It has been suggested that over-expression of FasL could provide immune-privileged sites where transfected genes or/and grafted cells could exert their therapeutic action without inducing an immune response. Given the good transfectability of skeletal muscle, such tissue has been considered as a prime candidate for FasL engineerization. However, attempts to create FasL-expressing myoblasts have so far generated conflicting results. In this paper we set out to further investigate such approach, both in vitro and in vivo. We have designed a chimeric expression plasmid in which the addition of a GFP moiety to the C-terminal of FasL coding sequence rendered the final protein incapable of inducing apoptosis. Such construct has then been used for in vitro and in vivo transfection of rat muscle cells and its effects were compared with those obtained with a plasmid expressing normal FasL. The vast majority of primary myoblasts transfected in vitro with the FasL-expressing plasmid underwent apoptosis within the first 48 hours. Conversely, transfections with the FasL-GFP plasmid failed to induce cell death and allowed the formation of FasL-GFP positive myotubes, thereby demonstrating the inability of the chimeric product to activate the FasL-Fas pathway. In vivo, muscles co-injected with LacZ- and FasL-encoding plasmids developed a leukocytes infiltrate and transfected fibers were rapidly cleared out. When muscles were injected with a mixture of FasL-GFP and LacZ plasmids, transfected fibers persisted for at least two weeks. The results presented here demonstrate that Fas-FasL interaction is responsible for both the apoptosis of FasL transfected myoblasts in vitro and disappearance of FasL-engineered muscle fibers in vivo when transfections are carried out in regenerating muscles. However, our findings also suggest that it might be possible to obtain FasL-engineered mature muscle fibers by appropriately regulating the expression of the exogenous DNA during muscle differentiation.

Immune-Privileged Sites within Skeletal Muscle: Apoptosis of FasL-Transfected Myoblasts Prevents the Formation of FasL-Expressing Muscle Fibers In Vivo

SANDRI, MARCO;VITIELLO, LIBERO;CANTINI, MARCELLO
2002

Abstract

It has been suggested that over-expression of FasL could provide immune-privileged sites where transfected genes or/and grafted cells could exert their therapeutic action without inducing an immune response. Given the good transfectability of skeletal muscle, such tissue has been considered as a prime candidate for FasL engineerization. However, attempts to create FasL-expressing myoblasts have so far generated conflicting results. In this paper we set out to further investigate such approach, both in vitro and in vivo. We have designed a chimeric expression plasmid in which the addition of a GFP moiety to the C-terminal of FasL coding sequence rendered the final protein incapable of inducing apoptosis. Such construct has then been used for in vitro and in vivo transfection of rat muscle cells and its effects were compared with those obtained with a plasmid expressing normal FasL. The vast majority of primary myoblasts transfected in vitro with the FasL-expressing plasmid underwent apoptosis within the first 48 hours. Conversely, transfections with the FasL-GFP plasmid failed to induce cell death and allowed the formation of FasL-GFP positive myotubes, thereby demonstrating the inability of the chimeric product to activate the FasL-Fas pathway. In vivo, muscles co-injected with LacZ- and FasL-encoding plasmids developed a leukocytes infiltrate and transfected fibers were rapidly cleared out. When muscles were injected with a mixture of FasL-GFP and LacZ plasmids, transfected fibers persisted for at least two weeks. The results presented here demonstrate that Fas-FasL interaction is responsible for both the apoptosis of FasL transfected myoblasts in vitro and disappearance of FasL-engineered muscle fibers in vivo when transfections are carried out in regenerating muscles. However, our findings also suggest that it might be possible to obtain FasL-engineered mature muscle fibers by appropriately regulating the expression of the exogenous DNA during muscle differentiation.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11577/1374520
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