Elucidating the interaction between stretch and stiffness using an agent-based spring network model of progressive pulmonary fibrosis.

Pulmonary fibrosis is a deadly disease that involves the dysregulation of fibroblasts and myofibroblasts, which are mechanosensitive. Previous computational models have succeeded in modeling stiffness-mediated fibroblasts behaviors; however, these models have neglected to consider stretch-mediated behaviors, especially stretch-sensitive channels and the stretch-mediated release of latent TGF-ß. Here, we develop and explore an agent-based model and spring network model hybrid that is capable of recapitulating both stiffness and stretch. Using the model, we evaluate the role of mechanical signaling in homeostasis and disease progression during self-healing and fibrosis, respectively. We develop the model such that there is a fibrotic threshold near which the network tends towards instability and fibrosis or below which the network tends to heal. The healing response is due to the stretch signal, whereas the fibrotic response occurs when the stiffness signal overpowers the stretch signal, creating a positive feedback loop. We also find that by changing the proportional weights of the stretch and stiffness signals, we observe heterogeneity in pathological network structure similar to that seen in human IPF tissue. The system also shows emergent behavior and bifurcations: whether the network will heal or turn fibrotic depends on the initial network organization of the damage, clearly demonstrating structure's pivotal role in healing or fibrosis of the overall network. In summary, these results strongly suggest that the mechanical signaling present in the lungs combined with network effects contribute to both homeostasis and disease progression.

Multiscale stiffness of human emphysematous precision cut lung slices.

Emphysema is a debilitating disease that remodels the lung leading to reduced tissue stiffness. Thus, understanding emphysema progression requires assessing lung stiffness at both the tissue and alveolar scales. Here, we introduce an approach to determine multiscale tissue stiffness and apply it to precision-cut lung slices (PCLS). First, we established a framework for measuring stiffness of thin, disk-like samples. We then designed a device to verify this concept and validated its measuring capabilities using known samples. Next, we compared healthy and emphysematous human PCLS and found that the latter was 50% softer. Through computational network modeling, we discovered that this reduced macroscopic tissue stiffness was due to both microscopic septal wall remodeling and structural deterioration. Lastly, through protein expression profiling, we identified a wide spectrum of enzymes that can drive septal wall remodeling, which, together with mechanical forces, lead to rupture and structural deterioration of the emphysematous lung parenchyma.

Antagonizing cholecystokinin A receptor in the lung attenuates obesity-induced airway hyperresponsiveness.

Obesity increases asthma prevalence and severity. However, the underlying mechanisms are poorly understood, and consequently, therapeutic options for asthma patients with obesity remain limited. Here we report that cholecystokinin-a metabolic hormone best known for its role in signaling satiation and fat metabolism-is increased in the lungs of obese mice and that pharmacological blockade of cholecystokinin A receptor signaling reduces obesity-associated airway hyperresponsiveness. Activation of cholecystokinin A receptor by the hormone induces contraction of airway smooth muscle cells. In vivo, cholecystokinin level is elevated in the lungs of both genetically and diet-induced obese mice. Importantly, intranasal administration of cholecystokinin A receptor antagonists (proglumide and devazepide) suppresses the airway hyperresponsiveness in the obese mice. Together, our results reveal an unexpected role for cholecystokinin in the lung and support the repurposing of cholecystokinin A receptor antagonists as a potential therapy for asthma patients with obesity.

Lysyl oxidase like 2 is increased in asthma and contributes to asthmatic airway remodelling.

BACKGROUND: Airway smooth muscle (ASM) cells are fundamental to asthma pathogenesis, influencing bronchoconstriction, airway hyperresponsiveness and airway remodelling. The extracellular matrix (ECM) can influence tissue remodelling pathways; however, to date no study has investigated the effect of ASM ECM stiffness and cross-linking on the development of asthmatic airway remodelling. We hypothesised that transforming growth factor-ß (TGF-ß) activation by ASM cells is influenced by ECM in asthma and sought to investigate the mechanisms involved. METHODS: This study combines in vitro and in vivo approaches: human ASM cells were used in vitro to investigate basal TGF-ß activation and expression of ECM cross-linking enzymes. Human bronchial biopsies from asthmatic and nonasthmatic donors were used to confirm lysyl oxidase like 2 (LOXL2) expression in ASM. A chronic ovalbumin (OVA) model of asthma was used to study the effect of LOXL2 inhibition on airway remodelling. RESULTS: We found that asthmatic ASM cells activated more TGF-ß basally than nonasthmatic controls and that diseased cell-derived ECM influences levels of TGF-ß activated. Our data demonstrate that the ECM cross-linking enzyme LOXL2 is increased in asthmatic ASM cells and in bronchial biopsies. Crucially, we show that LOXL2 inhibition reduces ECM stiffness and TGF-ß activation in vitro, and can reduce subepithelial collagen deposition and ASM thickness, two features of airway remodelling, in an OVA mouse model of asthma. CONCLUSION: These data are the first to highlight a role for LOXL2 in the development of asthmatic airway remodelling and suggest that LOXL2 inhibition warrants further investigation as a potential therapy to reduce remodelling of the airways in severe asthma.

Substrate stiffening promotes VEGF-A functions via the PI3K/Akt/mTOR pathway.

While it is now well-established that substrate stiffness regulates vascular endothelial growth factor-A (VEGF-A) mediated signaling and functions, causal mechanisms remain poorly understood. Here, we report an underlying role for the PI3K/Akt/mTOR signaling pathway. This pathway is activated on stiffer substrates, is amplified by VEGF-A stimulation, and correlates with enhanced endothelial cell (EC) proliferation, contraction, pro-angiogenic secretion, and capillary-like tube formation. In the settings of advanced age-related macular degeneration, characterized by EC and retinal pigment epithelial (RPE)-mediated angiogenesis, these data implicate substrate stiffness as a novel causative mechanism and Akt/mTOR inhibition as a novel therapeutic pathway.

CD38 plays an age-related role in cholinergic deregulation of airway smooth muscle contractility.

BACKGROUND: Allergen-induced airway hyperresponsiveness in neonatal mice, but not adult mice, is caused by elevated innervation and consequent cholinergic hyperstimulation of airway smooth muscle (ASM). Whether this inflammation-independent mechanism contributes to ASM hypercontraction in childhood asthma warrants investigation. OBJECTIVE: We aimed to establish the functional connection between cholinergic stimulation and ASM contractility in different human age groups. METHODS: First, we used a neonatal mouse model of asthma to identify age-related mediators of cholinergic deregulation of ASM contractility. Next, we conducted validation and mechanistic studies in primary human ASM cells and precision-cut lung slices from young (<5 years old) and adult (>20 years old) donor lungs. Finally, we evaluated the therapeutic potential of the identified cholinergic signaling mediators using culture models of human ASM hypercontraction. RESULTS: ASM hypercontraction due to cholinergic deregulation in early postnatal life requires CD38. Mechanistically, cholinergic signaling activates the phosphatidylinositol 3-kinase/protein kinase B pathway in immature ASM cells to upregulate CD38 levels, thereby augmenting the Ca2+ response to contractile agonists. Strikingly, this early-life, CD38-mediated ASM hypercontraction is not alleviated by the ß-agonist formoterol. CONCLUSIONS: The acetylcholine-phosphatidylinositol 3-kinase/protein kinase B-CD38 axis is a critical mechanism of airway hyperresponsiveness in early postnatal life. Targeting this axis may provide a tailored treatment for children at high risk for allergic asthma.

Stabilizing breathing pattern using local mechanical vibrations: comparison of deterministic and stochastic stimulations in rodent models of apnea of prematurity.

Mechanical stimulation has been shown to reduce apnea of prematurity (AOP), a major concern in preterm infants. Previous work suggested that the underlying mechanism is stochastic resonance, amplification of a subthreshold signal by stochastic stimulation. We hypothesized that the mechanism behind the reduction of apnea length may not be a solely stochastic phenomenon, and suggest that a purely deterministic, non-random mechanical stimulation could be equally as effective. Mice and rats were anesthetized, tracheostomized, and mechanically ventilated to halt spontaneous breathing. Two miniature motors controlled by a microcontroller were attached around the abdomen. Ventilation was paused, stimulations were applied, and the time to the rodent's first spontaneous breath (T) was measured. Six spectrally different signals were compared to one another and the no-stimulation control in mice. The most successful deterministic stimulation (D) at reducing apnea was then compared to a pseudo-random noise (PRN) signal of comparable amplitude and frequency. CO2%, CO2 stabilization time (Ts), O2 saturation (SpO2%), and T were also measured. D significantly reduced T compared to no stimulation for medium and high amplitudes. PRN also reduced T, without  a difference between D and PRN. Furthermore, both stimulations significantly reduced Ts with no significant differences between the respective stimulations. However, there was no effect of D or PRN on SpO2%. The lack of differences between D and PRN led to an additional series of experiment comparing the same D to a band-limited white noise (WN) signal in young rats. Both D and WN were shown to significantly reduce T, with D showing statistical superiority in reduction of apnea. We further speculate that both deterministic and stochastic mechanical stimulations induce some form of mechanotransduction which is responsible for their efficacy, and our findings suggest that mechanical stimulation may be effective in treating AOP. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13534-021-00203-x.

Cooperativity between ß-agonists and c-Abl inhibitors in regulating airway smooth muscle relaxation.

Current therapeutic approaches to avoid or reverse bronchoconstriction rely primarily on ß2 adrenoceptor agonists (ß-agonists) that regulate pharmacomechanical coupling/cross bridge cycling in airway smooth muscle (ASM). Targeting actin cytoskeleton polymerization in ASM represents an alternative means to regulate ASM contraction. Herein we report the cooperative effects of targeting these distinct pathways with ß-agonists and inhibitors of the mammalian Abelson tyrosine kinase (Abl1 or c-Abl). The cooperative effect of ß-agonists (isoproterenol) and c-Abl inhibitors (GNF-5, or imatinib) on contractile agonist (methacholine, or histamine) -induced ASM contraction was assessed in cultured human ASM cells (using Fourier Transfer Traction Microscopy), in murine precision cut lung slices, and in vivo (flexiVent in mice). Regulation of intracellular signaling that regulates contraction (pMLC20, pMYPT1, pHSP20), and actin polymerization state (F:G actin ratio) were assessed in cultured primary human ASM cells. In each (cell, tissue, in vivo) model, c-Abl inhibitors and ß-agonist exhibited additive effects in either preventing or reversing ASM contraction. Treatment of contracted ASM cells with c-Abl inhibitors and ß-agonist cooperatively increased actin disassembly as evidenced by a significant reduction in the F:G actin ratio. Mechanistic studies indicated that the inhibition of pharmacomechanical coupling by ß-agonists is near optimal and is not increased by c-Abl inhibitors, and the cooperative effect on ASM relaxation resides in further relaxation of ASM tension development caused by actin cytoskeleton depolymerization, which is regulated by both ß-agonists and c-Abl inhibitors. Thus, targeting actin cytoskeleton polymerization represents an untapped therapeutic reserve for managing airway resistance.

Cytokeratin 5 determines maturation of the mammary myoepithelium.

At invasion, transformed mammary epithelial cells expand into the stroma through a disrupted myoepithelial (ME) cell layer and basement membrane (BM). The intact ME cell layer has thus been suggested to act as a barrier against invasion. Here, we investigate the mechanisms behind the disruption of ME cell layer. We show that the expression of basal/ME proteins CK5, CK14, and α-SMA altered along increasing grade of malignancy, and their loss affected the maintenance of organotypic 3D mammary architecture. Furthermore, our data suggests that loss of CK5 prior to invasive stage causes decreased levels of Zinc finger protein SNAI2 (SLUG), a key regulator of the mammary epithelial cell lineage determination. Consequently, a differentiation bias toward luminal epithelial cell type was detected with loss of mature, α-SMA-expressing ME cells and reduced deposition of basement membrane protein laminin-5. Therefore, our data discloses the central role of CK5 in mammary epithelial differentiation and maintenance of normal ME layer.

A High-Throughput System for Cyclic Stretching of Precision-Cut Lung Slices During Acute Cigarette Smoke Extract Exposure.

RATIONALE: Precision-cut lung slices (PCLSs) are a valuable tool in studying tissue responses to an acute exposure; however, cyclic stretching may be necessary to recapitulate physiologic, tidal breathing conditions. OBJECTIVES: To develop a multi-well stretcher and characterize the PCLS response following acute exposure to cigarette smoke extract (CSE). METHODS: A 12-well stretching device was designed, built, and calibrated. PCLS were obtained from male Sprague-Dawley rats (N = 10) and assigned to one of three groups: 0% (unstretched), 5% peak-to-peak amplitude (low-stretch), and 5% peak-to-peak amplitude superimposed on 10% static stretch (high-stretch). Lung slices were cyclically stretched for 12 h with or without CSE in the media. Levels of Interleukin-1ß (IL-1ß), matrix metalloproteinase (MMP)-1 and its tissue inhibitor (TIMP1), and membrane type-MMP (MT1-MMP) were assessed via western blot from tissue homogenate. RESULTS: The stretcher system produced nearly identical normal Lagrangian strains (E xx and E yy , p > 0.999) with negligible shear strain (E xy < 0.0005) and low intra-well variability 0.127 ± 0.073%. CSE dose response curve was well characterized by a four-parameter logistic model (R 2 = 0.893), yielding an IC50 value of 0.018 cig/mL. Cyclic stretching for 12 h did not decrease PCLS viability. Two-way ANOVA detected a significant interaction between CSE and stretch pattern for IL-1ß (p = 0.017), MMP-1, TIMP1, and MT1-MMP (p < 0.001). CONCLUSION: This platform is capable of high-throughput testing of an acute exposure under tightly-regulated, cyclic stretching conditions. We conclude that the acute mechano-inflammatory response to CSE exhibits complex, stretch-dependence in the PCLS.

Cytokine-induced molecular responses in airway smooth muscle cells inform genome-wide association studies of asthma.

BACKGROUND: A challenge in the post-GWAS era is to assign function to disease-associated variants. However, available resources do not include all tissues or environmental exposures that are relevant to all diseases. For example, exaggerated bronchoconstriction of airway smooth muscle cells (ASMCs) defines airway hyperresponsiveness (AHR), a cardinal feature of asthma. However, the contribution of ASMC to genetic and genomic studies has largely been overlooked. Our study aimed to address the gap in data availability from a critical tissue in genomic studies of asthma. METHODS: We developed a cell model of AHR to discover variants associated with transcriptional, epigenetic, and cellular responses to two AHR promoting cytokines, IL-13 and IL-17A, and performed a GWAS of bronchial responsiveness (BRI) in humans. RESULTS: Our study revealed significant response differences between ASMCs from asthma cases and controls, including genes implicated in asthma susceptibility. We defined molecular quantitative trait loci (QTLs) for expression (eQTLs) and methylation (meQTLs), and cellular QTLs for contractility (coQTLs) and performed a GWAS of BRI in human subjects. Variants in asthma GWAS were significantly enriched for ASM QTLs and BRI-associated SNPs, and near genes enriched for ASM function, many with small P values that did not reach stringent thresholds of significance in GWAS. CONCLUSIONS: Our study identified significant differences between ASMCs from asthma cases and controls, potentially reflecting trained tolerance in these cells, as well as a set of variants, overlooked in previous GWAS, which reflect the AHR component of asthma.

Aspergillus fumigatus Protease Alkaline Protease 1 (Alp1): A New Therapeutic Target for Fungal Asthma.

We review three recent findings that have fundamentally altered our understanding of causative mechanisms underlying fungal-related asthma. These mechanisms may be partially independent of host inflammatory processes but are strongly dependent upon the actions of Alp1 on lung structural cells. They entail (i) bronchial epithelial sensing of Alp1; (ii) Alp1-induced airway smooth muscle (ASM) contraction; (iii) Alp1-induced airflow obstruction. Collectively, these mechanisms point to Alp1 as a new target for intervention in fungal asthma.

Inhibiting Airway Smooth Muscle Contraction Using Pitavastatin: A Role for the Mevalonate Pathway in Regulating Cytoskeletal Proteins.

Despite maximal use of currently available therapies, a significant number of asthma patients continue to experience severe, and sometimes life-threatening bronchoconstriction. To fill this therapeutic gap, we examined a potential role for the 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) inhibitor, pitavastatin. Using human airway smooth muscle (ASM) cells and murine precision-cut lung slices, we discovered that pitavastatin significantly inhibited basal-, histamine-, and methacholine (MCh)-induced ASM contraction. This occurred via reduction of myosin light chain 2 (MLC2) phosphorylation, and F-actin stress fiber density and distribution, in a mevalonate (MA)- and geranylgeranyl pyrophosphate (GGPP)-dependent manner. Pitavastatin also potentiated the ASM relaxing effect of a simulated deep breath, a beneficial effect that is notably absent with the ß2-agonist, isoproterenol. Finally, pitavastatin attenuated ASM pro-inflammatory cytokine production in a GGPP-dependent manner. By targeting all three hallmark features of ASM dysfunction in asthma-contraction, failure to adequately relax in response to a deep breath, and inflammation-pitavastatin may represent a unique asthma therapeutic.

Preserving Airway Smooth Muscle Contraction in Precision-Cut Lung Slices.

Precision-cut lung slices (PCLS) are ideal for measuring small airway contraction. However, these measurements are currently limited to acute exposure scenarios that typically last a few minutes to a few hours. Using an insulin-supplemented culture medium, we prolong the small airway contractility in mouse PCLS for up to two weeks. Compared to conventional culture medium, insulin-supplemented culture medium provides no additional benefit in preserving cellular viability or airway structure. However, it protects the airway smooth muscle (ASM) against a loss of smooth muscle myosin heavy chain (SMMHC) expression. We elucidate the significance of this new culture medium for chronic disease modeling of IL-13-induced airway hyper-responsiveness.

As the endothelial cell reorients, its tensile forces stabilize.

When adherent cells are subjected to uniaxial sinusoidal stretch at frequencies close to physiological, their body and their contractile stress fibers realign nearly perpendicularly to the stretch axis. A common explanation for this phenomenon is that stress fibers reorient along the direction where they are unaffected by the applied cyclic stretch and thus can maintain optimal (homeostatic) tensile force. The ability of cells to achieve tensional homeostasis in response to external disturbances is important for normal physiological functions of cells and tissues and it provides protection against diseases, including cancer and atherosclerosis. However, quantitative experimental data that support the idea that stretch-induced reorientation is associated with tensional homeostasis are lacking. We observed previously that in response to uniaxial cyclic stretch of 10% strain amplitudes, traction forces of single endothelial cells reorient in the direction perpendicular to the stretch axis. Here we carried out a secondary analysis of those data to investigate whether this reorientation of traction forces is associated with tensional homeostasis. Our analysis showed that stretch-induced reorientation of traction forces was accompanied by attenuation of temporal variability of the traction field to the level that was observed in the absence of stretch. These findings represent a quantitative experimental evidence that stretch-induced reorientation of cellular traction forces is associated with the cell's tendency to achieve tensional homeostasis.

Assembly of Peripheral Actomyosin Bundles in Epithelial Cells Is Dependent on the CaMKK2/AMPK Pathway.

Defects in the maintenance of intercellular junctions are associated with loss of epithelial barrier function and consequent pathological conditions, including invasive cancers. Epithelial integrity is dependent on actomyosin bundles at adherens junctions, but the origin of these junctional bundles is incompletely understood. Here we show that peripheral actomyosin bundles can be generated from a specific actin stress fiber subtype, transverse arcs, through their lateral fusion at cell-cell contacts. Importantly, we find that assembly and maintenance of peripheral actomyosin bundles are dependent on the mechanosensitive CaMKK2/AMPK signaling pathway and that inhibition of this route leads to disruption of tension-maintaining actomyosin bundles and re-growth of stress fiber precursors. This results in redistribution of cellular forces, defects in monolayer integrity, and loss of epithelial identity. These data provide evidence that the mechanosensitive CaMKK2/AMPK pathway is critical for the maintenance of peripheral actomyosin bundles and thus dictates cell-cell junctions through cellular force distribution.

Adipokine Leptin Co-operates With Mechanosensitive Ca2 +-Channels and Triggers Actomyosin-Mediated Motility of Breast Epithelial Cells.

In postmenopausal women, a major risk factor for the development of breast cancer is obesity. In particular, the adipose tissue-derived adipokine leptin has been strongly linked to tumor cell proliferation, migration, and metastasis, but the underlying mechanisms remain unclear. Here we show that treatment of normal mammary epithelial cells with leptin induces EMT-like features characterized by higher cellular migration speeds, loss of structural ordering of 3D-mammo spheres, and enhancement of epithelial traction forces. Mechanistically, leptin triggers the phosphorylation of myosin light chain kinase-2 (MLC-2) through the interdependent activity of leptin receptor and Ca2+ channels. These data provide evidence that leptin-activated leptin receptors, in co-operation with mechanosensitive Ca2+ channels, play a role in the development of breast carcinomas through the regulation of actomyosin dynamics.

Tissue traction microscopy to quantify muscle contraction within precision-cut lung slices.

In asthma, acute bronchospasm is driven by contractile forces of airway smooth muscle (ASM). These forces can be imaged in the cultured ASM cell or assessed in the muscle strip and the tracheal/bronchial ring, but in each case, the ASM is studied in isolation from the native airway milieu. Here, we introduce a novel platform called tissue traction microscopy (TTM) to measure ASM contractile force within porcine and human precision-cut lung slices (PCLS). Compared with the conventional measurements of lumen area changes in PCLS, TTM measurements of ASM force changes are 1) more sensitive to bronchoconstrictor stimuli, 2) less variable across airways, and 3) provide spatial information. Notably, within every human airway, TTM measurements revealed local regions of high ASM contraction that we call "stress hotspots". As an acute response to cyclic stretch, these hotspots promptly decreased but eventually recovered in magnitude, spatial location, and orientation, consistent with local ASM fluidization and resolidification. By enabling direct and precise measurements of ASM force, TTM should accelerate preclinical studies of airway reactivity.

Aspergillus fumigatus-Secreted Alkaline Protease 1 Mediates Airways Hyperresponsiveness in Severe Asthma.

The hallmark features of allergic asthma are type 2 (eosinophilic) inflammation and airways hyperresponsiveness (AHR). Although these features often comanifest in mouse lungs in vivo, we demonstrate in this study that the serine protease Alp1 from the ubiquitous mold and allergen, Aspergillus fumigatus, can induce AHR in mice unable to generate eosinophilic inflammation. Strikingly, Alp1 induced AHR in mice devoid of protease-activated receptor 2/F2 trypsin-like receptor 1 (PAR2/F2RL1), a receptor expressed in lung epithelium that is critical for allergic responses to protease-containing allergens. Instead, using precision-cut lung slices and human airway smooth muscle cells, we demonstrate that Alp1 directly increased contractile force. Taken together, these findings suggest that Alp1 induces bronchoconstriction through mechanisms that are largely independent of allergic inflammation and point to a new target for direct intervention of fungal-associated asthma.

Extracellular matrix stiffness regulates human airway smooth muscle contraction by altering the cell-cell coupling.

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