Thalidomide attenuates nitric oxide mediated angiogenesis by blocking migration of endothelial cells

Abstract

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.

Thalidomide‐induced teratogenesis: History and mechanisms

“…In the time‐sensitive window many organs/tissues are undergoing growth and maturation and could be affected in this manner, either directly or through loss of vessels, resulting in hypoxia and cell death. This work supports that of other studies which showed disruption of actin cytoskeleton dynamics in HUVEC cells following exposure to thalidomide (Tamilarasan et al, 2006) and an antiangiogenic analog of thalidomide, CPS49 (Therapontos et al, 2009), resulting in angiogenesis failure. Thalidomide binding to tubulin could prevent cell proliferation and migration, resulting in failed tissue morphogenesis, the severity of which could be time dependent depending on vascular and tissue maturation.…”

“…Early exposure resulted in severely truncated limbs, even Amelia, whereas later exposure caused less severe damage, for example loss of a digit or digit tips. In fact the damage caused by CPS49 in chicken embryos was consistent with damage seen in other studies using thalidomide (Somers, 1962; Boylen et al, 1963; Merker et al, 1988; Tamilarasan et al, 2006; Therapontos et al, 2009; Vargesson, 2009, 2013; Ema et al, 2010). …”

“…Other studies have also demonstrated that blood vessels are targeted and destroyed by thalidomide (D'Amato et al, 1994; Hallene et al, 2006; Tamilarasan et al, 2006; Siamwala et al, 2012; Vargesson, 2013). These include rodent and rabbit corneal assays where blood vessels induced in response to a FGF soaked bead are destroyed following thalidomide application (D'Amato et al, 1994; Kenyon et al, 1997).…”

“…Thalidomide inhibits nitric oxide expression resulting in vessel loss. Overexpression of nitric oxide can rescue and/or prevent thalidomide‐induced damage (Tamilarasan et al, 2006; Majumder et al, 2009; Siamwala et al, 2012). In zebrafish embryos, thalidomide causes blood vessel defects through VEGF receptor depletion (Yabu et al, 2005).…”

“…cGMP levels are reduced following thalidomide administration, which causes failed angiogenesis in HUVEC cell cultures (Tamilarasan et al, 2006). When cGMP levels were increased this prevented thalidomide induced antiangiogenesis.…”