ADAM15 Deficiency Attenuates Pulmonary Hyperpermeability and Acute Lung Injury in Lipopolysaccharide-Treated Mice

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vascular permeability, metalloproteinase, endothelial dysfunction, inflammation

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ADAM15 is a disintegrin and metalloprotease recently implicated in cancer and chronic immune disorders. We have recently characterized ADAM15 as a mediator of endothelial barrier dysfunction. Whether this molecule contributes to acute inflammation has not been evaluated. The purpose of this study was to investigate the role of ADAM15 in mediating pulmonary microvascular leakage during acute inflammatory injury. Immunofluorescent staining and Western blotting revealed that the endothelium was the main source of ADAM15 in lung tissue. In a mouse model of acute lung injury induced by lipopolysaccharide (LPS), upregulation of ADAM15 was observed in association with pulmonary edema and neutrophil infiltration. The LPS-induced inflammatory injury, as demonstrated by bronchoalveolar lavage neutrophil count, lung wet-to-dry weight ratio, and myeloperoxidase activity, was significantly attenuated in Adam15−/− mice. Studies with primary cell culture demonstrated abundant ADAM15 expression in endothelial cells (ECs) of mouse lung but not in neutrophils. Deficiency of ADAM15 in ECs had no obvious effect on basal permeability but significantly attenuated hyperpermeability response to LPS as evidenced by albumin flux assay and measurements of transendothelial electrical resistance, respectively. ADAM15 deficiency also reduced neutrophil chemotactic transmigration across endothelial barriers in the presence or absence of formyl-methionyl-leucyl-phenylalanine (fMLP). Rescue expression of ADAM15 in Adam15−/− ECs restored neutrophil transendothelial migration. These data indicate that ADAM15 upregulation contributes to inflammatory lung injury by promoting endothelial hyperpermeability and neutrophil transmigration.

the adams (a disintegrin and metalloproteinase) are multifunctional transmembrane glycoproteins that are involved in a variety of biological processes (5, 17). In general, ADAMs are metalloproteinases that are capable of cleaving cell surface protein ectodomains and inducing signaling events through receptor shedding, and/or outside-in signaling (8, 26). ADAM15 is a less studied ADAM isoform that is found in human vein endothelium (13) and is associated with certain types of cancer and chronic inflammatory or immunological disorders (5, 14, 21). In particular, ADAM15 supports cancer metastasis by promoting tumor cell migration and angiogenesis (21, 24). Increased ADAM15 expression is also detected in atherosclerotic lesions, rheumatoid synovium, angiogenic retinas, and the intestines of patients with inflammatory bowl disease (1, 35). This suggests that increased ADAM15 expression is involved in a variety of inflammatory conditions.

Our recent studies of the effects of thrombin on human umbilical vein endothelial cells (HUVECs) has identified ADAM15 as an important mediator of endothelial barrier dysfunction. In general, endothelial barriers are formed by a layer of closely apposed endothelial cells that are anchored to the extracellular matrix at the basement membrane. The cell-cell and cell-matrix adhesion structures act coordinately with the cytoskeleton to maintain the integrity of the barrier with a low basal permeability (22, 31). However, these structures can be degraded or shed by proteases like ADAMs (8, 26) or may undergo conformational changes in response to pathological signaling (12, 18, 31). In HUVEC monolayers, ADAM15 is necessary for both endothelial hyperpermeability and increased neutrophil transmigration in response to thrombin exposure (28). Moreover, these ADAM15-dependent effects are mediated largely by endothelial intracellular signaling through Src kinase and extracellular regulated kinase (ERK)-1/2, and not through receptor ectodomain shedding. These interesting findings prompted us to further investigate the role of ADAM15 in pathological states involving endothelial barrier dysfunction.

Acute lung injury (ALI) is a condition that involves pulmonary microvascular leakage in response to systemic or local lung inflammation. Unresolved ALI can progress into respiratory distress syndrome, a major cause of mortality and morbidity in patients with infectious disease or sepsis. Despite theories advanced regarding its pathogenesis, there has been no major breakthrough in treatment or prevention of ALI. This is largely owing to the complexity of the disease and our incomplete understanding of its endpoint cellular mechanisms (6, 15). Microvascular barrier dysfunction is a common endpoint of inflammatory response characterized by endothelial hyperpermeability, plasma leakage, and leukocyte infiltration in the lungs, leading to respiratory distress and multiple organ failure (7, 16). On the basis of the observation that ADAM15 is involved in several inflammatory conditions, coupled with our results showing thrombin induced barrier dysfunction in umbilical vein endothelium is ADAM15 dependent, we infer that ADAM15 plays a role in endothelial barrier dysfunction during inflammation in other tissues including the lungs.

In the present study we examined the role of ADAM15 in the lungs and in pulmonary endothelium, under experimental inflammatory conditions [exposure to bacterial lipopolysaccharide (LPS)]. LPS exposure is known to induce edema formation and neutrophil infiltration in the lungs. We find that LPS exposure increases ADAM15 expression in the murine lungs. LPS-induced pulmonary edema and neutrophil infiltration are greatly attenuated in gene knockout Adam15−/− mice, and similarly ADAM15 deficiency decreases LPS-induced endothelial hyperpermeability and neutrophil transendothelial migration in vitro in primary cultured mouse lung endothelial cell (MLEC) monolayers. Together the data support a causal role for ADAM15 in endothelial barrier dysfunction during LPS-induced inflammation in the lungs.

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Citation / Publisher Attribution

American Journal of Physiology-Lung Cellular and Molecular Physiology, v. 304, issue 3, p. L135-L142