Fas ligand/Fas-mediated apoptosis in human coronary artery smooth muscle cells: therapeutic implications of fratricidal mode of action

Objective: This study aimed to determine the mode of action of Fas ligand ( FasL)/Fas at mediating apoptosis so as to evaluate the potential of FasL in gene therapy for restenosis. Methods: Passaged human coronary artery smoot h muscle (HCASM) cells were infected with recombinant adenoviral vectors ex pressing murine FasL. Various parameters of FasL expression and apoptosis, were measured using FACS, immunofluorescence, calorimetric, and cytotoxicit y assays. Results: Most HCASM cells under normal growth conditions expresse d Fas and were shown to be susceptible to membrane bound but not soluble Fa sL. However, some FasL expressing cells survived for up to 7 days. These su rviving cells were observed to be spatially distributed and were not in dir ect physical contact with each other. Upon examination, it was determined t hat although the majority of the surviving cells expressed FasL, only 30% e xpressed both Fas and FasL. These cells were capable of inducing apoptosis of target cells and some were also susceptible to FasL expressing cells, pr ovided that the effector and target cells were in close physical contact. F asL/Fas-mediated apoptosis was inhibited by p35, a baculovirus gene that in hibits caspases. Additionally, in contrast to HCASM cells, neither membrane -bound nor soluble FasL induced apoptosis in coronary artery endothelial ce lls. Conclusions: Fa-L expressing HCASM cells do not undergo FasL/Fas media ted "suicide" but kill neighboring cells bearing Fas in a "fratricidal" man ner. A small population of HCASM cells expresses no surface Fas. These resu lts imply that HCASM cells transduced in vivo with FasL may serve as "scave ngers" and exert a bystander effect on surrounding cells that may be enhanc ed by co-expression of p35. As FasL-mediated apoptosis occurs in coronary a rterial smooth muscle but not endothelial cells, FasL may also offer an adv antage over other genes for use in restenosis since the latter may indiscri minately delay re-endothelialization at the sites of gene. (C) 2001 Elsevie r Science B.V. All rights reserved.