Distribution of particulate matter and tissue remodeling in the human lung.

We examined the relationship between intrapulmonary particle distribution of carbonaceous and mineral dusts and remodeling of the airways along anatomically distinct airway paths in the lungs of Hispanic males from the central valley of California. Lung autopsy specimens from the Fresno County Coroner's Office were prepared by intratracheal instillation of 2% glutaraldehyde at 30 cm H(2)O pressure. Two distinct airway paths into the apico-posterior and apico-anterior portions of the left upper lung lobe were followed. Tissue samples for histologic analysis were generally taken from the intrapulmonary second, fourth, sixth, and ninth airway generations. Parenchymal tissues beyond the 12th airway generation of each airway path were also analyzed. There was little evidence of visible particle accumulation in the larger conducting airways (generations 2-6), except in bronchial-associated lymphoid tissues and within peribronchial connective tissue. In contrast, terminal and respiratory bronchioles arising from each pathway revealed varying degrees of wall thickening and remodeling. Walls with marked thickening contained moderate to heavy amounts of carbonaceous and mineral dusts. Wall thickening was associated with increases in collagen and interstitial inflammatory cells, including dust-laden macrophages. These changes were significantly greater in first-generation respiratory bronchioles compared to second- and third-generation respiratory bronchioles. These findings suggest that accumulation of carbonaceous and mineral dust in the lungs is significantly affected by lung anatomy with the greatest retention in centers of lung acini. Furthermore, there is significant remodeling of this transitional zone in humans exposed to ambient particulate matter

Quantification of Spatial Parameters in 3D Cellular Constructs Using Graph Theory

Multispectral three-dimensional (3D) imaging provides spatial information for biological structures that cannot be measured by traditional methods. This work presents a method of tracking 3D biological structures to quantify changes over time using graph theory. Cell-graphs were generated based on the pairwise distances, in 3D-Euclidean space, between nuclei during collagen I gel compaction. From these graphs quantitative features are extracted that measure both the global topography and the frequently occurring local structures of the “tissue constructs.” The feature trends can be controlled by manipulating compaction through cell density and are significant when compared to random graphs. This work presents a novel methodology to track a simple 3D biological event and quantitatively analyze the underlying structural change. Further application of this method will allow for the study of complex biological problems that require the quantification of temporal-spatial information in 3D and establish a new paradigm in understanding structure-function relationships

Asthma: a comparison of animal models using stereological methods

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Lung adenocarcinoma originates from retrovirus infection of proliferating type 2 pneumocytes during pulmonary post-natal development or tissue repair

Jaagsiekte sheep retrovirus (JSRV) is a unique oncogenic virus with distinctive biological properties. JSRV is the only virus causing a naturally occurring lung cancer (ovine pulmonary adenocarcinoma, OPA) and possessing a major structural protein that functions as a dominant oncoprotein. Lung cancer is the major cause of death among cancer patients. OPA can be an extremely useful animal model in order to identify the cells originating lung adenocarcinoma and to study the early events of pulmonary carcinogenesis. In this study, we demonstrated that lung adenocarcinoma in sheep originates from infection and transformation of proliferating type 2 pneumocytes (termed here lung alveolar proliferating cells, LAPCs). We excluded that OPA originates from a bronchioalveolar stem cell, or from mature post-mitotic type 2 pneumocytes or from either proliferating or non-proliferating Clara cells. We show that young animals possess abundant LAPCs and are highly susceptible to JSRV infection and transformation. On the contrary, healthy adult sheep, which are normally resistant to experimental OPA induction, exhibit a relatively low number of LAPCs and are resistant to JSRV infection of the respiratory epithelium. Importantly, induction of lung injury increased dramatically the number of LAPCs in adult sheep and rendered these animals fully susceptible to JSRV infection and transformation. Furthermore, we show that JSRV preferentially infects actively dividing cell in vitro. Overall, our study provides unique insights into pulmonary biology and carcinogenesis and suggests that JSRV and its host have reached an evolutionary equilibrium in which productive infection (and transformation) can occur only in cells that are scarce for most of the lifespan of the sheep. Our data also indicate that, at least in this model, inflammation can predispose to retroviral infection and cancer

Coupled Analysis of In Vitro and Histology Tissue Samples to Quantify Structure-Function Relationship

The structure/function relationship is fundamental to our understanding of biological systems at all levels, and drives most, if not all, techniques for detecting, diagnosing, and treating disease. However, at the tissue level of biological complexity we encounter a gap in the structure/function relationship: having accumulated an extraordinary amount of detailed information about biological tissues at the cellular and subcellular level, we cannot assemble it in a way that explains the correspondingly complex biological functions these structures perform. To help close this information gap we define here several quantitative temperospatial features that link tissue structure to its corresponding biological function. Both histological images of human tissue samples and fluorescence images of three-dimensional cultures of human cells are used to compare the accuracy of in vitro culture models with their corresponding human tissues. To the best of our knowledge, there is no prior work on a quantitative comparison of histology and in vitro samples. Features are calculated from graph theoretical representations of tissue structures and the data are analyzed in the form of matrices and higher-order tensors using matrix and tensor factorization methods, with a goal of differentiating between cancerous and healthy states of brain, breast, and bone tissues. We also show that our techniques can differentiate between the structural organization of native tissues and their corresponding in vitro engineered cell culture models

Acidic Extracellular pH Promotes Activation of Integrin αvβ3

Acidic extracellular pH is characteristic of the cell microenvironment in several important physiological and pathological contexts. Although it is well established that acidic extracellular pH can have profound effects on processes such as cell adhesion and migration, the underlying molecular mechanisms are largely unknown. Integrin receptors physically connect cells to the extracellular matrix, and are thus likely to modulate cell responses to extracellular conditions. Here, we examine the role of acidic extracellular pH in regulating activation of integrin [alpha]v[beta]3. Through computational molecular dynamics simulations, we find that acidic extracellular pH promotes opening of the [alpha]v[beta]3 headpiece, indicating that acidic pH can thereby facilitate integrin activation. This prediction is consistent with our flow cytometry and atomic force microscope-mediated force spectroscopy assays of integrin [alpha]v[beta]3 on live cells, which both demonstrate that acidic pH promotes activation at the intact cell surface. Finally, quantification of cell morphology and migration measurements shows that acidic extracellular pH affects cell behavior in a manner that is consistent with increased integrin activation. Taken together, these computational and experimental results suggest a new and complementary mechanism of integrin activation regulation, with associated implications for cell adhesion and migration in regions of altered pH that are relevant to wound healing and cancer.National Institute of Biomedical Imaging and Bioengineering (U.S.) (Award Number T32EB006348)Massachusetts Institute of Technology (Collamore-Rogers Fellowship)National Institutes of Health (U.S.) (NIH Cell Migration Consortium Grant U54-GM069668)National Science Foundation (U.S.) (CAREER Award)Singapore-MIT Alliance for Research and Technology (BioSystem and Micromechanics (BioSyM) Interdisciplinary Research Group

Integrin-Specific Mechanoresponses to Compression and Extension Probed by Cylindrical Flat-Ended AFM Tips in Lung Cells

Cells from lung and other tissues are subjected to forces of opposing directions that are largely transmitted through integrin-mediated adhesions. How cells respond to force bidirectionality remains ill defined. To address this question, we nanofabricated flat-ended cylindrical Atomic Force Microscopy (AFM) tips with ∼1 µm2 cross-section area. Tips were uncoated or coated with either integrin-specific (RGD) or non-specific (RGE/BSA) molecules, brought into contact with lung epithelial cells or fibroblasts for 30 s to form focal adhesion precursors, and used to probe cell resistance to deformation in compression and extension. We found that cell resistance to compression was globally higher than to extension regardless of the tip coating. In contrast, both tip-cell adhesion strength and resistance to compression and extension were the highest when probed at integrin-specific adhesions. These integrin-specific mechanoresponses required an intact actin cytoskeleton, and were dependent on tyrosine phosphatases and Ca2+ signaling. Cell asymmetric mechanoresponse to compression and extension remained after 5 minutes of tip-cell adhesion, revealing that asymmetric resistance to force directionality is an intrinsic property of lung cells, as in most soft tissues. Our findings provide new insights on how lung cells probe the mechanochemical properties of the microenvironment, an important process for migration, repair and tissue homeostasis

Multiway modeling and analysis in stem cell systems biology

<p>Abstract</p> <p>Background</p> <p>Systems biology refers to multidisciplinary approaches designed to uncover emergent properties of biological systems. Stem cells are an attractive target for this analysis, due to their broad therapeutic potential. A central theme of systems biology is the use of computational modeling to reconstruct complex systems from a wealth of reductionist, molecular data (e.g., gene/protein expression, signal transduction activity, metabolic activity, etc.). A number of deterministic, probabilistic, and statistical learning models are used to understand sophisticated cellular behaviors such as protein expression during cellular differentiation and the activity of signaling networks. However, many of these models are bimodal i.e., they only consider row-column relationships. In contrast, multiway modeling techniques (also known as tensor models) can analyze multimodal data, which capture much more information about complex behaviors such as cell differentiation. In particular, tensors can be very powerful tools for modeling the dynamic activity of biological networks over time. Here, we review the application of systems biology to stem cells and illustrate application of tensor analysis to model collagen-induced osteogenic differentiation of human mesenchymal stem cells.</p> <p>Results</p> <p>We applied Tucker1, Tucker3, and Parallel Factor Analysis (PARAFAC) models to identify protein/gene expression patterns during extracellular matrix-induced osteogenic differentiation of human mesenchymal stem cells. In one case, we organized our data into a tensor of type protein/gene locus link × gene ontology category × osteogenic stimulant, and found that our cells expressed two distinct, stimulus-dependent sets of functionally related genes as they underwent osteogenic differentiation. In a second case, we organized DNA microarray data in a three-way tensor of gene IDs × osteogenic stimulus × replicates, and found that application of tensile strain to a collagen I substrate accelerated the osteogenic differentiation induced by a static collagen I substrate.</p> <p>Conclusion</p> <p>Our results suggest gene- and protein-level models whereby stem cells undergo transdifferentiation to osteoblasts, and lay the foundation for mechanistic, hypothesis-driven studies. Our analysis methods are applicable to a wide range of stem cell differentiation models.</p

The epithelial cholinergic system of the airways

Acetylcholine (ACh), a classical transmitter of parasympathetic nerve fibres in the airways, is also synthesized by a large number of non-neuronal cells, including airway surface epithelial cells. Strongest expression of cholinergic traits is observed in neuroendocrine and brush cells but other epithelial cell types—ciliated, basal and secretory—are cholinergic as well. There is cell type-specific expression of the molecular pathways of ACh release, including both the vesicular storage and exocytotic release known from neurons, and transmembrane release from the cytosol via organic cation transporters. The subcellular distribution of the ACh release machineries suggests luminal release from ciliated and secretory cells, and basolateral release from neuroendocrine cells. The scenario as known so far strongly suggests a local auto-/paracrine role of epithelial ACh in regulating various aspects on the innate mucosal defence mechanisms, including mucociliary clearance, regulation of macrophage function and modulation of sensory nerve fibre activity. The proliferative effects of ACh gain importance in recently identified ACh receptor disorders conferring susceptibility to lung cancer. The cell type-specific molecular diversity of the epithelial ACh synthesis and release machinery implies that it is differently regulated than neuronal ACh release and can be specifically targeted by appropriate drugs