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.