Enhanced Notch3 signaling contributes to pulmonary emphysema in a Murine Model of Marfan syndrome

Marfan syndrome (MFS) is a heritable disorder of connective tissue, caused by mutations in the fibrillin-1 gene. Pulmonary functional abnormalities, such as emphysema and restrictive lung diseases, are frequently observed in patients with MFS. However, the pathogenesis and molecular mechanism of pulmonary involvement in MFS patients are underexplored. Notch signaling is essential for lung development and the airway epithelium regeneration and repair. Therefore, we investigated whether Notch3 signaling plays a role in pulmonary emphysema in MFS. By using a murine model of MFS, fibrillin-1 hypomorphic mgR mice, we found pulmonary emphysematous-appearing alveolar patterns in the lungs of mgR mice. The septation in terminal alveoli of lungs in mgR mice was reduced compared to wild type controls in the early lung development. These changes were associated with increased Notch3 activation. To confirm that the increased Notch3 signaling in mgR mice was responsible for structure alterations in the lungs, mice were treated with N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglucine t-butyl ester (DAPT), a γ\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\gamma$$\end{document}-secretase inhibitor, which inhibits Notch signaling. DAPT treatment reduced lung cell apoptosis and attenuated pulmonary alteration in mice with MFS. This study indicates that Notch3 signaling contributes to pulmonary emphysema in mgR mice. Our results may have the potential to lead to novel strategies to prevent and treat pulmonary manifestations in patients with MFS.

www.nature.com/scientificreports/ Notch signaling is essential for lung development and is required for maintaining the integrity of both the epithelial and smooth muscle layers of the distal airway [14][15][16] . Notch genes encode large single-transmembrane receptors responsible for mediating communication between neighboring cells; this communication is crucial to direct cell fate decisions during organ development 15 . Four Notch receptors have been identified in mammals: Notch1, Notch2, Notch3, and Notch4. They are composed of a large extracellular domain, which mediates ligand interaction; a transmembrane domain; and an intracellular domain. In the canonical signaling pathway, Notch receptors are proteolytically cleaved by several proteinases upon receptor-ligand binding at the cell surface. One major proteinase responsible for Notch cleavage is γ-secretase 15 . Proteolytic cleavage of Notch releases the Notch intracellular domain (NICD) and allows it to translocate to the nucleus, where it interacts with transcription factor CBF-1 to promote transcriptional activation of downstream effectors.
Notch signaling plays a critical role in differentiation, regeneration, and repair of the airway epithelium [16][17][18][19][20][21][22][23] . In the airway epithelium of the developing embryo, Notch signaling maintains a balance between ciliated, secretory, and neuroendocrine cells 17,18 . Constitutive Notch signaling inhibits the differentiation of alveolar epithelium 16 . Postnatally, Notch signaling in the epithelium is responsible for a host of processes involved in development of the distal lung 19 . It also prevents epithelial club cells from differentiating into goblet cells and plays a critical role in recovery of the airway epithelium following injury [20][21][22] . Of the four Notch receptors, Notch3 has been found to play the most critical role in regulating alveolar epithelium. A gain of function in Notch interferes with distal alveolar formation by disrupting differentiation processes 16 . Constitutive Notch3 expression in the peripheral epithelium inhibits type II pneumocytes from differentiation into type I pneumocytes; this results in an altered lung morphology 23 . Histological and functional changes were observed in both lung development and maturation of patients and a murine model of MFS 10 . However, the mechanism by which Notch3 exerts its function in pulmonary emphysema in MFS remains unknown.
To investigate the influence of Notch signaling in MFS alveolar development, we studied the role of Notch3 on pulmonary morphogenesis in MFS mice. Here we show the pulmonary emphysematous changes were observable at a very early stage in the development of MFS mice. This change is correlated with increased Notch3 activation. We found that inhibition of Notch signaling rescued development of the distal alveoli in MFS mice.

Results
Distal airspace was progressively increased in the lungs of mgR mice. Disorders of the respiratory system have been noted in a distinct subgroup of patients with MFS. Previous studies of mouse models of Marfan syndrome (Fbn1 mgΔ/mgΔ and Fbn1 mgR/mgR ) have shown that Fbn1 mgΔ/mgΔ and Fbn1 mgR/mgR mice developed pulmonary emphysema as early as postnatal day (PD)1 and PD14, respectively 10 . Because they are severely affected, Fbn1 mgΔ/mgΔ mice have a short lifespan, 2-3 weeks, which makes it hard to study the pathogenesis of emphysema and test pharmacologic agents. For this reason, we used a widely accepted murine model of MFS (Fbn1 mgR/mgR or mgR) which is less severely affected allowing later assessment of interventions. Histological sections of lungs were analyzed by H&E staining. Compared to their wild type (WT) littermates, lungs of mgR mice had enlarged distal airspaces at PD7 and PD56 (Fig. 1B, D) which is consistent with previous findings 10 . The mean linear intercept (L m ), as a measure of interalveolar wall distance, was significantly increased in mgR mice compared with WT littermate controls (Fig. 1E). The progressive distal airspace enlargement was seen between PD7 and PD56 in mgR mouse lungs.
Notch3 signaling was increased in the lungs of mgR mice. Notch signaling is essential for lung development. We examined Notch1-4 mRNA levels by real-time PCR. No notable difference in mRNA expression of www.nature.com/scientificreports/ Notch1, 2, and 4 was observed between WT and mgR mice (Supplemental Fig. 1). However, the expression of Notch3 mRNA was significantly increased in the lungs of mgR mice at PD7 and PD56 ( Fig. 2A). The increased Notch3 expression in the lung of mgR mice was confirmed with immunofluorescence staining (Fig. 2B). Constitutive activation of Notch3 resulted in altered lung morphology and inhibition of pneumocyte differentiation and proliferation 23 . We examined Notch3 activation using Western blot analysis. As shown in Fig. 2C-E, the levels of active Notch3 (Notch3 intracellular domain, N3ICD) in mgR mice were increased in both PD7 and PD56. These data don't directly implicate Notch3 in emphysema but identify a putative therapeutic target we believe to have a role in lung development. Since Notch3 is involved in proliferation and differentiation of alveolar epithelial cells (AEC) and vascular smooth muscle cells (SMCs) [23][24][25] , we characterized which cell types were responsible for high production of Notch3 in mgR mice using immunofluorescence staining of lungs from WT and mgR mice at PD56 (Fig. 3A). Notch3 was co-expressed with SMC marker (SMA), type I AEC (AEC-I) marker (AQP5), and type II AEC (AEC-II) marker (SP-C) (Fig. 3A). However, there was more intense staining of Notch3 in the SMCs and AEC-I of mgR mice compared to WT controls. We further assessed cell proliferation in the lungs of WT and mgR mice by immunostaining with Ki67, a proliferation marker. We found no significant difference between WT and mgR mice at PD7 and PD56 (Supplemental Fig. 2). It was reported that pneumocyte apoptosis contributed to emphysematous changes in the lungs of Fbn1 mgΔ/mgΔ mice 10 . We assessed cell apoptosis in the lungs of WT and mgR mice by immunostaining of caspase 3 (CC3), a cell apoptosis marker. We found more caspase 3-positive cells in the lungs of mgR mice compared to that of WT mice at PD7 (Fig. 3B).

Inhibition of Notch activation remarkably reduced pulmonary emphysematous changes in mgR mice.
To determine whether inhibition of Notch3 activation in mgR mice could prevent pulmonary morphological changes, we treated WT and mgR mice with ϒ-secretase inhibitor, DAPT, to inhibit Notch sign-  www.nature.com/scientificreports/ aling. The treatment started at PD10 and mice were sacrificed at PD56. Histological changes of lung tissue were evaluated using H&E staining ( Fig. 4A-D). Quantitation of changes in alveolar space was performed with the mean linear intercept analysis. DAPT treatment had no effect on WT mice but remarkably reduced distal airspace enlargement in mgR mice (Fig. 4E). Notch3 activation levels in lungs were examined by Western blot. Active Notch3 was significantly higher in DMSO-treated mgR mice compared to DMSO-treated controls, but lower in DAPT-treated mgR mice compared to DMSO treatment (Fig. 4F, G).
To further confirm the role of Notch3 activation in pneumocytes, lung cells were isolated from WT and mgR mice. AEC identity was confirmed with immunofluorescent staining (Supplemental Fig. 3). Cells were treated with DAPT for 48 h. As shown in Fig. 5A and B, cells from mgR mice had higher levels of active Notch3 compared with cells from WT mice. DAPT treatment significantly inhibited Notch3 activation in the cells from mgR mice. Furthermore, increased Notch3 translocation to nuclei (white arrowheads) was observed in DMSO-treated cells from mgR mice. DAPT treatment inhibited Notch3 nuclear translocation (Fig. 5C). These data suggest that increased activation of Notch3 in lung cells contributes to emphysematous changes in mgR mice. Inhibition of Notch3 signaling may attenuate pulmonary emphysema in MFS.
To determine whether inhibition of Notch activation signaling has effects on AEC apoptosis and elastin degradation in the lungs of mgR mice, we evaluated apoptosis of AECs in mgR mice with or without DAPTtreated mice using immunostaining of caspase 3 ( Fig. 6A-D). We found fewer caspase3-positive cells in the lungs of DAPT-treated mgR mice compared to DMSO-treated mgR mice (Fig. 6D, E). VVG staining of lung tissue showed elastin degradation and fragmentation in the lungs of mgR mice (Fig. 6G). DAPT treatment preserved lung elastic fiber integrity in mgR mice (Fig. 6I). These results demonstrate that enhanced Notch3 activation stimulates AEC apoptosis and elastic fiber degradation in mgR mice.

Discussion
Pulmonary dysfunction is one of the common manifestations in MFS. However, the pathogenesis and molecular mechanisms of pulmonary alterations in MFS are unknown. Notch3 signaling plays an important role in regulating the formation and development of alveolar epithelium. The purpose of the present study was to investigate www.nature.com/scientificreports/ the role of Notch signaling in the pathogenesis of pulmonary emphysema in MFS. By using mgR mice, a murine model of MFS, we have shown that emphysematous changes are apparent at an early age, PD7. The distal airspace enlargement was progressively increased with age. We have identified increased Notch3 activation in the lungs of mgR mice. Furthermore, Notch inhibition using a γ-secretase inhibitor, DAPT, attenuated lung morphological changes. These data suggest that enhanced Notch3 activation contributes to pulmonary morphological changes in mgR mice. MFS is chiefly caused by mutations in the FBN1 gene, which encodes for the extracellular matrix (ECM) glycoprotein fibrillin-1 26,27 . Fibrillin-1 is the main component of microfibrils, and it associates with elastin to form elastic fibers in the ECM. Currently, there are three commonly used murine models of MFS: Fbn1 mgΔ/mgΔ , Fbn1 mgR/mgR (mgR), and Fbn1 C1039G/+ . Fbn1 mgΔ/mgΔ mice represent a severe form of the disease, expressing approximately 10% of normal fibrillin-1. These mice die of cardiovascular complications within 2-3 weeks of age 12 . The mgR model demonstrates a hypomorphic mutation of FBN1, and homozygous mice produce approximately 20% of normal fibrillin-1. These mice display clinical features and manifestations similar to classical MFS patients, and they die naturally at an average age of 3-6 months 13 . C1039G/+ is a heterozygous missense mutation of  27 . Previous studies showed that dysregulated TGF-β activation contributed to MFS pathogenesis 28,29 . Inhibiting TGF-β activation with a TGF-β-neutralizing antibody improved alveolar septation in the lungs of Fbn1 mgΔ/mgΔ mice 10 . However, this pharmacological inhibition of TGF-β underscores the complex and context-dependent roles of TGF-β in MFS. While previous studies investigating systemic neutralization of TGF-β in the Fbn1 C1039G/+ model prevented the formation of thoracic aortic aneurysm (TAA) 28 , later studies using the mgR model demonstrated that TGF-β exerted an opposing effect on TAA pathology: TGF-β neutralization broadly correlated with both early-and late-stage TAA progression 30 . The authors showed that early (PD16) treatment with TGF-β-neutralizing antibodies exacerbated TAA formation, while later (PD45) treatment demonstrated a contrasting beneficial effect 30 . Our previous study supported that the initial consequence of FBN1 mutation was not accompanied by a significant increase in TGF-β activation 31 .
These contrasting examples of TGF-β contribution to aortic physiology in both early and late MFS stages provide a strong rationale for this study. The identification of new biomarkers independent of the TGF-β signaling pathway, such as Notch, is crucial to better understand the origin of pulmonary emphysema development in MFS. Notch signaling plays a critical role in the development of the respiratory system 19 . Notch2 is the primary receptor involved in Clara/ciliated cell fate selection 32 . Notch1-3 are involved in regulating pulmonary neuroendocrine cell fate selection 14 . Notch4 is an endothelial cell-specific mammalian Notch gene 33 . In order to determine the role of Notch signaling in lung development and pulmonary impairment related to MFS, we examined the expression of Notch1-4 receptors in the lungs of mgR and WT mice. We found that Notch3 expression was significantly increased in the lungs of mgR mice. To isolate the role of Notch3 in MFS-related pulmonary morphological changes, we evaluated the histological differences between lungs of WT and mgR mice. Apparent structural alterations were observed in the lungs of 1-week-old mgR mice compared to WT controls. Alveolar septation was progressively reduced in mgR mice with age. This change is associated with increased Notch3 activation. Previous studies demonstrate that fibrillin-1 regulates Notch expression, and abnormal fibrillin-1 expression impacts Notch signaling 34 . Fibrillin-1 expression is critical for latent TGF-β binding protein (LTBP)-1, -3, and -4 to incorporate into lung tissue 35 . LTBP-1 deposition in the ECM was shown to be significantly decreased in www.nature.com/scientificreports/ association with fibrillin-1 knockdown in retinal epithelial cells 36 . LTBP-1 can directly bind to the Notch3 extracellular domain 37 . Therefore, we hypothesized that fibrillin-1 mutation leads to enhanced activation/signaling of Notch3 which in turn impairs lung development, specifically distal airspace septation. We administered DAPT, a γ-secretase inhibitor, to mgR mice starting at PD10. DAPT inhibits Notch3 signaling by preventing the proteolysis of Notch3. Histological analysis of the lungs in mgR mice at PD56 showed that DAPT treatment attenuated pulmonary alteration and suppressed the Notch3 activity in lung cells of mgR mice. Furthermore, fibrillin-1 deficiency stimulated lung cell apoptosis. Notch3 activation can induce the intrinsic apoptotic cascade 38 . Our results showed that inhibition of Notch3 by DAPT treatment reduced lung cell apoptosis and preserved the integrity of elastic fibers in the lungs of mgR mice. These results demonstrate that increased Notch3 activation in fibrillin-1 deficient mgR mice contributes to lung emphysematous changes by inducing apoptosis and elastin degradation. However, further studies are required to investigate potential crosstalk between Notch3 and fibrillin-1.
Several studies have suggested that in the mammalian lung, Notch signaling is linked to a variety of diseases, including chronic obstructive pulmonary disease (COPD), asthma, pulmonary fibrosis, and lung lesions in some congenital disease 23,[39][40][41] . Notch3 has been show to play a role in regulating alveolar epithelium; specifically, its constitutive expression alters lung morphology by inhibiting type II pneumocytes from differentiating into type I pneumocytes 23 . Conversely, another study demonstrated that Notch3 was downregulated in both adult smokers and smokers with COPD 42 . These studies suggest that Notch3 is critical to maintain normal epithelial cell fate decision pathways in the airway epithelium. Differential thresholds of Notch3 signaling activation may determine normal or abnormal lung development. Our data demonstrate a direct link between enhanced Notch3 activation/signaling and impairment of distal alveolar septation. This pulmonary pathology can be attenuated with treatment of a drug to inhibit Notch signaling. Our results suggest that inhibition of Notch3 signaling in the MFS lung may prove to be an effective strategy in prevention and treatment of MFS-related pulmonary alterations.

Methods
Animals. All animal protocols in this study were reviewed and approved by the Institutional Animal Care and Use Committee for the University of Nebraska Medical Center (Permission number: 17-074-08FC). All experiments and procedures were performed in accordance with the regulations and guidelines set forth by the University of Nebraska Medical Center Animal Care Committee for the use and care of laboratory animals. Heterozygous mutant mice (Fbn1 mgR/+ or mgR/+) in a mixed C57BL/6J;129 SvEv background were mated to generate homozygous Fbn1 mutant mice (Fbn1 mgR/mgR or mgR) and wild type (WT) littermates. Mice were genotyped at postnatal day (PD)7 by PCR 43,44 . Because male and female mice were equally affected, both sexes were used in this study. WT littermates and homozygous mgR mice were sacrificed at PD7 (n = 20/group) and PD56 (n = 16/group). Typically these mice weigh 4-5 g at PD7 and 20-25 g at PD56. Mouse lungs were inflated and perfusion-fixed with 10% neutral buffered formalin 45 or collected for RNA and protein extraction. For histological studies, the lower left lobe of the lungs was ligated, excised, immersed in formalin, and fixed for histological studies. In order to identify the role of Notch3, WT (n = 8/group) and mgR (n = 8/group) mice were treated with γ-secretase inhibitor, N-[N-(3,5-Difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT) (Abcam, Cambridge, UK). DAPT was suspended in DMSO. Mice were injected subcutaneously with 10 mg/kg of DAPT daily beginning at PD10 until sacrifice at PD56. A control group received DMSO only. After treatment, mice were sacrificed. Mouse lungs were perfusion-fixed for histology or collected for protein extraction. All mice were housed in the pathogen-free animal facility for the duration of the protocol.
Histology and morphometric analysis. After 24 h fixation in formalin, the lungs were embedded in paraffin following our previous protocol 31 . Lung sections were cut and stained with hematoxylin for 1.5 min and eosin for 30 s (H&E) according to the manufacturer protocol (Abcam). Morphometric analyses utilized two 4-μm paraffin-embedded, H&E-stained sections of the lower left lobe from each of the 8-10 mice. Each section was subjected to mean linear intercept analysis according to previously published methods 46,47 . The mean linear intercept, a measure of interalveolar wall distance, was determined by light microscopy at a total magnification of × 100 and obtained by dividing the total length of a line drawn across each lung section by the total number of intercepts encountered in 20 lines per section 47 . As previously performed by our lab, mouse lungs were stained for connective tissue using Verhoeff solution, ferric chloride, sodium thiosulfate, and Van Gieson solution (BBC Biochemical, Mt. Vernon, WA) 48 . Staining cycles alternated between fixing and washing procedures, as described previously 48 . Each slide was examined and photographed using light microscopy (× 40; Nikon).
Isolation and culture of pulmonary epithelial cells. The pulmonary epithelial cell isolation was done by following the methods from Jansing et al. 49 with minor modifications. WT and mgR mice (n = 6/group) were anesthetized. The lungs were perfused with PBS via the right ventricle to remove red blood cells. Then, a 22G cannula was inserted into the trachea. The digestion buffer, dispase (5 units/ml) and DNase (0.01%) (Sigma), was instilled into the lungs through the cannula. After 5 min, the lungs were dissected and incubated with digestion buffer for an hour at 37 °C. The cells were washed with PBS. Erythrocytes were lysed with erythrocyte lysis buffer (Gibco, Gaithersburg, MD). The cells were washed again and re-suspended in lung epithelial cell specific medium (Gibco), 40% RPMI-1640, 40% LHC-9, 20% fetal bovine serum. The cells were cultured on bovine collagen-coated plates (Advanced Biomatrix, San Diego, CA). Cells were stained with CD326 (EpCAM) and AQP5 antibodies (Invitrogen, Waltham, MA) to confirm the purity of alveolar epithelial cells. To study notch signaling in lung cells, we treated cells from WT and mgR mice with 20 μM DAPT for 48 h. DMSO treatment was used as a control. After treatment, cells were harvested for protein extraction.
Western blot. The right lungs were homogenized. The protein from the lung tissue and cells was extracted with RIPA lysis and extraction buffer (Thermo Scientifi, Waltham, MA). Nuclear protein from fresh lung tissues was isolated using Nuclear Extraction Kit (Abcam), according to the manufacturer's protocol. The protein concentration was standardized with the Bio-Rad Protein Assay Dye Reagent Concentrate #5,000,006 (Bio-Rad Laboratories, Hercules, CA). Thirty-five to seventy μg of protein extracts were loaded into 4-20% Criterion TGX precast gels (Bio-Rad Laboratories). Following electrophoresis, the gel was transferred onto a 0.45 μm PVDF membrane (Bio-Rad Laboratories). The membrane was incubated overnight at 4 °C with antibodies directed against Notch3 (1:1,000), β-tubulin (1:1,000), GAPDH (1:1,000), or TBP (1:1,000) (Cell Signaling, Beverly, MA). Bound primary antibodies were detected with HRP-conjugated, species specific, secondary antibodies (1:1,000) (Cell Signaling) using the Clarity Western ECL system (Bio-Rad Laboratories). The quantification was done using NIH imageJ software and standardized by internal loading controls.
Statistical analyses. Data are expressed as mean value ± the standard error (SE) of the mean. For continuous variables, if the data were normally distributed, the Student's t test (comparison between two groups) or ANOVA with the appropriate post hoc test (comparison among groups of three or more) were used. Statistical significance was accepted at a P < 0.05. www.nature.com/scientificreports/