Stress amplifies lung tissue mechanics, inflammation and oxidative stress induced by chronic inflammation

Background: Mechanisms linking behavioral stress and inflammation are poorly understood, mainly in distal lung tissue. Objective: We have investigated whether the forced swim stress (FS) could modulate lung tissue mechanics, iNOS, cytokines, oxidative stress activation, eosinophilic recruitment, and remodeling in guinea pigs (GP) with chronic pulmonary inflammation. Methods: The GP were exposed to ovalbumin or saline aerosols (2x/wk/4wks, OVA, and SAL). Twenty-four hours after the 4th inhalation, the GP were submitted to the FS protocol (5x/wk/2wks, SAL-S, and OVA-S). Seventy-two hours after the 7th inhalation, lung strips were cut and tissue resistance (Rt) and elastance (Et) were obtained (at baseline and after OVA and Ach challenge). Strips were submitted to histopathological evaluation. Results: The adrenals' weight, the serum cortisol, and the catecholamines were measured. There was an increase in IL-2, IL-5, IL-13, IFN-gamma, iNOS, 8-iso-PGF2 alpha, and in %Rt and %Et after Ach challenge in the SAL-S group compared to the SAL one. The OVA-S group has had an increase in %Rt and %Et after the OVA challenge, in %Et after the Ach and in IL-4, 8-iso-PGF2 alpha, and actin compared to the OVA. Adrenal weight and cortisol serum were increased in stressed animals compared to nonstressed ones, and the catecholamines were unaltered. Conclusion & clinical relevance: Repeated stress has increased distal lung constriction, which was associated with an increase of actin, IL-4, and 8-iso-PGF2 alpha levels. Stress has also induced an activation of iNOS, cytokines, and oxidative stress pathways.

A two-compartment mechanochemical model of the roles of transforming growth factor-beta and tissue tension in dermal wound healing

The repair of dermal tissue is a complex process of interconnected phenomena, where cellular, chemical and mechanical aspects all play a role, both in an autocrine and in a paracrine fashion. Recent experimental results have shown that transforming growth factor-beta (TGF-beta) and tissue mechanics play roles in regulating cell proliferation, differentiation and the production of extracellular materials. We have developed a 1D mathematical model that considers the interaction between the cellular, chemical and mechanical phenomena, allowing the combination of TGF-beta and tissue stress to inform the activation of fibroblasts to myofibroblasts. Additionally, our model incorporates the observed feature of residual stress by considering the changing zero-stress state in the formulation for effective strain. Using this model, we predict that the continued presence of TGF-beta in dermal wounds will produce contractures due to the persistence of myofibroblasts; in contrast, early elimination of TGF-beta significantly reduces the myofibroblast numbers resulting in an increase in wound size. Similar results were obtained by varying the rate at which fibroblasts differentiate to myofibroblasts and by changing the myofibroblast apoptotic rate. Taken together, the implication is that elevated levels of myofibroblasts is the key factor behind wounds healing with excessive contraction, suggesting that clinical strategies which aim to reduce the myofibroblast density may reduce the appearance of contractures.

Volume dependence of high-frequency respiratory mechanics in healthy adults

Respiratory system input impedance (Zrs) at low to medium frequencies below 100 Hz, and study of its volume dependence, have been used extensively to quantify airway and tissue mechanics. Zrs at high oscillation frequencies including the first antiresonant frequency (far,1) may contain important information about airway mechanics. Changes in high-frequency Zrs with lung volume have not been studied. The volume-dependent behavior of high-frequency Zrs, specifically far,1 and respiratory system resistance at first antiresonance (Rrs(far,1)), was characterized in 16 healthy adults. Zrs was measured with a forced oscillation signal (5-302.5 Hz) through a wavetube setup. To track Zrs, subjects performed slow deep inspiratory and expiratory maneuvers over 30-s measurements, during which average impedance was calculated over 0.4-s intervals, with successive overlapping estimates every 0.156 s. Flow was measured using a pneumotachometer and integrated to obtain volume. Transpulmonary pressure dependence (Ptp) of Zrs was separately determined in five subjects. Both far,1 and Rrs(far,1) decreased with increasing lung volume and Ptp, consistent with an increase in airway caliber and decreased airway wall compliance as volume increased. These characterizations provide insight into airway mechanics, and are furthermore a necessary first step toward determining whether volume dependence of the first antiresonance is altered in disease.

Mathematical investigation of the interactions between the inflammatory response and mechanical aspects of dermal wound repair

Dermal wound repair involves complex interactions between cells, cytokines and mechanics to close injuries to the skin. In particular, we investigate the contribution of fibroblasts, myofibroblasts, TGFβ, collagen and local tissue mechanics to wound repair in the human dermis. We develop a morphoelastic model where a realistic representation of tissue mechanics is key, and a fibrocontractive model that involves a reasonable approximation to the true kinetics of the important bioactive species. We use each of these descriptions to elucidate the mechanisms that generate pathologies such as hypertrophic scars, contractures and keloids. We find that for hypertrophic scar and contracture development, factors regulating the myofibroblast phenotype are critical, with heightened myofibroblast activation, reduced myofibroblast apoptosis or prolonged inflammation all predicted as mediators for scar hypertrophy and contractures. Prevention of these pathologies is predicted when myofibroblast apoptosis is induced, myofibroblast activation is blocked or TGFβ is neutralised. To investigate keloid invasion, we develop a caricature representation of the fibrocontractive model and find that TGFβ spread is the driving factor behind keloid growth. Blocking activation of TGFβ is found to cause keloid regression. Thus, we recommend myofibroblasts and TGFβ as targets for clinicians when developing intervention strategies for prevention and cure of fibrotic scars.

Ultra-structural defects cause low bone matrix stiffness despite high mineralization in osteogenesis imperfecta mice.

Bone is a complex material with a hierarchical multi-scale organization from the molecule to the organ scale. The genetic bone disease, osteogenesis imperfecta, is primarily caused by mutations in the collagen type I genes, resulting in bone fragility. Because the basis of the disease is molecular with ramifications at the whole bone level, it provides a platform for investigating the relationship between structure, composition, and mechanics throughout the hierarchy. Prior studies have individually shown that OI leads to: 1. increased bone mineralization, 2. decreased elastic modulus, and 3. smaller apatite crystal size. However, these have not been studied together and the mechanism for how mineral structure influences tissue mechanics has not been identified. This lack of understanding inhibits the development of more accurate models and therapies. To address this research gap, we used a mouse model of the disease (oim) to measure these outcomes together in order to propose an underlying mechanism for the changes in properties. Our main finding was that despite increased mineralization, oim bones have lower stiffness that may result from the poorly organized mineral matrix with significantly smaller, highly packed and disoriented apatite crystals. Using a composite framework, we interpret the lower oim bone matrix elasticity observed as the result of a change in the aspect ratio of apatite crystals and a disruption of the crystal connectivity.

Contractile and mechanical properties of epithelia with perturbed actomyosin dynamics.

Mechanics has an important role during morphogenesis, both in the generation of forces driving cell shape changes and in determining the effective material properties of cells and tissues. Drosophila dorsal closure has emerged as a reference model system for investigating the interplay between tissue mechanics and cellular activity. During dorsal closure, the amnioserosa generates one of the major forces that drive closure through the apical contraction of its constituent cells. We combined quantitation of live data, genetic and mechanical perturbation and cell biology, to investigate how mechanical properties and contraction rate emerge from cytoskeletal activity. We found that a decrease in Myosin phosphorylation induces a fluidization of amnioserosa cells which become more compliant. Conversely, an increase in Myosin phosphorylation and an increase in actin linear polymerization induce a solidification of cells. Contrary to expectation, these two perturbations have an opposite effect on the strain rate of cells during DC. While an increase in actin polymerization increases the contraction rate of amnioserosa cells, an increase in Myosin phosphorylation gives rise to cells that contract very slowly. The quantification of how the perturbation induced by laser ablation decays throughout the tissue revealed that the tissue in these two mutant backgrounds reacts very differently. We suggest that the differences in the strain rate of cells in situations where Myosin activity or actin polymerization is increased arise from changes in how the contractile forces are transmitted and coordinated across the tissue through ECadherin-mediated adhesion. Altogether, our results show that there is an optimal level of Myosin activity to generate efficient contraction and suggest that the architecture of the actin cytoskeleton and the dynamics of adhesion complexes are important parameters for the emergence of coordinated activity throughout the tissue.

Uso de la matriz extracelular como material para ingeniería de tejidos

Introducción: La evaluación de injertos vasculares de submucosa de intestino delgado para la regeneración de vasos sanguíneos ha producido una permeabilidad variable (0-100%) que ha sido concurrente con la variabilidad en las técnicas de fabricación. Metodología: Investigamos los efectos de fabricación en permeabilidad y regeneración en un diseño experimental de 22factorial que combino: 1) preservación (P) o remoción (R) de la capa estratum compactum del intestino, y 2) deshidratada (D) o hidratada (H), dentro de cuatro grupos de estudio (PD, RD, PH, RH). Los injertos fueron implantados en las Arterias Carótidas de porcinos (ID 4.5mm, N=4, 7d). Permeabilidad, trombogenicidad, reacción inflamatoria, vascularización, infiltración de fibroblastos, perfil de polarización de macrófagos y fuerza tensil biaxial fueron evaluadas. Resultados: Todos los injertos PD permanecieron permeables (4/4), pero tuvieron escasa vascularización e infiltración de fibroblastos. El grupo RD permaneció permeable (4/4), presentó una extensa vascularización e infiltración de fibroblastos, y el mayor número del fenotipo de macrófagos (M2) asociado a regeneración. El grupo RH presentó menor permeabilidad (3/4), una extensa vascularización e infiltración de fibroblastos, y un perfil dominante de M2. El grupo PH presentó el menor grado de permeabilidad, y a pesar de mayor infiltración celular que PD, exhibió un fenotipo de macrófagos dominante adverso. La elasticidad de los injertos R evolucionó de una manera similar a las Carótidas nativas (particularmente RD, mientras que los injertos P mantuvieron su rigidez inicial. Discusión: Concluimos que los parámetros de fabricación afectan drásticamente los resultados, siendo los injertos RD los que arrojaron mejores resultados.

Rho-kinase inhibition attenuates airway responsiveness, inflammation, matrix remodeling, and oxidative stress activation induced by chronic inflammation

Possa SS, Charafeddine HT, Righetti RF, da Silva PA, Almeida-Reis R, Saraiva-Romanholo BM, Perini A, Prado CM, Leick-Maldonado EA, Martins MA, Tiberio ID. Rho-kinase inhibition attenuates airway responsiveness, inflammation, matrix remodeling, and oxidative stress activation induced by chronic inflammation. Am J Physiol Lung Cell Mol Physiol 303: L939-L952, 2012. First published September 21, 2012; doi:10.1152/ajplung.00034.2012.-Several studies have demonstrated the importance of Rho-kinase in the modulation of smooth muscle contraction, airway hyperresponsiveness, and inflammation. However, the effects of repeated treatment with a specific inhibitor of this pathway have not been previously investigated. We evaluated the effects of repeated treatment with Y-27632, a highly selective Rho-kinase inhibitor, on airway hyperresponsiveness, oxidative stress activation, extracellular matrix remodeling, eosinophilic inflammation, and cytokine expression in an animal model of chronic airway inflammation. Guinea pigs were subjected to seven ovalbumin or saline exposures. The treatment with Y-27632 (1 mM) started at the fifth inhalation. Seventy-two hours after the seventh inhalation, the animals' pulmonary mechanics were evaluated, and exhaled nitric oxide (E-NO) was collected. The lungs were removed, and histological analysis was performed using morphometry. Treatment with Y-27632 in sensitized animals reduced E-NO concentrations, maximal responses of resistance, elastance of the respiratory system, eosinophil counts, collagen and elastic fiber contents, the numbers of cells positive for IL-2, IL-4, IL-5, IL-13, inducible nitric oxide synthase, matrix metalloproteinase-9, tissue inhibitor of metalloproteinase-1, transforming growth factor-beta, NF-kappa B, IFN-gamma, and 8-iso-prostaglandin F2 alpha contents compared with the untreated group (P < 0.05). We observed positive correlations among the functional responses and inflammation, remodeling, and oxidative stress pathway activation markers evaluated. In conclusion, Rho-kinase pathway activation contributes to the potentiation of the hyperresponsiveness, inflammation, the extracellular matrix remodeling process, and oxidative stress activation. These results suggest that Rho-kinase inhibitors represent potential pharmacological tools for the control of asthma.

Inducible Nitric Oxide Synthase Inhibition Attenuates Physical Stress-Induced Lung Hyper-Responsiveness and Oxidative Stress in Animals with Lung Inflammation

Mechanisms involved in stress-induced asthmatic alterations have been poorly characterised. We assessed whether inducible nitric oxide synthase (iNOS) inhibition modulates the stress-amplified lung parenchyma responsiveness, oxidative stress and extracellular matrix remodelling that was previously increased by chronic lung inflammation. Guinea pigs were subjected to 7 exposures to ovalbumin (1-5 mg/ml) or saline (OVA and SAL groups) over 4 weeks. To induce behavioural stress, animals were subjected to a forced swimming protocol (5 times/week, over 2 weeks; SAL-Stress and OVA-Stress groups) 24 h after the 4th inhalation. 1400W (iNOS-specific inhibitor) was administered intraperitoneally in the last 4 days of the protocol (SAL-1400W, OVA-1400W, SAL-Stress+1400W and OVA-Stress+1400W groups). Seventy-two hours after the last inhalation, animals were anaesthetised and exsanguinated, and adrenal glands were removed. Lung tissue resistance and elastance were evaluated by oscillatory mechanics and submitted for histopathological evaluation. Stressed animals had higher adrenal weights compared to non-stressed groups, which were reduced by 1400W treatment. Behavioural stress in sensitised animals amplified the resistance and elastance responses after antigen challenge, numbers of eosinophils and iNOS+ cells, actin content and 8-iso-PGF2 alpha density in the distal lung compared to the OVA group. 1400W treatment in ovalbumin-exposed and stressed animals reduced lung mechanics, iNOS+ cell numbers and 8-iso-PGF2a density compared to sensitised and stressed animals that received vehicle treatment. We concluded that stress amplifies the distal lung constriction, eosinophilic inflammation, iNOS expression, actin content and oxidative stress previously induced by chronic lung inflammation. iNOS-derived NO contributes to stress-augmented lung tissue functional alterations in this animal model and is at least partially due to activation of the oxidative stress pathway. copyright (C) 2012S. Karger AG, Basel

Mechanisms of force-mediated regulation of transcription, chromatin remodeling and cell fate decisions

Tissue mechanics and cellular interactions influence every single cell in our bodies to drive morphogenesis. However, little is known about mechanisms by which cells sense physical forces and transduce them from the cytoskeleton to the nucleus to control gene expression and stem cell fate. We have identified a novel nuclear-mechanosensor complex, consisting of the nuclear membrane protein emerin (Emd), actin and non-muscle myosin IIA (NMIIA), that regulates transcription, chromatin remodeling and lineage commitment. Force-induced enrichment of Emd at the outer nuclear membrane leads to a compensation between H3K9me2,3 and H3K27me3 on constitutive heterochromatin. This strain-induced epigenetic switch is accompanied by the global rearrangement of chromatin. In parallel, forces promote local F-actin polymerization at the outer nuclear membrane, which limits the availability of nuclear G-actin. Subsequently, the reduction of nuclear G-actin results in attenuated global transcription and therefore increased H3K27me3 occupancy to reinforce gene silencing. Restoring nuclear actin levels in the presence of mechanical strain counteracts PRC2-mediated silencing of transcribed genes. This mechanosensory circuit is also observed in vivo. Depletion of NMIIA in mouse epidermis leads to decreased H3K27me3 levels and precocious lineage commitment, thus abrogating organ growth and patterning. Our results reveal how mechanical signals regulate nuclear architecture, chromatin organization and transcription to control cell fate decisions.

Mechanics of biological networks: from the cell cytoskeleton to connective tissue.

From the cell cytoskeleton to connective tissues, fibrous networks are ubiquitous in metazoan life as the key promoters of mechanical strength, support and integrity. In recent decades, the application of physics to biological systems has made substantial strides in elucidating the striking mechanical phenomena observed in such networks, explaining strain stiffening, power law rheology and cytoskeletal fluidisation - all key to the biological function of individual cells and tissues. In this review we focus on the current progress in the field, with a primer into the basic physics of individual filaments and the networks they form. This is followed by a discussion of biological networks in the context of a broad spread of recent in vitro and in vivo experiments.

Assessment of the mechanics of a tissue-engineered rat trachea in an image-processing environment

OBJECTIVES: Despite the recent success regarding the transplantation of tissue-engineered airways, the mechanical properties of these grafts are not well understood. Mechanical assessment of a tissue-engineered airway graft before implantation may be used in the future as a predictor of function. The aim of this preliminary work was to develop a noninvasive image-processing environment for the assessment of airway mechanics.METHOD: Decellularized, recellularized and normal tracheas (groups DECEL, RECEL, and CONTROL, respectively) immersed in Krebs-Henseleit solution were ventilated by a small-animal ventilator connected to a Fleisch pneumotachograph and two pressure transducers (differential and gauge). A camera connected to a stereomicroscope captured images of the pulsation of the trachea before instillation of saline solution and after instillation of Krebs-Henseleit solution, followed by instillation with Krebs-Henseleit with methacholine 0.1 M (protocols A, K and KMCh, respectively). The data were post-processed with computer software and statistical comparisons between groups and protocols were performed.RESULTS: There were statistically significant variations in the image measurements of the medial region of the trachea between the groups (two-way analysis of variance [ANOVA], p<0.01) and of the proximal region between the groups and protocols (two-way ANOVA, p<0.01).CONCLUSIONS: The technique developed in this study is an innovative method for performing a mechanical assessment of engineered tracheal grafts that will enable evaluation of the viscoelastic properties of neo-tracheas prior to transplantation.