Kisspeptin/KISS1R Signaling Modulates Human Airway Smooth Muscle Cell Migration.

Airway remodeling is a cardinal feature of asthma, associated with increased airway smooth muscle (ASM) cell mass and upregulation of extracellular matrix deposition. Exaggerated ASM cell migration contributes to excessive ASM mass. Previously, we demonstrated the alleviating role of Kp (kisspeptin) receptor (KISS1R) activation by Kp-10 in mitogen (PDGF [platelet-derived growth factor])-induced human ASM cell proliferation in vitro and airway remodeling in vivo in a mouse model of asthma. Here, we examined the mechanisms by which KISS1R activation regulates mitogen-induced ASM cell migration. KISS1R activation using Kp-10 significantly inhibited PDGF-induced ASM cell migration, further confirmed using KISS1R shRNA. Furthermore, KISS1R activation modulated F/G actin dynamics and the expression of promigration proteins like CDC42 (cell division control protein 42) and cofilin. Mechanistically, we observed reduced ASM RhoA-GTPAse with KISS1R activation. The antimigratory effect of KISS1R was abolished by PKA (protein kinase A)-inhibitory peptide. Conversely, KISS1R activation significantly increased cAMP and phosphorylation of CREB (cAMP-response element binding protein) in PDGF-exposed ASM cells. Overall, these results highlight the alleviating properties of Kp-10 in the context of airway remodeling.

Downregulation of protein phosphatase 2Aα in asthmatic airway smooth muscle.

Airway smooth muscle cell (ASM) is renowned for its involvement in airway hyperresponsiveness through impaired ASM relaxation and bronchoconstriction in asthma, which poses a significant challenge in the field. Recent studies have explored different targets in ASM to alleviate airway hyperresponsiveness, however, a sizeable portion of patients with asthma still experience poor control. In our study, we explored protein phosphatase 2 A (PP2A) in ASM as it has been reported to regulate cellular contractility by controlling intracellular calcium ([Ca2+]i), ion channels, and respective regulatory proteins. We obtained human ASM cells and lung tissues from healthy and patients with asthma and evaluated PP2A expression using RNA-Seq data, immunofluorescence, and immunoblotting. We further investigated the functional importance of PP2A by determining its role in bronchoconstriction using mouse bronchus and human ASM cell [Ca2+]i regulation. We found robust expression of PP2A isoforms in human ASM cells with PP2Aα being highly expressed. Interestingly, PP2Aα was significantly downregulated in asthmatic tissue and human ASM cells exposed to proinflammatory cytokines. Functionally, FTY720 (PP2A agonist) inhibited acetylcholine- or methacholine-induced bronchial contraction in mouse bronchus and further potentiated isoproterenol-induced bronchial relaxation. Mechanistically, FTY720 inhibited histamine-evoked [Ca2+]i response and myosin light chain (MLC) phosphorylation in the presence of interleukin-13 (IL-13) in human ASM cells. To conclude, we for the first time established PP2A signaling in ASM, which can be further explored to develop novel therapeutics to alleviate airway hyperresponsiveness in asthma.NEW & NOTEWORTHY This novel study deciphered the expression and function of protein phosphatase 2Aα (PP2Aα) in airway smooth muscle (ASM) during asthma and/or inflammation. We showed robust expression of PP2Aα in human ASM while its downregulation in asthmatic ASM. Similarly, we demonstrated reduced PP2Aα expression in ASM exposed to proinflammatory cytokines. PP2Aα activation inhibited bronchoconstriction of isolated mouse bronchi. In addition, we unveiled that PP2Aα activation inhibits the intracellular calcium release and myosin light chain phosphorylation in human ASM.

Nicotinic receptors in airway disease.

With continued smoking of tobacco products and expanded use of nicotine delivery devices worldwide, understanding the impact of smoking and vaping on respiratory health remains a major global unmet need. Although multiple studies have shown a strong association between smoking and asthma, there is a relative paucity of mechanistic understanding of how elements in cigarette smoke impact the airway. Recognizing that nicotine is a major component in both smoking and vaping products, it is critical to understand the mechanisms by which nicotine impacts airways and promotes lung diseases such as asthma. There is now increasing evidence that α7 nicotinic acetylcholine receptors (α7nAChRs) are critical players in nicotine effects on airways, but the mechanisms by which α7nAChR influences different airway cell types have not been widely explored. In this review, we highlight and integrate the current state of knowledge regarding nicotine and α7nAChR in the context of asthma and identify potential approaches to alleviate the impact of smoking and vaping on the lungs.

House dust mite-induced asthma exacerbates Alzheimer's disease changes in the brain of the AppNL-G-F mouse model of disease.

Alzheimer's disease (AD) is an age-related neurodegenerative disorder characterized by the accumulation of amyloid-ß (Aß) plaques, neuroinflammation, and neuronal death. Besides aging, various comorbidities increase the risk of AD, including obesity, diabetes, and allergic asthma. Epidemiological studies have reported a 2.17-fold higher risk of dementia in asthmatic patients. However, the molecular mechanism(s) underlying this asthma-associated AD exacerbation is unknown. This study was designed to explore house dust mite (HDM)-induced asthma effects on AD-related brain changes using the AppNL-G-F transgenic mouse model of disease. Male and female 8-9 months old C57BL/6J wild type and AppNL-G-F mice were exposed to no treatment, saline sham, or HDM extract every alternate day for 16 weeks for comparison across genotypes and treatment. Mice were euthanized at the end of the experiment, and broncho-alveolar lavage fluid (BALF), blood, lungs, and brains were collected. BALF was used to quantify immune cell phenotype, cytokine levels, total protein content, lactate dehydrogenase (LDH) activity, and total IgE. Lungs were sectioned and stained with hematoxylin and eosin, Alcian blue, and Masson's trichrome. Serum levels of cytokines and soluble Aß1-40/42 were quantified. Brains were sectioned and immunostained for Aß, GFAP, CD68, and collagen IV. Finally, frozen hippocampi and temporal cortices were used to perform Aß ELISAs and cytokine arrays, respectively. HDM exposure led to increased levels of inflammatory cells, cytokines, total protein content, LDH activity, and total IgE in the BALF, as well as increased pulmonary mucus and collagen staining in both sexes and genotypes. Levels of serum cytokines increased in all HDM-exposed groups. Serum from the AppNL-G-F HDM-induced asthma group also had significantly increased soluble Aß1-42 levels in both sexes. In agreement with this peripheral change, hippocampi from asthma-induced male and female AppNL-G-F mice demonstrated elevated Aß plaque load and increased soluble Aß 1-40/42 and insoluble Aß 1-40 levels. HDM exposure also increased astrogliosis and microgliosis in both sexes of AppNL-G-F mice, as indicated by GFAP and CD68 immunoreactivity, respectively. Additionally, HDM exposure elevated cortical levels of several cytokines in both sexes and genotypes. Finally, HDM-exposed groups also showed a disturbed blood-brain-barrier (BBB) integrity in the hippocampus of AppNL-G-F mice, as indicated by decreased collagen IV immunoreactivity. HDM exposure was responsible for an asthma-like condition in the lungs that exacerbated Aß pathology, astrogliosis, microgliosis, and cytokine changes in the brains of male and female AppNL-G-F mice that correlated with reduced BBB integrity. Defining mechanisms of asthma effects on the brain may identify novel therapeutic targets for asthma and AD.

Kisspeptin regulates airway hyperresponsiveness and remodeling in a mouse model of asthma.

Asthma is a multifactorial disease of origin characterized by airway hyperresponsiveness (AHR) and airway remodeling. Several pieces of evidence from other pathologies suggest that Kisspeptins (Kp) regulate cell proliferation, migration, and invasion, mechanisms that are highly relevant to asthma. Our recent in vitro studies show Kp-10 (active peptide of Kp), via its receptor, KISS1R, inhibits human airway smooth muscle cell proliferation. Here, we hypothesize a crucial role for Kp-10 in regulating AHR and airway remodeling in vivo. Utilizing C57BL/6J mice, we assessed the effect of chronic intranasal Kp-10 exposure on mixed allergen (MA)-induced mouse model of asthma. MA-challenged mice showed significant deterioration of lung function compared to those exposed to vehicle (DPBS); Kp-10 treatment significantly improved the MA-altered lung functions. Mice treated with Kp-10 alone did not show any notable changes in lung functions. MA-exposed mice showed a significant reduction in KISS1R expression as compared to vehicle alone. MA-challenged mice showed significant alterations in immune cell infiltration in the airways and remodeling changes. Proinflammatory cytokines were significantly increased upon MA exposure, an effect abrogated by Kp-10 treatment. Furthermore, biochemical and histological studies showed Kp-10 exposure significantly reduced MA-induced smooth muscle mass and soluble collagen in the lung. Overall, our findings highlight the effect of chronic Kp-10 exposure in regulating MA-induced AHR and remodeling. © 2023 The Pathological Society of Great Britain and Ireland.

Nicotine affects mitochondrial structure and function in human airway smooth muscle cells.

Exposure to cigarette smoke and e-cigarettes, with nicotine as the active constituent, contributes to increased health risks associated with asthma. Nicotine exerts its functional activity via nicotinic acetylcholine receptors (nAChRs), and the alpha7 subtype (α7nAChR) has recently been shown to adversely affect airway dynamics. The mechanisms of α7nAChR action in airways, particularly in the context of airway smooth muscle (ASM), a key cell type in asthma, are still under investigation. Mitochondria have garnered increasing interest for their role in regulating airway tone and adaptations to cellular stress. Here mitochondrial dynamics such as fusion versus fission, and mitochondrial Ca2+ ([Ca2+]m), play an important role in mitochondrial homeostasis. There is currently no information on effects and mechanisms by which nicotine regulates mitochondrial structure and function in ASM in the context of asthma. We hypothesized that nicotine disrupts mitochondrial morphology, fission-fusion balance, and [Ca2+]m regulation, with altered mitochondrial respiration and bioenergetics in the context of asthmatic ASM. Using human ASM (hASM) cells from nonasthmatics, asthmatics, and smokers, we examined the effects of nicotine on mitochondrial dynamics and [Ca2+]m. Fluorescence [Ca2+]m imaging of hASM cells with rhod-2 showed robust responses to 10 µM nicotine, particularly in asthmatics and smokers. In both asthmatics and smokers, nicotine increased the expression of fission proteins while decreasing fusion proteins. Seahorse analysis showed blunted oxidative phosphorylation parameters in response to nicotine in these groups. α7nAChR siRNA blunted nicotine effects, rescuing [Ca2+]m, changes in mitochondrial structural proteins, and mitochondrial dysfunction. These data highlight mitochondria as a target of nicotine effects on ASM, where mitochondrial disruption and impaired buffering could permit downstream effects of nicotine in the context of asthma.NEW & NOTEWORTHY Asthma is a major healthcare burden, which is further exacerbated by smoking. Recognizing the smoking risk of asthma, understanding the effects of nicotine on asthmatic airways becomes critical. Surprisingly, the mechanisms of nicotine action, even in normal and especially asthmatic airways, are understudied. Accordingly, the goal of this research is to investigate how nicotine influences asthmatic airways in terms of mitochondrial structure and function, via the a7nAChR.

Th1 cytokines synergize to change gene expression and promote corticosteroid insensitivity in pediatric airway smooth muscle.

BACKGROUND: Corticosteroids remain a key therapy for treating children with asthma. Patients with severe asthma are insensitive, resistant, or refractory to corticosteroids and have poorly controlled symptoms that involve airway inflammation, airflow obstruction, and frequent exacerbations. While the pathways that mediate corticosteroid insensitivity in asthma remain poorly defined, recent studies suggest that enhanced Th1 pathways, mediated by TNFα and IFNγ, may play a role. We previously reported that the combined effects of TNFα and IFNγ promote corticosteroid insensitivity in developing human airway smooth muscle (ASM). METHODS: To further understand the effects of TNFα and IFNγ on corticosteroid sensitivity in the context of neonatal and pediatric asthma, we performed RNA sequencing (RNA-seq) on human pediatric ASM treated with fluticasone propionate (FP), TNFα, and/or IFNγ. RESULTS: We found that TNFα had a greater effect on gene expression (~ 1000 differentially expressed genes) than IFNγ (~ 500 differentially expressed genes). Pathway and transcription factor analyses revealed enrichment of several pro-inflammatory responses and signaling pathways. Interestingly, treatment with TNFα and IFNγ augmented gene expression with more than 4000 differentially expressed genes. Effects of TNFα and IFNγ enhanced several pro-inflammatory genes and pathways related to ASM and its contributions to asthma pathogenesis, which persisted in the presence of corticosteroids. Co-expression analysis revealed several gene networks related to TNFα- and IFNγ-mediated signaling, pro-inflammatory mediator production, and smooth muscle contractility. Many of the co-expression network hubs were associated with genes that are insensitive to corticosteroids. CONCLUSIONS: Together, these novel studies show the combined effects of TNFα and IFNγ on pediatric ASM and implicate Th1-associated cytokines in promoting ASM inflammation and hypercontractility in severe asthma.

Apelin-Induced Relaxation of Coronary Arteries Is Impaired in a Model of Second-Hand Cigarette Smoke Exposure.

ABSTRACT: Apelin, an endogenous ligand for APJ receptors, causes nitric oxide (NO)-dependent relaxation of coronary arteries. Little is known about the effects of apelin/APJ receptor signaling in the coronary circulation under pathological conditions. Here, we tested the hypothesis that the vasorelaxing effect of apelin is impaired by cigarette smoke extract (CSE), an established model for second-hand smoke exposure. Isolated rat coronary arteries were treated with 2% CSE for 4 hours. Apelin-induced relaxation of coronary arteries was abolished by CSE exposure, while relaxations to acetylcholine (ACh) (endothelium-dependent relaxation) and to diethyl amine NONOate (NO donor) were similar in control and CSE-treated arteries. Immunoblot analysis demonstrated that apelin increased eNOS ser1177 phosphorylation under control conditions but had no effect after exposure to CSE. Moreover, GRK2 expression was increased in CSE-exposed coronary endothelial cells. Pretreatment with CMPD101, a GRK2 inhibitor, improved the relaxation response to apelin in CSE-exposed coronary arteries. CSE treatment failed to inhibit relaxations evoked by CMF-019, an APJ receptor biased agonist that has little effect on GRK2. In arteries exposed to CSE, apelin impaired the response to ACh but not to diethyl amine NONOate. ACh-induced relaxation was unaffected by CMF-019 in either control or CSE-treated coronary arteries. The results suggest that APJ receptor signaling using the GRK2 pathway contributes to both loss of relaxation to apelin itself and the ability of apelin to inhibit endothelium-dependent relaxation to ACh in CSE-exposed coronary arteries, likely because of impaired production of NO from endothelial cells. These changes in apelin/APJ receptor signaling under pathological conditions (eg, exposure to second-hand smoke) could create an environment that favors increased vasomotor tone in coronary arteries.

Glial-derived neurotrophic factor in human airway smooth muscle.

Airway smooth muscle (ASM) cells modulate the local airway milieu via production of inflammatory mediators and growth factors including classical neurotrophins, such as brain-derived neurotrophic factor (BDNF). The glial cell-derived neurotrophic factor (GDNF) family of ligands (GFLs) are nonclassical neurotrophins and their role in the airway is barely understood. The major GFLs, GDNF and Neurturin (NRTN) bind to GDNF family receptor (GFR) α1 and α2 respectively that pair with Ret receptor to accomplish signaling. In this study, we found GDNF is expressed in human lung and increased in adult asthma, while human ASM expresses GDNF and its receptors. Accordingly, we used human ASM cells to test the hypothesis that ASM expression and autocrine signaling by GFLs regulate [Ca2+ ]i . Serum-deprived ASM cells from non-asthmatics were exposed to 10 ng/ml GDNF or NRTN for 15 min (acute) or 24 h (chronic). In fura-2 loaded cells, acute GDNF or NRTN alone induced [Ca2+ ]i responses, and further enhanced responses to 1 µM ACh or 10 µM histamine. Ret inhibitor (SPP86; 10 µM) or specific GDNF chelator GFRα1-Fc (1 µg/ml) showed roles of these receptors in GDNF effects. In contrast, NRTN did not enhance [Ca2+ ]i response to histamine. Furthermore, conditioned media of nonasthmatic and asthmatic ASM cells showed GDNF secretion. SPP86, Ret inhibitor and GFRα1-Fc chelator markedly decreased [Ca2+ ]i response compared with vehicle, highlighting autocrine effects of secreted GDNF. Chronic GDNF treatment increased histamine-induced myosin light chain phosphorylation. These novel data demonstrate GFLs particularly GDNF/GFRα1 influence ASM [Ca2+ ]i and raise the possibility that GFLs are potential targets of airway hyperresponsiveness.

Androgen receptor activation alleviates airway hyperresponsiveness, inflammation, and remodeling in a murine model of asthma.

Epidemiological studies demonstrate an apparent sex-based difference in the prevalence of asthma, with a higher risk in boys than girls, which is reversed postpuberty, where women become more prone to asthma than men, suggesting a plausible beneficial role for male hormones, especially androgens as a regulator of pathophysiology in asthmatic lungs. Using a murine model of asthma developed with mixed allergen (MA) challenge, we report a significant change in airway hyperresponsiveness (AHR), as demonstrated by increased thickness of epithelial and airway smooth muscle layers and collagen deposition, as well as Th2/Th17-biased inflammation in the airways of non-gonadectomized (non-GDX) and gonadectomized (GDX) male mice. Here, compared with non-GDX mice, MA-induced AHR and inflammatory changes were more prominent in GDX mice. Activation of androgen receptor (AR) using 5α-dihydrotestosterone (5α-DHT, AR agonist) resulted in decreased Th2/Th17 inflammation and remodeling-associated changes, resulting in improved lung function compared with MA alone challenged mice, especially in GDX mice. These changes were not observed with Flutamide (Flut, AR antagonist). Overall, we show that AR exerts a significant and beneficial role in asthma by regulating AHR and inflammation.

Nicotinic α7 acetylcholine receptor (α7nAChR) in human airway smooth muscle.

Diseases such as asthma are exacerbated by inflammation, cigarette smoke and even nicotine delivery devices such as e-cigarettes. However, there is currently little information on how nicotine affects airways, particularly in humans, and changes in the context of inflammation or asthma. Here, a longstanding assumption is that airway smooth muscle (ASM) that is key to bronchoconstriction has muscarinic receptors while nicotinic receptors (nAChRs) are only on airway neurons. In this study, we tested the hypothesis that human ASM expresses α7nAChR and explored its profile in inflammation and asthma using ASM of non-asthmatics vs. mild-moderate asthmatics. mRNA and western analysis showed the α7 subunit is most expressed in ASM cells and further increased in asthmatics and smokers, or by exposure to nicotine, cigarette smoke or pro-inflammatory cytokines TNFα and IL-13. In these effects, signaling pathways relevant to asthma such as NFκB, AP-1 and CREB are involved. These novel data demonstrate the expression of α7nAChR in human ASM and suggest their potential role in asthma pathophysiology in the context of nicotine exposure.

Sex-Steroid Signaling in Lung Diseases and Inflammation.

Sex/gender difference exists in the physiology of multiple organs. Recent epidemiological reports suggest the influence of sex-steroids in modulating a wide variety of disease conditions. Sex-based discrepancies have been reported in pulmonary physiology and various chronic inflammatory responses associated with lung diseases like asthma, chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, and rare lung diseases. Notably, emerging clinical evidence suggests that several respiratory diseases affect women to a greater degree, with increased severity and prevalence than men. Although sex-specific differences in various lung diseases are evident, such differences are inherent to sex-steroids, which are major biological variables in men and women who play a central role to control these differences. The focus of this chapter is to comprehend the sex-steroid biology in inflammatory lung diseases and to understand the mechanistic role of sex-steroids signaling in regulating these diseases. Exploring the roles of sex-steroid signaling in the regulation of lung diseases and inflammation is crucial for the development of novel and effective therapy. Overall, we will illustrate the importance of differential sex-steroid signaling in lung diseases and their possible clinical implications for the development of complementary and alternative medicine to treat lung diseases.

Neurotrophin Regulation and Signaling in Airway Smooth Muscle.

Structural and functional aspects of bronchial airways are key throughout life and play critical roles in diseases such as asthma. Asthma involves functional changes such as airway irritability and hyperreactivity, as well as structural changes such as enhanced cellular proliferation of airway smooth muscle (ASM), epithelium, and fibroblasts, and altered extracellular matrix (ECM) and fibrosis, all modulated by factors such as inflammation. There is now increasing recognition that disease maintenance following initial triggers involves a prominent role for resident nonimmune airway cells that secrete growth factors with pleiotropic autocrine and paracrine effects. The family of neurotrophins may be particularly relevant in this regard. Long recognized in the nervous system, classical neurotrophins such as brain-derived neurotrophic factor (BDNF) and nonclassical ligands such as glial-derived neurotrophic factor (GDNF) are now known to be expressed and functional in non-neuronal systems including lung. However, the sources, targets, regulation, and downstream effects are still under investigation. In this chapter, we discuss current state of knowledge and future directions regarding BDNF and GDNF in airway physiology and on pathophysiological contributions in asthma.

Estrogen receptors differentially regulate intracellular calcium handling in human nonasthmatic and asthmatic airway smooth muscle cells.

Asthma is defined as chronic inflammation of the airways and is characterized by airway remodeling, hyperresponsiveness, and acute bronchoconstriction of airway smooth muscle (ASM) cells. Clinical findings suggest a higher incidence and severity of asthma in adult women, indicating a concrete role of sex steroids in modulating the airway tone. Estrogen, a major female sex steroid mediates its role through estrogen receptors (ER) ERα and ERß, which are shown to be expressed in human ASM, and their expression is upregulated in lung inflammation and asthma. Previous studies suggested rapid, nongenomic signaling of estrogen via ERs reduces intracellular calcium ([Ca2+]i), thereby promoting relaxation of ASM. However, long-term ER activation on [Ca2+]i regulation in human ASM during inflammation or in asthma is still not known. In Fura-2-loaded nonasthmatic and asthmatic human ASM cells, we found that prolonged (24 h) exposure to ERα agonist (PPT) increased [Ca2+]i response to histamine, whereas ERß activation (WAY) led to decreased [Ca2+] compared with vehicle. This was further confirmed by ER overexpression and knockdown studies using various bronchoconstrictor agents. Interestingly, ERß activation was more effective than 17ß-estradiol in reducing [Ca2+]i responses in the presence of TNF-α or IL-13, while no observable changes were noticed with PPT in the presence of either cytokine. The [Ca2+]i-reducing effects of ERß were mediated partially via L-type calcium channel inhibition and increased Ca2+ sequestration by sarcoplasmic reticulum. Overall, these data highlight the differential signaling of ERα and ERß in ASM during inflammation. Specific ERß activation reduces [Ca2+]i in the inflamed ASM cells and is likely to play a crucial role in regulating ASM contractility, thereby relaxing airways.

Sex steroids skew ACE2 expression in human airway: a contributing factor to sex differences in COVID-19?

The incidence, severity, and mortality of ongoing coronavirus infectious disease 19 (COVID-19) is greater in men compared with women, but the underlying factors contributing to this sex difference are still being explored. In the current study, using primary isolated human airway smooth muscle (ASM) cells from normal males versus females as a model, we explored the effect of estrogen versus testosterone in modulating the expression of angiotensin converting enzyme 2 (ACE2), a cell entry point for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Using confocal imaging, we found that ACE2 is expressed in human ASM. Furthermore, Western analysis of ASM cell lysates showed significantly lower ACE2 expression in females compared with males at baseline. In addition, ASM cells exposed to estrogen and testosterone for 24 h showed that testosterone significantly upregulates ACE2 expression in both males and females, whereas estrogen downregulates ACE2, albeit not significant compared with vehicle. These intrinsic and sex steroids induced differences may help explain sex differences in COVID-19.

Differential estrogen-receptor activation regulates extracellular matrix deposition in human airway smooth muscle remodeling via NF-κB pathway.

Altered airway smooth muscle (ASM) mass and extracellular matrix (ECM) deposition in airways are characteristic features of remodeling in asthma. Increased ECM production modulates ASM cell proliferation and leads to airway remodeling. Our previous studies showed that ASM from patients with asthma exhibited increased expression of estrogen receptor (ER)-ß, which upon activation down-regulated ASM proliferation, implicating an important role for estrogen signaling in airway physiology. There is no current information on the effect of differential ER activation on ECM production. In this study, we evaluated the effect of ER-α vs. ER-ß activation on ECM production, deposition, and underlying pathways. Primary human ASM cells isolated from asthmatics and nonasthmatics were treated with E2, an ER-α agonist [propylpyrazoletriol (PPT)], and an ER-ß agonist [WAY-200070 (WAY)] with TNF-α or platelet-derived growth factor (PDGF) followed by evaluation of ECM production and deposition. Expression of proteins and genes corresponding to ECM were measured using Western blotting and quantitative RT-PCR with subsequent matrix metalloproteinase (MMP) activity. Molecular mechanisms of ER activation in regulating ECM were evaluated by luciferase reporter assays for activator protein 1 (AP-1) and NF-κB. TNF-α or PDGF significantly (P < 0.001) increased ECM deposition and MMP activity in human ASM cells, which was significantly reduced with WAY treatment but not with PPT. Furthermore, TNF-α- or PDGF-induced ECM gene expression in ASM cells was significantly reduced with WAY (P < 0.001). Moreover, WAY significantly down-regulated the activation of NF-κB (P < 0.001) and AP-1 (P < 0.01, P < 0.05) in ASM cells from asthmatics and nonasthmatics. Overall, we demonstrate differential ER signaling in controlling ECM production and deposition. Activation of ER-ß diminishes ECM deposition via suppressing the NF-κB pathway activity and might serve as a novel target to blunt airway remodeling.-Ambhore, N. S., Kalidhindi, R. S. R., Pabelick, C. M., Hawse, J. R., Prakash, Y. S., Sathish, V. Differential estrogen-receptor activation regulates extracellular matrix deposition in human airway smooth muscle remodeling via NF-κB pathway.

Hypoxia-Responsive, Polymeric Nanocarriers for Targeted Drug Delivery to Estrogen Receptor-Positive Breast Cancer Cell Spheroids.

Uncontrolled cell growth, division, and lack of enough blood supply causes low oxygen content or hypoxia in cancerous tumor microenvironments. 17ß-Estradiol (E2), an estrogen receptor (ER) ligand, can be incorporated on the surface of nanocarriers for targeted drug delivery to breast cancer cells overexpressing ER. In the present study, we synthesized estradiol-conjugated hypoxia-responsive polymeric nanoparticles (polymersomes) encapsulating the anticancer drug doxorubicin (E2-Dox-HRPs) for targeted delivery into the hypoxic niches of estrogen-receptor-positive breast cancer microtumors. Estradiol-conjugated polymersomes released over 90% of their encapsulated Dox in a sustained manner within hypoxia (2% oxygen) after 12 h. However, they released about 30% of Dox in normal oxygen partial pressure (21% oxygen, normoxia) during this time. Fluorescence microscopic studies demonstrated higher cytosolic and nuclear internalization of E2-Dox-HRPs (targeted polymersomes) compared to those of Dox-HRPs (nontargeted polymersomes). Monolayer cell viability studies on ER-positive MCF7 cells showed higher cytotoxicity of targeted polymersomes in hypoxia compared to in normoxia. Cytotoxicity studies with hypoxic three-dimensional spheroid cultures of MCF7 cells treated with targeted polymersomes indicated significant differences compared to those of normoxic spheroids. The novel estradiol-conjugated hypoxia-responsive polymersomes described here have the potential for targeted drug delivery in estrogen-receptor-positive breast cancer therapy.

Growth inhibitory and anti-metastatic activity of epithelial cell adhesion molecule targeted three-way junctional delta-5-desaturase siRNA nanoparticle for breast cancer therapy.

8-Hydroxyoctanoic acid (8-HOA) produced through cyclooxygenase-2 (COX-2) catalyzed dihomo-γ-linolenic acid (DGLA) peroxidation in delta-5-desaturase inhibitory (D5D siRNA) condition showed an inhibitory effect on breast cancer cell proliferation and migration. However, in vivo use of naked D5D siRNA was limited by off-target silencing and degradation by endonucleases. To overcome the limitation and deliver the D5D siRNA in vivo, we designed an epithelia cell adhesion molecule targeted three-way junctional nanoparticle having D5D siRNA. In this study, we have hypothesized that 3WJ-EpCAM-D5D siRNA will target and inhibit the D5D enzyme in cancer cells leading to peroxidation of supplemented DGLA to 8-HOA resulting in growth inhibitory effect in the orthotopic breast cancer model developed by injecting 4T1 cells. On analysis, we observed a significant reduction in tumor size and metastatic lung nodules in animals treated with a combination of 3WJ-EpCAM-D5D siRNA and DGLA through activating intrinsic apoptotic signaling pathway and by reducing endothelial-mesenchymal damage.

Role of Differential Estrogen Receptor Activation in Airway Hyperreactivity and Remodeling in a Murine Model of Asthma.

Evidence suggests that airway hyperresponsiveness (AHR) is a characteristic feature of asthma. Epidemiological studies have confirmed that the severity of asthma is greater in women, suggesting a critical role of female sex steroid hormones (especially estrogen). Very few in vivo studies have examined the role of sex steroid hormones in asthma, and the sequence of events that occur through differential activation of estrogen receptors (ERs) remains to be determined in asthmatic airways. Our recent in vitro findings indicated that ERß had increased expression in asthmatic airway smooth muscle (ASM), and that its activation by an ERß-specific agonist downregulated airway remodeling. In this study, we translated the in vitro findings to a murine asthma model and examined the differential role of ER activation in modulating lung mechanics. C57BL/6J male, female, and ovariectomized mice were exposed to mixed allergen (MA) and subcutaneously implanted with sustained-release pellets of placebo, an ERα agonist (4,4',4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol [PPT]), and/or an ERß agonist (WAY-200070). We then evaluated the effects of these treatments on airway mechanics, biochemical, molecular, and histological parameters. Mice exposed to MA showed a significant increase in airway resistance, elastance, and tissue damping, and a decrease in compliance; pronounced effects were observed in females. Compared with PPT, WAY treatment significantly reversed the MA-induced changes. The increased mRNA/protein expression of ERα, ERß, and remodeling genes observed in MA-treated mice was significantly reversed in WAY-treated mice. This novel study indicates that activation of ERß signaling downregulates AHR and airway remodeling, and is a promising target in the development of treatments for asthma.

Androgen Receptor-Mediated Regulation of Intracellular Calcium in Human Airway Smooth Muscle Cells.

BACKGROUND/AIMS: With the prevalence of asthma being greater in women, detrimental effects of female sex steroids have been explored, but potential protective effects of androgens are not established. Airway smooth muscle (ASM) is a key cell type in contractility and remodelling of asthma. There are no data on expression and functionality of androgen receptor (AR) in human ASM cells. METHODS: We used primary human ASM cells from non-asthmatics vs. asthmatics to determine AR expression at baseline and with inflammation measured using Western blotting/qRT-PCR, and the role of AR in regulating intracellular Ca2+ ([Ca2+]i) measured using Fluo-3 loaded real time [Ca2+]i imaging. RESULTS: We found that compared to females, baseline AR is greater in male ASM and increases with inflammation/asthma. Androgens, via AR, blunted TNFα or IL-13-induced enhancement of ASM [Ca2+]i in both males and females, with retained efficacy in asthmatics. AR effects involve reduced Ca2+ influx via L-type channels and store-operated Ca2+ entry, the latter by downregulating STIM1 and Orai1 and increasing TMEM66. CONCLUSION: Our data show AR expression is increased in female ASM with asthma, but has retained functionality that could be used to reduce [Ca2+]i towards alleviating airway hyperresponsiveness.