A) Deletion and mutation analysis of the TM promoter.
HUVECs were transiently transfected with luciferase reporter constructs with different parts of the human TM 5′-flanking region deleted. Cell were treated with vehicle (V), atorvastatin (A), or pre-treated with mevalonate for 30 minutes before being treated with atorvastatin (M+A) for 24 hours.
Deletion/mutation of HSE1 and HSE3, or of SP1/KLF element partly inhibits statin-induced TM upregulation.
B) Mutation analysis of HSE and/or SP1/KLF element.
HUVECs, transiently transfected with luciferase reporter constructs with HSE3, SP1/KLF, or both elements mutated, were treated with vehicle (V), atorvastatin (A) or pravastatin (P) for 24 hours.
Mutation of either HSE3 or SP1/KLF element only partly inhibited statin-induced TM upregulation, whereas, mutation of both elements together completely abolished statin-induced TM upregulation.
Relative light units (RLU) were determined.
Atorvastatin: 1×10−5M; mevalonate: 5×10−4M; pravastatin: 4×10−5M
* p < 0.05 versus vehicle.