Adventitial versus intimal liposome-mediated ex vivo transfection of canine saphenous vein grafts with endothelial nitric oxide synthase gene.
OBJECTIVE
Experiments were designed (1) to evaluate liposome-mediated endothelial constitutive nitric oxide synthase (ecNOS) transfection in vein grafts and (2) to compare intimal and adventitial routes of transfection.


METHODS
Male mongrel dogs (N = 36) underwent bilateral femoral artery bypass grafting with the lateral saphenous vein. In each animal one vein was transfected with plasmid (pVR1012) containing the ecNOS gene, and another vein was transfected with plasmid alone (control). Gene transfer was performed from either the intimal surface (Group I, n = 18) or the adventitial surface (Group II, n = 18). In each group there were three transfection subgroups (n = 6 each): (a ) 10 microg/mL naked plasmid DNA, (b ) 10 microg/mL plasmid DNA + liposome (LipofectAMINE PLUS), and (c ) 100 microg/mL plasmid DNA + LipofectAMINE PLUS. Grafts were harvested on the third postoperative day, and the transfection was assessed with molecular techniques and enzyme assay for activity of NOS by conversion of tritiated l-arginine to tritiated l-citrulline. Proliferating cells were quantified with a digital analysis of histologic sections after nuclear antigen Ki-67 (MIB1) immunohistochemistry.


RESULTS
Transgene was identified with polymerase chain reaction in all ecNOS-transfected grafts, regardless of transfection modality. However, significant transcription of the ecNOS transgene was observed only in Group IIc (mean ecNOS messenger RNA, 8.7+/-1.7 vs. 3.1+/-0.7 x 10(-2) attomole/microL, in transfected compared with control grafts, respectively, P =.01). NOS activity increased approximately twofold in this group (11.58+/-2.1 and 6.3+/-1.0 pmol tritiated l-citrulline per milligram protein per hour in transfected and control grafts, respectively, P = .05). Numbers of proliferating cells did not differ among ecNOS-transfected and control grafts in any transfection group.


CONCLUSIONS
These results suggest that ecNOS transfection of vein grafts is feasible through intimal and adventitial routes with naked DNA or a liposomal vector. However, efficient transcription of the transgene is evident at postoperative day 3 only after adventitial transfection of 100 microg/mL of the gene.
Kalra
M
M
Department of Surgery and Biophysics and Physiology, Mayo Clinic and Foundation, Rochester, MN, USA.
Jost
C J
CJ
Severson
S R
SR
Miller
V M
VM
D000818:Animals; D001483:Base Sequence; D019917:Blood Vessel Prosthesis Implantation; D002455:Cell Division; D004247:DNA / Q000032:analysis / Q000235:genetics; D018076:DNA, Complementary / Q000032:analysis / Q000235:genetics; D003627:Data Interpretation, Statistical; D004285:Dogs; D004730:Endothelium, Vascular / Q000201:enzymology*; D005263:Femoral Artery / Q000601:surgery; D007150:Immunohistochemistry; D008081:Liposomes; D008297:Male; D008969:Molecular Sequence Data; D009569:Nitric Oxide / Q000097:blood; D019001:Nitric Oxide Synthase / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D012106:Research; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012501:Saphenous Vein / Q000166:cytology / Q000637:transplantation*; D013997:Time Factors; D014162:Transfection; D017539:Tunica Intima / Q000201:enzymology; D018608:Ultrasonography, Doppler
D018076:DNA, Complementary; D008081:Liposomes; D012333:RNA, Messenger; D009569:Nitric Oxide; D004247:DNA; D019001:Nitric Oxide Synthase