Fig 1.
Increased BALF ATX/LPA levels upon LPS-induced ALI.
Mice were administered aerosolized LPS and were sacrificed 6, 12, 24 and 48 hours later (n = 3–5; a representative experiment out of two is shown). A. Hematoxylin & Eosin staining of lung tissue sections following LPS exposure for the indicated times resulted in alveolar wall thickening and leukocyte infiltration into the lung interstitium and alveolar space. B. Increased BALF cellularity upon LPS/ALI. C. Pulmonary microvascular leakage and edema induced by LPS was reflected in BALF protein content. D. Increased ATX activity in LPS/ALI BALFs, as measured with the TOOS assay. E. IHC for ATX in lung tissue showing constitutive expression from the bronchial epithelium, as well as a weak diffuse staining pattern in the lung parenchyma. F-G. BALF total LPC/LPA levels respectively upon LPS/ALI, as measured with HPLC-MS/MS.
Fig 2.
Genetic deletion of ATX from the bronchial epithelium has minor effects in ALI development.
Aerosolized LPS was administered in mice where ATX was genetically deleted specifically from bronchial epithelial cells (CC10Enpp2-/-), as well as wild type littermates. Mice were sacrificed 24 hours later (n = 3–8; a representative experiment out of two is shown). A-C. Histological analysis (A) and BALF measurements (B & C) indicated no significant changes in the lungs of mice lacking ATX expression in the bronchial epithelium in comparison to their wild type littermates. D. Conditional deletion of ATX from the cells of the bronchial epithelium led to decreased enzyme activity of ATX in the BALF.
Fig 3.
Generation of TgCC10Enpp2 mice.
A. Schematic representation of the construct used for the generation of the transgenic mice. B. Genotyping PCR of the 4 offsprings that carried the transgene, out of the 54 that were generated after the injections of the trangene-microinjected zygotes in surrogate mothers. C. All four transgenic lines contained equal copy numbers, as identified with Real-Time PCR. D. Real-Time RT-PCR confirmed the expression of the transgene (L39 is shown). E. Total ATX activity levels in the BALFs of TgCC10Enpp2 mice (L39) were found moderately upregulated with the TOOS assay. F. In the same mice, BALF LPA was also found elevated, as measured with HPLC-MS/MS. (C-F n = 3–8).
Fig 4.
Genetic overexpression of ATX from the bronchial epithelium has minor effects in ALI development.
Transgenic (TgCC10hEnpp2+/+) mice overexpressing hATX in the bronchial epithelium and littermate wild type mice were administered aerosolized LPS to induce ALI and were sacrificed 24 hours later (n = 3–10; a representative experiment out of two is shown). A-C. Histological analysis (A) and BALF measurements (B & C) indicated no significant changes in the lungs of mice overexpressing ATX expression in the bronchial epithelium in comparison to their wild type littermates. D. Increased enzyme activity of ATX in the transgenic mice.
Fig 5.
Deletion of ATX from myeloid cells had no effects in ALI development.
Aerosolized LPS was administered in mice where ATX was deleted from the myeloid cells (LysMEnpp2-/-) and wild type littermate mice. Mice were sacrificed 24 hours later. A-C. Histological analysis (A) and BALF measurements (B & C) showed no differences in inflammation and edema between mice lacking ATX expression from myeloid cells and their wild type littermates. D. Conditional deletion of ATX from myeloid cells had no effects in BALF ATX activity.
Fig 6.
Systemic overexpression of ATX exacerbates ALI.
Transgenic (Tga1t1Enpp2+/+) or heterozygous complete knock-out (Enpp2+/-) mice and their corresponding wild type littermate mice were administered with aerosolized LPS to induce lung injury and were sacrificed 24 hours later (n = 2–6; a representative experiment out of two is shown). A-D. Histological analysis (A) and BALF measurements (B & C) showed increased inflammation and edema in the lungs of the transgenic mice with systemic overexpression of ATX as a result of increased BALF ATX activity (D). On the contrary, histological analysis (A) and BALF measurements (E-G) showed non-significant effects in the lungs of the heterozygous knock-out mice as a result of decreased ATX activity levels.
Fig 7.
Pharmacologic inhibition of ATX has no effects in ALI development.
GWJ-A-23 was injected intraperitoneally before challenging mice with aerosolized LPS. Mice were sacrificed 24 hours later (n = 3–7; a representative experiment out of two is shown). A-C. Histological analysis (A) and indicated BALF measurements (B,C) suggested minor effects in ALI development, despite decreased BALF ATX activity (D) and the corresponding BALF LPA levels (E).