Epigenetic toxicity of heavy metals - implications for embryonic stem cells.

Exposure to heavy metals, such as cadmium, nickel, mercury, arsenic, lead, and hexavalent chromium has been linked to dysregulated developmental processes, such as impaired stem cell differentiation. Heavy metals are well-known modifiers of the epigenome. Stem and progenitor cells are particularly vulnerable to exposure to potentially toxic metals since these cells rely on epigenetic reprogramming for their proper functioning. Therefore, exposure to metals can impair stem and progenitor cell proliferation, pluripotency, stemness, and differentiation. In this review, we provide a comprehensive summary of current evidence on the epigenetic effects of heavy metals on stem cells, focusing particularly on DNA methylation and histone modifications. Moreover, we explore the underlying mechanisms responsible for these epigenetic changes. By providing an overview of heavy metal exposure-induced alterations to the epigenome, the underlying mechanisms, and the consequences of those alterations on stem cell function, this review provides a foundation for further research in this critical area of overlap between toxicology and developmental biology.

Cavum Septum Pellucidum in Former American Football Players: Findings From the DIAGNOSE CTE Research Project.

Background and Objectives: Exposure to repetitive head impacts (RHI) is linked to the development of chronic traumatic encephalopathy (CTE), which can only be diagnosed at post-mortem. The presence of a cavum septum pellucidum (CSP) is a common finding in post-mortem studies of confirmed CTE and in neuroimaging studies of individuals exposed to RHI. This study examines CSP in living former American football players, investigating its association with RHI exposure, traumatic encephalopathy syndrome (TES) diagnosis, and provisional levels of certainty for CTE pathology. Methods: Data from the DIAGNOSE CTE Research Project were used to compare the presence and ratio of CSP in former American football players (n = 175), consisting of former college (n = 58) and former professional players (n = 117), and asymptomatic unexposed controls without RHI exposure (n = 55). We further evaluated potential associations between CSP measures and cumulative head impact index (CHII) measures (frequency, linear acceleration, and rotational force), a TES diagnosis (yes/no), and a provisional level of certainty for CTE pathology (suggestive, possible, and probable). Results: Former American football players exhibited a higher CSP presence and ratio than unexposed asymptomatic controls. Among player subgroups, professional players showed a greater CSP ratio than former college players and unexposed asymptomatic controls. Among all football players, CHII rotational forces correlated with an increased CSP ratio. No significant associations were found between CSP measures and diagnosis of TES or provisional levels of certainty for CTE pathology. Discussion: This study confirms previous findings, highlighting a greater prevalence of CSP and a greater CSP ratio in former American football players compared with unexposed asymptomatic controls. In addition, former professional players showed a greater CSP ratio than college players. Moreover, the relationship between estimates of CHII rotational forces and CSP measures suggests that cumulative frequency and strength of rotational forces experienced in football are associated with CSP. However, CSP does not directly correlate with TES diagnosis or provisional levels of certainty for CTE, indicating that it may be a consequence of RHI associated with rotational forces. Further research, especially longitudinal studies, is needed for confirmation and to explore changes over time.

An Introduction to the Human Connectome Project for Early Psychosis.

BACKGROUND: The time following a recent onset of psychosis is a critical period during which intervention may be maximally effective. Studying individuals in this period also offers an opportunity to investigate putative brain biomarkers of illness prior to the long-term effects of chronicity and medication. The Human Connectome Project for Early Psychosis (HCP-EP) was funded by the National Institutes of Mental Health (NIMH) as an extension of the original Human Connectome Project's approach to understanding the human brain and its structural and functional connections. DESIGN: The HCP-EP data were collected at 3 sites in Massachusetts (Beth Israel Deaconess Medical Center, McLean Hospital, and Massachusetts General Hospital), and one site in Indiana (Indiana University). Brigham and Women's Hospital served as the data coordination center and as an imaging site. RESULTS: The HCP-EP dataset includes high-quality clinical, cognitive, functional, neuroimaging, and blood specimen data acquired from 303 individuals between the ages of 16-35 years old with affective psychosis (n = 75), non-affective psychosis (n = 148), and healthy controls (n = 80). Participants with early psychosis were within 5 years of illness onset (mean duration = 1.9 years, standard deviation = 1.4 years). All data and novel or modified analytic tools developed as part of the study are publicly available to the research community through the NIMH Data Archive (NDA) or GitHub (https://github.com/pnlbwh). CONCLUSIONS: This paper provides an overview of the specific HCP-EP procedures, assessments, and protocols, as well as a brief characterization of the study participants to make it easier for researchers to use this rich dataset. Although we focus here on discussing and comparing affective and non-affective psychosis groups, the HCP-EP dataset also provides sufficient information for investigators to group participants differently.

Hepatotoxicity of titanium dioxide nanoparticles.

The food additive E171 (titanium dioxide, TiO2), is widely used in foods, pharmaceuticals and cosmetics. It is a fine white powder, with at least one third of its particles sized in the nanoparticulate (˂100 nm range, TiO2 NPs). The use of E171 is controversial as its relevant risk assessment has never been satisfactorily accomplished. In vitro and in vivo studies have shown dose-dependent toxicity in various organs including the liver. TiO2 NPs have been shown to induce inflammation, cell death and structural and functional changes within the liver. The toxicity of TiO2 NPs in experimental models varies between organs and according to their physiochemical characteristics and parameters such as dosage and route of administration. Among these factors, ingestion is the most significant exposure route, and the liver is a key target organ. The aim of this review is to highlight the reported adverse effects of orally administered TiO2 NPs on the liver and to discuss the controversial state of its toxicity.

Brain morphometry in former American football players: findings from the DIAGNOSE CTE research project.

Exposure to repetitive head impacts in contact sports is associated with neurodegenerative disorders including chronic traumatic encephalopathy (CTE), which currently can be diagnosed only at post-mortem. American football players are at higher risk of developing CTE given their exposure to repetitive head impacts. One promising approach for diagnosing CTE in vivo is to explore known neuropathological abnormalities at post-mortem in living individuals using structural MRI. MRI brain morphometry was evaluated in 170 male former American football players ages 45-74 years (n = 114 professional; n = 56 college) and 54 same-age unexposed asymptomatic male controls (n = 54, age range 45-74). Cortical thickness and volume of regions of interest were selected based on established CTE pathology findings and were assessed using FreeSurfer. Group differences and interactions with age and exposure factors were evaluated using a generalized least squares model. A separate logistic regression and independent multinomial model were performed to predict each traumatic encephalopathy syndrome (TES) diagnosis, core clinical features and provisional level of certainty for CTE pathology using brain regions of interest. Former college and professional American football players (combined) showed significant cortical thickness and/or volume reductions compared to unexposed asymptomatic controls in the hippocampus, amygdala, entorhinal cortex, parahippocampal gyrus, insula, temporal pole and superior frontal gyrus. Post hoc analyses identified group-level differences between former professional players and unexposed asymptomatic controls in the hippocampus, amygdala, entorhinal cortex, parahippocampal gyrus, insula and superior frontal gyrus. Former college players showed significant volume reductions in the hippocampus, amygdala and superior frontal gyrus compared to the unexposed asymptomatic controls. We did not observe Age × Group interactions for brain morphometric measures. Interactions between morphometry and exposure measures were limited to a single significant positive association between the age of first exposure to organized tackle football and right insular volume. We found no significant relationship between brain morphometric measures and the TES diagnosis core clinical features and provisional level of certainty for CTE pathology outcomes. These findings suggested that MRI morphometrics detect abnormalities in individuals with a history of repetitive head impact exposure that resemble the anatomic distribution of pathological findings from post-mortem CTE studies. The lack of findings associating MRI measures with exposure metrics (except for one significant relationship) or TES diagnosis and core clinical features suggested that brain morphometry must be complemented by other types of measures to characterize individuals with repetitive head impacts.

ENIGMA's simple seven: Recommendations to enhance the reproducibility of resting-state fMRI in traumatic brain injury.

Resting state functional magnetic resonance imaging (rsfMRI) provides researchers and clinicians with a powerful tool to examine functional connectivity across large-scale brain networks, with ever-increasing applications to the study of neurological disorders, such as traumatic brain injury (TBI). While rsfMRI holds unparalleled promise in systems neurosciences, its acquisition and analytical methodology across research groups is variable, resulting in a literature that is challenging to integrate and interpret. The focus of this narrative review is to address the primary methodological issues including investigator decision points in the application of rsfMRI to study the consequences of TBI. As part of the ENIGMA Brain Injury working group, we have collaborated to identify a minimum set of recommendations that are designed to produce results that are reliable, harmonizable, and reproducible for the TBI imaging research community. Part one of this review provides the results of a literature search of current rsfMRI studies of TBI, highlighting key design considerations and data processing pipelines. Part two outlines seven data acquisition, processing, and analysis recommendations with the goal of maximizing study reliability and between-site comparability, while preserving investigator autonomy. Part three summarizes new directions and opportunities for future rsfMRI studies in TBI patients. The goal is to galvanize the TBI community to gain consensus for a set of rigorous and reproducible methods, and to increase analytical transparency and data sharing to address the reproducibility crisis in the field.

Nickel-induced transcriptional memory in lung epithelial cells promotes interferon signaling upon nicotine exposure.

Exposure to nickel, an environmental respiratory toxicant, is associated with lung diseases including asthma, pulmonary fibrosis, bronchitis and cancers. Our previous studies have shown that a majority of the nickel-induced transcriptional changes are persistent and do not reverse even after the termination of exposure. This suggested transcriptional memory, wherein the cell 'remembers' past nickel exposure. Transcriptional memory, due to which the cells respond more robustly to a previously encountered stimulus has been identified in a number of organisms. Therefore, transcriptional memory has been described as an adaptive mechanism. However, transcriptional memory caused by environmental toxicant exposures has not been well investigated. Moreover, how the transcriptional memory caused by an environmental toxicant might influence the outcome of exposure to a second toxicant has not been explored. In this study, we investigated whether nickel-induced transcriptional memory influences the outcome of the cell's response to a second respiratory toxicant, nicotine. Nicotine, an addictive compound in tobacco, is associated with the development of chronic lung diseases including chronic obstructive pulmonary disease (COPD) and pulmonary fibrosis. Our results show that nicotine exposure upregulated a subset of genes only in the cells previously exposed to nickel. Furthermore, our analyses indicate robust activation of interferon (IFN) signaling in these cells. IFN signaling is a driver of inflammation, which is associated with many chronic lung diseases. Therefore, our results suggest that nicotine exposure of lung cells that retain the transcriptional memory of previous nickel exposure could result in increased susceptibility to developing chronic inflammatory lung diseases.

Prorelaxant E-type Prostanoid Receptors Functionally Partition to Different Procontractile Receptors in Airway Smooth Muscle.

Prostaglandin E2 imparts diverse physiological effects on multiple airway cells through its actions on four distinct E-type prostanoid (EP) receptor subtypes (EP1-EP4). Gs-coupled EP2 and EP4 receptors are expressed on airway smooth muscle (ASM), yet their capacity to regulate the ASM contractile state remains subject to debate. We used EP2 and EP4 subtype-specific agonists (ONO-259 and ONO-329, respectively) in cell- and tissue-based models of human ASM contraction-magnetic twisting cytometry (MTC), and precision-cut lung slices (PCLSs), respectively-to study the EP2 and EP4 regulation of ASM contraction and signaling under conditions of histamine or methacholine (MCh) stimulation. ONO-329 was superior (<0.05) to ONO-259 in relaxing MCh-contracted PCLSs (log half maximal effective concentration [logEC50]: 4.9 × 10-7 vs. 2.2 × 10-6; maximal bronchodilation ± SE, 35 ± 2% vs. 15 ± 2%). However, ONO-259 and ONO-329 were similarly efficacious in relaxing histamine-contracted PCLSs. Similar differential effects were observed in MTC studies. Signaling analyses revealed only modest differences in ONO-329- and ONO-259-induced phosphorylation of the protein kinase A substrates VASP and HSP20, with concomitant stimulation with MCh or histamine. Conversely, ONO-259 failed to inhibit MCh-induced phosphorylation of the regulatory myosin light chain (pMLC20) and the F-actin/G-actin ratio (F/G-actin ratio) while effectively inhibiting their induction by histamine. ONO-329 was effective in reversing induced pMLC20 and the F/G-actin ratio with both MCh and histamine. Thus, the contractile-agonist-dependent differential effects are not explained by changes in the global levels of phosphorylated protein kinase A substrates but are reflected in the regulation of pMLC20 (cross-bridge cycling) and F/G-actin ratio (actin cytoskeleton integrity, force transmission), implicating a role for compartmentalized signaling involving muscarinic, histamine, and EP receptor subtypes.

Cells in the polyaneuploid cancer cell (PACC) state have increased metastatic potential.

Although metastasis is the leading cause of cancer deaths, it is quite rare at the cellular level. Only a rare subset of cancer cells (~ 1 in 1.5 billion) can complete the entire metastatic cascade: invasion, intravasation, survival in the circulation, extravasation, and colonization (i.e. are metastasis competent). We propose that cells engaging a Polyaneuploid Cancer Cell (PACC) phenotype are metastasis competent. Cells in the PACC state are enlarged, endocycling (i.e. non-dividing) cells with increased genomic content that form in response to stress. Single-cell tracking using time lapse microscopy reveals that PACC state cells have increased motility. Additionally, cells in the PACC state exhibit increased capacity for environment-sensing and directional migration in chemotactic environments, predicting successful invasion. Magnetic Twisting Cytometry and Atomic Force Microscopy reveal that cells in the PACC state display hyper-elastic properties like increased peripheral deformability and maintained peri-nuclear cortical integrity that predict successful intravasation and extravasation. Furthermore, four orthogonal methods reveal that cells in the PACC state have increased expression of vimentin, a hyper-elastic biomolecule known to modulate biomechanical properties and induce mesenchymal-like motility. Taken together, these data indicate that cells in the PACC state have increased metastatic potential and are worthy of further in vivo analysis.

Rhinovirus C15 Attenuates Relaxation and cAMP Production in Human Airways and Smooth Muscle.

Rhinoviruses (RVs) evoke as many as 85% of acute asthma exacerbations in children and 50% in adults and can induce airway hyperresponsiveness and decrease efficacy of current therapeutics to provide symptom relief. Using human precision-cut lung slices (hPCLSs), primary human air-liquid interface-differentiated airway epithelial cells (HAECs), and human airway smooth muscle (HASM) as preclinical experimental models, we demonstrated that RV-C15 attenuates agonist-induced bronchodilation. Specifically, airway relaxation to formoterol and cholera toxin, but not forskolin (Fsk), was attenuated following hPCLS exposure to RV-C15. In isolated HASM cells, exposure to conditioned media from RV-exposed HAECs decreased cellular relaxation in response to isoproterenol and prostaglandin E2, but not Fsk. Additionally, cAMP generation elicited by formoterol and isoproterenol, but not Fsk, was attenuated following HASM exposure to RV-C15-conditioned HAEC media. HASM exposure to RV-C15-conditioned HAEC media modulated expression of components of relaxation pathways, specifically GNAI1 and GRK2. Strikingly, similar to exposure to intact RV-C15, hPCLS exposed to UV-inactivated RV-C15 showed markedly attenuated airway relaxation in response to formoterol, suggesting that the mechanism(s) of RV-C15-mediated loss of bronchodilation is independent of virus replication pathways. Further studies are warranted to identify soluble factor(s) regulating the epithelial-driven smooth muscle loss of ß2-adrenergic receptor function.

Cutaneous Mucormycosis of the Upper Extremity: Case Reports Highlighting Rapid Diagnosis and Management.

ABSTRACT: Mucormycosis is a devastating fungal infection known for its angioinvasive spread and hematogenous dissemination, quickly resulting in multiorgan involvement and potentially fatal complications. Timely diagnosis is essential to facilitate early, aggressive debridement, yet the diagnosis remains difficult to obtain because of the need for culture and microscopy diagnosis. We provide case examples, which highlight diagnostic pearls and the multidisciplinary approach that are critical for improving local outcomes, preventing systemic spread, and reducing mortality.

ß2 -Adrenoceptor agonist profiling reveals biased signalling phenotypes for the ß2 -adrenoceptor with possible implications for the treatment of asthma.

BACKGROUND AND PURPOSE: ß-Adrenoceptor agonists relieve airflow obstruction by activating ß2 -adrenoceptors, which are G protein-coupled receptors (GPCRs) expressed on human airway smooth muscle (HASM) cells. The currently available ß-adrenoceptor agonists are balanced agonists, however, and signal through both the stimulatory G protein (Gs )- and ß-arrestin-mediated pathways. While Gs signalling is beneficial and promotes HASM relaxation, ß-arrestin activation is associated with reduced Gs efficacy. In this context, biased ligands that selectively promote ß2 -adrenoceptor coupling to Gs signalling represent a promising strategy to treat asthma. Here, we examined several ß-adrenoceptor agonists to identify Gs -biased ligands devoid of ß-arrestin-mediated effects. EXPERIMENTAL APPROACH: Gs -biased ligands for the ß2 -adrenoceptor were identified by high-throughput screening and then evaluated for Gs interaction, Gi interaction, cAMP production, ß-arrestin interaction, GPCR kinase (GRK) phosphorylation of the receptor, receptor trafficking, ERK activation, and functional desensitization of the ß2 -adrenoceptor. KEY RESULTS: We identified ractopamine, dobutamine, and higenamine as Gs -biased agonists that activate the Gs /cAMP pathway upon ß2 -adrenoceptor stimulation while showing minimal Gi or ß-arrestin interaction. Furthermore, these compounds did not induce any receptor trafficking and had reduced GRK5-mediated phosphorylation of the ß2 -adrenoceptor. Finally, we observed minimal physiological desensitization of the ß2 -adrenoceptor in primary HASM cells upon treatment with biased agonists. CONCLUSION AND IMPLICATIONS: Our work demonstrates that Gs -biased signalling through the ß2 -adrenoceptor may prove to be an effective strategy to promote HASM relaxation in the treatment of asthma. Such biased compounds may also be useful in identifying the molecular mechanisms that determine biased signalling and in design of safer drugs.

Thunderstorm asthma: a review, risks for Aotearoa New Zealand, and health emergency management considerations.

AIM: To provide an up-to-date review of thunderstorm asthma (TA), identifying causative factors, and to discuss implications for management of TA in New Zealand. METHODS: A literature search was carried out to identify articles that investigate the characteristics and causative factors of TA. Nine electronic databases were searched, yielding 372 articles, reduced to 30 articles after screening for duplication and relevance. RESULTS: TA is globally rare, with 29 reported events since 1983, but is expected to increase in frequency as Earth warms. Triggers include both pollen (particularly ryegrass pollen) and fungal spores. Individual risk factors include outdoor exposure, sensitivity to triggering allergens and history of seasonal allergic rhinitis. History of asthma is not a strong risk factor but is associated with severity of outcome. Limited data on demographic characteristics suggests that individuals aged between 20 and 60 and (in Australasia) of Asian/Indian ethnicity are at higher risk. A single TA event has been reported in New Zealand to date, but much of New Zealand may be at risk of future events given that ryegrass pastures are widely distributed, and summer thunderstorms can occur anywhere. CONCLUSIONS: We recommend developing rapidly deployable public messaging to support the health emergency management response to future TA events, together with the instigation of routine aeroallergen monitoring.

Radiation Doses in Patients Undergoing Computed Tomographic Coronary Artery Calcium Evaluation With a 64-Slice Scanner Versus a 256-Slice Scanner.

Computed tomographic coronary artery calcium scanning enables cardiovascular risk stratification; however, exposing patients to high radiation levels is an ongoing concern. New-generation computed tomographic systems use lower radiation doses than older systems do. To quantify comparative doses of radiation exposure, we prospectively acquired images from 220 patients with use of a 64-slice GE LightSpeed VCT scanner (control group, n=110) and a 256-slice GE Revolution scanner (study group, n=110). The groups were matched for age, sex, and body mass index; statistical analysis included t tests and linear regression. The mean dose-length product was 21% lower in the study group than in the control group (60.2 ± 27 vs 75.9 ± 22.6 mGy·cm; P <0.001) and also in each body mass index subgroup. Similarly, the mean effective radiation dose was 21% lower in the study group (0.84 ± 0.38 vs 1.06 ± 0.32 mSv) and lower in each weight subgroup. After adjustment for sex, women in the study group had a lower dose-length product (50.4 ± 23.4 vs 64.7 ± 27.6 mGy·cm) than men did and received a lower effective dose (0.7 ± 0.32 vs 0.9 ± 0.38 mSv) (P=0.009). As body mass index and waist circumference increased, so did doses for both scanners. Our study group was exposed to radiation doses lower than the previously determined standard of 1 mSv, even after adjustment for body mass index and waist circumference. In 256-slice scanning for coronary artery calcium, radiation doses are now similar to those in lung cancer screening and mammography.

Triglyceride-derived fatty acids reduce autophagy in a model of retinal angiomatous proliferation.

Dyslipidemia and autophagy have been implicated in the pathogenesis of blinding neovascular age-related macular degeneration (NV-AMD). VLDL receptor (VLDLR), expressed in photoreceptors with a high metabolic rate, facilitates the uptake of triglyceride-derived fatty acids. Since fatty acid uptake is reduced in Vldlr-/- tissues, more remain in circulation, and the retina is fuel deficient, driving the formation in mice of neovascular lesions reminiscent of retinal angiomatous proliferation (RAP), a subtype of NV-AMD. Nutrient scarcity and energy failure are classically mitigated by increasing autophagy. We found that excess circulating lipids restrained retinal autophagy, which contributed to pathological angiogenesis in the Vldlr-/- RAP model. Triglyceride-derived fatty acid sensed by free fatty acid receptor 1 (FFAR1) restricted autophagy and oxidative metabolism in photoreceptors. FFAR1 suppressed transcription factor EB (TFEB), a master regulator of autophagy and lipid metabolism. Reduced TFEB, in turn, decreased sirtuin-3 expression and mitochondrial respiration. Metabolomic signatures of mouse RAP-like retinas were consistent with a role in promoting angiogenesis. This signature was also found in human NV-AMD vitreous. Restoring photoreceptor autophagy in Vldlr-/- retinas, either pharmacologically or by deleting Ffar1, enhanced metabolic efficiency and suppressed pathological angiogenesis. Dysregulated autophagy by circulating lipids might therefore contribute to the energy failure of photoreceptors driving neovascular eye diseases, and FFAR1 may be a target for intervention.

Identification and characterization of an atypical Gαs-biased ß2AR agonist that fails to evoke airway smooth muscle cell tachyphylaxis.

G protein-coupled receptors display multifunctional signaling, offering the potential for agonist structures to promote conformational selectivity for biased outputs. For ß2-adrenergic receptors (ß2AR), unbiased agonists stabilize conformation(s) that evoke coupling to Gαs (cyclic adenosine monophosphate [cAMP] production/human airway smooth muscle [HASM] cell relaxation) and ß-arrestin engagement, the latter acting to quench Gαs signaling, contributing to receptor desensitization/tachyphylaxis. We screened a 40-million-compound scaffold ranking library, revealing unanticipated agonists with dihydroimidazolyl-butyl-cyclic urea scaffolds. The S-stereoisomer of compound C1 shows no detectable ß-arrestin engagement/signaling by four methods. However, C1-S retained Gαs signaling-a divergence of the outputs favorable for treating asthma. Functional studies with two models confirmed the biasing: ß2AR-mediated cAMP signaling underwent desensitization to the unbiased agonist albuterol but not to C1-S, and desensitization of HASM cell relaxation was observed with albuterol but not with C1-S These HASM results indicate biologically pertinent biasing of C1-S, in the context of the relevant physiologic response, in the human cell type of interest. Thus, C1-S was apparently strongly biased away from ß-arrestin, in contrast to albuterol and C5-S C1-S structural modeling and simulations revealed binding differences compared with unbiased epinephrine at transmembrane (TM) segments 3,5,6,7 and ECL2. C1-S (R2 = cyclohexane) was repositioned in the pocket such that it lost a TM6 interaction and gained a TM7 interaction compared with the analogous unbiased C5-S (R2 = benzene group), which appears to contribute to C1-S biasing away from ß-arrestin. Thus, an agnostic large chemical-space library identified agonists with receptor interactions that resulted in relevant signal splitting of ß2AR actions favorable for treating obstructive lung disease.

The Management of Complications of Feminizing Gender Affirming Genital Surgery.

Penile inversion vaginoplasty is the most common technique used for gender affirming genital surgery in the treatment of gender dysphoria among transwomen. As vaginoplasty becomes more widely available, the management of associated complications has become its own field. There is a relative dearth of literature on surgery for complications following vaginoplasty. This review illustrates surgical technique and management options for patient reported complaints and complications following vaginoplasty. The goal of this manuscript is 2-fold (1) to introduce community surgeons to common postoperative issues they may encounter and (2) provide a systematic operative approach to complications for reconstructive surgeons who see transgender patients regularly.

FFAR1 activation attenuates histamine-induced myosin light chain phosphorylation and cortical tension development in human airway smooth muscle cells.

BACKGROUND: Activation of free fatty acid receptors (FFAR1 and FFAR4) which are G protein-coupled receptors (GPCRs) with established (patho)physiological roles in a variety of obesity-related disorders, induce human airway smooth muscle (HASM) cell proliferation and shortening. We reported amplified agonist-induced cell shortening in HASM cells obtained from obese lung donors. We hypothesized that FFAR1 modulate excitation-contraction (EC) coupling in HASM cells and play a role in obesity-associated airway hyperresponsiveness. METHODS: In HASM cells pre-treated (30 min) with FFAR1 agonists TAK875 and GW9508, we measured histamine-induced Ca2+ mobilization, myosin light chain (MLC) phosphorylation, and cortical tension development with magnetic twisting cytometry (MTC). Phosphorylation of MLC phosphatase and Akt also were determined in the presence of the FFAR1 agonists or vehicle. In addition, the effects of TAK875 on MLC phosphorylation were measured in HASM cells desensitized to ß2AR agonists by overnight salmeterol treatment. The inhibitory effect of TAK875 on MLC phosphorylation was compared between HASM cells from age and sex-matched non-obese and obese human lung donors. The mean measurements were compared using One-Way ANOVA with Dunnett's test for multiple group comparisons or Student's t-test two-group comparison. For cortical tension measurements by magnetic twisted cytometry, mixed effect model using SAS V.9.2 was applied. Means were considered significant when p ≤ 0.05. RESULTS: Unexpectedly, we found that TAK875, a synthetic FFAR1 agonist, attenuated histamine-induced MLC phosphorylation and cortical tension development in HASM cells. These physiological outcomes were unassociated with changes in histamine-evoked Ca2+ flux, protein kinase B (AKT) activation, or MLC phosphatase inhibition. Of note, TAK875-mediated inhibition of MLC phosphorylation was maintained in ß2AR-desensitized HASM cells and across obese and non-obese donor-derived HASM cells. CONCLUSIONS: Taken together, our findings identified the FFAR1 agonist TAK875 as a novel bronchoprotective agent that warrants further investigation to treat difficult-to-control asthma and/or airway hyperreactivity in obesity.

The odorant receptor OR2W3 on airway smooth muscle evokes bronchodilation via a cooperative chemosensory tradeoff between TMEM16A and CFTR.

The recent discovery of sensory (tastant and odorant) G protein-coupled receptors on the smooth muscle of human bronchi suggests unappreciated therapeutic targets in the management of obstructive lung diseases. Here we have characterized the effects of a wide range of volatile odorants on the contractile state of airway smooth muscle (ASM) and uncovered a complex mechanism of odorant-evoked signaling properties that regulate excitation-contraction (E-C) coupling in human ASM cells. Initial studies established multiple odorous molecules capable of increasing intracellular calcium ([Ca2+]i) in ASM cells, some of which were (paradoxically) associated with ASM relaxation. Subsequent studies showed a terpenoid molecule (nerol)-stimulated OR2W3 caused increases in [Ca2+]i and relaxation of ASM cells. Of note, OR2W3-evoked [Ca2+]i mobilization and ASM relaxation required Ca2+ flux through the store-operated calcium entry (SOCE) pathway and accompanied plasma membrane depolarization. This chemosensory odorant receptor response was not mediated by adenylyl cyclase (AC)/cyclic nucleotide-gated (CNG) channels or by protein kinase A (PKA) activity. Instead, ASM olfactory responses to the monoterpene nerol were predominated by the activity of Ca2+-activated chloride channels (TMEM16A), including the cystic fibrosis transmembrane conductance regulator (CFTR) expressed on endo(sarco)plasmic reticulum. These findings demonstrate compartmentalization of Ca2+ signals dictates the odorant receptor OR2W3-induced ASM relaxation and identify a previously unrecognized E-C coupling mechanism that could be exploited in the development of therapeutics to treat obstructive lung diseases.