Search

The morphometric analysis of lung structure using the principles of stereology has emerged as a powerful tool to describe the structural changes in lung architecture that accompany the development of lung disease that is experimentally modelled in adult mice. These stereological principles are now being applied to the study of the evolution of the lung architecture over the course of prenatal and postnatal lung development in mouse neonates and adolescents. The immature lung is structurally and functionally distinct from the adult lung, and has a smaller volume than does the adult lung. These differences have raised concerns about whether the inflation fixation of neonatal mouse lungs with the airway pressure (Paw) used for the inflation fixation of adult mouse lungs may cause distortion of the neonatal mouse lung structure, leading to the generation of artefacts in subsequent analyses. The objective of this study was to examine the impact of a Paw of 10, 20 and 30 cmH2O on the estimation of lung volumes and stereologically assessed parameters that describe the lung structure in developing mouse lungs. The data presented demonstrate that low Paw (10 cmH2O) leads to heterogeneity in the unfolding of alveolar structures within the lungs, and that high Paw (30 cmH2O) leads to an overestimation of the lung volume, and thus, affects the estimation of volume-dependent parameters, such as total alveoli number and gas-exchange surface area. Thus, these data support the use of a Paw of 20 cmH2O for inflation fixation in morphometric studies on neonatal mouse lungs. [ABSTRACT FROM AUTHOR]

Background and Purpose: Recruitment and involvement of bone-/blood-derived circulating fibrocytes (CF) in the promotion of fibrotic tissue remodelling processes have been shown. However, their direct contribution to pathological changes is not clear. The present study investigates the causal role of CF in the pathogenesis of pulmonary hypertension (PH).Experimental Approach: For selective ablation of CF, we applied the suicidal gene strategy with herpes simplex virus thymidine kinase (HSV-TK) and ganciclovir. The transgenic mice were generated, having HSV-TK-GFP transgene under the collagen 1 promoter. To selectively target CF, HSV-TK-GFP+ bone marrow transplanted into irradiated wild type mice. These chimera mice were subjected to hypoxia for PH induction and ganciclovir for CF ablation.Key Results: In vivo CF ablation reduced right ventricular hypertrophy and vascular remodelling with reduced total collagen content. We quantified the CF recruited in the perivascular area and arterial wall of small pulmonary arteries. There was significant recruitment of CF in the lung in response to hypoxia. The characterization of CF showed the expression of CD45 and collagen1 (GFP) along with α-smooth muscle actin (αSMA).Conclusion and Implications: Our data demonstrated that CF ablation has a potential impact on right ventricular hypertrophy and vascular remodelling in the setting of experimental pulmonary hypertension induced by hypoxia. The beneficial effects may be related to the direct contribution of fibrocytes or its paracrine effect on other resident cell types. Thus, clinical manipulation of CF may represent a novel therapeutic approach to ameliorate the disease state in pulmonary hypertension. [ABSTRACT FROM AUTHOR]

Summary: Accurate estimation of the absolute number of a particular cell‐type in whole organs is increasingly important in studies on organogenesis, and the remodelling and repair of diseased tissues. The unbiased estimation of the absolute number of cells in an organ is complicated, and design‐based stereology remains the method of choice. This has led investigators to explore alternative approaches – such as flow cytometry – as a faster and less labour‐intensive replacement for stereology. To address whether flow cytometry might substitute stereology, design‐based stereology was compared with microfluorosphere‐controlled flow cytometry, for estimation of the absolute number of alveolar epithelial type 2 cells (AEC2) in the lungs of two mouse strains: wild‐type C57BL/6J mice and Sftpc‐YFP mice. Using design‐based stereology, ≈10.7 million and ≈9.0 million AEC2 were estimated in the lungs of wild‐type C57BL/6J mice and Sftpc‐YFP mice, respectively. Substantially fewer AEC2 were estimated using flow cytometry. In wild‐type C57/BL6J mouse lungs, 59% of the AEC2 estimated by design‐based stereology were estimated by flow cytometry (≈6.3 million), using intracellular staining for pro‐surfactant protein C. Similarly, in Sftpc‐YFP mouse lungs, 23% of the AEC2 estimated by design‐based stereology were estimated by flow cytometry (≈2.1 million), using yellow fluorescent protein fluorescence. Our data suggest that flow cytometry underestimates AEC2 number, possibly due to impaired recoverability of AEC2 from dissociated lung tissue. These data suggest design‐based stereology as the method of choice for the unbiased estimation of the absolute number of cells in an organ. Lay Description: There is much interest in studies on the pathological changes that accompany disease, to be able to count or estimate the number of a particular cell‐type in solid tissue, such as an organ. The easiest way to do this is to make liquid suspensions of single cells from solid tissue, and then to count the number of cells of interest, using either a microscope, or automated cell counting (for example, a flow cytometer). Alternatively, solid tissue may be examined microscopically, where the cell‐type of interest might also be counted 'by eye' or in an automated manner using software (called planimetry). All of these approaches to counting cells in solid organs come with serious drawbacks, and estimation of the cell number may thus be inaccurate. To overcome this, we have employed a combination of mathematical tools and statistical principles together with microscopy (called 'design‐based stereology') that permits the unbiased counting of cells in microscopic fields, which can then be extrapolated to the entire solid tissue volume, to accurately estimate the number of a cell‐type of interest in the solid tissue. We have compared this method with the estimation of cell number using a flow cytometer. Our data reveal that flow cytometry appreciably underestimates the total number of cells in solid tissue, where we used the lung as an example of solid tissue, and estimated the number of a unique cell‐type in the lung: the alveolar epithelial type 2 cell, to compare stereology with flow cytometry. We believe that flow cytometry underestimates the cell number due to the difficulty of breaking up solid tissue into single cells, and being able to recover all of those single cells for analysis. Our data supports the recommendation to use stereology, not flow cytometry, to accurately estimate the number of a particular cell‐type in solid tissue. Accurate estimation of the absolute number of a particular cell‐type in whole organs is increasingly important in studies on organogenesis, and the remodelling and repair of diseased tissues. Although estimation of the relative number of cells might be straightforward, unbiased estimation of the absolute number of cells in an organ is complicated, and design‐based stereology remains the method of choice. This has led investigators to explore alternative approaches – such as flow cytometry – as a faster and less labour‐intensive replacement for stereology. To address whether flow cytometry might substitute stereology, design‐based stereology was compared with microfluorosphere‐controlled flow cytometry, for estimation of the absolute number of alveolar epithelial type 2 cells (AEC2) in the lungs of two mouse strains: wild‐type C57BL/6J mice and Sftpc‐YFP mice. Using design‐based stereology, ≈10.7 million and ≈9.0 million AEC2 were estimated in the lungs of wild‐type C57BL/6J mice and Sftpc‐YFP mice, respectively. Substantially fewer AEC2 were estimated using flow cytometry. In wild‐type C57/BL6J mouse lungs, 59% of the AEC2 estimated by design‐based stereology were estimated by flow cytometry (≈6.3 million), using intracellular staining for pro‐surfactant protein C. Similarly, in Sftpc‐YFP mouse lungs, 23% of the AEC2 estimated by design‐based stereology were estimated by flow cytometry (≈2.1 million), using yellow fluorescent protein fluorescence. Our data suggest that flow cytometry underestimates AEC2 number, possibly due to impaired recoverability of AEC2 from dissociated lung tissue. These data suggest design‐based stereology as the method of choice for the unbiased estimation of the absolute number of cells in an organ. [ABSTRACT FROM AUTHOR]

Transcription factor 21 (Tcf21) is a basic helix-loop-helix transcription factor required for mesenchymal development in several organs. Others have demonstrated that Tcf21 is expressed in embryonic lung mesenchyme and that loss of Tcf21 results in a pulmonary hypoplasia phenotype. Although recent singlecell transcriptome analysis has described multiple mesenchymal cell types in the lung, few have characterized the Tcf21 expressing population. To explore the Tcf21 mesenchymal lineage, we traced Tcf21-expressing cells during embryogenesis and in the adult. Our results showed that Tcf21 progenitor cells at embryonic day (E)11.5 generated a subpopulation of fibroblasts and lipofibroblasts and a limited number of smooth muscle cells. After E15.5, Tcf21 progenitor cells exclusively become lipofibroblasts and interstitial fibroblasts. Lipid metabolism genes were highly expressed in perinatal and adult Tcf21 lineage cells. Overexpression of Tcf21 in primary neonatal lung fibroblasts led to increases in intracellular neutral lipids, suggesting a regulatory role for Tcf21 in lipofibroblast function. Collectively, our results reveal that Tcf21 expression after E15.5 delineates the lipofibroblast and a population of interstitial fibroblasts. The Tcf21 inducible Cre mouse line provides a novel method for identifying and manipulating the lipofibroblast. [ABSTRACT FROM AUTHOR]

The objective of lung development is to generate an organ of gas exchange that provides both a thin gas diffusion barrier and a large gas diffusion surface area, which concomitantly generates a steep gas diffusion concentration gradient. As such, the lung is perfectly structured to undertake the function of gas exchange: a large number of small alveoli provide extensive surface area within the limited volume of the lung, and a delicate alveolo-capillary barrier brings circulating blood into close proximity to the inspired air. Efficient movement of inspired air and circulating blood through the conducting airways and conducting vessels, respectively, generates steep oxygen and carbon dioxide concentration gradients across the alveolo-capillary barrier, providing ideal conditions for effective diffusion of both gases during breathing. The development of the gas exchange apparatus of the lung occurs during the second phase of lung development--namely, late lung development--which includes the canalicular, saccular, and alveolar stages of lung development. It is during these stages of lung development that preterm-born infants are delivered, when the lung is not yet competent for effective gas exchange. These infants may develop bronchopulmonary dysplasia (BPD), a syndrome complicated by disturbances to the development of the alveoli and the pulmonary vasculature. It is the objective of this review to update the reader about recent developments that further our understanding of the mechanisms of lung alveolarization and vascularization and the pathogenesis of BPD and other neonatal lung diseases that feature lung hypoplasia. [ABSTRACT FROM AUTHOR]

Postnatal lung maturation generates a large number of small alveoli, with concomitant thinning of alveolar septal walls, generating a large gas exchange surface area but minimizing the distance traversed by the gases. This demand for a large and thin gas exchange surface area is not met in disorders of lung development, such as bronchopulmonary dysplasia (BPD) histopathologically characterized by fewer, larger alveoli and thickened alveolar septal walls. Diseases such as BPD are often modeled in the laboratory mouse to better understand disease pathogenesis or to develop new interventional approaches. To date, there have been no stereology-based longitudinal studies on postnatal mouse lung development that report dynamic changes in alveoli number or alveolar septal wall thickness during lung maturation. To this end, changes in lung structure were quantified over the first 22 mo of postnatal life of C57BL/6J mice. Alveolar density peaked at postnatal day (P)39 and remained unchanged at 9 mo (P274) but was reduced by 22 mo (P669). Alveoli continued to be generated, initially at an accelerated rate between P5 and P14, and at a slower rate thereafter. Between P274 and P669, loss of alveoli was noted, without any reduction in lung volume. A progressive thinning of the alveolar septal wall was noted between P5 and P28. Pronounced sex differences were observed in alveoli number in adult (but not juvenile) mice, when comparing male and female mouse lungs. This sex difference was attributed exclusively to the larger volume of male mouse lungs. [ABSTRACT FROM AUTHOR]

ACTA2 expression identifies pulmonary airway and vascular smooth muscle cells (SMCs) as well as alveolar myofibroblasts (MYF). Mesenchymal progenitors expressing fibroblast growth factor 10 ( Fgf10), Wilms tumor 1 ( Wt1), or glioma-associated oncogene 1 ( Gli1) contribute to SMC formation from early stages of lung development. However, their respective contribution and specificity to the SMC and/or alveolar MYF lineages remain controversial. In addition, the contribution of mesenchymal cells undergoing active WNT signaling remains unknown. Using Fgf10CreERT2, Wt1CreERT2, Gli1CreERT2, and Axin2CreERT2 inducible driver lines in combination with a tdTomatoflox reporter line, the respective differentiation of each pool of labeled progenitor cells along the SMC and alveolar MYF lineages was quantified. The results revealed that while FGF10+ and WT1+ cells show a minor contribution to the SMC lineage, GLI1+ and AXIN2+ cells significantly contribute to both the SMC and alveolar MYF lineages, but with limited specificity. Lineage tracing using the Acta2-CreERT2 transgenic line showed that ACTA2+ cells labeled at embryonic day (E)11.5 do not expand significantly to give rise to new SMCs at E18.5. However, ACTA2+ cells labeled at E15.5 give rise to the majority (85%-97%) of the SMCs in the lung at E18.5 as well as alveolar MYF progenitors in the lung parenchyma. Fluorescence-activated cell sorting-based isolation of different subpopulations of ACTA2+ lineage-traced cells followed by gene arrays, identified transcriptomic signatures for alveolar MYF progenitors versus airway and vascular SMCs at E18.5. Our results establish a new transcriptional landscape for further experiments addressing the function of signaling pathways in the formation of different subpopulations of ACTA2+ cells. S tem C ells 2017;35:1566-1578 [ABSTRACT FROM AUTHOR]

Bronchopulmonary dysplasia (BPD) is the most common complication of preterm birth characterized by blunted post-natal lung development. BPD can be modelled in mice by exposure of newborn mouse pups to elevated oxygen levels. Little is known about the mechanisms of perturbed lung development associated with BPD. The advent of transgenic mice, where genetic rearrangements can be induced in particular cell-types at particular time-points during organogenesis, have great potential to explore the pathogenic mechanisms at play during arrested lung development. Many inducible, conditional transgenic technologies available rely on the application of the estrogen-receptor modulator, tamoxifen. While tamoxifen is well-tolerated and has been widely employed in adult mice, or in healthy developing mice; tamoxifen is not well-tolerated in combination with hyperoxia, in the most widely-used mouse model of BPD. To address this, we set out to establish a safe and effective tamoxifen dosing regimen that can be used in newborn mouse pups subjected to injurious stimuli, such as exposure to elevated levels of environmental oxygen. Our data reveal that a single intraperitoneal dose of tamoxifen of 0.2 mg applied to newborn mouse pups in 10 μl Miglyol vehicle was adequate to successfully drive Cre recombinase-mediated genome rearrangements by the fifth day of life, in a murine model of BPD. The number of recombined cells was comparable to that observed in regular tamoxifen administration protocols. These findings will be useful to investigators where tamoxifen dosing is problematic in the background of injurious stimuli and mouse models of human and veterinary disease. [ABSTRACT FROM AUTHOR]

A reduced number of alveoli is the structural hallmark of diseases of the neonatal and adult lung, where alveoli either fail to develop (as in bronchopulmonary dysplasia), or are progressively destroyed (as in chronic obstructive pulmonary disease). To correct the loss of alveolar septa through therapeutic regeneration, the mechanisms of septa formation must first be understood. The present study characterized platelet-derived growth factor receptor-positive (PDGFRα+) cell populations during late lung development in mice. PDGFRα+ cells (detected using a PDGFRαGFP reporter line) were noted around the proximal airways during the pseudoglandular stage. In the canalicular stage, PDGFRα+ cells appeared in the more distal mesenchyme, and labeled α-smooth muscle actin-positive tip cells in the secondary crests and lipofibroblasts in the primary septa during alveolarization. Some PDGFRα+ cells appeared in the mesenchyme of the adult lung. Over the course of late lung development, PDGFRα+ cells consistently expressed collagen I, and transiently expressed markers of mesenchymal stem cells. With the use of both, a constitutive and a conditional PDGFRαCre line, it was observed that PDGFRα+ cells generated alveolar myofibroblasts including tip cells of the secondary crests, and lipofibroblasts. These lineages were committed before secondary septation. The present study provides new insights into the time-dependent commitment of the PDGFRα+ cell lineage to lipofibroblasts and myofibroblasts during late lung development that is needed to better understand the cellular contribution to the process of alveolarization. [ABSTRACT FROM AUTHOR]

Although the pulmonary interstitial lipofibroblast (LF) has been widely recognized in rat and mouse lungs, their presence in human lungs remains controversial. In a recent issue of the Journal, Tahedl and associates (Tahedl D, Wirkes A, Tschanz SA, Ochs M, Mühlfeld C. Am J Physiol Lung Cell Mol Physiol 307: L386-L394, 2014) address this controversy and provide the most detailed stereological analysis of LFs in mammals other than rodents. Strikingly, their observations demonstrate that LFs were only observed in rodents, which contrasts with earlier reports. This editorial reviews the anatomical, physiological, and biochemical characteristics of the LF to better understand the significance of LFs for lung development and disease. Although lipid droplets are a signature of the LF cell type, it remains unclear whether lipid storage is the defining characteristic of LFs, or whether other less overt properties determine the importance of LFs. Are lipid droplets an adaptation to the neonatal environment, or are LFs a surrogate for other properties that promote alveolar development, and do lipid droplets modify physiology or disease in adults?. [ABSTRACT FROM AUTHOR]

Background: Gas exchange represents the key physiological function of the lung, and is dependent upon proper formation of the delicate alveolar structure. Malformation or destruction of the alveolar gas-exchange regions are key histopathological hallmarks of diseases such as bronchopulmonary dysplasia (BPD), chronic obstructive pulmonary disease (COPD), and pulmonary fibrosis; all of which are characterized by perturbations to the alveolo-capillary barrier structure. Impaired gas-exchange is the primary initial consequence of these perturbations, resulting in severe clinical symptoms, reduced quality of life, and death. The pronounced morbidity and mortality associated with malformation or destruction of alveoli underscores a pressing need for new therapeutic concepts. The re-induction of alveolarization in diseased lungs is a new and exciting concept in a regenerative medicine approach to manage pulmonary diseases that are characterized by an absence of alveoli.Main Text: Mechanisms of alveolarization first need to be understood, to identify pathways and mediators that may be exploited to drive the induction of alveolarization in the diseased lung. With this in mind, a variety of candidate cell-types, pathways, and molecular mediators have recently been identified. Using lineage tracing approaches and lung injury models, new progenitor cells for epithelial and mesenchymal cell types - as well as cell lineages which are able to acquire stem cell properties - have been discovered. However, the underlying mechanisms that orchestrate the complex process of lung alveolar septation remain largely unknown.Conclusion: While important progress has been made, further characterization of the contributing cell-types, the cell type-specific molecular signatures, and the time-dependent chemical and mechanical processes in the developing, adult and diseased lung is needed in order to implement a regenerative therapeutic approach for pulmonary diseases. [ABSTRACT FROM AUTHOR]

Previous studies point to protein kinase C (PKC) isozyme η as a resistance factor in cancer cells. Therefore, we investigated whether down-regulation of PKCη with second generation antisense oligonucleotides (ODNs) would sensitise A549 human lung carcinoma cells to cytostatics. The effects were compared to the outcome of Bcl-xL down-regulation. Upon treatment with antisense ODNs, PKCη and Bcl-xL were both significantly reduced on mRNA and protein level. Down-regulation of either PKCη or Bcl-xL in combination with vincristine or paclitaxel resulted in a significant increase in caspase-3 activity compared to that in the control oligonucleotide treated cells. In addition, PKCη down-regulation augmented vincristine-induced dissipation of mitochondrial transmembrane potential. In conclusion, these results confirm that PKCη might represent a considerable resistance factor and an interesting target to improve anticancer chemotherapy. [Copyright &y& Elsevier]