Improved survival and reduced vascular permeability by eliminating or blocking 12/15-lipoxygenase in mouse models of acute lung injury (ALI) - PubMed (original) (raw)

Improved survival and reduced vascular permeability by eliminating or blocking 12/15-lipoxygenase in mouse models of acute lung injury (ALI)

Alexander Zarbock et al. J Immunol. 2009.

Abstract

Acute lung injury (ALI) is a prevalent disease associated with high mortality. 12/15-lipoxygenase (12/15-LO) is an enzyme producing 12-hydroxyeicosatetraenoic acid (HETE) and 15-HETE from arachidonic acid. To test whether 12/15-LO is involved in increasing vascular permeability in the lung, we investigated the role of 12/15-LO in murine models of LPS-induced pulmonary inflammation and clinically relevant acid-induced ALI. The vascular permeability increase upon LPS inhalation was abolished in Alox15(-/-) mice lacking 12/15-LO and in wild-type mice after pharmacological blockade of 12/15-LO. Alox15(-/-) mice also showed improved gas exchange, reduced permeability increase, and prolonged survival in the acid-induced ALI model. Bone marrow chimeras and reconstitution experiments revealed that 12-HETE produced by hematopoietic cells regulates vascular permeability through a CXCR2-dependent mechanism. Our findings suggest that 12/15-LO-derived 12-HETE is a key mediator of vascular permeability in acute lung injury.

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Figures

Figure 1. 12/15-lipoxygenase is involved in LPS-induced regulation of vascular permeability

(A) Vascular permeability of WT mice and _Alox15_−/− (_Alox15_−/−) mice was measured 6h after LPS inhalation by the Evans blue method (n=6-7). (B) After pharmacological blockade of 12/15-LO by the inhibitor CDC (Cinnamyl-3,4-Dihydroxy-a-Cyanocinnamate, open bars) or in vehicle control mice (black bars), vascular permeability (6h after LPS) was measured by the Evans blue method. Vascular permeability in WT mice pre-treated with the 12/15-LO inhibitor or vehicle (n=4). Dotted line indicates Evans blue in saline-treated WT mice for comparison. #P < 0.05.

Figure 2. 12/15-LO produced in lung macrophages is up-regulated following LPS inhalation

(A) 12/15-LO mRNA expression in lungs of unstimulated and stimulated WT mice and Alox15 deficient mice was investigated by quantitative RT-PCR (n.d., not detectable). 12/15-LO protein expression shown by immunohistochemistry in lungs from untreated WT (B) mice and WT mice 3h after LPS inhalation (C+D). (E-H) Fluorescence microscopy images of monocytes/macrophages in the lung upon LPS stimulation. Nuclei (blue, DAPI, E), Mac-2 (red, clone M3/38, F), and 12/15-LO (green, polyclonal rabbit anti-porcine, G). (H) The merged image demonstrates co-localization (yellow) of Mac-2 and 12/15-LO in one of the two monocytes/macrophages. Scale bar equals 20 μm. (E-H)

Figure 3. Hematopoietic 12/15-LO is responsible for regulation of vascular permeability following LPS-inhalation

_Alox15_−/− mice on a CD45.2 background and CD45.1 WT mice were used in bone marrow transplant experiments. (A) Monocytes and macrophages in single cell lung preparations were identified by gating on CD45+F4/80+ cells and the percentage of reconstituted cells were shown in the histogram by CD45.2 expression (donor-derived). (B) Chimeric mice lacking hematopoietic Alox15 are protected from increased vascular permeability as measured by Evans Blue extravasation upon LPS inhalation (n ≥ 4). #P < 0.05.

Figure 4. 12-HETE regulates endothelial and epithelial integrity and vascular permeability

Endothelial (A) and alveolar (B) cell response to 12-HETE activation as reflected by F-actin localization. Human pulmonary endothelial (A) and alveolar (B) cells were treated with 0.1 nM of 12-HETE, and F-actin was localized by phalloidin staining. Images are representative of 3 experiments with similar results. (C) Vascular permeability was measured by the Evans blue method 6h after intratracheal injection of 12-HETE or vehicle into WT or _Alox15_−/− mice (n = 4). #P < 0.05. (D) CXCL1 levels in the lung of WT and _Alox15_−/− mice after 12-HETE application.

Figure 5. 12-HETE-induced vascular leakage requires CXCL1 and CXCR2

CXCL1 levels in the BAL (A) and CXCR2 mRNA expression in the lung of WT mice and _Alox15_−/− mice (B). Vascular permeability was measured by the Evans blue method 6h after intratracheal injection of 12-HETE (open bars) or vehicle (black bars) into WT or _Cxcr2_−/− mice, mice (n = 4). Neutralization of CXCL1 by antibody (n = 3-4) reduced the vascular permeability increase in response to 12-HETE, similar to the level seen in _Cxcr2_−/− mice.

Figure 6. Absence of 12/15-LO improves acid-induced acute lung injury

Two hours after initiation of acid-induced ALI, gas exchange was measured in _Alox15_−/− and wild-type mice before and after HCl instillation (A). Permeability measured by BAL protein contents (B) compared to control mice (n ≥ 3 mice). #P < 0.05. (C) _Alox15_−/− mice showed significantly prolonged survival after induction of acid-induced ALI compared to HCl-treated WT mice (n = 6 per group). P < 0.001 by log rank test.

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Improved survival and reduced vascular permeability by eliminating or blocking 12/15-lipoxygenase in mouse models of acute lung injury (ALI) - PubMed (original) (raw)