Inhibition of EZH2 prevents acute respiratory distress syndrome (ARDS)-associated pulmonary fibrosis by regulating the macrophage polarization phenotype.

BACKGROUND: We recently reported histone methyltransferase enhancer of zeste homolog 2 (EZH2) as a key epigenetic regulator that contributes to the dysfunction of innate immune responses to sepsis and subsequent lung injury by mediating the imbalance of macrophage polarization. However, the role of EZH2 in acute respiratory distress syndrome (ARDS)-associated fibrosis remains poorly understood. METHODS: In this study, we investigated the role and mechanisms of EZH2 in pulmonary fibrosis in a murine model of LPS-induced ARDS and in ex-vivo cultured alveolar macrophages (MH-S) and mouse lung epithelial cell line (MLE-12) by using 3-deazaneplanocin A (3-DZNeP) and EZH2 the small interfering (si) RNA. RESULTS: We found that treatment with 3-DZNeP significantly ameliorated the LPS-induced direct lung injury and fibroproliferation by blocking EMT through TGF-ß1/Smad signaling pathway and regulating shift of macrophage phenotypes. In the ex-vivo polarized alveolar macrophages cells, treatment with EZH2 siRNA or 3-DZNeP suppressed the M1 while promoted the M2 macrophage differentiation through modulating the STAT/SOCS signaling pathway and activating PPAR-γ. Moreover, we identified that blockade of EZH2 with 3-DZNeP suppressed the epithelial to mesenchymal transition (EMT) in co-cultured bronchoalveolar lavage fluid (BALF) and mouse lung epithelial cell line through down-regulation of TGF-ß1, TGF-ßR1, Smad2 while up-regulation of Smad7 expression. CONCLUSIONS: These results indicate that EZH2 is involved in the pathological process of ARDS-associated pulmonary fibrosis. Targeting EZH2 may be a potential therapeutic strategy to prevent and treat pulmonary fibrosis post ARDS.

Interkinetic nuclear movements promote apical expansion in pseudostratified epithelia at the expense of apicobasal elongation.

Pseudostratified epithelia (PSE) are a common type of columnar epithelia found in a wealth of embryonic and adult tissues such as ectodermal placodes, the trachea, the ureter, the gut and the neuroepithelium. PSE are characterized by the choreographed displacement of cells' nuclei along the apicobasal axis according to phases of their cell cycle. Such movements, called interkinetic movements (INM), have been proposed to influence tissue expansion and shape and suggested as culprit in several congenital diseases such as CAKUT (Congenital anomalies of kidney and urinary tract) and esophageal atresia. INM rely on cytoskeleton dynamics just as adhesion, contractility and mitosis do. Therefore, long term impairment of INM without affecting proliferation and adhesion is currently technically unachievable. Here we bypassed this hurdle by generating a 2D agent-based model of a proliferating PSE and compared its output to the growth of the chick neuroepithelium to assess the interplay between INM and these other important cell processes during growth of a PSE. We found that INM directly generates apical expansion and apical nuclear crowding. In addition, our data strongly suggest that apicobasal elongation of cells is not an emerging property of a proliferative PSE but rather requires a specific elongation program. We then discuss how such program might functionally link INM, tissue growth and differentiation.

Patterning of papillae on the mouse tongue: A system for the quantitative assessment of planar cell polarity signaling.

The dorsal surface of the mouse tongue is covered by ~7000 papillae, asymmetric epithelial protrusions that are precisely oriented to create a stereotyped macroscopic pattern. Within the context of this large-scale pattern, neighboring papillae exhibit a high degree of local order that minimizes the differences in their orientations. We show here that the orientations of lingual papillae are under the control of the core planar cell polarity (PCP) genes Vangl1, Vangl2, and Celsr1. Using K14-Cre and Nkx2.5-Cre to induce conditional knockout of Vangl1 and/or Vangl2 in the tongue epithelium, we observe more severe disruptions to local order among papillae with inactivation of larger numbers of Vangl genes, a greater role for Vangl2 than Vangl1, and a more severe phenotype with the Vangl2 Looptail (Lp) allele than the Vangl2 null allele, consistent with a dominant negative mode of action of the Vangl2Lp allele. Interestingly, Celsr1-/- tongues show disruption of both local and global order, with many papillae in the anterior tongue showing a reversed orientation. To quantify each of these phenotypes, we have developed and applied three procedures for sampling the orientations of papillae and assessing the degree of order on different spatial scales. The experiments reported here establish the dorsal surface of the mouse tongue as a favorable system for studying PCP control of epithelial patterning.

Modelling planar polarity of epithelia: the role of signal relay in collective cell polarization.

Collective cell polarization is an important characteristic of tissues. Epithelia commonly display cellular structures that are polarized within the plane of the tissue. Establishment of this planar cell polarity requires mechanisms that locally align polarized structures between neighbouring cells, as well as cues that provide global information about alignment relative to an axis of a tissue. In the Drosophila ovary, the cadherin Fat2 is required to orient actin filaments located at the basal side of follicle cells perpendicular to the long axis of the egg chamber. The mechanisms directing this orientation of actin filaments, however, remain unknown. Here we show, using genetic mosaic analysis, that fat2 is not essential for the local alignment of actin filaments between neighbouring cells. Moreover, we provide evidence that Fat2 is involved in the propagation of a cue specifying the orientation of actin filaments relative to the tissue axis. Monte Carlo simulations of actin filament orientation resemble the results of the genetic mosaic analysis, if it is assumed that a polarity signal can propagate from a signal source only through a connected chain of wild-type cells. Our results suggest that Fat2 is required for propagating global polarity information within the follicle epithelium through direct cell-cell contact. Our computational model might be more generally applicable to study collective cell polarization in tissues.

Quantitative assessment of the response of primary derived human osteoblasts and macrophages to a range of nanotopography surfaces in a single culture model in vitro.

The effect of nanotopography on a range of Ti oxide surfaces was determined. Flat Ti, 3%, 19%, 30% and 43% topography densities of 110 nm high hemispherical protrusions were cultured in contact with primary derived human macrophages and osteoblasts in single culture models. Prior to introduction of the test substrate the phenotype and optimum conditions for in vitro cell culture were established. The cellular response was investigated and quantified by assessments of cytoskeletal development and orientation, viable cell adhesion, cytokine production and release and RT-PCR analysis of osteogenic markers. The tested nanotopographies did not have a statistically significant effect on viable cell adhesion and subsequent cytoskeletal formation. Surface chemistry was the dominant factor as established via incorporation of a tissue culture polystyrene, TCPS, control. The topography surfaces induced a release of chemotactic macrophage activation agents at 1 day in conjunction with stress fibre formation and a subsequent fibronectin network formation. Osteoblasts migrated away from the topography surfaces to the exposed TCPS within the wells during the 7-day period.

Connective tissue polarity unraveled by a markov-chain mechanism of collagen fibril segment self-assembly.

The well-established occurrence of pyroelectricity (Lang, 1966) in tissues of living organisms has found a first explanation by a Markov-chain mechanism taking place during collagen fibril self-assembly in extracytoplasmic channels. Recently reported biochemical findings on the longitudinal fusion reactivity of small fibril segments (which undergo C-, N- and C-, C- but not N-, N-terminal fusions; see Graham et al., 2000; Kadler et al., 1996) may provide a mechanism by which a difference in the fusion probabilities P(CC), P(NN) drives the self-assembly into partial macroscopic polar order. In principle, a Markov-chain growth process can lower the noncentrosymmetric infinity 2 symmetry describing dielectric properties of a growing limb (as managed by fibroblasts) into the polar infinity group. It is proposed that macroscopically polar properties enter the biological world by a stochastic mechanism of unidirectional growth. Polarity formation in organisms shows similarity to effects reported for molecular crystals (Hulliger et al., 2002).

Markov-type evolution of materials into a polar state.

Assembling polar building blocks into a solid material by a Markov-chain process of unidirectional growth principally results in a metastable state that shows effects of macroscopic polarity. Stochastic polarity formation can be described by probabilities for the attachment of building blocks to a surface. Because of the polar symmetry of the building blocks, there is a fundamental difference in the probabilities for attaching them "tip-first" or "back-first" to growth sites at a surface. A difference in the corresponding probabilities drives the evolution of a vectorial property through a gain in configurational entropy. Examples from the mechanical, the crystalline and the biological world demonstrate growth-induced macroscopic polarity. In crystals, growth upon centrosymmetric seeds can produce twinned crystals with a "sectorwise" pyroelectric effect. Polarity formation in connective tissues is explained by a Markov-chain mechanism, which drives the self-assembly of collagen fibril segments. An unified stochastic growth model brings up a general concept for the formation of materials with polar properties.

Assessment of f-actin organization and apical-basal polarity during in vivo cat endothelial wound healing.

PURPOSE: To assess the relationships between cytoskeletal changes and apical-basal polarity during healing of mechanical scrape injuries in the cat corneal endothelium. METHODS: Ten cats (20 eyes) were used in this study. One mechanical scrape injury was created in the corneal endothelium of each eye using a blunt olive tip cannula. Tandem scanning confocal microscopy (TSCM) was performed at sequential time points after injury for in vivo assessment of cell morphology and wound healing rates. In two eyes, scanning electron microscopy was performed to allow verification of TSCM observations. Ten eyes were collected between 6 and 48 hours after wounding for in situ labeling of f-actin, ZO-1, or both. RESULTS: Cat endothelial cell morphology observed using in vivo microscopy was identical to that shown using scanning electron microscopy. During healing, endothelial cells always remained attached to the endothelial sheet, although some showed extensions of lamellipodia into the open wound area. The in situ localization of f-actin also correlated with the TSCM in vivo wound morphology. Quantitative analysis showed that there was a decrease in the intensity of phalloidin-fluorescein isothiocyanate staining at the leading edge of the wound, suggesting a decrease in f-actin; a significant correlation was found between the relative intensity of f-actin staining and the distance from the wound margin (R = 0.98, P < 0.01). At 24 and 48 hours after injury, both ZO-1 and f-actin maintained an apical localization within cells immediately adjacent to the leading edge, despite the considerable distance of movement and dramatic decrease in the intensity of f-actin staining. CONCLUSIONS: Overall, these data demonstrate that after scrape injury in the cat, endothelial cells exhibit a pattern of healing in which total intracellular f-actin is reduced, but normal cell connectivity and apical-basal polarity are maintained throughout.

The fundamental motor of the human neutrophil is not random: evidence for local non-Markov movement in neutrophils.

The search for a fundamental mechano-chemical process that results in net cell motion has led investigators to fit neutrophil tracking data to well described physical models in hopes of understanding the functional form of the driving force. The Ornstein-Uhlenbeck (OU) equation for mean square displacement describes a locally persistent and globally random process and is often used as a starting point for analysis of neutrophil displacements. Based upon the apparently close fit of neutrophil tracking data to this equation and the nature of its derivation, biologists have inferred that the motor of the neutrophil is best represented as a random process. However, 24 of 37 neutrophil paths that we investigated preferentially display programmatic rather than Markov short term correlations between displacements or turn angles. These correlations reflect a bimodal rather than a uniform distribution of subpath correlations in the two variables, and are strongly sampling rate-dependent. Significant periodic components of neutrophil shape change are also detected at the same time scale using either Fourier or elliptical Fourier transform-based descriptors of the neutrophil perimeter. Oscillations in neutrophil velocity have the same period. Taken together, these data suggest a nonstochastic, and perhaps periodic, component to the process driving neutrophil movement.