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Postischemic improvement of hepatic microhemodynamics by a mixed ETA-, ETB-receptor antagonist (RO-47-0203)
ELSEVIER
Postischemic Improvement of Hepatic Microhemodynamics Mixed ETA-, ET,-Receptor Antagonist (RO-47-0203) T.A. Koeppel,
T. Kraus, J.C. Thies, M.-M. Gebhard,
I
SCHEMIA/reperfusion injury can be a major cause of structural and functional damage to livers in patients undergoing hepatic surgery and liver transplantation.‘~2 In particular, neutrophils and activated liver macrophages mediate toxic effects of reperfusion, and the resulting postischemic liver damage is further aggravated by disturbances of hepatic microvascular blood flo~.~,~ There is strong evidence that the potent vasoconstrictor endothelin-l (ET-l) impairs hepatic macro- and microhemodynamits in postischemic livers.‘,’ Application of monoclonal antibodies directed against ET-l have been shown to reduce parenchymal cell injury and improve hepatic perfusion.” In this study we evaluated whether specific blockage of ET-receptors with Bosentan (Ro 47-0203) a mixed ET-,, ET-,-receptor antagonist, improves hepatic microhemodynamics and reduces leukocyte-endothelium cell interactions in reperfused livers by using intravital fluorescence microscopy (IVM). MATERIALS Experimental
AND
METHODS
Protocol
Male Wistar rats (n = 12) were included in the study. A laparotomy was performed by a transverse abdominal incision. After mobilization of the left liver lobe a clip was placed in the hilus of the left liver lobe to occlude the supplying artery, vein, and draining bile duct. The treatment group (n = 6) received 15 mg X kg bw-’ Bosentan (Ro-47-0203, Hoffman LaRoche, Basel, Switzerland), infused slowly (1 mL/min) intravenously 1 minute prior to reperfusion. Control animals (n = 6) received an equivalent volume of Ringer’s solution. After 70 minutes of ischemia, declamping was performed, and 10 minutes postreperfusion the liver lobe was exteriorized onto a specially designed stage.’ During the experiment, arterial blood pressure was monitored. Central body temperature was kept constant, between 36.5” to 375°C. by a heating pad.
In Vivo Microscopy To evaluate postischemic hepatic microvascular blood flow, sinusoidal perfusion within perfused hepatic acini, sinusoidal diameters, and flow velocity of intraarterially injected latex beads (3 X 10’ beads x kg bw-‘, diameter 1.1 Frn (Polysciences Inc., Warrington, PA) in sinusoids were assessed between 20 to 90 minutes postreperfusion.*,” Furthermore, leukocyte-endothelial cell interactions were analyzed in postsinusoidal venules.
0 1997 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
by a
G. Otto. and S. Post
Statistics All data are presented as mean 2 SEM. P-values <.05 were considered to prove statistical significance of differences. For all microscopy data including several measurements for each animal, nested design analysis of variance was performed using SAS software for personal computers (SAS Institute, Cary, NC).
RESULTS
Systemic application of Bosentan did not affect macrohemodynamic parameters compared to controls. Disturbances of hepatic microvascular blood flow could be observed during reperfusion in hepatic acini in both experimental groups. Treatment with Bosentan was followed by an increase of 25% in mean velocity of latex particles analyzed in zone 2 of hepatic acini, indicating an improved sinusoidal blood flow (Bosentan: 638 ? 11 pm/s vs. Controls: 521 + 10 pm/s, P < .OS). Quantitative analysis of sinusoidal perfusion within perfused acini revealed a significant reduction of the percentage of nonperfused sinusoids from 10.6 5 1% to 2.8 t- 0.7% in the treatment group (P < .05). Assessment of sinusoidal diameters in midzonal areas of analyzed acini showed an increase in mean sinusoidal width in zone 2 from 9.9 +- 0.1 pm to 10.6 ? 0.1 pm in treated animals (P < .OS). Moreover, the number of rolling leukocytes in venules was reduced by 30% after Bosentan application (P < .OS). CONCLUSION
Our results show that a pharmacological blockage of ETreceptors with Bosentan attenuates manifestations of microvascular perfusion failure during reperfusion after warm ischemia. The beneficial impact of Bosentan on hepatic microhemodynamics was characterized by an increased sinusoidal blood flow velocity, increased sinusoidal diame-
From Department of General Surgery and Institute of Experimental Surgery, University of Heidelberg, Heidelberg; and Department of General Surgery, University of Gottingen, Gottingen, Germany. This work was supported by grants from the Forschungsschwerpunkt Transplantation Heidelberg. Address reprint requests to Thomas A. Koeppel, MD, Yale University, Department of Internal Medicine, Section of Digestive Diseases, 1080 LMP, PO 208019, New Haven, CT 06520-8019.
0041-1345/97/$17.00 PII SO041 -1345(96)00598-2
1365
Transplantation
Proceedings,
29, 1365-l 366 (1997)
KOEPPEL, KRAUS, THIES ET AL
1366
ters, and by a reduction of nonperfused sinusoids, indicating improved functional microvascular perfusion. ET has been shown to be responsible for alterations in hepatic macro- and microcirculation after ischemia.5y6 In the liver, a variety of cells express ET-receptors, including smooth-muscle cells in pre- and postsinusoidal vessels as well as contractile, perisinusoidal Ito ce1ls.r’ Therefore, specific blockage of ET-receptors with Bosentan could have prevented constriction of the hepatic vasculature and sinusoids. Furthermore, decreased numbers of rolling leukocytes were observed in postsinusoidal venules. It has been reported that ET induces the expression of specific adhesion molecules.‘l The observed decrease in rolling of leukocytes in postsinusoidal venules could be a result of a decreased adhesion molecule expression during reperfusion. Our results provide further evidence that ET is involved in the complex pathophysiology of ischemia/reperfusion injury of the liver.
REFERENCES
1. Huguet C, Gavelli A, Chieco PA, et al: Surgery 111:251, 1992 2. Thurman RG, Marzi I, Seitz G, et al: Transplantation 46:502, 1988 3. Jaeschke H, Farhood A: Am J Physiol 260:G355, 1991 4. Vollmar B, Glasz J, Leiderer R, et al: Am J Path01 145(6): 1421, 1994 5. Goto M, Takei Y, Kawano S, et al: Hepatology 19:675, 1994 6. Nakamura S, Nishiyama R, Serizawa A, et al: Transplantation 59:679, 1995 7. Post S, Palma P, Gonzalez AP, et al: Ann Surg 220:691, 1994 8. Post S, Palma P, Rentsch M, et al: Hepatology 18:1490, 1993 9. Marzi I, Bauer M, Secchi A, et al: Shock 3:27, 1995 10. Zhang JX, Bauer M, Clemens MG: Am J Physio1269:G269, 1994 11. McCarron RM, Wang L, Stanimirovic DB, et al: Neurosci Lett 156:31, 1993
Postischemic Improvement of Hepatic Microhemodynamics Mixed ETA-, ET,-Receptor Antagonist (RO-47-0203) T.A. Koeppel,
T. Kraus, J.C. Thies, M.-M. Gebhard,
I
SCHEMIA/reperfusion injury can be a major cause of structural and functional damage to livers in patients undergoing hepatic surgery and liver transplantation.‘~2 In particular, neutrophils and activated liver macrophages mediate toxic effects of reperfusion, and the resulting postischemic liver damage is further aggravated by disturbances of hepatic microvascular blood flo~.~,~ There is strong evidence that the potent vasoconstrictor endothelin-l (ET-l) impairs hepatic macro- and microhemodynamits in postischemic livers.‘,’ Application of monoclonal antibodies directed against ET-l have been shown to reduce parenchymal cell injury and improve hepatic perfusion.” In this study we evaluated whether specific blockage of ET-receptors with Bosentan (Ro 47-0203) a mixed ET-,, ET-,-receptor antagonist, improves hepatic microhemodynamics and reduces leukocyte-endothelium cell interactions in reperfused livers by using intravital fluorescence microscopy (IVM). MATERIALS Experimental
AND
METHODS
Protocol
Male Wistar rats (n = 12) were included in the study. A laparotomy was performed by a transverse abdominal incision. After mobilization of the left liver lobe a clip was placed in the hilus of the left liver lobe to occlude the supplying artery, vein, and draining bile duct. The treatment group (n = 6) received 15 mg X kg bw-’ Bosentan (Ro-47-0203, Hoffman LaRoche, Basel, Switzerland), infused slowly (1 mL/min) intravenously 1 minute prior to reperfusion. Control animals (n = 6) received an equivalent volume of Ringer’s solution. After 70 minutes of ischemia, declamping was performed, and 10 minutes postreperfusion the liver lobe was exteriorized onto a specially designed stage.’ During the experiment, arterial blood pressure was monitored. Central body temperature was kept constant, between 36.5” to 375°C. by a heating pad.
In Vivo Microscopy To evaluate postischemic hepatic microvascular blood flow, sinusoidal perfusion within perfused hepatic acini, sinusoidal diameters, and flow velocity of intraarterially injected latex beads (3 X 10’ beads x kg bw-‘, diameter 1.1 Frn (Polysciences Inc., Warrington, PA) in sinusoids were assessed between 20 to 90 minutes postreperfusion.*,” Furthermore, leukocyte-endothelial cell interactions were analyzed in postsinusoidal venules.
0 1997 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
by a
G. Otto. and S. Post
Statistics All data are presented as mean 2 SEM. P-values <.05 were considered to prove statistical significance of differences. For all microscopy data including several measurements for each animal, nested design analysis of variance was performed using SAS software for personal computers (SAS Institute, Cary, NC).
RESULTS
Systemic application of Bosentan did not affect macrohemodynamic parameters compared to controls. Disturbances of hepatic microvascular blood flow could be observed during reperfusion in hepatic acini in both experimental groups. Treatment with Bosentan was followed by an increase of 25% in mean velocity of latex particles analyzed in zone 2 of hepatic acini, indicating an improved sinusoidal blood flow (Bosentan: 638 ? 11 pm/s vs. Controls: 521 + 10 pm/s, P < .OS). Quantitative analysis of sinusoidal perfusion within perfused acini revealed a significant reduction of the percentage of nonperfused sinusoids from 10.6 5 1% to 2.8 t- 0.7% in the treatment group (P < .05). Assessment of sinusoidal diameters in midzonal areas of analyzed acini showed an increase in mean sinusoidal width in zone 2 from 9.9 +- 0.1 pm to 10.6 ? 0.1 pm in treated animals (P < .OS). Moreover, the number of rolling leukocytes in venules was reduced by 30% after Bosentan application (P < .OS). CONCLUSION
Our results show that a pharmacological blockage of ETreceptors with Bosentan attenuates manifestations of microvascular perfusion failure during reperfusion after warm ischemia. The beneficial impact of Bosentan on hepatic microhemodynamics was characterized by an increased sinusoidal blood flow velocity, increased sinusoidal diame-
From Department of General Surgery and Institute of Experimental Surgery, University of Heidelberg, Heidelberg; and Department of General Surgery, University of Gottingen, Gottingen, Germany. This work was supported by grants from the Forschungsschwerpunkt Transplantation Heidelberg. Address reprint requests to Thomas A. Koeppel, MD, Yale University, Department of Internal Medicine, Section of Digestive Diseases, 1080 LMP, PO 208019, New Haven, CT 06520-8019.
0041-1345/97/$17.00 PII SO041 -1345(96)00598-2
1365
Transplantation
Proceedings,
29, 1365-l 366 (1997)
KOEPPEL, KRAUS, THIES ET AL
1366
ters, and by a reduction of nonperfused sinusoids, indicating improved functional microvascular perfusion. ET has been shown to be responsible for alterations in hepatic macro- and microcirculation after ischemia.5y6 In the liver, a variety of cells express ET-receptors, including smooth-muscle cells in pre- and postsinusoidal vessels as well as contractile, perisinusoidal Ito ce1ls.r’ Therefore, specific blockage of ET-receptors with Bosentan could have prevented constriction of the hepatic vasculature and sinusoids. Furthermore, decreased numbers of rolling leukocytes were observed in postsinusoidal venules. It has been reported that ET induces the expression of specific adhesion molecules.‘l The observed decrease in rolling of leukocytes in postsinusoidal venules could be a result of a decreased adhesion molecule expression during reperfusion. Our results provide further evidence that ET is involved in the complex pathophysiology of ischemia/reperfusion injury of the liver.
REFERENCES
1. Huguet C, Gavelli A, Chieco PA, et al: Surgery 111:251, 1992 2. Thurman RG, Marzi I, Seitz G, et al: Transplantation 46:502, 1988 3. Jaeschke H, Farhood A: Am J Physiol 260:G355, 1991 4. Vollmar B, Glasz J, Leiderer R, et al: Am J Path01 145(6): 1421, 1994 5. Goto M, Takei Y, Kawano S, et al: Hepatology 19:675, 1994 6. Nakamura S, Nishiyama R, Serizawa A, et al: Transplantation 59:679, 1995 7. Post S, Palma P, Gonzalez AP, et al: Ann Surg 220:691, 1994 8. Post S, Palma P, Rentsch M, et al: Hepatology 18:1490, 1993 9. Marzi I, Bauer M, Secchi A, et al: Shock 3:27, 1995 10. Zhang JX, Bauer M, Clemens MG: Am J Physio1269:G269, 1994 11. McCarron RM, Wang L, Stanimirovic DB, et al: Neurosci Lett 156:31, 1993