Thalidomide attenuates nitric oxide mediated angiogenesis by blocking migration of endothelial cells
- KP Tamilarasan†1,
- Gopi Krishna Kolluru†1,
- Megha Rajaram1,
- M Indhumathy1, 2,
- R Saranya1, 2 and
- Suvro Chatterjee1Email author
© Tamilarasan et al; licensee BioMed Central Ltd. 2006
Received: 08 September 2005
Accepted: 04 April 2006
Published: 04 April 2006
Thalidomide is an immunomodulatory agent, which arrests angiogenesis. The mechanism of anti-angiogenic activity of thalidomide is not fully understood. As nitric oxide is involved in angiogenesis, we speculate a cross-talk between thalidomide and nitric oxide signaling pathway to define angiogenesis. The aim of present study is to understand the mechanistic aspects of thalidomide-mediated attenuation of angiogenesis induced by nitric oxide at the cellular level.
To study the cellular mechanism of thalidomide-mediated blocking of angiogenesis triggered by nitric oxide, we used two endothelial cell based models: 1) wound healing and 2) tube formation using ECV 304, an endothelial cell line. These cell-based models reflect pro-angiogenic events in vivo. We also studied the effects of thalidomide on nitric oxide mediated egg yolk angiogenesis. Thalidomide could block the formation of blood vessels both in absence and presence of nitric oxide. Thalidomide effects on migration of, and actin polymerization in, ECV 304 cells were studied at the single cell level using live cell imaging techniques and probes to detect nitric oxide.
Results demonstrate that thalidomide blocks nitric oxide-mediated angiogenesis in egg yolk model and also reduces the number of tubes formed in endothelial cell monolayers. We also observed that thalidomide arrests wound healing in presence and absence of nitric oxide in a dose-dependent fashion. Additionally, thalidomide promotes actin polymerization and antagonizes the formation of membrane extensions triggered by nitric oxide in endothelial cells. Experiments targeting single tube structure with thalidomide, followed by nitric oxide treatment, show that the tube structures are insensitive to thalidomide and nitric oxide. These observations suggest that thalidomide interferes with nitric oxide-induced migration of endothelial cells at the initial phase of angiogenesis before cells co-ordinate themselves to form organized tubes in endothelial cells and thereby inhibits angiogenesis.
Thalidomide exerts inhibitory effects on nitric oxide-mediated angiogenesis by altering sub-cellular actin polymerization pattern, which leads to inhibition of endothelial cell migration.
Thalidomide, α-(N-phthalimido) glutarimide is an immunomodulatory agent, which is used as a drug to treat multiple myeloma and other types of cancers . The drug thalidomide, first synthesized in 1954 [2, 3], was widely prescribed to treat morning sickness in pregnant women in the early 60s. However, thalidomide became anathema when it was found to be seriously teratogenic having caused serious birth defects in more than 10,000 newborns. It was subsequently banned in Europe. As scientists probed further into the causes for teratogenicity, they realized the innate potency of this drug to treat other diseases . Now, a quarter of a century later, it appears that it may be a miracle drug for such diseases as cancer, AIDS and SLE. In August 1998, Food and Drug Administration (FDA) approved Thalidomide for sale in the USA for chronic treatment of erythema nodosum leprosum (ENL), a painful inflammatory dermatological reaction of lepromatous leprosy .
Thalidomide has anti-angiogenic properties that are independent of its immunomodulatory effects [5, 6]. Its involvement in angiogenesis is being studied extensively as angiogenesis is one of the most important developments in metastazing tumors. Thalidomide has been shown to inhibit the expression of vascular endothelial growth factor (VEGF) and IL-6, which is believed to be the mechanism for the attenuation of angiogenesis by thalidomide 
Nitric oxide (NO), produced by the enzyme nitric oxide synthase (NOS), an important second messenger in many signaling pathways, is a potent vasodilator . NO is produced by the action of NOS in the transformation of arginine to citrulline and activates soluble guanylate cyclase to produce cGMP . NO is involved in angiogenesis and endothelial cells (EC) migration . Recent publications suggest that endothelial-derived NO is required for Ang1-induced angiogenesis and that the PI3-kinase signaling mediates the activation of eNOS and NO release in response to Ang1 . NO is also instrumental in promoting Ang1-induced angiogenesis in combination with HSP90 and Akt in coronary artery endothelium .
As thalidomide interferes with angiogenesis, a process in which NO also plays a crucial role, we speculate a cross talk between thalidomide and NO signaling pathway. Currently, the involvement and interaction of thalidomide and the NO signaling pathway is not known. The mechanistic aspects of thalidomide action need to be elucidated further. The aim of present work was to study the mechanistic aspects of the attenuation of NO-driven angiogenesis due to thalidomide at the cellular level. Our study indicates that thalidomide attenuates NO-driven angiogenesis by blocking migration of ECs even before any tube structure had been formed, which further hints an interaction between thalidomide and NO signaling.
Dulbecco's modified Eagle's medium (DMEM), from Hi-Media, Mumbai, India. Fetal bovine serum (FBS) was from Invitrogen Life technologies (Gaithersburg, MD). Thalidomide was purchased from Sigma Chemical Co (St. Louis, MO), and phalloidin Alexa Fluor 568 (phalloidin) from Molecular Probes (Portland, OR, USA). All other chemicals were at least of the reagent grade.
Human umbilical vein endothelial cells (ECV 304) were cultured in DMEM supplemented with 10 % FBS (v/v) and 1% penicillin (w/v) and streptomycin (w/v).
Endothelial tube formation assay
ECV 304 cells were seeded on collagen (collagen type I) plated 12-well plates with 60% cell density. After 7 hours of incubation, 500 μmol of sodium nitroprusside (SNP), a NO donor, was added to the cells. After another 17 hour period, cells were treated with thalidomide at different concentrations (50, 100 and 150 μg/ml) and incubated for 8 hours. All the incubations were performed in an incubator at 37°C and under 5% CO2 95% air. The number of tubes was counted under bright field phase contrast microscope. Only the complete ring structures created by 3 to 5 ECs were counted as tubes.
Single cell migration assay
Cell migration was assessed by a wound healing method . One million ECV 304 cells in 2 ml of DMEM/10% FBS were seeded in a 35 mm dish. Twenty-four hours later, when the cells reached confluence, a linear wound was made by scratching the monolayer with a 1 mm wide sterile plastic scraper. As per the experimental protocol described elsewhere , cells were washed with PBS, treated with SNP (500 μmol) and incubated for a fixed time period (8 hours unless otherwise stated) with and without thalidomide at different concentrations. Light microscopy images were taken with 10× and 40× magnifications.
Boyden's chamber based migration assay
Trypsinized ECV 304 cells were used for migration assay using Boyden's chamber, which is a two-chamber system. The upper and lower chambers are separated by a collagen coated 8-μm pore size polycarbonate membranes. ECV 304 cells were loaded in the upper well with thalidomide alone or thalidomide plus SNP. Lower well was filled with DMEM. The chambers were then incubated at 37°C, 5% CO2 for 3 hours. Cells were migrated across the membrane and stuck to the lower part of the membrane. After the incubation, the polycarbonate membrane was fixed and stained with propidium iodide, a fluorescent nuclear probe. Endothelial cell migration activity was quantified as the number of migrated cells on the lower surface of the membrane. Cell number was counted before and after experiments to quantify the proliferation status of the loaded cells in Boyden's chamber.
Egg yolk angiogenesis assay
Fourth day incubated eggs were collected from the Poultry Research Station, Nandanam, Chennai. Eggs were broken and gently plated on a cellophane bed in Petri dishes under sterile conditions. Egg yolks were incubated with 500 μmol SNP on a filter paper disc for 6 hours. Thalidomide discs (25, 50,75 μg/ml) were then placed on the egg yolks and were incubated for another 6 hours. Images were taken using a Kodak digital camera at 0, 6 and 12 hours of incubation. Quantification of angiogenesis was performed by using Scion Image, Release Alpha 4.0 3.2 and Adobe Photoshop version 6.0.
ECV 304 cells were cultured on cover glasses in 12-well plates till they reach 40% confluency before starting the experiments. ECV 304 cells were incubated with 500 μmol SNP for 15 minutes. Next, thalidomide was added at different concentrations (25, 50, 75 μg/ml) to the cells. After 15 minutes of incubation at 37°C, the cover slips were washed gently with PBS and cells were fixed in 2% paraformaldehyde for 7 minutes, permeabilized with 0.1% Triton X -100 for 2 minutes and incubated with phalloidin alexa fluor 568 (phalloidin) (0.5 μM final concentration) for 1 hour. The fluorescence of phalloidin bound to F-actin was viewed under NIKON TE2000-U fluorescent microscope at 560 nm emission. Photographs were taken with an Andor CCD camera attached to the microscope.
Data is presented as mean + SE. Data was analyzed using paired and unpaired Student's t-tests as appropriate. A P value smaller or equal to 0.05 was selected as a criterion for a statistically significant difference.
Thalidomide blocks angiogenesis
Thalidomide attenuates tube formation in EC monolayer
Thalidomide arrests wound healing in EC monolayer
Thalidomide induces cytoskeletal rearrangements in ECs
Thalidomide attenuates NO-mediated angiogenesis in egg yolk models and tube formation in EC monolayers
Thalidomide arrests NO mediated wound healing in ECs
Thalidomide antagonizes the formation of SNP induced lamellipodia and filopodia
The known anti-angiogenic effects of thalidomide might be attributed to its anti-migratory effects [20, 21]. Because no detailed study has been performed so far to understand the anti-migratory effects of thalidomide at the cellular level, we studied the pattern of actin polymerization in ECs treated with SNP and SNP plus thalidomide. SNP induced the formation of filopodia and lamellipodia in EC membrane while subsequent treatments with thalidomide (25, 50 and 75 μg/ml) either inhibited the formation or reduced the number of filopodia and lamellipodia on the EC surface (data not shown).
Thalidomide arrests SNP induced migration of ECs
Thalidomide reduces sub-population of the SNP treated cells with highermembrane actin polymerization pattern
In 1994, an ophthalmologist at Harvard Medical School discovered that thalidomide inhibits angiogenesis when tissues become deficient in oxygen . The specific mechanism by which thalidomide inhibits angiogenesis is, as of yet, not known . In our present work, we tested the anti-angiogenic potential of thalidomide by using egg yolk vascular bed. Results indicate that thalidomide blocks growth of the blood vessels at the periphery of the vascular bed (figure 1). We speculate that thalidomide affect the EC migration to attenuate angiogenesis at the cellular level. Thalidomide is also involved in the process of inflammation [24, 25, and 26]. Inflammation and cellular migration are two indispensable cellular mechanisms that complement each other . Through experimentation in rabbits and in doing case studies on people being treated with thalidomide, researchers determined that thalidomide modulates the production of the inflammatory cytokine, TNF . TNF-alpha is a cytokine produced by immune cells in the blood stream that acts as pro-angiogenic factor . Thalidomide inhibits TNF-alpha by amplifying the degradation of messenger RNA (mRNA), and decreases the production of interleukin-12, which is involved in immunity responses, the stimulation of inflammation, and suppression of certain cytokines [29–31]. From these observations we delineate an association between cellular migration and thalidomide actions in ECs.
Our present work documents that thalidomide attenuates basal and NO-mediated EC migration in a dose-dependent manner (figure 3, 4, 9). Hence, in addition to NO signaling thalidomide appears to affect the migratory pattern of ECs by interfering other signaling pathway/s as well. Contrary to our assumption, single tube structure in the monolayer of ECs was resistant to thalidomide and SNP treatment in short-term experimental models (figure 10). We can speculate that the level of maturity of tubes is important for the effects of SNP and thalidomide as our results showed that nascent tubes were resistant to the effects of thalidomide (figure 10). Experiments in which phalloidin labeling of actin was performed indicate that thalidomide inhibits actin polymerization at the cell-cell interface (figure 5 and 6) and sensitizes cells to extend cell surface protrusions before completing a tube structure (upper panel, figure 10). Therefore, it is a plausible hypothesis that significant re-arrangements of actin cytoskeleton pattern during tube formation triggers membrane changes in ECs to protect the prima facie tube structures from the effects of excess growth factors and autacoids such as NO. This observation also supports the concept that thalidomide restricts blood vessel formation by inhibiting cellular proliferation and migration at the single cell level before they organize into a tube or ring like structure.
Dynamic rearrangement of the cytoskeleton is the key to migration of the cells . Signaling pathways attributed to the migration of cells form a complex network of small GTPase-driven local networks that ultimately converge into the controlling nodes for actin polymerization pattern in the down-stream events . Signaling pathways involving small GTPases and down stream activators such as ROCK, cdc42, MLCK tune the actin polymerization pattern in the cell membrane to define filopodia, a parallel arrangements of F-actins and lamellipodia, a diagonal arrangements of F-actins . Dynamics of sub-cellular actin polymerization defines the cell surface extensions such as filopodia and lamellipodia structures, which are crucial to cellular migration . With respect to actin polymerization, VEGF-treatment leads to rapid phosphorylation of actin, depolymerization factor cofilin, and its upstream regulator, LIM-kinase (LIMK). Pharmacological inhibitors of phosphoinositide-3 kinase (PI3-K) and the rho-activated kinase (ROCK) attenuate VEGF-induced LIMK phosphorylation that indicates a role for (PI3-K) and ROCK in the signaling pathways leading to regulation of LIMK activity . We observed an altered phalloidin staining pattern, which represents the actin polymerization status of thalidomide treated ECs (figure 5 and 6). The number of lamellipodia was reduced significantly in thalidomide-treated cells while a strong pattern of central microfilament and membrane dense actin plates appeared in the cytosol and membrane respectively (figure 5). With respect to sub-cellular actin polymerization, a recent publication suggests that the sub-cellular localization of actin-related protein 2/3 (Arp2/3) complex, a prime actin polymerization nucleator, is crucial to the migration of the cells . These observations prompt us to speculate that thalidomide signals to downstream effectors to impair migratory mechanisms of ECs by changing the actin polymerization pattern during the initial phase of angiogenesis by blocking individual cells to organize a tube like structure.
Does thalidomide cross-talk with NO downstream signaling pathway to interfere with cellular migration? To check the role of thalidomide in NO driven endothelial rearrangements we delivered NO via SNP prior to thalidomide treatments of the cells. We found that thalidomide neutralizes NO induced changes in the cytoskeleton of ECs (data not shown). This prima facie observation supports our hypothesis that thalidomide may cross talk with NO signaling pathway. Bioavailability and functions of NO are critically controlled by the redox status of the system . Reports suggest that thalidomide enhances superoxide anion release from human polymorphonuclear and mononuclear leukocytes . Therefore, relationship of NO and thalidomide with other oxygen species such as peroxynitrite, hydrogen peroxide, superoxide radicals and nitrite oxide would determine the nature of cross talk between thalidomide and NO down stream signaling. Recent reports claim that thalidomide exhibits NOS-inhibitory activity . Although the results of our study support the thought of an interplay between thalidomide and NO downstream signaling pathway, it is possible that thalidomide exerts its anti-migratory effects, at least in part, by inhibiting NOS at the NO upstream events. The possible explanation is that inactivation of NOS by thalidomide is associated with the re-localization of NOS due to cytoskeletal rearrangements. Reorientation of actin-based infra-structure could place NOS in discrete membrane compartments or cytoplasmic regions causing insufficient access to other signaling proteins, which are responsible for NOS activation. There is an inverse relationship between the concentration of G-actin and eNOS expression in endothelial cells subjected to pharmacological alteration of their cytoskeleton . The increase in eNOS activity caused by G-actin was much higher than that caused by F-actin . However, our results document a higher degree of polymerization of actin in thalidomide-treated cells, which lead to expanded cell surface and perimeter as well (Figure 12). We speculate a possible mechanism of thalidomide mediated blocking of NO driven endothelial functions, which attributes, at least in part, to the thalidomide associated cytoskeleton rearrangement causing NOS inactivation. Hence, results of our present work strongly indicate that thalidomide blocks NO driven endothelial functions by interfering with the actin polymerization pattern of ECs. We deem that thalidomide attenuates NO driven endothelial functions, in part, in ECs by interplaying with the NO down stream signaling molecules.
Present work evidences a novel cellular mechanism of thalidomide mediated endothelial dysfunction, which attributes to the altered pattern of EC migration. Further investigation into the cause of thalidomide-mediated disruption in NO-induced EC migration shows that thalidomide induces dose-dependent actin polymerization in EC in the early phase of angiogenesis, which might be the basic mechanism of the attenuation of angiogenesis. An extended study aiming at the dissection of biochemical signaling pathway, which will unravel the cross talk between thalidomide and NO down stream signaling would help to understand further the thalidomide actions in endothelium, an area of immense clinical importance.
Acquired Immunodeficiency syndrome
Actin related protein
Cyclic guanosine monophosphate
Dulbecco's modified Eagle's medium
erythema nodosum leprosum
Endothelial Nitric oxide synthase
Fetal Bovine serum.
Food and Drug Administration
Heat shock Protein 90
Myosin Light Chain Kinase
Murine Tumor Necrosis Factor-alpha
nitric oxide synthase
Phosphate buffer Saline.
Systemic lupus erythematosus
Tumor Necrosis Factor
Vascular Endothelial Growth Factor
The authors wish to thank Dr. Eve de Lamirande for her insight reading and assistance in preparation of the manuscript. The authors also acknowledge Ms. S. Geetha Priya and Mr. Ajit Muley for their assistance in preparation of the manuscript. This work was partially supported by Wellcome Trust Grant (#070069).
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