chapter 3 simulated microgravity and...
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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,
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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
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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
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* *
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).
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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).
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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
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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.
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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)
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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.
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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
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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).
80
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”
90
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).
94
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
95
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
97
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.