Loss of extracellular superoxide dismutase 3 (SOD3) contributes to inflammatory and fibrotic lung diseases. The human SOD3 R213G polymorphism decreases matrix binding, redistributing SOD3 from the lung to extracellular fluids, and protects against LPS-induced alveolar inflammation. We used R213G mice expressing a naturally occurring single-nucleotide polymorphism, rs1799895, within the heparin-binding domain of SOD3, which results in an amino acid substitution at position 213 to test the hypothesis that the redistribution of SOD3 into the extracellular fluids would impart protection against bleomycin-induced lung fibrosis and secondary pulmonary hypertension (PH). In R213G mice, SOD3 content and activity was increased in extracellular fluids and decreased in lung at baseline, with greater increases in bronchoalveolar lavage fluid (BALF) SOD3 compared with wild-type mice 3 days after bleomycin. R213G mice developed less fibrosis based on pulmonary mechanics, fibrosis scoring, collagen quantification, and gene expression at 21 days, and less PH by right ventricular systolic pressure and pulmonary arteriole medial wall thickening at 28 days. In wild-type mice, macrophages, lymphocytes, neutrophils, proinflammatory cytokines, and protein increased in BALF on Day 7 and/or 21. In R213G mice, total BALF cell counts increased on Day 7 but resolved by 21 days. At 1 or 3 days, BALF pro- and antiinflammatory cytokines and BALF protein were higher in R213G mice, resolving by 21 days. We conclude that the redistribution of SOD3 as a result of the R213G single-nucleotide polymorphism protects mice from bleomycin-induced fibrosis and secondary PH by improved resolution of alveolar inflammation.

Aims TGF-β signaling is required for chronic hypoxia-induced pulmonary hypertension (PH). The activation of TGF-β by thrombospondin-1 (TSP-1) contributes to the pathogenesis of hypoxia-induced PH. However, neither the cellular source of pathologic TSP-1 nor the downstream signaling pathway that link activated TGF-β to PH have been determined. In this study, we hypothesized that circulating monocytes, which are recruited to become interstitial macrophages, are the major source of TSP-1 in hypoxia-exposed mice, and TSP-1 activates TGF-β with increased Rho kinase signaling, causing vasoconstriction. Methods and Results Flow cytometry revealed that a specific subset of interstitial macrophages is the major source of pathologic TSP-1 in hypoxia. Intravenous depletion and parabiosis experiments demonstrated that these cells are circulating prior to recruitment into the interstitium. Rho kinase mediated vasoconstriction was a major downstream target of active TGF-β. Thbs1 deficient bone marrow protected against hypoxic-PH by blocking TGF-β activation and Rho kinase-mediated vasoconstriction. Conclusions In hypoxia-challenged mice, bone marrow derived and circulating monocytes are recruited to become interstitial macrophages which express TSP-1, resulting in TGF-β activation and Rho kinase-mediated vasoconstriction. Translational Perspectives Inflammation contributes to the pathogenesis of many forms of pulmonary hypertension, but blocking inflammation has not been a successful therapeutic strategy to date. Here we found that mice with experimental hypoxia-induced pulmonary hypertension have recruitment of circulating, classical monocytes into the lungs, and that these cells express the protein thrombospondin-1 that causes activation of TGF-β and results in Rho-kinase mediated vasoconstriction. These data suggest that more precise targeting of inflammation, such as blocking specific cells like monocytes or cytokines like TGF-β, would be a more effective future therapeutic approach for pulmonary hypertension etiologies where these pathways underlie disease pathogenesis.

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