Apoptosis in vascular endothelial cells caused by serum deprivation, oxidative stress and transforming growth factor-beta

Vascular endothelial cell apoptosis has previously been shown to play a rol e in the pathogenesis of hypertension-induced vessel deletion and damage. I n the present in vitro study we analyse several possible relevant causative factors of vascular endothelial cell apoptosis, namely, serum deprivation and nutrient depletion, oxidative stress in the forms of hypoxia, hyperoxia or free radical damage, and altered levels of transforming growth factor-b eta 1 (TGF-beta 1) protein. An established cell line, bovine aortic endothe lial cells (BAEC), was maintained in complete growth medium (RPMI-1640 plus 15% fetal calf serum and antibiotics, abbreviated as RPMI) in 25cm(2) flas ks or in 12-well plates on glass coverslips. Confluent but actively-growing cultures were treated with either hypoxia (PO2 of RPMI = 50mmHg), serum-fr ee media (SFM), SFM plus hypoxia, hyperoxia (PO2 of RPMI = 450mmHg), hydrog en peroxide (H2O2, 1 mM) in SFM, or TGF-beta 1 protein (10ng/mL) in SFM. Ap propriate control Cultures were used. BAEC were collected 48h or 72h after all treatments except for TGF-beta 1 and H2O2 treatments that were collecte d at 16-18h. Cell death was assessed using morphological characteristics or in situ end labeling (ISEL), cell proliferation assessed using proliferati ng cell nuclear antigen (PCNA), and TGF-beta 1 expression assessed using tr anscript levels or immunohistochemistry. All treatments significantly incre ased levels of apoptosis over control cultures (P<0.05), and decreased leve ls of cell proliferation. Treatment with TGF-beta 1 protein or SFM plus hyp oxia induced greatest levels of apoptosis. TGF-beta 1 protein and transcrip t levels were decreased in treated cultures, results suggesting that a para crine source of TGF-beta 1 protein would be needed as a cause of endothelia l cell apoptosis in vivo. Future therapies against inappropriate vessel del etion in disease states may use the known gene-driven nature of apoptosis t o modify this sort of cell death in endothelial cells.