Gene Therapy With Endothelial Nitric Oxide Synthase Protects Aortic Allografts From Arteriosclerosis

Cardiac allograft arterosclerosis poses a major limitation for long-term survival of heart transplan recipients. Owing to the lacking of effective therapy, how to prevent the development of the cardiac allograft arterosclerosis become more important. We constructed PLXSN-eNOS plasmid, then investigated whether gene delivery of Enos into donor arteries before transplantation would surppress intimal hyperplasia of allgrafts .eNOS gene was linked to PLXSN plasmid to construct PLXSN-eNOS plasmid with the help of intermediary plasmid Pblue by ECORI, hindIIIand Xhol enzyme. The plasmid PLXSN-eNOS was constructed correctly and confrimed by ECORI and Xhol enzyme digestion, then was furtherly confirmed by PCR.Orthotopic carotid allograft transplantation was performed in male rats using SD rats as donors and wister rats as recipients. Carotids have been soaked in the PLXSN-LF2000 or PLXSN-eNOS-LF2000 solution for 30min before they end-to-end anastomoses was performed by 11/0 nylone medical suture with standard microvascular surgical techniques. The wistars were randomly divided into control and treatment groups.The three following parts were studied: (1) The liposome-mediated gene transfection of PLXSN-eNOS in the donor carotids onpost-operative days 1, 7, 14 and 28 was investiged by RT-PCR. (2) The allografts were harvested and cross-section of the vascular tissues were used for immuohistochemical staining of eNOS, iNOS, NF-kB, ICAM-land VCAM-1. (3) The allograft pathologic changes were observed by electron microscopy and the average intimal/medical ratio of the allografts were measured.The majore results and conclusion were obtained as follows: (l)eNOS gene transfection of allografts was confirmed by RT-PCR on post-operative days 7,14 and 18. (2) The expression of eNOS was significantly discreased and the expression of iNOS significantly increased in the intima of the allografts on post-operatively days 1,7,14 and 28 in the control group. The expression of iNOS significantly increased on post-operative days 1 and 7, but its expression decreased on post-operative days 14 and 28 in the media of the allografts. Liposome-mediated gene transfection of eNOS preserved eNOS expression in the endothelial cells and surpressed iNOS expression in the intima and media of the allografts. (2) ICAM-1 and VCAM-1 expression were all significantly reduced in the intima in eNOS-transfected carotids compared with control groups. (3) NF-kB expression was significantly reduced in the intima in eNOS-transfected carotids compared with control groups.(4)The diffuse intima thickening involving the entire circumference of allograft was due to proliferation of smooth muscle cells, associated with a densification and increase of extracell matrix (fibrous tissue).Diffuse infiltration by lymphocyte and mononuclear cells in intima, media and advertitia was observed. The media appeared thinned,with some pachy destruction of elastic lamella.The intima of 28-day allograft showed abundant extracellular matrix and collagen fibril surrounding the smooth muscle cells by the electron microscopy. Conclusion:1. The plasmid PLXSN-eNOS is constructed correctly and confrimed by ECORI and Xhol enzyme digestion and PCR.2. plasmid PLXSN-eNOS can transfect rat carotid by soaking method.3. eNOS gene transfection of preserved eNOS expression in the endothelial cells and surpressed iNOS, ICAM-1, VCAM-1, NF-kB expression in the intima or/and media of the allografts to prevent the allgraft arterosclerosis.