Kazuhiko Nishizaki

Gene Transfer Into the Donor Heart During Cold Preservation for Heart Transplantation

BACKGROUND Ex vivo gene transfer to heart grafts may hold promise as a means of changing alloreactivity or xenoreactivity after transplantation. However, it remains to be determined how effectively gene transfer can be accomplished within a short time in cold-stored grafts that are ready to be transplanted. METHODS We performed an experimental study using a replication-defective adenovirus (Adex1CALacZ) encoding the Escherichia coli beta-galactosidase (beta-gal) gene to perform gene transfer to heart grafts awaiting transplantation. Thirty hearts of Wistar rats were removed and their coronary arteries were perfused with University of Wisconsin solution containing 1 x 10(9), 1 x 10(10), or 1 x 10(11) plaque-forming units of the recombinant adenovirus at 4 degrees C for 60 minutes. As a control, other hearts were perfused with University of Wisconsin solution with an adenoviral vector that did not contain the beta-gal gene (Adex1w1) for the same period. After perfusion, the grafts were implanted in the necks of syngeneic adult rats. The grafts were removed each week after transplantation and their expression of beta-gal was assessed by 5-bromo-4-chloro-3-indoyl-beta-D-galactoside staining. RESULTS Successful gene transfer and expression of the beta-gal gene were demonstrated in adenovirus-perfused hearts. Gene transfer occurred preferentially in the cardiomyocytes over the endothelial cells and smooth muscle cells of the coronary vessels. In hearts perfused with 1 x 10(9) plaque-forming units of the adenovirus, gene expression persisted for 4 weeks after transfer, but it diminished gradually and was minimal by day 28. Histologic analyses revealed slight inflammatory reactions in the myocardium. In hearts perfused with 1 x 10(10) and 1 x 10(11) plaque-forming units of the adenovirus, beta-gal diminished 3 weeks after transplantation and a prominent infiltration of leukocytes was recognized in the myocardium. CONCLUSIONS This study demonstrated that the cardiomyocytes of heart grafts express an exogenous gene product after adenovirus-mediated gene transfer under hypothermic preservation conditions. However, immune or inflammatory reactions to recombinant adenoviruses must be taken into account when a large number of adenoviruses are injected into the coronary arteries.

In vivo gene gun–mediated transduction into rat heart with Epstein-Barr virus-based episomal vectors

BACKGROUND Gene guns have been used to transfer genes into various organs, but there has been no report of successful gene gun-mediated gene transfer into the heart. In this study, we assessed the possibility of gene therapy using a gene gun and an episomal plasmid vector. METHODS Gene transfer was performed using two sizes of gold particles and two plasmids (an episomal vector and a conventional plasmid vector). From the first to eighth week after the bombardment, rats were sacrificed. The excised hearts were subjected to X-gal staining and histologic examination. To ensure that plasmid was not distributed to organs other than the heart, the presence of the beta-gal sequence was examined by polymerase chain reaction analyses. RESULTS Gene expression persisted for 6 weeks. The episomal vector apparently contributed to long-lasting expression. Infiltration of monocytes or leukocytes was very faint. The beta-gal DNA was detected in bombarded hearts but not other organs. CONCLUSIONS Gene gun-mediated transfer of the episomal vector into beating heart may provide a simple, efficient, and useful strategy for gene therapy.

Tracheal allotransplantation maintaining cartilage viability with long-term cryopreserved allografts

BACKGROUND Cartilage viability of a cryopreserved tracheal allograft seems to affect graft function and durability. We previously reported the influence of warm ischemia and cryopreservation on cartilage viability of tracheal allografts. For the clinical application of tracheal allotransplantation, it is essential to preserve grafts for a long time. In this study, we assessed cartilage viability of tracheal allografts after long-term cryopreservation in transplantation models. METHODS The tracheas were harvested from Lewis rats. The grafts were frozen to -80 degrees C in a programmable freezer immediately after being harvested and were then stored in liquid nitrogen (-196 degrees C) for different lengths of preservation (1, 2, 6, 9, 12, 18, and 24 months; n for each group = 8). Cartilage viability was evaluated by estimating proteoglycan synthesis. After harvest or thawing of the tracheas, the cartilage was labeled with 4 muCi/mL of Na2 35SO4. Specimens were then hydrolyzed in 0.5 mol/L NaOH, and a solution of the extracts was then counted by a liquid scintillation counter. 35Sulfur incorporation before and after cryopreservation was examined in each group. Tracheal allotransplantation was performed using Lewis rats as donors and Brown Norway rats as recipients. RESULTS The average 35S incorporation in the cartilage before cryopreservation was 224 +/- 17 disintegrations per minute per milligram of tissue protein. The average 35S incorporation in the cartilage after cryopreservation decreased to 67% to 76% compared with that before cryopreservation. There were no significant differences among the groups in 35S incorporations after cryopreservation. Histologic examination after transplantation revealed normal tracheal cartilage in all groups. CONCLUSIONS The viability of tracheal cartilage after cryopreservation decreased to 67% to 76%. There were no significant differences in viability of cartilage among the tracheas after different lengths of cryopreservation. Tracheal allotransplantation after long-term cryopreservation can be safely performed in the rat model.

Effects of warm ischemia and cryopreservation on cartilage viability of tracheal allografts

BACKGROUND For clinical use of a cryopreserved tracheal allograft, it is important to evaluate cartilage viability. We assessed cell viability of the cartilage in a cryopreserved tracheal allograft by measurement of Na2 35SO4 incorporation. We also investigated the effects of warm ischemic time on tracheal cartilage viability. METHODS The tracheas from Lewis rats were harvested and preserved at different warm ischemic times from cardiac death to preservation (0, 1, 2, 4, 6, 9, and 12 hours, each group n = 8). The cartilage was labeled with 4 muCi/mL of Na2 35SO4. The specimen was hydrolyzed in 0.5 mol/L NaOH, and a solution of the extracts was then counted by liquid scintillation counter. Tracheas were transplanted into Brown Norway rats. RESULTS 35Sulfur incorporation in the cartilage decreased as warm ischemic time increased. In addition, 35Sulfur incorporation decreased from 76% to 67% after cryopreservation. Histologic examinations of the normal tracheal cartilage before preservation and after thawing were done in all the groups. After transplantation, the cartilage had severe fibrous changes, and its layer was almost nonobservable in the 9- and 12-hour groups. CONCLUSIONS The viability of the tracheal cartilage decreased with warm ischemic time and from 76% to 67% after cryopreservation. In the rat tracheal transplantation model, a cryopreserved tracheal allotransplant could be done safely with a graft that was cryopreserved within 6 hours of warm ischemic time.

Enhancement of Cell Viability in Cryopreserved Rat Vascular Grafts by Administration of Regenerating Gene (Reg) Inducers

The regenerative capacity of viable cells remaining in cryopreserved vascular allografts is still unclear. Recently, the regenerating gene (Reg) has been documented to play an important role in various regenerating tissues. Here we show the possibility of Reg induction for the enhancement of cryopreserved vascular allograft viability. Cryopreserved rat aortae were isografted or allografted heterotopically. Fresh isografts were also tested. The transplants were retrieved 3, 6, 9, and 12 days after implantation and the intragraft Reg mRNA was measured by a real-time quantitative reverse transcriptional polymerase chain reaction method. Reg expression was not detected before implantation. Reg expression in cryopreserved isografts gradually increased after transplantation, whereas in fresh isografts or cryopreserved allografts it decreased over time after initial expression. Daily administration of 0.5 g/kg nicotinamide (an agent known to be a potent inducer of Reg) induced intragraft Reg mRNA in cryopreserved allografts (p < 0.05) accompanied by augmentation of the intragraft cell population. Daily administration of 0.5 mg/kg FK506 (an immunosuppressant) induced intragraft Reg mRNA both in cryopreserved isografts and allografts (p < 0.01). We conclude that Reg-inductive therapy shows promise as a novel strategy for enhancing the viability of vascular allografts. Moreover, FK506 may be involved in tissue regeneration as well as immunosuppression.