Selective activation and proliferation of a quiescent stem cell population in the neuroepithelial body microenvironment.

BACKGROUND: The microenvironment (ME) of neuroepithelial bodies (NEBs) harbors densely innervated groups of pulmonary neuroendocrine cells that are covered by Clara-like cells (CLCs) and is believed to be important during development and for adult airway epithelial repair after severe injury. Yet, little is known about its potential stem cell characteristics in healthy postnatal lungs. METHODS: Transient mild lung inflammation was induced in mice via a single low-dose intratracheal instillation of lipopolysaccharide (LPS). Bronchoalveolar lavage fluid (BALF), collected 16 h after LPS instillation, was used to challenge the NEB ME in ex vivo lung slices of control mice. Proliferating cells in the NEB ME were identified and quantified following simultaneous LPS instillation and BrdU injection. RESULTS: The applied LPS protocol induced very mild and transient lung injury. Challenge of lung slices with BALF of LPS-treated mice resulted in selective Ca2+-mediated activation of CLCs in the NEB ME of control mice. Forty-eight hours after LPS challenge, a remarkably selective and significant increase in the number of divided (BrdU-labeled) cells surrounding NEBs was observed in lung sections of LPS-challenged mice. Proliferating cells were identified as CLCs. CONCLUSIONS: A highly reproducible and minimally invasive lung inflammation model was validated for inducing selective activation of a quiescent stem cell population in the NEB ME. The model creates new opportunities for unraveling the cellular mechanisms/pathways regulating silencing, activation, proliferation and differentiation of this unique postnatal airway epithelial stem cell population.

Selective gene expression analysis of the neuroepithelial body microenvironment in postnatal lungs with special interest for potential stem cell characteristics.

BACKGROUND: The pulmonary neuroepithelial body (NEB) microenvironment (ME) consists of innervated cell clusters that occur sparsely distributed in the airway epithelium, an organization that has so far hampered reliable selective gene expression analysis. Although the NEB ME has been suggested to be important for airway epithelial repair after ablation, little is known about their potential stem cell characteristics in healthy postnatal lungs. Here we report on a large-scale selective gene expression analysis of the NEB ME. METHODS: A GAD67-GFP mouse model was used that harbors GFP-fluorescent NEBs, allowing quick selection and pooling by laser microdissection (LMD) without further treatment. A panel of stem cell-related PCR arrays was used to selectively compare mRNA expression in the NEB ME to control airway epithelium (CAE). For genes that showed a higher expression in the NEB ME, a ranking was made based on the relative expression level. Single qPCR and immunohistochemistry were used to validate and quantify the PCR array data. RESULTS: Careful optimization of all protocols appeared to be essential to finally obtain high-quality RNA from pooled LMD samples of NEB ME. About 30% of the more than 600 analyzed genes showed an at least two-fold higher expression compared to CAE. The gene that showed the highest relative expression in the NEB ME, Delta-like ligand 3 (Dll3), was investigated in more detail. Selective Dll3 gene expression in the NEB ME could be quantified via single qPCR experiments, and Dll3 protein expression could be localized specifically to NEB cell surface membranes. CONCLUSIONS: This study emphasized the importance of good protocols and RNA quality controls because of the, often neglected, fast RNA degradation in postnatal lung samples. It was shown that sufficient amounts of high-quality RNA for reliable complex gene expression analysis can be obtained from pooled LMD-collected NEB ME samples of postnatal lungs. Dll3 expression, which has also been reported to be important in high-grade pulmonary tumor-initiating cells, was used as a proof-of-concept to confirm that the described methodology represents a promising tool for further unraveling the molecular basis of NEB ME physiology in general, and its postnatal stem cell capacities in particular.

Novel Small Molecule-Derived, Highly Selective Substrates for Fibroblast Activation Protein (FAP).

Fibroblast activation protein (FAP) is a proline-selective serine protease. It is hardly expressed in healthy adult tissue but upregulated in tissue remodeling sites associated with several diseases including epithelial cancer types, atherosclerosis, arthritis and fibrosis. Ongoing research aims at clinical implementation of FAP as a biomarker for these diseases. Several immunochemical methods that quantify FAP expression have been reported. An alternative/complementary approach focuses on quantification of FAP's enzymatic activity. Developing an activity-based assay for FAP has nonetheless proven challenging because of selectivity issues with respect to prolyl oligopeptidase (PREP). Here, we present substrate-type FAP probes that are structurally derived from a FAP-inhibitor (UAMC1110) that we published earlier. Both cleavage efficiency and FAP-selectivity of the best compounds in the series equal or surpass the most advanced peptide-based FAP substrates reported to date. Finally, proof-of-concept is provided that 4-aminonaphthol containing probes can spatially localize FAP activity in biological samples.