Y S. Prakash - US grants

DESCRIPTION: Cardiac depression is more pronounced in the neonatal heart than in the adult organ. The studies described in this project are designed to achieve the overall goal of identifying the mechanism underlying age-related (neonate versus adult) differences in the cardiac depressant effects of volatile anesthetics. This proposal addresses two of the three possible factors altering cardiac function: intracellular calcium and the sensitivity of the contractile apparatus to intracellular calcium. Using an adult versus neonatal rat model, the mechanisms underlying the effect of volatile anesthetics on 1) intracellular calcium regulation related specifically to influx through the L-type channel sand calcium release from the sarcoplasmic reticulum, and 2) Ca-sensitivity of force generation will be addressed, with the underlying hypothesis that both factors contribute to cardiodepression secondary to volatile anesthetics.

Airway hyperreactivity (AHR) and remodeling in asthma involve increased airway smooth muscle (ASM) contractility, mass, and extracellular matrix (ECM) driven by inflammation. ASM actively secretes growth factors that modulate airway structure/function via autocrine/paracrine influences. In previous cycles, we identified brain-derived neurotrophic factor (BDNF) as an ASM-derived factor with autocrine enhancement of ASM contractility, proliferation and fibrosis. Within this purview, we discovered glial-derived neurotrophic factor (GDNF) and a related member neurturin (NRTN) as novel growth factors in the airway that promote inflammation effects. GDNF and NRTN have protective roles in the nervous system but there is minimal to no information on GDNF or NRTN in airway biology or asthma, particularly for ASM. Preliminary studies show that A) Human ASM expresses and secretes GDNF and NRTN in response to agonist, with increased release by TNF? or TGF? and in asthmatic ASM; B) GDNF and NRTN receptors Ret, GFR?1 and GFR?2 are present in ASM with increased expression in inflammation/asthma; C) Exogenous GDNF and NRTN have pleiotropic effects on ASM, enhancing [Ca2+]cyt and contractility, promoting ECM formation, and intriguingly ER stress, mitochondrial fission, mitochondrial Ca2+ and respiration; D) GDNF and NRTN can interact via GFR?1. In vivo studies in mixed allergen (MA) mouse models of asthma show 1) GDNF enhances airway reactivity; 2) Ret inhibition or chelation of GDNF blunt MA effects on AHR and remodeling. Based on these data, we propose an overall hypothesis that ASM expression and autocrine signaling by GDNF ligand family contributes to AHR and remodeling in asthma. We will test this concept via four Aims, focusing particularly on the novel role of ASM-derived GDNF and NRTN. Our Aims are: Aim 1: To examine mechanisms of upstream regulation of GDNF vs. NRTN in human ASM; Aim 2: To examine mechanisms by which GDNF vs. NRTN enhance Ca2+/contractility in human ASM in the context of inflammation and asthma; Aim 3: To examine mechanisms by which GDNF vs. NRTN enhance remodeling in human ASM in the context of inflammation and asthma; Aim 4: To examine in vivo importance of GDNF vs. NRTN in the context of AHR and remodeling using a mixed allergen mouse model of asthma. Aims 1-3 utilize human epithelium-denuded ASM tissues and isolated ASM cells from mild or moderate asthmatics vs. non-asthmatics to examine signaling mechanisms by which inflammatory mediators enhance GDNF/NRTN production (Aim 1), the receptor and intracellular pathways by which these ligands influence contractility (Aim 2) vs. ER stress, mitochondrial structure/function and proliferation/ECM (Aim 3). Aim 4 applies the MA model to mice where GDNF vs. NRTN is enhanced or inhibited, particularly in smooth muscle and explores changes in airway structure, ECM composition, and mechanics. Clinical significance lies in establishing the role of ASM-derived growth factors such as GDNF or NRTN that influence multiple aspects of asthma pathophysiology and are appealing therapeutic targets.

ABSTRACT Supplemental oxygen (hyperoxia) with/without continuous nasal positive airway pressure (CPAP) to preterm infants is associated with airway hyperreactivity (AHR) proceeding to wheezing and asthma. Understanding mechanisms by which hyperoxia and CPAP induce sustained AHR represents our long-term goal, and an unmet clinical need. We propose the initial stretch imposed by CPAP on more compliant bronchial airways in premature infants, particularly with added hyperoxia, is contributory. AHR involves greater [Ca2+]i and contractility of airway smooth muscle (ASM), and remodeling mediated partly by ASM proliferation. Our published studies and preliminary work using human fetal airway cells and neonatal mouse models show moderate hyperoxia (50% O2) and mechanical stretch not only enhance ASM contractility and proliferation, but also bronchial epithelial arginase, raising the question of whether and how epithelial arginase and neonatal AHR are linked. We propose the novel and intriguing idea that arginase-derived polyamines (e.g. spermine) have downstream effects on a novel ASM target: extracellular Ca2+ sensing receptor (CaSR). Although well-known for regulating body Ca2+, there is limited information on CaSR in lung, and none in postnatal airways. Preliminary data in developing human airways show high expression of ASM CaSR that responds to extracellular Ca2+ ([Ca2+]o) or spermine, and enhances [Ca2+]I, contractility and proliferation. Hyperoxia and stretch each increase CaSR expression and function. In neonatal mice exposed to 50% O2 and/or CPAP which show sustained AHR, epithelial arginase and ASM CaSR are increased, while inhibitors of arginase (nor-NOHA) and CaSR (calcilytic NPS2143) blunt AHR. Our overall hypothesis is that the epithelial arginase-ASM CaSR axis contributes to AHR in the context of hyperoxia and CPAP exposure in prematurity. We will examine this idea via 3 Aims. Aim 1: In developing bronchial epithelium, determine the effect of hyperoxia and/or stretch on the arginase pathway; Aim 2: In developing ASM, determine the role of CaSR in enhanced [Ca2+]i/contractility and proliferation induced by hyperoxia and/or stretch; Aim 3: In neonatal mouse models of hyperoxia and/or CPAP exposure, determine the role of the arginase-CaSR axis in AHR and airway remodeling. 18-22 week human fetal bronchial epithelial cells (fBECs) and ASM cells (fASM) are exposed to hyperoxia (<60% O2) +/- 0-15% stretch (representing 0 to high CPAP) with continuous 5% oscillations, mimicking clinical hyperoxia +/- CPAP in spontaneously breathing premies. fBEC arginase pathway and downstream effects (Aim 1), and role of fASM CaSR in [Ca2+]i/contractility and proliferation following hyperoxia +/- stretch (Aim 2) are examined, with signaling pathways such as RhoA/Rho kinase and MAPKs. In Aim 3, airway structural, functional and molecular changes are assessed in neonatal WT and smooth muscle CaSR KO mice exposed to 50% O2 +/- intermittent CPAP (3, 6, 9 cmH2O) for 7 days, with 14 days recovery (mimicking human conditions). Effects of inhibiting arginase (nor-NOHA) vs. CaSR (NPS2143) are tested towards establishing clinical significance.

? DESCRIPTION (provided by applicant): The proposed studies will address fundamental questions regarding the mechanisms underlying inflammation-induced enhancement of both hyperactive (contractile) and proliferative (synthetic) states of human airway smooth muscle (hASM), which are hallmarks of asthma. Identifying these mechanisms is the key to developing novel therapeutic targets for asthma. Our central hypothesis is that in hASM, inflammatory cytokines induce sarco-endoplasmic reticulum (SR/ER) stress leading to reduced expression of the mitochondrial fusion protein mitofusin 2 (Mfn2), and that this pathway plays a central role in asthma by triggering both hyper reactive (contractile) and proliferative (synthetic) states. Four Specific Aims are proposed: Specific Aim 1: To determine the impact of inflammatory cytokines on SR/ER stress, Mfn2 expression and mitochondrial fragmentation. In this aim, we will use dissociated hASM cells and tissue from normal and asthmatic patients to test the hypothesis that inflammatory cytokines trigger SR/ER stress due at least in part to an increase in ROS generation, and that SR/ER stress leads to reduced Mfn2 expression and increased mitochondrial fragmentation. Specific Aim 2: To determine the functional impact of SR/ER stress and reduced Mfn2 expression. In this aim, we will use dissociated hASM cells and tissue from normal and asthmatic patients to test the hypothesis that in hASM, inflammatory cytokine-induced SR/ER stress and reduced Mfn2 expression uncouples mitochondria and the SR/ER, thereby reducing mitochondrial Ca2+ buffering leading to elevated [Ca2+]cyt and force responses to agonist stimulation. Specific Aim 3: To determine the impact of SR/ER stress and reduced Mfn2 on hASM cell proliferation. In this aim, we will use dissociated hASM cells and tissue from normal and asthmatic patients to test the hypothesis that inflammatory cytokines increase hASM cell proliferation as a result of decreased Mfn2 expression. Specific Aim 4: To determine the efficacy of therapeutic approaches targeting SR/ER stress in alleviating airway hyper reactivity and remodeling in a mouse model of asthma. In this aim, we will use a mixed allergen mouse model to test the hypothesis that targeting SR/ER stress using chemical chaperones (e.g., 4-PBA, TUDCA) will provide an effective therapeutic strategy to reverse airway hyperactivity and blunt remodeling associated with asthma.

The Department of Physiology & Biomedical Engineering (BME) at Mayo Clinic has a long and rich history of preparing pre- and postdoctoral students for academic careers in a biomedical research environment that is increasingly more technological and complex. We strongly believe that a training grant that takes the novel approach of encouraging and nurturing biomedical research skills alongside computational, mathematical and engineering skills will create a unique cadre of future leaders in biomedical research related to lung disease. Under the auspices of the Training Program in Lung Physiology and Biomedical Engineering we successfully implemented a multifaceted program to train the next generation of biomedical researchers in lung physiology and disease. We recruited highly competitive predoctoral and postdoctoral trainees from different backgrounds (including clinicians (MDs)) who highlighted the success of our multidisciplinary approach. Accordingly, in this first renewal of our T32, the primary objectives of the training program will continue to be to train three groups of trainees for biomedical research careers in lung physiology and disease: 1) Predoctoral PhD (or MD/PhD) students with undergraduate backgrounds in engineering, mathematics or physics; 2) Postdoctoral PhD scientists with backgrounds in engineering, mathematics, physics or basic biomedical sciences; and 3) Postdoctoral MD or MD/PhD clinician-scientists. To achieve our objectives, we are requesting support for 2 predoctoral students (PhD or MD/PhD) and 6 postdoctoral trainees (with PhD and/or MD). From a pool of highly competitive eligible candidates with diverse backgrounds, we plan to recruit: 1) Predoctoral students via the Mayo Graduate (PhD students) and Medical (MD/PhD students) Schools; 2) Postdoctoral PhD scientists from applicants working in or applying to faculty laboratories; and 3) Postdoctoral MD or MD/PhD clinician-scientists from the large pool of residents or fellows from participating clinical departments (especially Anesthesiology, Pulmonary/Critical Care, Radiology and Surgery). A total of 20 highly-funded faculty mentors were selected based on their outstanding pre- and postdoctoral training records and their abilities to support trainees via extramural funding. The program is jointly led by Drs. Gary Sieck, PhD (Prof of Physiology, BME, and Anesthesiology; contact PI) and Y.S. Prakash, MD, PhD (Professor and Chair of Physiology and BME, Vice-Chair, Anesthesiology; Co-PI) who have extensive experience in biomedical research and demonstrated leadership in graduate and postgraduate education as well as administration at departmental, institutional and national levels. Individual trainee needs are met by common formal didactic program in lung physiology and BME, customized elective coursework, and training in writing manuscripts, grant applications, presentations, professional networking and interview skills. Success of the training program will be determined by retention and placement of trainees in academia at all levels of career development and ultimately as established, extramurally-funded biomedical researchers.

ABSTRACT 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.

The impact of acute airway inflammation is mediated by pro-inflammatory cytokines (e.g., TNF?), and underlies a number of respiratory diseases. A fundamental question is why are some individuals more susceptible than others to the negative impact of airway inflammation. We will explore a novel homeostatic mechanism, which protects airway smooth muscle (hASM) cells from the negative impact of inflammation-induced reactive oxygen species (ROS) formation and protein unfolding (endoplasmic reticulum (ER) stress). We believe that a failure in this homeostatic mechanism leads to increased ROS formation thereby exacerbating oxidative and ER stress. Overall Hypothesis: TNF?-induced ROS formation and protein unfolding activates the pIRE1?/XBP1s ER stress pathway in hASM, which initiates a homeostatic response directed towards increasing mitochondrial biogenesis and mitochondrial volume density to reduce O2 consumption and ROS formation by individual mitochondrion, while still meeting the increase in ATP demand ? sharing the energetic load across mitochondria. Furthermore, reduced Mfn2 disrupts mitochondrial tethering to the ER, thereby decreasing mitochondrial Ca2+ influx and maximum respiratory capacity of mitochondria. Aim 1: TNF?-induced activation of pIRE1?/XBP1s ER stress pathway increases mitochondrial volume density and reduces O2 consumption and ROS formation per mitochondrion. In hASM cells, the downstream impact of TNF?-induced activation of the pIRE1?/XBP1s ER stress pathway will be explored using transfection of a non-phosphorylatable IRE1? mutant plasmid (DP-IRE1?) or an unspliceable XBP1 (uXBP1) mRNA. In addition, we will examine the effects of siRNA knockdown of PGC1? and Mfn2 overexpression on TNF?-induced changes in mitochondrial biogenesis, mitochondrial volume density, O2 consumption and ROS formation. Aim 2: TNF?-induced reduction in Mfn2 disrupts mitochondrial tethering to ER, decreases mitochondrial Ca2+ influx and reduces maximum respiratory capacity of mitochondria. In hASM cells, we will examine the impact of DP-IRE1? or uXBP1 mRNA transfection and siRNA Mfn2 knockdown on TNF?-induced disruption of mitochondrial/ ER tethering, decreased mitochondrial Ca2+ influx and reduced maximum respiratory capacity of mitochondria. Aim 3: The impact of TNF? on activation of the pIRE1?/XBP1s ER stress pathway and downstream effects are mitigated by ROS scavenging and chemical chaperone treatment. In hASM cells, the mitigating effects of ROS scavenging and chemical chaperone treatment on TNF?-induced activation of the pIRE1?/XBP1s ER stress pathway will be examined.