Annals of Biomedical Engineering, Vol. 33, No. 10, October 2005 (© 2005) pp. 1405–1410
DOI: 10.1007/s10439-005-6153-5
Simulated Microgravity Impairs Leukemic Cell Survival Through
Altering VEGFR-2/VEGF-A Signaling Pathway
LOÏC VINCENT,1 PATRICIA AVANCENA,2 JOSEPH CHENG,1 SHAHIN RAFII,1 and SINA Y. RABBANY1,2
1
Department of Genetic Medicine, Cornell University Medical College, New York, NY and 2 Bioengineering Program, Hofstra
University, Hempstead, New York, NY
(Received 18 February 2005; accepted 26 May 2005)
that link the cytoskeleton to the extracellular matrix and to
the other cells.6 The mechanism by which these mechanical signals are transduced and converted to biochemical
responses, however, are not clearly understood.
The rotating cell culture system, also known as the rotating wall vessel (RWV) system (Synthecon Inc., Houston,
TX) developed by NASA provides us with a novel way
to see inside a cell by understanding how gravitational
forces alter cell function. In this rotating wall bioreactor,
the liquid media and the cells rotate with the walls of the
container. This action suspends the cells in the media so that
the effects of gravity-driven convection and sedimentation
are significantly reduced. The two factors governing the
simulated microgravity environment are low shear stress
that promotes close apposition of the cells, and randomized
gravitational vectors, which either affect gene expression or
indirectly facilitate paracrine/autocrine intercellular signaling through diffusion of differentiative humoral factors.7,10
Through solid body rotation and viscous coupling, the RWV
bioreactor subjects suspended cells to a continual state of
free fall, hence simulating microgravity.
Several studies suggest that culture of cells in microgravity may reduce cell proliferation2,3 and differentiation.1 For
example, simulated microgravity was found to inhibit the
proliferation of bone marrow-derived CD34+ cells, while
preserving their self-renewal capabilities.8 To observe the
effects of simulated microgravity on leukemic cell cultures,
we have examined the effect of simulated microgravity
on the proliferation rates and the tyrosine kinase vascular
endothelial growth factor receptor-2 (VEGFR-2) expression levels of the rapidly proliferating promyelomonocytic
leukemic cell line HL60. Autocrine as well as paracrine
activations of VEGFR-2 and vascular endothelial growth
factor-A (VEGF-A) signaling pathways have been shown
to support the proliferation and survival of leukemic cells.4,5
Membrane localization of VEGFR-2 has been shown to be
critical in conferring functionality to this receptor.
The aim of our study was to investigate how simulated
microgravity might affect leukemic cell proliferation, and
to attempt to determine the relation between the alteration
Abstract—Motile cells capable of undergoing transendothelial
migration, such as hematopoietic and leukemic cells, have been
shown to sense and respond to a decrease in their surrounding
gravity. In this study, we investigated the effects of microgravity
on human leukemic cell proliferation and expression of receptors
that control cell survival, such as the tyrosine kinase vascular
endothelial growth factor receptor-2 (VEGFR-2). VEGFR-2 is
shuttled between the nucleus and membrane, and through an autocrine activation of its ligand, VEGF-A, conveys signals that control cell survival. Autocrine or paracrine stimulation of VEGFR2 facilitates localization of this receptor from the membrane to
the nucleus—a process that results in increased survival of the
leukemic cells. Here, we provide evidence that the mechanical
forces altered by simulated microgravity localize and maintain
VEGFR-2 in the membrane, and also block VEGF-A expression.
This interferes with the shuttling of VEGFR-2 to the nucleus,
resulting in a decrease in signaling and enhanced leukemic cell
death. These data suggest that microgravity modulates cell survival through altering the cellular trafficking and activation state
of tyrosine kinase receptors. This study has potential implications
for understanding the regulation of receptor biology in pathophysiology, particularly VEGFR trafficking, thereby providing for the
development of appropriate therapeutic strategies to abrogate intracrine stimulation triggered by VEGFR internalization.
Keywords—Cell survival, Receptor trafficking, Mechanotransduction, Mechanical forces.
INTRODUCTION
Circulating cells in the blood stream have the ability
to sense multiple, simultaneous inputs. The integration of
these signals inside the cell ultimately dictates its biological
behavior. Physical forces along with biochemical signals
mediated by growth factors and adhesion molecules are the
fundamental regulators of tissue development. Cells may
sense mechanical stresses in their local environment, such
as those due to gravity, through the balance of forces that
are transmitted across transmembrane adhesion receptors
Address correspondence to Sina Y. Rabbany, Bioengineering Program,
104 Weed Hall, Hofstra University, Hempstead, NY 11549. Electronic
mail: sina.y.rabbany@hofstra.edu
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C 2005 Biomedical Engineering Society
0090-6964/05/1000-1405/1 1406
VINCENT et al.
of VEGFR-2 trafficking and the inhibition of cell proliferation. Indeed, VEGFR-2 tyrosine kinase receptor provides
for an ideal receptor to study the role of biomechanical
forces, since localization of VEGFR-2 to the membrane is
the major determinant in modulating VEGFR-2 signaling.
The data presented here suggest that the mechanical forces
imparted by simulated microgravity localize and maintain
VEGFR-2 to the membrane and block VEGF-A expression
thereby decreasing the survival of HL60 cells.
METHODS
Cell Culture
HL60 leukemic cells were cultured in Iscove’s Modified Dulbecco’s Medium (IMDM) (Gibco BRL, Rockville,
MD) supplemented with 10% fetal calf serum (FCS), penicillin (100 U/ml), streptomycin (100 µg/ml), and Fungizone (0.25 µg/ml).
Simulated Microgravity Conditions
The RWV system consists of a rotator base, power supply, and a culturing vessel (Synthecon Inc., Houston, TX).
Simulated microgravity conditions were created using a
reusable 55 ml slow-turning lateral vessel (STLV). An air
pump draws air through a 0.22 µm filter and discharges it
into the vessel. Residual air was removed through a syringe
port. The STLV was seeded with 5 × 104 cells/ml in IMDM,
supplemented with 10% FCS and the antibiotics, and rotated at a speed of 8 rpm. As control for the experiment, a
static vented T75 flask was used with identical volume and
cell density, and low fluid shear stress was maintained by
avoiding movement of the flask, keeping it in a static 1g
condition. In addition, a motion control where the bioreactor is rotated (8 rpm) along an axis that is parallel to the
gravity vector was used as control and compared to the T75
flask control. However, a comparison of two gravity controls (T75 flask vs. motion control) yielded a statistically
insignificant 5.9% difference in the number of viable cells,
and the localization and expression of VEGFR-2 were not
affected (data not shown). Therefore, the T75 flask was
further used as the static control. Both the microgravity and
static cultures were then left in a 37◦ C, 5% CO2 humidified
environment for 48 h without replenishing the medium.
Growth Evaluation and Apoptosis
For growth evaluation, the rotating STLV was stopped
and the cells were immediately collected by aspiration,
centrifuged at 1200 rpm for 5 min, and cell viability was
determined by trypan blue exclusion after a 48-h incubation.
HL60 cells were collected and analyzed for the presence
of apoptotic cells by flow cytometry (Coulter Elite Flow
Cytometer, Beckman Coulter, Inc., Fullerton, CA) using
the ApoAlert Annexin V-fluorescein isothiocyanate (FITC)
propidium iodide (PI) Apoptosis Kit (Becton Dickinson,
Palo Alto, CA), following the manufacturer’s instructions.
Cell Cycle Analysis
Cell cycle determination was performed according to
Vindelhov’s technique12 as previously described.11 HL60
cells were cultured for 2 days, and the percentage of cells
in each cell cycle phase was determined by flow cytometry.
Immunofluorescent Staining of VEGFR-2
After culture for 2 days, HL60 cells were spun onto glass
microscope slides and VEGFR-2 expression was determined by immunofluorescent staining. Briefly, HL60 cells
were fixed in 3.7% (v/v) formaldehyde/phosphate-buffered
saline (PBS) and permeabilized with 90% methanol/PBS
(v/v). Then, HL60 cells were incubated with 1 µg/ml of
primary monoclonal antibodies (mAb) against VEGFR-2
(clone 1121, ImClone Systems, Inc., NY), then washed
and incubated with secondary FITC-conjugated antibodies (1/1000, Vector Laboratories, Burlingame, CA). The
samples were mounted in Vectashield containing 4 ,6Diamidino-2-Phenylindole (DAPI) to visualize the nuclei
and analyzed by fluorescence microscopy at 400× magnification (Olympus, NJ).
Flow Cytometry Determination of VEGFR-2
HL60 cells were collected following the 2-day culture,
centrifuged for 5 min at 1200 rpm, and cells were immediately fixed using 3.7% paraformaldehyde. Nonpermeabilized HL60 were incubated with the mAb for VEGFR2 or an unspecific, isotype-matched murine antibody as
a control, washed, and were subsequently incubated with
secondary FITC-conjugated antibodies. The total number
of VEGFR-2 on the cell surface was assessed by the determination of the mean of fluorescence intensity (MFI) as
compared to that determined under normal gravity using a
Coulter Elite Flow Cytometer.
Western Blot Analysis
Cells were lysed in radioimmune precipitation buffer
(50 mM Tris pH 7.5, 150 mM NaCl, 1% nonidet P-40,
0.1% sodium dodecyl sulfate, and 0.5% deoxycholate). Insoluble debris was removed, and the protein concentration
of the supernatant was determined by BCA protein assay
kit (Pierce Biotechnology). Cell lysates (100 µg) were
separated on 15% SDS–PAGE gels. The protein samples
were then transferred to nitrocellulose membrane. Protein
expression was confirmed by immunoblotting with antibodies raised against VEGF-A (Santa Cruz Biotechnology,
Santa Cruz, CA) or β-actin (Sigma, St. Louis, MO). After incubation with the appropriate primary antibodies and
horseradish peroxidase-conjugated secondary antibodies,
Simulated Microgravity Impairs Leukemic Cell Survival
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FIGURE 1. Simulated microgravity impairs cell proliferation and induces apoptosis. HL60 leukemic cells (5 × 104 cells/ml) were
seeded in the slow-turning lateral vessel corresponding to simulated microgravity condition or in regular cell culture flask for
static condition. (A) After a 2-day culture, cells were harvested, homogenized, and the number of viable cells was determined
by trypan blue exclusion. Results of three independent experiments are expressed as the mean of the number of viable cells ±
SEM(∗ P < 0.05, as compared with static condition, n = 3). (B) After a 2-day culture, cells were harvested and analyzed for the
presence of apoptotic cells. Results are shown as the percentage of viable cells (Annexin V− PI− ), early apoptotic cells (Annexin
V+ PI−) , late apoptotic cells (Annexin V+ PI+ ), and dead cells (Annexin V− PI+ ). Under static conditions, leukemic cells were mostly
alive, whereas the leukemic cells were prone to death under simulated microgravity. The data presented are representative of three
independent experiments. (PI = propidium iodide).
Results were statistically analyzed using a two-tailed
nonparametric Mann–Whitney test. The results are expressed as mean value ± standard error of the mean (SEM).
P < 0.05 was considered significant.
< 0.05). The number of HL60 cells after simulated microgravity decreased by 50 ± 6.63% as compared to that
obtained in the static condition (n = 3, P < 0.05). Flow
cytometry analysis of Annexin V and PI staining revealed
that the majority of the leukemic cells (97.1% of the total cells) were alive (Annexin V− PI− ) when they were
cultured under static conditions (Fig. 1B). However, under
simulated microgravity, 25.9% of HL60 underwent apoptosis (AnnexinV+ ) after the 2-day culture.
RESULTS
Simulated Microgravity Induces G1 Cell Cycle Arrest
the membranes were developed with enhanced chemiluminescent reagent (Amersham Biosciences, Piscataway, NJ).
Statistical Analysis
Simulated Microgravity Decreases Number of Viable Cells
After a 2-day culture in the RWV, simulated microgravity markedly decreased the number of viable cells (Fig. 1A,
4.26 ± 0.52-fold increase after simulated microgravity vs.
8.52 ± 0.70-fold increase after static condition, n = 3, P
Concomitant with induction of apoptosis under simulated microgravity, the reduced HL60 cell proliferation after
simulated microgravity was characterized by a slower rate
of exit from G1 phase and by a decrease of the percentage
of cells in S and G2/M phases of cell cycle as compared
with those in static conditions (Fig. 2, n = 3). In addition,
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VINCENT et al.
FIGURE 2. Simulated microgravity induces G1 cell cycle arrest. HL60 leukemic cells (5 × 104 cells/ml) were seeded in the slowturning lateral vessel corresponding to simulated microgravity condition or in a regular cell culture flask for static condition. After
a 2-day culture, cells were harvested and the percentage of cells in each phase of the cell cycle was determined by flow cytometry.
Note the augmentation of the percentage of cells in the sub-G0/G1 phase, and the decrease in the S and G2/M phases. The data
presented are representative of three independent experiments.
the apoptotic effect induced by simulated microgravity observed by the apoptosis assay was confirmed by the cell
cycle analysis, since 28.2% of the HL60 cells were in subG0/G1 phase of the cell cycle corresponding to apoptotic
cells (Fig. 2, n = 3).
Simulated Microgravity Impairs Activation of
VEGFR-2/VEGF-A Cell Signaling Pathway
Since VEGFR-2 activation and VEGF-A autocrine and
paracrine signaling pathways are sufficient to support the
proliferation and survival of HL60 cells, we assessed
whether simulated microgravity-induced cell death might
be due to dysregulation of membrane-bound localization
of VEGFR-2 and/or VEGF-A expression. In static conditions, VEGFR-2 is mainly present in the nuclei of proliferating HL60 cells, as demonstrated by immunofluorescent
costaining of nuclei with DAPI and VEGFR-2 using mAb of
permeabilized cells (Fig. 3A). On the other hand, VEGFR-2
is mainly observed as a membrane-bound localization after
simulated microgravity (Fig. 3A). In addition, flow cytometry detection of VEGFR-2 using mAb for VEGFR-2 on
nonpermeabilized HL60 cells showed that simulated microgravity strongly increased the total number of VEGFR-2 on
the cell surface assessed by the determination of the MFI
as compared to that determined under normal gravity (13
vs. 7.15, respectively, n = 3, P < 0.05) (Fig. 3B). Concomitant with membrane-bound localization of VEGFR-2,
VEGF-A expression was strongly downregulated under microgravity conditions as compared to the amount evidenced
in HL60 cells cultured under static control conditions
(Fig. 4). Western blot analysis of protein samples probed
with anti-β-actin antibody confirmed equal loading of the
samples.
DISCUSSION
Autocrine as well as paracrine activation loops of
VEGFR-2/VEGF-A signaling pathway in leukemic cells
have been shown to be essential signaling mechanisms
that support leukemic cell proliferation and malignancy.4,5
However, it is uncertain as to whether gravitational stresses
are involved in these autocrine/paracrine loops. In this
study, we investigated whether simulated microgravity
may alter autocrine/paracrine activation loops involved in
leukemic cell proliferation. Here, we have provided evidence that simulated microgravity decreases leukemic cell
growth and survival by interfering with VEGFR-2 localization and VEGF-A expression.
One possible explanation to understand how simulated
microgravity impairs cell proliferation might be the fact that
cell proliferation may be dependent on mechanical stresses
such as gravity. Under normal gravity via the conventional
cell culture method, cells are not suspended in the culture
media, and they can sense convection and sedimentation,
which may afford mitogenic signals necessary to induce
cell proliferation. In contrast, simulated microgravity decreases shear stress and randomized gravitational vectors,
which may impair gene expression and intercellular activation through paracrine and/or autocrine loops. However, the
mechanism by which mechanical signals due to simulated
microgravity are transduced and converted to biochemical
responses resulting in leukemic cell survival and growth
are not understood, and our study aimed to elucidate the
effect of simulated microgravity on VEGFR-2 intracrine
signaling.
VEGFR-2 activation occurs via binding to its ligand
VEGF-A that mediates HL60 cell proliferation through
autocrine signaling. However, functional VEGFR-2 was
recently found to be expressed not only on acute leukemia
Simulated Microgravity Impairs Leukemic Cell Survival
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FIGURE 3. Simulated microgravity induces membrane-bound VEGFR-2 expression. HL60 leukemic cells (5 × 104 cells/ml) were
seeded in the slow-turning lateral vessel corresponding to simulated microgravity condition or in a regular cell culture flask for
static condition. After a 2-day culture, cells were harvested and stained for VEGFR-2. (A) The leukemic cells were permeabilized
and subjected to immunofluorescent staining using mAb against VEGFR-2 (FITC) and DAPI to stain the DNA (nucleus). VEGFR2 expression was detected inside the leukemic cells (in green) and the DAPI staining (in blue) showed the nuclear expression
of VEGFR-2 under static conditions. In contrast, VEGFR-2 was mainly detected as a membrane-bound receptor after simulated
microgravity. Results were obtained from three independent experiments. Magnification: 400×. Scale bar = 10 µm. (B) The leukemic
cells were stained for VEGFR-2 and analyzed by flow cytometry. The total number of VEGFR-2 on the cell surface was assessed by
the determination of the mean of fluorescence intensity (MFI). VEGFR-2 expression was analyzed in three independent experiments.
cell surface but also predominantly in the nuclei of activated cells,9 suggesting that the pathophysiological role
of VEGFR-2 signaling may not be exclusively mediated by the conventional paradigm of membrane-bound
ligand–receptor interaction. The intracellular trafficking
and nuclear localization of VEGFR-2 in highly proliferating cells under static conditions suggest that VEGFR-2
intracrine signaling is a potent mediator of mitogenesis.
Once inside the nucleus, the potential activity of VEGFR2 may include recruitment of other signaling partners or
FIGURE 4. Simulated microgravity blocks VEGF-A expression. HL60 leukemic cells (5 × 104 cells/ml were seeded in the slow-turning
lateral vessel corresponding to simulated microgravity condition or in a regular cell culture flask for static condition. After a 2-day
culture, cells were harvested and VEGF-A exon in cell lysates was analyzed by western blot using anti-VEGF-bodies. Equal amount
of protein loading was assessed by immunoblot using β-actin antibodies.
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VINCENT et al.
functioning as a transcription factor itself. Remarkably,
this intracrine trafficking is reversed under simulated microgravity, and VEGFR-2 is confined only to the cell
membrane. In addition, simulated microgravity decreased
the expression of VEGF-A, which abrogates VEGF-A autocrine and paracrine VEGFR-2 stimulation absolutely necessary for leukemic cell survival. Since the growth of HL60
leukemic cells is dependent on both paracrine and autocrine
activation loops involving VEGFR-2/VEGF-A signaling
in which VEGFR-2 might traffic between the membrane
and the nucleus of highly proliferating cells,9 we provide
evidence that simulated microgravity alters VEGFR-2 intracrine trafficking through VEGFR-2 membrane-bound
stabilization, impairing leukemic cell survival and growth.
In summary, microgravity may be an important tool of
broad interest to elucidate behavior of cells in culture conditions. Our data provide evidence that the precise mechanism
for this intriguing regulation of VEGFR-2 localization after
simulated microgravity is mainly related to the interference
of VEGF-A autocrine and paracrine activation. The results
shown here demonstrate that simulated microgravity decreases cell proliferation of mitogenic cells. Manipulation
of the gravitational forces may provide a novel means to
identify the dynamic biomechanical pathways that are involved in the survival of motile and migratory cells such
as leukemic and stem cells. Furthermore, this study provides the impetus for manipulating biophysical parameters
involved in expansion of VEGFR-2 positive cells given that
one of the major hurdles in therapeutic organ regeneration is
the lack of adequate pluripotent cells such as VEGFR-2 positive stem and progenitor cells. Therefore, we can exploit
microgravity-induced manipulation of VEGFR-2 positive
cells as a platform for utilizing a larger number of cells
for generating tissue-engineered organs. Ongoing studies
are in progress to further characterize whether simulated
microgravity interferes with expression of genes involved
in cell proliferation and survival.
ACKNOWLEDGMENTS
The authors thank Zhenping Zhu and Daniel J. Hicklin
from ImClone Systems Inc. (New York) for providing relevant antibodies.
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