Crucial role for ecto-5'-nucleotidase (CD73) in vascular leakage during hypoxia

Abstract

Extracellular adenosine has been widely implicated in adaptive responses to hypoxia. The generation of extracellular adenosine involves phosphohydrolysis of adenine nucleotide intermediates, and is regulated by the terminal enzymatic step catalyzed by ecto-5'-nucleotidase (CD73). Guided by previous work indicating that hypoxia-induced vascular leakage is, at least in part, controlled by adenosine, we generated mice with a targeted disruption of the third coding exon of Cd73 to test the hypothesis that CD73-generated extracellular adenosine functions in an innate protective pathway for hypoxia-induced vascular leakage. Cd73(-/-) mice bred and gained weight normally, and appeared to have an intact immune system. However, vascular leakage was significantly increased in multiple organs, and after subjection to normobaric hypoxia (8% O(2)), Cd73(-/-) mice manifested fulminant vascular leakage, particularly prevalent in the lung. Histological examination of lungs from hypoxic Cd73(-/-) mice revealed perivascular interstitial edema associated with inflammatory infiltrates surrounding larger pulmonary vessels. Vascular leakage secondary to hypoxia was reversed in part by adenosine receptor agonists or reconstitution with soluble 5'-nucleotidase. Together, our studies identify CD73 as a critical mediator of vascular leakage in vivo.

Figure 1.

Restriction maps of CD73 genomic DNA, targeting vector, and recombined allele. (A) CD73 genomic DNA, including exon 3 and intronic sequences used to construct the short (1 kb) and long arms (4.2 kb) of the targeting vector. (B) CD73 targeting vector showing the antisense orientation of the CD73 sequences relative to the neo cassette. (C) Recombined, gene-targeted CD73 allele. The flanking probe used for Southern blots is shown, as are the positions of the PCR primers (arrows) used for screening the ES cell clones. (D) Southern blot of genomic DNA from Cd73 ^+/+, Cd73 ^−/+, and Cd73 ^−/− mice. Genomic DNA was purified from mouse tails, digested with BamHI, and subjected to Southern blotting by conventional methods. The probe was from intronic sequence 3′ to the long arm as shown in C. The wild-type BamHI fragment is 8.9 kb, whereas that from the gene-targeted mice is 7.4 kb. (E) Northern blot of kidney RNA from Cd73 ^+/+ and Cd73 ^−/− mice. Triplicate Northern blots were performed with 15 μg of kidney RNA in each lane. Blots were hybridized to the following probes: exon 2 (5′ probe), exon 3, or exon 4 (3′ probe). Ethidium bromide staining was used to confirm equal RNA loading.

Figure 2.

CD73 expression and function on leukocytes from Cd73 ^+/+ and Cd73 ^−/− mice. CD73 expression was evaluated on leukocytes from lymph node, spleen, peripheral blood, bone marrow, and thymus from Cd73 ^+/+ (left) and Cd73 ^−/− (right) mice with monoclonal antibody TY/23 + PE–goat anti–rat IgG. Staining with an isotype-matched control antibody is shown in the shaded histograms.

Figure 3.

Vascular leakage in Cd73-deficient mice in vivo. Cd73 ^−/− mice (black bars) and age-, weight-, and gender-matched littermate controls (white bars) were administered intravenous Evan's blue (0.2 ml of 0.5% in PBS per mouse) and exposed to room temperature air (A) or normobaric hypoxia (B, 8% O₂, 92% N₂) for 4 h. Animals were killed, and the colon (Co), lung (Lg), liver (Lv), muscle (Mu), heart (Ht), kidney (Kd), and brain (Br) were harvested. Evan's blue concentrations in organs were quantified as described in Materials and Methods. Data are expressed as mean ± SD Evan's blue OD/50 mg wet tissue and are pooled from four to six animals per condition, where * indicates P < 0.025 between Cd73 ^+/+ and Cd73 ^−/− mice and # indicates P < 0.025 between hypoxia and normoxia. (C) Images of abdominal dissections from wild-type and Cd73 ^−/− mice subjected to normoxia and hypoxia for 4 h.

Figure 4.

Influence of CD73 inhibition on vascular leakage in vivo. Age-, weight-, and gender-matched mice were administered APCP (20 mg/kg i.p.) or an equal volume of PBS followed by intravenous Evan's blue solution (0.2 ml of 0.5% in PBS per mouse) and exposed to room temperature air (A) or to normobaric hypoxia (B, 8% O₂, 92% N₂) for 4 h. Animals were killed and the colon (Co), lung (Lg), liver (Lv), muscle (Mu), heart (Ht), kidney (Kd), and brain (Br) were harvested. Evan's blue concentrations in organs were quantified as described in Materials and Methods. Data are expressed as mean ± SD Evan's blue OD/50 mg wet tissue and are pooled from four to six animals per condition where * indicates P < 0.05 in comparisons between APCP and PBS, and # indicates P < 0.025 between hypoxia and normoxia. (C) HPLC analysis of CD73 5′-NT enzyme activity (conversion of 1 mM E-AMP to E-Ado) in serum harvested at 4 h from normoxic animals administered −APCP (top) or +APCP (bottom).

Figure 5.

Vascular leakage and reconstitution of Cd73 ^−/− mice with 5′-NT. Cd73 ^−/− mice (B and D) and age-, weight-, and gender-matched littermate controls (A and C) were administered 5′-NT purified from C. atrox venom (500 U/kg i.p.; black bars) or PBS (white bars) followed by intravenous Evan's blue solution (0.2 ml of 0.5% in PBS per mouse) and exposed to room temperature air (A and B) or normobaric hypoxia (C and D, 8% O₂, 92% N₂) for 4 h. Animals were killed and the colon (Co), lung (Lg), liver (Lv), muscle (Mu), heart (Ht), kidney (Kd), and brain (Br) were harvested. Evan's blue concentrations in organs were quantified as described in Materials and Methods. Data are expressed as mean ± SD Evan's blue OD/50 mg wet tissue and are pooled from four to six animals per condition where, in comparisons between 5′-NT and PBS, * indicates P < 0.05 and # indicates P < 0.025.

Figure 6.

Influence of adenosine receptor antagonists on hypoxia-induced vascular leakage. Wild-type mice were administered either PBS (Control), the A_2B receptor antagonist MRS1754 (1 mg/kg i.p. plus 1 mg/kg s.c.) or the A_2A receptor antagonist ZM241385 (1 mg/kg i.p. plus 1 mg/kg s.c.) followed by intravenous Evan's blue solution (0.2 ml of 0.5% in PBS per mouse) and exposed to room air (black bars) or normobaric hypoxia (gray bars, 8% O₂, 92% N₂) for 4 h. Animals were killed, and lungs were harvested. (A) Evan's blue concentrations in organs were quantified as described in Materials and Methods. Data are expressed as mean ± SD Evan's blue OD/50 mg wet tissue and are pooled from four animals per condition where * indicates P < 0.01 between normoxia and hypoxia and # indicates P < 0.01 between treatment and control. (B) Assessment of lung water content. Data are expressed as mean ± SD mg H₂O/mg dry tissue, and are pooled from four animals per condition where, in comparisons between hypoxia and normoxia, * indicates P < 0.01, # indicates P < 0.05 between treatment and control, and ## indicates P < 0.025 between treatment and control.

Figure 7.

Influence of the adenosine receptor agonist NECA on vascular leakage of Cd73 ^−/− mice. Cd73 ^−/− mice (B and D) and age-, weight-, and gender-matched littermate controls (A and C) were administered the adenosine analogue NECA (0.1 mg/kg i.p. plus 0.1 mg/kg s.c., black bars) or PBS (white bars) followed by intravenous Evan's blue solution (0.2 ml of 0.5% in PBS per mouse) and exposed to room temperature air (A and B) or normobaric hypoxia (C and D, 8% O₂, 92% N₂) for 4 h. Animals were killed, and the colon (Co), lung (Lg), liver (Lv), muscle (Mu), heart (Ht), kidney (Kd), and brain (Br) were harvested. Evan's blue concentrations in organs were quantified as described in Materials and Methods. Data are expressed as mean ± SD Evan's blue OD/50 mg wet tissue and are pooled from four to six animals per condition where * indicates P < 0.025 and # indicates P < 0.01 between NECA and PBS.

Figure 8.

Characterization of lungs from Cd73 ^−/− mice. Wild-type (A and D) or Cd73 ^−/− (B and C) mice were subjected to normoxia (A and B) or hypoxia (C and D). Whole lungs were fixed with 10% formalin at total lung capacity, sectioned, and stained with hematoxylin and eosin. (A) A representative image of a wild-type control animal (magnification, 100). (C) Perivascular interstitial edema (line arrows) and epithelial disruption (block arrow) in Cd73 ^−/− hypoxic mice (magnification, 100). (D) Subtle perivascular interstitial edema (line arrow) associated with wild-type hypoxic mice (magnification, 100). (E) Assessment of lung water content in normoxia (black bars) and hypoxia (gray bars) in the presence and absence of NECA administration (0.1 mg/mg i.p. plus 0.1 mg/kg s.c.). Data are expressed as mean ± SD mg H₂O/mg dry tissue and are pooled from three to four animals per condition where, in comparisons between hypoxia and normoxia, * indicates P < 0.025 and, in comparisons between NECA and PBS, # indicates P < 0.025.