Airway Smooth Muscle Responsiveness Research Articles

Testosterone Enhances KV Currents and Airway Smooth Muscle Relaxation Induced by ATP and UTP through P2Y4 Receptors and Adenylyl Cyclase Pathway.

Numerous studies suggest the involvement of adenosine-5'-triphosphate (ATP) and similar nucleotides in the pathophysiology of asthma. Androgens, such as testosterone (TES), are proposed to alleviate asthma symptoms in young men. ATP and uridine-5'-triphosphate (UTP) relax the airway smooth muscle (ASM) via purinergic P2Y2 and P2Y4 receptors and K+ channel opening. We previously demonstrated that TES increased the expression of voltage-dependent K+ (KV) channels in ASM. This study investigates how TES may potentiate ASM relaxation induced by ATP and UTP. Tracheal tissues treated with or without TES (control group) from young male guinea pigs were used. In organ baths, tracheas exposed to TES (40 nM for 48 h) showed enhanced ATP- and UTP-evoked relaxation. Tetraethylammonium, a K+ channel blocker, annulled this effect. Patch-clamp experiments in tracheal myocytes showed that TES also increased ATP- and UTP-induced K+ currents, and this effect was abolished with flutamide (an androgen receptor antagonist). KV channels were involved in this phenomenon, which was demonstrated by inhibition with 4-aminopyridine. RB2 (an antagonist of almost all P2Y receptors except for P2Y2), as well as N-ethylmaleimide and SQ 22,536 (inhibitors of G proteins and adenylyl cyclase, respectively), attenuated the enhancement of the K+ currents induced by TES. Immunofluorescence and immunohistochemistry studies revealed that TES did not modify the expression of P2Y4 receptors or COX-1 and COX-2, while we have demonstrated that this androgen augmented the expression of KV1.2 and KV1.5 channels in ASM. Thus, TES leads to the upregulation of P2Y4 signaling and KV channels in guinea pig ASM, enhancing ATP and UTP relaxation responses, which likely limits the severity of bronchospasm in young males.

The gut metagenome harbors metabolic and antibiotic resistance signatures of moderate-to-severe asthma.

Asthma is a common allergic airway disease that has been associated with the development of the human microbiome early in life. Both the composition and function of the infant gut microbiota have been linked to asthma risk, but functional alterations in the gut microbiota of older patients with established asthma remain an important knowledge gap. Here, we performed whole metagenomic shotgun sequencing of 95 stool samples from a cross-sectional cohort of 59 healthy and 36 subjects with moderate-to-severe asthma to characterize the metagenomes of gut microbiota in adults and children 6 years and older. Mapping of functional orthologs revealed that asthma contributes to 2.9% of the variation in metagenomic content even when accounting for other important clinical demographics. Differential abundance analysis showed an enrichment of long-chain fatty acid (LCFA) metabolism pathways, which have been previously implicated in airway smooth muscle and immune responses in asthma. We also observed increased richness of antibiotic resistance genes (ARGs) in people with asthma. Several differentially abundant ARGs in the asthma cohort encode resistance to macrolide antibiotics, which are often prescribed to patients with asthma. Lastly, we found that ARG and virulence factor (VF) richness in the microbiome were correlated in both cohorts. ARG and VF pairs co-occurred in both cohorts suggesting that virulence and antibiotic resistance traits are coselected and maintained in the fecal microbiota of people with asthma. Overall, our results show functional alterations via LCFA biosynthetic genes and increases in antibiotic resistance genes in the gut microbiota of subjects with moderate-to-severe asthma and could have implications for asthma management and treatment.

High-fat Western diet alters crystalline silica-induced airway epithelium ion transport but not airway smooth muscle reactivity

ObjectivesSilicosis is an irreversible occupational lung disease resulting from crystalline silica inhalation. Previously, we discovered that Western diet (HFWD)-consumption increases susceptibility to silica-induced pulmonary inflammation and fibrosis. This study investigated the potential of HFWD to alter silica-induced effects on airway epithelial ion transport and smooth muscle reactivity.MethodsSix-week-old male F344 rats were fed a HFWD or standard rat chow (STD) and exposed to silica (Min-U-Sil 5®, 15 mg/m3, 6 h/day, 5 days/week, for 39 d) or filtered air. Experimental endpoints were measured at 0, 4, and 8 weeks post-exposure. Transepithelial potential difference (Vt), short-circuit current (ISC) and transepithelial resistance (Rt) were measured in tracheal segments and ion transport inhibitors [amiloride, Na+ channel blocker; NPPB; Clˉ channel blocker; ouabain, Na+, K+-pump blocker] identified changes in ion transport pathways. Changes in airway smooth muscle reactivity to methacholine (MCh) were investigated in the isolated perfused trachea preparation.ResultsSilica reduced basal ISC at 4 weeks and HFWD reduced the ISC response to amiloride at 0 week compared to air control. HFWD + silica exposure induced changes in ion transport 0 and 4 weeks after treatment compared to silica or HFWD treatments alone. No effects on airway smooth muscle reactivity to MCh were observed.

IgE-dependent human basophil responses are inversely associated with the sarcoplasmic reticulum Ca2+-ATPase (SERCA).

Basophils crucially contribute to allergies and other Th2-driven diseases by rapidly releasing inflammatory and immunomodulatory mediators following high-affinity IgE-receptor crosslinking. Although these basophil-mediated responses depend on sensitization with antigen-specific IgE, this does not necessarily predict clinical symptom severity. It is thought that the balance of early stimulatory (e.g. SYK) and inhibitory (e.g. SHIP-1) intracellular signals are associated with basophil responsiveness, which is also critically dependent on calcium mobilization. Previous studies suggest that the sarcoplasmic reticulum Ca2+-ATPase (SERCA2), which regulates cytosolic calcium levels, may be inversely associated with airway smooth muscle reactivity in asthma. Since basophils are implicated in asthma severity, our aims were to address whether SERCA2 is implicated in human basophil responses, especially following IgE-mediated activation. Human basophils were obtained from buffy coats, following research ethics approval, and further purified by immunomagnetic cell sorting. Expressions of SERCA2, and other isoforms, were determined by Western blotting in parallel to measuring IgE-dependent histamine releases from the same donors. The effects of a SERCA-activator and inhibitor were also assessed on their abilities to modulate basophil histamine release. We observed an inverse correlation between basophil responsiveness to IgE-dependent stimulation and SERCA2 expression. Thapsigargin, a highly-specific SERCA inhibitor, stimulated basophil histamine release and potentiated IgE-dependent secretion of the amine. Conversely, disulfiram, a SERCA activator, inhibited IgE-dependent basophil activation. The results obtained from this exploratory study indicate that SERCA2 may be an additional regulator of basophil reactivity alongside early excitatory or inhibitory signal transduction pathways.

CEBPD modulates the airway smooth muscle transcriptomic response to glucocorticoids

BackgroundCCAAT/Enhancer Binding Protein D (CEBPD), a pleiotropic glucocorticoid-responsive transcription factor, modulates inflammatory responses. Of relevance to asthma, expression of CEBPD in airway smooth muscle (ASM) increases with glucocorticoid exposure. We sought to characterize CEBPD-mediated transcriptomic responses to glucocorticoid exposure in ASM by measuring changes observed after knockdown of CEBPD and its impact on asthma-related ASM function.MethodsPrimary ASM cells derived from four donors were transfected with CEBPD or non-targeting (NT) siRNA and exposed to vehicle control, budesonide (100 nM, 18 h), TNFα (10 ng/ml, 18 h), or both budesonide and TNFα. Subsequently, RNA-Seq was used to measure gene expression levels, and pairwise differential expression results were obtained for exposures versus vehicle and knockdown versus control conditions. Weighted gene co-expression analysis was performed to identify groups of genes with similar expression patterns across the various experimental conditions (i.e., CEBPD knockdown status, exposures).ResultsCEBPD knockdown altered expression of 3037 genes under at least one exposure (q-value < 0.05). Co-expression analysis identified sets of 197, 152 and 290 genes that were correlated with CEBPD knockdown status, TNFα exposure status, and both, respectively. JAK-STAT signaling pathway genes, including IL6R and SOCS3, were among those influenced by both TNFα and CEBPD knockdown. Immunoblot assays revealed that budesonide-induced IL-6R protein expression and augmented IL-6-induced STAT3 phosphorylation levels were attenuated by CEBPD knockdown in ASM.ConclusionsCEBPD modulates glucocorticoid responses in ASM, in part via modulation of IL-6 receptor signaling.

Interactions of a high‐fat Western diet and crystalline silica inhalation on airway epithelial ion transport and airway reactivity

Silicosis, an irreversible occupational lung disease caused by crystalline silica inhalation, is a serious health risk for silica‐exposed workers. NIOSH reports that Appalachian coal miners have higher rates of obesity and metabolic dysfunction (MetDys) compared to the general U.S. adult population. MetDys is a risk factor for lung function impairment, pulmonary hypertension, and asthma. Consumption of a high‐fat Western diet (HFWD) is associated with obesity and MetDys. In this study, we investigated the effects of, and determine interactions between, HFWD‐consumption and silica‐exposure on airway epithelial ion transport and smooth muscle reactivity in the F344 rat. Six‐week‐old male F344 rats were fed either a HFWD [40.6% fat (19.5% lard), 40.6% total carbohydrate (20% sucrose), 14.8 % protein] or standard rat chow (STD) [6.2 % fat, 44.2 % carbohydrate (grain sources), 18.6 % protein] for the duration of the study. Following 16 weeks of diet‐consumption, inhalation exposure to respirable crystalline silica (Min‐U‐Sil 5®, 15 mg/m3, 6 h/d, 5 d/wk, for 39 d) or filtered air began, with endpoint experiments conducted at 0, 4, and 8 wk post‐exposure. Airway epithelial ion transport maintains airway surface liquid osmolarity and depth required for effective cilia motility and clearance of xenogens. Changes in ion transport were determined ex vivo by measurement of transepithelial potential difference (Vt), short‐circuit current (ISC) and transepithelial resistance (Rt) in rat tracheal segments mounted in Ussing chambers, and administered the ion transport inhibitors amiloride (Na+ channel blocker; apical), 5‐nitro‐2‐(3‐phenylpropylamino) benzoic acid (NPPB; Clˉ channel blocker; apical), and ouabain (Na+, K+‐pump blocker; basolateral). Airway hyperresponsiveness is associated with obesity and pulmonary diseases such as asthma and COPD; thus, the isolated perfused trachea apparatus was employed to ascertain whether silica or HFWD altered airway smooth muscle reactivity to serosal or mucosal applied methacholine (MCh). HFWD‐consumption had no effect on basal Vt. Silica exposure increased Na+ transport at 0 wk, decreased basal ISC at 4 wk, and reduced Cl‐ channel and Na+, K+‐pump activity at both 4 wk and 8 wk compared to STD+AIR controls. HFWD‐consumption caused a reduction in Cl‐ transport and Na+, K+‐pump activity at 4 wk, while increasing Rt in response to ouabain at 0 wk and NPPB at 8 wk compared to STD+AIR. HFWD+SIL increased basal ISC at 0 and 4 wk, caused reduction in Cl‐ transport and Na+, K+‐pump activity at 4 wk, while reducing Rtin response to ouabain at 4 wk compared to STD+SIL. No significant changes in tracheal reactivity to MCh were observed. In conclusion, HFWD and silica altered epithelial ion transport, but the combined effects of HFWD+SIL were not synergistic.

Measurement of Airway Hyperresponsiveness in Mice.

Asthma has been the most prevalent chronic respiratory disease (Mensah et al. J Allergy Clin Immunol 142:744-748, 2018). To explore pathogenic mechanism or new treatments of asthma, mice have been utilized to model the disease. Eosinophilic airway inflammation, allergen specific-IgE, and airway hyperresponsiveness have been characteristic features of allergic asthma (Drake et al. Pulm Ther 5:103-115, 2019). In mouse models, airway hyperresponsiveness to inhaled broncho-constrictor agents such as methacholine chloride (MCh) has been a key disease marker (Alessandrini et al. Front Immunol 11:575936, 2020). A variety of systems to assess airway reactivity in mice are currently available. Here, three distinct systems are described as these have been used in many publications. In the first system, an invasive system in which mice are anesthetized and intubated followed by mechanical ventilation, lung resistance (R), dynamic compliance (C), and other respiratory parameters with MCh challenge are measured. In the second system, a noninvasive system equipped with a chamber in which mice can move freely and spontaneously breathe, changes in airways with MCh challenge are measured as enhanced pause (Penh) values. In the third system, in vitro airway smooth muscle (ASM) reactivity is monitored in an extracted mouse tracheal duct with a cholinergic agonist challenge or electrical stimulation. Each of these systems has unique features, benefits, or disadvantages.

Dynamic cytosolic Ca2+ and force responses to muscarinic stimulation in airway smooth muscle.

During agonist stimulation of airway smooth muscle (ASM), agonists such as ACh induce a transient increase in cytosolic Ca2+ concentration ([Ca2+]cyt), which leads to a contractile response [excitation-contraction (E-C) coupling]. Previously, the sensitivity of the contractile response of ASM to elevated [Ca2+]cyt (Ca2+ sensitivity) was assessed as the ratio of maximum force to maximum [Ca2+]cyt. However, this static assessment of Ca2+ sensitivity overlooks the dynamic nature of E-C coupling in ASM. In this study, we simultaneously measured [Ca2+]cyt and isometric force responses to three concentrations of ACh (1, 2.6, and 10 μM). Both maximum [Ca2+]cyt and maximum force responses were ACh concentration dependent, but force increased disproportionately, thereby increasing static Ca2+ sensitivity. The dynamic properties of E-C coupling were assessed in several ways. The temporal delay between the onset of ACh-induced [Ca2+]cyt and onset force responses was not affected by ACh concentration. The rates of rise of the ACh-induced [Ca2+]cyt and force responses increased with increasing ACh concentration. The integral of the phase-loop plot of [Ca2+]cyt and force from onset to steady state also increased with increasing ACh concentration, whereas the rate of relaxation remained unchanged. Although these results suggest an ACh concentration-dependent increase in the rate of cross-bridge recruitment and in the rate of rise of [Ca2+]cyt, the extent of regulatory myosin light-chain (rMLC20) phosphorylation was not dependent on ACh concentration. We conclude that the dynamic properties of [Ca2+]cyt and force responses in ASM are dependent on ACh concentration but reflect more than changes in the extent of rMLC20 phosphorylation.

5-HT2A, 5-HT1B/D, 5HT3 and 5-HT7 receptors as mediators of serotonin-induced direct contractile response of bovine airway smooth muscle.

Background: Serotonin (5-hydroxytryptamine; 5-HT) performs a variety of functions in the body including the modulation of muscle tone in respiratory airways. Several studies indicate a possible role of 5-HT in the pathophysiology of bronchial hyperresponsiveness. However, the receptors and the molecular mechanisms by which 5-HT acts on airway smooth muscle (ASM) continue to be controversial. Most of the evidence suggests the participation of different subtypes of receptors in an indirect response. This study supports the proposal that 5-HT directly contracts ASM and characterizes pharmacologically the subtypes of serotonergic receptors involved. The characterization was carried out by using selective antagonists in an organ bath model allowing study of the smooth muscle of segments of bovine trachea. Results: The results obtained show that 5-HT2A receptors are the main mediators of the direct contractile response of bovine ASM, with the cooperation of the 5-HT7, 5-HT3 and 5-HT1B/D receptors. Also, it was observed that the muscle response to serotonin is developed more slowly and to a lesser extent in comparison with the response to cholinergic stimulation. Conclusion: Overall, the receptors that mediate the direct serotonergic contraction of the smooth muscle of the bovine trachea are 5-HT2A, 5-HT7, 5-HT3 and 5-HT1B/D receptors.

DM-induced Hypermethylation of IR and IGF1R attenuates mast cell activation and airway responsiveness in rats.

Diabetes has been reported to modulate the airway smooth muscle reactivity and lead to attenuation of allergic inflammatory response in the lungs. In this study, we aimed to study the effect of insulin on cell activation and airway responsiveness in patients with diabetes mellitus (DM). The airway contraction in rat model groups including a non‐DM group, a non‐DM+INDUCTION group, a DM+INDUCTION group and a DM+INDUCTION+INSULIN group was measured to observe the effect of insulin on airway responsiveness. Radioenzymatic assay was conducted to measure the levels of histamine, and ELISA assay was conducted to measure bronchial levels of interleukin (IL)‐1b, tumour necrosis factor (TNF)‐a, cytokine‐induced neutrophil chemoattractant (CINC)‐1, P‐selectin and β‐hexosaminidase. The tension in the main and intrapulmonary bronchi of DM+INDUCTION rats was lower than that of the non‐DM+INDUCTION rats, whereas the treatment of insulin partly restored the normal airway responsiveness to OA in DM rats. The release of histamine was remarkably suppressed in DM+INDUCTION rats but was recovered by the insulin treatment. Also, OA significantly increased the levels of IL‐1b, TNF‐a, CINC‐1 and P‐selectin in non‐DM rats, whereas insulin treatment in DM+INDUCTION rats partly restored the normal levels of IL‐1b, TNF‐a, CINC‐1 and P‐selectin in DM rats. Moreover, the expression of IR and IGF1R was evidently suppressed in DM rats, with the methylation of both IR and IGF1R promoters was aggravated in DM rats. Therefore, we demonstrated that DM‐induced hypermethylation inhibited mast cell activation and airway responsiveness, which could be reversed by insulin treatment.

Validation of A 3D Bioprinted Model of Airway Smooth Muscle – A Novel Tool to Study the Effects of Airway Stiffening in Asthma

BackgroundAsthma is a chronic obstructive airway disease characterized by airway wall remodeling and aberrant contraction of airway smooth muscle (ASM), which each contribute to clinical symptoms by restricting airflow. However, the precise link between airway mechanics and ASM contractility remains elusive, because ASM cell biology is routinely studied in 2D models that inadequately replicate the complex in‐vivo 3D microenvironment of healthy or diseased airways. We used a technique called 3D bioprinting to develop an experimental model of ASM in which we could characterize the response of ASM to altered mechanical loads in a realistic 3D structure.MethodsHuman ASM were resuspended at 2.5×107 cells/mL in a bio‐ink consisting of RGD‐coupled‐alginate (0.375% w/v), fibrinogen (5 mg/mL) and collagen‐I (1 mg/mL). Cells were then bioprinted using an Aspect Biosystems RX‐1 Bioprinter, as a ring‐shaped bundle of muscle constrained within a stiffness‐modifiable acellular alginate support (0.75–1.25% w/v). After bioprinting, constructs were thrombin treated (1.25 U/mL, 30 min) to initiate fibrin polymerization and maintained in standard ASM culture conditions. Cell health, viability and morphology were assessed with LDH assays, Hoechst/propidium‐iodide and filamentous‐actin staining respectively. Expression of ASM relevant genes was measured by qPCR. Reduction in lumen area representing contractile tone was tracked by live cell microscopy.ResultsASM bioprinted without acellular supports shortened excessively and rapidly lost structural integrity. Comparatively, including supports provided a mechanical load that controlled baseline shortening; cells supported by 0.75%, 1% and 1.25% alginate exhibited &gt;45%, &gt;25% and &lt;15% lumen reduction respectively. Constructs bioprinted in 1% supports exhibited high cell viability (&gt;80%) for up to 14 days in culture. Histology 4 days after printing revealed development of a highly organized tissue with evidence of cell‐cell contacts and alignment of actin fibres. Nuclei were evenly distributed in all axes indicating formation of a true 3D structure. Compared with 2D, cells in 3D had a higher mRNA abundance of myosin heavy chain, but vimentin and matrix metalloproteinase‐3 were lower (all p&lt;0.05). Importantly, administration of acetylcholine resulted in muscle contraction, while cytochalasin D caused a dramatic decrease in tone for all tissues with structural supports.ConclusionWe have validated a novel tool for studying ASM biomechanics. ASM displayed varying levels of baseline shortening to different mechanical loads, supporting that mechanical cues may have profound effects on cellular function. Further, higher abundance of contractile genes in 3D demonstrates adoption of a physiologically relevant phenotype. Moving forward, contraction, relaxation and calcium dynamics will be quantified across a range of stiffnesses. These data will help unmask mechanisms by which airway mechanics regulate ASM contractility, with important clinical relevance to asthma.Support or Funding InformationFunding: NSERC Discovery Grant (ARW), Research Manitoba Studentship (JO, SS)

Crucial role for lung iron level and regulation in the pathogenesis and severity of asthma.

Accumulating evidence highlights links between iron regulation and respiratory disease. Here, we assessed the relationship between iron levels and regulatory responses in clinical and experimental asthma.We show that cell-free iron levels are reduced in the bronchoalveolar lavage (BAL) supernatant of severe or mild-moderate asthma patients and correlate with lower forced expiratory volume in 1 s (FEV1). Conversely, iron-loaded cell numbers were increased in BAL in these patients and with lower FEV1/forced vital capacity (FVC) ratio. The airway tissue expression of the iron sequestration molecules divalent metal transporter 1 (DMT1) and transferrin receptor 1 (TFR1) are increased in asthma, with TFR1 expression correlating with reduced lung function and increased Type-2 (T2) inflammatory responses in the airways. Furthermore, pulmonary iron levels are increased in a house dust mite (HDM)-induced model of experimental asthma in association with augmented Tfr1 expression in airway tissue, similar to human disease. We show that macrophages are the predominant source of increased Tfr1 and Tfr1+ macrophages have increased Il13 expression. We also show that increased iron levels induce increased pro-inflammatory cytokine and/or extracellular matrix (ECM) responses in human airway smooth muscle (ASM) cells and fibroblasts ex vivo and induce key features of asthma in vivo, including airway hyper-responsiveness (AHR) and fibrosis, and T2 inflammatory responses.Together these complementary clinical and experimental data highlight the importance of altered pulmonary iron levels and regulation in asthma, and the need for a greater focus on the role and potential therapeutic targeting of iron in the pathogenesis and severity of disease.

Design for a multicenter, randomized, sham-controlled study to evaluate safety and efficacy after treatment with the Nuvaira\xae lung denervation system in subjects with chronic obstructive pulmonary disease (AIRFLOW-3)

BackgroundTargeted lung denervation (TLD) is a bronchoscopically delivered ablation therapy that selectively interrupts pulmonary parasympathetic nerve signaling. The procedure has the potential to alter airway smooth muscle tone and reactivity, decrease mucous secretion, and reduce airway inflammation and reflex airway hyperresponsiveness. Secondary outcome analysis of a previous randomized, sham-controlled trial showed a reduction in moderate-to-severe exacerbations in patients with COPD after TLD treatment. A pivotal trial, AIRFLOW-3 has been designed to evaluate the safety and efficacy of TLD combined with optimal medical therapy to reduce moderate or severe exacerbations throughout 1 year, compared with optimal medical therapy alone.MethodsThe study design is a multicenter, randomized, full sham bronchoscopy controlled, double-blind trial that will enroll 400 patients (1:1 randomization). Key inclusion criteria are FEV1/FVC < 0.7, FEV1 30 to 60% of predicted, post-bronchodilator, ≥ 2 moderate or 1 severe COPD exacerbations in the prior year, and COPD assessment test (CAT) ≥ 10. Primary objective will be the comparison of moderate or severe COPD exacerbations through 12 months of TLD therapy with optimal medical therapy versus optimal medical therapy alone. The sham group will be allowed to cross over at 1 year. Patients will be followed for up to 5 years.DiscussionThe multicenter, randomized, full sham bronchoscopy controlled, double-blind AIRFLOW-3 trial will evaluate the efficacy of TLD to reduce moderate or severe COPD exacerbations beyond optimal medical therapy alone. The target population are patients with COPD, who suffer persistent symptoms and exacerbations despite optimal treatment, defining an unmet medical need requiring novel therapeutic solutions. This trial is registered at clinicaltrials.gov: NCT03639051.

The effect of pulmonary surfactant on the airway smooth muscle after lipopolysaccharide exposure and its mechanisms.

Pulmonary surfactant has a relaxing effect on the airway smooth muscle (ASM), which suggests its role in the pathogenesis of respiratory diseases associated with hyperreactivity of the ASM, such as asthma and chronic obstructive pulmonary disease (COPD). The ASM tone may be directly or indirectly modified by bacterial wall component lipopolysaccharide (LPS). This study elucidated the effect of LPS on the ASM reactivity and the role of surfactant in this interaction. The experiments were performed using ASM of adult guinea pigs by in vitro method of tissue organ bath (ASM unexposed-healthy or exposed to LPS under in vitro conditions) and ASM of animals intraperitoneally injected with LPS at a dose 1 mg/kg of b.w. once a day during 4-day period. Variable response of LPS was controlled by cyclooxygenase inhibitor indomethacin and relaxing effect of exogenous surfactant was studied using leukotriene and histamine receptor antagonists. The exogenous surfactant has relaxing effect on the ASM, but does not reverse LPS-induced smooth muscle contraction. The results further indicate participation of prostanoids and potential involvement of leukotriene and histamine H1 receptors in the airway smooth muscle contraction during LPS exposure.

The Effect of Chronic Hypoxia on Airway Remodeling in Murine Model of Asthma

Background and Hypothesis: Chronic hypoxia during growth and development results in adaptive responses to increase oxygen transport to tissues, such as increasing lung volume and lung diffusion. Asthma is characterized by airway hyper-reactivity, which can result from increased airway smooth muscle (ASM) and airway remodeling following airway inflammation. Chronic hypoxia may attenuate airway inflammation, airway reactivity and airway remodeling in patients with asthma. Measured by in vivo airway resistance in response to 50 mg/ml Ach, our preliminary data suggests that rats conceived and raised under chronic hypoxic conditions (15% O2) exhibit lower airway reactivity, compared to room air controls (20% O2) at baseline (0.397, 0.789 respectively) and following Ova sensitization/challenge (0.696, 1.159 respectively). We hypothesized lower airway reactivity was associated with less ASM in response to hypoxic conditions. &#x0D; Methods: Sprague Dawley rats were conceived, raised and evaluated in 4 groups: hypoxia/PBS challenge (HA-PBS); hypoxia/Ova challenge (HA-Ova); normoxia/PBS challenge (RA-PBS); normoxia/Ova challenge (RA-Ova). The lung tissue (4 animals/group) was fixed, sectioned, and ASM was quantified by immunohistochemistry using imageJ software.&#x0D; Results: Measured ASM (mm2) from each group was log transformed to normalize data acquired from various lumen sizes and the least square means of each group were calculated. Airways from HA-PBS animals (N= 59) tended to have the lowest ASM, and ASM progressively increased in RA-PBS (N=41), HA-Ova (N=58) and RA-Ova groups (N=38 airways) [-6.41, -6.3, -6.26, -6.17]. There was an excellent correlation between ASM and airway resistance; more ASM was associated with greater airway reactivity (Pearson correlation of 0.99).&#x0D; Conclusions and Potential Impact: Chronic hypoxia may suppress ASM development, as well as suppress the increase in ASM associated with atopic inflammation. Understanding the mechanisms that inhibit ASM growth under conditions of chronic hypoxia may provide insight into new therapies for asthma.

Airway Smooth Muscle-Specific Transcriptomic Signatures of Glucocorticoid Exposure.

Glucocorticoids, commonly used asthma controller medications, decrease symptoms in most patients, but some remain symptomatic despite high-dose treatment. The physiological basis underlying the glucocorticoid response, especially in asthma patients with severe, refractory disease, is not fully understood. We sought to identify differences between the transcriptomic response of airway smooth muscle (ASM) cells derived from donors with fatal asthma and donors without asthma to glucocorticoid exposure and to compare ASM-specific changes with those observed in other cell types. In cells derived from nine donors with fatal asthma and eight donors without asthma, RNA sequencing was used to measure ASM transcriptome changes after exposure to budesonide (100 nM 24 h) or control vehicle (DMSO). Differential expression results were obtained for this dataset, as well as 13 publicly available glucocorticoid-response transcriptomic datasets corresponding to seven cell types. Specific genes were differentially expressed in response to glucocorticoid exposure (7,835 and 6,957 in ASM cells derived from donors with fatal asthma and donors without asthma, respectively; adjusted P value < 0.05). Transcriptomic changes in response to glucocorticoid exposure were similar in ASM derived from donors with fatal asthma and donors without asthma, with enriched ontological pathways that included cytokine- and chemokine-related categories. A comparison of glucocorticoid-induced changes in the nonasthma ASM transcriptome with those observed in six other cell types showed that ASM has a distinct glucocorticoid-response signature that is also present in ASM cells from donors with fatal asthma.

Ventilasi dan Perfusi, serta Hubungan antara Ventilasi dan Perfusi

Lung is a place for gas exchange where ventilation and perfusion occurs. Ventilation is the first step where sequential process of inhalation and exhalation take place. Meanwhile perfusion as the other step facilitates the gas exchange and tissue supply need. Blood flows through the lungs are equals as the amount of cardiac output where the factors that control cardiac output are mainly peripheral factors, also control pulmonary blood flow. In general condition, pulmonary blood vessels act as a passive tube, which can be increased with the increasing pressure and narrowed the pressure drop. Oxygen absorption level from lungs into bloodstream is a critical determinant for functional capacity, and an important factor wheter in normal conditions (including exercise) or even in illness state. Lung diffusion capacity is influenced by several geometric and functional factors. Gravitation influence systematic gradient in ventilation and perfusion distribution. Ventilation and blood flow variations at horizontal level also occur due to intrinsic anatomic variations and vascular geometry, as well as the differences in airway and vascular smooth muscle response which modifies the distribution. The change of integrity intrapleural chamber, hydrostatic pressure and osmotic imbalance, malfunction of surfactants, other intrinsic weakness of the branching system in the form of a progressive airway, and all the things that could potentially damage the structure of the lung can cause ventilation and diffusion dysfunction.

CME Accreditation Page

Meconium aspiration syndrome (MAS) is a major cause of respiratory insufficiency in the term and post-term newborns. There are several pathomechanisms participating in this disorder, particularly the airway obstruction, surfactant dysfunction, inflammation, lung edema, pulmonary vasoconstriction and bronchoconstriction. Inflammatory changes resulting from meconium aspiration cause severe impairment of lung parenchyma and surfactant, and influence the reactivity of both vascular and airway smooth muscle. Therefore, anti-inflammatory drugs may be of benefit in the management of MAS. This article reviews the pharmacological actions and side effects of various anti-inflammatory drugs used up to now in the experimental models of MAS and in the treatment of newborns with meconium aspiration.

Saponins of Dioscorea Nipponicae Inhibits IL-17A-Induced Changes in Biomechanical Behaviors of In Vitro Cultured Human Airway Smooth Muscle Cells.

Airway hyperresponsiveness (AHR) is one of the main pathologic features of bronchial asthma, which is largely attributable to enhanced contractile response of asthmatic airway smooth muscle. Although β2 adrenergic receptor agonists are commonly used to relax airway smooth muscle for treating AHR, there are side effects such as desensitization of long-term use. Therefore, it is desirable to develop alternative relaxant for airway smooth muscle, preferably based on natural products. One potential candidate is the inexpensive and widely available natural herb saponins of Dioscorea nipponicae (SDN), which has recently been reported to suppress the level of inflammatory factor IL-17A in ovalbumin-induced mice, thereby alleviating the inflammation symptoms of asthma. Here, we evaluated the biomechanical effect of SDN on IL-17A-mediated changes of cultured human airway smooth muscle cells (HASMCs) in vitro. The stiffness and traction force of the cells were measured by optical magnetic twisting cytometry (OMTC), and Fourier transform traction microscopy (FTTM), respectively. The cell proliferation was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetry, the cell migration was measured by cell scratch test, and the changes of cell cytoskeleton were assessed by laser confocal microscopy. We found that the stiffness and traction force of HASMCs were enhanced along with the increases of IL-17A concentration and exposure time, and SDN treatment dose-dependently reduced these IL-17A-induced changes in cell mechanical properties. Furthermore, SDN alleviated IL-17A-mediated effects on HASMCs proliferation, migration, and cytoskeleton remodeling. These results demonstrate that SDN could potentially be a novel drug candidate as bronchodilator for treating asthma-associated AHR.

Prestrain and cholinergic receptor-dependent differential recruitment of mechanosensitive energy loss and energy release elements in airway smooth muscle.

We tested the hypothesis that oscillatory airway smooth muscle (ASM) mechanics is governed by mechanosensitive energy loss and energy release elements that can be recruited by prestrain and cholinergic stimulation. We measured mechanical energy loss and mechanical energy release in unstimulated and carbachol-stimulated bovine ASM held at prestrains ranging from 0.3 to 1.0 Lo (reference length) and subjected to sinusoidal length oscillation at 1 hz with oscillatory strain amplitudes ranging from 0.1 to 1.5% Lo. We found that oscillatory ASM mechanics during sinusoidal length oscillation is governed predominantly by one class of nonlinear mechanosensitive energy loss element and one class of nonlinear mechanosensitive energy release element with differential mechanosensitivities to oscillatory strain amplitude. The greater mechanosensitivity of the energy loss element than energy release element may explain the bronchodilatory effect of deep inspiration. Prestrain, an important determinant of ASM responsiveness, differentially increased energy loss and energy release in unstimulated and carbachol-stimulated ASM. Cholinergic stimulation, an important cause of bronchoconstriction and airway inflammation, also differentially increased energy loss and energy release. When prestrain and cholinergic stimulation were combined, we found that prestrain and cholinergic stimulation synergistically increased energy loss and energy release by ASM. The relationship between recruitment of energy loss elements and recruitment of energy release elements was nonlinear, suggesting that energy loss and energy release elements are not coupled in ASM cells. These findings imply that large lung volume and cholinergic ASM activation would synergistically increase mechanical energy expenditure during inspiration and mechanical recoil of ASM during expiration. NEW & NOTEWORTHY We report for the first time that oscillatory airway smooth muscle mechanics is governed predominantly by one class of nonlinear mechanosensitive energy loss element and one class of nonlinear mechanosensitive energy release element with differential mechanosensitivities to oscillatory strain amplitude. Prestrain and cholinergic stimulation synergistically and differentially recruit energy loss and energy release elements. The greater mechanosensitivity of the energy loss element than the energy release element may explain the bronchodilatory effect of deep inspiration.