ORIGINAL ARTICLE

Simulated microgravity perturbs actin polymerizationto promote nitric oxide-associated migrationin human immortalized Eahy926 cells

Jamila H. Siamwala & S. Himabindu Reddy &

Syamantak Majumder & Gopi Krishna Kolluru &

Ajit Muley & Swaraj Sinha & Suvro Chatterjee

Received: 16 November 2009 /Accepted: 18 January 2010 /Published online: 20 February 2010# Springer-Verlag 2010

Abstract Microgravity causes endothelium dysfunctionsand vascular endothelium remodeling in astronauts return-ing from space flight. Cardiovascular deconditioning occursas a consequence of an adaptive response to microgravitypartially due to the effects exerted at cellular level.Directional migration of endothelial cell which are centralin maintaining the structural integrity of vascular walls isregulated by chemotactic, haptotactic, and mechanotacticstimuli which are essential for vasculogenesis. We exploredthe migration property of transformed endothelial cells(EC) exposed to 2-h microgravity, simulated using a three-dimensional clinostat constructed based on blueprint pub-lished by the Fokker Space, Netherlands. Migration of ECwas measured using the scrap wound healing in thepresence or absence of actin polymerization inhibitor—cytochalasin D (CD) in Eahy926 cell lines. Simulatedmicrogravity increased cellular migration by 25% whileCD-blocked microgravity induced cellular migration. Thekey migratory structures of cells, filopodia and lamellipo-dia, formed by EC were more in simulated microgravitycompared to gravity. Parallel experiments with phalloidinand diaminorhodamine-4M (DAR-4M) showed that simulat-ed microgravity caused actin rearrangements that lead to25% increase in nitric oxide production. Further nitric oxidemeasurements showed a higher nitric oxide production which

was not abrogated by phosphoinositol 3 kinase inhibitor(Wortmanin). Bradykinin, an inducer of nitric oxide, promp-ted two folds higher nitric oxide production along withsimulated microgravity in a synergistic manner. We suggestthat limited exposure to simulated microgravity increasesEahy926 cell migration by modulating actin and releasingnitric oxide.

Keywords Simulated microgravity . Endothelial cells .

Actin . Nitric oxide .Migration

Introduction

Microgravity is known to cause a plethora of cardiacdysfunctions by interfering with endothelial physiology.Changes like muscle atrophy, cardiovascular decondition-ing, disturbances in pulmonary function, and fluid regulat-ing systems occur in the body under low gravity. Thesechanges can cause adaptation problems when astronautsreturn back to earth, especially after long-duration spaceflights (Yates and Kerman 1998).

Endothelial cells play a crucial role in the maintenanceof the functional integrity of the vascular wall. They play anactive role in control of various components of homeosta-sis, vascular tone and permeability, and cardiovascularconditioning (Girn et al. 2007).

Low gravitational forces alter various cellular processesin cultured mammalian cells (Carlsson et al. 2002).Endothelium remodeling is the foremost in adapting tomicrogravity. Endothelial cells which form the inner liningof blood vessels are affected by the reduction of gravity(Cines et al. 1998; van Hinsbergh 1996; Ross 1999;Buravkova et al. 2005).

J. H. Siamwala : S. H. Reddy : S. Majumder :G. K. Kolluru :A. Muley : S. Sinha : S. Chatterjee (*)Vascular Biology Lab, AU-KBC Research Centre,Anna University,MIT Campus, Chromepet,Chennai 600044, Indiae-mail: soovro@yahoo.ca

Protoplasma (2010) 242:3–12DOI 10.1007/s00709-010-0114-z

The mechanisms of physiological adaptations in spacemust be understood in order to develop effective counter-measures. Cellular mechanisms involved in adaptationprocesses to microgravity are not known. Recent advancesin the area of microgravity biology confer faster migrationof endothelial cells (Romanov et al. 2001) under lowgravity environment. The migration of endothelial cellsrequires activation of several signaling pathways thatconverge on cytoskeletal remodeling. Actin microfilamentsystem constitutes the gravity-sensitive cell component(Martin and Cotter 1990). Previous reports have demonstrat-ed actin remodeling in space-flown cells on spaceflight andground-based simulation of microgravity (Hughes-Fulford etal. 1993). However, downstream events of actin-basedremodeling are not known yet.

Nitric oxide (NO), produced by the enzyme nitric oxidesynthase (NOS), an important second messenger in manysignaling pathways, is a potent vasodilator (Sessa 2005),thereby responsible for vascular conditioning. Recentreports have shown that actin activates NO productionthrough the induction of NOS (Zeng and Morrison 2001).We hypothesize that simulated microgravity modulatesactin, thereby activating NOS and producing NO which inturn promotes the migration of endothelial cells.

The aim of the study was to identify the exact changescaused by simulated microgravity on the endothelium inrelation to actin remodeling. We found that limitedexposure to microgravity activates endothelium by actinremodeling followed by NO production.

Materials and methods

Materials

Dulbecco’s Modified Eagle Medium (DMEM), fetal bovineserum (FBS), penicillin, and streptomycin were purchasedfrom PAN Biotech. Phalloidin, DAR-4M, 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI), and cytochalasinD (CD) were purchased from Sigma Chemical Co., St.Louis, MO, NOS inhibitor L-NIO (Enzo Life Sciences).Posphoinositol 3 kinase (PI3K) inhibitor Wortmanin(Santa Cruz Biotechnology, Inc.) and nitric oxide inducerBradykinin (Sigma-Aldrich) were obtained commercially.

Methods

Cells

As microgravity is implicated in migration of immortalizedcells, we used an immortalized endothelial hybrid cell lineEAhy926 obtained by fusing human endothelial cells withthe lung carcinoma cell line A549, a kind gift from Dr C.J.

S. Edgell. The cells were cultured in DMEM supplementedwith 10% FBS (v/v) and 1% penicillin (w/v) and strepto-mycin (w/v). Another immortalized human umbilical veinendothelial cell line (ECV 304), which was identified to bea bladder cancer derived EJ1/T24 cells by STR-PCRanalysis, was used as a representative of cancerous ECsand was cultured in DMEM supplemented with 10% FBS(v/v) and 1% penicillin (w/v) and streptomycin (w/v).

Simulation of microgravity

A three-dimensional (3D) clinostat is an apparatus thatnullifies the effect of gravity, and it has been used insubstitution studies on microgravity effects (Gruener et al.1994; Grimm et al. 2002; Hirasaka et al. 2005; Li et al. 2002;Sarkar et al. 2000; Uva et al. 2002; Woods et al. 2003;Versari et al. 2007).

The 3D clinostat is constructed based on design ofclinostat used by the Fokker Space, Netherlands (Fig. 1).The clinostat is capable of randomizing motion whichtheoretically cancels the uniform gravity influence andsubjects an object to weightlessness referred commonly assimulated microgravity. The clinostat consists of an innerchamber containing the samples which rotate clockwise,anticlockwise, vertically, as well as horizontally. Thehorizontal and vertical motions are provided by an outerchamber. All the chambers are operated by small motors.The cycles of rotation were controlled by the computer atan outer frame rotation of 20 rpm and an inner framerotation of 13 rpm to cancel the dynamic simulation ofgravity in any direction. The formula for microgravity

AB

C

Fig. 1 3D Clinostat. Photograph of 3D clinostat which was used inthe present study to simulate microgravity conditions. AThe inner ringis capable of rotating in both clockwise and anti-clockwise direction.B Outer ring can rotate bidirectionally. C Removable sample holder

4 J.H. Siamwala et al.

(g′) is g′=ω2R/g where g = 9.8 m/s2, R=radius from thecenter of rotation, and the clinostat rotates with a constantangular velocity (ω) where angular velocity is equalto angular displacement in radians/time taken (θ/τ). Theangular velocity obtained using the clinostat was 2 rads/s. Atthis angular velocity, the simulated microgravity is 10−3

(Huijser 2000). All the samples were kept exactly in thecenter of the clinostat. For the motion controls, sampleswere kept in customized vertical shaker to providemovements. Quality control experiments were performedusing different cell lines to verify the microgravity effects aspreviously reported (Ichigi and Asashima 2000). Themicrogravity effects obtained were similar to those obtainedin space labs. The cells were grown on coverslip fitted in cellchambers or flask filled completely with media, thusdiminishing the likelihood of turbulence and shear forcesduring rotation of cultures. The cells were placed in mediasupplemented with sodium bicarbonate which was capableof providing the cells with sufficient carbon dioxide requiredduring 2-h exposure. The temperature was maintained at37°C using a sensor system.

Migration assay

Cell migration was assessed by a scrape wound assay on24-well plates. Eahy926 (1 × 106) cells were seeded toconfluence, and a scratch injury was made using a sterile20-μl pipette tip. The cells were incubated for every 2-h timeperiods till 8 h under gravity and simulated microgravity.After the respective incubations at 37°C/5% CO2, lightmicroscopy images were taken with Nikon Coolpix cameraunder Motic phase contrast microscope. Images werequantified using Scion Image, Release Alpha 4.0 3.2.

NO, actin, and nucleus imaging with DAR-4FM, phalloidin,and DAPI, respectively

Eahy926 cells were grown on the cover glass and wereexposed to gravity or simulated microgravity for 2 h. The cellswere incubated with fluorescent probe DAR-4FM (Sigma-Aldrich) for 15 min. Time-lapse images were taken every5 min. Fluorescence intensity of the cells was calculated byusing image analysis module of Adobe Photoshop ver. 7.0.The cells were then fixed using 2% cold paraformaldehyde.Cells were permeabilized with 0.1% Triton X-100. Cells wereincubated at 37°C for 15 min with phalloidin Alexa Flour(dilution 1:1,000) and 10 min with nuclear stain (DAPI).Images were taken with Olympus DP71 digital camera.

Tube and collateral formation assay

EAhy926 (1 × 105) cells were seeded on Matrigel (8 mg/ml;BD Biosciences, USA)-coated cover glasses in 24-well

plates. After 48-h incubation, the cells were placed undergravity or simulated microgravity treatments for 2 h.Motic phase contrast microscope attached with NikonCoolpix camera was used to take images of collateralsand tubes and were quantified using Scion Image,Release Alpha 4.0 3.2.

NO estimation by Griess assay

EAhy926 cells were treated under gravity or simulatedmicrogravity for 2 h. NO was measured by the Griess assayprotocol, as described elsewhere (Nims et al. 1996).

Statistical analysis

Data are expressed as mean ± SEM. Each experiment wasperformed in triplicate unless otherwise specified. Statisti-cal analyses were performed by one-way ANOVA, t test asappropriate, followed by Tukey’s post hoc test. Values ofP < 0.05 were considered statistically significant.

Results

Microgravity induces endothelial wound healing

To evaluate endothelial migration under microgravitytreatment, endothelial wound healing assay was per-formed. After creating a scratch wound, EAhy926 cellmonolayers were exposed to microgravity for 2 h in thepresence of actin polymerization blocker CD. Resultdepicted that microgravity caused a 25% increase inwound healing (Fig. 2) which was abrogated by CDtreatment.

Microgravity promotes migratory extensions in EC

Lamellipodia and filopodia are two key migratory bodiesfor cellular migration. The lamellipodium (pl. lamellipodia)is a cytoskeletal actin projection on the mobile edge of thecell. It contains a two-dimensional actin mesh; the wholestructure pulls the cell across a substrate (Bruce et al. 2002).Within the lamellipodia are ribs of actin called microspikes,which, when they spread beyond the lamellipodiumfrontier, are called filopodia (Small et al. 2002). In orderto determine if microgravity promotes the formation offilopodia and lamellipodia-like structures, EAhy926 wereexposed to 2-h microgravity in the presence of CD.Microgravity-exposed cells showed twofold increase inthe number of filopodia after 2-h microgravity treatment(Fig. 3a). Cells were stained with phalloidin, and data wereprepared based on the number of central microfilaments,stress fibers, locomotory structures like filopodia, lamelli-

Simulated microgravity to promote migration in Eahy926 cells 5

podia. It was observed that blocking actin polymerizationwith CD under gravity treatment promoted formation of anaverage 19 stress fibers (Fig. 3b) compared to an average of13 stress fibers seen under microgravity treatment. Whilethe locomotory structures of cell lamellipodia and filopodiawere more in microgravity-exposed cells and less ingravity-exposed cells.

Microgravity modulates actin-dependent NOproduction by EC

Cell migration is associated with regulation of the actincytoskeleton. Cells were exposed to microgravity for 2 hfollowed by time scan NO imaging using DAR-4FMfluorescent probe. Nitric oxide production by EC increasedunder microgravity temporally. To explore the possibilitythat simulated microgravity increase nitric oxide production

by actin remodeling, in the presence of CD, gravity- andmicrogravity-exposed cells were initially checked for nitricoxide production followed by dual staining of actin andnucleus with phalloidin and DAPI, respectively. The numberof central microfilaments was more in microgravity-exposedcells in comparison to that of gravity-exposed cells. Thecentral microfilaments were well structured and directed ingravity-exposed cells, while directionless, shorter centralmicrofilaments were observed in microgravity-exposed cells(Fig. 4a). Importantly, CD-stimulated stress fiber formationwas markedly attenuated in cells exposed to microgravity(Fig. 4b).

Microgravity-induced collateral formation

When plated on Matrigel, ECV304 invades the Matrigelforming characteristic well like depressions which will be

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Fig. 2 Migration of EC. Wound healing experiments to evaluate theeffect of microgravity on endothelial cell migration. Monolayer ofEAhy926 cells with scratch wounds were exposed to gravity ormicrogravity in the presence or absence of CD (2 uM) for 2 h.Representative images were taken at zeroth and second hour of

treatment. Wound healing was quantified by processing the imagesusing Image J software. Bright field images were taken with 10×magnifications under an inverted bright field microscope. Datapresented as percentage wound healing. *P < 0.05 vs gravity

6 J.H. Siamwala et al.

hereafter called as pits. Extensions from pits resemble themigratory structures of EC and were termed as collaterals.We used this model to examine the functions of ECactivated by simulated microgravity. ECV304 cells wereplated in Matrigel-coated 24-well plate. Formed pits werethen treated under microgravity for 2 h and studied for

collateral formation. Microgravity elevated collateral for-mation by 60% (Fig. 5).

Microgravity-driven endothelial capillary formation is lesssensitive to L-NIO, a nonselective eNOS blocker

Microgravity-mediated endothelial tube formation wasstudied under L-NIO treatment. Counting of formed tubessuggested that L-NIO administration completely attenuatedendothelial tube formation under gravity treatment (Fig. 6).However, microgravity-driven endothelial tubes were lesssensitive to L-NIO, thus blocking only 55% of theendothelial tube formation under microgravity (Fig. 6).

Bradykinin promoted microgravity-induced nitric oxideproduction in EC

NO agonists were used to evaluate the production of nitricoxide under microgravity. Microgravity treated Eahy926cells were incubated with or without bradykinin to evaluatethe cumulative effect of microgravity and bradykinin onendothelial nitric oxide production. Microgravity aloneincreased NO production in EC by 69%. Bradykinintreatment under gravity condition elevated NO productionin EC by 45% while microgravity and bradykinin showedsynergistic effects on NO production (Fig. 7).

Wortmanin, PI3K blocker partially attenuated endothelialnitric oxide production under microgravity

Wortmanin was administered to quantify the level ofendothelial nitric oxide production under microgravity.Wortmanin blocked cellular nitric oxide production by70% under gravity condition (Fig. 8). As observed earlier,microgravity elevated endothelial nitric oxide productionby 50% while administration of Wortmanin blockedendothelial NO production by 40% (Fig. 8).

Discussions

Gravity is exerted permanently on organisms which are inconstant orientation in the gravity field (static stimulation)as well as if their orientation is changed with respect to thegravity vector (dynamic stimulation; Buchen et al. 1993).The only practicable way of achieving microgravity is touse parabolic flights of rockets or space labs. Alternativelyon earth, experiments with clinostats have been performedsince 1882 (Sachs 1882; Pfeffer 1990). We designed the 3Dclinostat to perform experiments on single cells and cellculture. Single cells and cell culture are good models forstudying primary effects of gravitational changes oncellular functions. EAhy926 and ECV304 cells which are

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Fig. 3 Actin remodeling under microgravity. a Representative imagesof actin in cells exposed to gravity and microgravity in the presence orabsence of CD. Photographs taken with an Andor CCD cameraattached to the fluorescence microscope Olympus IX71. Arrowsindicate microfilaments in the upper panel and the sharp processesin the lower panel. b Migratory structures filopodia, lamellipodia,microfilaments, and stress fibers of cells treated with gravity andmicrogravity in the presence or absence of CD are expressed mean ±SE. Data is representative of three independent experiments. f=Lamellipodia vs gravity, ==filopodia vs gravity, *=microfilament vsgravity, #=stress fibers vs gravity

Simulated microgravity to promote migration in Eahy926 cells 7

immortalized endothelial cells were used to study themicrogravity effects on cellular migration and actinremodeling. These cells have been frequently used byworkers to demonstrate effects on endothelial cells sincethese cell lines retain partial characteristics of endothelialcells and functionally behave as endothelial cells (Edgellet al. 1983; Takahashi et al. 1990)

Cell cytoskeleton plays a key role in mechanisms ofadaptation particularly under gravity (Ingber 1999). Cyto-skeletal components such as microtubules are changed inmicrogravity, and this is presumed to explain the effect ofmicrogravity on cells (Papaseit et al. 2000). Migration ofcells due to cytoskeletal changes is a very importantphysiological phenomenon which if disturbed can lead toseveral detrimental effects on the body. Bone loss and

orthostatic intolerance are common problems in astronautsreturning from space flight. Sarkar et al. (1999) showed thataltered cytoskeleton leads to death of osteoblasts undersimulated microgravity. Also Plett et al. (2004) showedsignificant reduction of stromal cell-derived factor 1 (SDF-1alpha)-directed migration, which correlated with decreasedexpression of F-actin and decreased migration after 2–3-dayrotation in clinostat in bone marrow progenitor cells.Inflammatory adherence and locomotion through theinterstitium are important components of the immuneresponse. Lewis et al. (1998) and Sundaresan et al. (2002)further showed disorganized microtubules and decreasedlocomotion in human T lymphocytes in space. Changes incytoskeleton were also observed in muscle myocytestreated with 9.5 days simulated in clinostat (Greuner et al.

Actin NucleusNO Overlap

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Fig. 4 Actin-associated nitricoxide production. a Representa-tive images of actin pattern,nitric oxide, nucleus, and amerge of all three. Images arethe representative of threeindividual experiments. b Datapresented as fold increase ofmembrane ruffles, filopodia, andnitric oxide production in cellsexposed to gravity andmicrogravity in the presence orabsence of CD. Data isrepresentative of three indepen-dent experiments. *=Membraneruffles vs gravity, ==filopodiavs gravity, #=nitric oxideproduction vs gravity

8 J.H. Siamwala et al.

1994). In EC Carlsson et al. (2003) showed reduction in theamounts of actin in response to microgravity which couldbe an adaptative mechanism to avoid the accumulation ofredundant actin fibers. Kumei et al. (2006) showed thatactin mRNA levels did not change in flight cultures on days4 and 5 of spaceflight. Recently, Versari et al. showed thatalterations of the actin cytoskeleton and increased nitricoxide synthesis in human primary endothelial cell respondto changes in gravity (Versari et al. 2007). We further showactin remodeling in response to limited exposure tosimulated microgravity using a clinostat. Further, wedelineate that microgravity-mediated remodeling of cyto-

skeleton induces NO production, which in turn leads tomigration of cells by a PI3K-independent pathway.

The EC migrate in response to different stimuli undervaried processes like angiogenesis, inflammation, andthrombosis. It is known that migration of endothelialcells is a molecular process that involves modulation incell adhesion, signal transduction, and reorganization ofcytoskeleton (Rubanyi 1993). The present study depictsthat 2-h exposure to microgravity induces endothelial cellmigration and faster wound healing by promoting mecha-notaxis of EC at the leading edge (Fig. 2). Filopodiacontain a tight bundle of long actin filaments oriented inthe direction of protrusion. Endothelial cell migrationinvolves reorganization of cytoskeleton and actin remod-eling (Li et al. 2005). As shown previously, space-flowncells showed irregular cytoskeletal fiber pattern. Grueneret al. (1994) and Hughes-Fulford et al. (1993) reportedthat actin reorganization responded to the gravity level andshowed abnormal assembly of actin stress fibers. Similarly,we observed that simulated microgravity disturbs the organi-zation of actin filaments (Fig. 3a). Phalloidin is known tostabilize actin filaments by inhibiting subunit dissociationat the barbed ends, while CD inhibits polymerization atthe pointed ends of actin filaments (Sampath and Pollardet al. 1991). Microtubule filaments extended from a poorlydefined centrosome in human lymphocytes (Jurkat cells;Lewis et al. 1998). Among other effects cytochalasins,paralysis of locomotion and membrane movements arereported (Carter 1967; Gail and Boone 1971; Spooner et al.1971; Wessells et al. 1971). We observed that CD treatmentsblocked the migration of gravity-exposed EAhy926 cells thatcould be the consequence of dilution of microtubulefilaments under microgravity. Similarly, CD blocked themigration of EAhy926 cells under microgravity, but still the

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Fig. 6 eNOS independent acti-vation of nitric oxide. Eahy926cells were exposed to micro-gravity and plated with orwithout L-NIO (10 μM) inMatrigel-coated 24-well plates.After 24 h of incubation at37°C/5% CO2, images offormed tubes were taken.Number of tubes formed perfield were counted and plotted.Tubes formed undermicrogravity were less sensitiveto L-NIO. Bright field imageswere taken with 20× magnifica-tions under an inverted brightfield microscope. Data presentedas percentage of L-NIOsensitive inhibition of tubes*P < 0.05 vs gravity

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Fig. 5 Functional effects of microgravity on EC. Measurement of thenumber of collaterals formed per pits in ECV304 exposed to 2-h gravity or microgravity. Bright field images taken at 40×magnification. Images are the representative of three individualexperiments. *P < 0.05 vs gravity

Simulated microgravity to promote migration in Eahy926 cells 9

filopodia and lamellipodia structures were intact possiblywithout motor action, which was further confirmed bymigration assays (Figs. 2 and 3a). Gravity-exposed cellsappeared rounded and had long, thin spikes extendingthroughout the surface. However, in microgravity-exposedcells, the long thin spikes were reduced considerably.However, in the absence of CD, gravity-exposed cells hadorganized and stabilized actin filaments while microgravityhad disorganized; short actin filaments concentrated in thecenter (Fig. 3a, b). We infer that simulated microgravitycauses actin reorganization which could be blocked with CD(Fig. 4a). We further delineate that microgravity stimulatescell surface mechanosensors, possibly a group of mechano-transduction proteins, to feel the differential and associatedmechanical strain in the cytoskeletal arrangements due tomicrogravity and further transmits the signal to engineerdownstream events such as actin polymerization in favor ofcellular migration.

Simulated microgravity increases myogenic tone ofcerebral arteries through both NOS-dependent and inde-pendent mechanisms (Geary et al. 1998). Microgravityinhibits proinflammatory responses by activating NOproducing machinery (Walther et al. 1998). Vessels fromhindlimb suspension rat showed an increase in cerebralarteries, a decrease in mesenteric arteries, and no changein carotid artery (Ma et al. 2003). Actin cytoskeleton isknown to regulate eNOS expression at a posttranslationalphase (Searles et al. 2004). Reorganization of the actincytoskeleton may affect eNOS activity leading to thealteration of NO production (Su et al. 2003). Actincytoskeleton disruption increases iNOS expression in

vascular smooth muscle (Zeng and Morrison 2001) andglomerular mesangial cell (Hattori and Kasai 2004).Polymerization state of β-actin crucially regulates theactivation state of NOS-3 and, hence, NO formationthrough altering its binding of heat shock protein 90(Hsp90; Ji et al. 2007). NO is also known to crosstalk withRho GTPase family members in modulation of actindynamics (Lee et al. 2005). As evident from our finding,microgravity modulates actin dynamics and increases NOproduction (Fig. 4b).

The PI3K–Akt pathway is an upstream signalingpathway for the activation of eNOS via serine-specificphosphorylation (Dimmeler et al. 1999). Phosphorylation ofvarious different proteins like paxillin, FAK, and MLCK bymitogen-activated protein kinase (MAPK) regulates thereorganization of microtubules and filamentous actin. These

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Fig. 8 Microgravity-driven NO production is partially dependent onPI3K-MAPK pathway. NO production from microgravity-treated ECwere measured with and without Wortmanin (5 nM). Monolayers ofEahy926 were treated under microgravity for 2 h followed by nitric oxideproduction measurement following Griess assay protocol. Cells wereincubated withWortmanin during the period of treatment. Data presentedas nitrite equivalent to nitric oxide production. *P < 0.05 vs gravity

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Fig. 7 Synergistic effects of bradykinin and microgravity on NOproduction. Nitric oxide production by Eahy926 measured undermicrogravity and bradykinin (5 μM) combination treatment. Cellswere exposed to microgravity for 2 h followed by bradykinintreatment for 15 min, and NO production was measured followingthe Griess assay protocol which measures the nitrite level in a solution.Data presented as nitrite equivalent to NO production. *P< 0.05 vsgravity

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Fig. 9 Schematic representation of the pathway

10 J.H. Siamwala et al.

modulators play key roles in cell spreading, lamellipodiumextension, and tail retraction during cell migration (Huanget al. 2004). In addition, NO regulates the activation of thep38 MAPK/MAPK-activated protein kinase/Hsp27 pathwaywhich is crucial for endothelial cell chemotaxis (Rousseauet al. 1997). It has also been reported that NO promotesendothelial cell migration and neovascularization via cGMP-dependent activation of PI3K (Kawasaki et al. 2003). Weused bradykinin as a positive modulator of DAG/IP3pathway under microgravity treatment. Surprisingly, NOproduction was observed to be four folds higher undermicrogravity + bradykinin treatment as compared to onlymicrogravity-treated cells (Fig. 7). This finding propoundedthe synergistic effect of microgravity and bradykinin onendothelial NO production. Because of this synergistic effectof BK and microgravity, we postulate that microgravity andBK promotes NO production in EC by two differentpathways. We further used PI3K inhibitor Wortmanin andfound that blocking PI3K did not attenuate simulatedmicrogravity-mediated NO production, thus indicating thatmicrogravity-induced NO production is independent of PI3Kpathway.

The present work concludes that microgravity causesactin remodeling and thereby activates NOS to induce NO-driven endothelial migration. Present work also provides apartial mechanistic insight into microgravity-mediatedendothelial activation by demonstrating PI3K-independentNO production which promotes migration in EC (Figs. 7,8 and 9).

Acknowledgment We are grateful to Mr. K.P. Tamilarasan and Mr.Karthikeyan Pasupathy for their technical assistance in fabricating themicrogravity machine. We also acknowledge the K.B. ChandrashekarResearch Foundation for the financial support.

Conflict of interest The authors declare that they have no conflict ofinterest.

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