Cynthia J. Koziol-White

Soluble guanylate cyclase as an alternative target for bronchodilator therapy in asthma

Significance Asthmatics depend on β-agonist bronchodilator drugs, but a majority develop resistance to these drugs in their lifetime, and new ways to bronchodilate are needed. We show that brochodilation can be triggered in normal human and asthmatic mouse airways through an alternative signaling pathway, using new pharmacologic agents that directly activate the soluble guanylate cyclase (sGC) enzyme. Because an sGC-based drug was recently approved to treat pulmonary arterial hypertension, our findings imply that such drugs could become new bronchodilators in asthma. Our work also provides insight on how the sGC signaling enzyme becomes desensitized toward NO in inflammatory asthma, and thus helps to explain why NO is an ineffective bronchodilator in this disease. Asthma is defined by airway inflammation and hyperresponsiveness, and contributes to morbidity and mortality worldwide. Although bronchodilation is a cornerstone of treatment, current bronchodilators become ineffective with worsening asthma severity. We investigated an alternative pathway that involves activating the airway smooth muscle enzyme, soluble guanylate cyclase (sGC). Activating sGC by its natural stimulant nitric oxide (NO), or by pharmacologic sGC agonists BAY 41–2272 and BAY 60–2770, triggered bronchodilation in normal human lung slices and in mouse airways. Both BAY 41–2272 and BAY 60–2770 reversed airway hyperresponsiveness in mice with allergic asthma and restored normal lung function. The sGC from mouse asthmatic lungs displayed three hallmarks of oxidative damage that render it NO-insensitive, and identical changes to sGC occurred in human lung slices or in human airway smooth muscle cells when given chronic NO exposure to mimic the high NO in asthmatic lung. Our findings show how allergic inflammation in asthma may impede NO-based bronchodilation, and reveal that pharmacologic sGC agonists can achieve bronchodilation despite this loss.

Inhibition of PI3K promotes dilation of human small airways in a rho kinase‐dependent manner

Asthma manifests as a heterogeneous syndrome characterized by airway obstruction, inflammation and hyperresponsiveness (AHR). Although the molecular mechanisms remain unclear, activation of specific PI3K isoforms mediate inflammation and AHR. We aimed to determine whether inhibition of PI3Kδ evokes dilation of airways and to elucidate potential mechanisms.

Inhibition of Phosphoinositide 3‐Kinase (PI3K) promotes dilation of human small airways in a Rho kinase‐dependent manner

Asthma manifests as a heterogeneous syndrome characterized by airway obstruction, inflammation and hyperresponsiveness (AHR). Although the molecular mechanisms remain unclear, activation of specific PI3K isoforms mediate inflammation and AHR. We aimed to determine whether inhibition of PI3Kδ evokes dilation of airways and to elucidate potential mechanisms.

PDE8 Is Expressed in Human Airway Smooth Muscle and Selectively Regulates cAMP Signaling by β2-Adrenergic Receptors and Adenylyl Cyclase 6

&NA; Two cAMP signaling compartments centered on adenylyl cyclase (AC) exist in human airway smooth muscle (HASM) cells, one containing &bgr;2‐adrenergic receptor AC6 and another containing E prostanoid receptor AC2. We hypothesized that different PDE isozymes selectively regulate cAMP signaling in each compartment. According to RNA‐sequencing data, 18 of 24 PDE genes were expressed in primary HASM cells derived from age‐ and sex‐matched donors with and without asthma. PDE8A was the third most abundant of the cAMP‐degrading PDE genes, after PDE4A and PDE1A. Knockdown of PDE8A using shRNA evoked twofold greater cAMP responses to 1 &mgr;M forskolin in the presence of 3‐isobutyl‐1‐methylxanthine. Overexpression of AC2 did not alter this response, but overexpression of AC6 increased cAMP responses an additional 80%. We examined cAMP dynamics in live HASM cells using a fluorescence sensor. PF‐04957325, a PDE8‐selective inhibitor, increased basal cAMP concentrations by itself, indicating a significant basal level of cAMP synthesis. In the presence of an AC inhibitor to reduce basal signaling, PF‐04957325 accelerated cAMP production and increased the inhibition of cell proliferation induced by isoproterenol, but it had no effect on cAMP concentrations or cell proliferation regulated by prostaglandin E2. Lipid raft fractionation of HASM cells revealed PDE8A immunoreactivity in buoyant fractions containing caveolin‐1 and AC5/6 immunoreactivity. Thus, PDE8 is expressed in lipid rafts of HASM cells, where it specifically regulates &bgr;2‐adrenergic receptor AC6 signaling without effects on signaling by the E prostanoid receptors 2/4‐AC2 complex. In airway diseases such as asthma and chronic obstructive pulmonary disease, PDE8 may represent a novel therapeutic target to modulate HASM responsiveness and airway remodeling.

Gα12 facilitates shortening in human airway smooth muscle by modulating phosphoinositide 3‐kinase‐mediated activation in a RhoA‐dependent manner

PI3K‐dependent activation of Rho kinase (ROCK) is necessary for agonist‐induced human airway smooth muscle cell (HASMC) contraction, and inhibition of PI3K promotes bronchodilation of human small airways. The mechanisms driving agonist‐mediated PI3K/ROCK axis activation, however, remain unclear. Given that G12 family proteins activate ROCK pathways in other cell types, their role in M3 muscarinic acetylcholine receptor‐stimulated PI3K/ROCK activation and contraction was examined.

Elastomeric sensor surfaces for high-throughput single-cell force cytometry

As cells with aberrant force-generating phenotypes can directly lead to disease, cellular force-generation mechanisms are high-value targets for new therapies. Here, we show that single-cell force sensors embedded in elastomers enable single-cell force measurements with ~100-fold improvement in throughput than was previously possible. The microtechnology is scalable and seamlessly integrates with the multi-well plate format, enabling highly parallelized time-course studies. In this regard, we show that airway smooth muscle cells isolated from fatally asthmatic patients have innately greater and faster force-generation capacity in response to stimulation than healthy control cells. By simultaneously tracing agonist-induced calcium flux and contractility in the same cell, we show that the calcium level is ultimately a poor quantitative predictor of cellular force generation. Finally, by quantifying phagocytic forces in thousands of individual human macrophages, we show that force initiation is a digital response (rather than a proportional one) to the proper immunogen. By combining mechanobiology at the single-cell level with high-throughput capabilities, this microtechnology can support drug-discovery efforts for clinical conditions associated with aberrant cellular force generation.A microtechnology involving force sensors embedded in elastomers for cell culture enables the high-throughput measurement of single-cell force generation from contractile cells in a scalable and highly parallelized manner.

Inhibition of spleen tyrosine kinase attenuates IgE‐mediated airway contraction and mediator release in human precision cut lung slices

Asthma presents as a heterogeneous syndrome characterized by airway obstruction, inflammation and hyper‐reactivity (AHR). Spleen tyrosine kinase (Syk) mediates allergen‐induced mast cell degranulation, a central component of allergen‐induced inflammation and AHR. However, the role of Syk in IgE‐mediated constriction of human small airways remains unknown. In this study, we addressed whether selective inhibition of Syk attenuates IgE‐mediated constriction and mast cell mediator release in human small airways.

Publisher Correction: Elastomeric sensor surfaces for high-throughput single-cell force cytometry

In the version of this Article originally published, in Fig. 1a, all cells in the top schematic were missing, and in the bottom-left schematic showing multiple pattern shapes, two cells were missing in the bottom-right corner. This figure has now been updated in all versions of the Article.

Oxidative damage of SP-D abolishes control of eosinophil extracellular DNA trap formation

The asthmatic airways are highly susceptible to inflammatory injury by air pollutants such as ozone (O3), characterized by enhanced activation of eosinophilic granulocytes and a failure of immune protective mechanisms. Eosinophil activation during asthma exacerbation contributes to the proinflammatory oxidative stress by high levels of nitric oxide (NO) production and extracellular DNA release. Surfactant protein‐D (SP‐D), an epithelial cell product of the airways, is a critical immune regulatory molecule with a multimeric structure susceptible to oxidative modifications. Using recombinant proteins and confocal imaging, we demonstrate here that SP‐D directly bound to the membrane and inhibited extracellular DNA trap formation by human and murine eosinophils in a concentration and carbohydrate‐dependent manner. Combined allergic airway sensitization and O3 exposure heightened eosinophilia and nos2 mRNA (iNOS) activation in the lung tissue and S‐nitrosylation related de‐oligomerisation of SP‐D in the airways. In vitro reproduction of the iNOS action led to similar effects on SP‐D. Importantly, S‐nitrosylation abolished the ability of SP‐D to block extracellular DNA trap formation. Thus, the homeostatic negative regulatory feedback between SP‐D and eosinophils is destroyed by the NO‐rich oxidative lung tissue environment in asthma exacerbations.