Transmural pressure signals through retinoic acid to regulate lung branching.

During development, the mammalian lung undergoes several rounds of branching, the rate of which is tuned by the relative pressure of the fluid within the lumen of the lung. We carried out bioinformatics analysis of RNA-sequencing of embryonic mouse lungs cultured under physiologic or sub-physiologic transmural pressure and identified transcription factor-binding motifs near genes whose expression changes in response to pressure. Surprisingly, we found retinoic acid (RA) receptor binding sites significantly overrepresented in the promoters and enhancers of pressure-responsive genes. Consistently, increasing transmural pressure activates RA signaling, and pharmacologically inhibiting RA signaling decreases airway epithelial branching and smooth muscle wrapping. We found that pressure activates RA signaling through the mechanosensor Yap. A computational model predicts that mechanical signaling through Yap and RA affects lung branching by altering the balance between epithelial proliferation and smooth muscle wrapping, which we test experimentally. Our results reveal that transmural pressure signals through RA to balance the relative rates of epithelial growth and smooth muscle differentiation in the developing mouse lung and identify RA as a previously unreported component in the mechanotransduction machinery of embryonic tissues.

Negative Transpulmonary Pressure Disrupts Airway Morphogenesis by Suppressing Fgf10.

Mechanical forces are increasingly recognized as important determinants of cell and tissue phenotype and also appear to play a critical role in organ development. During the fetal stages of lung morphogenesis, the pressure of the fluid within the lumen of the airways is higher than that within the chest cavity, resulting in a positive transpulmonary pressure. Several congenital defects decrease or reverse transpulmonary pressure across the developing airways and are associated with a reduced number of branches and a correspondingly underdeveloped lung that is insufficient for gas exchange after birth. The small size of the early pseudoglandular stage lung and its relative inaccessibility in utero have precluded experimental investigation of the effects of transpulmonary pressure on early branching morphogenesis. Here, we present a simple culture model to explore the effects of negative transpulmonary pressure on development of the embryonic airways. We found that negative transpulmonary pressure decreases branching, and that it does so in part by altering the expression of fibroblast growth factor 10 (Fgf10). The morphogenesis of lungs maintained under negative transpulmonary pressure can be rescued by supplementing the culture medium with exogenous FGF10. These data suggest that Fgf10 expression is regulated by mechanical stress in the developing airways. Understanding the mechanical signaling pathways that connect transpulmonary pressure to FGF10 can lead to the establishment of novel non-surgical approaches for ameliorating congenital lung defects.

Bioengineered in Vitro Tissue Model of Fibroblast Activation for Modeling Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a complex disease of unknown etiology with no current curative treatment. Modeling pulmonary fibrotic (PF) tissue has the potential to improve our understanding of IPF disease progression and treatment. Rodent animal models do not replicate human fibroblastic foci (Hum-FF) pathology, and current iterations of in vitro model systems (e.g., collagen hydrogels, polyacrylamide hydrogels, and fibrosis-on-chip systems) are unable to replicate the three-dimensional (3D) complexity and biochemical composition of human PF tissue. Herein, we fabricated a 3D bioengineered pulmonary fibrotic (Eng-PF) tissue utilizing cell laden silk collagen type I dityrosine cross-linked hydrogels and Flexcell bioreactors. We show that silk collagen type I hydrogels have superior stability and mechanical tunability compared to other hydrogel systems. Using customized Flexcell bioreactors, we reproduced Hum-FF-like pathology with airway epithelial and microvascular endothelial cells. Eng-PF tissues can model myofibroblast differentiation and permit evaluation of antifibrotic drug treatments. Further, Eng-PF tissues could be used to model different facets of IPF disease, including epithelial injury with the addition of bleomycin and cellular recruitment by perfusion of cells through the hydrogel microchannel.

Engineered cell and tissue models of pulmonary fibrosis.

Pulmonary fibrosis includes several lung disorders characterized by scar formation and Idiopathic Pulmonary Fibrosis (IPF) is a particularly severe form of pulmonary fibrosis of unknown etiology with a mean life expectancy of 3years' post-diagnosis. Treatments for IPF are limited to two FDA approved drugs, pirfenidone and nintedanib. Most lead candidate drugs that are identified in pre-clinical animal studies fail in human clinical trials. Thus, there is a need for advanced humanized in vitro models of the lung to improve candidate treatments prior to moving to human clinical trials. The development of 3D tissue models has created systems capable of emulating human lung structure, function, and cell and matrix interactions. The specific models accomplish these features and preliminary studies conducted using some of these systems have shown potential for in vitro anti-fibrotic drug testing. Further characterization and improvements will enable these tissue models to extend their utility for in vitro drug testing, to help identify signaling pathways and mechanisms for new drug targets, and potentially reduce animal models as standard pre-clinical models of study. In the current review, we contrast different in vitro models based on increasing dimensionality (2D, 2.5D and 3D), with added focus on contemporary 3D pulmonary models of fibrosis.

Automated quantification of three-dimensional organization of fiber-like structures in biological tissues.

Fiber-like structures are prevalent in biological tissues, yet quantitative approaches to assess their three-dimensional (3D) organization are lacking. We develop 3D directional variance, as a quantitative biomarker of truly 3D fibrillar organization by extending the directional statistics formalism developed for describing circular data distributions (i.e. when 0° and 360° are equivalent) to axial ones (i.e. when 0° and 180° are equivalent). Significant advantages of this analysis include its time efficiency, sensitivity and ability to provide quantitative readouts of organization over different size scales of a given data set. We establish a broad range of applications for this method by characterizing collagen fibers, neuronal axons and fibroblasts in the context of cancer diagnostics, traumatic brain injury and cell-matrix interactions in developing engineered tissues. This method opens possibilities for unraveling in a sensitive, and quantitative manner the organization of essential fiber-like structures in tissues and ultimately its impact on tissue function.

Phenol red-silk tyrosine cross-linked hydrogels.

UNLABELLED: Phenol red is a cytocompatible pH sensing dye that is commonly added to cell culture media, but removed from some media formulations due to its structural mimicry of estrogen. Phenol red free media is also used during live cell imaging, to avoid absorbance and fluorescence quenching of fluorophores. To overcome these complications, we developed cytocompatible and degradable phenol red-silk tyrosine cross-linked hydrogels using horseradish peroxidase (HRP) enzyme and hydrogen peroxide (H2O2). Phenol red added to silk during tyrosine crosslinking accelerated di-tyrosine formation in a concentration-dependent reaction. Phenol red diffusion studies and UV-Vis spectra of phenol red-silk tyrosine hydrogels at different pHs showed altered absorption bands, confirming entrapment of dye within the hydrogel network. LC-MS of HRP-reacted phenol red and N-acetyl-l-tyrosine reaction products confirmed covalent bonds between the phenolic hydroxyl group of phenol red and tyrosine on the silk. At lower phenol red concentrations, leak-proof hydrogels which did not release phenol red were fabricated and found to be cytocompatible based on live-dead staining and alamar blue assessments of encapsulated fibroblasts. Due to the spectral overlap between phenol red absorbance at 415nm and di-tyrosine fluorescence at 417nm, phenol red-silk hydrogels provide both absorbance and fluorescence-based pH sensing. With an average pKa of 6.8 and good cytocompatibiltiy, phenol red-silk hydrogels are useful for pH sensing in phenol red free systems, cellular microenvironments and bioreactors. STATEMENT OF SIGNIFICANCE: Phenol red entrapped within hydrogels facilitates pH sensing in phenol red free environments. Leak-proof phenol red based pH sensors require covalent binding techniques, but are complicated due to the lack of amino or carboxyl groups on phenol red. Currently, there is no simple, reliable technique to covalently link phenol red to hydrogel matrices, for real-time pH sensing in cell culture environments. Herein, we take advantage of phenolic groups for covalent linkage of phenol red to silk tyrosine in the presence of HRP and H2O2. The novelty of the current system stems from its simplicity and the use of silk protein to create a cytocompatible, degradable sensor capable of real-time pH sensing in cell culture microenvironments.