α-1 Antitrypsin (AAT) deficiency is the leading genetic cause of emphysema; however, until recently, no genuine animal models of AAT deficiency existed, hampering the development of new therapies. This shortcoming is now addressed by both AAT-null and antisense oligonucleotide mouse models. The goal of this study was to more fully characterize the antisense oligonucleotide model. Both liver AAT mRNA and serum AAT levels were lower in anti-AAT versus control oligonucleotide-treated mice after 6, 12, and 24 wk. Six and twelve weeks of anti-AAT oligonucleotide therapy induced emphysema that was worse in female than male mice: mean linear intercept 73.4 versus 62.5 μm ( P = 0.000003). However, at 24 wk of treatment, control oligonucleotide-treated mice also developed emphysema. After 6 wk of therapy, anti-AAT male and female mice demonstrated a similar reduction serum AAT levels, and there were no sex or treatment-specific alterations in inflammatory, serine protease, or matrix metalloproteinase mRNAs, with the exception of chymotrypsin-like elastase 1 ( Cela1), which was 7- and 9-fold higher in anti-AAT versus control male and female lungs, respectively, and 1.6-fold higher in female versus male anti-AAT-treated lungs ( P = 0.04). While lung AAT protein levels were reduced in anti-AAT-treated mice, lung AAT mRNA levels were unaffected. These findings are consistent with increased emphysema susceptibility of female patients with AAT-deficiency. The anti-AAT oligonucleotide model of AAT deficiency is useful for compartment-specific, in vivo molecular biology, and sex-specific studies of AAT-deficient emphysema, but it should be used with caution in studies longer than 12-wk duration.
We have shown that Chymotrypsin-like Elastase 1 (CELA1) is expressed in human lung, neutralized by alpha-1 antitrypsin (AAT), and important for emphysema in a mouse model of AAT-deficiency. Here, we show a role for CELA1 in late lung remodeling using human specimens and mouse models. Methods: Studies were approved by CCHMC IRB and IACUC. In mice, emphysema following 2 units of tracheal PPE was assessed at 21, 42, and 84 days and in aged 72-75 week-old WT and Cela1-/-lungs. Lungs of non-lung organ donors, COPD, and aged healthy subjects (n=38 total) were used for PCR, IHC, IF, PLISH, enzymatic assays, and stretch-binding assays. DESeq2 of major airway, small airway, and BAL mRNA-seq was performed. Results: In the mouse PPE model, Cela1 mRNA and protein were increased 3-fold at 21 and 42 days post-PPE (p<0.05). Cela1-/-and WT mice had similar emphysema at 21-days, but WT demonstrated progressive emphysema at 42 and 84-days (p<0.01) while Cela1-/-did not (p<0.01 vs WT). PLISH demonstrated Cela1 mRNA in the epithelium of small conducting airways. Cela1-/-mice had less age-related airspace enlargement than WT (p<0.05). CELA1 protein was increased 3-fold in COPD specimens compared to healthy (p<0.05), and PLISH showed CELA1 mRNA in human conducting airway epithelium. Comparing mRNA-seq of large airway, conducting airway, and BAL, CELA1 mRNA was present only in small conducing airway of COPD and controls subjects. PCA showed CELA1-expressing small airway specimens of normal and COPD clustered with enrichment in secretory pathways. In healthy and COPD specimens, CELA1 mRNA levels correlated with MMP12, but not MMP2, MMP8, MMP9, MMP14, PRTN3, CTSG, or ELANE. Among these mRNAs, only CELA1 and MMP12 correlated with lung elastolytic, gelatinase, and proteinase activity, and only CELA1 was significant (p<0.001). anti-CELA1 antibody and serine proteinase inhibition reduced the elastolytic activity of CELA1 mRNA-high lung homogenate. CELA1 mRNA levels increased exponentially with age, and WB of aged and young lung showed reduced AAT neutralized CELA1 in aged, smoker lung. Biaxial stretching of human lung sections using fluorophore-labeled CELA1 and albumin showed 20-fold increased binding of CELA1 but not albumin with stretch (p<0.05) in healthy, but not COPD lung. Conclusions: We propose a 3-hit model for CELA1mediated airspace destruction in human emphysema. 1-CELA1 expression in small conducting airways increases with age, 2-smoking reduces AAT-neutralization of CELA1, and 3-increased strain from disruption of adjacent alveolar units allows CELA1 binding to lung elastin leading to feed-forward alveolar destruction.
Emphysema is an important element of many progressive lung diseases, with chronic obstructive pulmonary disease (COPD) being the most common. With the exception of α 1-antitrypsin (AAT) replacement therapy there are no disease modifying therapies for progressive emphysema. We previously reported that alveolar type 2 (AT2)-cell synthesized CELA1 is neutralized by AAT and that CELA1 is necessary for emphysema in AAT-deficiency. Here, we use mouse models and human tissues to show that CELA1 is required for progressive emphysema. In mice, lung injury was induced with tracheal porcine pancreatic elastase. Cela1 began increasing at 21-days, and Cela1-/- mice were protected from continued airspace enlargement at 42 and 84 days (p<0.01). Aged Cela1-/- mice had less airspace simplification than aged WT mice (p<0.05). In humans and mice, CELA1 mRNA and protein were present in subsets of conducing airway epithelial and AT2 cells. COPD lungs had 3-fold more CELA1 protein than control (p<0.05). Among COPD-associated proteases, only CELA1 was positively and significantly correlated with lung elastolytic activity (p<0.001). Rabbit polyclonal and mouse monoclonal anti-CELA1 antibodies inhibited elastolytic activity of CELA1 mRNA-high but not CELA1 mRNA-low human lungs. CELA1 mRNA levels increased exponentially with age, and smoking reduced that ratio of AAT-neutralized:native CELA1 (p<0.05). CELA1 binding to lung tissue increased 6-fold with biaxial strain (p<0.05). We propose that CELA1 predisposes to progressive emphysema via (1) increased expression with age, (2) reduced AAT neutralization with smoking, and (3) increased CELA1-binding to lung matrix with strain. Anti-CELA1 therapies may represent a novel disease modifying therapy to prevent emphysema progression.
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