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CHAPTER 3

SIMULATED MICROGRAVITY AND ANGIOGENESIS

3.1 OVERVIEW

The two main components of tissue regeneration are to restore the

blood supply in the damaged area through angiogenesis and another is to

maintain a renewable supply of stem cells through stem cell differentiation.

The creation of vascularized tissue is the first step to engineer more complex

tissue architecture (Jain et al 2005). For the growth of the tissue it is essential

to create functional blood vessels to supply cells with oxygen and nutrients

and to remove waste products. Angiogenesis or the formation of blood vessels

occurs in a mechanically dynamic environment through intussusception and

sprouting mechanisms in response to a physiological need in an embryo and

adult. Tissue engineered blood vessel have limited use because

1) High-flow/low-resistance conditions because of poor elasticity

2) Low compliance

3) Thrombogenicity of their synthetic surfaces (Griese et al

2003).

Angiogenesis is a multistep process which involves activation of

ECs in response to factors leading to endothelial cell migration, proliferation,

ring formation and finally tube formation. A biophysical stimulus,

microgravity can yield three-dimensional tissue specimens that can serve as

tubes for growth and development of biological transplants (Freed et al 1997,

51

Unsworth and Lelkes 1998). Recent advances in the area of microgravity

biology confer faster migration (Romanov et al 2001) and proliferation of EC

(Ziche et al 1997) under low gravity environment. However there has been no

direct evidence of angiogenesis in microgravity environment. In this chapter

the effect of simulated microgravity on each step of angiogenesis is explored

in detail. Directional migration in response to a stimulus requires actin

polymerization, resulting in the formation of migratory structures viz.

lamellipodia and fillopodia. Also described in detail is the effect of simulated

microgravity on actin polymerization pattern, stress fibres formation and

nitric oxide production. ECs have different subtypes depending on the

localization, organ type and functions. Hence it was quintessential to

investigate the effects of simulated microgravity on different endothelial

subtypes such as endocardial, microvascular and macrovascular cells. Finally,

attempts were also made to gain mechanistic insights into microgravity

mediated in vitro and in ovo angiogenesis (Figure 3.1).

Figure 3.1 An overview of work presented in chapter 3 and 4

Microgravity

52

3.2 RESULTS

Angiogenesis is a dynamic process that occurs in response to an

external stimulus such as hypoxia, growth factors and biophysical forces such

as shear stress. Although angiogenesis modulation in the presence of external

stimuli is well documented, the effects of microgravity on angiogenesis are

not yet known. In this module we analysed the angiogenic response of

capillaries, ECs and caprine aortic tissues in the presence of microgravity. A

scheme of experiments performed in the present chapter is given in

Figure 3.2.

Figure 3.2 Plan of study of in vitro and ex ovo angiogenesis

3.2.1 Effects of Microgravity on EC Activation and Angiogenesis

3.2.1.1 Microgravity Effects on in ovo Vascular Growth

Angiogenesis is easily studied in the early stages of a developing

embryo at the onset of vasculature and angiogenesis. To determine the effects

53

* *

of simulated microgravity on the early stage of vasculogenesis, fertilized eggs

were incubated at 37oC during early days (day 0) of vasculogenesis. After day

2 of development, eggs (day 2 and day 0) were exposed to 2 h of simulated

microgravity using the RPM. By rotating eggs three-dimensionally at random

rates on the RPM, their dynamic stimulation by gravity in every direction was

minimized (Hoson et al 1997). The 2 h microgravity treatment of 3 day old

eggs was associated with a significant and marked stimulation of

neovascularization (Figure 3.3). The length, size, junctions and the number of

complexes formed as determined from the pictographs using angioquant

software was found to be significantly higher in microgravity treated eggs

(Figure 3.3). Vascular system is capable of remodeling its structure over

surprisingly short time frame (Unsworth and Lelkes 1998, Kamiya et al 1998,

Langille 1993, Mulvany and Aalkjaer 1990) and adaptation to new

environment i.e., microgravity.

Figure 3.3 Microgravity stimulates neovascularisationTwo days incubated eggs were treated under microgravity and gravity andincubated further for 24h. Quantitative analysis of capillary density in 24h

microgravity treated CAM was evaluated by using stereomicroscope of

magnification 400x. Results are mean ± SEM of 10 experiments.** and * compared to gravity (**p<0.001 and *p<0.05).

54

3.2.1.2 Microgravity Effects on in vitro Capillary-like Tube Formation

To verify the results in vitro, EC suspension was subjected to 2 h of

simulated microgravity. The cell suspension was then placed on matrigel and

allowed to form tubes for 8 h. The result demonstrates that microgravity

induced a significant (*p<0.05) increase in capillary like-tube formation in

EC (Figure 3.4).

Figure 3.4 Microgravity induces capillary like tubes on 3D matrigelEndothelial cell suspension was subjected to microgravity for

2 h. 30,000 cells were seeded in each of matrigel-coated wells and thenumber of tubes formed after 2 h of simulated microgravity was measured

after 24 h incubation in 37oC CO2 incubator. Images were captured in a

bright field phase contrast microscope. Representative phase-contrastimages of experimental conditions are shown in the upper panels and

compiled data are shown in the graph (lower panel). Differences are

statistically significant (**p<0.01).

55

3.2.1.3 Live Cell Tracking of Tubes under Simulated Microgravity

To follow live cell tube formation, the treated cells were plated on

matrigel and course of the formation of the tube was followed for 24 h. At the

6th

h number of circular pits were formed in the matrigel. The cells migrated

from both the pits to form tube like structure. At the end of the 14th

h the tube

shaped structure was complete and another smaller tube branched out from

the bigger tube to form a branch vessel at the end of 24 h (Figure 3.5)

Figure 3.5 Live cell tube formationEC suspension was exposed to microgravity for 2 h and plated on a matrigel

coated 96 well plate and placed in 37oC CO2 incubator. Individual pits

formed were tracked for 24 h and images taken at different time pointsusing a bright field phase contrast microscope. Representative phase-

contrast images of experimental conditions are shown of experiments

repeated 2 times.

3.2.1.4 Microgravity Effects on Ring Formation

One of the unique features of the EC is to form ring shaped

structures. The ring is the fundamental unit of early angiogenesis. The rings

stack one on top of the other to form vessels. EC suspension was exposed to

simulated microgravity for 2 h in the presence of NOS inhibitor L-NAME.

The EC formed rings after 8 h of incubation. The results depict significant

increase in number of rings in microgravity compared to gravity. The number

of rings were reduced in the presence of NOS inhibitor in both gravity and

microgravity (Figure 3.6).

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Figure 3.6 Microgravity increases EC ring numberEC cells suspension was treated with 2 h microgravity in the presence of

NOS inhibitor, L-NAME. The numbers of rings formed were counted manually

after 8 h of incubation in 37oC CO2 incubator. Images were captured in a

bright field phase contrast microscope. Representative phase-contrast imageof experimental conditions are shown in the upper panels and compiled data

are shown in the graph (lower panel). Differences are statistically

significant (**p<0.01) and are the results of 3 independent experiments.

Summary

The results show that microgravity increases the number of

capillaries in a developing chick embryo. Microgravity also stimulates the

formation of capillary like structures on a 3D matrigel which mimics the

basement membrane. Further microgravity stimulates ECs to form rings

which are the basic structure of any blood vessel.

3.2.2 Endothelial Cell Activation under Microgravity

3.2.2.1 Microgravity Effects on Cell Proliferation

Next we looked at the effects of microgravity on the endothelial

cells lining the inner walls of the blood vessels. Physiologically angiogenesis

is a highly organized sequence of cellular events comprising of vascular

initiation, formation, maturation, remodeling and regression, controlled to

meet the requirements of damaged tissue. Within the process of sprouting

angiogenesis, EC undergo proliferation. EC proliferation is a basic

mechanism in regulation of angiogenesis (Ausprunk and Folkman 1977 and

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Djonov et al 2000). We investigated microgravity effect on endothelial cell

proliferation by incubating EC suspension with MTT (0.2mg/ml) for 2 h. EC

proliferation was also measured using a simple haemocytometer and

fluorimetry. EC viability was also measured using a cell tracker, CMFDA.

Microgravity promoted EC proliferation and cells were viable after

microgravity treatment (Figure 3.7).

Figure 3.7 Microgravity induced EC proliferation thus not compromise

the EC viabilityA. EC suspension treated with simulated microgravity for 2 h showed an

increase in metabolic rate of cells, correlated to an increased in cellularproliferation as observed by MTT assay. B. EC suspension treated with

simulated microgravity for 2 h and plated equally in 35mm dishes. After 24

h incubation EC monolayer was probed with CMFDA and reading wastaken at excitation/emission of 495nm/515nm. A certain increase in the cell

number was observed under microgravity while images (inset) showed no

cell death under microgravity treatment. Simple hemocytometer counting using

a haemocytometer showed increase in cell number under microgravity. C.CMFDA flourimetry also showed an increase in cell intensity in cells

exposed to microgravity. *p<0.05, in comparison to gravity controls tested by

one way ANOVA. Results are the mean ± SEM. of 4 individual experiments.

B C

A

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3.2.2.2 Microgravity Effects on EC Migration

During the angiogenesis process, ECs are stimulated to degrade the

basement membrane and migrate into the perivascular stroma in response to a

gradient of angiogenesis inducing factors including VEGF. Wound healing

assay for migration of the endothelial cell monolayer revealed that wound

healed 20% faster in 2 h microgravity treated endothelial cell monolayer

(Figure 3.8). In order to determine if the EC retain microgravity memory, the

cells were pretreated with microgravity for 2 h and kept for incubation for

another 24 h in incubator at 37oC. Next, the EC monolayer was wounded and

images taken at 2 h and 4 h respectively (Figure 3.8). Chemokinesis is an

important property for migration of EC in response to pro-angiogenic factors.

Once the EC is activated by the proangiogenic factors they migrate to form

blood vessels (Tsurumi et al 1996). Boyden chamber was used to determine

chemokinesis of 2 h microgravity treated endothelial cell suspension. The

number of cells that migrated from the upper chamber to the lower chamber

was significantly more in microgravity treated group compared to that of the

control (*p<0.05) (Figure 3.8).

Figure 3.8 (Continued)

A

59

Figure 3.8 Microgravity increases EC migrationMigration assays were performed by wound healing and Boyden’s chamber

assays (n = 3).A EC were treated with simulated microgravity for 2 h. Next,migration responses were examined after 2 h migration. B The EC were

treated with microgravity for 2 h. After 24 h the wound healing was

calculated for 2 h and 4 h respectively. Results expressed as mean ± SEM*p<0.05 relative to migration of control cells. Monolayer migration

assessed by scraping a wound. Results represent the area of wound healed

after 2 h simulated microgravity in 4 independent experiments. ††p<0.01

compared to gravity.

3.2.2.3 Microgravity Effects on EC Migratory Structures

The lamellipodia and filopodia are two key migratory extensions

for migration. The lamellipodium (pl. Lamellipodia) is a cytoskeletal actin

projection on the mobile edge of the cell. It contains a two-dimensional actin

mesh, the whole structure 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 lamellipodium frontier, are called filopodia (Small et al

2002). EC were spread at 40% confluency on a cover slip and treated with

simulated microgravity for 2 h. The migratory structures viz. Filopodia and

Lamellipodia were counted from the pictographs taken using an inverted

phase contrast microscope.Single cells showed an increase in the number of

B

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filopodia of single cell plated on matrigel after 2 h treatment with microgravity

(Figure 3.9).

Figure 3.9 Cellular extensions under microgravityFilopodia extend in response to microgravity (higher magnification image is

indicated by square). Phase contrast images of cells 2 h hours after

simulated microgravity are shown at the top. Micrographs are representativefrom three independent experiments. The graph (below) depicts the

significantly higher number of filopodial structures per cell in 2 h simulated

microgravity as compared to gravity (**p < 0.01), n=25 individual pictures.

3.2.2.4 Microgravity Induces Wound Healing: Cell Migration or

Proliferation?

To ascertain if microgravity induced increase in cell migration did

not involve cell proliferation, a blocker of cell proliferation 5FU was used.

Eahy926 cell suspension treated with 2 h microgravity showed (1.3 folds)

higher proliferation than cell suspension kept in normal gravity when

estimated after 24 h incubation. Blocking proliferation with 5FU showed

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convincingly that the result of faster wound healing was a cumulative effect

of EC migration and proliferation (Figure 3.10).

Figure 3.10 Microgravity mediated increase is through cell migrationCell migration assessed in the presence or absence of an inhibitor ofproliferation, 5-Fluorouracil. Graph showed no significant difference in

wound healing between gravity control and 5FU set, while significant

difference in wound healing between microgravity control and 5FU set.

*p<0.05 compared to microgravity control (n=4).

3.2.2.5 Microgravity Effects on Collateral Formation

So far the experiments were performed in 2D. An attempt was

made to evaluate the EC migration characteristics on 3D lattice. When plated

on matrigel, ECV304 invades the matrigel forming characteristic well like

depressions which will be hereafter called as pits. Extensions from pits

resembling the migratory structures of ECs and were termed as collaterals.

We used this model to examine the functions of ECs activated by simulated

microgravity. ECV304 cells were plated in matrigel coated 24 well plate.

Formed pits were then treated under microgravity for 2 h and studied for

collateral formation. Microgravity elevated collateral formation by 60%

(Figure 3.11).

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Figure 3.11 Collateral formation and migration is due to change in actin

polymerization pattern

Measurement of the number of collaterals formed per pits in EC exposed to

gravity or microgravity. Bright-field images taken at 40x magnification.Images are the representative of 3 individual experiments. *p<0.05 vs

gravity.

Summary

Microgravity increases endothelial cell migration and proliferation

which is quintessential for angiogenesis. The surge in endothelial cell

migration in microgravity is due to migration alone and not as a function of

proliferation. The molecular details of microgravity mediated cell migration

was yet to be investigated.

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3.2.3 Molecular Mechanisms of EC Migration under Microgravity

3.2.3.1 Microgravity Effects on Actin Polymerization

In this module the mechanism through which microgravity

stimulates endothelial cell migration was characterized. Migratory properties

of EC are solely dependant on actin polymerization at the edge of the cells. In

this portion the exact mechanism of microgravity driven EC migration was

explored. To evaluate the simulated microgravity implications in actin

polymerization, a wound healing experiment was performed. After creating a

scratch wound, Eahy926 cell monolayer were exposed to microgravity for 2 h

in the presence of actin polymerization blocker CD. Result depicted that

microgravity promoted endothelial wound healing by 5% (Figure 3.12) which

was abrogated by CD.

Figure 3.12 Microgravity induced EC migration is actin dependant Monolayer of Eahy926 cells with scratch wounds were exposed to gravity

or microgravity in the presence or absence of CD (2 M) for 2 h.

Representative images were taken at 0th and 2

nd hour of treatment. Wound

healing was quantified by processing the images using Image J software.

Bright-field images were taken with 10X magnifications under an inverted

bright field microscope. Data presented as percentage wound healing in 2 h.*p<0.05 vs gravity.

64

3.2.3.2 Microgravity Effects on Actin Dependent Stress Fibre Formation

In order to determine if microgravity induced formation of

filopodia and lamellipodia are actin dependant, Eahy926 were exposed to 2 h

microgravity in the presence of CD. Microgravity exposed cells showed

2 folds increase in the number of filopodia after 2 h microgravity treatment

(Figure 3.13). Cells were stained with phalloidin and graph prepared based on

the number of central microfilaments, stress fibres, locomotory structures like

filopodia, lamellipodia. It was observed that blocking actin polymerization

with CD under gravity treatment promoted formation of an average 19 stress

fibers (Figure 3.13) compared to an average of 13 stress fibers seen under

microgravity treatment. Whereas the locomotory structures of cell-

lamellipodia, filopodia were more in microgravity exposed cells and less in

gravity exposed cells.

Figure 3.13 (Continued)

65

Figure 3.13 Stress fibre formation is actin dependantRepresentative images of actin in cells exposed to gravity and microgravity

in the presence or absence of CD.Photographs taken with an Andor CCD

camera attached to the fluorescence microscope Olympus IX71. Arrows

indicate microfilaments in the upper panel and the sharp processes in thelower panel (B) Migratory structures fillopodia, lamellipodia, microfilaments,

stress fibres of cells treated with gravity and microgravity in the presence or

absence of CD are expressed mean +/- SEM. Data is representative of

3 independent experiments. =Lamellipodiavs gravity, = Filopodiavs

gravity, * = Microfilament vs gravity, # = Stress fibresvs gravity.

3.2.3.3 Microgravity Effects on Actin Polymerization Pattern and NO

Production

Cell migration is associated with regulation of the actin

cytoskeleton. Cells were exposed to microgravity for 2 h followed by time

scan NO imaging using DAR-4FM fluorescent probe. Nitric oxide production

by EC was increased under microgravity exposure with time. To explore the

possibility that simulated microgravity increase nitric oxide production by

actin remodelling, in the presence of CD gravity and microgravity exposed

cells were initially checked for nitric oxide production followed by dual

staining of actin and nucleus with phalloidin and DAPI respectively. The

number of central microfilaments was more in microgravity exposed cells in

comparison to that of gravity exposed cells. The central microfilaments were

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well structured and directed in gravity exposed cells, while directionless,

shorter central microfilaments were seen in microgravity exposed cells

(Figure 3.14). Importantly, CD stimulated stress fiber formation was

markedly attenuated in cells exposed to microgravity (Figure 3.14).

Figure 3.14 Actin dependant NO production under microgravityRepresentative images of actin pattern, nitric oxide, nucleus and a merge ofall three. Images are the representative of 3 individual experiments.

Data presented as fold increase of membrane ruffles, filopodia and nitric

oxide production in cells exposed to gravity and microgravity in the

presence or absence of CD. Data is representative of 3 independent

experiments =Membrane ruffles vs gravity, = Filopodia vs gravity,# = Nitric oxide production vs gravity.

67

Summary

The results showed that microgravity induced EC migration was

actin dependant as shown in Figure 3.15. Further actin polymerization was

attributed to increased NO production and NO was sufficient for changing EC

low migration status in gravity to EC high migration status in microgravity.

The source of NO production still required further investigation.

Figure 3.15 Diagrammatic representations of events occurring in a cell

during their transition from a gravity state to microgravity

state

3.2.4 NO Signalling in the Endothelium under Microgravity

3.2.4.1 Microgravity Effects on NO Production

Vascular relaxation, mediated by NO is a prerequisite for EC to

enter the angiogenic cascade (Griffioen and Molema 2000). The primary

blood vessel (Caprine aorta) was stimulated for 2 h microgravity. Caprine

aorta was dissected and placed in Krebs-Heinsleits buffer (pH 7.4). The tissue

was then removed and the buffer examined for nitric oxide production by

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Greiss assay. NO production in microgravity was significantly more

(**p<0.05) (2.5 folds) than in gravity (Figure 3.16). NO production from the

monolayer of EC also achieved similar results (**p<0.05) (Figure 3.16). The

protein levels detected using Lowry’s assay was found to be equal (Figure 3.16).

Figure 3.16 Microgravity induces NO productionA. NO production from thoracic goat aorta. Microgravity induces NO

production. Caprine aorta was dissected and placed in Krebs-Heinsleits(pH 7.4). This was subjected to microgravity for 2 h. The tissue was then

removed and the solution examined for nitric oxide production by Greiss

assay. NO production determined from endothelial cell monolayer. Theculture supernatants were assayed for nitrite production. Nitrite production

was more in microgravity treated thoracic goat aorta and EC monolayer in

comparison to gravity. B. Total proteins levels were quantified using

Lowry’s assay. The levels of significance of treated vs control wasdetermined by one way ANOVA †p<0.05,**p<0.001 compared to gravity.

Results are the mean ± SEM of 4 individual experiments.

A

B

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3.2.4.2 Microgravity Effects on Intracellular NO Production

To confirm if microgravity promotes cellular NO production, cell

membrane permeable fluorescent probe DAF-2DA was used. Eahy926 and

BPAEC cells were cultured on cover slips in different sets and subjected to

microgravity treatment for 2 h and culture medium was replaced with PBS

containing L-arginine (0.1mM) 30 min and loaded with DAF-2DA. Relative

NO production was assessed by quantitation of fluorescence intensity from

captured microscopic images of DAF-2DA experiments. BPAEC treated for

2 h microgravity produced a higher level of NO as detected by DAF-NO

fluorescence intensity of the cells (Figure 3.17). Eahy926 cells also produced

a higher level of NO under microgravity (Figure 3.17).

Figure 3.17 Microgravity stimulates sub-cellular NO productionMicrogravity mediated sub-cellular NO production was measured from BPAEC

by using the cell membrane permeable fluorescent probes, DAF-2DA. Cells were

cultured in cover slips for 24 hours and then culture medium was replaced with

PBS containing L-arginine (0.1 mM) loaded with DAF-2DA. Relative NOproduction was assessed by quantitation of fluorescence intensity from captured

microscopic images for DAF-2DA experiments. EC produced NO in a time-

dependent manner after microgravity stimulation. The cells pre-incubated withL-arginine produced NO in a time-dependent manner after microgravity

stimulation, as assessed by increased intracellular DAF-2DA fluorescence

(n=10). Selected areas from each micrograph from 3 independent experiments.

*p<0.05 compared to 15 minutes gravity and **p compared to 20 min gravity.

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3.2.4.3 Microgravity Effects on NO Agonist, Bradykinin

Microgravity treated EC were incubated with bradykinin to

evaluate the cumulative effect of microgravity and bradykinin on endothelial

nitric oxide production. Microgravity alone increased NO production in EC

by 69%. Bradykinin treatment under gravity condition elevated NO production in

EC by 45% while microgravity and bradykinin showed synergistic effects on

NO production (Figure 3.18). This effect was retained even after the

microgravity treated cells were incubated for another 2 h at 37°C.

Figure 3.18 Synergistic effects of Bradykinin (BK) and microgravity on

NO production

NO production by EC was measured using a microgravity and BK (5 M)combination treatment. EC were exposed to microgravity for 2 h followed

by BK treatment for 15min, and NO production was measured followingthe Griess assay protocol. Data presented as nitrite equivalent to NO

production from 3 independent experiments. *p<0.05 compared to gravity.

Summary

Different techniques of NO estimation confirmed that both

intracellular and extracellular NO was increased in the presence of

microgravity. Bradykinin and microgravity had synergistic effects on NO

production in ECs. Next the source of NO production was investigated.

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3.2.5 Exploring the Source of NO Production in Microgravity

3.2.5.1 Source of NO Production in EC under Microgravity

So far microgravity was implicated in angiogenesis through

increase in NO production. However the source of NO production was not yet

known. To investigate the source of NO production eNOS and iNOS

pharmaceutical blockers, inhibitors and siRNA were used. The results from greiss

assay and DAF-2DA conclude that microgravity increases the NO production in

EC (Figure 3.16 and 3.17). When an iNOS inhibitor 1400W ( ) was used NO

levels decreased significantly under microgravity (Figure 3.19) hinting that the

increase in NO production was iNOS dependent. This also explained significant

increase (**p<0.05) in NO production in micro gravity compared to gravity.

Direct NO measurements (Figure 3.19) using NO electrode fortified the

results obtained from the Greiss assay. The treatments with 1400W

significantly (**p<0.01) reduced the NO production under microgravity.

Figure 3.19 Microgravity elevates iNOS dependent NO production by ECEC were exposed to microgravity for 2 h in the presence or absence of

iNOS inhibitor 1400W (25 M). NO production by EC was measured directlyusing a NO sensitive electrode. Data is representated by mean of 3 independent

experiments. *p<0.05 vs gravity control and †p<0.05 vs microgravity control.

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3.2.5.2 Effects of 1400W on Intracellular NO Production Induced by

Microgravity

BPAEC were exposed to simulated microgravity for 2 h in the

presence of 1400W, an iNOS inhibitor. The BPAEC were then incubated with

DAF 2DA which is an intracellular NO probe for 15min. There was an

increase in NO intensity in microgravity which was inhibited by 1400W

(Figure 3.20). A slope calculated from the images also showed that there is a

2 fold increase in NO in microgravity treated cells. This increase was

abolished using 1400W (Figure 3.20)

Figure 3.20 (Continued)

Bright field 0 5 15 (min)A

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Figure 3.20 Microgravity mediated NO production is blunted by iNOS

inhibitor

Microgravity mediated sub-cellular NO production was measured from

BPAEC by using the cell membrane permeable fluorescent probes, DAF-

2DA. A and B Cells were cultured in cover slips for 24 hours and thenculture medium was replaced with PBS containing L-arginine (0.1 mM)

loaded with DAF-2DA in presence or absence of 1400W. Relative NO

production was assessed by quantization of fluorescence intensity fromcaptured microscopic images for DAF-2DA experiments. (n=10) selected

areas from each micrograph from 3 independent experiments. *p<0.05

compared to 15 minutes gravity and **p compared to 20min gravity.

Summary

iNOS and not eNOS was responsible for microgravity induced NO

production in the ECs. Microgravity specifically increased iNOS expression

levels. The next question was if iNOS is implicated at a functional level also.

3.2.6 iNOS Implications at a Functional Level

3.2.6.1 Effects of iNOS Inhibition on Capillary Like Tube Formation

Next the question if iNOS is expressed at a functional level also

was addressed. Microgravity induced capillary-like tube formation was

examined by treating Eahy926 and BPAEC cells for 2 h under simulated

B

74

microgravity in the presence of iNOS inhibitor 1400W. The number of tubes

in in gravity +1400W and microgravity +1400W treated reduced 38% and

40% compared to gravity and microgravity without 1400W (Figure 3.21)

Figure 3.21 Formation of microgravity induced tubes on 3D matrigel is

iNOS dependent

Endothelial cell suspension was subjected to microgravity for different time

intervals. 30,000 cells were seeded in each of matrigel-coated wells and thenumber of tubes formed after 2 h of simulated microgravity was measured

after 24 h incubation in 37oC CO2 incubator in the presence or absence of

iNOS inhibitor (1400W). Data presented as percentage difference in the

number of tubes after 2 h of microgravity. Control represents percentagedifference between gravity and microgravity without inhibitors.

Representative phase-contrast images of experimental conditions are shown

in the upper panels. *p<0.05 and †p<0.05 compared to control.

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3.2.6.2 Effects of iNOS Knockdown on Microgravity Induced Tube

Formation

The EC were transfected with iNOS siRNA and incubated for 24h.

The cells were then exposed to simulated microgravity for 2 h followed by

plating on matrigel. While tubes were formed in cells exposed to

microgravity, they were completely abolished in cells transfected with iNOS

siRNA (Figure 3.22)

Figure 3.22 Tube formation under microgravity was checked using

iNOS siRNA transfected cells

After transfection, EC cell suspension was treated under simulated

microgravity for 2 h. Next, cells were plated in 12 well plate and incubatedfor 24 h. Tube formation was reduced in siRNA transfected cells under

microgravity treatment. ** significantly different than only microgravity

(p<0.001).

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3.2.6.3 Effects of iNOS knockdown on microgravity induced NO

production

To investigate the effect of microgravity on iNOS dependent NO

production, iNOS siRNA transfected cells were treated under microgravity for

2 h and NO production was quantified using DAF fluorimetric protocol.

Result suggested that NO levels dropped by half a fold in siRNA transfected

cells than control (Figure 3.23).

Figure 3.23 iNOS siRNA blunts NO productionNO production from siRNA transfected cells was carried out under

microgravity treatment. Cells were first transfected with siRNA using

lipofectin transfection protocol. Next, siRNA transfected cells were treatedunder simulated microgravity for 2 h and NO production was measured

using DAF flurometric protocol. NO production was reduced under

microgravity in iNOS siRNA transfected cells. ** significantly differentcompared to microgravity (**p<0.001)

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3.2.6.4 Microgravity Effects on iNOS Transcription

The level of iNOS mRNA was probed in microgravity treated cells.

Total mRNA was isolated from microgravity treated and untreated samples

followed by RT-PCR using iNOS primer. Result showed a two fold increase

in the level of iNOS mRNA in microgravity treated cells than control cells

(Figure 3.24).

Figure 3.24 Level of iNOS mRNA in microgravity treated cells was

measured using RT-PCR technique

Cells were treated under simulated microgravity for 2 h. Next, mRNA from

the microgravity treated cells were isolated using spin prep kit (MedoxInc).

cDNA synthesis was performed on 200ng of RNA using mulv reversetranscriptase (Finzymes) and PCR was performed using 50ng of cDNA

concentration. Microgravity elevated iNOS mRNA level in EC than the

gravity treated cells. ** significantly different than gravity (**p<0.001).

Summary

The source of microgravity induced NO production was found to be

iNOS. Both iNOS expression levels and iNOS activity were high in the

presence of microgravity. Blunting iNOS using iNOS siRNA resulted in

inhibition of microgravity induced NO production. Thus iNOS was found to

be the key player in microgravity induced NO production at both cellular and

functional level. Microgravity effects on macrovascular endothelial cells was

addressed, but its effects on microvascular and endocardial cells was yet to be

explored.

316 bp

480 bp

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3.2.7 Effects of Microgravity on Different Endothelial Subtypes

3.2.7.1 Macrovascular Cells, Endocardial, Microvascular Cells

Response to Microgravity Stimulus

ECs have been reported to show differential effects in response to a

stimulus depending on their subtype. To evaluate the overall effects of

simulated microgravity on ECs, two other subtypes of ECs were used-

endocardial cells and microvascular cells. Migration of the endothelial

subtypes was determined using wound healing assay in the presence or

absence of 1400W. Further, quantification of wound healing in macrovascular

and microvascular EC under microgravity treatment depicted higher wound

healing property of macrovascular EC under microgravity which is sensitive

to 1400W (Figure 3.25) as opposed to less wound healing in microgravity

treated microvascular EC (Figure 3.25).

Figure 3.25 Microgravity induced differential effects on EC migrationWound healing with and without 1400W was quantified in microgravity

treated macrovascular, endocardial and microvascular EC. Microgravity

elevated macrovacular EC wound healing which was attenuated with

1400W administration. Microgravity showed no effect on wound healing ofendocardial and microvascular EC. **p<0.001 vs gravity; ††p<0.001 vs

microgravity.

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3.2.7.2 NOS Protein Levels in Different Endothelial Subtypes in

microgravity

Further, expression of iNOS protein in BPAEC was independently

examined by western blot. The data revealed a higher expression of iNOS

protein in 2 h microgravity treated cells. Expression of iNOS level in

macrovascular and microvascular cells remained similar under gravity and

microgravity (Figure 3.26). Quantification of iNOS expression in

microvascular, macrovascular and endocardial cells demonstrated that

microgravity treatments elevated iNOS level in macrovascular EC while no

change was observed in microvascular and endocardial cells under

microgravity (Figure 3.26).

Figure 3.26 iNOS expression levels in micro and macrovascular EC cellsiNOS protein expression was measured in microgravity treated

macrovascular, endocardial vascular and microvascular EC using westernblot technique. Data representative of three individual experiments. Mean

+/- SEM, *p<0.05 vs gravity by one way ANOVA.

3.2.7.3 Microgravity Effects on eNOS Expression Levels in EC Subtypes

Further, expression of eNOS protein in BPAEC was independently

examined by immunofluorescence, which revealed a higher expression of

eNOS protein in 2 h microgravity treated cells. Expression of eNOS level

remained similar under gravity and microgravity (Figure 3.27).

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Figure 3.27 Immunofluorescence localization of eNOS in EC spread on

the coverslips

EC were treated with simulated microgravity for 2 h, fixed, permeabilised

and stained with anti eNOS antibodies. Representative cells were

photographed with Andor CCD camera attached to the fluorescencemicroscope Olympus IX71. The graph is representative of intensities of

individual cells (n=50). **p<0.01vs gravity using one way ANOVA.

81

3.2.7.4 Microgravity Effects on iNOS Expression Levels in EC Subtypes

Quantification of iNOS expression in microvascular, macrovascular

and EEC demonstrated that microgravity treatments elevated iNOS level in

macrovascular EC while no change was observed in microvascular and EEC

under microgravity (Figure 3.28). Quantification of iNOS expression in

microvascular, macrovascular and EEC demonstrated that microgravity

treatments elevated iNOS level in macrovascular EC while no change was

observed in microvascular and EEC under microgravity (Figure 3.28).

Figure 3.28 Immunofluorescence localization of eNOS and iNOS in EC

spread on the coverslips

EC were treated with simulated microgravity for 2 h, fixed, permeabilisedand stained with anti iNOS or anti eNOS antibodies. Representative cells

were photographed withAndor CCD camera attached to the fluorescence

microscope Olympus IX71. The graph is representative of intensities ofindividual cells (n=50). **p<0.01vs gravity using one way ANOVA.

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3.2.7.5 Microgravity Effects on NO Production in Different EC Subtypes

NO production by macrovascular and microvascular EC quantified

using DAR-4M fluorescence probe illustrated a higher level 1400W sensitive

NO production in macrovascular EC under microgravity while no change in

NO levels were observed in microvascular EC and endocardial vascular cells

(Figure 3.29).

Figure 3.29 NO production in different EC subtypesNO production by macrovascular, endocardial and microvascular EC was

measured using NO sensitive DAR-4M fluorescence probe. Microgravity

promoted NO production by macrovascular EC is sensitive to 1400W.Microgravity did not modulate NO production by microvascular EC.

**p<0.001 vs gravity; ††p<0.001 vs microgravity.

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Summary

Microgravity has heterogeneous effects on different endothelial

subtypes. Microgravity increases NO production through iNOS activation in

macrovascular cells but not microvascular and endocardial cells. To

summarize microgravity increase NO production in macrovascular cells

through iNOS activation (Figure 3.30). Next the molecular mechanism of

microgravity induced angiogenesis was dissected (Figure 3.31).

Figure 3.30 Summary of results of microgravity effects on angiogenesis

Increased Increased Promoted Promoted Promoted

Polymerization

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Figure 3.31 Schematic representation of the angiogenesis pathway

3.2.8 Mechanism of Microgravity Induced Angiogenesis

Although it was clear that microgravity increases NO production

through iNOS activation, the mechanism of NO driven angiogenesis was still

unknown.

3.2.8.1 Wortmanin, a Phospho Inositol 3 Kinase (PI3K) Blocker

Effects on Microgravity Induced NO Production

In order to explore the involvement of PI3K pathway in

microgravity induced angiogenesis, a PI3K blocker, Wortmanin was

administered to quantify the level of endothelial nitric oxide production under

microgravity. Wortmanin blocked cellular nitric oxide production by 70%

under gravity condition (Figure 3.32). As observed earlier, microgravity

elevated endothelial nitric oxide production by 50% while administration of

Wortmanin blocked endothelial NO production by 40% (Figure 3.32).

Angiogenesis

?

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Figure 3.32 Microgravity driven NO production is partially dependent

on PI3K-MAPK pathway

NO production from microgravity treated EC were measured with and with

outwortmanin (5 M). Monolayers of EC were treated under microgravityfor 2 h followed by nitric oxide production measurement following Griess

assay protocol. Cells were incubated with wortmanin during the period of

treatment. Data presented as nitrite equivalent to nitric oxide production.*p<0.05 vs gravity

3.2.8.2 Microgravity Effects on cGMP Levels in EC

Nitric oxide acts downstream through the cGMP/PKG pathway. In

order to determine if the iNOS derived nitric oxide was dependant on cGMP,

the EC were treated with microgravity for 2 h and cGMP levels measured

using the manufacturers protocol. The cGMP levels were significantly higher

in microgravity compared to gravity. The cGMP levels were reduced in the

presence of iNOS inhibitor (1400W) (Figure 3.33). Thus microgravity

induced NO was found to be cGMP dependant (Figure 3.34).

86

Figure 3.33 Microgravity increases cGMP levelscGMP levels were measured from EC treated with 2 h microgravity using a

cGMP detection kit. The cells were incubated with iNOS inhibitor duringthe treatment period. The cGMP levels of 3 independent experiments

calculated from the standard graph are presented as mean +/- SEM *p<0.05

vs gravity.

Figure 3.34 cGMP is downstream of microgravity induced NO production

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3.2.8.3 Microgravity Acts Via cGMP/PKG Pathway to Promote Tube

Formation

To dissect the NO down stream pathway under microgravity

induced nitric oxide production, we performed tube formation assay using

pharmacological blockers ODQ and KT5823 for sGC and PKG respectively

(Figure 3.35). Result revealed a significant drop in the number of tubes under

ODQ and KT5823 treatments respectively in microgravity environment.

Combination treatments with ODQ + 8Bromo-cGMP and ODQ + Sildenafil

citrate partially recovered ODQ mediated inhibition of tube formation under

microgravity (Figure 3.35).

Figure 3.35 Microgravity induced tube formation is cGMP/PKG

dependent phenomenon

Tubes formed in the presence or absence of ODQ (10 M), ODQ + 8

Bromo-cGMP (10 M), ODQ + Sildenafil citrate (1 M) and KT 5823

(1 M) were counted from the images. **p<0.05vs gravity control, †p<0.05vs microgravity control, #p<0.05 vs microgravity + ODQ.

Summary

Microgravity stimulates angiogenesis by activating iNOS which in

turn increase NO levels. NO then activates guanylate cyclase resulting in

elevation of cGMP levels. cGMP inturns switches on the protein kinase G

further initiates blood vessel formation.

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Figure 3.36 Summary of nitric oxide downstream pathway in

macrovascular cells

Summarizing the Results of the Present Chapter

Microgravity induces in vitro and in ovo angiogenesis.

Microgravity also activates endothelial cell migration,proliferation, ring

formation and tube formation. Further microgravity increases endothelial cell

function through activating iNOS and increasing NO production. iNOS is the

key player in modulating NO production in different endothelial subtypes in

the presence of microgravity. Finally microgravity dependant angiogenesis is

mediated through iNOS-cGMP-PKG pathway.

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3.3 DISCUSSIONS

Microgravity and nitric oxide has been implicated in influencing

several physiological processes raising the intriguing question whether

microgravity is proangiogenic. Our observations reveal that angiogenesis is

stimulated by simulated microgravity via iNOS responsive NO production.

Treatment of endothelial monolayer and embryo vascular plexus with

simulated microgravity resulted in an increase in tube formation and

capillaries. To substantiate our findings that microgravity induces endothelial

activation we used an array of cells and tissues including transformed

secondary ECs, primary macro vascular endothelial cells, caprine aorta and

CAM angiogenesis models. Further dissection of downstream events of NOS-

expression confirmed that simulated microgravity promotes angiogenesis in

the matrigel scaffolds via a cGMP dependent pathway.

The process of blood vessel formation from pre-existing capillaries,

angiogenesis is a sequence of events required for normal tissue growth,

wound healing, embryonic development and menstrual cycle. Angiogenesis is

initiated in response to hypoxic or ischemic conditions (Griffioen and

Molema 2000). Vascular relaxation mediated by NO is a prerequisite for the

endothelial cells to enter the angiogenic cascade. Vascular endothelial growth

factor (VEGF) is a major player in initiation of angiogenesis via endothelial

NO production and increasing endothelial cell permeability. Simulation of

microgravity by using a horizontally rotating bioreactor was shown by Dutt et

al (2003) to up regulate VEGF and basic fibroblast growth factor and

promotes three-dimensional assembly and differentiation which are the

prerequisite of blood vessel formation (Dutt et al. 2003). Recently Infanger et

al (2006) showed that after 72 hours of clinorotation, EC assembled to three-

dimensional tubular structures when the cells were in suspension (Infanger

et al 2006). The concept of microgravity as “angiogenesis-modulating force”

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was further fortified when a 14-day space mission with rats revealed that

microgravity induces capillary formation in forepaw of the rats as an adaptive

reaction along with ultra structural changes in musculature (Volodina and

Pozdnyakov 2006). The results of our present work evidence that EC, either

immortalized transformed or primary, rearrange themselves in a microgravity

environment to form tubes in 3-D matrigel scaffolds (Figure 3.4). The CAM

assay results rationalize that simulated microgravity prompted vasculogenesis

(Figure 3.3), which may be the outcome of the endothelial activation under

microgravity. Simulated microgravity promotes endothelial activation by

promoting pro-angiogenic molecules such as VEGF, collagen type I,

fibronectin, osteopontin, laminin and flk-1 protein in cell based models

(Infanger et al 2006). Moreover, a variation in NOS expression and activity

levels was observed in hind limb un-weighing (HU) rat model. Vessels from

HU rat showed an increase in cerebral arteries, a decrease in mesenteric

arteries, and no change in carotid artery (Ma et al 2003). Implications of

microgravity in endothelial monolayer experiment were planned on the basis

of these observations.

Since angiogenesis, relies essentially on the ability of capillary

endothelium to migrate and proliferate, the study was designed to understand

the core effect of simulated microgravity on endothelial activation, which

may be the cause of angiogenesis in microgravity treated scaffolds. We

observed that a limited exposure to microgravity promoted endothelial

migration (Figure 3.8A and 3.8B). We presume that microgravity stimulates

cell surface mechanosensors, possibly a group of mechano-transduction

proteins, to feel the differential and associated mechanical strain in the

cytoskeletal arrangements due to microgravity, and further transmits the

signal to engineer down-stream event such as actin polymerization in favor of

cellular migration. The work of (Spisni et al 2006) claims that tyrosine

phosphorylation of caveolin-1 constitutes the early mechanosensor for 24-48

91

h microgravity exposure. Filamin, a common binding protein for caveolin and

actin, cross-links actin filaments into orthogonal networks, bundles actin

filaments, and connects the actin network to specific transmembrane proteins.

Since down-regulation of caveolin-1 in association with tyrosine

phosphorylation is predicted to promote iNOS activity, we postulate that

microgravity introduces iNOS activation and actin remodeling for endothelial

tube formation by using mechanosensing property of caveolin-1. It has been

shown that simulated microgravity influences actin fiber remodeling in

association with small GTPase Rho in bovine brain microvascular cells

(Higashibata et al 2006), while NO is known to interplay with Rho GTPase

family members in modulation of actin dynamics (Lee et al 2005).

NO also enhances endothelial migration by stimulating endothelial

cell podokinesis (Noiri 1998), increasing vß3 (Murohara 1999) expression

and increasing dissolution of the extracellular matrix via the bFGF induced

upregulation of urokinase-type plasminogen activator (Ziche 1997). The ECs

migrate in response to different stimuli under varied processes like

angiogenesis, inflammation and thrombosis. It is known that migration of ECs

is a molecular process that involves modulation in cell adhesion, signal

transduction and reorganization of cytoskeleton (Rubanyi 1993). Our study

depicts that a 2 h exposure to microgravity inducts endothelial cell migration

and faster wound healing by promoting mechanotaxis of endothelial cells at

the leading edge (Figure 3.8). Filopodia contain a tight bundle of long actin

filaments oriented in the direction of protrusion. Endothelial cell migration

involves reorganization of cytoskeleton and actin remodeling (Li et al 2005).

As shown previously, space flown cells showed irregular cytoskeletal fibre

pattern. Microtubule filaments extended from a poorly defined centrosome in

human lymphocytes (Jurkat cells) (Lewis et al 1998). Gruener and Hughes-

Fulford reported that actin reorganization responded to the gravity level and

showed abnormal assembly of actin stress fibers (Gruener 1994, Hughes-

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Fulford 1993). Similarly we observed that simulated microgravity disturbs the

organization of actin filaments (Figure 3.12). Phalloidin is known to stabilize

actin filaments by inhibiting subunit dissociation at the barbed ends, while CD

inhibits polymerization at the pointed ends of actin filaments (Prakash et al

1991). Among other effects cytochalasins, paralysis of locomotion and

membrane movements are reported (Carter 1967, Gail et al 1971, Spooner

et al 1971, Wessells et al 1971). We found that in gravity exposed cells, in the

presence of CD cell migration was blocked while under microgravity

although cell migration was blocked the migratory structures namely

filopodia, lamellipodia were still intact, possibly without motor actions

(Figure 3.12). Gravity exposed cells appeared rounded and had long, thin

spikes extending throughout the surface. However in microgravity exposed

cells, the long thin spikes were reduced considerably. However in the absence

of CD, gravity exposed cells had organized and stabilized actin filaments

while microgravity had disorganized, short actin filaments concentrated in the

centre (Figure 3.13). Based on our observation that simulated microgravity

causes actin reorganization which can be blocked with CD (Figures 3.12 and

3.13) we infer that microgravity stimulates cell surface mechanosensors,

possibly a group of mechanotransduction proteins, to feel the differential and

associated mechanical strain in the cytoskeletal arrangements due to

microgravity, and further transmits the signal to engineer down-stream event

such as actin polymerization in favour of cellular migration.

Simulated microgravity increases myogenic tone of cerebral

arteries through both NOS-dependent and independent mechanisms (Geary

et al 1998). Microgravity inhibits pro-inflammatory responses by activating

NO producing machinery (Walther et al 1998) Vessels from hindlimb

suspension rat showed an increase in cerebral arteries, a decrease in

mesenteric arteries, and no change in carotid artery (Ma et al 2003). Actin

cytoskeleton is known to regulate eNOS expression at post translational phase

93

(Searles et al 2004). Reorganization of the actin cytoskeleton may affect

eNOS activity leading to the alteration of NO production (Su et al 2003).

Actin cytoskeleton disruption increases iNOS expression in vascular smooth

muscle (Zeng et al 2001) and glomerular mesanglial cell (Hattori et al 2004).

Polymerization state of -actin crucially regulates the activation state of

NOS-3, and hence NO formation, through altering its binding of heat shock

protein 90 (Hsp90) (Ji et al 2007). NO is also known to crosstalk with Rho

GTPase family members in modulation of actin dynamics (Lee et al 2005). As

evident from our finding, microgravity modulates actin dynamics and increase

nitric oxide production (Figure 3.14). Bradykinin is also a known inducer of

nitric oxide that activates PI3K leading to eNOS phosphorylation (Bernier

et al 2000). However, NO production was increased 4 folds when bradykinin

treated ECs were subjected to microgravity (Figure 3.18) showing the

synergistic effect of microgravity and bradykinin on endothelial NO

production. Microgravity causes actin remodeling and thereby activates NOS

to induce NO driven endothelial migration. Although the exact mechanism is

not known, it has been suggested that NO increases EC proliferation in part

by increasing the VEGF (Dulak 2000) or FGF (Ziche 1997) expression. Also

a hemodynamic force such as shear stress in the microcirculation has been

associated with EC proliferation (Hudlicka 1998). In our studies also we

found that exposing cells to simulated microgravity resulted in an increase in

NO mediated endothelial cell proliferation (Figure 3.7).

The mechanisms by which NO promotes endothelial cell migration,

proliferation and angiogenesis are not fully elucidated. NO is an endothelial

survival factor, inhibiting apoptosis (Rossig 1999), and enhancing endothelial

cell proliferation (Cooke 2002). Microgravity promotes gene expression of

EC as early as 10 minutes of microgravity treatment (Infanger et al 2007).

A selected set of genes, VEGF, osteopontin, Fas, TGF-beta-1, caspase-3 were

expressed under microgravity in time dependent fashion (Infanger et al 2007).

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Microgravity inhibits pro-inflammatory responses by reducing synthesis of

interleukin and thereby possibly activating NO producing machinery (Walther

et al 1998). BPAEC in the rotating wall vessel (RWV) produces higher level

of basal NO (Ai et al 2002). NO produced by endothelial cells varies with

different endothelial subpopulations.

Endothelial cells have several subtypes depending on their location

in the body, organ microenvironment, cellular components, basement

membrane and extracellular matrix. The response of endothelial cells to

microgravity is debated because of heterogeneous experimental approaches

such as variation in duration of treatments, levels of the gravity and origin of

the cell types. In particular, Mariotti has recently shown a modest decrease of

iNOS and an increase of eNOS in human microvascular endothelial cells,

while Versari has demonstrated the upregulation of eNOS in HUVEC in

microgravity. HUVEC and bovine aortic endothelial cells grow faster

(Carlsson et al 2003) while microvascular endothelial cells grow slower under

microgravity (Cotrupi et al 2005). However, the response of endothelial cells

to microgravity is debated because of heterogeneous experimental approaches

such as variation in duration of exposure, levels of the gravity and origin of

the cell types. HUVEC and BAEC grow faster (Infanger et al 2006), while

microvascular endothelial cells grow slower under microgravity (Carlsson

et al 2003). Endocardial EC is another endothelial cell type that demonstrates

unique endothelial phenotypes (Misfeldt et al 2009). Work of Mebaza et al

showed that endocardial EC is a greater source of PGI2 than macrovascular

EC (Mebaza et al 1995). It has also been reported that endocardial EC

contains Weibel-Palade antibodies dispersed all over the cytosol which is not

observed in macrovascular cells (Andries and Brutsaert 1991). We tested the

hypothesized that iNOS is the key to the heterogeneous endothelial functions

in macrovascular EC, endocardial EC and microvascular EC under

microgravity. We observed higher iNOS expression levels in macrovascular

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EC but no significant change in iNOS expression in endocardial and

microvascular endothelial cells (Figure 3.25, 3.26 and 3.28). Other

investigators furnished functional evidence, in rat cerebral artery, that hind

limb unloading reduced nitric oxide activity, and increased myogenic tone of

the blood vessels (Geary et al 1998). The present study also depicts that

simulated microgravity retards endothelial migration in microvascular

endothelial and endocardial vascular monolayers (Figure 3.25). However,

iNOS and nitric oxide are possibly and not exclusive determinates of

endothelial functions. Cotrupi et al. showed that microgravity reversibly

inhibits endothelial growth and this correlates with an upregulation of p21, a

cyclin-dependent kinases inhibitor along with a down regulation of

interleukin 6, which contribute to growth retardation (Cotrupi et al 2005).

Therefore, it emerges that microgravity positively modulates macrovascular

originated cells, while retards endothelial functions in microvascular

originated cells. Our defined approach demonstrates that the iNOS is a key

modulator in the heterogeneous effects of limited microgravity exposure (2 h)

on endothelial cells. However, the question comes again, why does

microgravity induce iNOS in macrovascular endothelial cells, particularly

when specialized eNOS is present in the cells? The work of Quaschning et al

(2008) showed that additional knockout of iNOS results in impaired

endothelium-dependent vasodilatation thus contributing to elevated blood

pressure in endothelin (ET)+/+

iNOS-/-

animals (Quaschning et al 2008). The

work established the concept that iNOS could be a crucial player along with

eNOS in the EC. We deem that microgravity de-couples mainly iNOS from

caveolin-1 in the EC via a mechanosensor pathway, which in turn produces

bulk NO to stimulate angiogenic cascades.

Some studies claim that expression and activation of iNOS

promotes angiogenesis (Song et al 2002), which we observed as the core

mechanism for the microgravity induced angiogenesis (Figures 3.19 and 3.20).

96

(Jianghui et al 2003) indicated that regulative effect of simulated microgravity

on iNOS expression is mediated at least partially via activation of protein

kinase C (Jianghui et al 2003). The present work confirms that microgravity

induced NOS expression, and an elevated NO level promoted cellular

migration and tube formation (Figure 3.21). LPS and TNF-alpha induced

expression of iNOS is associated with the apoptosis of EC (Arai et al 2008).

However, the question comes again, why microgravity induces iNOS in

endothelial cells, particularly when specialized eNOS is present in the EC?

The work of Quaschning et al (2008) showed that additional knockout of

iNOS results in impaired endothelium-dependent vasodilatation thus

contributing to elevated blood pressure in endothelin (ET)+/+ iNOS-/-

animals. The work established the concept that iNOS could be a crucial player

along with eNOS in the EC. We postulate that microgravity de-couples

mainly iNOS from caveolin-1 in the EC via a mechanosensor pathway, which

in turn produces bulk NO to stimulate angiogenic cascades. Our unpublished

data, which shows that a 15 minutes bulk NO exposure signals the EC to form

endothelial tubes in matrigel, also supports the postulation. Vascular

endothelial growth factor (VEGF) has been shown to protect EC from

programmed cell death under microgravity (Infanger et al 2004).

The PI3K –Akt pathway is an upstream signalling pathway for the

activation of eNOS via serine specific phosphorylation (Dimmeler et al 1999).

Phosphorylation of paxillin, FAK, calpain, MAP1B and MAP2, MAPKAPK

2/3, and MLCK by MAPKs might regulate the reorganization of microtubules

and lamentous actin. These modulators play key roles in cell spreading,

lamellipodium extension and tail retraction during cell migration (Huang et al

2004). In addition, NO regulate the activation of the p38 mitogen-activated

protein kinase (MAPK)/MAPK-activated protein kinase/Hsp27 pathway

which is crucial for endothelial cell chemotaxis (Rousseau et al 1997). It has

also been reported that NO promotes endothelial cell migration and

neovascularization via cGMP-dependent activation of PI3K (Kawasaki et al

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2003). When we used PI3K inhibitor wortmanin we found that blocking PI3

kinase did not attenuate simulated microgravity mediated NO production, thus

indicating PI3K independent NO production under microgravity treatment.

NO possibly worked through a cGMP dependant pathway.

Targeting key steps of the NO downstream signaling with sGC inhibitor and

8-bromo cGMP, a cGMP analog we could convincingly demonstrate that

microgravity induced tube formation from endothelial monolayer is cGMP

dependent (Figure 3.35). It has been shown that sGC-PKG pathway is

implicated in angiogenesis events in various physiological and pathological

situations. Recently Senthilkumar demonstrated that sildenafil therapy results

in increased angiogenic activity through a PKG-dependent pathway in critical

limb ischemia (Senthilkumar et al 2007). In downstream to cGMP, cGMP-

dependent protein kinases (PKG) are key enzymes of nitric oxide–cGMP

signaling cascade.

In summary, the experiments performed in this study established

that simulated microgravity increases the number of EC tubes by activating

endothelial iNOS in macrovascular endothelial cells. The work also

established that iNOS is the key molecular switch in the heterogeneous effects

of microgravity on macro and microvascular endothelial cells. Finally, the

dissection of NO-downstream signaling brought to light the key role of

cGMP-PKG pathway in the modulation of angiogenesis in microgravity

environment.

The work done so far offers ample evidence that microgravity

supports angiogenesis by activating NO machinery in endothelial cells. This

knowledge will be helpful to regenerate damaged tissue. However as

hypothesized, we further studied microgravity implications in stem cell

differentiation which is another functional pillar of tissue regeneration. The

following chapter will address this issue.