Orthotopic Ferret Tracheal Transplantation Using a Recellularized Bioengineered Graft Produces Functional Epithelia.

Tracheal grafts may be necessary to bridge long-segment defects after curative resection for airway obstructions. Bioengineered grafts have emerged as an appealing option, given the possibilities of altering the histologic and cellular profile of the conduit. We previously designed a bioreactor capable of luminally decellularizing and recellularizing a ferret trachea with surface airway epithelia (SAE) basal cells (BCs), and we sought to assess the fate of these grafts when transplanted in an orthotopic fashion. As adjuncts to the procedure, we investigated the use of a vascular endothelial growth factor (VEGF)-laden hydrogel and of immunosuppression (IS) in graft revascularization and viability. IS was shown to limit early graft revascularization, but this effect could be counteracted with VEGF supplementation. Submucosal gland (SMG) loss was shown to be inevitable regardless of the revascularization strategy. Lastly, the bioengineered tracheas survived one month after transplant with differentiation of our implanted BCs that then transitioned into a recipient-derived functional epithelium. The work presented in this manuscript has important implications for future cellular and regenerative therapies.

Krt14 and Krt15 differentially regulate regenerative properties and differentiation potential of airway basal cells.

Keratin expression dynamically changes in airway basal cells (BCs) after acute and chronic injury, yet the functional consequences of these changes on BC behavior remain unknown. In bronchiolitis obliterans (BO) after lung transplantation, BC clonogenicity declines, which is associated with a switch from keratin15 (Krt15) to keratin14 (Krt14). We investigated these keratins' roles using Crispr-KO in vitro and in vivo and found that Krt14-KO and Krt15-KO produce contrasting phenotypes in terms of differentiation and clonogenicity. Primary mouse Krt14-KO BCs did not differentiate into club and ciliated cells but had enhanced clonogenicity. By contrast, Krt15-KO did not alter BC differentiation but impaired clonogenicity in vitro and reduced the number of label-retaining BCs in vivo after injury. Krt14, but not Krt15, bound the tumor suppressor stratifin (Sfn). Disruption of Krt14, but not of Krt15, reduced Sfn protein abundance and increased expression of the oncogene dNp63a during BC differentiation, whereas dNp63a levels were reduced in Krt15-KO BCs. Overall, the phenotype of Krt15-KO BCs contrasts with Krt14-KO phenotype and resembles the phenotype in BO with decreased clonogenicity, increased Krt14, and decreased dNp63a expression. This work demonstrates that Krt14 and Krt15 functionally regulate BC behavior, which is relevant in chronic disease states like BO.

Ferret Lung Transplantation Models Differential Lymphoid Aggregate Morphology Between Restrictive and Obstructive Forms of Chronic Lung Allograft Dysfunction.

BACKGROUND: Long-term survival after lung transplantation remains limited by chronic lung allograft dysfunction (CLAD). CLAD has 2 histologic phenotypes, namely obliterative bronchiolitis (OB) and restrictive alveolar fibroelastosis (AFE), which have distinct clinical presentations, pathologies, and outcomes. Understanding of OB versus AFE pathogenesis would improve with better animal models. METHODS: We utilized a ferret orthotopic single-lung transplantation model to characterize allograft fibrosis as a histologic measure of CLAD. Native lobes and "No CLAD" allografts lacking aberrant histology were used as controls. We used morphometric analysis to evaluate the size and abundance of B-cell aggregates and tertiary lymphoid organs (TLOs) and their cell composition. Quantitative RNA expression of 47 target genes was performed simultaneously using a custom QuantiGene Plex Assay. RESULTS: Ferret lung allografts develop the full spectrum of human CLAD histology including OB and AFE subtypes. While both OB and AFE allografts developed TLOs, TLO size and number were greater with AFE histology. More activated germinal center cells marked by B-cell lymphoma 6 Transcription Repressor, (B-cell lymphoma 6) expression and fewer cells expressing forkhead box P3 correlated with AFE, congruent with greater diffuse immunoglobulin, plasma cell abundance, and complement 4d staining. Furthermore, forkhead box P3 RNA induction was significant in OB allografts specifically. RNA expression changes were seen in native lobes of animals with AFE but not OB when compared with No CLAD native lobes. CONCLUSIONS: The orthotopic ferret single-lung transplant model provides unique opportunities to better understand factors that dispose allografts to OB versus AFE. This will help develop potential immunomodulatory therapies and antifibrotic approaches for lung transplant patients.

A Novel Bioreactor for Reconstitution of the Epithelium and Submucosal Glands in Decellularized Ferret Tracheas.

Tracheal grafts introduce the possibility to treat airway pathologies that require resection. While there has been success with engraftment of the surface airway epithelium (SAE) onto decellularized tracheas, there has been minimal advancement in regenerating the submucosal glands (SMGs). We designed a cost-effective open-system perfusion bioreactor to investigate the engraftment potential of ferret SAEs and murine myoepithelial cells (MECs) on a partly decellularized ferret trachea with the goal of creating a fully functional tracheal replacement. An air-liquid interface was also arranged by perfusing humidified air through the lumen of a recellularized conduit to induce differentiation. Our versatile bioreactor design was shown to support the successful partial decellularization and recellularization of ferret tracheas. The decellularized grafts maintained biomechanical integrity and chondrocyte viability, consistent with other publications. The scaffolds supported SAE basal cell engraftment, and early differentiation was observed once an air-liquid interface had been established. Lastly, MEC engraftment was sustained, with evidence of diffuse SMG reconstitution. This model will help shed light on SMG regeneration and basal cell differentiation in vitro for the development of fully functional tracheal grafts before transplantation.

Direct cysteine sulfenylation drives activation of the Src kinase.

The Src kinase controls aspects of cell biology and its activity is regulated by intramolecular structural changes induced by protein interactions and tyrosine phosphorylation. Recent studies indicate that Src is additionally regulated by redox-dependent mechanisms, involving oxidative modification(s) of cysteines within the Src protein, although the nature and molecular-level impact of Src cysteine oxidation are unknown. Using a combination of biochemical and cell-based studies, we establish the critical importance of two Src cysteine residues, Cys-185 and Cys-277, as targets for H2O2-mediated sulfenylation (Cys-SOH) in redox-dependent kinase activation in response to NADPH oxidase-dependent signaling. Molecular dynamics and metadynamics simulations reveal the structural impact of sulfenylation of these cysteines, indicating that Cys-277-SOH enables solvent exposure of Tyr-416 to promote its (auto)phosphorylation, and that Cys-185-SOH destabilizes pTyr-527 binding to the SH2 domain. These redox-dependent Src activation mechanisms offer opportunities for development of Src-selective inhibitors in treatment of diseases where Src is aberrantly activated.

Avian lungs: A novel scaffold for lung bioengineering.

Allogeneic lung transplant is limited both by the shortage of available donor lungs and by the lack of suitable long-term lung assist devices to bridge patients to lung transplantation. Avian lungs have different structure and mechanics resulting in more efficient gas exchange than mammalian lungs. Decellularized avian lungs, recellularized with human lung cells, could therefore provide a powerful novel gas exchange unit for potential use in pulmonary therapeutics. To initially assess this in both small and large avian lung models, chicken (Gallus gallus domesticus) and emu (Dromaius novaehollandiae) lungs were decellularized using modifications of a detergent-based protocol, previously utilized with mammalian lungs. Light and electron microscopy, vascular and airway resistance, quantitation and gel analyses of residual DNA, and immunohistochemical and mass spectrometric analyses of remaining extracellular matrix (ECM) proteins demonstrated maintenance of lung structure, minimal residual DNA, and retention of major ECM proteins in the decellularized scaffolds. Seeding with human bronchial epithelial cells, human pulmonary vascular endothelial cells, human mesenchymal stromal cells, and human lung fibroblasts demonstrated initial cell attachment on decellularized avian lungs and growth over a 7-day period. These initial studies demonstrate that decellularized avian lungs may be a feasible approach for generating functional lung tissue for clinical therapeutics.

Development and Applications of a Calmodulin-Based Fusion Protein System for the Expression and Purification of WW and Zinc Finger Modules.

Calmodulin from Homo sapiens is an α-helical calcium-binding protein that expresses to high levels in E. coli. When the N-terminus of a calmodulin variant is bound to Ca2+, it undergoes a conformational change, exposing hydrophobic pockets. This property can be utilized for purification purposes, as these pockets bind to phenyl sepharose resin with high affinity. Washing with EDTA chelates the Ca2+ ions from the protein, inducing a conformational change back to the more folded state and eluting the protein from the column. We describe herein the use of a protein expression and purification technique using the calmodulin variant and a short linker for proteolytic cleavage by the mutant NIa-Pro tobacco etch virus protease. We have shown this approach to be useful in obtaining purified quantities of various small proteins that could not be expressed using other methods, including high enough concentrations of a designed WW domain protein for NMR structural analysis. We have also obtained promising results on the usefulness of this procedure to express and purify zinc finger proteins without the addition of zinc ions or other cofactors.

Disruption of a cystine transporter downregulates expression of genes involved in sulfur regulation and cellular respiration.

Cystine and cysteine are important molecules for pathways such as redox signaling and regulation, and thus identifying cellular deficits upon deletion of the Saccharomyces cerevisiae cystine transporter Ers1p allows for a further understanding of cystine homeostasis. Previous complementation studies using the human ortholog suggest yeast Ers1p is a cystine transporter. Human CTNS encodes the protein Cystinosin, a cystine transporter that is embedded in the lysosomal membrane and facilitates the export of cystine from the lysosome. When CTNS is mutated, cystine transport is disrupted, leading to cystine accumulation, the diagnostic hallmark of the lysosomal storage disorder cystinosis. Here, we provide biochemical evidence for Ers1p-dependent cystine transport. However, the accumulation of intracellular cystine is not observed when the ERS1 gene is deleted from ers1-Δ yeast, supporting the existence of modifier genes that provide a mechanism in ers1-Δ yeast that prevents or corrects cystine accumulation. Upon comparison of the transcriptomes of isogenic ERS1+ and ers1-Δ strains of S. cerevisiae by DNA microarray followed by targeted qPCR, sixteen genes were identified as being differentially expressed between the two genotypes. Genes that encode proteins functioning in sulfur regulation, cellular respiration, and general transport were enriched in our screen, demonstrating pleiotropic effects of ers1-Δ. These results give insight into yeast cystine regulation and the multiple, seemingly distal, pathways that involve proper cystine recycling.