Identification of Macrophage Genotype and Key Biological Pathways in Circulating Angiogenic Cell Transcriptome.

BACKGROUND: Circulating angiogenic cells (CAC) have been identified as important regulators of vascular biology. However, there is still considerable debate about the genotype and function of CAC. METHODS AND RESULTS: Data from publicly available gene expression data sets were used to analyse the transcriptome of in vitro cultured CAC (CACiv). Genes and pathways of interest were further evaluated using qPCR comparing CACiv versus CD14+ monocytic cells. The CACiv transcriptome strongly related to tissue macrophages, and more specifically to regulatory M2c macrophages. The cytokine expression profile of CACiv was predominantly immune modulatory and resembled the cytokine expression of tumor-associated macrophages (TAM). Pathway analysis revealed previously unrecognized biological processes in CACiv, such as riboflavin metabolism and liver X receptor (LXR)/retinoid X receptor (RXR) and farnesoid X receptor (FXR)/retinoid X receptor (RXR) pathways. Analysis of endothelial-specific genes did not show evidence for endothelial transdifferentiation. CONCLUSIONS: CACiv are genotypically similar to regulatory M2c macrophages and lack signs of endothelial differentiation.

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.

Functional expression of the multimodal extracellular calcium-sensing receptor in pulmonary neuroendocrine cells.

The Ca(2+)-sensing receptor (CaSR) is the master regulator of whole-body extracellular free ionized [Ca(2+)]o. In addition to sensing [Ca(2+)]o, CaSR integrates inputs from a variety of different physiological stimuli. The CaSR is also expressed in many regions outside the [Ca(2+)]o homeostatic system, including the fetal lung where it plays a crucial role in lung development. Here, we show that neuroepithelial bodies (NEBs) of the postnatal mouse lung express a functional CaSR. NEBs are densely innervated groups of neuroendocrine epithelial cells in the lung representing complex sensory receptors in the airways and exhibiting stem cell characteristics. qRT-PCR performed on laser microdissected samples from GAD67-GFP mouse lung cryosections revealed exclusive expression of the CaSR in the NEB microenvironment. CaSR immunoreactivity was present at NEB cells from postnatal day 14 onwards. Confocal imaging of lung slices revealed that NEB cells responded to an increase of [Ca(2+)]o with a rise in intracellular Ca(2+) ([Ca(2+)]i); an effect mimicked by several membrane-impermeant CaSR agonists (e.g. the calcimimetic R-568) and that was blocked by the calcilytic Calhex-231. Block of TRPC channels attenuated the CaSR-dependent increases in [Ca(2+)]i, suggesting that Ca(2+) influx through TRPC channels contributes to the total [Ca(2+)]i signal evoked by the CaSR in NEBs. CaSR also regulated baseline [Ca(2+)]i in NEBs and, through paracrine signaling from Clara-like cells, coordinated intercellular communication in the NEB microenvironment. These data suggest that the NEB CaSR integrates multiple signals converging on this complex chemosensory unit, and is a key regulator of this intrapulmonary airway stem cell niche.

GABAergic signaling in the pulmonary neuroepithelial body microenvironment: functional imaging in GAD67-GFP mice.

Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the central nervous system (CNS) of vertebrates, but has also been reported in multiple cell types outside the CNS. A GABAergic system has been proposed in neuroepithelial bodies (NEBs) in monkey lungs. Pulmonary NEBs are known as complex intraepithelial sensory airway receptors and are part of the NEB microenvironment. Aim of the present study was to unravel a GABAergic signaling system in the NEB microenvironment in mouse lungs, enabling the use of genetically modified animals for future functional studies. Immunostaining of mouse lungs revealed that glutamic acid decarboxylase 65/67 (GAD65/67), a rate-limiting enzyme in the biosynthesis of GABA, and the vesicular GABA transporter (VGAT) were exclusively expressed in NEB cells. In GAD67-green fluorescent protein (GFP) knock-in mice, all pulmonary NEBs appeared to express GFP. For confocal live cell imaging, ex vivo vibratome lung slices of GAD67-GFP mice can be directly loaded with fluorescent functional probes, e.g. a red-fluorescent calcium dye, without the necessity of time-consuming prior live visualization of NEBs. RT-PCR of the NEB microenvironment obtained by laser microdissection revealed the presence of both GABAA and GABAB (R1 and R2) receptors, which was confirmed by immunostaining. In conclusion, the present study not only revealed the presence of a GABAergic signaling pathway, but also the very selective expression of GFP in pulmonary NEBs in a GAD67-GFP mouse model. Different proof of concept experiments have clearly shown that adoption of the GAD67-GFP mouse model will certainly boost future functional imaging and gene expression analysis of the mouse NEB microenvironment.

Precision-cut vibratome slices allow functional live cell imaging of the pulmonary neuroepithelial body microenvironment in fetal mice.

We recently developed an ex vivo lung slice model that allows for confocal live cell imaging (LCI) of neuroepithelial bodies (NEBs) in postnatal mouse lungs (postnatal days 1-21 and adult). NEBs are morphologically well-characterized, extensively innervated groups of neuroendocrine cells in the airway epithelium, which are shielded from the airway lumen by 'Clara-like' cells. The prominent presence of differentiated NEBs from early embryonic development onwards, strongly suggests that NEBs may exert important functions during late fetal and neonatal life. The main goal of the present study was to adapt the current postnatal LCI lung slice model to enable functional studies of fetal mouse lungs (gestational days 17-20).In vibratome lung slices of prenatal mice, NEBs could be unequivocally identified with the fluorescent stryryl pyridinium dye 4-Di-2-ASP. Changes in the intracellular free calcium concentration and in mitochondrial membrane potential could be monitored using appropriate functional fluorescent indicators (e.g. Fluo-4).It is clear that the described fetal mouse lung slice model is suited for LCI studies of Clara cells, ciliated cells, and the NEB microenvironment, and offers excellent possibilities to further unravel the significance of NEBs during the prenatal and perinatal period.

Neuroepithelial bodies as mechanotransducers in the intrapulmonary airway epithelium: involvement of TRPC5.

In rodent lungs, a major part of the myelinated vagal airway afferents selectively contacts pulmonary neuroepithelial bodies (NEBs). Because most myelinated vagal airway afferents concern physiologically characterized mechanoreceptors, the present study aimed at unraveling the potential involvement of NEB cells in transducing mechanosensory information from the airways to the central nervous system. Physiological studies were performed using confocal Ca(2+) imaging of airway epithelium in murine lung slices. Mechanical stimulation by short-term application of a mild hypoosmotic solution (230 mosmol) resulted in a selective, fast, reversible, and reproducible Ca(2+) rise in NEB cells. Other airway epithelial cells could only be activated using more severe hypoosmotic stimuli (< 200 mosmol). NEB cells selectively expressed the Ca(2+)-permeable osmo- and mechanosensitive transient receptor potential canonical channel 5 (TRPC5) in their apical membranes, whereas immunoreactivity for TRP vanilloid-4 and TRP melastatin-3 was abundant in virtually all other airway epithelial cells. Hypoosmotic activation of NEB cells was prevented by GsMTx-4, an inhibitor of mechanosensitive ion channels, and by SKF96365, an inhibitor of TRPC channels. Short application of gadolinium, reported to activate TRPC5 channels, evoked a transient Ca(2+) rise in NEB cells. Osmomechanical activation of NEB cells gave rise to a typical delayed activation of Clara-like cells due to the release of ATP from NEB cells. Because ATP may activate the NEB-associated P2X(2/3) ATP receptor expressing myelinated vagal afferents, the current observations strongly suggest that pulmonary NEB cells are fully equipped to initiate mechanosensory signal transduction to the central nervous system via a purinergic signaling pathway.

Expression of mechanogated two-pore domain potassium channels in mouse lungs: special reference to mechanosensory airway receptors.

Afferent activities arising from sensory nerve terminals located in lungs and airways are carried almost exclusively by fibres travelling through the vagus nerve. Based on electrophysiological investigations, intrapulmonary airway-related vagal afferent receptors have been classified into three main subtypes, two of which are myelinated and mechanosensitive, i.e., rapidly and slowly adapting receptors. To allow for a full functional identification of the distinct populations of airway receptors, morphological and neurochemical characteristics still need to be determined. Nerve terminals visualised using markers for myelinated vagal afferents seem to be almost uniquely associated with two morphologically well-formed airway receptor end organs, smooth muscle-associated airway receptors (SMARs) and neuroepithelial bodies (NEBs), localised in airway smooth muscle and epithelium, respectively. Due to the lack of a selective marker for SMARs in mice, no further neurochemical coding is available today. NEBs are extensively innervated diffusely spread groups of neuroendocrine cells in the airway epithelium, and are known to receive at least two separate populations of myelinated vagal afferent nerve terminals. So far, however, no evidence has been reported for the expression of channels that may underlie direct sensing and transduction of mechanical stimuli by the receptor terminals in NEBs and SMARs. This study focused on the expression of mechanogated two-pore domain K(+) (K(2P)) channels, TREK-1 and TRAAK, in mouse airways and more particular in the NEB micro-environment and in SMARs by multiple immunostaining. TREK-1 could be detected on smooth muscle cells surrounding intrapulmonary airways and blood vessels, while TRAAK was expressed on myelinated vagal afferents terminating both in SMARs and in the NEB micro-environment. Co-stainings with known markers for subpopulations of myelinated vagal afferents and general neuronal markers revealed that all identified SMARs exhibit TRAAK immunoreactivity, and that at least three subpopulations exist in mouse airways. Also, the intraepithelial terminals of both subpopulations of NEB-associated myelinated vagal sensory nerve fibres were shown to express TRAAK. In conclusion, the present study finally characterised an intrinsically mechanosensitive ion channel, the K(2P) channel TRAAK, on the terminals of identified myelinated vagal nodose airway afferents, organised as SMARs and as components of the innervation of NEBs. These data support the hypothesis that both SMARs and NEBs harbour the morphological counterparts of electrophysiologically identified myelinated vagal airway mechanoreceptors. TRAAK appears to be strongly involved in regulating airway mechanosensing since it was found to be expressed on the terminals of all subpopulations of potential vagal mechanosensors.

Neurochemical pattern of the complex innervation of neuroepithelial bodies in mouse lungs.

As best characterized for rats, it is clear that pulmonary neuroepithelial bodies (NEBs) are contacted by a plethora of nerve fiber populations, suggesting that they represent an extensive group of multifunctional intraepithelial airway receptors. Because of the importance of genetically modified mice for functional studies, and the current lack of data, the main aim of the present study was to achieve a detailed analysis of the origin and neurochemical properties of nerve terminals associated with NEBs in mouse lungs. Antibodies against known selective markers for sensory and motor nerve terminals in rat lungs were used on lungs from control and vagotomized mice of two different strains, i.e., Swiss and C57-Bl6. NEB cells were visualized by antibodies against either the general neuroendocrine marker protein gene-product 9.5 (PGP9.5) or calcitonin gene-related peptide (CGRP). Thorough immunohistochemical examination of NEB cells showed that some of these NEB cells also exhibit calbindin D-28 k (CB) and vesicular acetylcholine transporter (VAChT) immunoreactivity (IR). Mouse pulmonary NEBs were found to receive intraepithelial nerve terminals of at least two different populations of myelinated vagal afferents: (1) Immunoreactive (ir) for vesicular glutamate transporters (VGLUTs) and CB; (2) expressing P2X(2) and P2X(3) ATP receptors. CGRP IR was seen in varicose vagal nerve fibers and in delicate non-vagal fibers, both in close proximity to NEBs. VAChT immunostaining showed very weak IR in the NEB-related intraepithelial vagal sensory nerve terminals. nNOS- or VIP-ir nerve terminals could be observed at the base of pulmonary NEBs. While a single NEB can be contacted by multiple nerve fiber populations, it was clear that none of the so far characterized nerve fiber populations contacts all pulmonary NEBs. The present study revealed that mouse lungs harbor several populations of nerve terminals that may selectively contact NEBs. Although at present the physiological significance of the innervation pattern of NEBs remains enigmatic, it is likely that NEBs are receptor-effector end-organs that may host complex and/or multiple functional properties in normal airways. The neurochemical information on the innervation of NEBs in mouse lungs gathered in the present study will be essential for the interpretation of upcoming functional data and for the study of transgenic mice.