Pulmonary epithelial neuropilin-1 deletion enhances development of cigarette smoke-induced emphysema

Abstract

Rationale: Cigarette smoke (CS) exposure is an important risk factor for chronic obstructive pulmonary disease; however, not all smokers develop disease, suggesting that other factors influence disease development.

Objectives: We sought to determine whether neuropilin-1 (Nrp1), an integral component of receptor complexes mediating alveolar septation and vascular development, was involved in maintenance of normal alveolar structure, and/or altered susceptibility to the effects of CS.

Methods: Transgenic mice were generated to achieve inducible lung-specific deletion of epithelial Nrp1. We determined whether conditional Nrp1 deletion altered airspace size, then compared the effects of chronic CS or filtered air exposure on airspace size, inflammation, and the balance between cell death and proliferation in conditionally Nrp1-deficient adult mice and littermate controls. Finally, we evaluated the effects of Nrp1 silencing on cell death after acute exposure of A549 cells to cigarette smoke extract or short chain ceramides.

Measurements and main results: Genetic deletion of epithelial Nrp1 in either postnatal or adult lungs resulted in a small increase in airspace size. More notably, both airspace enlargement and apoptosis of type I and type II alveolar epithelial cells were significantly enhanced following chronic CS exposure in conditionally Nrp1-deficient adult mice. Silencing of Nrp1 in A549 cells did not alter cell survival after vehicle treatment but significantly augmented apoptosis after exposure to cigarette smoke extract or ceramide.

Conclusions: These data support a role for epithelial Nrp1 in the maintenance of normal alveolar structure and suggest that dysregulation of Nrp1 expression may promote epithelial cell death in response to CS exposure, thereby enhancing emphysema development.

Figure 1.

Lung morphometry after conditional pulmonary epithelial Nrp1 deletion in adult mice. (A) Representative sections (10×) of lungs from littermate controls (top panel), CCSP-rtTA/tetO-Cre/Nrp1^flox/+ (bottom left) and CCSP-rtTA/tetO-Cre/Nrp1^flox/flox (bottom right) mice treated with doxycycline chow from 6 weeks of age for 12 weeks (Bar = 100 μm). (B) Average (± SE) mean linear intercept (MLI) and mean chord length (MCL) increased significantly in CCSP-rtTA/tetO-Cre/Nrp1^flox/flox mice after 12 weeks of conditional Nrp1 deletion (n = 6 mice/group). *P ≤ 0.0l vs. littermates; ^φP ≤ 0.01 vs. CCSP-rtTA/tetO-Cre/Nrp1^flox/+; black bars = MLI; gray bars = MCL.

Figure 2.

Lung morphometry after cigarette smoke (CS) exposure and conditional pulmonary epithelial Nrp1 deletion. (A) Representative histology (10×) from CCSP-rtTA/tetO-Cre/Nrp1^flox/flox mice (bottom panels) and littermates (top panels) after conditional pulmonary epithelial Nrp1 deletion and simultaneous CS or filtered air (FA) exposure for 12 weeks. Prominent intraalveolar macrophages in CS-exposed Nrp1–deficient mice are highlighted (insert, lower right panel) (Bar = 100 μm). (B) Average (± SE), mean linear intercept (MLI) increased and surface-to-volume ratio (S:V) decreased after simultaneous pulmonary epithelial Nrp1 deletion and CS exposure (n = 8–9 mice per group). White bars = CCSP-rtTA/tetO-Cre/Nrp1^flox/flox; black bars = littermates. *P ≤ 0.05 vs. littermates; ^φP ≤ 0.05 vs. CCSP-rtTA/tetO-Cre/Nrp1^flox/flox, FA.

Figure 3.

Proliferating cell nuclear antigen (PCNA) immunostaining after cigarette smoke (CS) exposure and pulmonary epithelial Nrp1 deletion. (A) Representative PCNA (green fluorescence; left panel) and nuclear 4′-6′-diamidino-2-phenylindole (DAPI) (blue fluorescence; middle panel), with merged image (right panel), following CS exposure and conditional Nrp1 deletion (60× magnification). (B) Relative expression (mean ± SE) of PCNA-positive nuclei, normalized to total cell number (assessed by nuclear staining with DAPI). Data were averaged from 10 random fields per mouse, and a minimum of 500 nuclei were counted for each animal. There tended to be fewer PCNA positive cells in CCSP-rtTA/tetO-Cre/Nrp1^flox/flox mice compared with littermate controls, but these differences did not achieve statistical significance. Relative expression of PCNA was not altered by CS exposure in mice of either genotype. White bars = CCSP-rtTA/tetO-Cre/Nrp1^flox/flox; black bars = littermates.

Figure 4.

Immunostaining for cleaved caspase 3 after cigarette smoke (CS) exposure and pulmonary epithelial Nrp1 deletion. (A) Representative sections (60×) of lungs from littermate controls (top panels), and CCSP-rtTA/tetO-Cre/Nrp1^flox/flox mice (bottom panels) after simultaneous CS or filtered air (FA) exposure and doxycycline-induced pulmonary epithelial Nrp1 deletion for 12 weeks. Green fluorescence demonstrates cytoplasmic staining for cleaved caspase 3 (white arrows). The number of cells expressing cleaved caspase 3 was normalized to total cell number estimated by nuclear staining with 4′-6′-diamidino-2-phenylindole (DAPI) (blue fluorescence). (B) Relative expression (mean ± SE) of cleaved caspase 3, normalized to DAPI, increased following CS exposure in CCSP-rtTA/tetO-Cre/Nrp1^flox/flox mice. Data were averaged from 10 random fields per mouse, and a minimum of 500 nuclei were counted for each animal. White bars = CCSP-rtTA/tetO-Cre/Nrp1^flox/flox; black bars = littermates; *P < 0.05.

Figure 5.

Increased DNA fragmentation after cigarette smoke (CS) exposure and conditional pulmonary epithelial Nrp1 deletion. (A) Representative images obtained using confocal microscopy following terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining (white fluorescence), and coimmunostaining for type I (T1-α; red fluorescence) and type II (SP-C; green fluorescence) alveolar epithelial cells. TUNEL-positive type I cells are marked by yellow arrows, and type II cells by white arrows. DNA fragmentation following CS exposure was seen in both type I and II alveolar epithelial cells. TUNEL positive cells were quantified after nuclear staining 4′-6′-diamidino-2-phenylindole (DAPI) (blue fluorescence). Five random fields were captured for each animal. Top panels = littermates; bottom panels = CCSP-rtTA/tetO-Cre/Nrp1^flox/flox. (B) Relative expression (mean ± SD) of TUNEL positive type I and type II alveolar epithelial cells increased following CS exposure in CCSP-rtTA/tetO-Cre/Nrp1^flox/flox mice. Data were normalized to values in littermate controls exposed to filtered air (FA). Black bars = FA-exposed littermates; gray bars = CS-exposed littermates; white bars = FA-exposed CCSP-rtTA/tetO-Cre/Nrp1^flox/flox; striped bars = CS-exposed CCSP-rtTA/tetO-Cre/Nrp1^flox/flox; *P < 0.05.

Figure 6.

Effects of cigarette smoke (CS) exposure and conditional pulmonary epithelial Nrp1 deletion on vascular endothelial growth factor (VEGF) concentration. VEGF concentration (mean ± SE), measured using ELISA, in lung lavage fluid (top panel; n = 4–5/group) and lung tissue homogenates (bottom panel; n = 6–7/group). In littermate control mice, VEGF concentration increased significantly after CS exposure compared with filtered air (FA). In contrast, no CS-induced increase in VEGF concentration was seen in Nrp1–deficient mice. A similar trend was seen when VEGF concentration was measured in lung tissue homogenate, although these differences did not reach statistical significance. Top panel, *P < 0.05 vs. littermates; bottom panel, ^φP < 0.05 vs. FA. White bars = CCSP-rtTA/tetO-Cre/Nrp1^flox/flox; black bars = littermates.

Figure 7.

Effects of conditional pulmonary epithelial Nrp1 deletion and cigarette smoke (CS) exposure on expression of Nrp1, p-vascular endothelial growth factor (VEGF)R2, and VEGFR2 in lung lysates. Representative expression of Nrp1, p-VEGFR2, and VEGFR2 by Western blot analysis of lung lysates (top panel), and mean (± SE) changes in expression, assessed by densitometry (lower panel; n = 4–6/group). Expression of Nrp1 was significantly lower in lung lysate from CCSP-rtTA/tetO-Cre/Nrp1^flox/flox mice after filtered air (FA) or chronic CS when compared with FA-exposed littermates. Exposure of littermate control mice to chronic CS also resulted in decreased Nrp1 expression when compared with FA. There were no significant differences detected in expression of either p-VEGFR2 or total VEGFR2 as a result of conditional pulmonary epithelial Nrp1 deletion, or chronic CS exposure in either littermate controls or CCSP-rtTA/tetO-Cre/Nrp1^flox/flox mice. 1 = FA, littermate; 2 = FA, CCSP-rtTA/tetO-Cre/Nrp1^flox/flox; 3 = CS, littermate; 4 = CS, CCSP-rtTA/tetO-Cre/Nrp1^flox/flox. White bars = CCSP-rtTA/tetO-Cre/Nrp1^flox/flox; black bars = littermates; *P < 0.05.

Figure 8.

Effects of Nrp1 silencing on (top panel) cigarette smoke extract (CSE)- and (bottom panel) ceramide-induced apoptosis of A549 cells. Apoptosis of A549 cells following 24 hours of exposure to CSE (10%; n = 6/condition; top panel) or ceramide 8:0 (10 μM; n = 5/condition, bottom panel) was augmented after silencing of Nrp1. Shown for comparison are results in A549 cells transfected with nontargeting (NT) siRNA. In contrast, silencing of Nrp1 had no effect on apoptosis (white bars, both panels) of vehicle-treated cells for either series of experiments. Cell death was assessed using the Apopercentage assay, and results were normalized to vehicle treated, mock-transfected cells. Results shown are mean ± SE. *P < 0.05 vs. vehicle; ^φP < 0.05 vs. NT siRNA. Top panel, white bars = vehicle; gray bars = 10% CSE. Bottom panel, white bars = vehicle; black bars = ceramide 10 μM.

Figure 9.

Effects of ZVAD-fmk on cigarette smoke extract (CSE)- or ceramide-induced apoptosis of A549 cells. Exposure to either 10% CSE (n = 4/condition; top panel, gray bars = 10% CSE; black bars = 10% CSE + ZVAD; white bars = vehicle + ZVAD), or 10 μM ceramide 8:0 (n = 8 /condition; middle panel, gray bars = 10% ceramide 8:0; black bars = ceramide 8:0 + ZVAD; white bars = vehicle + ZVAD) for 24 hours resulted in apoptosis, which was augmented by silencing of Nrp1 and attenuated by pretreatment of cells for 30 minutes with the pan-caspase inhibitor ZVAD-fmk (50 μM). Cell death was assessed using the apopercentage assay (top and middle panels), and results were normalized to vehicle-treated, mock-transfected cells. *P < 0.05 vs. vehicle + ZVAD, CSE + ZVAD; ^φP < 0.05 vs. NT siRNA (top panel). *P < 0.05 vs. vehicle + ZVAD, ceramide + ZVAD; ^φP < 0.05 vs. NT siRNA (middle panel). To confirm that Nrp1 silencing enhanced CSE-induced apoptosis, we also evaluated cell death after exposure to 10% CSE for 24 hours by measurement of nucleosome release in cell lysates using the Cell Death ELISA^PLUS kit (n = 8/condition; bottom panel, gray bars = 10% CSE; black bars = 10% CSE + ZVAD; white bars = vehicle + ZVAD). Results for this assay were normalized to vehicle-treated, mock-transfected cells. Results shown are mean ± SE. *P < 0.05 vs. vehicle + ZVAD, CSE + ZVAD; ^φP < 0.05 vs. NT siRNA (bottom panel).