Adrian Shifren

Mechanisms of Remodeling in Asthmatic Airways

Asthma is a chronic inflammatory airway disorder characterized by airway hyperresponsiveness and reversible airflow obstruction. Subgroups of asthma patients develop airflow obstruction that is irreversible or only partially reversible and experience an accelerated rate of lung function decline. The structural changes in the airways of these patients are referred to as airway remodeling. All elements of the airway wall are involved, and remodeled airway wall thickness is substantially increased compared to normal control airways. Airway remodeling is thought to contribute to the subphenotypes of irreversible airflow obstruction and airway hyperresponsiveness, and it has been associated with increased disease severity. Reversal of remodeling is therefore of paramount therapeutic importance, and mechanisms responsible for airway remodeling are feasible therapeutic targets for asthma treatment. This paper will focus on our current understanding of the mechanisms of airway remodeling in asthma and potential targets for future intervention.

Matrix modulation of compensatory lung regrowth and progenitor cell proliferation in mice

Mechanical stress is an important modulator of lung morphogenesis, postnatal lung development, and compensatory lung regrowth. The effect of mechanical stress on stem or progenitor cells is unclear. We examined whether proliferative responses of epithelial progenitor cells, including dually immunoreactive (CCSP and proSP-C) progenitor cells (CCSP+/SP-C+) and type II alveolar epithelial cells (ATII), are affected by physical factors found in the lung of emphysematics, including loss of elastic recoil, reduced elastin content, and alveolar destruction. Mice underwent single lung pneumonectomy (PNY) to modulate transpulmonary pressure (mechanical stress) and to stimulate lung regeneration. Control mice underwent sham thoracotomy. Plombage of different levels was employed to partially or completely abolish this mechanical stress. Responses to graded changes in transpulmonary pressure were assessed in elastin-insufficient mice (elastin +/-, ELN+/-) and elastase-treated mice with elastase-induced emphysema. Physiological regrowth, morphometry (linear mean intercept; Lmi), and the proliferative responses of CCSP+/SP-C+, Clara cells, and ATII were evaluated. Plombage following PNY significantly reduced transpulmonary pressure, regrowth, and CCSP+/SP-C+, Clara cell, and ATII proliferation following PNY. In the ELN+/- group, CCSP+/SP-C+ and ATII proliferation responses were completely abolished, although compensatory lung regrowth was not significantly altered. In contrast, in elastase-injured mice, compensatory lung regrowth was significantly reduced, and ATII but not CCSP+/SP-C+ proliferation responses were impaired. Elastase injury also reduced the baseline abundance of CCSP+/SP-C+, and CCSP+/SP-C+ were found to be displaced from the bronchioalveolar duct junction. These data suggest that qualities of the extracellular matrix including elastin content, mechanical stress, and alveolar integrity strongly influence the regenerative capacity of the lung, and the patterns of cell proliferation in the lungs of adult mice.

Mice Lacking the Extracellular Matrix Protein MAGP1 Display Delayed Thrombotic Occlusion Following Vessel Injury

Mice lacking the extracellular matrix protein microfibril-associated glycoprotein-1 (MAGP1) display delayed thrombotic occlusion of the carotid artery following injury as well as prolonged bleeding from a tail vein incision. Normal occlusion times were restored when recombinant MAGP1 was infused into deficient animals prior to vessel wounding. Blood coagulation was normal in these animals as assessed by activated partial thromboplastin time and prothrombin time. Platelet number was lower in MAGP1-deficient mice, but the platelets showed normal aggregation properties in response to various agonists. MAGP1 was not found in normal platelets or in the plasma of wild-type mice. In ligand blot assays, MAGP1 bound to fibronectin, fibrinogen, and von Willebrand factor, but von Willebrand factor was the only protein of the 3 that bound to MAGP1 in surface plasmon resonance studies. These findings show that MAGP1, a component of microfibrils and vascular elastic fibers, plays a role in hemostasis and thrombosis.

Elastin insufficiency predisposes to elevated pulmonary circulatory pressures through changes in elastic artery structure

Elastin is a major structural component of large elastic arteries and a principal determinant of arterial biomechanical properties. Elastin loss-of-function mutations in humans have been linked to the autosomal-dominant disease supravalvular aortic stenosis, which is characterized by stenotic lesions in both the systemic and pulmonary circulations. To better understand how elastin insufficiency influences the pulmonary circulation, we evaluated pulmonary cardiovascular physiology in a unique set of transgenic and knockout mice with graded vascular elastin dosage (range 45-120% of wild type). The central pulmonary arteries of elastin-insufficient mice had smaller internal diameters (P < 0.0001), thinner walls (P = 0.002), and increased opening angles (P = 0.002) compared with wild-type controls. Pulmonary circulatory pressures, measured by right ventricular catheterization, were significantly elevated in elastin-insufficient mice (P < 0.0001) and showed an inverse correlation with elastin level. Although elastin-insufficient animals exhibited mild to moderate right ventricular hypertrophy (P = 0.0001) and intrapulmonary vascular remodeling, the changes were less than expected, given the high right ventricular pressures, and were attenuated compared with those seen in hypoxia-induced models of pulmonary arterial hypertension. The absence of extensive pathological cardiac remodeling at the high pressures in these animals suggests a developmental adaptation designed to maintain right-sided cardiac output in a vascular system with altered elastin content.

Sequencing of idiopathic pulmonary fibrosis-related genes reveals independent single gene associations

Background Previous studies investigating a genetic basis for idiopathic pulmonary fibrosis (IPF) have focused on resequencing single genes in IPF kindreds or cohorts to determine the genetic contributions to IPF. None has investigated interactions among the candidate genes. Objective To compare the frequencies and interactions of mutations in six IPF-associated genes in a cohort of 132 individuals with IPF with those of a disease-control cohort of 192 individuals with chronic obstructive pulmonary disease (COPD) and the population represented in the Exome Variant Server. Methods We resequenced the genes encoding surfactant proteins A2 (SFTPA2), and C (SFTPC), the ATP binding cassette member A3 (ABCA3), telomerase (TERT), thyroid transcription factor (NKX2-1) and mucin 5B (MUC5B) and compared the collapsed frequencies of rare (minor allele frequency <1%), computationally predicted deleterious variants in each cohort. We also genotyped a common MUC5B promoter variant that is over-represented in individuals with IPF. Results We found 15 mutations in 14 individuals (11%) in the IPF cohort: (SFTPA2 (n=1), SFTPC (n=5), ABCA3 (n=4) and TERT (n=5)). No individual with IPF had two different mutations, but one individual with IPF was homozygous for p.E292V, the most common ABCA3 disease-causing variant. We did not detect an interaction between any of the mutations and the MUC5B promoter variant. Conclusions Rare mutations in SFTPA2, SFTPC and TERT are collectively over-represented in individuals with IPF. Genetic analysis and counselling should be considered as part of the IPF evaluation.

Mechanisms of emphysema in autosomal dominant cutis laxa

Heterozygous elastin gene mutations cause autosomal dominant cutis laxa associated with emphysema and aortic aneurysms. To investigate the molecular mechanisms leading to cutis laxa in vivo, we generated transgenic mice by pronuclear injection of minigenes encoding normal human tropoelastin (WT) or tropoelastin with a cutis laxa mutation (CL). Three independent founder lines of CL mice showed emphysematous pulmonary airspace enlargement. No consistent dermatological or cardiovascular pathologies were observed. One CL and one WT line were selected for detailed studies. Both mutant and control transgenic animals showed elastin deposition into pulmonary elastic fibers, indicated by increased desmosine levels in the lung and by colocalization of transgenic and endogenous elastin by immunostaining. CL mice showed increased static lung compliance and decreased stiffness of lung tissue. In addition, markers of transforming growth factor-β (TGFβ) signaling and the unfolded protein response (UPR) were elevated together with increased apoptosis in the lungs of CL animals. We conclude that the synthesis of mutant elastin in CL activates multiple downstream disease pathways by triggering a UPR, altered mechanical signaling, increased release of TGFβ and apoptosis. We propose that the combined effects of these processes lead to the development of an emphysematous pulmonary phenotype in CL.

Long-term outcomes of bronchial thermoplasty in subjects with severe asthma: a comparison of 3-year follow-up results from two prospective multicentre studies

Bronchial thermoplasty is an endoscopic therapy for severe asthma. The previously reported, randomised sham-controlled AIR2 (Asthma Intervention Research 2) trial showed a significant reduction in severe asthma exacerbations, emergency department visits and hospitalisations after bronchial thermoplasty. More “real-world” clinical outcome data is needed. This article compares outcomes in bronchial thermoplasty subjects with 3 years of follow-up from the ongoing, post-market PAS2 (Post-FDA Approval Clinical Trial Evaluating Bronchial Thermoplasty in Severe Persistent Asthma) study with those from the AIR2 trial. 279 subjects were treated with bronchial thermoplasty in the PAS2 study. We compared the first 190 PAS2 subjects with the 190 bronchial thermoplasty-treated subjects in the AIR2 trial at 3 years of follow-up. The PAS2 subjects were older (mean age 45.9 versus 40.7 years) and more obese (mean body mass index 32.5 versus 29.3 kg·m−2) and took higher doses of inhaled corticosteroids (mean dose 2301 versus 1961 μg·day−1). More PAS2 subjects had experienced severe exacerbations (74% versus 52%) and hospitalisations (15.3% versus 4.2%) in the 12 months prior to bronchial thermoplasty. At year 3 after bronchial thermoplasty, the percentage of PAS2 subjects with severe exacerbations, emergency department visits and hospitalisations significantly decreased by 45%, 55% and 40%, respectively, echoing the AIR2 results. The PAS2 study demonstrates similar improvements in asthma control after bronchial thermoplasty compared with the AIR2 trial despite enrolling subjects who may have had poorer asthma control. A comparison of the results obtained thus far in PAS2 with AIR2 evaluating bronchial thermoplasty in severe asthma http://ow.ly/sB0X30csDuE

Alternative splicing and tissue-specific elastin misassembly act as biological modifiers of human elastin gene frameshift mutations associated with dominant cutis laxa.

Background: A humanized mouse was developed to study elastin assembly and the pathogenesis of cutis laxa. Results: Mutant transcripts incorporate into elastic fibers of skin and lung with adverse effects but not aorta. Conclusion: Elastin frameshift mutations alter elastin assembly domains. Significance: The mechanism of elastic fiber assembly may not be the same in all tissues. Elastin is the extracellular matrix protein in vertebrates that provides elastic recoil to blood vessels, the lung, and skin. Because the elastin gene has undergone significant changes in the primate lineage, modeling elastin diseases in non-human animals can be problematic. To investigate the pathophysiology underlying a class of elastin gene mutations leading to autosomal dominant cutis laxa, we engineered a cutis laxa mutation (single base deletion) into the human elastin gene contained in a bacterial artificial chromosome. When expressed as a transgene in mice, mutant elastin was incorporated into elastic fibers in the skin and lung with adverse effects on tissue function. In contrast, only low levels of mutant protein incorporated into aortic elastin, which explains why the vasculature is relatively unaffected in this disease. RNA stability studies found that alternative exon splicing acts as a modifier of disease severity by influencing the spectrum of mutant transcripts that survive nonsense-mediated decay. Our results confirm the critical role of the C-terminal region of tropoelastin in elastic fiber assembly and suggest tissue-specific differences in the elastin assembly pathway.

Modification and functional inactivation of the tropoelastin carboxy-terminal domain in cross-linked elastin

The carboxy-terminus of tropoelastin is a highly conserved, atypical region of the molecule with sequences that define both cell and matrix interactions. This domain also plays a critical but unknown role in the assembly and crosslinking of tropoelastin during elastic fiber maturation. Using a competitive ELISA with an antibody to an elastase-resistant epitope in the carboxy-terminus of tropoelastin (domain-36), we quantified levels of the domain-36 sequence in elastase-derived peptides from mature, insoluble elastin. We found that the amount of carboxy-terminal epitope in elastin is approximately 0.2% of the expected value, assuming each tropoelastin monomer that is incorporated into the insoluble polymer has an intact carboxy-terminus. The low levels suggest that the majority of domain-36 sequence is either removed at some stage of elastin assembly or that the antigenic epitope is altered by posttranslational modification. Biochemical evidence is presented for a potential lysine-derived cross-link in this region, which would alter the extractability and antigenicity of the carboxy-terminal epitope. These results show that there is little or no unmodified domain-36 in mature elastin, indicating that the cell and matrix binding activities associated with this region of tropoelastin are lost or modified as elastin matures. A crosslinking function for domain-36 may serve to help register the multiple crosslinking sites in elastin and explains why mutations that alter the domain-36 sequence have detrimental effects on elastic fiber assembly.

A Phase II Clinical Trial of an Aromatase Inhibitor for Postmenopausal Women with Lymphangioleiomyomatosis

Rationale: Lymphangioleiomyomatosis (LAM) is a progressive cystic lung disease that predominantly affects women and can worsen with pregnancy, estrogen treatment, and the menstrual cycle, suggesting an important role for estrogen in disease pathogenesis. Objectives: To assess the efficacy and safety of the aromatase inhibitor letrozole in the treatment of LAM. Methods: Seventeen postmenopausal women with LAM were enrolled in this phase II trial and randomized to receive letrozole 2.5 mg daily (n = 9) or placebo (n = 8) for a period of 12 months. Five patients in each group were also taking sirolimus at baseline and remained on the drug throughout the treatment period. Lung function, exercise capacity, quality of life, and serum vascular endothelial growth factor D (VEGF‐D) were measured at baseline and at 3‐month intervals. Results: Fifteen patients completed the study. Two patients withdrew. There were no differences in adverse events in the letrozole and placebo groups. The target enrollment of 25 patients per arm was not met, so the efficacy of letrozole could not be assessed as planned. After adjusting for sirolimus use, we found that the rate of change in FEV1 for all subjects was ‐3 ± 3 ml/mo (P = 0.4), and for serum VEGF‐D, the rate of change was ‐0.024 ± 0.009 pg/ml/mo (P = 0.015), showing a steeper decline in the letrozole group (‐0.029 ± 0.013; P = 0.025). All patients who were taking sirolimus had a reduction in VEGF‐D levels from baseline to the last visit, compared with only half of the patients who were not taking sirolimus. In a post hoc analysis, eight matched letrozole‐treated‐placebo‐treated pairs were constructed, six of which demonstrated better FEV1 improvement for the letrozole‐treated patients. Conclusions: Letrozole treatment appears to be safe and well tolerated in postmenopausal patients with LAM, including those taking sirolimus. Enrollment in this trial was compromised by the publication of an effective treatment (sirolimus) in the same month as the study opened, resulting in limited power to detect treatment effects. Post hoc matched pairs exploration studies provide tentative support for additional studies of letrozole in LAM. Considering the reduced rate of lung function decline in postmenopausal patients, future studies will likely require enhanced study designs, such as selective enrollment of those with prognostic biomarkers predictive of decline. Clinical trial registered with www.clinicaltrials.gov (NCT01353209).