027Blocking/Knocking out Smad3 Alleviates Doxorubicin Effects on Endothelial Cells

Recent studies have shown that patients with early-stage cancers are now more likely to die from complications of anticancer therapy than cancer. Doxorubicin (Dox), a drug used to treat numerous cancers, is known to cause delayed cardiomyopathy. We have previously reported reduced microvascular density and increased levels of TGFbeta related transcripts in Dox treated mouse hearts. We hypothesized that microvascular remodeling in Dox treated hearts is mediated by enhanced TGFbeta/Smad3 signaling. We observed the inhibition of human umbilical vein endothelial cell (HUVEC) proliferation and suppression of the formation of vessel-like structures by Dox. TGFbeta pathway inhibitor SB431542 (SB), which selectively blocks Smad2/3 signaling, alleviated the effects of Dox in vitro. In aortic ring experiments, Dox decreased the number of sprouts in aortas from wild type mice while aortic preparations from Smad3 knockout animals were more resistant to the deleterious effect of Dox and produced increased numbers of sprouts. In experiments with co-cultures of cardiac fibroblasts and HUVEC, we identified the detachment of endothelial cells as an additional putative mechanism of microvascular remodeling by Dox. Live imaging of co-cultures treated with Dox in the presence of caspase-3/7 substrate revealed that detaching endothelial cells were not undergoing apoptosis prior to detachment. SB prevented detachment and increased the number of endothelial cells in co-cultures treated with Dox. Treatment with Dox led to the reduced adhesion of endothelial cells to laminin, a major constituent of the endothelial basement membrane while SB improved adhesion of Dox treated cells to laminin. In conclusion, Dox causes inhibition of sprouting angiogenesis in aortic rings and detachment of endothelial cells in an in vitro co-culture model of formation of vessel-like structures. These deleterious processes appear to be mediated by TGFbeta/Smad3 signaling as they were alleviated by both a pathway inhibitor and a genetic Smad3 knockout.