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From the Department of Neurophysiology, University of Cologne, Cologne, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The modes of action of thalidomide in embryopathy have been a matter of controversy for more than 30 years.4 Recently, it has been shown that thalidomide is an inhibitor of angiogenesis induced by basic fibroblast growth factor in rabbit5 and mouse6 cornea micropocket assays. In further studies thalidomide has been shown to generate reactive oxygen species (ROS), which may be responsible for the teratogenic effects of this compound.7,8 The present study was undertaken to evaluate whether ROS formation by thalidomide and the inhibition of angiogenesis are correlated. Furthermore, the anti-angiogenic properties of thalidomide have not yet been investigated in an embryonic tissue where both vasculogenesis, ie, the de novo formation of blood vessel structures from in situ differentiating endothelial progenitors (angioblasts), and angiogenesis, ie, sprouting of new capillaries from the preexisting network, occur. Previous studies on the anti-angiogenic effects of thalidomide did not offer any information on the molecular events that resulted in the impairment of blood vessel formation with its consequence of teratogenesis in the embryo.
In our experiments we used embryoid bodies, which were previously introduced by us as a novel in vitro model of anti-angiogenesis.9 By means of this in vitro model we evaluated the mechanisms of the anti-angiogenic capacity of thalidomide. Embryoid bodies are spheroidal three-dimensional embryonic tissues grown from pluripotent murine embryonic stem (ES) cells and have been shown to differentiate vessel-like structures,10,11 effectively improving the diffusion properties of the tissue.9 When cultivated in spinner flask technique, embryoid bodies attain a diameter of 1 to 2 mm.9 We investigated whether thalidomide inhibited endothelial cell growth and impaired the diffusion properties in this embryonic tissue. Because the teratogenicity of many xenobiotics, including thalidomide, is thought to depend at least in part on their bioactivation by embryonic cytochrome P-450, prostaglandin H synthase, and lipoxygenases to electrophilic and/or free radical intermediates, which in turn may oxidize DNA, proteins, and lipids,7,8 we evaluated the induction of ROS generation by thalidomide. We demonstrate that thalidomide exerts its anti-angiogenic effects by the generation of hydroxyl radicals, in that scavengers of hydroxyl radicals abolished the inhibitory effects of thalidomide on capillary structure formation in embryoid bodies.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The ES cell line CCE12 was grown on mitotically inactivated feeder layers of primary murine embryonic fibroblasts for a maximum of eight passages in Iscove’s medium (Life Technologies, Rockville, MD) supplemented with 20% heat-inactivated (56°C, 30 minutes) fetal calf serum (FCS) (Boehringer Mannheim, Mannheim, Germany), 2 mmol/L Glutamax, (Life Technologies), 100 µmol/L 2-mercaptoethanol (Sigma, Deisenhofen, Germany), 1% MEM non-essential amino acids stock solution, 100 IU/ml penicillin, and 100 µg/ml streptomycin (all from Life Technologies) in a humidified environment containing 5% CO2 at 37°C and passaged every 2 to 3 days. At day 0 of differentiation adherent cells were enzymatically dissociated using 0.2% trypsin and 0.05% EDTA in PBS (Life Technologies) and seeded at a density of 1·107 ml-1 cells in 250 ml siliconized spinner flasks (Live Technologies, Fernwald, Germany) containing 100 ml Iscove’s medium supplemented with the same additive as described above. After 24 hours, 150 ml medium was added to give a final volume of 250 ml. The spinner flask medium was stirred at 20 rpm using a stirrer system (Integra Biosciences, Fernwald, Germany). Every day, 125 ml of the medium was removed and replaced by fresh cell culture medium.
Treatment with Thalidomide, Thalidomide Analogues, and Hydroxyl Radical Scavengers
At day 3 of cell culture embryoid bodies were removed from spinner culture flasks. 20–30 embryoid bodies were subcultivated in liquid overlay culture for 5 days in bacteriological petri dishes (6.4 cm; Greiner, Solingen, Germany) containing 6 ml cell culture medium. Thalidomide ((±)-2-(2,6-Dioxo-3-piperidinyl)-1H-isoindole-1,3(2H)-dione), purchased from Biotrend (Cologne, Germany), and phthaloyl glutamic acid (PGA), purchased from Sigma, were dissolved in dimethyl formamide (DMF) and added to the cell culture medium in concentrations as indicated. Phthalimide (PI; Sigma) was dissolved in cell culture medium. Cell culture medium supplemented with the agents was completely changed every day. In experiments with hydroxyl radical scavengers either 10 mmol/L mannitol (Merck, Darmstadt, Germany) or 200 µmol/L 2-mercaptoethanol (Sigma, Deisenhofen, Germany) (final concentration 300 µmol/L) were coadministered with thalidomide. The control samples were treated with only the solvent DMF (final concentration 0.4%). Embryoid body diameters were determined every 24 hours.
Antibody Staining
The monoclonal antibody anti-mouse platelet endothelial cell adhesion molecule-1 (PECAM-1; CD31; Endogen, Woburn, MA) was used at a concentration of 5 µg/ml. Embryoid bodies were fixed in ice-cold methanol/acetone (7:3) for 60 minutes at -20°C, and washed with phosphate-buffered saline (PBS) containing 0.1% Triton X-100 (PBST; Sigma). Blocking against unspecific binding was performed for 60 minutes with 10% fat-free milk powder (Heirler, Radolfzell, Germany) dissolved in PBS. Embryoid bodies were subsequently incubated for 90 minutes at room temperature with PECAM-1 primary antibody dissolved in PBS supplemented with 10% milk powder. Embryoid bodies were then washed three times with PBST (0.01% Triton) and reincubated with a Cy5-conjugated rabbit anti-syrian hamster IgG (H + L; Dianova, Hamburg, Germany) at a concentration of 3.8 µg/ml in PBS containing 10% milk powder. After washing three times in PBST (0.01%) embryoid bodies were stored in PBS until inspection. For the excitation of the Cy5 fluorochrome the 633-nm band of a helium/neon laser of the confocal setup was used. Emission was recorded using a long-pass 655-nm filter set.
Confocal Laser Scanning Microscopy
Fluorescence recordings were performed by means of a confocal laser scanning setup (LSM 410, Zeiss, Jena, Germany) connected to an inverted microscope (Axiovert 135, Zeiss). The confocal setup was equipped with a 0.5 mW helium/neon laser, single excitation at 543 nm (excitation of doxorubicin) and a 5 mW helium/neon laser single excitation 633 nm (excitation of Cy5). Emission was recorded using long-pass filter sets LP570 and RG665, respectively. A 16x numerical aperture (N.A.) 0.5, oil immersion-corrected objective (Neofluar, Zeiss) was used.
Full frame images (512 x 512 pixels) were acquired from whole-mount embryoid bodies using the extended depth of focus algorithm of the confocal setup. In brief, 5 full frame images separated by a distance of 20 µm in the z-direction were recorded that included the information of the capillary area and spatial organization in a tissue slice 100 µm thick. From the acquired images an overlay image giving a three-dimensional projection of the vascular structures in the scanned tissue slice of 100 µm thickness was generated. The pixel intensity in optical sections of embryoid bodies treated only with the secondary antibody (background fluorescence) was separately evaluated and subtracted from the overlay image. The mean area (in mm2) of PECAM-1-positive cell structures was determined as the integrated sum of pixel values above background level in the overlay image and was calculated by the image analysis software of the confocal setup.
Diffusion Studies with Doxorubicin
For diffusion studies embryoid bodies were incubated at room temperature for 10, 30, and 60 minutes with 10 µmol/L doxorubicin (Sigma). They were subsequently washed and doxorubicin fluorescence was determined using the 543-nm line of a helium/neon laser of the confocal setup and the optical probe technique as previously described.13,14 In brief, optical sections with a thickness of 10 µm were performed from the periphery of embryoid bodies toward the center and doxorubicin fluorescence was determined in selected regions of interest (600 µm2, 40 x 40 pixels). The depth-intensity curves were fitted using a four-parameter exponential equation and a computer-based least squares fitting routine.13 For the determination of diffusion coefficients the doxorubicin distribution was evaluated with the optical sectioning routine of the confocal setup after 5, 20, and 30 minutes of incubation with doxorubicin. From the maximum diffusion distance x of doxorubicin after 30 minutes of incubation, diffusion coefficients were calculated according to the Einstein-Smoluchovski equation D = x2/2t where D is the diffusion coefficient, x is the maximum diffusion distance of doxorubicin from the embryoid body periphery, and t is the diffusion time of 30 minutes.
Determination of the Intracellular Redox State
The intracellular redox state was measured using the fluorescent dye 2'7'-dichlorodihydrofluorescein diacetate (H2DCF-DA; Molecular Probes, Eugene, OR), a nonpolar compound that is converted into a nonfluorescent polar derivative by cellular esterases after incorporation into cells. H2DCF is membrane impermeable and is rapidly oxidized to the highly fluorescent 2',7'-dichlorofluorescein (DCF) in the presence of intracellular ROS.15 For the experiments, embryoid bodies were incubated in E1 medium containing 135 mmol/L NaCl, 5.4 mmol/L KCl, 1.8 mmol/L CaCl2, 1 mmol/L MgCl2, 10 mmol/L glucose, and 10 mmol/L Hepes, pH 7.4. Subsequently they were incubated in 20 µmol/L H2DCF-DA for 50 minutes at room temperature. For fluorescence excitation, the 488-nm band of the argon ion laser of the confocal setup was used. Emission was recorded using a long-pass LP515 filter set.
Statistical Analysis
Data are given as mean values ± SE with n denoting the number of experiments performed with embryoid bodies from different spinner flasks. Student’s t-test for unpaired data was applied as appropriate. A value of P < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Thalidomide has been previously described as exerting
anti-angiogenic properties in the corneal micropocket
assay.5,6
To evaluate whether thalidomide likewise
displayed anti-angiogenic effects in the embryoid body model, embryoid
bodies were treated from day 3 to day 8 of development with either 10
or 100 µg/ml thalidomide. On day 8, angiogenesis progression was
evaluated by confocal laser scanning microscopy and
immunohistochemistry of PECAM-1 expression in five consecutive optical
sections of 20 µm thickness. This procedure yielded information on
the capillary area and spatial organization of capillaries in a tissue
slice of 100 µm thickness. As previously reported endothelial cell
differentiation was absent in 3-day-old embryoid bodies, whereas the
extension of the capillary area was maximal after 8 days of cell
culture (data not shown).9
Thalidomide significantly
reduced the extension of the capillary area from 0.10 ± 0.016
mm2
in the control to 0.026 ± 0.012
mm2
in the treated sample
(n = 10 embryoid bodies for each experimental
condition in five independent experiments; Figure 1, A and B
). When embryoid bodies were
treated with 10 µg/ml thalidomide, no significant effect on
endothelial cell differentiation was observed (data not shown).
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The anti-angiogenic effects of thalidomide may be due to the
generation of toxic ROS, which increase during long term
incubation with the compound. Moreover, ROS generation may be more
efficient with hydrolysis products of thalidomide. To evaluate this
issue, embryoid bodies were treated for either 4 or 24 hours with 10
and 100 µg/ml thalidomide, 10 and 100 µg/ml PGA, which is a
hydrolysis product of thalidomide, and 100 µg/ml PI, which is the
inactive major component of the thalidomide molecule. Subsequent ROS
generation was monitored using the fluorescent ROS indicator DCF. Our
data show that in control embryoid bodies ROS were continuously
generated, owing to the activity of a diphenylene iodonium
chloride-inhibitable NADPH-oxidase present in embryoid bodies (Sauer H,
Rahimi G, Hescheler J, Wartenberg M, unpublished observations).
However, the kinetics of H2DCF oxidation to
fluorescent DCF was significantly increased in thalidomide treated
embryoid bodies as compared to the untreated control
(n = 4 embryoid bodies for each experimental
condition in three independent experiments; Figure 2A
). An increase in ROS generation was
observed after a 4-hour incubation with 100 µg/ml thalidomide and 100
µg/ml PGA, which elevated DCF fluorescence to 200 ± 38%
(n = 3) and 211 ± 46%
(n = 4), respectively, of the untreated control.
Incubation with either 10 µg/ml thalidomide (n
= 4), 10 µg/ml PGA (n = 3) or 100 µg/ml PI
(n = 3) was without significant effect on ROS
generation (Figure 2B)
. An incubation for 24 hours with 100 µg/ml of
either thalidomide or PGA did not result in a further increase of ROS
formation. When 10 µg/ml of either thalidomide or PGA was
administered for 24 hours, ROS formation was increased to 131 ±
7.5% (n = 5) and 128 ± 20%
(n = 3), which was, however, not significantly
different from the untreated control. The nature of the ROS generated
by thalidomide was evaluated by coincubating embryoid bodies with 100
µg/ml thalidomide and either 10 mmol/L mannitol or 300 µmol/L
2-mercaptoethanol, both of which are known to be specific scavengers of
hydroxyl radicals.16,17
Figure 2C
shows that after
treatment of embryoid bodies for 4 hours with thalidomide the
intracellular redox state as indicated by DCF fluorescence was elevated
to 184 ± 23% of the control level (n = 4
embryoid bodies for each experimental condition in five independent
experiments). Coadministration of either mannitol or 2-mercaptoethanol
with thalidomide lowered the intracellular redox state to 120 ±
11% and 115 ± 8%, respectively, which was not statistically
significant from the control (set to 100%). Comparable results were
achieved when embryoid bodies were incubated with 100 µg/ml PGA in
the presence of either mannitol or 2-mercaptoethanol (not shown). From
these data we concluded that thalidomide specifically generated
hydroxyl radicals.
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If generation of hydroxyl radicals is the cause of the
anti-angiogenic effects of thalidomide, these effects should be absent
when ROS formation by thalidomide is inhibited by coadministration of
hydroxyl radical scavengers. We therefore incubated embryoid bodies
from day 3 to day 8 with 100 µg/ml thalidomide in the presence of
either 10 mmol/L mannitol or 300 µmol/L 2-mercaptoethanol, and
subsequently evaluated the capillary areas in embryoid bodies. We
observed that the anti-angiogenic effect of thalidomide was abolished
when hydroxyl radicals were scavenged during the incubation period. The
capillary area amounted to 0.08 ± 0.037 mm2
and 0.11 ± 0.06 mm2
in the samples treated
with mannitol and 2-mercaptoethanol, respectively, which is not
significantly different from control (0.10 ± 0.05
mm2; n = 10 embryoid bodies for
each experimental condition in three independent experiments; Figures 3 and 4
).
Incubation with radical scavengers in the absence of thalidomide did
not exert significant effects on capillary structure formation
(n = 10 embryoid bodies for each experimental
condition in four independent experiments). Therefore, we concluded
that the anti-angiogenic effects of thalidomide were due to the
generation of highly reactive hydroxyl radicals. To evaluate the
specificity of thalidomide on the inhibition of angiogenesis and to
exclude that the agents applied in the present study exerted cytotoxic
effects, the growth kinetics of embryoid bodies was followed during the
time of thalidomide treatment (n 
20 embryoid
bodies for each experimental condition). Neither thalidomide nor the
applied hydroxyl radical scavengers, either alone or in
coadministration with thalidomide, exerted any significant effects on
the growth kinetics of embryoid bodies, indicating that thalidomide may
act specifically on vasculogenesis and angiogenesis in embryoid bodies
(Figure 5)
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demonstrates that the diffusion
coefficient D amounted to 9.12 · 10-8 ± 1.5
· 10-8 cm2
s-1 in the control (treated only with the
solvent DMF). Thalidomide significantly reduced D to 1.37 ·
10-8 ± 0.19 · 10-8
cm2
s-1, which indicates
that inhibition of capillary formation impaired the diffusion
properties of the tissue. In the presence of mannitol and
2-mercaptoethanol the inhibitory effect of thalidomide on the tissue
diffusion properties was significantly reversed. D amounted to 7.5 ·
10-8 ± 1.7 · 10-8
cm2
s-1 for mannitol and
and 6.5 · 10-8 ± 1.7 ·
10-8 cm2
s-1 for 2-mercaptoethanol, which was not
significantly different from control (n = 4
embryoid bodies for each experimental condition in three independent
experiments). From these data we concluded that the anti-angiogenic
effects of thalidomide could be reversed by hydroxyl radical
scavengers. Hydroxyl radical scavengers restored not only the extension
of the capillary area but also the functionality of the capillaries
developed in embryoid bodies.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study thalidomide in a concentration of 100 µg/ml significantly inhibited blood vessel development in embryoid bodies, when the compound was administered during the period of vasculogenesis and angiogenesis which has been previously shown to occur between day 4 and day 8 of embryoid body development.9 However, 10 µg/ml of the compound did not exert significant effects on the formation of capillary structures. The relatively high concentrations of thalidomide that were necessary to achieve the anti-angiogenic effect may be due to species-dependent differences in the susceptibility toward thalidomide teratogenicity. This assumption is underscored by the recent observations of Parman and colleagues8 of a lack of DNA oxidation by thalidomide in mice. Furthermore, previous studies have demonstrated an absence of susceptibility of rodents to orally administered concentrations of thalidomide that are teratogenic in humans.19,20
The inhibition of blood vessel formation had the consequence of impaired diffusion properties of the tissue as evaluated by determination of the diffusion constant D of the fluorescent anthracycline doxorubicin using the recently developed optical probe technique.13,14 The impaired diffusion properties of embryonic tissue as a consequence of limited blood supply may result in the limb defects seen in children after thalidomide treatment of their pregnant mothers. It has been previously mentioned that the limb bud is a particularly vulnerable target to a teratogen that inhibited endothelial cell function.5 Besides its effect on endothelial cell differentiation, thalidomide did not exert any overall toxicity in the embryoid body assay, in that embryoid body growth was not impaired after thalidomide treatment. From this we concluded that the antiproliferative effect of thalidomide appeared to be restricted to endothelial cells. The low toxicity of thalidomide has been known for long time and was one of the reasons to recommend this drug for the treatment of indisposition of pregnant women.4
The experiments of the present study demonstrate that thalidomide exerts its anti-angiogenic properties via the generation of ROS. Interestingly, prolonged incubation (24 hours) with 100 µg/ml thalidomide and PGA did not result in an accumulation of ROS, as compared to an incubation for 4 hours, indicating the short lifetimes of ROS and the effective antioxidant defense systems present in embryoid bodies. Furthermore, PGA did not form ROS more efficiently than thalidomide, excluding the possibility that ROS are generated exclusively by hydrolysis products of thalidomide. A slight but not significant increase in ROS was achieved after a 24-hour incubation with 10µg/ml thalidomide and PGA, indicating that lower concentrations of the compounds may also be bioactive.
The toxicity of ROS on endothelial cells has been extensively investigated in recent years, because oxidative damage of blood vessels is a frequent event during reoxygenation after hypoxia. Moreover, endothelial cell injury is observed during acute inflammatory processes when activated neutrophils generate reactive oxygen metabolites within the target cell.21 Because embryoid bodies constitutively generate ROS by an enzyme closely related to the respiratory burst nicotinamide adenine dinucleotide phosphate (NADPH) oxidase of neutrophils (Sauer H, Rahimi G, Hescheler J, Wartenburg M, unpublished observations), it can be ruled out that low levels of ROS per se impair vasculogenesis and angiogenesis in the embryoid body model.
Our data indicate that the reactive oxygen intermediate generated by thalidomide is the hydroxyl radical. Mannitol and 2-mercaptoethanol, which specifically scavenge hydroxyl radicals, abolished the increased DCF fluorescence observed after thalidomide treatment. Likewise, the anti-angiogenic effect of thalidomide was absent, and capillary formation apparently not different from the untreated control was observed when thalidomide was administered in the presence of hydroxyl radical scavengers. This was further corroborated by diffusion studies, which demonstrated that in the presence of hydroxyl radical scavengers the diffusion properties of the embryoid body tissue were restored to levels not significantly different from control. From these data we concluded that hydroxyl radical scavengers reestablished both the extension of the capillary network differentiated in embryoid bodies and the functionality of the capillaries.
ROS formation, including hydroxyl radicals, by thalidomide has been recently demonstrated and was counteracted by antioxidants and inhibitors of lipoxygenases and prostaglandin H synthase,22 indicating that these peroxidases may bioactivate the compound. Highly reactive hydroxyl radicals may induce DNA oxidation in either endothelial cell precursors or endothelial cells during vasculogenesis and angiogenesis in the embryo. This notion is supported by recent experiments that demonstrate that phenytoin and structurally related drugs including thalidomide initiated the oxidation of DNA in embryonic tissues.8,22 Phenytoin has been previously demonstrated to cause phalangeal hypoplasia in rabbit fetuses by a mechanism that is preceded by ischemia and vascular disruption.23,24
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Acknowledgements |
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Footnotes |
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Accepted for publication September 15, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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