Stem Cells, Cell Therapies and Bioengineering in Lung Biology and Diseases 2023.

Repair and regeneration of a diseased lung using stem cells or bioengineered tissues is an exciting therapeutic approach for a variety of lung diseases and critical illnesses. Over the past decade increasing evidence from preclinical models suggests that cells, which are not normally resident in the lung can be utilized to modulate immune responses after injury, but there have been challenges in translating these promising findings to the clinic. In parallel, there has been a surge in bioengineering studies investigating the use of artificial and acellular lung matrices as scaffolds for three-dimensional lung or airway regeneration, with some recent attempts of transplantation in large animal models. The combination of these studies with those involving stem cells, induced pluripotent stem cell derivatives, and/or cell therapies is a promising and rapidly developing research area. These studies have been further paralleled by significant increases in our understanding of the molecular and cellular events by which endogenous lung stem and/or progenitor cells arise during lung development and participate in normal and pathologic remodeling after lung injury. For the 2023 Stem Cells, Cell Therapies, and Bioengineering in Lung Biology and Diseases Conference, scientific symposia were chosen to reflect the most cutting-edge advances in these fields. Sessions focused on the integration of "-omics" technologies with function, the influence of immune cells on regeneration, and the role of the extracellular matrix in regeneration. The necessity for basic science studies to enhance fundamental understanding of lung regeneration and to design innovative translational studies was reinforced throughout the conference.

Clustering of phosphatase RPTPα promotes Src signaling and the arthritogenic action of synovial fibroblasts.

Receptor-type protein phosphatase α (RPTPα) promotes fibroblast-dependent arthritis and fibrosis, in part, by enhancing the activation of the kinase SRC. Synovial fibroblasts lining joint tissue mediate inflammation and tissue damage, and their infiltration into adjacent tissues promotes disease progression. RPTPα includes an ectodomain and two intracellular catalytic domains (D1 and D2) and, in cancer cells, undergoes inhibitory homodimerization, which is dependent on a D1 wedge motif. Through single-molecule localization and labeled molecule interaction microscopy of migrating synovial fibroblasts, we investigated the role of RPTPα dimerization in the activation of SRC, the migration of synovial fibroblasts, and joint damage in a mouse model of arthritis. RPTPα clustered with other RPTPα and with SRC molecules in the context of actin-rich structures. A known dimerization-impairing mutation in the wedge motif (P210L/P211L) and the deletion of the D2 domain reduced RPTPα-RPTPα clustering; however, it also unexpectedly reduced RPTPα-SRC association. The same mutations also reduced recruitment of RPTPα to actin-rich structures and inhibited SRC activation and cellular migration. An antibody against the RPTPα ectodomain that prevented the clustering of RPTPα also inhibited RPTPα-SRC association and SRC activation and attenuated fibroblast migration and joint damage in arthritic mice. A catalytically inactivating RPTPα-C469S mutation protected mice from arthritis and reduced SRC activation in synovial fibroblasts. We conclude that RPTPα clustering retains it to actin-rich structures to promote SRC-mediated fibroblast migration and can be modulated through the extracellular domain.

Saracatinib, a Selective Src Kinase Inhibitor, Blocks Fibrotic Responses in Preclinical Models of Pulmonary Fibrosis.

Rationale: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and often fatal disorder. Two U.S. Food and Drug Administration-approved antifibrotic drugs, nintedanib and pirfenidone, slow the rate of decline in lung function, but responses are variable and side effects are common. Objectives: Using an in silico data-driven approach, we identified a robust connection between the transcriptomic perturbations in IPF disease and those induced by saracatinib, a selective Src kinase inhibitor originally developed for oncological indications. Based on these observations, we hypothesized that saracatinib would be effective at attenuating pulmonary fibrosis. Methods: We investigated the antifibrotic efficacy of saracatinib relative to nintedanib and pirfenidone in three preclinical models: 1) in vitro in normal human lung fibroblasts; 2) in vivo in bleomycin and recombinant Ad-TGF-ß (adenovirus transforming growth factor-ß) murine models of pulmonary fibrosis; and 3) ex vivo in mice and human precision-cut lung slices from these two murine models as well as patients with IPF and healthy donors. Measurements and Main Results: In each model, the effectiveness of saracatinib in blocking fibrogenic responses was equal or superior to nintedanib and pirfenidone. Transcriptomic analyses of TGF-ß-stimulated normal human lung fibroblasts identified specific gene sets associated with fibrosis, including epithelial-mesenchymal transition, TGF-ß, and WNT signaling that was uniquely altered by saracatinib. Transcriptomic analysis of whole-lung extracts from the two animal models of pulmonary fibrosis revealed that saracatinib reverted many fibrogenic pathways, including epithelial-mesenchymal transition, immune responses, and extracellular matrix organization. Amelioration of fibrosis and inflammatory cascades in human precision-cut lung slices confirmed the potential therapeutic efficacy of saracatinib in human lung fibrosis. Conclusions: These studies identify novel Src-dependent fibrogenic pathways and support the study of the therapeutic effectiveness of saracatinib in IPF treatment.

Why So Salty? Transient Diabetes Insipidus After Discontinuation of Vasopressin.

In recent years, vasopressin has been increasingly used as an early treatment of vasopressor-refractory septic shock. In this article, we describe 2 episodes of transient diabetes insipidus after vasopressin for the treatment of septic shock was discontinued, which adds to a modest number of case studies reporting the same phenomenon. With the anticipated continued use of vasopressin in intensive care units, it can be expected that this adverse effect will occur with some frequency. Awareness and early recognition of this phenomenon can lead to prompt diagnosis and treatment.

PTPα promotes fibroproliferative responses after acute lung injury.

The acute respiratory distress syndrome (ARDS) is a major healthcare problem, accounting for significant mortality and long-term disability. Approximately 25% of patients with ARDS will develop an overexuberant fibrotic response, termed fibroproliferative ARDS (FP-ARDS) that portends a poor prognosis and increased mortality. The cellular pathological processes that drive FP-ARDS remain incompletely understood. We have previously shown that the transmembrane receptor-type tyrosine phosphatase protein tyrosine phosphatase-α (PTPα) promotes pulmonary fibrosis in preclinical murine models through regulation of transforming growth factor-ß (TGF-ß) signaling. In this study, we examine the role of PTPα in the pathogenesis of FP-ARDS in a preclinical murine model of acid (HCl)-induced acute lung injury. We demonstrate that although mice genetically deficient in PTPα (Ptpra-/-) are susceptible to early HCl-induced lung injury, they exhibit markedly attenuated fibroproliferative responses. In addition, early profibrotic gene expression is reduced in lung tissue after acute lung injury in Ptpra-/- mice, and stimulation of naïve lung fibroblasts with the BAL fluid from these mice results in attenuated fibrotic outcomes compared with wild-type littermate controls. Transcriptomic analyses demonstrate reduced extracellular matrix (ECM) deposition and remodeling in mice genetically deficient in PTPα. Importantly, human lung fibroblasts modified with a CRISPR-targeted deletion of PTPRA exhibit reduced expression of profibrotic genes in response to TGF-ß stimulation, demonstrating the importance of PTPα in human lung fibroblasts. Together, these findings demonstrate that PTPα is a key regulator of fibroproliferative processes following acute lung injury and could serve as a therapeutic target for patients at risk for poor long-term outcomes in ARDS.

Protein tyrosine phosphatase-α amplifies transforming growth factor-ß-dependent profibrotic signaling in lung fibroblasts.

Idiopathic pulmonary fibrosis (IPF) is a progressive, often fatal, fibrosing lung disease for which treatment remains suboptimal. Fibrogenic cytokines, including transforming growth factor-ß (TGF-ß), are central to its pathogenesis. Protein tyrosine phosphatase-α (PTPα) has emerged as a key regulator of fibrogenic signaling in fibroblasts. We have reported that mice globally deficient in PTPα (Ptpra-/-) were protected from experimental pulmonary fibrosis, in part via alterations in TGF-ß signaling. The goal of this study was to determine the lung cell types and mechanisms by which PTPα controls fibrogenic pathways and whether these pathways are relevant to human disease. Immunohistochemical analysis of lungs from patients with IPF revealed that PTPα was highly expressed by mesenchymal cells in fibroblastic foci and by airway and alveolar epithelial cells. To determine whether PTPα promotes profibrotic signaling pathways in lung fibroblasts and/or epithelial cells, we generated mice with conditional (floxed) Ptpra alleles (Ptpraf/f). These mice were crossed with Dermo1-Cre or with Sftpc-CreERT2 mice to delete Ptpra in mesenchymal cells and alveolar type II cells, respectively. Dermo1-Cre/Ptpraf/f mice were protected from bleomycin-induced pulmonary fibrosis, whereas Sftpc-CreERT2/Ptpraf/f mice developed pulmonary fibrosis equivalent to controls. Both canonical and noncanonical TGF-ß signaling and downstream TGF-ß-induced fibrogenic responses were attenuated in isolated Ptpra-/- compared with wild-type fibroblasts. Furthermore, TGF-ß-induced tyrosine phosphorylation of TGF-ß type II receptor and of PTPα were attenuated in Ptpra-/- compared with wild-type fibroblasts. The phenotype of cells genetically deficient in PTPα was recapitulated with the use of a Src inhibitor. These findings suggest that PTPα amplifies profibrotic TGF-ß-dependent pathway signaling in lung fibroblasts.

The Importance of Tyrosine Phosphorylation Control of Cellular Signaling Pathways in Respiratory Disease: pY and pY Not.

Reversible phosphorylation of proteins on tyrosine residues is an essential signaling mechanism by which diverse cellular processes are closely regulated. The tight temporal and spatial control of the tyrosine phosphorylation status of proteins by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs) is critical to cellular homeostasis as well as to adaptations to the external environment. Via regulation of cellular signaling cascades involving other protein kinases and phosphatases, receptors, adaptor proteins, and transcription factors, PTKs and PTPs closely control diverse cellular processes such as proliferation, differentiation, migration, inflammation, and maintenance of cellular barrier function. Given these key regulatory roles, it is not surprising that dysfunction of PTKs and PTPs is important in the pathogenesis of human disease, including many pulmonary diseases. The roles of various PTKs and PTPs in acute lung injury and repair, pulmonary fibrosis, pulmonary vascular disease, and inflammatory airway disease are discussed in this review. It is important to note that although there is overlap among many of these proteins in various disease states, the mechanisms by which they influence the pathogenesis of these conditions differ, suggesting wide-ranging roles for these enzymes and their potential as therapeutic targets.

Transforming Growth Factor-ß: Master Regulator of the Respiratory System in Health and Disease.

In this article, we review the biology and physiological importance of transforming growth factor-ß (TGF-ß) to homeostasis in the respiratory system, its importance to innate and adaptive immune responses in the lung, and its pathophysiological role in various chronic pulmonary diseases including pulmonary arterial hypertension, chronic obstructive pulmonary disease, asthma, and pulmonary fibrosis. The TGF-ß family is responsible for initiation of the intracellular signaling pathways that direct numerous cellular activities including proliferation, differentiation, extracellular matrix synthesis, and apoptosis. When TGF-ß signaling is dysregulated or essential control mechanisms are unbalanced, the consequences of organ and tissue dysfunction can be profound. The complexities and myriad checkpoints built into the TGF-ß signaling pathways provide attractive targets for the treatment of these disease states, many of which are currently being investigated. This review focuses on those aspects of TGF-ß biology that are most relevant to pulmonary diseases and that hold promise as novel therapeutic targets.

Matrix metalloproteinases and protein tyrosine kinases: potential novel targets in acute lung injury and ARDS.

Acute lung injury (ALI) and ARDS fall within a spectrum of pulmonary disease that is characterized by hypoxemia, noncardiogenic pulmonary edema, and dysregulated and excessive inflammation. While mortality rates have improved with the advent of specialized ICUs and lung protective mechanical ventilation strategies, few other therapies have proven effective in the management of ARDS, which remains a significant clinical problem. Further development of biomarkers of disease severity, response to therapy, and prognosis is urgently needed. Several novel pathways have been identified and studied with respect to the pathogenesis of ALI and ARDS that show promise in bridging some of these gaps. This review will focus on the roles of matrix metalloproteinases and protein tyrosine kinases in the pathobiology of ALI in humans, and in animal models and in vitro studies. These molecules can act independently, as well as coordinately, in a feed-forward manner via activation of tyrosine kinase-regulated pathways that are pivotal in the development of ARDS. Specific signaling events involving proteolytic processing by matrix metalloproteinases that contribute to ALI, including cytokine and chemokine activation and release, neutrophil recruitment, transmigration and activation, and disruption of the intact alveolar-capillary barrier, will be explored in the context of these novel molecular pathways.

The importance of matrix metalloproteinase-3 in respiratory disorders.

The matrix metalloproteinases (MMPs) encompass a family of zinc-dependent endopeptidases that are secreted into the extracellular environment or remain bound to the cell surface. While MMPs were initially identified based on their ability to degrade collagen and other components of the extracellular matrix, recent studies indicate that their non-degradative functions are physiologically paramount. In particular, MMPs are now known to participate in diverse physiological processes that control key aspects of inflammatory and immune responses and neoplasia, in part by selective triggering of cellular signaling pathways via limited proteolytic processing of extracellular and membrane-associated proteins, including cytokines and cell surface receptors. Herein, we focus on the unique roles of MMP-3 (stromelysin-1) in acute lung injury and repair, pulmonary fibrosis, and lung cancer.

Protein tyrosine phosphatase α mediates profibrotic signaling in lung fibroblasts through TGF-ß responsiveness.

Fibrotic lung diseases represent a diverse group of progressive and often fatal disorders with limited treatment options. Although the pathogenesis of these conditions remains incompletely understood, receptor type protein tyrosine phosphatase α (PTP-α encoded by PTPRA) has emerged as a key regulator of fibroblast signaling. We previously reported that PTP-α regulates cellular responses to cytokines and growth factors through integrin-mediated signaling and that PTP-α promotes fibroblast expression of matrix metalloproteinase 3, a matrix-degrading proteinase linked to pulmonary fibrosis. Here, we sought to determine more directly the role of PTP-α in pulmonary fibrosis. Mice genetically deficient in PTP-α (Ptpra(-/-)) were protected from pulmonary fibrosis induced by intratracheal bleomycin, with minimal alterations in the early inflammatory response or production of TGF-ß. Ptpra(-/-) mice were also protected from pulmonary fibrosis induced by adenoviral-mediated expression of active TGF-ß1. In reciprocal bone marrow chimera experiments, the protective phenotype tracked with lung parenchymal cells but not bone marrow-derived cells. Because fibroblasts are key contributors to tissue fibrosis, we compared profibrotic responses in wild-type and Ptpra(-/-) mouse embryonic and lung fibroblasts. Ptpra(-/-) fibroblasts exhibited hyporesponsiveness to TGF-ß, manifested by diminished expression of αSMA, EDA-fibronectin, collagen 1A, and CTGF. Ptpra(-/-) fibroblasts exhibited markedly attenuated TGF-ß-induced Smad2/3 transcriptional activity. We conclude that PTP-α promotes profibrotic signaling pathways in fibroblasts through control of cellular responsiveness to TGF-ß.

Role of ß-catenin-regulated CCN matricellular proteins in epithelial repair after inflammatory lung injury.

Repair of the lung epithelium after injury is integral to the pathogenesis and outcomes of diverse inflammatory lung diseases. We previously reported that ß-catenin signaling promotes epithelial repair after inflammatory injury, but the ß-catenin target genes that mediate this effect are unknown. Herein, we examined which ß-catenin transcriptional coactivators and target genes promote epithelial repair after inflammatory injury. Transmigration of human neutrophils across cultured monolayers of human lung epithelial cells resulted in a fall in transepithelial resistance and the formation of discrete areas of epithelial denudation ("microinjury"), which repaired via cell spreading by 96 h. In mice treated with intratracheal (i.t.) LPS or keratinocyte chemokine, neutrophil emigration was associated with increased permeability of the lung epithelium, as determined by increased bronchoalveolar lavage (BAL) fluid albumin concentration, which decreased over 3-6 days. Activation of ß-catenin/p300-dependent gene expression using the compound ICG-001 accelerated epithelial repair in vitro and in murine models. Neutrophil transmigration induced epithelial expression of the ß-catenin/p300 target genes Wnt-induced secreted protein (WISP) 1 and cysteine-rich (Cyr) 61, as determined by real-time PCR (qPCR) and immunostaining. Purified neutrophil elastase induced WISP1 upregulation in lung epithelial cells, as determined by qPCR. WISP1 expression increased in murine lungs after i.t. LPS, as determined by ELISA of the BAL fluid and qPCR of whole lung extracts. Finally, recombinant WISP1 and Cyr61 accelerated repair, and Cyr61-neutralizing antibodies delayed repair of the injured epithelium in vitro. We conclude that ß-catenin/p300-dependent expression of WISP1 and Cyr61 is critical for epithelial repair and represents a potential therapeutic target to promote epithelial repair after inflammatory injury.

Racial differences in selected cytokine allelic and genotypic frequencies among healthy, pregnant women in North Carolina.

BACKGROUND: Genetic susceptibility to diseases is likely influenced by common DNA variants in the form of single nucleotide polymorphisms (SNPs). The value of SNPs for linkage and association mapping studies may depend on the distribution of SNP allele frequencies across populations. OBJECTIVES: To establish the SNP allelic frequencies among Caucasian and African American women for tumor necrosis factor (TNF)alpha, transforming growth factor (TGF)beta1, interleukin-10 (IL10), interleukin-6 (IL6), and interferon (IFN)gamma. MATERIALS AND METHODS: DNA was extracted from whole blood from 123 healthy, pregnant women. PCR-based genotyping was performed for the genes encoding TNFalpha (-308G/A), TGFbeta1 (codon 10C/T, codon 25C/G), IL10 (-1082A/G, -819T/C, -592A/C), IL6 (-174C/G) and IFNgamma (874T/A). Allele frequencies were determined by Hardy-Weinberg Equilibrium and Linkage Disequilibrium tests. Differences in the SNP allelic frequencies between Caucasians and African Americans were assessed by the chi(2) of Amitage trend test. RESULTS: SNP allelic and genotypic frequencies for IL6 and IFNgamma, but not for TNFalpha, TGFbeta1, and IL10, differed significantly between the Caucasian and African American women. CONCLUSIONS: Recognition of racial differences in SNP allelic and genotypic frequencies for selected cytokines is important for designing and powering future linkage and association mapping studies investigating the role of cytokines in human disease.

The Neuron-Glia Unit in Neuropathology: Is it a Double-Edged Sword?

For decades, astrocytes were primarily recognized as structural components of the CNS. Only recently have we begun to understand their other functions within the context of CNS homeostasis. Astrocytes were previously thought of as passive cells, due primarily to the fact that they did not appear to respond to electrical stimulation or synaptic activity and showed only slight changes in membrane potential. It is now appreciated that astrocytes can, in fact, depolarize in response to neural activity [Haydon, 2001]. Additionally, astrocytes play an important role in distributing energy substrates to neurons [Dienel and Hertz 2001], Astrocyte-astrocyte, as well as astrocyte - neuron cross talk have been documented [Haydon, 2001). Other astrocytic functions include the production of trophic factors, the regulation of neurotransmitters and ion concentration, and the removal of debris from the extracellular environment [Aschner et al" 2002]. Thus, astrocytes play a number of essential roles in the CNS. When functioning optimally, they act the part of the charming astrocyte. However, astrocytes also play a role in generating reactive intermediates or impede brain remodeling and regeneration upon injury shifting them into the contrasting position of the "villain" astrocyte.