Response to hypoxia involves transforming growth factor-beta 2 and Smad proteins in human endothelial cells

Oxygen deprivation (hypoxia) is a consistent component of ischemia that ind uces an inflammatory and prothrombotic response in the endothelium. In this report, it is demonstrated that exposure of endothelial cells to hypoxia ( 1% O-2) increases messenger RNA and protein levels of transforming growth f actor-beta2 (TGF-beta2), a cytokine with potent regulatory effects on vascu lar inflammatory responses. Messenger RNA levels of the TGF-beta2 type II m embrane receptor, which is a serine threonine kinase, also increased. The s timulatory effect of hypoxia was found to occur at the level of transcripti on of the TGF-beta2 gene and involves Smad proteins, a class of intracellul ar signaling proteins that mediates the downstream effects of TGF-beta rece ptors. Transient transfection studies showed that the region spanning -77 a nd -40 base pairs within the TGF-beta2 promoter (harboring a Smad-binding " CAGA box") is activated in hypoxic cells compared with nonhypoxic controls (P < .01). Hypoxia also stimulated transcription from another promoter, 3TP -Lux, a reporter construct responsive to Smads and TGF-<beta>. In addition, specific binding to a Smad-binding oligonucleotide was observed with nucle ar extracts from hypoxic endothelial cells but not from nonhypoxic cells. I t is concluded that Smad proteins, which can regulate endothelial responses to mechanical and inflammatory stress, also may play an important role in vascular responses to hypoxia and ischemia. (Blood. 2001;98:3324-3331) (C) 2001 by The American Society of Hematology.