Thalidomide, a sedative drug given to pregnant women, unfortunately caused limb deformities in thousands of babies. Recently the drug was revived because of its therapeutic potential; however the search is still ongoing for an antidote against thalidomide induced limb deformities. In the current study we found that nitric oxide (NO) rescues thalidomide affected chick (Gallus gallus) and zebrafish (Danio rerio) embryos. This study confirms that NO reduced the number of thalidomide mediated limb deformities by 94% and 80% in chick and zebrafish embryos respectively. NO prevents limb deformities by promoting angiogenesis, reducing oxidative stress and inactivating caspase-3 dependent apoptosis. We conclude that NO secures angiogenesis in the thalidomide treated embryos to protect them from deformities.

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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 concentrationdependent 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.

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Cadmium, a ubiquitous heavy metal, interferes with endothelial functions and angiogenesis. Bradykinin is a Ca-mobilizing soluble peptide that acts via nitric oxide to promote vasodilation and capillary permeability. The objective of the present study was to explore the Cd implications in bradykinin-dependent endothelial functions. An egg yolk angiogenesis model was employed to evaluate the effect of Cd on bradykinin-induced angiogenesis. The results demonstrate that 100 nmol/L Cd attenuated bradykinin-dependent angiogenesis. The results of the in vitro wound healing and tube formation assays by using EAhy 926, a transformed endothelial cell line, suggest that Cd blocked bradykinin-mediated endothelial migration and tube formation by 38% and 67%, respectively, while nitric oxide supplementation could reverse the effect of Cd on bradykinin-induced endothelial migration by 94%. The detection of nitric oxide by using a DAF-2DA fluorescent probe, Griess assay, and ultrasensitive electrode suggests that Cd blocked bradykinin-induced nitric oxide production. Fluorescence imaging of eNOS-GFP transfected endothelial cells, immunofluorescence, and Western blot studies of Cd and bradykinin-treated cells show that Cd interfered with the localization pattern of eNOS, which possibly attenuates nitric oxide production in part. Additionally, Ca imaging of Cd- and bradykinin-treated cells suggests that Cd blocked bradykinin-dependent Ca influx into the cells, thus partially blocking Ca-dependent nitric oxide production in endothelial cells. The results of this study conclude that Cd blunted the effect of bradykinin by interfering with the Ca-associated NOS activity specifically by impeding subcellular trafficking of eNOS.