Junctional adhesion molecule-A deficiency increases hepatic ischemia-reperfusion injury despite reduction of neutrophil transendothelial migration by Andrej Khandoga, Julia S. Kessler, Herbert Meissner, Marc Hanschen, Monica Corada, Toshiyuki Motoike, Georg Enders, Elisabetta Dejana, and Fritz Krombach Blood Volume 106(2):725-733 July 15, 2005 ©2005 by American Society of Hematology

JAM-A expression in the hepatic microvasculature. JAM-A expression in the hepatic microvasculature. Immunostaining for JAM-A was performed in cryosections of the murine liver. (A) Under control conditions, JAM-A expression was found in cells localized in liver parenchyma (arrow), but not in vascular endothelial cells. Magnification, × 200 (objective 20×/0.5). (B) In the postischemic liver, JAM-A expression was strongly expressed in the vessel wall of hepatic venules (arrow). Magnification, × 200. (C) Examination of JAM-A staining at higher magnification (× 1000 oil immersion objective 100×/1.30) shows JAM-A positively stained endothelial cells (arrows indicate spindle-shaped nuclei of endothelial cells) as well as JAM-A-expressing adherent leukocytes in the liver after I/R (arrowhead). (D) In bile duct epithelial cells (arrow), postischemic JAM-A expression was very weak and hardly detectable. Magnification, × 1000 oil. (E) No JAM-A expression was detected in JAM-A-/- mice after I/R. Magnification, × 200. (F) In postischemic livers of endothelial JAM-A-/- mice, JAM-A expression was found only in parenchymal (arrow) but not in vascular endothelial (arrowhead) cells. Magnification, × 200. Andrej Khandoga et al. Blood 2005;106:725-733 ©2005 by American Society of Hematology

JAM-A mRNA expression. JAM-A mRNA expression. Representative RT-PCR gels (left) and the results of densitometric analysis presented as ratio of JAM-A to β-actin (right) show mRNA expression of JAM-A in hepatic tissue of sham-operated mice and mice after 90 minutes of hepatic ischemia and 140 minutes of reperfusion. The number of cycles was 35 for JAM-A and 23 for β-actin. n = 6 animals per group; mean ± SEM. Andrej Khandoga et al. Blood 2005;106:725-733 ©2005 by American Society of Hematology

Leukocyte-endothelial cell interactions. Leukocyte-endothelial cell interactions. Numbers of leukocytes rolling (A) and adherent (B) in postsinusoidal venules as well as number of leukocytes intravascularly accumulated in sinusoids (C) were quantitatively analyzed using intravital video fluorescence microscopy in sham-operated mice, JAM-A+/+ mice after I/R (I/R-JAM+/+), JAM-A-/- mice after I/R (I/R-JAM-/-), and in endothelial JAM-A-/- mice after I/R (I/R-eJAM-/-). Ischemia time: 90 minutes; reperfusion time: 30 minutes (▪) and 120 minutes (□). n = 6 animals per group; mean ± SEM; *P < .05 versus sham-operated group; #P < .05 versus IR-JAM-A+/+ group. Andrej Khandoga et al. Blood 2005;106:725-733 ©2005 by American Society of Hematology

Transendothelial migration of total leukocytes and neutrophils. Transendothelial migration of total leukocytes and neutrophils. Microphotographs demonstrate immunostaining for the common leukocyte antigen CD45 (left) and staining for granulocyte naphthol-ASD chloroacetate esterase (right) in the liver tissue of a sham-operated mouse (A), a JAM-A+/+ mouse after I/R (90/140 minutes) (B), and a JAM-A-/- mouse after I/R (C, top). Extravascular localized cells (arrows) were quantified in 10 high-power fields at microscope magnification × 400 (objective 40×/0.75) and (bottom) expressed as number of cells per square millimeter of liver surface (I/R-JAM+/+: JAM-A+/+ mice after I/R; I/R-JAM-/-: JAM-A-/- mice after I/R; and I/R-eJAM-/-: endothelial JAM-A-/- mice after I/R). n = 6 animals per group; mean ± SEM; *P < .05 versus sham-operated group; #P < .05 versus IR-JAM-A+/+ group. Andrej Khandoga et al. Blood 2005;106:725-733 ©2005 by American Society of Hematology

Transendothelial migration of T cells. Transendothelial migration of T cells. Microphotographs demonstrate immunostaining for the T-cell marker CD3 in the liver tissue of a sham-operated mouse (A), a JAM-A+/+ mouse after I/R (90/140 minutes) (B), and a JAM-A-/- mouse after I/R (C, left). Extravascularly localized cells (arrows) were quantified in 10 high-power fields at microscope magnification × 400 (objective 40×/0.75) and (right) expressed as number of cells per square millimeter of liver surface (I/R-JAM+/+: JAM-A+/+ mice after I/R; I/R-JAM-/-: JAM-A-/- mice after I/R; and I/R-eJAM-/-: endothelial JAM-A-/- mice after I/R). n = 6 animals per group; mean ± SEM; *P < .05 versus sham-operated group. Andrej Khandoga et al. Blood 2005;106:725-733 ©2005 by American Society of Hematology

Platelet-endothelial cell interactions. Platelet-endothelial cell interactions. The role of JAM-A for postischemic platelet-endothelial cell interactions was assessed using intravital fluorescence microscopy. Microphotographs in panels A and B demonstrate rhodamine 6G-labeled JAM-A+/+ platelets in the hepatic microcirculation of a sham-operated JAM-A+/+ mouse (A) and a JAM-A+/+ mouse after hepatic I/R (B). Photomicrograph in panel C shows JAM-A-/- platelets in the postischemic microvessels of a JAM-A+/+ mouse. Platelets adherent in postsinusoidal venules are marked by arrows; platelets accumulated in sinusoids are labeled by arrowheads. Monitor magnification, × 500 (water immersion objective 25×/0.6 W). Andrej Khandoga et al. Blood 2005;106:725-733 ©2005 by American Society of Hematology

Microvascular and hepatocellular injury. Microvascular and hepatocellular injury. (A) Sinusoidal perfusion was measured as a parameter of microvascular hepatic I/R injury. Microvascular perfusion failure is presented as the number of nonperfused sinusoids in sham-operated mice, JAM-A+/+ mice after I/R (I/R-JAM+/+), JAM-A-/- mice after I/R (I/R-JAM-/-), and endothelial JAM-A-/- mice after I/R (I/R-eJAM-/-). Ischemia time: 90 minutes; reperfusion time: 30 minutes (▪) and 120 minutes (□). (B) Serum activity of the liver enzymes AST and ALT was determined as a marker of hepatocellular necrotic injury after 90 minutes of ischemia followed by 140 minutes of reperfusion. (C) TUNEL-positive apoptotic hepatocytes were quantified in 10 high-power fields at microscope magnification × 400 in livers undergoing 90 minutes of ischemia followed by 140 minutes of reperfusion. n = 6 animals per group; mean ± SEM; *P < .05 versus sham-operated group; #P < .05 versus IR-JAM-A+/+ group. Andrej Khandoga et al. Blood 2005;106:725-733 ©2005 by American Society of Hematology