Effects of human tissue plasminogen gene transfer on allograft coronary atherosclerosis

Background: Transplant coronary atherosclerosis is a major limiting factor to successful long-term cardiac transplantation. The depletion of tissue pl asminogen activator (tPA) in the arteriolar smooth muscle cells has been as sociated with a higher incidence of accelerated graft atherosclerosis. In v ivo overexpression of tPA may inhibit accelerated graft atherosclerosis and improve the long-term results of heart transplantation. We evaluated the f easibility, distribution, and effects of intracoronary transfer of the huma n tPA (htPA) gene in a rabbit heterotopic cardiac transplant model, using a novel cationic liposome compound designed for improved delivery to vascula r endothelium. Methods: Human tPA cDNA under the control of the SV40 promoter (100 mug) wa s complexed with the novel cationic liposome (+/-)-N-(3-aminopropyl)-N,N-di methyl-2,3-bis(dodecyloxy)-1-propanaminium bromide (GAP: DLRIE) (50 mug), a nd delivered ex vivo to the donor heart by slow intracoronary infusion. Con trol hearts received an "empty" liposome preparation. Grafts were then impl anted into recipient rabbits in the heterotopic cervical position. For the analysis of gene expression, beating donor hearts were collected at 4 days. To examine the effects of htPA expression on graft atherosclerosis, animal s received a 0.5% cholesterol diet for 30 days posttransplant, as well as 1 0 mg/kg cyclosporine A daily. Beating hearts were collected at 30 days post transplant and analyzed for the development of transplant atherosclerosis b y image analysis. Results: Northern blot analysis for the htPA messenger RNA (mRNA) transcrip ts showed significantly higher counts in hearts receiving the htPA gene as compared to controls. The distribution of these transcripts favored the lef t ventricle (LV) and septal regions over the right ventricle (RV). Scintill ation analysis of specimens stained by immunoflourescence showed expression of htPA throughout the perivascular myocardium that was significantly high er in grafts transduced with the htPA gene than in control or native hearts . Expression in the vascular wall was also significantly enhanced. Scintill ation counts per x 200 field were 262 +/- 145 in htPA-transduced hearts and 20 +/- 27 in controls (p = 0.001), and mean luminescence was 83.7 +/- 12.5 in htPA-transduced hearts and 62.9 +/- 12.8 in controls (p = 0.01). Intima l hyperplasia was assessed by mean percent luminal stenosis in small- and m edium-sized arteries and was 31.12 +/- 23.5% in htPA-transduced hearts and 86.59 +/- 17.5% in control hearts (p < 0.0001). Conclusions: These results demonstrate that expression of the htPA gene can be induced by ex vivo intracoronary gene transfer at the time of allograft preservation. Liposome-mediated delivery of the htPA gene at the time of t ransplantation results in significant early transgene expression, and signi ficantly inhibits the development of graft coronary atherosclerosis.