Varisco Lab

Stretch is necessary for alveolarization of the peripheral lung.^1-2 If the adult lung is subjected to increased stretch, then the alveolarization program can be re-initiated. In adult mice following left pneumonectomy, the right lung-principally the right accessory lobe, stretches and regenerates with complete recovery of lung volume and alveolar number by 6 weeks post-surgery. We have developed a novel fluorescent elastin in situ zymography technique which can quantify and localize lung elastin remodeling (Figure 1A). During development, elastin remodeling peaks during the alveolar stage of lung development (Figure 1B) and is equally distributed between parenchymal, major arteries, and major airways (Figure 1C). During post-pneumonectomy lung regeneration, lung elastin remodeling is re-induced (Figure 1D) but only in the lung parenchyma (Figure 1E). We have developed a novel technique to section and stretch living lung (Figure 1F-G) and have demonstrated that the induction of elastin remodeling in the adult lung occurs in response to strain and occurs within minutes of strain application (Figure 1H). We are seeking to understand the mechanisms underlying this stretch-regulated matrix remodeling response.

¹Ysasi AB, et al. Effect of unilateral diaphragmatic paralysis on postpneumonecomy lung growth. Am J Physiol Lung Cell Mol Physiol. (305)6:L439-45. Sept 2013.

²Dane DM, et al. Separating in vivo mechanical stimuli for postpneumonectomy compensation: physiological assessment. J Appl Physiol (1985). (114)1:99-106. Jan 2013.

Chymotrypsin-like elastase 1 (Cela1) is a pancreatic elastase which we identified as being expressed in the lung in a developmentally regulated manner (Figure 1A). Cela1 is expressed in areas of lung elastin remodeling during development (Figure 1B-D). We developed a flow cytometry panel to identify the cells expression Cela1. In the prenatal lung, Cela1 is expressed principally in epithelial cells (Figure 2E), but postnatally it is expressed predominantly in CD90-positive lung fibroblasts (Figure 2F). During post-pneumonectomy lung regeneration, the accessory lobe experiences the most growth. Accessory lobe Cela1 expression increases after pneumonectomy (Figure 2G), and this increased expression is largely limited to the accessory lobe (Figure 2H). Since expression profile of Cela1 suggested that stretch regulated its expression, we developed wax lungs to prevent post-pneumonectomy lung expansion but not the increased blood flow and inflammatory changes that are associated with pneumonectomy (Figure 2I). The post-pneumonectomy increase in Cela1 expression is dependent upon stretch (Figure 2J). Using CRISPR-Cas9 technology, we created a premature stop codon in Cela1 exon-2 and are breeding these mice to determine whether Cela1 has a critical role in lung development.

Previous reports indicated that strain increased the binding of pancreatic elastase to lung elastin in a strain-dependent manner.¹ To test whether Cela1 possessed similar binding kinetics, we synthesized and fluorophore-labeled recombinant Cela1. We then used a 3D printer to make a confocal microscope compatible lung stretching device (Figure 3A) and developed a technique for sectioning and sectioning live lung sections. We quantified elastase activity and Cela1 binding with sequential stretch (Figure 3B-C and Video) and found that Cela1 binding was enhanced by stretch (Figure 3D). Incubation with soluble elastin competitively inhibited Cela1 binding (Figure 3E). Binding of Cela1 co-localized with areas of stretch-induced elastase activity (Figure 3F). To differentiate central from distal lung matrix remodeling, we masked alveolar ducts and respiratory bronchioles on cropped lung sections (Figure 3G-H) and quantified the stretch-dependent kinetics of Cela1 binding. Cela1 binding in the peripheral lung was enhanced by stretch, and binding was inhibited by soluble elastin (Figure 3I).

Our current studies are focusing on the molecular biology of how and where Cela1 binds to tropoelastin and how strain may permit Cela1 access to its preferred proteolytic site.

¹Jesudason R, et al. Mechanical forces regulate elastase activity and binding site availability in lung elastin. Biophys J (99) 9:3076-83. Sept 2010.

Previous studies have linked elastin remodeling with the development of new blood vessels from existing ones (angiogenesis).^1-2 In the lung, elastin remodeling was associated with proliferating (angiogenin positive) cells.³ To test whether Cela1 regulated angiogenesis, we first silenced Cela1 in an embryonic mouse lung mesenchymal/endothelial cell line (MFLM4). Cela1 silencing inhibited tubulogenesis (Figure 4A-B). In vitro gain of function experiments demonstrated that overexpression of Cela1 could trigger tubulogenesis in lung fibroblasts (Figure 4C-D). Using vascular-labeled Danio, silencing of a Cela1 orthologue impaired angiogenesis of the segmental vasculature and co-injecting murine Cela1 mRNA rescued this phenotype (Figure 4E-G). Anti-Cela1 antibody inhibited microvascular invasion in a subcutaneous matrigel plug assay (Figure 4H-I). Demonstrating therapeutic potential, Cela1 expression was associated with the increased microvascular density in a mouse model of lung adenocarcinoma (Figure 4J-K). All of these data support an important role for Cela1 in regulating lung microvascular morphogenesis.

Our current studies are investigating the mechanisms by which Cela1 exerts its angiogenic effects and whether these mechanisms can be targeted to improve pulmonary health.

¹Pocza P, et al. Locally generative VGVAGP and VAPG elastin-derived peptides amplify melanoma invasion via the galectin-3 receptor. Int J Cancer. (122)9:1972-80. May 2008.

²Robinet A, et al. Elastin-derived peptides enhance angiogenesis by promoting endothelial cell migration and tubulogenesis through upregulation of MT1-MMP. J Cell Sci. (118)2:L343-56. Jan 2005.

³Liu S, Young SM, Varisco BM. Dynamic expression of chymotrypsin-like elastase 1 over the course of murine lung development. Am J Physiol Lung Cell Mol Physiol. (306)12:L1104-16. Jun 2014.