Motohiko Yasutomi

Liberation of vasoactive substances and its prevention with thromboxane A2 synthase inhibitor in pig liver transplantation

There are multiple causes of liver graft nonfunction in the early post-transplant period. Since a severe microcirculatory disturbance based on ischemia-reperfusion liver injury is considered to be the main underlying pathophysiology, it is suspected that various vasoactive substances are liberated after reperfusion of the graft. In order to investigate this matter, we conducted an experimental study with pig liver allotransplantation. Two groups of animals received donor grafts with or without thromboxane synthase inhibitor (sodium ozagrel), 1.25 mg/kg body weight intravenously, given at the time of liver harvesting. All of the recipient animals in the treatment group (n=10) survived longer than 7 days whereas three of ten animals in the control group died within 7 days. Serum lactate dehydrogenase (LDH) in the recipient serum at 1 h after reperfusion was significantly lower in the treatment group (915.1±167.3 U/l) than in the control group (1264.4±134.7 U/l). Serum thromboxane B2 (2261.7±1055.7 pg/ml) and endothelin-1 (6.3±2.2 pg/ml) after reperfusion in the treatment group were significantly lower than those in the control group (4220.0±1711.0 pg/ml and 11.2±3.1 pg/ml, respectively). Although serum angiotensin II after reperfusion tended to be lower in the treatment group than in the controls serum renin activity was less than 3 ng/ml in both groups of animals. There were no differences in the plasma endotoxin levels between the two groups. We conclude that the administration of sodium ozagrel to the donor animals provided better graft function in recipients than no such treatment. We speculate that the inhibition of thromboxane A2 production suppresses the liberation of other vasoconstrictive substances, preventing microcirculatory disturbance and, thereby, contributing to improved graft function after liver transplantation.

Prevention of experimental hepatic metastasis with thromboxane synthase inhibitor.

To investigate the effectiveness of thromboxane (Tx) synthase inhibitor in the prevention of experimental hepatic metastasis, an in vivo study was designed. Hepatic metastasis was brought about by injection of 1×105 cells of colon 38 tumor into the portal vein of C57 B1/65 mice. Seven groups (n=16 in each group) received different treatments: with TxA2 synthase inhibitor (sodium ozagrel), 5, 10 or 15 mg/kg BW before tumor inoculation, and daily for the following 3 days, (groups A, B and C, respectively); with acetyl salicylic acid (aspirin), 1.0, 1.5 or 2.0 mg/kg BW (groups C, D, and E, respectively); a control group, inoculated with vehicle only. Serum TxB2, a stable metabolite of TxA2, and prostaglandin F1α were measured. Labeling index for tumor proliferation by bromodeoxy-uridine radioimmuno-assay was also studied. Incidence of metastasis in groups A (60.5%), B (49.5%), C (43.0%), D (80.5%), E (66.0%) and F (58.4%) was less than that in the control group (100%). Tumor size, number or labeling index did not differ among the groups. Serum TxB2 (pg/ml) levels were significantly lower in all of the groups than in the control. Serum PGF1α levels in the groups with aspirin were lower than those in sodium ozagrel. Tx synthase inhibitor is effective in the prevention of experimental hepatic metastasis when it is given before and immediately after tumor inoculation. As Tx synthase inhibitor leaves metabolic pathway to PGI2 productionintact, it is more effective in the prevention of metastasis than aspirin since aspirin inhibits both thromboxane and PGI2.

Evidence that the adenoviral vector containing the CTLA4-Ig gene improves transgene expression and graft survival

Abstract Rejection of organ allografts is dependent on T cell activation, which requires T-cell recepter engagement by antigen and costimulatory signals delivered by T-cell surface molecules such as CD28. CTLA4-Ig is a recombinant fusion protein that contains the extracellular domain of human CTLA4 (a homologue of CD28) fused to a human IgG 1 heavy chain. It has previously been shown to block the CD28-mediated costimulatory signal and to inhibit immune responses in vitro and in vivo. 1,2 Recently, the replication-defective adenovirus vector has been shown to be efficient in transferring exogenous genes into a variety of cells both in vitro and in vivo. 3 In this study, we examined the ability of adenovirus vector containing CTLA4-Ig (Adex/CTLA4-1g) to modify the rejection process observed in ACI to LEW rats liver transplantation.

Immunosuppressive drugs and their effect on experimental tumor growth

The effect of cyclosporin (CyA), FK 506, and mycophenolate mofetil (MPM) on tumor growth was investigated using syngeneic mouse colon carcinoma 38. Mice were laparotomized and the tumor cells were injected into the portal vein to establish liver metastasis. The animals were grouped as follows: groups A-1, B-1, and C-1 were given CyA [15 mg/kg body weight (BW)], FK 506 (0.15 mg/kg BW), and MPM (100 mg/kg BW), respectively, 30 min before tumor inoculation and daily for 5 days by gavage; groups A-2, B-2, and C-2 were given CyA (30 mg/kg BW), FK 506 (0.3 mg/kg BW), and MPM (200 mg/kg BW), respectively, with the same dose timing; and groups A-3, B-3, and C-3 received CyA (30 mg/kg BW), FK 506 (0.3 mg/kg BW), and MPM (200 mg/kg BW), respectively, on the 7th post-tumor inoculation day and on the following 5 days. The mean tumor diameter in groups A-1 and A-2 was greater than that in the control group and in groups C-1 and C-2 at 3 weeks (P<0.05). The mean tumor numbers in groups A-1 and A-2 were greater than those in the control group and in groups C-1 and C-2 at 4 weeks (P<0.05). With in vitro MTT assay, all three drugs acted cytostatically on tumor cells with a higher concentration (10-6–10-4 mol/l), while no cytostatic effect was noted with CyA at a lower concentration (10-9–10-7 mol/l). Labeling indexes (%) by bromodeoxyuridine (BrdUrd) immunohistochemistry in groups A-1, A-2, and B-1 were significantly greater than those in the control group and in groups C-1 and C-2 (P<0.05). Although the mechanism of cytoproliferative action of CyA and FK 506 is not well understood, a decrease in immunosurveillance capability by natural kill cells due to suppression of interleukin-2, their direct action as growth factors, and/or enhanced tumor cell adhesion can be considered.

Superoxide scavenging activity in experimental liver transplantation.

Superoxide dismutase (SOD) activity was evaluated by measuring superoxide scavenging capability with the aid of an electron spin resonance (ESR) spin trapping method in a swine orthotopic liver transplantation (OLT) model. The animals were divided into two groups, depending on the length of the survival periods: the short survival group (n=8) survived less than 6 days and the long survival group (n=15) 6 days or longer. SOD activity was significantly lower in the short survival group than in the long survival group after reperfusion (P<0.01). During the period of cold preservation, a minimal change in SOD activity was noted, regardless of the length of preservation. Serum aspartate aminotransferase (AST) levels after reperfusion and serum lactate dehydrogenase (LDH) levels 1 h after reperfusion were significantly higher in the short survival group than in the long survival group (P<0.01 and P<0.05, respectively). The difference in polymorphonuclear leukocytes (PMN) was significantly greater in the short survival group at 1 h after reperfusion (P<0.01). The authors conclude that superoxide scavenging activities in the graft reflect the magnitude of reperfusion injury, which can be a reliable parameter for the estimation of graft outcome.

Endothelin-1 and in vitro evidence of renal artery vasoconstriction after liver transplantation

Twelve pigs underwent orthotopic liver transplantation. The mean endothelin-1 (ET-1) levels in the serum samples of the recipient animals 1 h after reperfusion of the graft (6.2±1.5 pg/ml) was significantly higher (P<0.05) than in pretransplantation samples (3.2±0.6 pg/ml). Serum blood urea nitrogen (BUN) 24 h after transplantation was 13.8±5.9 mg/dl, which was significantly higher than before transplantation (6.4±2.2 mg/dl). There was a positive correlation between the serum BUn and ET-1 (r=0.62,P<0.05). An in vitro isometric tension study was performed for the contractility response rate of the intact renal artery in the bath chamber containing the serum of the corresponding recipient animals. The mean contractility response rates were higher with the serum obtained after reperfusion of the graft (66.9±32.4%) than with those obtained before transplantation (18.3±9.2%) when compared to a standard contractility rate of 100% with 40 mM KCl. Moreover, these contractility response rates were significantly reduced (32.8±21.0%) with the addition of the ET-1 receptor antagonist FR139 317. The results of the present study demonstrated that the liver transplantation was associated with elevation of ET-1 in the serum of the recipient animals. It was considered that ET-1 in the serum caused a direct vasoconstriction of the renal artery in vitro. This may help to explain the renal dysfunction that is often seen in the recipients of clinical liver transplantation.