Effects of human tissue plasminogen gene transfer on allograft coronary atherosclerosis

Transplant coronary atherosclerosis is a major limiting factor to successful long-term cardiac transplantation. The depletion of tissue plasminogen activator (tPA) in the arteriolar smooth muscle cells has been associated with a higher incidence of accelerated graft atherosclerosis. In vivo overexpression of tPA may inhibit accelerated graft atherosclerosis and improve the long-term results of heart transplantation. We evaluated the feasibility, distribution, and effects of intracoronary transfer of the human tPA (htPA) gene in a rabbit heterotopic cardiac transplant model, using a novel cationic liposome compound designed for improved delivery to vascular endothelium.

Human tPA cDNA under the control of the SV40 promoter (100 microg) was complexed with the novel cationic liposome (+/-)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propanaminium bromide (GAP: DLRIE) (50 microg), and delivered ex vivo to the donor heart by slow intracoronary infusion. Control hearts received an "empty" liposome preparation. Grafts were then implanted 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, animals received a 0.5% cholesterol diet for 30 days posttransplant, as well as 10 mg/kg cyclosporine A daily. Beating hearts were collected at 30 days posttransplant and analyzed for the development of transplant atherosclerosis by image analysis.

Northern blot analysis for the htPA messenger RNA (mRNA) transcripts showed significantly higher counts in hearts receiving the htPA gene as compared to controls. The distribution of these transcripts favored the left ventricle (LV) and septal regions over the right ventricle (RV). Scintillation analysis of specimens stained by immunoflourescence showed expression of htPA throughout the perivascular myocardium that was significantly higher in grafts transduced with the htPA gene than in control or native hearts. Expression in the vascular wall was also significantly enhanced. Scintillation 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). Intimal hyperplasia was assessed by mean percent luminal stenosis in small- and medium-sized arteries and was 31.12 +/- 23.5% in htPA-transduced hearts and 86.59 +/- 17.5% in control hearts (p < 0.0001). 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 transplantation results in significant early transgene expression, and significantly inhibits the development of graft coronary atherosclerosis.