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Lung resistance ( RL) is determined by airway and parenchymal tissue resistance, as well as the degree of heterogeneity in airway constriction. Deep inspirations (DIs) are known to reverse experimentally induced increase in RL, but the mechanism is not entirely clear. The first step toward understanding the effect of DI is to determine how each of the resistance components is affected by DI. In the present study, we measured RL and apparent airway resistance ( RAW, which combines the effects of airway resistance and airway heterogeneity) simultaneously before and after a DI in acetylcholine (ACh)-challenged ex vivo sheep lungs. We found that at normal breathing frequency (0.25 Hz) ACh-challenge led to a doubling of RL, 80.3% of that increase was caused by an increase in RAW; the increase in apparent tissue resistance ( RT) was insignificant. 57.7% of the increase in RAW was abolished by a single DI. After subtracting RAW from RL, the remaining RT was mostly independent of ACh-challenge and its reduction after a DI came mostly from the change in the mechanical properties of lung parenchyma. We conclude that at normal breathing frequency, RL in an unchallenged lung is mostly composed of RT, and the increase in RL due to ACh-challenge stems mostly from the increase in RAW and that both RAW and RT can be greatly reduced by a DI, likely due to a reduction in true airway resistance and heterogeneity, as well as parenchymal tissue hysteresis post DI.

Lung resistance ( RL) and elastance ( EL) can be measured during positive or negative pressure ventilation. Whether the different modes of ventilation produce different RL and EL is still being debated. Although negative pressure ventilation (NPV) is more physiological, positive pressure ventilation (PPV) is more commonly used for treating respiratory failure. In the present study, we measured lung volume, airway diameter, and airway volume, as well as RL and EL with PPV and NPV in explanted sheep lungs. We found that lung volume under a static pressure, either positive or negative, was not different. However, RL and EL were significantly higher in NPV at high inflation pressures. Interestingly, diameters of smaller airways (diameters L is higher in NPV (vs. PPV), because the peripheral parenchyma is a major source of tissue resistance, which is a part of the RL that increases with pressure. This explanation is consistent with the finding that during high frequency ventilation (>1 Hz, where RL reflects airway resistance more than tissue resistance), the difference in RL between NPV and PPV disappeared.

The development of orally administered protein drugs is challenging due to their intrinsic unfavourable features, including large molecular size and poor chemical stability, both of which limit gastrointestinal (GI) absorption efficiency. Nanoparticles can overcome the GI barriers effectively and improve the oral bioavailability of proteins in the GI tract. They possess large surface area to volume ratio, and can facilitate the GI absorption of nanoparticles via the paracellular and transcellular routes. Nanoparticles can be prepared by various fabrication techniques that can encapsulate the fragile therapeutic proteins via hydrophobic bonding and electrostatic interaction. A desirable technique should involve minimal harsh conditions and encapsulate therapeutic proteins with preserved functionalities. The current review examines the characteristics of each preparation technique, and illustrates the examples of insulin-loaded nanoparticles that have been developed in each fabrication method. The following techniques, which include nanoprecipitation, hydrophobic conjugation, flash nanocomplexation, double emulsion, ionotropic gelation, and layer-by-layer adsorption, have been used to formulate ligand-modified nanoparticles for targeted delivery of insulin. Other techniques, including reduction, complex coacervation (polyelectrolyte complexation), hydrophobic ion pairing and emulsion solvent diffusion method, and sol-gel technology, were also discussed in the latter part of the review due to their extensive use in fabrication of insulin nanoparticles. This review also discusses the strategies that have been utilised during the formulation process to improve the stability and bioactivity of therapeutic proteins.

Deep inspiration (DI)-induced bronchodilation is the first line of defense against bronchoconstriction in healthy subjects. A hallmark of asthma is the lack of this beneficial effect of DI. The mechanism underlying the bronchodilatory effect of DI is not clear. Understanding the mechanism will help us unravel the mystery of asthma pathophysiology. It has been postulated that straining airway smooth muscle (ASM) during a DI could lead to bronchodilation and bronchoprotection. The hypothesis is currently under debate, and a central question is whether ASM is sufficiently stretched during a DI for its contractility to be compromised. Besides bronchoconstriction, another contributor to lung resistance is airway heterogeneity. The present study examines changes in airway diameter and heterogeneity at different lung volumes. Freshly explanted sheep lungs were used in plethysmographic measurements of lung resistance and elastance at different lung volumes, whereas the airway dimensions were measured by computed tomography (CT). The change in airway diameter informed by CT measurements was applied to isolated airway ring preparations to determine the strain-induced loss of ASM contractility. We found that changing the transpulmonary pressure from 5 to 30 cmH2O led to a 51% increase in lung volume, accompanied by a 46% increase in the airway diameter with no change in airway heterogeneity. When comparable airway strains measured in the whole lung were applied to isolated airway rings in either relaxed or contracted state, a significant loss of ASM contractility was observed, suggesting that DI-induced bronchodilation and bronchoprotection can result from strain-induced loss of ASM contractility.

The cyclic interaction between myosin crossbridges and actin filaments underlies smooth muscle contraction. Phosphorylation of the 20-kDa myosin light chain (MLC20) is a crucial step in activating the crossbridge cycle. Our current understanding of smooth muscle contraction is based on observed correlations among MLC20 phosphorylation, maximal shortening velocity ( Vmax), and isometric force over the time course of contraction. However, during contraction there are changes in the extent of phosphorylation of many additional proteins as well as changes in activation of enzymes associated with the signaling pathways. As a consequence, the mechanical manifestation of muscle contraction is likely to change with time. To simplify the study of these relationships, we measured the mechanical properties of airway smooth muscle at different levels of MLC20 phosphorylation at a fixed time during contraction. A simple correlation emerged when time-dependent variables were fixed. MLC20 phosphorylation was found to be directly and linearly correlated with the active stress, stiffness, and power of the muscle; the observed weak dependence of Vmax on MLC20 phosphorylation could be explained by the presence of an internal load in the muscle preparation. These results can be entirely explained by the Huxley crossbridge model. We conclude that when the influence of time-dependent events during contraction is held constant, the basic crossbridge mechanism in smooth muscle is the same as that in striated muscle.

The gut microbiota plays multiple critical roles in maintaining the health of the host, especially in ruminants. However, our understanding of the establishment of gut microbiota from birth to adulthood is still limited. To address this, the bacterial ecology of the rumen, abomasum, duodenum, and rectum in Holstein cows ranging in age from 1 week to 5 years old was investigated using 16S rRNA gene sequencing in this study. A major change in the composition, diversity, and abundance of bacteria was observed with increased age (p 0.94) were applied. Importantly, the Gompertz model parameter differed between the gut segments, with the highest microbiota growth rate found in the rectum, followed by the rumen, abomasum, and duodenum. Compared to older animals, greater microbiota similarities were found in the adjacent gut segments of younger animals (p, Microbiota gradually matured followed the Gompertz model in each gut segment (rumen, abomasum, duodenum, and rectum) of cows. Microbiota in the rectum matured earliest followed by that in abomasum and rumen, and microbiota in the duodenum matured latest.

Major depressive disorder (MDD) is a prevalent psychiatric disorder that brings great harm and burden to both patients and society. This study aimed to examine the effects of whole-body vibration (WBV) training on a chronic restraint stress (CRS) induced depression rat model and provide an initial understanding of related molecular mechanisms. Adult Sprague-Dawley male rats were randomly divided into the following three groups: a) control group, b) depressive disorder group, and c) depression with WBV training treatment group. Daily food intake, body weight, sucrose preference test, open field test, elevated plus maze, forced swimming test, and Barnes maze task tests were performed. Immunofluorescence staining and ELISA analysis were used to assess neuronal damage, synaptic proteins, glial cells, and trophic factors. The data of behavioral tests and related biochemical indicators were statistically analyzed and compared between groups. Rats undergoing CRS showed increased anxiety-like behavior and memory impairment, along with synaptic atrophy and neuronal degeneration. WBV could reverse behavioral dysfunction, inhibit the degeneration of neurons, alleviate the damage of neurons and the pathological changes of glial cells, enhance trophic factor expression, and ameliorate the downregulation of dendritic and synaptic proteins after CRS. The effect of WBV in rats may be mediated via the reduction of hippocampal neuronal degeneration and by improving expression of synaptic proteins. WBV training exerts multifactorial benefits on MDD that supports its use as a promising new therapeutic option for improving depression-like behaviors in the depressive and/or potentially depressive.

Objectives The myoblast cell line, C2C12, has been utilised extensively in vitro as an examination model in understanding metabolic disease progression. Although it is indispensable in both preclinical and pharmaceutical research, a comprehensive review of its use in the investigation of insulin resistance progression and pharmaceutical development is not available. Key findings C2C12 is a well-documented model, which can facilitate our understanding in glucose metabolism, insulin signalling mechanism, insulin resistance, oxidative stress, reactive oxygen species and glucose transporters at cellular and molecular levels. With the aid of the C2C12 model, recent studies revealed that insulin resistance has close relationship with various metabolic diseases in terms of disease progression, pathogenesis and therapeutic management. A holistic, safe and effective disease management is highly of interest. Therefore, significant efforts have been paid to explore novel drug compounds and natural herbs that can elicit therapeutic effects in the targeted sites at both cellular (e.g. mitochondria, glucose transporter) and molecular level (e.g. genes, signalling pathway). Summary The use of C2C12 myoblast cell line is meaningful in pharmaceutical and biomedical research due to their expression of GLUT-4 and other features that are representative to human skeletal muscle cells. With the use of the C2C12 cell model, the impact of drug delivery systems (nanoparticles and quantum dots) on skeletal muscle, as well as the relationship between exercise, pancreatic β-cells and endothelial cells, was discovered.

The principle of mechanopharmacology of airway smooth muscle (ASM) is based on the premise that physical agitation, such as pressure oscillation applied to an airway, is able to induce bronchodilation by reducing contractility and softening the cytoskeleton of ASM. Although the underlying mechanism is not entirely clear, there is evidence to suggest that large-amplitude stretches are able to disrupt the actomyosin interaction in the crossbridge cycle and weaken the cytoskeleton in ASM cells. Rho-kinase is known to enhance force generation and strengthen structural integrity of the cytoskeleton during smooth muscle activation and plays a key role in the maintenance of force during prolonged muscle contractions. Synergy in relaxation has been observed when the muscle is subject to oscillatory length change while Rho-kinase is pharmacologically inhibited. In this review, inhibition of Rho-kinase coupled to therapeutic pressure oscillation applied to the airways is explored as a combination treatment for asthma.

Oral delivery of insulin-loaded therapeutics is challenging due to the extreme pH condition, enzymatic degradation, and limited permeation in the gastrointestinal (GI) tract. Nanoparticles can overcome various GI barriers and improve the oral bioavailability of insulin. Although a number of novel strategies have been reported to optimise the orally administered nano-formulation, its clinical translation remains unachievable. Animal studies are essential to establish the protective effect, transit behaviour, retention time, mucoadhesiveness degree, absorption mechanism and distribution of nanoparticle in the GI tract. In this review, we examine various instrumentations, such as Ussing chamber, fluorescence microscope, TEM, CLSM, that are available for investigating the ex vivo intestinal absorption and mucoadhesive capability of insulin-loaded nanoparticles. More importantly, a comprehensive understanding and evaluation of in vivo animal studies are crucial to clarify the physiological properties of insulin nanoparticles. In addition, the biocompatibility of nanoparticle is a critical prerequisite for short-term and long-term use of drug formulation. The success of oral nanomedicine should improve the bioavailability of insulin and elicit no damage to internal organs. Lastly, we reviewed the current status of animal assays, including mucoadhesiveness study, biocompatibility (integrity of intestinal mucosa, histological analysis, oxidative stress, physical symptoms), biodistribution (fluorescence imaging, SPECT) and in vivo efficacy study, for the evaluation of orally administered insulin-loaded nanoparticles in pre-clinical stage.

The oral bioavailability of therapeutic proteins is limited by the gastrointestinal barriers. Encapsulation of labile proteins into nanoparticles is a promising strategy. In order to improve the stability of nanoparticles, lyophilisation has been used to remove water molecules from the suspension. Although various cryoprotections were employed in the preparation of lyophilised nanoparticles, the selection of cryoprotectant type and concentration in majority of the developed formulation was not justified. In this study, nanoparticles were fabricated by cationic chitosan and anionic Dz13Scr using complex coacervation. The effect of cryoprotectant types (mannitol, sorbitol, sucrose and trehalose) and their concentrations (1, 3, 5, 7, 10% w/v) on physiochemical properties of nanoparticles were measured. Cellular assays were performed to investigate the impact of selected cryoprotectant on cytotoxicity, glucose consumption, oral absorption mechanism and gastrointestinal permeability. The obtained results revealed that mannitol (7% w/v) could produce nanoparticles with small size (313.2 nm), slight positive charge and uniform size distribution. The addition of cryoprotectant could preserve the bioactivity of entrapped insulin and improve the stability of nanoparticles against mechanical stress during lyophilisation. The gastrointestinal absorption of nanoparticles is associated with both endocytic and paracellular pathways. With the use of 7% mannitol, lyophilised nanoparticles induced a significant glucose uptake in C2C12 cells. This work illustrated the importance of appropriate cryoprotectant in conservation of particle physiochemical properties, structural integrity and bioactivity. An incompatible cryoprotectant and inappropriate concentration could lead to cake collapse and formation of heterogeneous particle size populations.

Diabetes mellitus is a chronic metabolic health disease affecting the homeostasis of blood sugar levels. However, subcutaneous injection of insulin can lead to patient non-compliance, discomfort, pain and local infection. Sub-micron sized drug delivery systems have gained attention in oral delivery of insulin for diabetes treatment. In most of the recent literature, the terms “microparticles” and “nanoparticle” refer to particles where the dimensions of the particle are measured in micrometers and nanometers respectively. For instance, insulin-loaded particles are defined as microparticles with size larger than 1 μm by most of the research groups. The size difference between nanoparticles and microparticles proffers numerous effects on the drug loading efficiency, aggregation, permeability across the biological membranes, cell entry and tissue retention. For instance, microparticulate drug delivery systems have demonstrated a number of advantages including protective effect against enzymatic degradation, enhancement of peptide stability, site-specific and controlled drug release. Compared to nanoparticulate drug delivery systems, microparticulate formulations can facilitate oral absorption of insulin by paracellular, transcellular and lymphatic routes. In this article, we review the current status of microparticles, microcapsules and microspheres for oral administration of insulin. A number of novel techniques including layer-by-layer coating, self-polymerisation of shell, nanocomposite microparticulate drug delivery system seem to be promising for enhancing the oral bioavailability of insulin. This review draws several conclusions for future directions and challenges to be addressed for optimising the properties of microparticulate drug formulations and enhancing their hypoglycaemic effects.

Therapeutic proteins are labile macromolecules that are prone to degradation during production, freeze-drying and storage. Recent studies showed that nanoparticles can enhance the stability and oral bioavailability of encapsulated proteins. Several conventional approaches (enzyme inhibitors, mucoadhesive polymers) and novel strategies (surface modification, ligand conjugation, flash nano-complexation, stimuli-responsive drug delivery systems) have been employed to improve the physiochemical properties of nanoparticles such as size, zeta potential, morphology, polydispersity index, drug release kinetics and cell-targeting capacity. However, clinical translation of protein-based nanoparticle is limited due to poor experimental design, protocol non-compliance and instrumentation set-up that do not reflect the physiological conditions, resulting in difficulties in mass production of nanoparticles and waste in research funding. In order to address the above concerns, we conducted a comprehensive review to examine the experimental designs and conditions for physical characterization of protein-based nanoparticles. Reliable and robust characterization is essential to verify the cellular interactions and therapeutic potential of protein-based nanoparticles. Importantly, there are a number of crucial factors, which include sample treatment, analytical method, dispersants, sampling grid, staining, quantification parameters, temperature, drug concentration and research materials, should be taken into careful consideration. Variations in research protocol and unreasonable conditions that are used in optimization of pharmaceutical formulations can have great impact in result interpretation. Last but not least, we reviewed all novel instrumentations and assays that are available to examine mucus diffusion capacity, stability and bioactivity of protein-based nanoparticles. These include circular dichroism, fourier transform infrared spectroscopy, X-ray diffractogram, UV spectroscopy, differential scanning calorimetry, fluorescence spectrum, Förster resonance energy transfer, NMR spectroscopy, Raman spectroscopy, cellular assays and animal models.

The adult olfactory mucosa, a highly regenerative tissue with unique life-long neurogenesis ability, is thought to harbor a naïve yet tightly controlled stem cell population. It will provide unique benefits in various stem cell-based therapies, such as stroke treatment. Here, we identified a subpopulation of adult pluripotent-like olfactory stem cells (APOSCs), which were modulated by an epigenetic repressor of CBX7. APOSCs form a floating sphere, express pluripotency markers Nanog, Oct-4, Sox-2, and SSEA-4 and show alkaline phosphatase activity. In addition, APOSCs display self-renewal and a pluripotent potential to differentiate into all three germ layers. Moreover, APOSCs coexpress pluripotency markers with CBX7. Within their natural niche, APOSCs from CBX7+/+ mice responded promptly to either spontaneous or injury-induced tissue regeneration. However, APOSCs from CBX7−/− mice manifested an impaired self-renewal and differentiation potential. Similarly, in vitro-cultivated CBX7−/− APOSCs underwent premature senescence, whereas CBX7+/+ APOSCs still actively divided, indicating that CBX7 is required for the self-renewal of APOSCs. Intracerebral implantation of APOSCs improved the stroke-mediated neurological dysfunction in rodents. These findings indicate that CBX7 plays a critical role in the regenerative properties of APOSCs and indicate the safety and feasibility of implantation of autologous APOSCs in stroke treatment.

Diabetes is a chronic medical condition, which is characterised by high blood sugar level. Exogenous insulin is commonly administered subcutaneously for the management of diabetes. However, daily injections of insulin could result in poor patient compliance and various side-effects. Although oral administration offers benefits, insulin is vulnerable to enzymatic degradation, chemical instability and poor gastrointestinal absorption. There is an absence of reviews on insulin-loaded liposomal drug carriers, despite that fact that liposomes have gained considerable attention recently for oral delivery of insulin. They demonstrate favourable characteristics, such as versatility, biocompatibility, protective effect against enzymatic degradation, and cell-specific targeting. In this review, we will explore the status quo for oral delivery of insulin-loaded liposomal formulations, followed by discussing the state of art of these vesicles. This review will provide a detailed overview on insulin-loaded conventional liposomes, and 7 types of current novel formulations. Lastly, the future direction for oral bioavailability enhancement and development of such nanoscale drug delivery systems will be discussed. Further optimisation in the drug entrapment efficiency and gastrointestinal absorption will be required to develop a clinically successful oral liposome-insulin formulation.

The standard method for measuring the phosphorylation of the regulatory myosin light chain (MLC20) in smooth muscle is extraction of the light chain using a urea extraction buffer, urea–glycerol gel electrophoresis of the soluble portion of the extract (supernatant) and Western blot analysis. The undissolved portion of the tissue during extraction (the pellet) is usually discarded. Because the pellet contains a finite amount of MLC20, omission of the pellet could result in inaccurate measurement of MLC20 phosphorylation. In this study we compared the level of tracheal smooth muscle MLC20 phosphorylation in the supernatant alone, with that in the complete tissue homogenate (supernatant and pellet) using the standard method. The supernatant fraction showed the well-known double bands representing phosphorylated and un-phosphorylated MLC20. The dissolved pellet fraction showed varying amounts of un-phosphorylated and phosphorylated MLC20. There was a small but statistically significant overestimation of the percent MLC20 phosphorylation if the pellet was not taken into consideration. The overestimation was 7% ± 2% (mean ± SEM) (p < 0.05) in unstimulated muscle and 2% ± 1% (p < 0.05) in acetylcholine (10−6mol/L) stimulated muscle. This finding suggests that for accurate estimation of the stoichiometry of MLC20 phosphorylation it is necessary to consider the contribution from the pellet portion of the muscle tissue homogenate.

Smooth muscle contraction can be divided into two phases: the initial contraction determines the amount of developed force and the second phase determines how well the force is maintained. The initial phase is primarily due to activation of actomyosin interaction and is relatively well understood, whereas the second phase remains poorly understood. Force maintenance in the sustained phase can be disrupted by strains applied to the muscle; the strain causes actomyosin cross-bridges to detach and also the cytoskeletal structure to disassemble in a process known as fluidization, for which the underlying mechanism is largely unknown. In the present study we investigated the ability of airway smooth muscle to maintain force after the initial phase of contraction. Specifically, we examined the roles of Rho-kinase and protein kinase C (PKC) in force maintenance. We found that for the same degree of initial force inhibition, Rho-kinase substantially reduced the muscle's ability to sustain force under static conditions, whereas inhibition of PKC had a minimal effect on sustaining force. Under oscillatory strain, Rho-kinase inhibition caused further decline in force, but again, PKC inhibition had a minimal effect. We also found that Rho-kinase inhibition led to a decrease in the myosin filament mass in the muscle cells, suggesting that one of the functions of Rho-kinase is to stabilize myosin filaments. The results also suggest that dissolution of myosin filaments may be one of the mechanisms underlying the phenomenon of fluidization. These findings can shed light on the mechanism underlying deep inspiration induced bronchodilation.

The central nervous system is known to play important roles in the regulation of renal sodium excretion. The present study was designed to reveal the interrelationship between cholinergic pathway in the magnocellular paraventricular nucleus (PVN) and the natriuresis induced by brain cholinergic stimuli. The results indicated that urinary sodium excretion was significantly increased at 40 min after intracerebroventricular (ICV) injection of carbachol (CBC). Immunohistochemical studies showed that CBC increased choline acetyltransferase-immunoreactivity (ChAT-IR) in the magnocellular PVN and renal proximal convoluted tubule (PCT), respectively. After pretreatment with atropine, urinary sodium excretion was significantly reduced, and carbachol-increased ChAT-IR in the magnocellular PVN and PCT was also significantly decreased. These results suggested that brain cholinergic stimuli induced the natriuresis and increased the activity of cholinergic neurons in the magnocellular PVN and cholinergic system in the PCT. The blockade of muscarinic receptor completely abolished the natriuresis and partially inhibited carbachol-exerted stimulatory effects in the magnocellular PVN and PCT. To summarize, brain cholinergic pathway and peripheral cholinergic system in kidney were found to contribute to the natriuresis following brain cholinergic stimulation. Our findings revealed novel evidence that PVN was involved in the natriuresis via humoral mechanisms.

Nanoparticles have demonstrated significant advancements in potential oral delivery of insulin. In this publication, we review the current status of polymeric, inorganic and solid-lipid nanoparticles designed for oral administration of insulin. Firstly, the structure and physiological function of insulin are examined. Then, the efficiency and shortcomings of insulin nanoparticle are discussed. These include the susceptibility to digestive enzyme degradation, instability in the acidic pH environment, poor mucus diffusion and inadequate permeation through the gastrointestinal epithelium. In order to optimise the nanocarriers, the following considerations, including polymer nature, surface charge, size, polydispersity index and morphology of nanoparticles, have to be taken into account. Some novel designs such as chitosan-based glucose-responsive nanoparticles, layer by layer technique-based nanoparticles and zwitterion nanoparticles are being adopted to overcome the physiological challenges. The review ends with some future directions and challenges to be addressed for the success of oral delivery of insulin-loaded nanoparticle formulation.

Smooth muscle is able to function over a much broader length range than striated muscle. The ability to maintain contractility after a large length change is thought to be due to an adaptive process involving restructuring of the contractile apparatus to maximize overlap between the contractile filaments. The molecular mechanism for the length-adaptive behavior is largely unknown. In smooth muscle adapted to different lengths we quantified myosin monomers, basal and activation-induced myosin light chain (MLC) phosphorylation, shortening-velocity, power-output and active force. The muscle was able to generate a constant maximal force over a 2-fold length range when it was allowed to go through isometric contraction/relaxation cycles after each length change (length adaptation). In the relaxed state myosin monomer concentration and basal MLC phosphorylation decreased linearly, while in the activated state activation-induced MLC phosphorylation and shortening-velocity/power-output increased linearly with muscle length. The results suggest that recruitment of myosin monomers and oligomers into the actin filament lattice (where they form force-generating filaments) occurs during muscle adaptation to longer length with the opposite occurring during adaptation to shorter length.

Owing to their spontaneous development of atherosclerosis, apolipoprotein E knockout mice (ApoEKO) are one of the best studied animal models for this disease. Little is known about the utility of various omega-3 fatty acid regimens, in particular fish oils, in preventing cardiac disease in ApoEKO mice. The purpose of this study was to determine the cardiovascular effects of omega-3 fatty acid supplementation with either safflower oil (control), fish oil, flaxseed oil, or designed oil in ApoEKO mice fed a high-fat diet for a total of 16 weeks. In-vivo cardiac function was assessed weekly using murine echocardiography. Blood pressure, plasma lipid levels, and brain natriuretic peptide (BNP) were serially measured. The results show that ApoEKO mice fed fish oil demonstrated an increase in left ventricular wall thickness as a result of increased afterload. Despite chronic treatment with fish oil over 16 weeks, blood pressure increased in ApoEKO mice by 20% compared with the baseline. Both echocardiographic evidence of left ventricular hypertrophy and biochemical increase in BNP levels confirmed diastolic dysfunction in ApoEKO mice fed fish oil. This suggests that high-fat diet supplemented with fish oil may lead to adverse cardiovascular effects in ApoE deficient mice.

Reduced airway distensibility due to increased airway stiffness is a characteristic of asthma. Airway stiffness is determined by the property and structural organization of the various elements of the airway wall, and is often divided into active and passive components. Active stiffness is thought to be associated with activation of muscle cells in the airway wall. This component of stiffness can be inhibited when active force produced by the muscle is abolished. Passive stiffness, on the other hand, is thought to stem from non-muscle component of the airway wall, especially the collagen/elastin fibrous network of the extracellular matrix within which the muscle cells are embedded. In this brief review, the notion that passive stiffness is exclusively extracellular in origin is challenged. Recent evidence suggests that a substantial portion of the passive stiffness of an in vitro preparation of tracheal smooth muscle is calcium sensitive and is regulated by Rho-kinase, although the underlying mechanism and the details of regulation for the development of this intracellular passive stiffness are still largely unknown. To reduce airway stiffness different lines of attack must be tailored to different components of the stiffness. The regulatable passive stiffness is distinct from the relatively permanent stiffness of the extracellular matrix and the stiffness associated with active muscle contraction. To improve airway distensibility during asthma exacerbation, a comprehensive approach to reduce overall airway stiffness should therefore include a strategy for targeting the regulatable passive stiffness.

Bronchospasm induced in non-asthmatic human subjects can be easily reversed by a deep inspiration (DI) whereas bronchospasm that occurs spontaneously in asthmatic subjects cannot. This physiological effect of a DI has been attributed to the manner in which a DI causes airway smooth muscle (ASM) cells to stretch, but underlying molecular mechanisms-and their failure in asthma-remain obscure. Using cells and tissues from wild type and zyxin-/- mice we report responses to a transient stretch of physiologic magnitude and duration. At the level of the cytoskeleton, zyxin facilitated repair at sites of stress fiber fragmentation. At the level of the isolated ASM cell, zyxin facilitated recovery of contractile force. Finally, at the level of the small airway embedded with a precision cut lung slice, zyxin slowed airway dilation. Thus, at each level zyxin stabilized ASM structure and contractile properties at current muscle length. Furthermore, when we examined tissue samples from humans who died as the result of an asthma attack, we found increased accumulation of zyxin compared with non-asthmatics and asthmatics who died of other causes. Together, these data suggest a biophysical role for zyxin in fatal asthma.

The airway smooth muscle (ASM) layer within the airway wall modulates airway diameter and distensibility. Even in the relaxed state, the ASM layer possesses finite stiffness and limits the extent of airway distension by the radial force generated by parenchymal tethers and transmural pressure. Airway stiffness has often been attributed to passive elements, such as the extracellular matrix in the lamina reticularis, adventitia, and the smooth muscle layer that cannot be rapidly modulated by drug intervention such as ASM relaxation by β-agonists. In this study, we describe a calcium-sensitive component of ASM stiffness mediated through the Rho-kinase signaling pathway. The stiffness of ovine tracheal smooth muscle was assessed in the relaxed state under the following conditions: 1) in physiological saline solution (Krebs solution) with normal calcium concentration; 2) in calcium-free Krebs with 2 mM EGTA; 3) in Krebs with calcium entry blocker (SKF-96365); 4) in Krebs with myosin light chain kinase inhibitor (ML-7); and 5) in Krebs with Rho-kinase inhibitor (Y-27632). It was found that a substantial portion of the passive stiffness could be abolished when intracellular calcium was removed; this calcium-sensitive stiffness appeared to stem from intracellular source and was not sensitive to ML-7 inhibition of myosin light chain phosphorylation, but was sensitive to Y-27632 inhibition of Rho kinase. The results suggest that airway stiffness can be readily modulated by targeting the calcium-sensitive component of the passive stiffness within the muscle layer.

Fibroblast growth factor (FGF) signaling regulates a multitude of cellular processes, including cell proliferation, survival, migration, and differentiation. In the vertebrate lens, FGF signaling regulates fiber cell differentiation characterized by high expression of crystallin proteins. However, a direct link between FGF signaling and crystallin gene transcriptional machinery remains to be established. Previously, we have shown that the bZIP proto-oncogene c-Maf regulates expression of αA-crystallin (Cryaa) through binding to its promoter and distal enhancer, DCR1, both activated by FGF2 in cell culture. Herein, we identified and characterized a novel FGF2-responsive region in the c-Maf promoter (-272/-70, FRE). Both c-Maf and Cryaa regulatory regions contain arrays of AP-1 and Ets-binding sites. Chromatin immunoprecipitation (ChIP) assays established binding of c-Jun (an AP-1 factor) and Etv5/ERM (an Ets factor) to these regions in lens chromatin. Analysis of temporal and spatial expression of c-Jun, phospho-c-Jun, and Etv5/ERM in wild type and ERK1/2 deficient lenses supports their roles as nuclear effectors of FGF signaling in mouse embryonic lens. Collectively, these studies show that FGF signaling up-regulates expression of αA-crystallin both directly and indirectly via up-regulation of c-Maf. These molecular mechanisms are applicable for other crystallins and genes highly expressed in terminally differentiated lens fibers.

Airway smooth muscle (ASM) is able to generate maximal force under static conditions, and this isometric force can be maintained over a large length range due to length adaptation. The increased force at short muscle length could lead to excessive narrowing of the airways. Prolonged exposure of ASM to submaximal stimuli also increases the muscle's ability to generate force in a process called force adaptation. To date, the effects of length and force adaptation have only been demonstrated under static conditions. In the mechanically dynamic environment of the lung, ASM is constantly subjected to periodic stretches by the parenchyma due to tidal breathing and deep inspiration. It is not known whether force recovery due to muscle adaptation to a static environment could occur in a dynamic environment. In this study the effect of length oscillation mimicking tidal breathing and deep inspiration was examined. Force recovery after a length change was attenuated in the presence of length oscillation, except at very short lengths. Force adaptation was abolished by length oscillation. We conclude that in a healthy lung (with intact airway-parenchymal tethering) where airways are not allowed to narrow excessively, large stretches (associated with deep inspiration) may prevent the ability of the muscle to generate maximal force that would occur under static conditions irrespective of changes in mean length; mechanical perturbation on ASM due to tidal breathing and deep inspiration, therefore, is the first line of defense against excessive bronchoconstriction that may result from static length and force adaptation.

Airway smooth muscle (ASM) in individuals with asthma is continuously stimulated by spasmogens released as part of chronic airway inflammation. This chronic submaximal stimulation of ASM produces "tone," which may or may not narrow airways sufficiently to induce respiratory symptoms. However, when coupled with a bronchoprovocative challenge with a nonspecific contractile agonist, this increased tone could contribute to the manifestation of airway hyperresponsiveness (AHR). In this study, we examined the effect of chronic acetylcholine (ACh) exposure at different muscle lengths to gain insights into the consequence of increased tone on the mechanical properties of ASM. The total force (the ACh-induced tone plus active force induced by a second stimulus-electric field stimulation [EFS]) increased immediately after induction of muscle tone, and increased further over time in the presence of the tone in a process termed "force adaptation." The phenomenon of force adaptation was observed over a wide range of muscle lengths and did not prevent length adaptation when the muscle was adapted to the tone before being subjected to a length change, suggesting that both length and force adaptations can occur sequentially and in an independent fashion in the same tissue. Together, these results suggest that adaptation of ASM to shortened length in the presence of muscle tone produced a condition that favored excessive force generation in response to a second stimulus (herein EFS) at reduced muscle length. In vivo these changes will be translated into excessive airway narrowing in response to naturally occurring and pharmacological bronchoconstricting stimuli.

Stress and strain are omnipresent in the lung due to constant lung volume fluctuation associated with respiration, and they modulate the phenotype and function of all cells residing in the airways including the airway smooth muscle (ASM) cell. There is ample evidence that the ASM cell is very sensitive to its physical environment, and can alter its structure and/or function accordingly, resulting in either desired or undesired consequences. The forces that are either conferred to the ASM cell due to external stretching or generated inside the cell must be borne and transmitted inside the cytoskeleton (CSK). Thus, maintaining appropriate levels of stress and strain within the CSK is essential for maintaining normal function. Despite the importance, the mechanisms regulating/dysregulating ASM cytoskeletal filaments in response to stress and strain remained poorly understood until only recently. For example, it is now understood that ASM length and force are dynamically regulated, and both can adapt over a wide range of length, rendering ASM one of the most malleable living tissues. The malleability reflects the CSK's dynamic mechanical properties and plasticity, both of which strongly interact with the loading on the CSK, and all together ultimately determines airway narrowing in pathology. Here we review the latest advances in our understanding of stress and strain in ASM cells, including the organization of contractile and cytoskeletal filaments, range and adaptation of functional length, structural and functional changes of the cell in response to mechanical perturbation, ASM tone as a mediator of strain-induced responses, and the novel glassy dynamic behaviors of the CSK in relation to asthma pathophysiology.

Extracts of the Chinese antirheumatic herbal remedy Tripterygium wilfordii Hook F (TWHF) have been shown to be of therapeutic benefit in patients with a variety of autoimmune and inflammatory diseases, including rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and psoriasis (1). Although TWHF has toxic potential, careful extraction procedures have generated preparations with an acceptable frequency of adverse reactions, which are largely related to the gastrointestinal tract and amenorrhea (1). Although the extracts of TWHF contain a variety of components, including diterpenoids, triterpenoids, and alkaloids, evidence suggests that most, if not all, of the therapeutic activity of extracts of TWHF can be accounted for by the content of 2 diterpenoids, triptolide and tripdiolide (2). However, it has been assumed that individual components do not work as well as the extracts because of unknown effects of mixtures of components. For example, one open clinical study compared triptolide with an ethyl acetate (EA) extract of TWHF in the treatment of RA and reported that triptolide was less effective in improving clinical manifestations of the disease and was associated with more side effects than the extract of TWHF (3). Despite the belief in a synergistic benefit from mixtures of components of TWHF extracts, investigation into the biologic activities of individual constituents of TWHF has proceeded. As one of the major active components of TWHF, triptolide has been reported to be able to suppress the production of a wide range of proinflammatory cytokines, including interleukin-2 (IL-2), interferon-γ (IFNγ), IL-6, and tumor necrosis factor (TNF), as well as inhibit the up-regulation of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) (4,5). Previous studies have shown that triptolide exerts its anti-inflammatory and immunosuppressive actions by directly suppressing the transcription of the genes that encode these proteins through interference with the function of transcription factors such as NF-κB, activator protein 1, nuclear factor of activated T cells, and Oct-1 (6–8). The immunosuppressive and anti-inflammatory activities of extracts of TWHF have been documented in several animal models, including collagen-induced arthritis (CIA) in mice and the air-pouch model of carageenan-induced inflammation in rats (9,10), whereas triptolide has been shown to suppress CIA when given prophylactically (11) or after the onset of disease (12). Moreover, triptolide has been claimed to be effective in the treatment of RA and psoriasis, although this has not been documented in randomized clinical trials (3,13), and the therapeutic effect in RA may be lower than that of the extract of TWHF (3). Structurally, tripdiolide is similar to triptolide, with an α,β-unsaturated lactone ring and 3 epoxide adducts. The only difference is that the hydrogen at the C2 position of triptolide is replaced by a hydroxide group in tripdiolide. Previous studies have shown that tripdiolide is as potent as triptolide in suppressing in vitro cell proliferation and IL-2 production by mitogenstimulated T cells (2). Only 1 study tested the in vivo biologic activity of tripdiolide, and prolonged survival in leukemia-bearing mice as a result of treatment with tripdiolide was reported (14). However, the impact of tripdiolide on animal models of autoimmunity or inflammation has not been examined. Previously, studies have shown that 2 extracts of TWHF improved the clinical manifestations of lupus in MRL/lpr mice (15,16). However, the composition of these extracts was not examined. In addition, the extracts were used prophylactically to prevent disease development but were not active as a therapy for established disease. Recently, we used the EA extract of TWHF in the treatment of established lupus nephritis in (NZB X NZW)F1 mice and found significant amelioration of disease activity (17). No previous study has examined the impact of purified components of TWHF on animal models of lupus nephritis. The current study was therefore performed to evaluate the therapeutic effects of the 2 individual diterpenoids on established nephritis in the (NZB X NZW)F1 mouse. We found that renal disease remarkably improved, and there was greater survival of (NZB X NZW)F1 mice treated with either triptolide or tripdiolide than mice treated with vehicle. These results support the conclusions that the therapeutic value of extracts of TWHF in lupus nephritis can be accounted for by the triptolide and tripdiolide contents, that cooperation between components is not required for therapeutic benefit, and that either diterpenoid could be effective therapy for lupus nephritis.

Smooth muscle myosin monomers self-assemble in solution to form filaments. Phosphorylation of the 20-kD regulatory myosin light chain (MLC20) enhances filament formation. It is not known whether the phosphorylated and non-phosphorylated filaments possess the same structural integrity.We purified myosin from bovine trachealis to form filaments, in ATP-containing zero-calcium solution during a slow dialysis that gradually reduced the ionic strength. Sufficient myosin light chain kinase and phosphatase, as well as calmodulin, were retained after the myosin purification and this enabled phosphorylation of MLC20 within 20-40s after addition of calcium to the filament suspension. The phosphorylated and non-phosphorylated filaments were then partially disassembled by ultrasonification. The extent of filament disintegration was visualized and quantified by atomic force microscopy.MLC20 phosphorylation reduced the diameter of the filaments and rendered the filaments more resistant to ultrasonic agitation. Electron microscopy revealed a similar reduction in filament diameter in intact smooth muscle when the cells were activated.Modification of the structural and physical properties of myosin filaments by MLC20 phosphorylation may be a key regulation step in smooth muscle where formation and dissolution of the filaments are required in the cells' adaptation to different cell length.

An asthma-like airway phenotype has been described in people with cystic fibrosis (CF). Whether these findings are directly caused by loss of CF transmembrane conductance regulator (CFTR) function or secondary to chronic airway infection and/or inflammation has been difficult to determine.Airway contractility is primarily determined by airway smooth muscle. We tested the hypothesis that CFTR is expressed in airway smooth muscle and directly affects airway smooth muscle contractility.Newborn pigs, both wild type and with CF (before the onset of airway infection and inflammation), were used in this study. High-resolution immunofluorescence was used to identify the subcellular localization of CFTR in airway smooth muscle. Airway smooth muscle function was determined with tissue myography, intracellular calcium measurements, and regulatory myosin light chain phosphorylation status. Precision-cut lung slices were used to investigate the therapeutic potential of CFTR modulation on airway reactivity.We found that CFTR localizes to the sarcoplasmic reticulum compartment of airway smooth muscle and regulates airway smooth muscle tone. Loss of CFTR function led to delayed calcium reuptake following cholinergic stimulation and increased myosin light chain phosphorylation. CFTR potentiation with ivacaftor decreased airway reactivity in precision-cut lung slices following cholinergic stimulation.Loss of CFTR alters porcine airway smooth muscle function and may contribute to the airflow obstruction phenotype observed in human CF. Airway smooth muscle CFTR may represent a therapeutic target in CF and other diseases of airway narrowing.

Airway smooth muscle (ASM) in vivo is constantly subjected to oscillatory strain due to tidal breathing and deep inspirations. ASM contractility is known to be adversely affected by strains, especially those of large amplitudes. Based on the cross-bridge model of contraction, it is likely that strain impairs force generation by disrupting actomyosin cross-bridge interaction. There is also evidence that strain modulates muscle stiffness and force through induction of cytoskeletal remodeling. However, the molecular mechanism by which strain alters smooth muscle function is not entirely clear. Here, we examine the response of ASM to iso-velocity stretches to probe the components within the muscle preparation that give rise to different features in the force response. We found in ASM that force response to a ramp stretch showed a biphasic feature, with the initial phase associated with greater muscle stiffness compared with that in the later phase, and that the transition between the phases occurred at a critical strain of ∼3.3%. Only strains with amplitudes greater than the critical strain could lead to reduction in force and stiffness of the muscle in the subsequent stretches. The initial-phase stiffness was found to be linearly related to the degree of muscle activation, suggesting that the stiffness stems mainly from attached cross bridges. Both phases were affected by the degree of muscle activation and by inhibitors of myosin light-chain kinase, PKC, and Rho-kinase. Different responses due to different interventions suggest that cross-bridge and cytoskeletal stiffness is regulated differently by the kinases.

This study demonstrates the fabrication and characterization of chicken egg albumen-based bio-memristors. By introducing egg albumen as an insulator to fabricate memristor devices comprising a metal/insulator/metal sandwich structure, significant bipolar resistive switching behavior can be observed. The 1/f noise characteristics of the albumen devices were measured and results suggested that their memory behavior results from the formation and rupture of conductive filaments. Oxygen diffusion and electrochemical redox reaction of metal ions under a sufficiently large electric field are the principal physical mechanisms of the formation and rupture of conductive filaments; these mechanisms were observed by analysis of the time-of-flight secondary ion mass spectrometry (TOF-SIMS) and resistance–temperature (R–T) measurement results. The switching property of the devices remarkably improved by heat-denaturation of proteins; reliable switching endurance of over 500 cycles accompanied by an on/off current ratio (Ion/off) of higher than 103 were also observed. Both resistance states could be maintained for a suitably long time (>104 s). Taking the results together, the present study reveals for the first time that chicken egg albumen is a promising material for nonvolatile memory applications.

Excessive narrowing of airways in response to contractile agonists is a characteristic feature of asthma. We hypothesized that airway smooth muscle (ASM) adaptation to short lengths could contribute to exaggerated airway narrowing during an acute attack of asthma by allowing the muscle to regain its ability to generate maximal force at a shortened length. To test this hypothesis we mimicked, in vitro, the sequence of contractile events that would occur during a spontaneous attack of asthma. Trachealis muscle was challenged with carbachol (300 nM, submaximal dose) and allowed to shorten to approximately half of its original length. After 30 min of adaptation at the shortened length in the presence of carbachol, muscle force, amount and rate of shortening in response to electrical stimulation were compared with corresponding values obtained from control experiments during which the ASM was not adapted to the short length. After adaptation at the shortened length the developed force, amount and rate of shortening increased by 1.93 +/- 0.08-, 1.57 +/- 0.12-, and 1.75 +/- 0.2-fold, respectively. Shortening of ASM in response to contractile agonists can lead to adaptation of the muscle to the shortened length that, in turn, can result in further shortening and the potential for airway closure.

It is believed that the contractile filaments in smooth muscle are organized into arrays of contractile units (similar to the sarcomeric structure in striated muscle), and that such an organization is crucial for transforming the mechanical activities of actomyosin interaction into cell shortening and force generation. Details of the filament organization, however, are still poorly understood. Several models of contractile filament architecture are discussed here. To account for the linear relationship observed between the force generated by a smooth muscle and the muscle length at the plateau of an isotonic contraction, a model of contractile unit is proposed. The model consists of 2 dense bodies with actin (thin) filaments attached, and a myosin (thick) filament lying between the parallel thin filaments. In addition, the thick filament is assumed to span the whole contractile unit length, from dense body to dense body, so that when the contractile unit shortens, the amount of overlap between the thick and thin filaments (i.e., the distance between the dense bodies) decreases in exact proportion to the amount of shortening. Assembly of the contractile units into functional contractile apparatus is assumed to involve a group of cells that form a mechanical syncytium. The contractile apparatus is assumed malleable in that the number of contractile units in series and in parallel can be altered to accommodate strains on the muscle and to maintain the muscle's optimal mechanical function.Key words: contraction model, ultrastructure, length adaptation, plasticity.

Excitation–contraction coupling (E–C coupling) in phenylephrine(PE)-stimulated rabbit inferior vena cava (IVC) depends on the generation of asynchronous recurring Ca 2+ waves in the in situ vascular smooth muscle cells (VSMC). Previous studies by our group have implicated a putative non-selective cationic store-operated channel and the reverse-mode Na + –Ca 2+ exchange in refilling of the intracellular Ca 2+ store via the sarco/endoplasmic reticulum ATPase (SERCA) and the maintenance of the recurring Ca 2+ waves. We hypothesize that for the proper functioning of these three Ca 2+ translocators in the process of SR refilling, the plasma membrane (PM) and the underlying superficial sarcoplasmic reticulum (SR) form specialized PM–SR junctions, which are essential for the maintenance of the recurring Ca 2+ waves. In order to test this hypothesis, calyculin-A, a serine/threonine phosphatase inhibitor that has been demonstrated to result in the disruption of the PM–SR junctions was used. In the control rabbit IVC, electron microscopy of the in situ VSMC indicates that 14.2 ± 0.7% of the PM is closely apposed by the prominent superficial SR network, forming numerous flattened PM–SR junctional cytoplasmic spaces. In the control IVC stimulation with 5 μM PE resulted in sustained recurring Ca 2+ waves with a frequency of 0.42 ± 0.02 Hz. In calyculin-A treated rabbit IVC, a concentration-dependent dissociation of the superficial SR and loss of PM–SR junctions was observed. This progressive loss of the PM–SR junctions occurs over the same concentration range as the inhibition of PE-induced recurring Ca 2+ waves. These findings offer support for the hypothesis that the presence of the PM–SR junctions is required for the generation of asynchronous recurring Ca 2+ waves, which underlie excitation–contraction coupling in the VSMC of the rabbit IVC.

properties and the therapy has been shown to have an acute and sustained beneficial effect on lung function (changes in FEV 1 ) in chronic persistent asthma [6] . Antileukotrienes do not have major side effects and appear to be well tolerated by most patients [7] , but a limitation of their widespread use may be due to the large percentage of nonresponders [8] . However, for those intolerant or resistant to corticosteroids but responsive to antileukotrienes, treatment with leukotriene inhibitors has become an invaluable therapeutic option. There are two types of leukotriene inhibitors: the leukotriene receptor antagonists such as montelukast and those that inhibit leukotriene formation such as zileuton. Zileuton is an inhibitor of the enzyme 5-lipoxygenase, which forms leukotrienes from arachidonic acid. The network of signaling pathways associated with leukotrienes is complex and is not completely understood [6] . The findings by Chen et al. [2] have shed light on this subject, specifically with regard to perturbations in the signaling pathways seen in allergen-sensitized animals. They have identified some potentially important downstream molecular players in leukotriene signaling in ovalbumin-sensitized rats with chronic inflammation, thickened airway walls and reduced lung function. They also showed that zileuton significantly reduced the expression of biomarkers of chronic inflammation, significantly reversed the airway remodeling and improved lung function in these sensitized, bronchohyperresponsive rats. A thickened airway wall with increased airway smooth muscle mass and extracellular matrix deposition as a result of airway remodeling is a hallmark of persistent asthma. The mechanism underlying the remodeling process is not entirely clear. It is thought that the chronic airway inflammation seen in asthma is a major contributor, but anti-inflammatory strategies often do not bring resolution to airway remodeling [1] . Findings reported by Chen et al. [2] in this issue of Respiration explore the effects of the leukotriene inhibitor (zileuton) on airway remodeling in an ovalbumin-sensitized rat model and revealed that downstream pathways of leukotriene stimulation contribute to airway remodeling. Furthermore, the leukotriene signaling appears to follow the same pathways as those considered to be linked to tumorigenesis [3, 4] . These findings suggest a common mechanism underlying the increase in airway tissue mass seen in chronic asthma and the tumor growth seen in malignancy. The primary components of asthma treatment separately target acute airway obstruction and chronic airway inflammation. Bronchodilators (predominately β 2 agonists) are used to treat the acute symptoms and corticosteroids are used to control inflammation. Increasingly, antileukotriene agents are being added to inhaled corticosteroids in combination therapy, especially for severe asthma. Antileukotrienes are also used on their own as a frontline therapy, mostly for mild asthma [5] . Antileukotrienes have both anti-inflammatory and bronchodilatory Published online: August 29, 2013

The observation that the length-force relationship in airway smooth muscle can be shifted along the length axis by accommodating the muscle at different lengths has stimulated great interest. In light of the recent understanding of the dynamic nature of length-force relationship, many of our concepts regarding smooth muscle mechanical properties, including the notion that the muscle possesses a unique optimal length that correlates to maximal force generation, are likely to be incorrect. To facilitate accurate and efficient communication among scientists interested in the function of airway smooth muscle, a revised and collectively accepted nomenclature describing the adaptive and dynamic nature of the length-force relationship will be invaluable. Setting aside the issue of underlying mechanism, the purpose of this article is to define terminology that will aid investigators in describing observed phenomena. In particular, we recommend that the term “optimal length” (or any other term implying a unique length that correlates with maximal force generation) for airway smooth muscle be avoided. Instead, the in situ length or an arbitrary but clearly defined reference length should be used. We propose the usage of “length adaptation” to describe the phenomenon whereby the length-force curve of a muscle shifts along the length axis due to accommodation of the muscle at different lengths. We also discuss frequently used terms that do not have commonly accepted definitions that should be used cautiously.

Induction of hypertrophy and inhibition of apoptosis may be important mechanisms contributing to increased airway smooth muscle (ASM) mass in asthma. Data from our laboratory indicate that cardiotrophin-1 (CT-1) induces hypertrophy and inhibits apoptosis in isolated human ASM cells. To determine whether these novel effects of CT-1 also occur in the airway tissue milieu and to determine whether structural changes are accompanied by functional changes, matched pairs of guinea pig airway explants were treated with or without CT-1 for 7 days, and structural features as well as isometric and isotonic contractile and relaxant mechanical properties were measured. CT-1 (0.2–5 ng/ml) increased both myocyte mass and extracellular matrix in a concentration-dependent fashion. CT-1 (10 ng/ml)-treated tissues exhibited a significant increase in passive tension at all lengths on day 7; at optimal length, passive tension generated by CT-1-treated tissues was 1.72 ± 0.12 vs. 1.0 ± 0.1 g for control. Maximal isometric stress was decreased in the CT-1-treated group on day 7 (0.39 ± 0.10 kg/cm2) vs. control (0.77 ± 0.15 kg/cm2, P < 0.05). Isoproterenol-induced relaxant potency was reduced in CT-1-treated tissues, log EC50 being −7.28 ± 0.34 vs. −8.12 ± 0.25 M in control, P < 0.05. These data indicate that CT-1 alters ASM structural and mechanical properties in the tissue environment and suggest that structural changes found in the airway wall in asthma are not necessarily associated with increased responsiveness.

Recent studies have shown that Cdk5, a member of the cyclin-dependent-kinase family, regulates adhesion and migration in a mouse corneal epithelial cell line. Here, we extend these findings to corneal wound healing in vivo and examine the mechanism linking Cdk5 to cytoskeletal reorganization and migration. Cdk5 was overexpressed in the corneal epithelium of transgenic mice under control of the ALDH3 promoter. Elevated Cdk5 expression retarded corneal debridement wound closure in these animals and suppressed remodeling of the actin cytoskeleton. Conversely, the Cdk5 inhibitor, olomoucine, accelerated debridement wound healing in organ cultured eyes of normal mice, caused migrating cells to separate from the epithelial cell sheet, and increased the level of activated Src(pY416) along the wound edge. To explore the relationship between Cdk5 and Src in greater detail, we examined scratch-wounded cultures of corneal epithelial cells. Src was activated in cells along the wound edge and blocking this activation with the Src kinase inhibitor, PP1, inhibited wound closure by 85%. Inhibiting Cdk5 activity with olomoucine or a dominant negative construct, Cdk5T33, increased the concentration of Src(pY416), shifted its subcellular localization to the cell periphery and enhanced wound closure. Cdk5(pY15), an activated form of Cdk5, also appeared along the wound edge. Inhibiting Src activity with PP1 blocked the appearance of Cdk5(pY15), suggesting that Cdk5 phosphorylation is Src dependent. Cdk5 and Src co-immunoprecipitated from scratch-wounded cultures, demonstrating that both kinases are part of an intracellular protein complex. These findings indicate that Cdk5 exerts its effects on cell migration during corneal epithelial wound healing by regulating the activation and localization of Src.

Actin polymerization as part of the normal smooth muscle response to various stimuli has been reported. The actin dynamics are believed to be necessary for cytoskeletal remodeling in smooth muscle in its adaptation to external stress and strain and for maintenance of optimal contractility. We have shown in our previous studies in airway smooth muscle that myosins polymerized in response to contractile activation as well as to adaptation at longer cell lengths. We postulated that the same response could be elicited from actins under the same conditions. In the present study, actin filament formation was quantified electron microscopically in cell cross sections. Nanometer resolution allowed us to examine regional distribution of filaments in a cell cross section. Airway smooth muscle bundles were fixed in relaxed and activated states at two lengths; muscle preparations were also fixed after a period of oscillatory strain, a condition known to cause depolymerization of myosin filaments. The results indicate that contractile activation and increased cell length nonsynergistically enhanced actin polymerization; the extent of actin polymerization was substantially less than that of myosin polymerization. Oscillatory strain increased thin filament formation. Although thin filament density was found higher in cytoplasmic areas near dense bodies, contractile activation did not preferentially enhance actin polymerization in these areas. It is concluded that actin thin filaments are dynamic structures whose length and number are regulated by the cell in response to changes in extracellular environment and that polymerization and depolymerization of thin filaments occur uniformly across the whole cell cross section.

Despite considerable investigation, the mechanisms underlying the functional properties of smooth muscle are poorly understood. This can be attributed, at least in part, to a lack of knowledge about the structure and organization of the contractile apparatus inside the muscle cell. Recent observations of the plasticity of smooth muscle and of morphometry of the cell have provided enough information for us to propose a quantitative, although highly simplified, model for the geometric arrangement of contractile units and their collective kinematic functions in smooth muscle, particularly airway smooth muscle. We propose that, to a considerable extent, contractile machinery restructures upon activation of the muscle and adapts to cell geometry at the time of activation. We assume that, under steady-state conditions, the geometric arrangement of contractile units and the filaments within these units determines the kinematic characteristics of the muscle. The model successfully predicts the results of experiments on airway smooth muscle plasticity relating to maximal force generation, maximal velocity of shortening, and the variation of compliance with adapted length. The model is also concordant with morphometric observations that show an increase in myosin filament density when muscle is adapted to a longer length. The model provides a framework for design of experiments to quantitatively test various aspects of smooth muscle plasticity in terms of geometric arrangement of contractile units and the muscle's mechanical properties.

Fluctuations in intracellular calcium concentration ([Ca2+]i) constitute the main link in excitation-contraction coupling (E-C coupling) in airway smooth muscle cells (ASMC). It has recently been reported that ACh induces asynchronous recurring Ca2+ waves in intact ASMC of murine bronchioles. With the use of a novel technique allowing us to simultaneously measure subcellular [Ca2+]i and force generation in ASMC located within an intact tracheal muscle bundle, we examined a similar pattern of Ca2+ signaling in the trachea. We found that application of ACh resulted in the generation of recurring intracellular Ca2+ waves progressing along the longitudinal axis of the ribbon-shaped intact ASMC. These Ca2+ waves were not synchronized between neighboring cells, and induction of wave-like [Ca2+]i oscillations was temporally associated with development of force by the tracheal muscle bundle. By comparing the concentration dependence of force generation and the parameters characterizing the [Ca2+]i oscillations, we found that the concentration-dependent increase in ACh-induced force development by the tracheal smooth muscle bundle is achieved by differential recruitment of intact ASMC to initiate Ca2+ waves and by enhancement in the frequency of [Ca2+]i oscillations and elevation of interspike [Ca2+]i once the cells are recruited. Our findings demonstrate that asynchronous recurring Ca2+ waves underlie E-C coupling in ACh-induced contraction of the intact tracheal smooth muscle bundle. Furthermore, in contrast to what was reported in enzymatically dissociated ASMC, Ca2+ influx through the L-type voltage-gated Ca2+ channel was not an obligatory requirement for the generation of [Ca2+]i oscillations and development of force in ACh-stimulated intact ASMC.

There is an abundance of ultrastructural data in the literature on vascular, visceral, and other smooth muscles; such data on airway smooth muscle, however, are conspicuously missing. Here we present a series of electron micrographs depicting contractile and cytoskeletal elements as well as organelles in porcine trachealis. Myosin thick filaments are present in the relaxed muscle; thick filament density increases substantially when the muscle is activated. Actin thin filaments are present in large excess over the thick filaments; the thin/thick filament ratio is about 31/1 in the relaxed state; this ratio is reduced to about 22/1 when the muscle is activated. The sarcoplasmic reticulum is often found associated with caveolae and mitochondria. Cells within a bundle are well connected by intermediate and gap junctions. The results demonstrate that quantitative morphological analysis of ultrastructure of airway smooth muscle fixed under different functional states is possible and will be essential in elucidating the structural basis of adaptation and contraction of the muscle.

The physically dynamic environment of the lung constantly modulates the mechanical properties of airway smooth muscle. In vitro experiments have shown that contractility of the muscle is compromised by oscillatory strains, perhaps through disruption of cross-bridge interaction and organization of the contractile filaments. To understand the mechanism by which oscillation affects contractility, functional changes of the muscle in terms of force-velocity relationship were assessed before and after imposition of length oscillation in both relaxed and activated states. The oscillation protocol was designed to reduce isometric force by 15–20%, followed by measurement of force-velocity properties. Maximal velocity and power changed by +8 and −14%, respectively, after oscillation applied in the relaxed state and changed by −15 and −25%, respectively, after oscillation applied during contraction. A simple model of reduced activation could not account for the results; neither could the results be explained satisfactorily by the current cross-bridge theory of contraction. The results, however, could be explained if the possibility of reorganization of the contractile filaments due to oscillatory strains was considered.

Vascular smooth muscle shows both plasticity and heterogeneity with respect to Ca2+signaling. Physiological perturbations in cytoplasmic Ca2+concentration ([Ca2+]i) may take the form of a uniform maintained rise, a transient uniform [Ca2+]ielevation, a transient localized rise in [Ca2+]i(also known as spark and puff), a transient propagated wave of localized [Ca2+]ielevation (Ca2+wave), recurring asynchronous Ca2+waves, or recurring synchronized Ca2+waves dependent on the type of blood vessel and the nature of stimulation. In this overview, evidence is presented which demonstrates that interactions of ion transporters located in the membranes of the cell, sarcoplasmic reticulum, and mitochondria form the basis of this plasticity of Ca2+signaling. We focus in particular on how the junctional complexes of plasmalemma and superficial sarcoplasmic reticulum, through the generation of local cytoplasmic Ca2+gradients, maintain [Ca2+]ioscillations, couple these to either contraction or relaxation, and promote Ca2+cycling during homeostasis.

Myosin thick filaments have been shown to be structurally labile in intact smooth muscles. Although the mechanism of thick filament assembly/disassembly for purified myosins in solution has been well described, regulation of thick filament formation in intact muscle is still poorly understood. The present study investigates the effect of resting calcium level on thick filament maintenance in intact airway smooth muscle and on thick filament formation during activation. Cross-sectional density of the thick filaments measured electron microscopically showed that the density increased substantially (144%) when the muscle was activated. The abundance of filamentous myosins in relaxed muscle was calcium sensitive; in the absence of calcium (with EGTA), the filament density deceased by 35%. Length oscillation imposed on the muscle under zero-calcium conditions produced no further reduction in the density. Isometric force and filament density recovered fully after reincubation of the muscle in normal physiological saline. The results suggest that in airway smooth muscle, filamentous myosins exist in equilibrium with monomeric myosins; muscle activation favors filament formation, and the resting calcium level is crucial for preservation of the filaments in the relaxed state.

Donor T cell-derived IL-17A can mediate late immunopathology in graft-versus-host disease (GVHD), however protective roles remain unclear. Using multiple cytokine and cytokine receptor subunit knockout mice we demonstrate that stem cell transplant (SCT) recipients lacking the ability to generate or signal IL-17 develop intestinal hyper-acute GVHD. This protective effect is restricted to the molecular interaction of IL-17A and/or IL-17F with the IL-17RA/C receptor. The protection from GVHD afforded by IL-17A required secretion from, and signaling in, both hematopoietic and non-hematopoietic host tissue. Given the intestinal-specificity of the disease in these animals, we hypothesized a microbiome contribution. Cohousing of WT with IL-17RA and IL-17RC deficient mice, dramatically enhanced the susceptibility of WT mice to acute GVHD. Furthermore, the gut microbiome of WT mice shifted towards that of the IL-17RA/C mice during cohousing prior to transplant, confirming that IL-17-sensitive gut microbiota controls susceptibility to acute GVHD. Finally, induced IL-17A deletion peri-transplant also enhanced acute GVHD, consistent with an additional protective role for this cytokine independent of effects on dysbiosis. Importantly, this implies that blocking IL-17 in a clinical trial could have adverse effects via dysbiosis, particularly in the early post-transplant setting and raises caution about their potential effects if used long term.

Deep inspirations (DIs) taken before an inhaled challenge with a spasmogen limit airway responsiveness in nonasthmatic subjects. This phenomenon is called bronchoprotection and is severely impaired in asthmatic subjects. The ability of DIs to prevent a decrease in forced expiratory volume in 1 s (FEV1) was initially attributed to inhibition of airway narrowing. However, DIs taken before methacholine challenge limit airway responsiveness only when a test of lung function requiring a DI is used (FEV1). Therefore, it has been suggested that prior DIs enhance the compliance of the airways or airway smooth muscle (ASM). This would increase the strain the airway wall undergoes during the subsequent DI, which is part of the FEV1 maneuver. To investigate this phenomenon, we used ovine tracheal smooth muscle strips that were subjected to shortening elicited by acetylcholine with or without prior strain mimicking two DIs. The compliance of the shortened strip was then measured in response to a stress mimicking one DI. Our results show that the presence of “DIs” before acetylcholine-induced shortening resulted in 11% greater relengthening in response to the third DI, compared with the prior DIs. This effect, although small, is shown to be potentially important for the reopening of closed airways. The effect of prior DIs was abolished by the adaptation of ASM to either shorter or longer lengths or to a low baseline tone. These results suggest that DIs confer bronchoprotection because they increase the compliance of ASM, which, consequently, promotes greater strain from subsequent DI and fosters the reopening of closed airways.