The Cellular and Physiological Basis for Lung Repair and Regeneration: Past, Present, and Future.

The respiratory system, which includes the trachea, airways, and distal alveoli, is a complex multi-cellular organ that intimately links with the cardiovascular system to accomplish gas exchange. In this review and as members of the NIH/NHLBI-supported Progenitor Cell Translational Consortium, we discuss key aspects of lung repair and regeneration. We focus on the cellular compositions within functional niches, cell-cell signaling in homeostatic health, the responses to injury, and new methods to study lung repair and regeneration. We also provide future directions for an improved understanding of the cell biology of the respiratory system, as well as new therapeutic avenues

BMP4 Sufficiency to Induce Choroid Plexus Epithelial Fate from Embryonic Stem Cell-Derived Neuroepithelial Progenitors

Choroid plexus epithelial cells (CPECs) have essential developmental and homeostatic roles related to the cerebrospinal fluid (CSF) and blood-CSF barrier they produce. Accordingly, CPEC dysfunction has been implicated in many neurological disorders, such as Alzheimer’s disease, and transplant studies have provided proof-of-concept for CPEC-based therapies. However, such therapies have been hindered by the inability to expand or generate CPECs in culture. During development, CPECs differentiate from preneurogenic neuroepithelial cells and require Bone Morphogenetic Protein (BMP) signaling, but whether BMPs suffice for CPEC induction is unknown. Here we provide evidence for BMP4 sufficiency to induce CPEC fate from neural progenitors derived from mouse embryonic stem cells (ESCs). CPEC specification by BMP4 was restricted to an early time period after neural induction in culture, with peak CPEC competency correlating to neuroepithelial cells rather than radial glia. In addition to molecular, cellular, and ultrastructural criteria, derived CPECs (dCPECs) had functions that were indistinguishable from primary CPECs, including self-assembly into secretory vesicles and integration into endogenous choroid plexus epithelium following intraventricular injection. We then used BMP4 to generate dCPECs from human ESC-derived neuroepithelial cells. These findings demonstrate BMP4 sufficiency to instruct CPEC fate, expand the repertoire of stem cell-derived neural derivatives in culture, and herald dCPEC-based therapeutic applications aimed at the unique interface between blood, CSF, and brain governed by CPECs

Contribution of Epithelial Hypoxia Signaling to Pulmonary Fibrosis: Role of FAK1 and Galectin-1 as Driver Molecules

Idiopathic Pulmonary Fibrosis (IPF) is a deadly disease of unknown origin, which causes 80,000 deaths every year in the US and Europe combined. Unknown etiology and late diagnosis, combined with limited treatment options, contribute to a dismal survival rate of 3-5 years post diagnosis. Although molecular mechanisms underlying IPF pathogenesis and progression have been studied for over two decades, lack of in vivo models that recapitulate chronic, progressive, and irreversible nature of IPF have contributed to limited therapeutic success in clinical trials. Currently, only two drugs, Pirfenidone and Nintedanib, are approved for IPF treatment in the US, with their efficacy yet to be completely determined. Patients with IPF often observe lung infections, alveolar collapse, and respiratory failure, which are associated with focal edema and local hypoxia and contribute to development of hypoxemia associated with acute exacerbation of IPF (AE-IPF). In my thesis, I posit that hypoxic injury to the lung epithelium can initiate profibrotic signaling that can contribute to pathogenesis and progression of pulmonary fibrosis in vitro and in vivo. In my in silico studies, I analyzed human protein kinases to identify structural peculiarities that diversify their functions and highlight central hub kinases governing cell signaling. Using this approach, I identified Focal Adhesion Kinase 1 (FAK1) as a central hub kinase contributing to cytoskeletal remodeling. My proteomics and transcriptional studies defined in vitro effect of hypoxia in activation of lung epithelial cells. Using systems biology approaches, I identified interplay between transforming growth factor – β (TGF–β) signaling, hypoxia signaling, and FAK1 signaling. Further, my studies identified Galectin-1 as a novel mediator of hypoxia-induced pulmonary fibrosis. To mimic exacerbation of PF in patients, I developed a novel mouse model of exacerbated pulmonary fibrosis using subclinical bleomycin injury with chronic hypoxia. Further, to fill the existing requirement of an in vivo model of chronic PF, I characterized a triple transgenic mouse model that conditionally activates hypoxia signaling in the lung epithelial cells and causes progressive PF over a span of 12 weeks. Lastly, I performed RNA-Seq experiments on primary AEC2s isolated from our transgenic mouse model to identify a hypoxia-mediated profibrotic role of microRNA-96 in down-regulation of PTEN, a tumor suppressor and anti-fibrotic protein. In conclusion, my studies established in vitro and in vivo roles of hypoxia in profibrotic activation of lung epithelium and identifies FAK1 and Gal-1 as key drivers of hypoxia-mediated fibrosis, which should be further evaluated in animal and human studies to determine their therapeutic potential

An inflammatory switch for stem cell plasticity

Tissue resident stem cells are capable of remarkable plasticity in areas of tissue damage, where inflammatory cells accumulate as part of the reparative response. A study in the lung now provides critical insight on how inflammatory signals alter cell-to-cell Notch signaling within the airway niche to drive stem cell plasticity

VEGF Drives the Car toward Better Gas Exchange

How lung epithelium and endothelium co-develop to maintain structural integrity of alveoli remains unclear. In this issue of Developmental Cell, Ellis et al. define how epithelial Vegfa directs development of a distinct endothelial cell population that ultimately plays a critical role in ensuring appropriate alveolar septation during alveologenesis

Distinct Airway Epithelial Stem Cells Hide among Club Cells but Mobilize to Promote Alveolar Regeneration

Lung injury activates specialized adult epithelial progenitors to regenerate the epithelium. Depending on the extent of injury, both remaining alveolar type II cells (AEC2s) and distal airway stem/progenitors mobilize to cover denuded alveoli and restore normal barriers. The major source of airway stem/progenitors other than basal-like cells remains uncertain. Here, we define a distinct subpopulation (∼5%) of club-like lineage-negative epithelial progenitors (LNEPs) marked by high H2-K1 expression critical for alveolar repair. Quiescent H2-K1high cells account for virtually all in vitro regenerative activity of airway lineages. After bleomycin injury, H2-K1 cells expand and differentiate in vivo to alveolar lineages. However, injured H2-K1 cells eventually develop impaired self-renewal with features of senescence, limiting complete repair. Normal H2-K1high cells transplanted into injured lungs differentiate into alveolar cells and rescue lung function. These findings indicate that small subpopulations of specialized stem/progenitors are required for effective lung regeneration and are a potential therapeutic adjunct after major lung injury

Presence and Utility of Intrinsically Disordered Regions in Kinases

Since aberrant cell signaling pathways underlie majority of pathophysiological morbidities, kinase inhibitors are routinely used for pharmacotherapy. However, most kinase inhibitors suffer from adverse off-target effects. Inhibition of one kinase in a pathogenic signaling pathway elicits multiple compensatory feedback signaling loops, reinforcing the pathway rather than inhibiting it, leading to chemoresistance. Thus, development of novel computational strategies providing predictive evidence to inhibit a specific set of kinases to mitigate an aberrant signaling pathway with minimum side-effects is imperative. First, our analyses reveal that many kinases contain intrinsically disordered regions, which may participate in facilitating protein–protein interactions at the kinome level. Second, we employ a kinome-wide approach to identify intrinsic disorder and streamline a methodology that adds to the knowledge of therapeutically targeting kinase cascades to treat diseases. Furthermore, we find that within the kinome network, some kinases with intrinsically disordered regions have a high topological score, likely acting as kinome modulators. Third, using network analysis, we demonstrate that 5 kinases emerge as topologically most significant, forming kinome sub-networks, comprising of other kinases and transcription factors that are known to serve as drivers of disease pathogenesis. To support these findings, we have biologically validated the interplay between kinome modulators SRC and AKT kinases and uncovered their novel function in regulating transcription factors of the SMAD family. Taken together, we identify novel kinome modulators driven by intrinsic disorder, and biologically validate the thesis that therapeutic disruption of the function of kinome modulators engaged in regulatory cross-talk between disparate pathways can lead to reduced oncogenic potential in cancer cells