While e-cigarettes are advertised as safer alternatives to smoking for asthmatics, there is increasing evidence of their pulmonary toxicity, necessitating better understanding of airway nicotine biology: a clinically-relevant unmet need, and major goal of this grant. In asthma, inflammation enhances airway smooth muscle (ASM) Ca2+ ([Ca2+]cyt) and contractility (airway hyperreactivity; AHR), along with remodeling involving ASM proliferation. Given that inhaled nicotine can directly influence ASM, nicotine effects on asthmatic ASM are critical, but little is known about nicotinic receptor (nAChR) expression, its role in human ASM, or in asthma. Preliminary data in human ASM show functional ?7 nAChRs that A) increase with inflammation and asthma, B) enhance ASM [Ca2+]cyt/contractility; C) enhance ER stress and mitochondrial fission, and respiration, and D) activate pro-proliferative pathways. Such effects are observed in a mixed-allergen mouse model of asthma exposed to acute or chronic inhaled nicotine, but blunted by ?7 inhibition and in ?7 KO mice. Thus, our hypothesis is that nicotine acts via ASM ?7 to promote AHR and remodeling in asthma.
Our Aims are:
Aim 1 : : In human ASM, determine mechanisms of ?7 expression and regulation in inflammation and asthma.
Aim 2 : In human ASM of non-asthmatics vs. asthmatics, to determine mechanisms by which ?7 contributes to nicotine enhancement of [Ca2+]cyt and contractility in the context of AHR.
Aim 3 : In human ASM of non-asthmatics vs. asthmatics, to determine mechanisms by which ?7 contributes to nicotine enhancement of cell proliferation.
Aim 4 : In a mouse model of allergic asthma, to determine the role of ?7 in effects of inhaled nicotine on AHR and remodeling. Studies use adult human ASM of non-asthmatics vs. mild-moderate asthmatics exposed to cytokines relevant to ASM and to asthma (TNF?, IL-6, IL-13), with/without nicotine.
Aim 1 explores expression and localization of ?7 nAChR, regulatory chaperones, and mechanisms by which ?7 is increased in inflammation/asthma (e.g. MAPKs, PI3/Akt, NF?B, Stats). ?7 functionality as a channel is tested using electrophysiology.
Aim 2 explores acute vs. chronic nicotine effects on [Ca2+]cyt regulation (influx, SR Ca2+ release) and contractility (traction force, organ bath).
Aim 3 explores nicotine effects on ASM proliferation, and the roles of ER stress, mitochondrial fission (Drp1, Fis1) vs. fusion (Mfn1/2, Opa1) and respiration (3a), and of cytokine-associated proliferative pathways (3b). In these Aims, role of ?7 is determined using broad vs. subunit- specific agonists, antagonists or siRNAs. In vitro results are integrated in Aim 4 using the adult mouse model of asthma with/without acute vs. chronic inhaled nicotine. Alleviating effects of ?7 inhibitor (MG624) and effects of ?7 KO in smooth muscle are tested. Studies assess airway reactivity, remodeling, and ASM biochemical changes. Clinical significance lies in identifying ASM ?7 as a novel target for alleviating AHR and remodeling of asthma as well as with nicotine.
With increasing use of nicotine delivery devices such as e-cigarettes that are advertised as safer for asthmatics, understanding the effects of inhaled nicotine on airways is highly relevant. However, the mechanisms of nicotine action even in normal airways are understudied, and unknown in asthma. Using human bronchial cells from asthmatics vs. non-asthmatics, and mouse models of asthma, we propose to test the novel idea that nicotinic acetylcholine receptors on airway smooth muscle contribute to increased airway reactivity and remodeling of asthma, thus identifying a potentially new avenue for therapeutic targeting.
