoA activity may inhibit cell migration as well. Here authors explore a hypothesis that RhoA-dependent actin cytoskeleton rearrangement is involved in angiogenesis promoted by simulated microgravity. Authors aim to test previous findings that both RhoA activity and actin filaments of HUVECs are reduced during simulated microgravity. They then determine whether these changes are responsible for the increased cell migration and tube formation during simulated microgravity.

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Authors used two main approaches: 1) loss of RhoA function by Rho inhibitor or genetic silencing of RhoA in cells under microgravity; 2) overexpression of RhoA in cells under normal gravity. The combined results provide a mechanistic link between RhoA inactivation, disorganization of actin filaments and enhanced angiogenic activities in HUVECs responding to simulated microgravity.

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Results Simulated microgravity in HUVECs induces both dissociation of F-actin and inactivation of RhoA To determine if simulated microgravity could alter the rearrangement of actin cytoskeleton in HUVECs, authors treated the cells with clinorotation to mimic microgravity conditions. Consistent with previous reports, a reduction in F-actin filaments was observed in cells following 24 hours of microgravity exposure (Fig. 1A, Clino), comparing to normal gravity-treated cells (NG). Approximately 80% decrease in F-actin assembly was seen based on the calculated arbitrary units of F-actin microfilaments by phalloidin staining(Fig. 1B).

Interestingly, this dramatic change in actin cytoskeleton networks coincided with the effect of simulated microgravity on RhoA signaling as well. First of all, protein levels of total RhoA were decreased in microgravitytreated HUVECs (Fig. 1C). In addition, authors found that the activity of RhoA, estimated by affinity-pulldown using effector, was attenuated by microgravity in comparison with NG-treated controls (Fig. 1D). To determine if there is a potential correlation between the reduction of F-actin and decreased RhoA activity in microgravity-treated HUVECs, authors first used C3 transferase, a RhoA inhibitor (Fig. 2). In the presence of the RhoA inhibitor, actin filaments in normal cells quickly disorganized as expected, whereas there was no further decrease on microgravity-treated cells (Fig. 2A and B). Thus, it is likely that the reduction of actin filaments in microgravity-treated HUVECs depends on the attenuation of RhoA activity. Meanwhile, protein levels of actin and GAPDH were not affected by RhoA inhibitor under either normal or microgravity conditions, indicating that it is indeed the organization, but not expression, of actin cytoskeleton that was affected by RhoA (Fig. 2C).

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RhoA is necessary and sufficient to mediate the rearrangement of actin cytoskeleton during simulated microgravity To confirm the functional significance of RhoA inactivation for the rearrangement of actin cytoskeleton during simulated microgravity, we used HUVECs that have either knockdown or overexpression of RhoA (Fig. 3). As shown as Fig. 3A, the levels of both RhoA transcript and protein were decreased in siRNA-knockdown cells (RhoA siRNA), comparing to control knockdown cells (control siRNA). On the other hand, overexpression plasmid for human RhoA gene was transfected into HUVECs, with cells transfected with empty vector as control.

The feasibility of the gain-of-the function approach was supported by the increased RhoA expression following transfection of RhoA plasmid in HUVECs (Fig. 3B). Immunofluorescent staining against RhoA in the above treated cells exhibited the same pattern (Fig. 3C).

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Consequently, actin filaments were markedly diminished in RhoA-knockdown HUVECs, bearing strong resemblance to the cells in microgravity condition (Fig. 4A). Similar to the findings using Rho inhibitor, the reduction of F-actin in microgravity-treated cells was not further decreased by RhoA knockdown (Fig. 4A and B).

In contrast, overexpression of RhoA in cells treated by simulated microgravity significantly restored the assembly of actin filaments, whereas their effects on normal gravity-treated cells were minimal (Fig. 4D and E). The amount of F-actin cytoskeleton microfilaments labeled by FITC-phalloidin was elevated by 3-fold in microgravity-treated cells, to comparable level of normal gravity condition. These data indicate that RhoA attenuation plays a key role on actin filament dissociation induced by simulated microgravity in HUVECs. Similarly, protein levels of actin and GAPDH were not affected by changes in RhoA expression under either normal or microgravity conditions, further validating that RhoA affected actin cytoskeleton organization rather than expression (Fig. 4C and F). RhoA inactivation plays a critical role in enhanced migration and tube formation of HUVECs responding to simulated microgravity Authors next examined whether the manipulations of RhoA could also alter the behaviors of microgravitytreated HUVECs in cell migration and angiogenesis, both of which are dependent upon cytoskeleton remodeling. First, in the presence of Rho inhibitor, the percentage of wound closure in normal gravity-treated cells was enhanced to a level similar to those under simulated microgravity (Fig. 5A). In contrast, treating cells with Rho inhibitor under microgravity condition had no effect, implicating a mechanistic association between the downregulation of RhoA signaling and increased cell migration induced by simulated microgravity.

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Accordingly, angiogenic activities under various gravity conditions, as indicated by tube formation capacity, showed an identical change in response to the Rho inhibitor (Fig.5B). RhoA inhibitor treatment increased the tube formation activity in cells under normal gravity, whereas the effects on microgravity-treated cells were insignificant.

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Authors then performed cell migration and tube formation assays in the cell models of genetically manipulated RhoA. Comparing to control knockdown (NG+control siRNA), HUVECs treated by RhoA siRNA (NG+RhoA siRNA) exhibited significantly increased abilities in both cell migration and tube formation (Fig. 6A and B). Notably, these effects closely resembled the cells induced by simulated microgravity (Clino+control siRNA). Once again, there were no significant changes in either cell migration or tube formation when RhoA knockdown was performed in cells stimulated by microgravity (Clino+RhoA siRNA). On the other hand as shown in Fig. 6C and D, when RhoA expression was increased in cells exposed to microgravity (Clino+RhoA plasmid), the angiogenic potential, including enhanced cell migration and increased tube formation, was normalized to similar levels of cells under normal gravity (NG), compared to the control cells (Clino+empty vector). These data indicate that repressed RhoA signaling is critical in the simulated microgravity-induced angiogenesis. Authors show that the attenuation of RhoA activity in HUVECs mediates the induced angiogenic activity during simulated microgravity. This conclusion is supported by the findings in three major types of models: â&#x20AC;˘ RhoA inhibitor treatment by C3 transferase; â&#x20AC;˘ Reduction of RhoA levels by siRNA knockdown;

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â&#x20AC;˘ Upregulation of RhoA levels by overexpression. Authors found that the actin filaments were greatly diminished in HUVECs by either clinorotation, a simulated condition for microgravity, or by RhoA inhibitions. Coincidently, RhoA activity is indeed suppressed by microgravity. Further manipulation of RhoA levels in cells demonstrates that RhoA is both necessary and sufficient for the disorganization of F-actin in HUVECs during simulated microgravity. Importantly, decreased RhoA is required for promoting cell migration and tube formation in microgravity-treated cells, suggesting that the RhoA-dependent cytoskeleton rearrangement may be intrinsically responsible for the stimulated angiogenesis by simulated microgravity. The physiological changes in the human body during spaceflight are initiated through an amazingly multitude of gravity sensing molecules for cell structural modulations. This research on angiogenesis induced by simulated microgravity has focused on one such signaling player, RhoA-dependent cytoskeleton rearrangement.

RhoA regulates downstream effectors by acting as a molecular switch between the active GTP-bound and inactive GDP-bound states. When activated by the family of guanine nucleotide exchange factors (GEFs), RhoA protein subsequently promotes the assembly of actin fibers in cells. Depending on the spatiotemporal dynamics of RhoA activity in cells, RhoA signaling is concerted with either cytoskeleton or membrane remodeling, playing distinct functions in cell migration. The data is consistent with prior findings that RhoA-associated signaling could be attenuated by simulated microgravity (Fig. 1). It was found that in the bovine brain microvascular endothelial cells, simulated microgravity decreased the amount of GTP-bound RhoA, which correlated with the downregulation of leukemia-associated Rho GEF, a Rho activator. In human mesenchymal stem cells cultured in simulated microgravity, both RhoA activity and subsequent cofilin phosphorylation were significant reduced. On the other hand, under simulated microgravity, upregulation of RhoA pathway was also observed in fibroblast cells and neural crest stem cells. Thus, it seems that RhoA activity could be influenced by variations in gravity environment in a cell- or contextspecific manner. In contrast, the organization of cytoskeleton is mostly found disrupted in cells exposed to microgravity. In general, the polymerizations of actin and microtubules are inhibited by microgravity, thus leading to the collapse of the cytoskeleton networks, changes in shape of cells and loss of matrix adhesions during microgravity. As a result, cells are often stimulated by microgravity towards a more migratory phenotype. The mechanisms underlying such gravity-dependent modulation of cytoskeleton have remained obscure, and the current results pinpoint one specific player in HUVECs, namely the downregulation of RhoA signaling in response to simulated microgravity. Overexpression of RhoA has rescued the reduced assembly of actin filaments in microgravity-treated cells, which strongly suggests the indispensable function of RhoA in the rearrangement of actin cytoskeleton during simulated microgravity (Fig. 3 and 4).

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