Prevention of alveolar destruction and airspace enlargement in a mouse model of pulmonary lymphangioleiomyomatosis (LAM)

Abstract

Pulmonary lymphangioleiomyomatosis (LAM) is a rare genetic disease characterized by neoplastic growth of atypical smooth muscle-like LAM cells, destruction of lung parenchyma, obstruction of lymphatics, and formation of lung cysts, leading to spontaneous pneumothoraces (lung rupture and collapse) and progressive loss of pulmonary function. The disease is caused by mutational inactivation of the tumor suppressor gene tuberous sclerosis complex 1 (TSC1) or TSC2. By injecting TSC2-null cells into nude mice, we have developed a mouse model of LAM that is characterized by multiple random TSC2-null lung lesions, vascular endothelial growth factor-D expression, lymphangiogenesis, destruction of lung parenchyma, and decreased survival, similar to human LAM. The mice show enlargement of alveolar airspaces that is associated with progressive growth of TSC2-null lesions in the lung, up-regulation of proinflammatory cytokines and matrix metalloproteinases (MMPs) that degrade extracellular matrix, and destruction of elastic fibers. TSC2-null lesions and alveolar destruction were differentially inhibited by the macrolide antibiotic rapamycin (which inhibits TSC2-null lesion growth by a cytostatic mechanism) and a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, simvastatin (which inhibits growth of TSC2-null lesions by a predominantly proapoptotic mechanism). Treatment with simvastatin markedly inhibited MMP-2, MMP-3, and MMP-9 levels in lung and prevented alveolar destruction. The combination of rapamycin and simvastatin prevented both growth of TSC2-null lesions and lung destruction by inhibiting MMP-2, MMP-3, and MMP-9. Our findings demonstrate a mechanistic link between loss of TSC2 and alveolar destruction and suggest that treatment with rapamycin and simvastatin together could benefit patients with LAM by targeting cells with TSC2 dysfunction and preventing airspace enlargement.

Figures

Fig. 1

TSC2-null lung lesions induce alveolar destruction. (A) Immunoblot analysis of TSC2-null and TSC2-positive LLC cells with specific antibodies to detect indicated proteins. (B) DNA synthesis and invasion of TSC2-null, control epithelial NMuMG (Contr), human LAM cells, and control human lung fibroblasts (HLF). Data are means ± SE from three independent measurements. **P < 0.001 for Contr versus TSC2-null and HLF versus LAM by analysis of variance (ANOVA) (Bonferroni-Dunn). (C) Lungs of vehicle-injected (Control) and TSC2-null cell–injected female NCr athymic nude (NCRNU-M) mice at day 15 after injection. (D) H&E analysis of lungs at day 15 after tail vein injection of vehicle (Control) or TSC2-null, LLC, or TSC2+/+p53−/− cells. Scale bars, 100 μm (top) and 1000 μm (bottom). (E and F) Analysis of MLI (E) and MAAA (F) of lungs from NCRNU-M and C57BL/6J mice at day 15 after injection of vehicle (Contr) or TSC2-null, LLC, or TSC2+/+p53−/− cells. Data are means ± SE of n > 8 in each group by ANOVA (Bonferroni-Dunn). **P < 0.001 for Contr versus TSC2-null cell–injected NCRNU-M mice; *P < 0.05 for Contr versus TSC2-null cell–injected C57BL/6J mice.

Fig. 2

Time-dependent alveolar airspace enlargement is associated with TSC2-null lesion growth in the lung. (A) Representative images of H&E-stained lungs collected at days 0, 10, 15, and 20 after injection. Scale bar, 200 μm. (B and C) Lesion/lung ratio and MAAA, calculated with Image-Pro Plus program. (B) Data are means (percentage of the lesion area to the total lung area) ± SE of n = 8 in each group. *P < 0.01 for day 15 versus day 0; **P < 0.001 for day 20 versus day 0 by Fisher. (C) Data are means ± SE of n > 5 in each group. *P < 0.01 for day 15 versus day 0; **P < 0.001 for day 20 versus day 0 by Fisher.

Fig. 3

SM α-actin–positive TSC2-null lesions show mTORC1 activation, increased VEGF-D, and lymphangiogenesis. (A) Fluorescence-activated cell sorting (FACS) analysis of epithelial NMuMG cells (Control), TSC2-null cells, control HLF cells, and human LAM cells with fluorescein isothiocyanate–conjugated SM α-actin antibody (purple) and control immunoglobulin G (green). Data are means ± SE from three independent experiments. *P < 0.001 for Control versus TSC2-null and HLF versus LAM by ANOVA (Bonferroni-Dunn). (B to E) Lung tissue sections stained for SM α-actin (B), P-S6 (C), VEGF-D (D), and LYVE-1 (E). Mouse lung specimens (Control, TSC2-null, and LLC) collected at day 20 after vehicle, TSC2-null, or LLC cell injection, and control and LAM human lung specimens were subjected to immunohistochemical analysis with anti–SM α-actin (green), anti–P-S6 (red), anti–VEGF-D (red), and anti–LYVE-1 (red) antibodies. 4',6-Diamidino-2-phenylindole staining (blue) indicates nuclei. Representative images were taken with a Nikon Eclipse TE-2000E microscope. V, vessel. Scale bars, 100 μm.

Fig. 4

Increases in MMPs, inflammatory cells, and cytokines and decrease in alveolar elastin are associated with TSC2-null lung lesion growth. (A to C) MMP expression assessed in the cell-free supernatant of the BAL fluid at the indicated time points. A multiplex assay was performed by Searchlight technology (Aushon Biosystems). Data are means ± SE of n = 11 in each group. ***P < 0.001 for days 15 and 20 versus day 0 by ANOVA (Bonferroni-Dunn). (D) Schematic representation of elastin and collagen disposition in the alveolar neck. (E) Volumes of elastin (Eln) and collagen (Col) in alveoli of Control and TSC2-null lesion–carrying mice. Elastin and collagen were analyzed in alveoli necks of vehicle-injected (Contr) and TSC2-null cell–injected (TSC2-null) mice and expressed as percentage of the total volume of the alveolar neck. Data are means ± SE. **P < 0.01 for Control versus TSC2-null lesion–carrying mice by ANOVA (Bonferroni-Dunn). (F and G) Representative images of alveoli necks of vehicle-injected (Control) (F) and TSC2-null cell–injected (TSC2-null) (G) mice. Verhoeff's elastin stain and Picro-Ponceau counterstain were used to detect elastin (black) and collagen (red), respectively. Arrowheads, elastin-enriched areas. Scale bar, 10 μm. (H to L) Proinflammatory cytokine and chemokine expression assessed with a multiplex assay in the cell-free supernatant of BAL fluid from mice at the indicated time points after tumor inoculation. (M) Inflammatory cell counts assessed from the BAL collected at times as indicated after tumor cell inoculation. Data are means ± SE of n = 11 in each group. *P < 0.05; **P < 0.01; ***P < 0.001 versus day 0 by ANOVA (Bonferroni-Dunn).

Fig. 5

Rapamycin plus simvastatin rescues animal survival, prevents lesion growth and lung destruction, and abrogates MMP induction. Mice injected with diluent (Control) or TSC2-null cells were treated with vehicle, rapamycin (Rapa), simvastatin (Simva), and rapamycin + simvastatin (Rapa + Simva) from day 3 after injection. (A) Weight of control (black) and TSC2-null cell–injected (red) mice were examined from day 3 (arrows) to day 20 of experiment. Data are means ± SE of n > 5 in each group. *P < 0.01 for Control versus TSC2-null cell–injected mice by ANOVA (Bonferroni-Dunn). Arrows, beginning of treatment. (B to D) H&E staining of murine lungs. Scale bar, 500 μm. (B) Lesion/lung ratio (C) and MAAA analysis (D) were performed at day 20 after injection. Data are means ± SE of n > 8 in each group. **P < 0.001 for Control versus TSC2-null cell–injected vehicle-treated mice and for Rapa, Simva, and Rapa + Simva versus vehicle for TSC2-null cell–injected mice by ANOVA (Bonferroni-Dunn). (E to G) Expression of MMP-2 (E), MMP-3 (F), and MMP-9 (G), assessed in the cell-free supernatant of BAL at day 20 by multiplex assay. Data are means ± SE of n > 6 in each group. **P < 0.001 for compound- versus vehicle-treated mice by ANOVA (Bonferroni-Dunn).

Fig. 6

Rapamycin and simvastatin differentially affect TSC2-null lesion growth and airspace enlargement. Mice, injected with TSC2-null cells, were treated with vehicle (−), rapamycin (Rapa), simvastatin (Simva), and rapamycin + simvastatin (Rapa + Simva) from day 10 after injection of TSC2-null cells. (A to D) Effects of drug treatment on lesion growth and airspace enlargement. (A) DNA synthesis (a percentage of Ki67-positive cells per total number of cells), (B) P-S6 [optical density (OD)], (C) apoptosis [a percentage of terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling (TUNEL)–positive cells per total number of cells], and (D) percentage of lesion tissue per total lung area at day 20 after injection. Data are means ± SE of n > 10 in each group. *P < 0.001 for compound- versus vehicle-treated animals by ANOVA (Bonferroni-Dunn). (E and F) MLI (E) and MAAA (F) analyses of lung tissue sections collected at day 20 after injection. Data are means ± SE of n > 8 in each group by ANOVA (Bonferroni-Dunn). Gray bars, naïve (non-injected vehicle-treated) animals.