Spectral monitoring of surfactant clearance during alveolar epithelial type II cell differentiation

In this study, we report on the noninvasive identification of spectral markers of alveolar type II (ATII) cell differentiation in vitro using Raman microspectroscopy. ATII cells are progenitor cells for alveolar type I (ATI) cells in vivo, and spontaneously differentiate toward an ATI-like phenotype in culture. We analyzed undifferentiated and differentiated primary human ATII cells, and correlated Raman spectral changes to cellular changes in morphology and marker protein synthesis (surfactant protein C, alkaline phosphatase, caveolin-1). Undifferentiated ATII cells demonstrated spectra with strong phospholipid vibrations, arising from alveolar surfactant stored within cytoplasmic lamellar bodies (Lbs). Differentiated ATI-like cells yielded spectra with significantly less lipid content. Factor analysis revealed a phospholipid-dominated spectral component as the main discriminator between the ATII and ATI-like phenotypes. Spectral modeling of the data revealed a significant decrease in the spectral contribution of cellular lipids—specifically phosphatidyl choline, the main constituent of surfactant, as ATII cells differentiate. These observations were consistent with the clearance of surfactant from Lbs as ATII cells differentiate, and were further supported by cytochemical staining for Lbs. These results demonstrate the first spectral characterization of primary human ATII cells, and provide insight into the biochemical properties of alveolar surfactant in its unperturbed cellular environment.     

Paracrine stimulation of surfactant secretion by extracellular ATP in response to mechanical deformation

We developed a heterologous system to study the effect of mechanical deformation on alveolar epithelial cells. First, isolated primary rat alveolar type II (ATII) cells were plated onto silastic substrata coated with fibronectin and maintained in culture under conditions where they become alveolar type I-like (ATI) cells. This was followed by a second set of ATII cells labeled with the nontransferable, vital fluorescent stain 5-chloromethylfluorescein diacetate to distinguish them from ATI cells. By morphometric analysis, equibiaxial deformation (stretch) of the silastic substratum induced comparable changes in cell surface area for both ATII and ATI cells. Surfactant lipid secretion was measured using cells metabolically labeled with [(3)H]choline. In response to 21% tonic stretch for 15 min, ATII cells seeded with ATI cells secreted nearly threefold more surfactant lipid compared with ATII cells seeded alone. ATI cells did not secrete lipid in response to stretch. The enhanced lipid secretion by ATII plus ATI cocultures was inhibited by treatment with apyrase and adenosine deaminase, suggesting that ATP release by ATI cells enhanced surfactant lipid secretion at 21% stretch. This was confirmed using a luciferase assay where, in response to 21% stretch, ATI cells released fourfold more ATP than ATII cells. Because ATI cells release significantly more ATP at a lower level of stretch than ATII cells, this supports the hypothesis that ATI cells are mechanosensors in the lung and that paracrine stimulation of ATII cells by extracellular ATP released from ATI cells plays a role in regulating surfactant secretion.     

Assessment of Cell Line Models of Primary Human Cells by Raman Spectral Phenotyping

Researchers have previously questioned the suitability of cell lines as models for primary cells. In this study, we used Raman microspectroscopy to characterize live A549 cells from a unique molecular biochemical perspective to shed light on their suitability as a model for primary human pulmonary alveolar type II (ATII) cells. We also investigated a recently developed transduced type I (TT1) cell line as a model for alveolar type I (ATI) cells. Single-cell Raman spectra provide unique biomolecular fingerprints that can be used to characterize cellular phenotypes. A multivariate statistical analysis of Raman spectra indicated that the spectra of A549 and TT1 cells are characterized by significantly lower phospholipid content compared to ATII and ATI spectra because their cytoplasm contains fewer surfactant lamellar bodies. Furthermore, we found that A549 spectra are statistically more similar to ATI spectra than to ATII spectra. The spectral variation permitted phenotypic classification of cells based on Raman spectral signatures with >99% accuracy. These results suggest that A549 cells are not a good model for ATII cells, but TT1 cells do provide a reasonable model for ATI cells. The findings have far-reaching implications for the assessment of cell lines as suitable primary cellular models in live cultures.     

Coexpression of RTI40 with alveolar epithelial type II cell proteins in lungs following injury: identification of alveolar intermediate cell types

Injured alveolar epithelial type (AT) I cells are replaced following the proliferation and transformation of ATII cells to new ATI cells. RTI(40) is an ATI cell-specific protein required for normal lung development. We hypothesized that intermediate cell types in the ATII-to-ATI cell transformation would coexpress RTI(40) and ATII cell-selective proteins. To test this hypothesis, we used a rat model of Staphylococcus aureus-induced acute lung injury and a panel of ATI and ATII cell-specific and -selective antibodies. S. aureus induced an acute inflammatory reaction that was resolving by day 3 postinoculation. At day 3 postinoculation, the alveolar wall was thickened secondary to ATII cell hyperplasia. With the use of confocal microscopy, there was a fivefold increase in the fractional surface area of alveolar walls stained with ATII cell membrane proteins (RTII(70) and MMC4) and a decrease in the fractional surface area associated with RTI(40)-expressing cells. S. aureus-treated lungs also contained unique cell types that coexpressed the RTI(40) and ATII markers RTI(40)/MMC4/RTII(70)- and RTI(40)/MMC4-positive cells. These cells were not observed in control lungs. RTI(40)/MMC4-positive cells were also found in cultured ATII cells before they transformed to an ATI-like phenotype. Our data suggest that RTI(40)/MMC4/RTII(70)- and RTI(40)/MMC4-positive cells are intermediates in the ATII-to-ATI cell transformation. These data also suggest that the coexpression of RTI(40) with ATII cell proteins may be used to identify and investigate ATII cell transdifferentiation to ATI cells following injury.     

In utero nicotine exposure alters fetal rat lung alveolar type II cell proliferation, differentiation, and metabolism

We recently suggested that alveolar interstitial fibroblast-to-myofibroblast transdifferentiation may be a key mechanism underlying in utero nicotine-induced lung injury. However, the effects of in utero nicotine exposure on fetal alveolar type II (ATII) cells have not been fully determined. Placebo, nicotine (1 mg/kg), or nicotine (1 mg/kg) + the peroxisome proliferator-activated receptor (PPAR)-gamma agonist prostaglandin J(2) (PGJ(2), 0.3 mg/kg) was administered intraperitoneally once daily to time-mated pregnant Sprague-Dawley rats from embryonic day 6 until their death on embryonic day 20. Fetal ATII cells were isolated, and ATII cell proliferation, differentiation (surfactant synthesis), and metabolism (metabolic profiling with the stable isotope [1,2-(13)C(2)]-d-glucose) were determined after nicotine exposure in utero or in vitro. In utero nicotine exposure significantly stimulated ATII cell proliferation, differentiation, and metabolism. Although the effects on ATII cell proliferation and metabolism were almost completely prevented by concomitant treatment with PGJ(2), the effects on surfactant synthesis were not. On the basis of in utero and in vitro data, we conclude that surfactant synthesis is stimulated by nicotine's direct effect on ATII cells, whereas cell proliferation and metabolism are affected via a paracrine mechanism(s) secondary to its effects on the adepithelial fibroblasts. These data provide evidence for direct and indirect effects of in utero nicotine exposure on fetal ATII cells that could permanently alter the "developmental program" of the developing lung. More importantly, concomitant administration of PPAR-gamma agonists can effectively attenuate many of the effects of in utero exposure to nicotine on ATII cells.     

Caveolin and its cellular and subcellular immunolocalisation in lung alveolar epithelium: implications for alveolar epithelial type I cell function

Caveolae are flask-shaped invaginations of the plasmalemma which pinch off to form discrete vesicles within the cell cytoplasm. Biochemically, caveolae may be distinguished by the presence of a protein, caveolin, that is the principal component of filaments constituting their striated cytoplasmic coat. Squamous alveolar epithelial type I (ATI) cells, comprising approximately 95% of the surface area of lung alveolar epithelium, possess numerous plasmalemmal invaginations and cytoplasmic vesicles ultrastructurally indicative of caveolae. However, an ultrastructural appearance does not universally imply the biochemical presence of caveolin. This immunocytochemical study has utilised a novel application of confocal laser scanning and electron microscopy unequivocally to localise caveolin-1 to ATI cells. Further, cytoplasmic vesicles and flask-shaped membrane invaginations in the ATI cell were morphologically identified whose membranes were decorated with anti-caveolin-1 immunogold label. Coexistent with this, however, in both ATI and capillary endothelial cells could be seen membrane invaginations morphologically characteristic of caveolae, but which lacked associated caveolin immunogold label. This could reflect a true biochemical heterogeneity in populations of morphologically similar plasmalemmal invaginations or an antigen threshold requirement for labelling. The cuboidal alveolar epithelial type II cell (ATII) also displayed specific label for caveolin-1 but with no ultrastructural evidence for the formation of caveolae. The biochemical association of caveolin with ATI cell vesicles has broad implications for the assignment and further study of ATI cell function.     

Assessing the efficacy of coherent anti‐Stokes Raman scattering microscopy for the detection of infiltrating glioblastoma in fresh brain samples

Coherent anti‐Stokes Raman scattering (CARS) microscopy is an emerging technique for identification of brain tumors. However, tumor identification by CARS microscopy on bulk samples and in vivo has been so far verified retrospectively on histological sections, which only provide a gross reference for the interpretation of CARS images without matching at cellular level. Therefore, fluorescent labels were exploited for direct assessment of the interpretation of CARS images of solid and infiltrative tumors. Glioblastoma cells expressing green fluorescent protein (GFP) were used for induction of tumors in mice (n = 7). The neoplastic nature of cells imaged by CARS microscopy was unequivocally verified by addressing two‐photon fluorescence of GFP on fresh brain slices and in vivo. In fresh unfixed biopsies of human glioblastoma (n = 10), the fluorescence of 5‐aminolevulinic acid‐induced protoporphyrin IX was used for identification of tumorous tissue. Distinctive morphological features of glioblastoma cells, i.e. larger nuclei, evident nuclear membrane and nucleolus, were identified in the CARS images of both mouse and human brain tumors. This approach demonstrates that the chemical contrast provided by CARS allows the localization of infiltrating tumor cells in fresh tissue and that the cell morphology in CARS images is useful for tumor recognition.

Experimental glioblastoma expressing green fluorescent protein.     

Stretch stimulation: its effects on alveolar type II cell function in the lung

Mechanical stimuli regulate cell function in much the same way as chemical signals do. This has been studied in various cell types, particularly those with defined mechanical roles. The alveolar type II cell (ATII) cell, which is part of the alveolar epithelium of the lung, is responsible for the synthesis and secretion of pulmonary surfactant. It is now widely believed that stretch of ATII cells, which occurs during breathing, is the predominant physiological trigger for surfactant release. To study this, investigators have used an increasingly sophisticated array of in vitro and in vivo models. Using various stretch devices and models of lung ventilation and expansion, it has been shown that stretch regulates multiple activities in ATII cells. In addition to surfactant secretion, stretch triggers the differentiation of ATII to alveolar type I cells, as well as ATII cell apoptosis. In doing so, stretch modulates the proportion of these cells in the lung epithelium during both development and maturation of the lung and following lung injury. From such studies, it appears that mechanical distortion plays an integral part in maintaining the overall structure and function of the lung.     

Label‐free quantitative chemical imaging and classification analysis of adipogenesis using mouse embryonic stem cells

Stem cells have received much attention recently for their potential utility in regenerative medicine. The identification of their differentiated progeny often requires complex staining procedures, and is challenging for intermediary stages which are a priori unknown. In this work, the ability of label‐free quantitative coherent anti‐Stokes Raman scattering (CARS) micro‐spectroscopy to identify populations of intermediate cell states during the differentiation of murine embryonic stem cells into adipocytes is assessed. Cells were imaged at different days of differentiation by hyperspectral CARS, and images were analysed with an unsupervised factorization algorithm providing Raman‐like spectra and spatially resolved maps of chemical components. Chemical decomposition combined with a statistical analysis of their spatial distributions provided a set of parameters that were used for classification analysis. The first 2 principal components of these parameters indicated 3 main groups, attributed to undifferentiated cells, cells differentiated into committed white pre‐adipocytes, and differentiating cells exhibiting a distinct protein globular structure with adjacent lipid droplets. An unsupervised classification methodology was developed, separating undifferentiated cell from cells in other stages, using a novel method to estimate the optimal number of clusters. The proposed unsupervised classification pipeline of hyperspectral CARS data offers a promising new tool for automated cell sorting in lineage analysis.      

Label‐free and non‐invasive monitoring of porcine trophoblast derived cells: differentiation in serum and serum‐free media

Traditional approaches to characterize stem cell differentiation are time‐consuming, lengthy and invasive. Here, Raman microspectroscopy (RM) and atomic force microscopy (AFM) – both considered as non‐invasive techniques – are applied to detect the biochemical and biophysical properties of trophoblast derived stem‐like cells incubated up to 10 days under conditions designed to induce differentiation. Significant biochemical and biophysical differences between control cells and differentiated cells were observed. Quantitative real time PCR was also applied to analyze gene expression. The relationship between cell differentiation and associated cellular biochemical and biomechanical changes were discussed.

Monitoring trophoblast cells differentiation     

Integration of diffraction phase microscopy and Raman imaging for label‐free morpho‐molecular assessment of live cells

Label‐free quantitative imaging is highly desirable for studying live cells by extracting pathophysiological information without perturbing cell functions. Here, we demonstrate a novel label‐free multimodal optical imaging system with the capability of providing comprehensive morphological and molecular attributes of live cells. Our morpho‐molecular microscopy (3M) system draws on the combined strength of quantitative phase microscopy (QPM) and Raman microscopy to probe the morphological features and molecular fingerprinting characteristics of each cell under observation. While the commonr‐path geometry of our QPM system allows for highly sensitive phase measurement, the Raman microscopy is equipped with dual excitation wavelengths and utilizes the same detection and dispersion system, making it a distinctive multi‐wavelength system with a small footprint. We demonstrate the applicability of the 3M system by investigating nucleated and nonnucleated cells. This integrated label‐free platform has a promising potential in preclinical research, as well as in clinical diagnosis in the near future.      

Fiber probe for nonlinear imaging applications

Over the past years it had been demonstrated that multimodal imaging combining the nonlinear modalities coherent anti‐Stokes Raman scattering (CARS), two‐photon excited auto‐fluorescence (TPEF) and second harmonic generation (SHG) show a great potential for tissue diagnosis and tumor identification. To extend the applicability of this multimodal imaging approach for in‐vivo tissue screening of difficult to access body regions the development of suitable fiber optic probes is required. Here we report about a novel CARS imaging fiber probe consisting of 10,000 coherent light guiding elements preserving the spatial relationship between the entrance and the output of the fiber. Therefore the scanning procedure can be shifted from the distal to the proximal end of the fiber probe and no moving parts or driving current are required to realize in‐vivo CARS endoscopy.

Back scattered CARS image of rabbit aorta with plaques (white) using a laser scanning microscope and an imaging fiber.     

The impact of cell fixation on coherent anti‐stokes Raman scattering signal intensity in neuronal and glial cell lines

A number of studies require sample fixation, aimed to preserve cells in a physiological state with minimal changes of morphology and intracellular molecular content. Sample fixation may significantly distort experimental data, which makes the data interpretation process more challenging. It is particularly important for study of lipid‐related diseases, where the biochemical and morphological characteristics of the cells need to be well preserved for an accurate data analysis. This study investigates the effects of formaldehyde and ethanol (EtOH) fixatives on coherent anti‐stokes Raman scattering (CARS) signal of proteins and lipids in major cellular compartments of neuronal and glial cells. We found that both fixatives induce alteration of proteins and lipids signal in studied cell lines. Furthermore, the impact of sample preservation methods on CARS signal varies between cell lines. For instance, our data reveals that EtOH fixation induces ~45% increase of CARS signal of proteins in the nucleolus of neuronal cells and ~35% decrease of CARS signal in glial cells. The results indicate that aldehyde fixation is a preferable method for preservation of neuronal and glial cells prior to CARS imaging, as it less affects both CARS signal and intracellular distribution of proteins and lipids.      

Alteration in the Wnt microenvironment directly regulates molecular events leading to pulmonary senescence

In the aging lung, the lung capacity decreases even in the absence of diseases. The progenitor cells of the distal lung, the alveolar type II cells (ATII), are essential for the repair of the gas‐exchange surface. Surfactant protein production and survival of ATII cells are supported by lipofibroblasts that are peroxisome proliferator‐activated receptor gamma (PPARγ)‐dependent special cell type of the pulmonary tissue. PPARγ levels are directly regulated by Wnt molecules; therefore, changes in the Wnt microenvironment have close control over maintenance of the distal lung. The pulmonary aging process is associated with airspace enlargement, decrease in the distal epithelial cell compartment and infiltration of inflammatory cells. qRT–PCR analysis of purified epithelial and nonepithelial cells revealed that lipofibroblast differentiation marker parathyroid hormone‐related protein receptor (PTHrPR) and PPARγ are reduced and that PPARγ reduction is regulated by Wnt4 via a β‐catenin‐dependent mechanism. Using a human in vitro 3D lung tissue model, a link was established between increased PPARγ and pro‐surfactant protein C (pro‐SPC) expression in pulmonary epithelial cells. In the senile lung, both Wnt4 and Wnt5a levels increase and both Wnt‐s increase myofibroblast‐like differentiation. Alteration of the Wnt microenvironment plays a significant role in pulmonary aging. Diminished lipo‐ and increased myofibroblast‐like differentiation are directly regulated by specific Wnt‐s, which process also controls surfactant production and pulmonary repair mechanisms.     

Single organelle dynamics linked to 3D structure by correlative live‐cell imaging and 3D electron microscopy

Live‐cell correlative light‐electron microscopy (live‐cell‐CLEM) integrates live movies with the corresponding electron microscopy (EM) image, but a major challenge is to relate the dynamic characteristics of single organelles to their 3‐dimensional (3D) ultrastructure. Here, we introduce focused ion beam scanning electron microscopy (FIB‐SEM) in a modular live‐cell‐CLEM pipeline for a single organelle CLEM. We transfected cells with lysosomal‐associated membrane protein 1‐green fluorescent protein (LAMP‐1‐GFP), analyzed the dynamics of individual GFP‐positive spots, and correlated these to their corresponding fine‐architecture and immediate cellular environment. By FIB‐SEM we quantitatively assessed morphological characteristics, like number of intraluminal vesicles and contact sites with endoplasmic reticulum and mitochondria. Hence, we present a novel way to integrate multiple parameters of subcellular dynamics and architecture onto a single organelle, which is relevant to address biological questions related to membrane trafficking, organelle biogenesis and positioning. Furthermore, by using CLEM to select regions of interest, our method allows for targeted FIB‐SEM, which significantly reduces time required for image acquisition and data processing.      

Signal generation and Raman‐resonant imaging by non‐degenerate four‐wave mixing under tight focusing conditions

The authors demonstrate Raman‐resonant imaging based on the simultaneous generation of several nonlinear frequency mixing processes resulting from a 3‐color coherent anti‐Stokes Raman scattering (CARS) experiment. The interaction of three coincident short‐pulsed laser beams simultaneously generates both 2‐color (degenerate) CARS and 3‐color (non‐degenerate) CARS signals, which are collected and characterized spectroscopically – allowing for resonant, doubly‐resonant, and non‐resonant contrast mechanisms. Images obtained from both 2‐color and 3‐color CARS signals are compared and found to provide complementary information. The 3‐color CARS microscopy scheme provides a versatile multiplexed modality for biological imaging, which may extend the capabilities of label‐free non‐linear microscopy, e.g. by probing multiple Raman resonances. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Internalization kinetics and cytoplasmic localization of functionalized diatomite nanoparticles in cancer cells by Raman imaging

Porous biosilica nanoparticles obtained from diatomites (DNPs) have been recently demonstrated to be non‐toxic nanovectors of therapeutic agents in cancer cells. In this work, the internalization kinetics and intracellular spatial distribution of functionalized DNPs incubated with human lung epidermoid carcinoma cell line (H1355) up to 72 hours are investigated by Raman imaging. The label‐free Raman results are compared with confocal fluorescence microscopy and photoluminescence (PL) data. Raman bands specifically assigned to DNPs and cellular components provide evidence that the nanovectors are internalized and co‐localize with lipid environments. A considerable DNPs uptake in cells is observed within 6 hours, with equilibrium being achieved after 18 hours. The obtained data show the presence of DNPs up to 72 hours, without damage to cell viability or morphology. The PL measurements performed on DNPs not penetrating the cells at different incubation times are strongly correlated with the results obtained by Raman imaging and confocal microscopy analyses.      

Caveolin-1 expression and caveolae biogenesis during cell transdifferentiation in lung alveolar epithelial primary cultures.

Caveolae are omega-shaped invaginations of the plasmalemma possessing a cytoplasmic membrane protein coat of caveolin. Caveolae are present in the in vivo alveolar epithelial type I (ATI) lung cell, but absent in its progenitor, the alveolar epithelial type II (ATII) cell. In primary culture ATII cells grown on a plastic substratum acquire with time an ATI-"like" phenotype. We demonstrate that freshly isolated rat ATII cells lack caveolae and expression of caveolin-1 (a critical caveolae structural protein). As the ATII cells acquire an ATI-like phenotype in primary culture caveolin-1 expression increases, with caveolin-1 signal at 192 h postseeding up to 50-fold greater than at 60 h; caveolae were morphologically evident only after 132 h. When maintaining the differentiated ATII phenotype with time, i.e., culture upon collagen with an apical interface of air, a temporal increase in caveolin-1 expression was not observed, with only very faint signals evident even at 192 h postseeding; at no time did these cultures display caveolae. In late primary ATII cultures caveolin-1 expression and caveolae biogenesis occur as a function of in vitro transformation from the ATII to the ATI-like phenotype. The results have broad implications for the in vitro study of the role of caveolae and caveolin in alveolar epithelial cell biology.     

Interpreting CARS images of tissue within the C–H‐stretching region

Single band coherent anti‐Stokes Raman scattering (CARS) microscopy is one of the fastest implementation of nonlinear vibrational imaging allowing for video‐rate image acquisition of tissue. This is due to the large Raman signal in the C—H‐stretching region. However, the chemical specificity of such images is conventionally assumed to be low. Nonetheless, CARS imaging within the C—H‐stretching region enables detection of single cells and nuclei, which allows for histopathologic grading of tissue. Relevant information such as nucleus to cytoplasm ratio, cell density, nucleus size and shape is extracted from CARS images by innovative image processing procedures. In this contribution CARS image contrast within the C—H‐stretching region is interpreted by direct comparison with Raman imaging and correlated to the tissue composition justifying the use of CARS imaging in this wavenumber region for biomedical applications. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Chronic WNT/β-catenin signaling induces cellular senescence in lung epithelial cells

The rapid expansion of the elderly population has led to the recent epidemic of age-related diseases, including increased incidence and mortality of chronic lung diseases, such as Idiopathic Pulmonary Fibrosis (IPF). Cellular senescence is a major hallmark of aging and has a higher occurrence in IPF. The lung epithelium represents a major site of tissue injury, cellular senescence and aberrant activity of developmental pathways such as the WNT/β-catenin pathway in IPF. The potential impact of WNT/β-catenin signaling on alveolar epithelial senescence in general as well as in IPF, however, remains elusive. Here, we characterized alveolar epithelial cells of aged mice and assessed the contribution of chronic WNT/β-catenin signaling on alveolar epithelial type (AT) II cell senescence. Whole lungs from old (16–24 months) versus young (3 months) mice had relatively less epithelial (EpCAM⁺) but more inflammatory (CD45⁺) cells, as assessed by flow cytometry. Compared to young ATII cells, old ATII cells showed decreased expression of the ATII cell marker Surfactant Protein C along with increased expression of the ATI cell marker Hopx, accompanied by increased WNT/β-catenin activity. Notably, when placed in an organoid assay, old ATII cells exhibited decreased progenitor cell potential. Chronic canonical WNT/β-catenin activation for up to 7 days in primary ATII cells as well as alveolar epithelial cell lines induced a robust cellular senescence, whereas the non-canonical ligand WNT5A was not able to induce cellular senescence. Moreover, chronic WNT3A treatment of precision-cut lung slices (PCLS) further confirmed ATII cell senescence. Simultaneously, chronic but not acute WNT/β-catenin activation induced a profibrotic state with increased expression of the impaired ATII cell marker Keratin 8. These results suggest that chronic WNT/β-catenin activity in the IPF lung contributes to increased ATII cell senescence and reprogramming. In the fibrotic environment, WNT/β-catenin signaling thus might lead to further progenitor cell dysfunction and impaired lung repair.