A2B adenosine receptor dampens hypoxia-induced vascular leak by Tobias Eckle, Marion Faigle, Almut Grenz, Stefanie Laucher, Linda F. Thompson, and Holger K. Eltzschig Blood Volume 111(4):2024-2035 February 15, 2008 ©2008 by American Society of Hematology

A2B adenosine receptor (A2BAR) on HMEC-1 cells promotes endothelial barrier function in vitro. A2B adenosine receptor (A2BAR) on HMEC-1 cells promotes endothelial barrier function in vitro. Inhibition of A2BAR mRNA expression by stable expression of A2BAR siRNA. HMEC-1 cells were stably transfected with a plasmid expressing A2BAR siRNA or a nonspecific siRNA (Control Transfected, SCR). Cells were exposed to hypoxia (pO2 = 20 torr) and real-time RT-PCR was used to confirm repression of the A2BAR (A) Data were calculated relative to β-actin and are expressed as fold change over control plus or minus SD after indicated time periods of hypoxia exposure. Results are derived from 3 experiments (*P < .05, different from normoxia; #P < .01, compared with normoxia and control transected; **P < .01 different from control transfected). Western blot analysis was used to confirm siRNA repression of the A2BAR protein (B). A representative blot of 3 is shown (a vertical line has been inserted to indicate a repositioned gel lane.), in addition to densitometric analysis of A2BAR protein levels relative to β-actin (*P < .01, different from normoxia; #P < .05, compared with normoxia and control transected; **P < .05 different from control transfected, n = 3). Repression of A2BAR expression inhibits adenosine-mediated enhancement of endothelial barrier function. Indicated concentrations of adenosine in HBSS were added to the apical surface of confluent normoxic (48-hour exposure to pO2 = 147 torr) or hypoxic (48-hour exposure to pO2 = 20 torr) HMEC-1 that were stably transfected with A2BAR (D) or control (C) siRNA. Permeability to FITC-dextran (70 kDa) was quantified by measuring transendothelial flux (3 samples over 60 minutes) and normalized as a percentage of control (HBSS). Baseline flux rates for normoxic conditions where approximately 200 to 250 pM/min per cm2 and 800 to 1000 pM/min per cm2 after hypoxia exposure (less than 5% of tracer). Data are derived from 6 monolayers in each condition (*P < .05, compared with baseline; #P < .05, compared with baseline and normoxia). (E) The A2BAR antagonist PSB1115 inhibits adenosine-mediated enhancement of endothelial barrier function. Adenosine elicited barrier responses were measured in normoxic endothelia (HMEC-1) with or without the addition of 1 μM PSB1115 (*P < .05, compared with baseline). (F) The A2BAR agonist BAY 60-6583 promotes barrier responses. Hypoxic endothelia (48-hour exposure to pO2 = 20 torr) that were stably transfected with A2BAR or control siRNA were assessed for BAY 60-6583–elicited barrier responses. Data are expressed as means plus or minus SD (*P < .05, compared with baseline. #P < .05, compared with baseline and untreated controls, n = 6). Tobias Eckle et al. Blood 2008;111:2024-2035 ©2008 by American Society of Hematology

Vascular permeability in A1, A2A, A2B and A3 adenosine receptor (AR)-deficient mice during hypoxia in vivo. Vascular permeability in A1, A2A, A2B and A3 adenosine receptor (AR)-deficient mice during hypoxia in vivo. (A) A1AR−/−, (B) A2AAR−/−, (C) A2BAR−/−, or (D) A3AR−/− mice and age-, weight-, and sex-matched littermate controls were exposed to room air (▭) or normobaric hypoxia (, 8% O2, 92% N2) for 4 hours and lung water content was measured. In additional studies, mice were administered intravenous Evan blue dye (0.2 mL of 0.5% in PBS) prior to hypoxia or normoxia exposure. Animals were killed and the heart (Ht), colon (Co), kidney (Kd), lung (Lg), spleen (Sp), brain (Br), muscle (Mu) and liver were harvested. Organ Evan blue dye concentrations were quantified as described in “In vivo hypoxia model.” Data are expressed as means plus or minus SD Evan blue OD/50 mg wet tissue; n = 6 animals/condition (*P < .05, compared with normoxia; **P < .05, compared with littermate controls; and #P < .05, compared with littermate controls and normoxia). Tobias Eckle et al. Blood 2008;111:2024-2035 ©2008 by American Society of Hematology

A2BAR and vascular permeability during hypoxia in vivo. A2BAR and vascular permeability during hypoxia in vivo. (A) A2BAR−/− mice or age-, weight-, and sex-matched littermate controls were administered intravenous Evan blue dye (0.2 mL of 0.5% in PBS per mouse) and exposed to room temperature air or normobaric hypoxia (8% O2, 92% N2) for 4 hours. Representative images of abdominal dissections are shown. Images were obtained using a Canon Power Shot G9 digital camera (Canon, Krefeld, Germany). (B) Age-, weight-, and sex-matched wild type mice were administered a selective A2BAR antagonist (PSB1115, 20 mg/kg intraperitoneally) or an equal volume of PBS and lung water content was measured. In other experiments, mice were injected with intravenous Evan blue dye (0.2 mL of 0.5% in PBS per mouse) after PBS1115 or vehicle treatment and exposed to room air or to normobaric hypoxia (8% O2, 92% N2) for 4 hours. Animals were killed and the heart (Ht), colon (Co), kidney (Kd), lung (Lg), spleen (Sp), brain (Br), muscle (Mu) and liver were harvested. Organ Evan blue dye concentrations were quantified as described in “In vivo hypoxia model.” Data are expressed as means plus or minus SD of Evan blue OD/50 mg wet tissue; n = 6 animals/condition (*P < .05, compared with normoxia; **P < .05, compared with normoxic −PSB1115 controls; and #P < .05, compared with normoxia and −PSB115 controls). (C) Age-, weight-, and sex-matched wild type mice were administered a selective A2BAR agonist (BAY 60-6583, 80 μg/kg.) or an equal volume of PBS and lung water content was measured. In other experiments, mice were injected with intravenous Evan blue dye (0.2 mL of 0.5% in PBS per mouse) after BAY 60-6583 or vehicle treatment and exposed to room air or to normobaric hypoxia (8% O2, 92% N2) for 4 hours. Data are expressed as means plus or minus SD of Evans blue OD/50 mg wet tissue; n = 6 animals/condition (*P < .05, compared with normoxia; **P < .05, compared with −BAY 60-6583 normoxic controls and #P < .01, compared with −BAY 60-6583 hypoxic controls). Tobias Eckle et al. Blood 2008;111:2024-2035 ©2008 by American Society of Hematology

Pulmonary A2BAR expression increases after exposure to hypoxia in vivo. Pulmonary A2BAR expression increases after exposure to hypoxia in vivo. Wild-type mice were subjected to normoxia or 4 hours of ambient hypoxia (8% oxygen). Whole lungs were fixed with 10% formalin at total lung capacity. Lungs were stained with antibody for the A2BAR. Normal goat IgG was used in controls at identical concentration and staining conditions as the target primary Ab (arrow marks vascular, asterisk alveolar structures). Evaluation of the histologic and immunohistochemical stainings and photographic documentation was performed using an Olympus Vanox AH-3 light microscope (Hamburg, Germany), original magnification, ×400. Tobias Eckle et al. Blood 2008;111:2024-2035 ©2008 by American Society of Hematology

Vascular permeability in A2BAR bone marrow chimeric mice. Vascular permeability in A2BAR bone marrow chimeric mice.A2BAR bone marrow chimeric mice (A: A2BAR+/+→A2BAR+/+ and A2BAR−/−→A2BAR−/−; B: A2BAR−/−→A2BAR+/+ and A2BAR+/+→A2BAR−/−) were administered intravenous Evan blue dye (0.2 mL of 0.5% in PBS) and exposed to room air or normobaric hypoxia (8% O2, 92% N2) for 4 hours. Animals were killed and the heart (Ht), colon (Co), kidney (Kd), lung (Lg), spleen (Sp), brain (Br), muscle (Mu) and liver were harvested. Organ Evan blue concentrations were quantified as described in “In vivo hypoxia model.” Data are expressed as means plus or minus SD of Evan blue OD/50 mg wet tissue (n = 6 animals/condition). (A: *P < .05, compared with normoxia; **P < .05, compared with A2BAR+/+→A2BAR+/+ bone marrow chimeric mice; and #P < .05, compared with A2BAR+/+→A2BAR+/+ bone marrow chimeric mice and normoxia. B: *P < .05, compared with normoxia; **P < .05, compared with A2BAR−/−→A2BAR+/+ bone marrow chimeric mice; and #P < .05, compared with A2BAR−/−→A2BAR+/+ bone marrow chimeric mice and normoxia). (C) Representative images of abdominal dissections are shown from A2BAR−/−→A2BAR+/+ and A2BAR+/+→A2BAR−/− bone marrow chimeric mice. Images were obtained using a Canon Power Shot G9 digital camera (Canon). Tobias Eckle et al. Blood 2008;111:2024-2035 ©2008 by American Society of Hematology

Influence of the A2BAR agonist (BAY 60-6583) on vascular permeability in bone marrow chimeric mice. Influence of the A2BAR agonist (BAY 60-6583) on vascular permeability in bone marrow chimeric mice.A2BAR bone marrow chimeric mice (A: A2BAR−/−→A2BAR+/+; B: A2BAR+/+→A2BAR−/−) were treated with the selective A2BAR agonist (BAY 60-6583, 80 μg/kg intraperitoneally) or an equal volume of PBS 30 minutes before intravenous Evan blue dye (0.2 mL of 0.5% in PBS) and exposed to room air or normobaric hypoxia (8% O2, 92% N2) for 4 hours. Animals were killed and the heart (Ht), colon (Co), kidney (Kd), lung (Lg), spleen (Sp), brain (Br), muscle (Mu) and liver (Lv) were harvested. Organ Evan blue dye concentrations were quantified after formamide extraction (55°C for 2 hours) as described in “In vivo hypoxia model.” Data are expressed as means plus or minus SD of Evans blue OD/50 mg wet tissue (n = 6 animals/condition). (A: *P < .05, compared with normoxia; **P < .05, compared with −BAY 60-6583 control; and #P < .05, compared with −BAY 60-6583 control and normoxia; B: *P < .05, compared with normoxia). Tobias Eckle et al. Blood 2008;111:2024-2035 ©2008 by American Society of Hematology

Hypoxia-induced increases in PMN-tissue accumulation in A2BAR bone marrow chimeric mice. Hypoxia-induced increases in PMN-tissue accumulation in A2BAR bone marrow chimeric mice.A2BAR bone marrow chimeric mice (A: A2BAR+/+→A2BAR+/+ and A2BAR−/−→A2BAR−/−; B: A2BAR−/−→A2BAR+/+ and A2BAR+/+→A2BAR−/−) were subjected to either normoxia (room air) or normobaric hypoxia (8% oxygen) for 4 hours, and the heart (Ht), colon (Co), kidney (Kd), lung (Lg), spleen (Sp), brain (Br), muscle (Mu) and liver (Lv) were assessed for PMN accumulation by myeloperoxidase activity (A: *P < .01, compared with normoxia and #P < .01, compared with A2BAR+/+→A2BAR+/+ bone marrow chimeric mice and normoxia; B: *P < .01, compared with normoxia and #P < .05, compared with A2BAR−/−→A2BAR+/+ bone marrow chimeric mice and normoxia). Error bars represent SD. Tobias Eckle et al. Blood 2008;111:2024-2035 ©2008 by American Society of Hematology