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The insertion and folding of proteins into membranes is crucial for cell viability. Yet, the detailed contributions of insertases remain elusive. Here, we monitor how the insertase YidC guides the folding of the polytopic melibiose permease MelB into membranes. In vivo experiments using conditionally depleted E. coli strains show that MelB can insert in the absence of SecYEG if YidC resides in the cytoplasmic membrane. In vitro single-molecule force spectroscopy reveals that the MelB substrate itself forms two folding cores from which structural segments insert stepwise into the membrane. However, misfolding dominates, particularly in structural regions that interface the pseudo-symmetric α-helical domains of MelB. Here, YidC takes an important role in accelerating and chaperoning the stepwise insertion and folding process of both MelB folding cores. Our findings reveal a great flexibility of the chaperoning and insertase activity of YidC in the multifaceted folding processes of complex polytopic membrane proteins., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The current therapeutic approach to asthma focuses exclusively on targeting inflammation and reducing airway smooth muscle force to prevent the recurrence of symptoms. However, even when inflammation is brought under control, airways in an asthmatic can still hyperconstrict when exposed to a low dose of agonist. This suggests that there are mechanisms at play that are likely triggered by inflammation and eventually become self-sustaining so that even when airway inflammation is brought back under control, these alternative mechanisms continue to drive airway hyperreactivity in asthmatics. In this study, we hypothesized that stiffening of the airway extracellular matrix is a core pathological change sufficient to support excessive bronchoconstriction even in the absence of inflammation. To test this hypothesis, we increased the stiffness of the airway extracellular matrix by photo-crosslinking collagen fibers within the airway wall of freshly dissected bovine rings using riboflavin (vitamin B2) and Ultraviolet-A radiation. In our experiments, collagen crosslinking led to a twofold increase in the stiffness of the airway extracellular matrix. This change was sufficient to cause airways to constrict to a greater degree, and at a faster rate when they were exposed to 10 -5 M acetylcholine for 5 min. Our results show that stiffening of the extracellular matrix is sufficient to drive excessive airway constriction even in the absence of inflammatory signals. NEW & NOTEWORTHY Targeting inflammation is the central dogma on which current asthma therapy is based. Here, we show that a healthy airway can be made to constrict excessively and at a faster rate in response to the same stimulus by increasing the stiffness of the extracellular matrix, without the use of inflammatory agents. Our results provide an independent mechanism by which airway remodeling in asthma can sustain airway hyperreactivity even in the absence of inflammatory signals.

In this study, we report the existence of a communication system among human smooth muscle cells that uses mechanical forces to frequency modulate long-range calcium waves. An important consequence of this mechanical signaling is that changes in stiffness of the underlying extracellular matrix can interfere with the frequency modulation of Ca 2+ waves, causing smooth muscle cells from healthy human donors to falsely perceive a much higher agonist dose than they actually received. This aberrant sensing of contractile agonist dose on stiffer matrices is completely absent in isolated smooth muscle cells, although the isolated cells can sense matrix rigidity. We show that the intercellular communication that enables this collective Ca 2+ response in smooth muscle cells does not involve transport across gap junctions or extracellular diffusion of signaling molecules. Instead, our data support a collective model in which mechanical signaling among smooth muscle cells regulates their response to contractile agonists., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

The interplay between cell-cell and cell-substrate interactions is complex yet necessary for the formation and healthy functioning of tissues. The same mechanosensing mechanisms used by the cell to sense its extracellular matrix also play a role in intercellular interactions. We used the discrete element method to develop a computational model of a deformable cell that includes subcellular components responsible for mechanosensing. We modeled a three-dimensional cell pair on a patterned (two-dimensional) substrate, a simple laboratory setup to study intercellular interactions. We explicitly modeled focal adhesions and adherens junctions. These mechanosensing adhesions matured, becoming stabilized by force. We also modeled contractile stress fibers that bind the discrete adhesions. The mechanosensing fibers strengthened upon stalling. Traction exerted on the substrate was used to generate traction maps (along the cell-substrate interface). These simulated maps are compared to experimental maps obtained via traction force microscopy. The model recreates the dependence on substrate stiffness of the tractions' spatial distribution, contractile moment of the cell pair, intercellular force, and number of focal adhesions. It also recreates the phenomenon of cell decoupling, in which cells exert forces separately when substrate stiffness increases. More importantly, the model provides viable molecular explanations for decoupling: mechanosensing mechanisms are responsible for competition between different fiber-adhesion configurations present in the cell pair. The point at which an increasing substrate stiffness becomes as high as that of the cell-cell interface is the tipping point at which configurations that favor cell-substrate adhesion dominate over those favoring cell-cell adhesion. This competition is responsible for decoupling., (Copyright © 2020 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Plasma cell leukemia (PCL) is an aggressive hematological malignancy characterized by an uncontrolled clonal proliferation of plasma cells (PCs) in the bone marrow and peripheral blood. PCL has been defined by an absolute number of circulating PCs exceeding 2.0 × 10 9 /L and/or >20% PCs in the total leucocyte count. It is classified as primary PCL, which develops de novo , and secondary PCL, occurring at the late and advanced stages of multiple myeloma (MM). Primary and secondary PCL are clinically and biologically two distinct entities. After the diagnosis, treatment should be immediate and should include a proteasome inhibitor and immunomodulator-based combination regimens as induction, followed by stem cell transplantation (SCT) in transplant-eligible individuals who have cleared the peripheral blood of circulating PCs. Due to the rarity of the condition, there have been very few clinical trials. Furthermore, virtually all of the myeloma trials exclude patients with active PCL. The evaluation of response has been defined by the International Myeloma Working Group and consists of both acute leukemia and MM criteria. With conventional chemotherapy, the prognosis of primary PCL has been ominous, with reported overall survival (OS) ranging from 6.8 to 12.6 months. The use of novel agents and autologous SCT appears to be associated with deeper response and an improved survival, although it still remains low. The PCL prognostic index provides a simple score to risk-stratify PCL. The prognosis of secondary PCL is extremely poor, with OS of only 1 month., Competing Interests: The authors declare they have no conflicts of interest., (© 2020 International Academy for Clinical Hematology. Publishing services by Atlantis Press International B.V.)

For an airway or a blood vessel to narrow, there must be a connected path that links the smooth muscle (SM) cells with each other, and transmits forces around the organ, causing it to constrict. Currently, we know very little about the mechanisms that regulate force transmission pathways in a multicellular SM ensemble. Here, we used extracellular matrix (ECM) micropatterning to study force transmission in a two-cell ensemble of SM cells. Using the two-SM cell ensemble, we demonstrate (a) that ECM stiffness acts as a switch that regulates whether SM force is transmitted through the ECM or through cell-cell connections. (b) Fluorescent imaging for adherens junctions and focal adhesions show the progressive loss of cell-cell borders and the appearance of focal adhesions with the increase in ECM stiffness (confirming our mechanical measurements). (c) At the same ECM stiffness, we show that the presence of a cell-cell border substantially decreases the overall contractility of the SM cell ensemble. Our results demonstrate that connectivity among SM cells is a critical factor to consider in the development of diseases such as asthma and hypertension.

Cells in the body are exposed to irregular mechanical stimuli. Here, we review the so-called fluctuation-driven mechanotransduction in which stresses stretching cells vary on a cycle-by-cycle basis. We argue that such mechanotransduction is an emergent network phenomenon and offer several potential mechanisms of how it regulates cell function. Several examples from the vasculature, the lung, and tissue engineering are discussed. We conclude with a list of important open questions., (©2016 Int. Union Physiol. Sci./Am. Physiol. Soc.)

Most tissues in the body are under mechanical tension, and while enzymes mediate many cellular and extracellular processes, the effects of mechanical forces on enzyme reactions in the native extracellular matrix (ECM) are not fully understood. We hypothesized that physiological levels of mechanical forces are capable of modifying the activity of collagenase, a key remodeling enzyme of the ECM. To test this, lung tissue Young's modulus and a nonlinearity index characterizing the shape of the stress-strain curve were measured in the presence of bacterial collagenase under static uniaxial strain of 0, 20, 40, and 80%, as well as during cyclic mechanical loading with strain amplitudes of ±10 or ±20% superimposed on 40% static strain, and frequencies of 0.1 or 1 Hz. Confocal and electron microscopy was used to determine and quantify changes in ECM structure. Generally, mechanical loading increased the effects of enzyme activity characterized by an irreversible decline in stiffness and tissue deterioration seen on both confocal and electron microscopic images. However, a static strain of 20% provided protection against digestion compared to both higher and lower strains. The decline in stiffness during digestion positively correlated with the increase in equivalent alveolar diameters and negatively correlated with the nonlinearity index. These results suggest that the decline in stiffness results from rupture of collagen followed by load transfer and subsequent rupture of alveolar walls. This study may provide new understanding of the role of collagen degradation in general tissue remodeling and disease progression.

Chronic obstructive pulmonary disease (COPD) is linked to both cigarette smoking and genetic determinants. We have previously identified iron-responsive element-binding protein 2 (IRP2) as an important COPD susceptibility gene and have shown that IRP2 protein is increased in the lungs of individuals with COPD. Here we demonstrate that mice deficient in Irp2 were protected from cigarette smoke (CS)-induced experimental COPD. By integrating RNA immunoprecipitation followed by sequencing (RIP-seq), RNA sequencing (RNA-seq), and gene expression and functional enrichment clustering analysis, we identified Irp2 as a regulator of mitochondrial function in the lungs of mice. Irp2 increased mitochondrial iron loading and levels of cytochrome c oxidase (COX), which led to mitochondrial dysfunction and subsequent experimental COPD. Frataxin-deficient mice, which had higher mitochondrial iron loading, showed impaired airway mucociliary clearance (MCC) and higher pulmonary inflammation at baseline, whereas mice deficient in the synthesis of cytochrome c oxidase, which have reduced COX, were protected from CS-induced pulmonary inflammation and impairment of MCC. Mice treated with a mitochondrial iron chelator or mice fed a low-iron diet were protected from CS-induced COPD. Mitochondrial iron chelation also alleviated CS-induced impairment of MCC, CS-induced pulmonary inflammation and CS-associated lung injury in mice with established COPD, suggesting a critical functional role and potential therapeutic intervention for the mitochondrial-iron axis in COPD.

Advancements in hyperpolarized helium-3 MRI (HP 3He-MRI) have introduced the ability to render and quantify ventilation patterns throughout the anatomic regions of the lung. The goal of this study was to establish how ventilation heterogeneity relates to the dynamic changes in mechanical lung function and airway hyperresponsiveness in asthmatic subjects. In four healthy and nine mild-to-moderate asthmatic subjects, we measured dynamic lung resistance and lung elastance from 0.1 to 8 Hz via a broadband ventilation waveform technique. We quantified ventilation heterogeneity using a recently developed coefficient of variation method from HP 3He-MRI imaging. Dynamic lung mechanics and imaging were performed at baseline, post-challenge, and after a series of five deep inspirations. AHR was measured via the concentration of agonist that elicits a 20% decrease in the subject's forced expiratory volume in one second compared to baseline (PC20) dose. The ventilation coefficient of variation was correlated to low-frequency lung resistance (R = 0.647, P < 0.0001), the difference between high and low frequency lung resistance (R = 0.668, P < 0.0001), and low-frequency lung elastance (R = 0.547, P = 0.0003). In asthmatic subjects with PC20 values <25 mg/mL, the coefficient of variation at baseline exhibited a strong negative trend (R = -0.798, P = 0.02) to PC20 dose. Our findings were consistent with the notion of peripheral rather than central involvement of ventilation heterogeneity. Also, the degree of AHR appears to be dependent on the degree to which baseline airway constriction creates baseline ventilation heterogeneity. HP 3He-MRI imaging may be a powerful predictor of the degree of AHR and in tracking the efficacy of therapy.

Periodic length fluctuations of airway smooth muscle during breathing are thought to modulate airway responsiveness in vivo. Recent animal and human intact airway studies have shown that pressure fluctuations simulating breathing can only marginally reverse airway narrowing and are ineffective at protecting against future narrowing. However, these previous studies were performed on relatively large (>5 mm diameter) airways, which are inherently stiffer than smaller airways for which a preponderance of airway constriction in asthma likely occurs. The goal of this study was to determine the effectiveness of breathing-like transmural pressure oscillations to reverse induced narrowing and/or protect against future narrowing of smaller, more compliant intact airways. We constricted smaller (luminal diameter = 2.92 ± 0.29 mm) intact airway segments twice with ACh (10(-6) M), once while applying tidal-like pressure oscillations (5-15 cmH2O) before, during, and after inducing constriction (Pre + Post) and again while only imposing the tidal-like pressure oscillation after induced constriction (Post Only). Smaller airways were 128% more compliant than previously studied larger airways. This increased compliance translated into 196% more strain and 76% greater recovery (41 vs. 23%) because of tidal-like pressure oscillations. Larger pressure oscillations (5-25 cmH2O) caused more recovery (77.5 ± 16.5%). However, pressure oscillations applied before and during constriction resulted in the same steady-state diameter as when pressure oscillations were only applied after constriction. These data show that reduced straining of the airways before a challenge likely does not contribute to the emergence of airway hyperreactivity observed in asthma but may serve to sustain a given level of constriction., (Copyright © 2015 the American Physiological Society.)

The purpose of this study was to determine how the initial distribution of elastase in mouse lungs determines the time course of tissue destruction and how structural heterogeneity at different spatial scales influences lung function. We evaluated lung function and alveolar structure in normal and emphysematous C57BL/6 mice at 2 and 21 days following orotracheal treatment with porcine pancreatic elastase (PPE). Initial distribution of elastase 1 h after treatment was assessed using red fluorescently labeled PPE (f-PPE) by laser scanning confocal microscopy. From measured input impedance of the respiratory system, the global lung compliance, and the variability of regional compliance were obtained. Lungs were fixed and equivalent airspace diameters were measured in four lobes of the right lung and three regions of the left lung. At day 2 and day 21, the mean airspace diameter of each region was significantly enlarged which was accompanied by an increased inter-regional heterogeneity. The deposition of f-PPE on day 0 was much more heterogeneous than the inter-regional diameters at both day 2 and day 21 and, at day 21, this reached statistical significance (p < 0.05). Microscale heterogeneity characterized by the overall variability of airspace diameters correlated significantly better with compliance than macroscale or inter-regional heterogeneity. Furthermore, while the spatial distribution of the inflammatory response does not seem to follow that of the elastase deposition, it correlates with the strongest regional determinant of lung function. These results may help interpret lung function decline in terms of structural deterioration in human patients with emphysema.

Extracellular matrix remodeling and tissue rupture contribute to the progression of emphysema. Lung tissue elasticity is governed by the tensile stiffness of fibers and the compressive stiffness of proteoglycans. It is not known how proteoglycan remodeling affects tissue stability and destruction in emphysema. The objective of this study was to characterize the role of remodeled proteoglycans in alveolar stability and tissue destruction in emphysema. At 30 days after treatment with porcine pancreatic elastase, mouse lung tissue stiffness and alveolar deformation were evaluated under varying tonicity conditions that affect the stiffness of proteoglycans. Proteoglycans were stained and measured in the alveolar walls. Computational models of alveolar stability and rupture incorporating the mechanical properties of fibers and proteoglycans were developed. Although absolute tissue stiffness was only 24% of normal, changes in relative stiffness and alveolar shape distortion due to changes in tonicity were increased in emphysema (P < 0.01 and P < 0.001). Glycosaminoglycan amount per unit alveolar wall length, which is responsible for proteoglycan stiffness, was higher in emphysema (P < 0.001). Versican expression increased in the tissue, but decorin decreased. Our network model predicted that the rate of tissue deterioration locally governed by mechanical forces was reduced when proteoglycan stiffness was increased. Consequently, this general network model explains why increasing proteoglycan deposition protects the alveolar walls from rupture in emphysema. Our results suggest that the loss of proteoglycans observed in human emphysema contributes to disease progression, whereas treatments that promote proteoglycan deposition in the extracellular matrix should slow the progression of emphysema.

Cells in the body exist in a dynamic mechanical environment where they are subject to mechanical stretch as well as changes in composition and stiffness of the underlying extracellular matrix (ECM). However, the underlying mechanisms by which cells sense and adapt to their dynamic mechanical environment, in particular to stretch, are not well understood. In this study, we hypothesized that emergent phenomena at the level of the actin network arising from active structural rearrangements driven by nonmuscle myosin II molecular motors play a major role in the cellular response to both stretch and changes in ECM stiffness. To test this hypothesis, we introduce a simple network model of actin-myosin interactions that links active self-organization of the actin network to the stiffness of the network and the traction forces generated by the network. We demonstrate that such a network replicates not only the effect of changes in substrate stiffness on cellular traction and stiffness and the dependence of rate of force development by a cell on the stiffness of its substrate, but also explains the physical response of adherent cells to transient and cyclic stretch. Our results provide strong indication that network phenomena governed by the active reorganization of the actin-myosin structure plays an important role in cellular mechanosensing and response to both changes in ECM stiffness and externally applied mechanical stretch.

Background: The formation of discrete elastin bands at the tips of secondary alveolar septa is important for normal alveolar development, but the mechanisms regulating the lung elastogenic program are incompletely understood. JNK suppress elastin synthesis in the aorta and is important in a host of developmental processes. We sought to determine whether JNK suppresses pulmonary fibroblast elastogenesis during lung development., Methods: Alveolar size, elastin content, and mRNA of elastin-associated genes were quantitated in wild type and JNK-deficient mouse lungs, and expression profiles were validated in primary lung fibroblasts. Tropoelastin protein was quantitated by Western blot. Changes in lung JNK activity throughout development were quantitated, and pJNK was localized by confocal imaging and lineage tracing., Results: By morphometry, alveolar diameters were increased by 7% and lung elastin content increased 2-fold in JNK-deficient mouse lungs compared to wild type. By Western blot, tropoelastin protein was increased 5-fold in JNK-deficient lungs. Postnatal day 14 (PND14) lung JNK activity was 11-fold higher and pJNK:JNK ratio 6-fold higher compared to PN 8 week lung. Lung tropoelastin, emilin-1, fibrillin-1, fibulin-5, and lysyl oxidase mRNAs inversely correlated with lung JNK activity during alveolar development. Phosphorylated JNK localized to pulmonary lipofibroblasts. PND14 JNK-deficient mouse lungs contained 7-fold more tropoelastin, 2,000-fold more emilin-1, 800-fold more fibrillin-1, and 60-fold more fibulin-5 than PND14 wild type lungs. Primarily lung fibroblasts from wild type and JNK-deficient mice showed similar differences in elastogenic mRNAs., Conclusions: JNK suppresses fibroblast elastogenesis during the alveolar stage of lung development.

Transpulmonary pressure and the mechanical stresses of breathing modulate many essential cell functions in the lung via mechanotransduction. We review how mechanical factors could influence the pathogenesis of emphysema. Although the progression of emphysema has been linked to mechanical rupture, little is known about how these stresses alter lung remodeling. We present possible new directions and an integrated multiscale view that may prove useful in finding solutions for this disease.

Fluctuating forces imposed on the airway smooth muscle due to breathing are believed to regulate hyperresponsiveness in vivo. However, recent animal and human isolated airway studies have shown that typical breathing-sized transmural pressure (Ptm) oscillations around a fixed mean are ineffective at mitigating airway constriction. To help understand this discrepancy, we hypothesized that Ptm oscillations capable of producing the same degree of bronchodilation as observed in airway smooth muscle strip studies requires imposition of strains larger than those expected to occur in vivo. First, we applied increasingly larger amplitude Ptm oscillations to a statically constricted airway from a Ptm simulating normal functional residual capacity of 5 cmH2O. Tidal-like oscillations (5-10 cmH2O) imposed 4.9 ± 2.0% strain and resulted in 11.6 ± 4.8% recovery, while Ptm oscillations simulating a deep inspiration at every breath (5-30 cmH2O) achieved 62.9 ± 12.1% recovery. These same Ptm oscillations were then applied starting from a Ptm = 1 cmH2O, resulting in approximately double the strain for each oscillation amplitude. When extreme strains were imposed, we observed full recovery. On combining the two data sets, we found a linear relationship between strain and resultant recovery. Finally, we compared the impact of Ptm oscillations before and after constriction to Ptm oscillations applied only after constriction and found that both loading conditions had a similar effect on narrowing. We conclude that, while sufficiently large strains applied to the airway wall are capable of producing substantial bronchodilation, the Ptm oscillations necessary to achieve those strains are not expected to occur in vivo.

A segmentation algorithm to isolate areas of ventilation from hyperpolarized helium-3 magnetic resonance imaging (HP (3)He MRI) is described. The algorithm was tested with HP (3)He MRI data from four healthy and six asthmatic subjects. Ventilated lung volume (VLV) measured using our semiautomated technique was compared to that obtained from manual outlining of ventilated lung regions and to standard spirometric measurements. VLVs from both approaches were highly correlated (R = 0.99; P < 0.0001) with a mean difference of 3.8 mL and 95% agreement indices of -30.8 mL and 38.4 mL. There was no significant difference between the VLVs obtained through the semiautomatic approach and the manual approach. A Dice coefficient which quantified the intersection of the two datasets was calculated and ranged from 0.95 to 0.97 with a mean of 0.96 ± 0.01 (mean ± SD). VLVs obtained through the semiautomatic algorithm were also highly correlated with measurements of forced expiratory volume in one second (FEV1) (R = 0.82; P = 0.0035) and forced vital capacity (FVC) (R = 0.95; P < 0.0001). The technique may open new pathways toward advancing more quantitative characterization of ventilation for routine clinical assessment for asthma severity as well as a number of other respiratory diseases.

The relation between the progression of chronic obstructive pulmonary disease (COPD) and exacerbations is unclear. Currently, no animal model of acute exacerbation of COPD (AECOPD) exists. The objectives of this study were to evaluate the effects of mechanical forces induced by deep inspirations (DIs) on short-term deterioration of lung structure and function to mimic AECOPD. At 2, 7, or 21 days after treatment with elastase, mice were ventilated with or without DIs (35 cmH(2)O airway pressure for 3 s, 2 times/min) for 1 h. Functional residual capacity (FRC) was measured with body plethysmography, and respiratory compliance, resistance, and hysteresivity were obtained via forced oscillations. From hematoxylin and eosin-stained sections, equivalent airspace diameters (D), alveolar wall thickness (W(t)), number of septal ruptures (N(sr)), and attachment density (A(d)) around airways were determined. FRC, compliance, and hysteresivity statistically significantly increased with time, and both increased due to DIs. Interestingly, DIs also had an effect on FRC, compliance, resistance, and hysteresivity in control mice. The development of emphysema statistically significantly increased D and W(t) in time, and the DIs caused subtle differences in D. At 21 days, the application of DIs changed the distribution of D, increased W(t) and N(sr), and decreased A(d). These results suggest that once a critical remodeling of the parenchyma has been reached, acute mechanical forces lead to irreversible changes in structure and function, mimicking COPD exacerbations. Thus, the acute application of DIs in mice with emphysema may serve as a useful model of AECOPD.

The aim of this study was to evaluate airway structure-function relations in elastase-induced emphysema in rats. Sprague-Dawley rats were treated intratracheally with 50 IU porcine pancreatic elastase (PPE, n = 8) or saline (controls, n = 6). Six weeks later, lung volumes [functional residual capacity (FRC), residual volume (RV), and total lung capacity (TLC)] and low-frequency impedance parameters (Newtonian resistance, R(N); tissue damping; tissue elastance, H) were measured, and tracheal sounds were recorded during slow inflation to TLC following in vivo degassing. The lungs were fixed and stained for standard morphometry, elastin, and collagen. In the PPE group, FRC and RV were higher [4.53 ± 0.7 (SD) vs. 3.28 ± 0.45 ml; P = 0.003 and 1.06 ± 0.35 vs. 0.69 ± 0.18 ml; P = 0.036, respectively], and H was smaller in the PPE-treated rats than in the controls (1,344 ± 216 vs. 2,178 ± 305 cmH(2)O/l; P < 0.001), whereas there was no difference in R(N). The average number of crackles per inflation was similar in the two groups; however, the crackle size distributions were different and the lower knee of the pressure-volume curves was higher in the PPE group. Microscopic images revealed different alveolar size distributions but similar bronchial diameters in the two groups. The treatment caused a slight but significant decrease in the numbers of alveolar attachments, no difference in elastin and slightly increased mean level and heterogeneity of collagen in the bronchial walls. These results suggest that tissue destruction did not affect the conventionally assessed airway resistance in this emphysema model, whereas the alterations in the recruitment dynamics can be an early manifestation of impaired airway function.

Secretion of pulmonary surfactant by alveolar epithelial type II cells is vital for the reduction of interfacial surface tension, thus preventing lung collapse. To study secretion dynamics, rat alveolar epithelial type II cells were cultured on elastic membranes and cyclically stretched. The amounts of phosphatidylcholine, the primary lipid component of surfactant, inside and outside the cells, were measured using radiolabeled choline. During and immediately after stretch, cells secreted less surfactant than unstretched cells; however, stretched cells secreted significantly more surfactant than unstretched cells after an extended lag period. We developed a model based on the hypothesis that stretching leads to jamming of surfactant traffic escaping the cell, similar to vehicular traffic jams. In the model, stretch increases surfactant transport from the interior to the exterior of the cell. This transport is mediated by a surface layer with a finite capacity due to the limited number of fusion pores through which secretion occurs. When the amount of surfactant in the surface layer approaches this capacity, interference among lamellar bodies carrying surfactant reduces the rate of secretion, effectively creating a jam. When the stretch stops, the jam takes an extended time to clear, and subsequently the amount of secreted surfactant increases. We solved the model analytically and show that its dynamics are consistent with experimental observations, implying that surfactant secretion is a fundamentally nonlinear process with memory representing collective behavior at the level of single cells. Our results thus highlight the importance of a jamming dynamics in stretch-induced cellular secretory processes.

Emphysema is a progressive disease characterized by the destruction of peripheral airspaces and subsequent decline in lung function. However, the relation between structure and function during disease progression is not well understood. The objective of this study was to assess the time course of the structural, mechanical, and remodeling properties of the lung in mice after elastolytic injury. At 2, 7, and 21 days after treatment with porcine pancreatic elastase, respiratory impedance, the constituents of lung extracellular matrix, and histological sections of the lung were evaluated. In the control group, no changes were observed in the structural or functional properties, whereas, in the treatment group, the respiratory compliance and its variability significantly increased by Day 21 (P < 0.001), and the difference in parameters decreased with increasing positive end-expiratory pressure. The heterogeneity of airspace structure gradually increased over time. Conversely, the relative amounts of elastin and type I collagen exhibited a peak (P < 0.01) at Day 2, but returned to baseline levels by Day 21. Structure-function relations manifested themselves in strong correlations between compliance parameters and both mean size and heterogeneity of airspace structure (r(2) > 0.9). Similar relations were also obtained in a network model of the parenchyma in which destruction was based on the notion that mechanical forces contribute to alveolar wall rupture. We conclude that, in a mouse model of emphysema, progressive decline in lung function is sensitive to the development of airspace heterogeneity governed by local, mechanical, force-induced failure of remodeled collagen.

We study the enzymatic degradation of an elastic fiber under tension using an anisotropic random-walk model coupled with binding-unbinding reactions that weaken the fiber. The fiber is represented by a chain of elastic springs in series along which enzyme molecules can diffuse. Numerical simulations show that the fiber stiffness decreases exponentially with two distinct regimes. The time constant of the first regime decreases with increasing tension. Using a mean field calculation, we partition the time constant into geometrical, chemical and externally controllable factors, which is corroborated by the simulations. We incorporate the fiber model into a multiscale network model of the extracellular matrix and find that network effects do not mask the exponential decay of stiffness at the fiber level. To test these predictions, we measure the force relaxation of elastin sheets stretched to 20% uniaxial strain in the presence of elastase. The decay of force is exponential and the time constant is proportional to the inverse of enzyme concentration in agreement with model predictions. Furthermore, the fragment mass released into the bath during digestion is linearly related to enzyme concentration that is also borne out in the model. We conclude that in the complex extracellular matrix, feedback between the local rate of fiber digestion and the force the fiber carries acts to attenuate any spatial heterogeneity of digestion such that molecular processes manifest directly at the macroscale. Our findings can help better understand remodeling processes during development or in disease in which enzyme concentrations and/or mechanical forces become abnormal.

Pulmonary emphysema is a connective tissue disease characterized by the progressive destruction of alveolar walls leading to airspace enlargement and decreased elastic recoil of the lung. However, the relationship between microscopic tissue structure and decline in stiffness of the lung is not well understood. In this study, we developed a 3D computational model of lung tissue in which a pre-strained cuboidal block of tissue was represented by a tessellation of space filling polyhedra, with each polyhedral unit-cell representing an alveolus. Destruction of alveolar walls was mimicked by eliminating faces that separate two polyhedral either randomly or in a spatially correlated manner, in which the highest force bearing walls were removed at each step. Simulations were carried out to establish a link between the geometries that emerged and the rate of decline in bulk modulus of the tissue block. The spatially correlated process set up by the force-based destruction lead to a significantly faster rate of decline in bulk modulus accompanied by highly heterogeneous structures than the random destruction pattern. Using the Karhunen-Loève transformation, an estimator of the change in bulk modulus from the first four moments of airspace cell volumes was setup. Simulations were then obtained for tissue destruction with different idealized alveolar geometry, levels of pre-strain, linear and nonlinear elasticity assumptions for alveolar walls and also mixed destruction patterns where both random and force-based destruction occurs simultaneously. In all these cases, the change in bulk modulus from cell volumes was accurately estimated. We conclude that microscopic structural changes in emphysema and the associated decline in tissue stiffness are linked by the spatial pattern of the destruction process.

Microtubules are structural components of the cytoskeleton that determine cell shape, polarity, and motility in cooperation with the actin filaments. In order to determine the role of microtubules in cell alignment, human airway smooth muscle cells were exposed to cyclic uniaxial stretch. Human airway smooth muscle cells, cultured on type I collagen-coated elastic silicone membranes, were stretched uniaxially (20% in strain, 30 cycles/min) for 2 h. The population of airway smooth muscle cells which were originally oriented randomly aligned near perpendicular to the stretch axis in a time-dependent manner. However, when the cells treated with microtubule disruptors, nocodazole and colchicine, were subjected to the same cyclic uniaxial stretch, the cells failed to align. Lack of alignment was also observed for airway smooth muscle cells treated with a microtubule stabilizer, paclitaxel. To understand the intracellular mechanisms involved, we developed a computational model in which microtubule polymerization and attachment to focal adhesions were regulated by the preexisting tensile stress, pre-stress, on actin stress fibers. We demonstrate that microtubules play a central role in cell re-orientation when cells experience cyclic uniaxial stretching. Our findings further suggest that cell alignment and cytoskeletal reorganization in response to cyclic stretch results from the ability of the microtubule-stress fiber assembly to maintain a homeostatic strain on the stress fiber at focal adhesions. The mechanism of stretch-induced alignment we uncovered is likely involved in various airway functions as well as in the pathophysiology of airway remodeling in asthma.

Many fundamental cellular and extracellular processes in the body are mediated by enzymes. At the single molecule level, enzyme activity is influenced by mechanical forces. However, the effects of mechanical forces on the kinetics of enzymatic reactions in complex tissues with intact extracellular matrix (ECM) have not been identified. Here we report that physiologically relevant macroscopic mechanical forces modify enzyme activity at the molecular level in the ECM of the lung parenchyma. Porcine pancreatic elastase (PPE), which binds to and digests elastin, was fluorescently conjugated (f-PPE) and fluorescent recovery after photobleach was used to evaluate the binding kinetics of f-PPE in the alveolar walls of normal mouse lungs. Fluorescent recovery after photobleach indicated that the dissociation rate constant (k(off)) for f-PPE was significantly larger in stretched than in relaxed alveolar walls with a linear relation between k(off) and macroscopic strain. Using a network model of the parenchyma, a linear relation was also found between k(off) and microscopic strain on elastin fibers. Further, the binding pattern of f-PPE suggested that binding sites on elastin unfold with strain. The increased overall reaction rate also resulted in stronger structural breakdown at the level of alveolar walls, as well as accelerated decay of stiffness and decreased failure stress of the ECM at the macroscopic scale. These results suggest an important role for the coupling between mechanical forces and enzyme activity in ECM breakdown and remodeling in development, and during diseases such as pulmonary emphysema or vascular aneurysm. Our findings may also have broader implications because in vivo, enzyme activity in nearly all cellular and extracellular processes takes place in the presence of mechanical forces., (Copyright © 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Chronic obstructive pulmonary disease (COPD) is a debilitating disease caused by chronic exposure to cigarette smoke (CS), which involves airway obstruction and alveolar loss (i.e., emphysema). The mechanisms of COPD pathogenesis remain unclear. Our previous studies demonstrated elevated autophagy in human COPD lung, and as a cellular and tissue response to CS exposure in an experimental model of emphysema in vivo. We identified the autophagic protein microtubule-associated protein 1 light chain-3B (LC3B) as a positive regulator of CS-induced lung epithelial cell death. We now extend these initial observations to explore the mechanism by which LC3B mediates CS-induced apoptosis and emphysema development in vivo. Here, we observed that LC3B(-/-) mice had significantly decreased levels of apoptosis in the lungs after CS exposure, and displayed resistance to CS-induced airspace enlargement, relative to WT littermate mice. We found that LC3B associated with the extrinsic apoptotic factor Fas in lipid rafts in an interaction mediated by caveolin-1 (Cav-1). The siRNA-dependent knockdown of Cav-1 sensitized epithelial cells to CS-induced apoptosis, as evidenced by enhanced death-inducing signaling complex formation and caspase activation. Furthermore, Cav-1(-/-) mice exhibited higher levels of autophagy and apoptosis in the lung in response to chronic CS exposure in vivo. In conclusion, we demonstrate a pivotal role for the autophagic protein LC3B in CS-induced apoptosis and emphysema, suggestive of novel therapeutic targets for COPD treatment. This study also introduces a mechanism by which LC3B, through interactions with Cav-1 and Fas, can regulate apoptosis.

Airways that collapse during deflation generate a crackle sound when they reopen during subsequent reinflation. Since each crackle is associated with the reopening of a collapsed airway, the likelihood of an airway to be a crackle source is identical to its vulnerability to collapse. To investigate this vulnerability of airways to collapse, crackles were recorded during the first inflation of six excised rabbit lungs from the collapsed state, and subsequent reinflations from 5, 2, 1, and 0 cmH(2)O end-expiratory pressure levels. We derived a relationship between the amplitude of a crackle sound at the trachea and the generation number (n) of the source airway where the crackle was generated. Using an asymmetrical tree model of the rabbit airways with elastic walls, airway vulnerability to collapse was also determined in terms of airway diameter D. During the reinflation from end-expiratory pressure = 0 cmH(2)O, the most vulnerable airways were estimated to be centered at n = 12 with a peak. Vulnerability in terms of D ranged between 0.1 and 1.3 mm, with a peak at 0.3 mm. During the inflation from the collapsed state, however, vulnerability was much less localized to a particular n or D, with maximum values of n = 8 and D = 0.75 mm. Numerical simulations using a tree model that incorporates airway opening and closing support these conclusions. Thus our results indicate that there are airways of a given range of diameters that can become unstable during deflation and vulnerable to collapse and subsequent injury.

In pulmonary emphysema, the alveolar structure progressively breaks down via a three-dimensional (3D) process that leads to airspace enlargement. The characterization of such structural changes has, however, been based on measurements from two-dimensional (2D) tissue sections or estimates of 3D structure from 2D measurements. In this study, we developed a novel silver staining method for visualizing tissue structure in 3D using micro-computed tomographic (CT) imaging, which showed that at 30 cmH20 fixing pressure, the mean alveolar airspace volume increased from 0.12 nl in normal mice to 0.44 nl and 2.14 nl in emphysematous mice, respectively, at 7 and 14 days following elastase-induced injury. We also assessed tissue structure in 2D using laser scanning confocal microscopy. The mean of the equivalent diameters of the alveolar airspaces was lower in 2D compared with 3D, while its variance was higher in 2D than in 3D in all groups. However, statistical comparisons of alveolar airspace size from normal and emphysematous mice yielded similar results in 2D and 3D: compared with control, both the mean and variance of the equivalent diameters increased by 7 days after treatment. These indexes further increased from day 7 to day 14 following treatment. During the first 7 days following treatment, the relative change in SD increased at a much faster rate compared with the relative change in mean equivalent diameter. We conclude that quantifying heterogeneity in structure can provide new insight into the pathogenesis or progression of emphysema that is enhanced by improved sensitivity using 3D measurements.

Adiponectin is an adipocyte-derived collectin that acts on a wide range of tissues including liver, brain, heart, and vascular endothelium. To date, little is known about the actions of adiponectin in the lung. Herein, we demonstrate that adiponectin is present in lung lining fluid and that adiponectin deficiency leads to increases in proinflammatory mediators and an emphysema-like phenotype in the mouse lung. Alveolar macrophages from adiponectin-deficient mice spontaneously display increased production of tumor necrosis factor-alpha (TNF-alpha) and matrix metalloproteinase (MMP-12) activity. Consistent with these observations, we found that pretreatment of alveolar macrophages with adiponectin leads to TNF-alpha and MMP-12 suppression. Together, our findings show that adiponectin leads to macrophage suppression in the lung and suggest that adiponectin-deficient states may contribute to the pathogenesis of inflammatory lung conditions such as emphysema.

The nature of the development of emphysema in the tight skin (Tsk) and the pallid (Pa) mice are not well understood. We assessed the mechanical and nonlinear properties of the respiratory system, the alveolar structure, and the levels of microfibril-associated glycoproteins (MAGP) 1 and 2 in Tsk mice with developmental emphysema; in Pa mice, which are thought to develop adult onset emphysema; and their background, the C57BL/6 mice, at an age of 7 wk. Minor differences between collagen-related elastic properties of the lungs of the Pa and C57BL/6 mice were seen at this early age. The lungs of the Tsk mice were significantly softer yet more nonlinear than those of the Pa and C57BL/6 mice. The MAGP-1 levels were similar in all three groups. However, the level of MAGP-2, which is associated with both fibrillin-1 and collagen, was higher in the Tsk than in the Pa mice, which also had more MAGP-2 than the C57BL/6. Both the mean and the variance of alveolar diameters were larger in the Tsk than in the other two groups, while the variance in the Pa was larger than in the C57BL/6 mice, implying early development of heterogeneity. Using a network model of the parenchyma, we linked the pathophysiologic changes in the Tsk mice to mechanical forces and failure of the alveolar walls. Our findings suggest the possibility that MAGP-2-related abnormal collagen assembly, combined with mechanical forces, is involved in the progression of emphysema in the Tsk mice.

The mean linear intercept (L(m)) can be used to estimate the surface area for gas exchange in the lung. However, in recent years, it is most commonly used as an index for characterizing the enlargement of airspaces in emphysema and the associated severity of structural destruction in the lung. Specifically, an increase in L(m) is thought to result from an increase in airspace sizes. In this paper, we examined how accurately L(m) measures the linear dimensions of airspaces from histological sections and a variety of computer-generated test images. To this end, we developed an automated method for measuring linear intercepts from digitized images of tissue sections and calculate L(m) as their mean. We examined how the shape of airspaces and the variability of their sizes influence L(m) as well as the distribution of linear intercepts. We found that, for a relatively homogeneous enlargement of airspaces, L(m) was a reliable index for detecting emphysema. However, in the presence of spatial heterogeneities with a large variability of airspace sizes, L(m) did not significantly increase and sometimes even decreased compared with its value in normal tissue. We also developed an automated method for measuring the area and computed an equivalent diameter of each individual airspace that is independent of shape. Finally, we introduced new indexes based on the moments of diameter that we found to be more reliable than L(m) to characterize airspace enlargement in the presence of heterogeneities.

Enlargement of the respiratory air spaces is associated with the breakdown and reorganization of the connective tissue fiber network during the development of pulmonary emphysema. In this study, a mouse (C57BL/6) model of emphysema was developed by direct instillation of 1.2 IU of porcine pancreatic elastase (PPE) and compared with control mice treated with saline. The PPE treatment caused 95% alveolar enlargement (P = 0.001) associated with a 29% lower elastance along the quasi-static pressure-volume curves (P < 0.001). Respiratory mechanics were measured at several positive end-expiratory pressures in the closed-chest condition. The dynamic tissue elastance was 19% lower (P < 0.001), hysteresivity was 9% higher (P < 0.05), and harmonic distortion, a measure of collagen-related dynamic nonlinearity, was 33% higher in the PPE-treated group (P < 0.001). Whole lung hydroxyproline content, which represents the total collagen content, was 48% higher (P < 0.01), and alpha-elastin content was 13% lower (P = 0.16) in the PPE-treated group. There was no significant difference in airway resistance (P = 0.7). The failure stress at which isolated parenchymal tissues break during stretching was 40% lower in the PPE-treated mice (P = 0.002). These findings suggest that, after elastolytic injury, abnormal collagen remodeling may play a significant role in all aspects of lung functional changes and mechanical forces, leading to progressive emphysema.

We developed a network model in an attempt to characterize heterogeneity of tissue elasticity of the lung. The model includes a parallel set of pathways, each consisting of an airway resistance, an airway inertance, and a tissue element connected in series. The airway resistance, airway inertance, and the hysteresivity of the tissue elements were the same in each pathway, whereas the tissue elastance (H) followed a hyperbolic distribution between a minimum and maximum. To test the model, we measured the input impedance of the respiratory system of ventilated normal and emphysematous C57BL/6 mice in closed chest condition at four levels of positive end-expiratory pressures. Mild emphysema was developed by nebulized porcine pancreatic elastase (PPE) (30 IU/day x 6 days). Respiratory mechanics were studied 3 wk following the initial treatment. The model significantly improved the fitting error compared with a single-compartment model. The PPE treatment was associated with an increase in mean alveolar diameter and a decrease in minimum, maximum, and mean H. The coefficient of variation of H was significantly larger in emphysema (40%) than that in control (32%). These results indicate that PPE treatment resulted in increased time-constant inequalities associated with a wider distribution of H. The heterogeneity of alveolar size (diameters and area) was also larger in emphysema, suggesting that the model-based tissue elastance heterogeneity may reflect the underlying heterogeneity of the alveolar structure.