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Background and Purpose: Nitric oxide (NO) promotes angiogenesis by activating endothelial cells. Thalidomide arrests angiogenesis by interacting with the NO pathway, but its putative targets are not known. Here, we have attempted to identify these targets., Experimental Approach: Cell-based angiogenesis assays (wound healing of monolayers and tube formation in ECV304, EAhy926 and bovine arterial endothelial cells), along with ex vivo and in vivo angiogenesis assays, were used to explore interactions between thalidomide and NO. We also carried out in silico homology modelling and docking studies to elucidate possible molecular interactions of thalidomide and soluble guanylyl cyclase (sGC)., Key Results: Thalidomide inhibited pro-angiogenic functions in endothelial cell cultures, whereas 8-bromo-cGMP, sildenafil (a phosphodiesterase inhibitor) or a NO donor [sodium nitroprusside (SNP)] increased these functions. The inhibitory effects of thalidomide were reversed by adding 8-bromo-cGMP or sildenafil, but not by SNP. Immunoassays showed a concentration-dependent decrease of cGMP in endothelial cells with thalidomide, without affecting the expression level of sGC protein. These results suggested that thalidomide inhibited the activity of sGC. Molecular modelling and docking experiments revealed that thalidomide could interact with the catalytic domain of sGC, which would explain the inhibitory effects of thalidomide on NO-dependent angiogenesis., Conclusion and Implications: Our results showed that thalidomide interacted with sGC, suppressing cGMP levels in endothelial cells, thus exerting its anti-angiogenic effects. These results could lead to the formulation of thalidomide-based drugs to curb angiogenesis by targeting sGC.

Background: 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., Methods: 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: 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., Conclusion: Thalidomide exerts inhibitory effects on nitric oxide-mediated angiogenesis by altering sub-cellular actin polymerization pattern, which leads to inhibition of endothelial cell migration.