Erythropoietin suppressed oxidized low density lipoprotein induced apoptosis of vascular smooth muscle cell

H Hu, XJ Yu, J Zhou, WJ Zang. College of medicine, Xi\'an Jiaotong university, Pharmacology, China

Background: Increasing evidence suggests that vascular smooth muscle cell (VSMC) apoptosis has been associated with a number of deleterious consequences of atherosclerosis, especially on plaque rupture. This emphasises that VSMC apoptosis is an essential target to develop therapy to treat atherosclerosis. Erythropoietin (EPO), a 165-amino acid glycoprotein hormone, has extra-hematopoietic and pleiotropic effects such as neuroprotective, tissue-protective, anti-cancer and immune-modulatory effects. As an anti-apoptosis factor, EPO plays anti-apoptosis effects on many types of cells to exert organ-protective effects. The purpose of this study was to investigate the role of EPO and its underlying mechanism in oxidized low density lipoprotein (ox-LDL) induced VSMC apoptosis.

Methods: VSMCs were isolated from thoracic aortas of Sprague-Dawley rats and cultured by using the explant technique. The identity and purity of the VSMCs were verified by immunostaining using antibody against smooth muscle α-actin. Apoptosis and necrosis of the VSMCs were distinguished by using Annexin V-FITC label and propidium iodide stain and analyzed by flow cytometry. Caspase-3 and caspase-8 activities were tested by spectrophotometry. The mRNA and proteins expression of related genes and proteins (Bax, Bcl-xL, Fas-L) were determined by real time PCR and western blot.

Results: Incubation of cultured VSMCs with ox-LDL (200μg/ml) for 24h resulted in significant cell death (apoptosis rate was about 17.8%). The reduced apoptosis rates after exposure to ox-LDL were reversed after pretreatment of 50, 100, 150U/ml EPO for 24 h by 15.7%, 12.6%, 11.2%, respectively. Caspase-3 activity was decreased after pretreatment of 100U/ml EPO, while caspase-8 activity was not changed obviously. The upregulation of Bax and Fas-L mRNA and proteins expression induced by ox-LDL was attenuated, and the downregulation of Bcl-xL was reversed after pretreatment of 100U/ml EPO.

Conclusion: These data demonstrated that EPO suppressed ox-LDL induced VSMC apoptosis by Fas-L pathway. These findings suggested that EPO maybe prevent atherosclerotic plaques instability and rupture to serve as a new drug for atherosclerosis treatment.