Endothelin(A) receptor blockade reduces ischemia/reperfusion injury in pig pancreas transplantation

Abstract

Objective: The effect of prophylactic administration of a selective endothelin(A) receptor antagonist (ET(A)-RA) on ischemia/reperfusion injury in an experimental model of graft pancreatitis after pancreas transplantation was evaluated.

Summary background data: It is well established that endothelin-1 (ET-1), a powerful vasoconstrictor, plays an important role in the development of pancreatitis. Recent studies have shown a beneficial effect of endothelin receptor antagonists in the therapy for experimental pancreatitis.

Methods: Relevant ischemia/reperfusion injury was induced in pig pancreas transplants after 6 hours hypothermic preservation in University of Wisconsin solution. The recipients were randomized into 2 groups: control pigs received isotonic saline and the treated group received the selective ET(A)-RA BSF 208075 at the beginning of reperfusion. On postoperative days 2 and 5, animals were relaparotomized to obtain tissue specimens. Blood monitoring included lipase, amylase, C-reactive protein, trypsinogen-activation peptide, thiobarbituric acid-reacting substances, and ET-1. Partial oxygen tension (p(ti)O(2)) was measured by a Clarke-type electrode and blood flow by laser doppler. A semiquantitative score index was used for assessment of histologic injury and for immunohistochemical analysis of ET-1 and ET(A) receptor expression. Tissue mRNA levels of prepro ET-1, ET(A) receptor, pro-interleukin (IL)-6, and pro-IL-1beta were quantified using TaqMan real-time reverse transcription-polymerase chain reaction (RT-PCR).

Results: Prophylactic treatment with ET(A)-RA significantly reduced the severity of graft pancreatitis evidenced by C-reactive protein. The finding of transient capillary perfusion at the beginning of reperfusion supports the application of the ET(A)-RA during this period. The dramatic increase of plasma ET-1 in the therapy group is a clear evidence of effective receptor blockade. Mean trypsinogen-activation peptide levels from the portal venous effluent, but not mean systemic plasma TAP values were significantly lower in the treated group. Analysis of p(ti)O(2) and blood flow revealed a significant improvement of capillary perfusion and blood flow in the treated group and was associated with relevant reduction of tissue injury. Intrapancreatic ET-1 and IL-6 mRNA expression and ET-1 protein levels were significantly lower in the therapy group as compared with the control group. In contrast, ET(A) mRNA showed a marked up-regulation by ET(A) receptor blockade.

Conclusion: Application of a ET(A)-RA reduces ischemia/reperfusion induced graft pancreatitis in a pig transplantation model by improving microcirculation and reducing tissue injury.

FIGURE 1. Partial oxygen tension (p_tiO₂) in the pancreatic grafts before ischemia in the donors and from 10 minutes to 5 days after reperfusion in the recipients given endothelin-A receptor antagonist (therapy group) or saline (control group). Values are means ± SD. *, significant difference control vs. therapy group (P < 0.05). p_tiO₂ was significantly higher at each measurement point in the therapy group as compared with the control group.

FIGURE 2. Mean plasma endothelin-1 (ET-1) values of the recipients given endothelin-A receptor antagonist (therapy group) or saline (control group). Values are means ± SD. Administration of the specific endothelin-A receptor antagonist led to significant higher ET-1 levels as compared with the control group during the period from 30 minutes to 12 hours after reperfusion. *, therapy group vs. control group, P < 0.05. **, before reperfusion vs. baseline, P < 0.05 (control and therapy group). ***, two hours after vs. before reperfusion, P < 0.05 (control and therapy group).

FIGURE 3. Semiquantitative histologic score indices of the pancreatic grafts at different time points of procurement and after transplantation. Recipients were given endotholin_A receptor antagonist (therapy group) or saline (control group). Score indices are calculated by means of score counts depending on the degree of tissue injury using 6 parameters (classification see Materials and Methods). The score indices of each group at each time of investigation are represented in the form of “box whisker plots” showing the median (line in box), the 25th and 75th percentiles (boxes) and the 95th percentiles (outer box). *Significant difference between control and therapy group (P < 0.05). Whereas histologic injury did not differ between the control and therapy group in the donors and before reperfusion, significantly less morphologic damage was seen at each measurement point after reperfusion in the group with endotholin_A receptor antagonist therapy.

FIGURE 4. Immunohistochemical score indices of ET-1 in the pancreatic grafts at different time points of procurement and after transplantation. Recipients were given endotholin_A receptor antagonist (therapy group) or saline (control group). Score values are calculated by multiplication of mean values of intensity of immunostaining with the mean number of positive cells. The score indices of each group at each time of investigation are represented in the form of “box whisker plots” showing the median (line in box), the 25th and 75th percentiles (boxes) and the 95th percentiles (outer box). *, significant difference between control and therapy group (P < 0.05). ET-1 expression was significantly higher 10 minutes and 1 hour after reperfusion in the control group as compared with the therapy group.

FIGURE 5. Immunohistochemical score indices of endothelin_A receptor of the pancreatic grafts at different time points of procurement and after transplantation. Recipients were given endotholin_A receptor antagonist (therapy group) or saline (control group). Score values are calculated by multiplication of mean values of intensity of immunostaining with the mean number of positive cells. The score indices of each group at each time of investigation are represented in the form of “box whisker plots” showing the median (line in box), the 25th and 75th percentiles (boxes) and the 95th percentiles (outer box). *, significant difference between control and therapy group (P < 0.05). ET_A receptor expression was significantly higher 10 minutes, 1 hour, and 2 days after reperfusion in the control group as compared with the therapy group.

FIGURE 6. Intrapancreatic mRNA expression of prepro endothelin-1 (ET-1), endothelin_A receptor (ET_A), pro-interleukin-6 (IL-6), and pro-interleukin-1β (IL-1β) 60 minutes after reperfusion in the recipients given ET_A receptor antagonist (therapy group) or saline (control group). mRNA levels are shown as fold increase/decrease of the baseline values in the corresponding donor group. *, significant difference between control and therapy group (P < 0.05). In comparison with the control group, the treated group showed a significant down-regulation of prepro ET-1 and pro IL-6 levels, whereas the ET_A value was significantly upregulated. The pro-IL-1β changes were not significantly different.