Surfactant Dysfunction and Lung Inflammation in the Female Mouse Model of Lymphangioleiomyomatosis

Abstract

Pulmonary lymphangioleiomyomatosis (LAM) is a rare lung disease caused by mutations of the tumor suppressor genes, tuberous sclerosis complex (TSC) 1 or TSC2. LAM affects women almost exclusively, and it is characterized by neoplastic growth of atypical smooth muscle-like TSC2-null LAM cells in the pulmonary interstitium, cystic destruction of lung parenchyma, and progressive decline in lung function. In this study, we hypothesized that TSC2-null lesions promote a proinflammatory environment, which contributes to lung parenchyma destruction. Using a TSC2-null female murine LAM model, we demonstrate that TSC2-null lesions promote alveolar macrophage accumulation, recruitment of immature multinucleated cells, an increased induction of proinflammatory genes, nitric oxide (NO) synthase 2, IL-6, chemokine (C-C motif) ligand 2 (CCL2)/monocyte chemotactic protein 1 (MCP1), chemokine (C-X-C motif) ligand 1 (CXCL1)/keratinocyte chemoattractant (KC), and up-regulation of IL-6, KC, MCP-1, and transforming growth factor-β1 levels in bronchoalveolar lavage fluid. Bronchoalveolar lavage fluid also contained an increased level of surfactant protein (SP)-D, but not SP-A, significant reduction of SP-B levels, and a resultant increase in alveolar surface tension. Consistent with the growth of TSC2-null lesions, NO levels were also increased and, in turn, modified SP-D through S-nitrosylation, forming S-nitrosylated SP-D, a known consequence of lung inflammation. Progressive growth of TSC2-null lesions was accompanied by elevated levels of matrix metalloproteinase-3 and -9. This report demonstrates a link between growth of TSC2-null lesions and inflammation-induced surfactant dysfunction that might contribute to lung destruction in LAM.

Pharmacological targeting of VEGFR signaling with axitinib inhibits Tsc2-null lesion growth in the mouse model of lymphangioleiomyomatosis

“…All animal procedures were performed according to a protocol approved by the University of Pennsylvania Animal Care and Use Committee. Briefly, 8-wk-old athymic nude female mice (NCRNU, Taconic) were injected with TMKOC cells (10 6 /100 l of PBS) into the tail vein, as previously described (4,13). Starting 3 days postinjection, mice received daily intraperitoneal injections of either diluent or 25 mg of axitinib (AG-013736; Tocris Bioscience, Bristol, UK; batch no.…”

“…Cells isolated from bronchoalveolar lavage (BAL) were collected, counted (Beckman-Coulter, Miami, FL), and differentiated, and the remaining cells, not used for differential analysis, were frozen and further analyzed by quantitative RT-PCR (qRT-PCR), as previously described (4,14). Aliquots of the cell-free BAL fluid were stored at Ϫ80°C and further analyzed for cytokine levels by Multiplex (Aushon, Billerica, MA), and nitric oxide (NO) metabolites by chemical reduction and chemiluminescence using the Ionics/Sievers Nitric Oxide Analyzer 280 (NOA 280; Ionics Instruments, Boulder, CO), as previously described (3,4). Following lavage, lungs were fixed in formalin for hematoxylin and eosin staining and immunohistochemical (IHC) analysis.…”

“…The reactions were carried out with SYBR Green PCR master mix on an ABI Prism 7300 sequence detection system (Applied Biosystems). Obtained Ct values were normalized to ␤-actin signals and further analyzed using the ⌬⌬Ct method, as previously described (4,14,28 t-test. In experiments with multiple treatment groups, significance tests were made within the confines of a significant two-way ANOVA and using Bonferroni correction for multiple comparisons.…”

Zoledronic acid inhibits TSC2-null cell tumor growth via RhoA/YAP signaling pathway in mouse models of lymphangioleiomyomatosis

“…Lymphangioleiomyomatosis (LAM) is a destructive cystic lung disease caused by LAM cells, which invade all the lung structures, including the lymphatics, airway wall, blood vessels, and interstitial spaces, therefore limiting the delivery of oxygen to the body. Moreover, LAM is a rare, progressive mesenchymal neoplasm mainly affecting childbearing age women [1][2][3][4]. The LAM cases are sometime difficult to diagnose because the disease symptoms are similar to other lung diseases (such as asthma, emphysema, and bronchitis).…”

Surfactant protein D multimerization and gene polymorphism in COPD and asthma

“…Previous studies have shown that multimerized (high molecular weight (HMW)) and non‐multimerized (low molecular weight (LMW)) SP‐D are interconvertible structures . The HMW/LMW ratio is dependent on several factors such as posttranslational modifications of SP‐D by glycosylation and, in surrounding inflammatory conditions, by nitrosylation, oxidative damage and proteolytic degradation which result in degradation of SP‐D multimers and release of LMW breakdown products …”