ERS SHORT-TERM RESEARCH TRAINING FELLOWSHIP

REYNAERT Niki

Fellowship Number STRTF 178-2010

ERS SHORT-TERM RESEARCH TRAINING FELLOWSHIPS APPLICATION

No. STRTF 178-2010 – Dr. Niki REYNAERT Section 1 – Fellowship Sought

Fellowship number: STRTF 178-2010

Title of proposed project: Stretch as a key mediator of oxidant production and inflammation in COPD

ERS Fellowship programme: STRTF

Keywords: stretch, oxidant, S-glutathionylation, inflammation

Expected starting date: 01.06.2011

Expected finishing date: 31.08.2011

Do you intend to return home after the Fellowship?

Yes

What are you plans after the Fellowship?

I will keep my post-doc appointment at Maastricht University during the fellowship. The methodology and established collaboration through this fellowship will be used in future grants to further develop my own research line within the department of Respiratory Medicine at the Maastricht University Medical Centre. I currently have my own funding until May of next year and I am a supervisor of multiple graduate students as can be seen in my cv.

Section 2 – Applicant personal details

Title:

Last name:

Dr.

REYNAERT

First names: Niki

Gender Female

Date of birth: 04.04.1979

Nationality: BELGIUM

Present position: post-doc

Since when: 2006

Name and address of the home institution:

Maastricht University Medical Centre Dept of Respiratory Medicine PO Box 616 6200AZ Maastricht

Country: NETHERLANDS

Telephone: -433882239

E-mail: n.reynaert@pul.unimaas.nl

ERS membership number: 113164

Are you now based in your Home country?:

Yes

Home/Host address: Weg naar Geneuth 95 3632 Maasmechelen Belgium

Since 01.04.2006 Section 3 – Home supervisor

Title:

Last name:

Prof.

WOUTERS

First names: Emiel FM

Present position: Head of Department of Respiratory Medicine, Maastricht University Medical Centre, the Netherlands

Since when: 1992 Name and address of the home institution:

Maastricht University Medical Centre NUTRIM school for nutrition, toxicology and metabolism Department of Respiratory Medicine PO Box 616 6200AZ Maastricht the Netherlands

Country: NETHERLANDS

Telephone: -433875013

Fax: -433875020

E-mail: e.wouters@mumc.nl

ERS member? Yes

ERS membership number: 12804 Section 4 – Host supervisor

Title:

Last name:

Prof.

BRIGHTLING

First names: Christopher

Present position: Professor of Respiratory Medicine, Wellcome Senior Clinical Research Fellow, honorary consultant, University of Leicester

Since when: 01.11.2007

Name and address of the home institution:

Department of Respiratory Medicine, Department of Infection, Immunity and Inflammation Glenfield Hospital Groby Road Glenfield Leicester LE3 9QP, UK

Country: UNITED KINGDOM

Telephone: +44 116 256 3998

Fax:

E-mail: ceb17@le.ac.uk

ERS member? Yes

ERS membership number: 71256

Section 5 – Professional qualifications and experience of the applicant

Postdoctoral researcher since:

2006

PhD holder since: 2006

MSc holder since 2001

MD holder since --

Other:

List degrees/diplomas/field of study/ years in which obtained and name of institutes):

*PhD cum laude from Maastricht University, the Netherlands in 2006, Thesis: Redox regulation of Inhibitory Kappa B Kinase. Oxidants in the pathogenesis of inflammatory lung disease. *m.sc. magna cum laude from Free University of Brussels, Belgium in 2001, Thesis: The role of hyaluronan fragments in chronic inflammation and lung pathology: studies into the pathogenesis of COPD.

Number of years fulltime research experience

9

Please provide other information on your research experience (part time, full time, while working, while studying, etc.)

*May 2006 - current: full time post-doctoral researcher at Maastricht University (except from Jan 2007 -April 2008 0.8 fte due to parent leave) *sept 2001- April 2006: PhD student at Maastricht University, the Netherlands - performed full-time research at University of Vermont, VT, US

Number of years professional experience (list: years, position, name of employer)

*May 2006 - current: full time post-doctoral researcher at Maastricht University (except from Jan 2007 -April 2008 0.8 fte due to parent leave)

Last two positions:

Position 1

Name of employer

From-To:

post doctoral researcher

Maastricht University Medical Centre+, Nutrim school for nutrition, toxicology and metabolism, Dept of Respiratory Medicine, PO Box 616, 6200 AZ Maastricht, the Netherlands

05-2006 to current

Do you have 1 first author publication:

Yes - Niki L. Reynaert, Albert van der Vliet, Amy S Guala, Toby McGovern, Milena Hristova, Cristen Pantano, Nicholas H. Heintz, John Heim, Ye-Shih Ho, Dwight E. Matthews, Emiel F.M. Wouters, and Yvonne M.W. Janssen-Heininger. Dynamic redox control of NF-kB through glutaredoxin-regulated S-glutathionylation of inhibitory kappa B kinase. Proc Natl Acad Sci U S A. 2006 Aug 29; 103(35) 13086-91.-

 

Section 6 – ERS Fellowship application details Number of publications in international peer-reviewed periodicals as per date of this application

In English: 16

In other language: 0

Professional societies or associations of which you are a member:

European Respiratory Society Netherlands Respiratory Society

Curriculum Vitae Niki L Reynaert

Personal historyPresent Position: Post doctoral fellow at the Department of Respiratory Medicine,(since 2006) Maastricht University Medical Centre, the NetherlandsPresent Address: Department of Respiratory Medicine,

Maastricht University PO Box 616

6202 AZ Maastrichtthe Netherlands

Phone: +31 43 3882270Fax: +31 43 3875051Email: N.Reynaert@pul.unimaas.nlHome Address: Weg naar Geneuth 95

3631 Maasmechelen, Belgium Phone: +32 89 248254

Citizenship: BelgianDate of birth: April 4th, 1979

Educational HistoryOctober 12th 2006 Ph.D cum laude, Thesis: Redox regulation of Inhibitory Kappa B Kinase.

Oxidants in the pathogenesis of inflammatory lung disease.2001 – 2006 Ph.D student at the Department of Respiratory Medicine, Maastricht

University, the NetherlandsResearch performed in the laboratory of Dr. Y. Janssen-Heininger at theDepartment of Pathology, University of Vermont, Vermont, USA

1997 – 2001 M.Sc. Magna cum laude – Biomedical Sciences, Free UniversityBrussels, Brussels, BelgiumThesis: The role of hyaluronan fragments in chronic inflammation and lungpathology: studies into the pathogenesis of COPD. Internship at theDepartment of Respiratory Medicine, Maastricht University, Maastricht, theNetherlands

Funding• Dutch Asthma foundation 3.2.09.049, €250.000 (2010-2014): Systemic effects and co-

morbidity in COPD are associated with markers of accelerated ageing• VENI grant, NWO/ZonMW (Netherlands organization for scientific research), 016.086.090,

€208.000 (01-06-2008 – 01-06-2011): Glutaredoxin 1 regulation of MMP activity as amechanism of emphysema development in COPD.

• Nutrition and Toxicology Research Institute Maastricht, primary funding Ph.D. project (2007– 2011): Glutaredoxin and protein S-glutathionylation in the aetiology of COPD.

• Kootstra Fellowship, Maastricht University (01-01-2007 - 31-12-2007)

Honors• Bursary recipient ERS Lung Science Conference 2009.• European Respiratory Society 2005, Cell and Molecular Biology Young Scientist Travel

Award.• Travel award from Society for Free Radicals, Biology and Medicine 2004.• Best abstract presentation and travel award at European Respiratory Society Lung Science

Conference 2003.

Lectures/PresentationsInvited speaker

• My Veni experience. PhD meeting FHML Maastricht University 2010• Glutaredoxins and protein S-glutathionylation in COPD. Helmholtz Centrum Munich April

19th 2010.• Vlag PhD week – guest alumnus 2009: Life after a PhD – My example.

• Oxidative stress in Asthma and COPD. European Allergy and Asthma Center Davossymposium with workshop: Asthma and COPD: chasing the differences. 2009.

• Glutaredoxins: balancing the imbalances in COPD. Research school for nutrition,toxicology and metabolism, Maastricht University. Research day 2008.

• Goede vrije radicalen en research beyond borders. Researcher’s night beyond borders2008. Maastricht University.

• Antioxidants and COPD: oxidants are not all bad. Maastricht University Dies Natalis 2008.• Signal transduction by reactive oxygen and nitrogen species in airway inflammation. Dutch

Asthma Foundation annual symposium 2004.Selected oral presentations at conferences

• GRX1 enhances wound repair of alveolar epithelial type II cells in vitro. EuropeanRespiratory Society Conference 2009.

• Association of Glutathione-S-Transferase Omega Haplotypes with Susceptibility to ChronicObstructive Pulmonary Disease. American Thoracic Society Conference 2009.

• Glutaredoxins in allergic airway disease and regulation by H2O2 in vivo. Society for FreeRadicals Biology and Medicine conference 2005.

• Reversible repression of IKKβ by H2O2 through S-glutathionylation. European RespiratorySociety Conference 2005.

• Inhibition of inhibitory kappa B kinase β by H2O2: A role for S-glutathionylation andirreversible oxidations. Society for Free Radicals, Biology and Medicine Conference 2004.

• Nitric oxide inhibits I kappa B kinase (IKK) through S-nitrosation and S-glutathiolation andprevents activation of Nuclear Factor Kappa B (NF-κB). Taormina 2003 EuropeanRespiratory Society Lung Science Conference.

Patents• Yvonne Janssen-Heininger, Niki Reynaert. Detection of glutathionylated proteins. U.S.

Pat. Apl. Ser. No.: 11/698,300.• Yvonne Janssen-Heininger, Niki Reynaert. Treatments Involving Glutaredoxins and

Similar Agents. U.S. Pat. Apl. Ser. No.: 60/934,129.• Yvonne Janssen-Heininger, Albert van der Vliet, Karina Ckless, Niki Reynaert. Detection

of nitrosylated proteins. US publication number 2005-02874. International publicationnumber WO2005/101019.

Peer reviewer for JournalsAmerican Journal of Respiratory and Critical Care Medicine, European Respiratory Journal,Journal of Gerontology, Plos One, Respiratory Research

Supervisor of PhD students - copromotor• Ine Kuipers 2007 – 2011: reversible protein oxidations in COPD• Paul Peeters 2009 – 2013: The inflammasome and EMT in silicosis• Poornima Gopal 2010-2014: Systemic effects and co-morbidity in COPD are associated

with markers of accelerated ageing• Irene Eurlings 2010 – 2014: Epithelial cell plasticity and alveolar wall remodeling in COPD

MembershipsEuropean Respiratory Society, Maastricht University Young Researchers Academy,Netherlands Respiratory Society

Extracurricular activities• Member of the organizing committee of the Netherlands Respiratory Society young

investigator symposium 2009: Linking clinical physiology to underlying airway remodeling inpulmonary disease: a challenge for clinical and basic scientists.

• Member of the organizing committee of the Netherlands Respiratory Society younginvestigator symposium 2010: Biomarker discovery and high throughput screening.

Niki L Reynaert

Publication list

Orginal peer reviewed§ D de Boer, AMJ Rousseau, DMM van Hedel-Bosch, NL Reynaert, WKWH Wodzig.

Analytical validation of the screening for glutathionylated haemoglobin (HbX1d3) byMALDI-TOFMS in order to monitor oxidative stress. Nederlands Tijdschrift voor KlinischeChemie en Laboratorium Geneeskunde 2010,35:175-177.

§ DG Yanbaeva, EFM Wouters, MA Dentener, MA Spruit and NL Reynaert. Association ofglutathione-S-transferase omega haplotypes with susceptibility to chronic obstructivepulmonary disease. Free Rad Res 2009, 43:738 - 743.

§ V. Anathy, S.W. Aesif, A.S. Guala, M. Havermans, N.L. Reynaert, Y.-S. Ho, R.C. Budd,and Y.M.W. Janssen-Heininger. Redox amplification of apoptosis by caspase dependentcleavage of glutaredoxin 1 and S-glutathionylation of Fas. J Cell Biol 2009, 184(2).

§ Niki L. Reynaert, Scott W. Aesif, Toby McGovern, Amy Brown, Emiel F.M. Wouters,Charles G. Irvin, and Yvonne M.W. Janssen-Heininger. Catalase overexpression doesnot attenuate inflammation and increases airway hyperresponsiveness in a murine modelof allergic airway disease. J Immunol 2007, 178(6).

§ Niki L Reynaert, Emiel FM Wouters and Yvonne Janssen-Heininger. Modulation ofglutaredoxin expression in a mouse model of allergic airway disease. Am J Respir CellMol Biol. 2007 ;36(2) 147-51.

§ Niki L. Reynaert, Albert van der Vliet, Amy S Guala, Toby McGovern, Milena Hristova,Cristen Pantano, Nicholas H. Heintz, John Heim, Ye-Shih Ho, Dwight E. Matthews, EmielF.M. Wouters, and Yvonne M.W. Janssen-Heininger. Dynamic redox control of NF-κBthrough glutaredoxin-regulated S-glutathionylation of inhibitory kappa B kinase. Proc NatlAcad Sci U S A. 2006 Aug 29; 103(35) 13086-91.

§ Niki L. Reynaert, Karina Ckless, Amy S. Guala, Emiel F.M. Wouters, Albert van der Vlietand Yvonne M.W. Janssen-Heininger. In situ detection of S-glutathionylated proteinsfollowing glutaredoxin-1 catalyzed cysteine derivatization. Biochim Biophys Acta. 2006Mar; 1760(3) 380-7.

§ Juanita HJ Vernooy, Niki Reynaert, Tim GAM Wolfs, Roy HE Cloots, Astrid Haegens,Bart de Vries, Mieke A Dentener, Wim A Buurman, Emiel FM Wouters. Rapid pulmonaryexpression of acute-phase reactants after local lipopolysaccharide exposure in mice isfollowed by an interleukin-6 mediated systemic acute-phase response. Exp Lung Res2005; 31(9-10) 855-871.

§ Karina Ckless, Niki L Reynaert, Douglas J Taatjes, Karen M Lounsbury, Albert van derVliet, Yvonne Janssen-Heininger. In situ detection and visualization of S-nitrosylatedproteins following chemical derivatization: identification of Ran GTPase as a target for S-nitrosylation. Nitric Oxide. 2004 Nov 11(3): 216-227.

§ Reynaert NL, Ckless K, Korn SH, Vos N, Guala AS, Wouters EF, Van Der Vliet A,Janssen-Heininger YM. Nitric oxide represses inhibitory kappa B kinase through S-nitrosylation. Proc Natl Acad Sci U S A. 2004 Jun 15;101(24): 8945-8950.

Review articles and commentaries§ Aesif SW, Janssen-Heininger YM, Reynaert NL. Protocols for the detection of S-

glutathionylated and S-nitrosylated proteins in situ. Methods Enzymol 2010, 474:289-96.§ Janssen-Heininger YM, Aesif SW, van der Velden J, Guala AS, Reiss JN, Roberson EC,

Budd RC, Reynaert NL, Anathy V. Ann N Y Acad Sci 2010; 1203:23-8.§ Wouters EF, Reynaert NL, Dentener MA, Vernooy JH. Systemic and local inflammation

in asthma and chronic obstructive pulmonary disease: is there a connection? Proc AmThorac Soc 2009, 6(8):638-647.

§ Janssen-Heininger YM, Poynter ME, Aesif SW, Pantano C, Ather JL, Reynaert NL,Ckless K, Anathy V, van der Velden J, Irvin CG, van der Vliet A. Nuclear factor kappa B,

airway epithelium and asthma: avenues for redox control. Proc Am Thorac Soc 2009,6(3):249-255.

§ Cristen Pantano, Niki L. Reynaert, Albert van der Vliet, and Yvonne M.W. Janssen-Heininger. Redox sensitive kinases of the nuclear factor kappa B signaling pathway.Antioxid Redox Signal. 2006 Sep-Oct; 8(9-10) 1791-1806.

§ Janssen-Heininger Y, Ckless K, Reynaert N, van der Vliet A. SOD Inactivation inAsthma: Bad News or NO News? Am J Pathol. 2005 Mar;166(3): 649-52.

§ Reynaert NL, Ckless K, Wouters EF, van der Vliet A, Janssen-Heininger YM. Nitric oxideand redox signaling in allergic airway inflammation. Antioxid Redox Signal. 2005 Jan-Feb;7(1-2): 129-43.

§ Janssen-Heininger YM, Persinger RL, Korn SH, Pantano C, McElhinney B, Reynaert NL,Langen RC, Ckless K, Shrivastava P, Poynter ME. Reactive nitrogen species and cellsignaling: implications for death or survival of lung epithelium. Am J Respir Crit Care Med2002 Dec 15;166(12 Pt 2):S9-S16.

Book chapters§ Involvement of oxidants in the aetiology of chronic airway diseases. Proteomic

approaches to identify redox processes in epithelial cell signaling and inflammation.Albert van der Vliet, Niki L Reynaert, Peter Bove, Karina Ckless, Anne-Katrin Greul,Milena Hristova, Yvonne Janssen-Heininger. Redox Proteomics: from ProteinModifications to Cellular Oxidative/Nitrosative Stress (I. Dalle-Donne, A. Scaloni, and A.Butterfield, Eds.), John Wiley & Sons, Inc. (July 2006).

Selected meeting abstracts§ Niki L. Reynaert, Ryanne JM Lemmens, Yvonne MW Janssen-Heininger and Emiel FM

Wouters. GRX1 enhances wound repair of alveolar epithelial type II cells in vitro. FreeRad Res. 2007, 41(suppl 1): S50.

§ Niki L. Reynaert, Albert van der Vliet, Milena Hristova, Cristen Pantano, Nicholas H.Heintz, John Heim, Dwight E. Matthews, Emiel F.M. Wouters, and Yvonne M.W.Janssen-Heininger, Inhibition of inhibitory kappa B kinase β by H2O2: A role for S-glutathionylation and irreversible oxidations. FRBM. 2004, 37(sup1): S70.

§ NL Reynaert, K Ckless, A van der Vliet, S Korn, N. Vos, EFM Wouters, Y Janssen-Heininger. Inhibition of I Kappa B Kinase (IKK) by S-nitrosation and S-glutathiolationprevents activation of Nuclear Factor Kappa B. Am J Respir Crit Care Med 2003, 167(7):A796.

Stretch as a key mediator of oxidant production and inflammation in COPD

SummaryA number of respiratory diseases, including COPD are characterized by remodeling and stretch pressures consequently rise upon normal breathing. The exact levels of stretch in diseased lungs have however not been measurable and it therefore remains to be determined whether these pressures cause inflammation and injury similar to what is observed after mechanical ventilation. Oxidative stress is an important hallmark of many chronic pulmonary inflammatory diseases, such as COPD and has been shown to occur as a result of cyclic stretch applied to lung epithelial cells in vitro. Importantly, oxidants do not only cause damage, but they also have the capacity to specifically induce or influence signal transduction. One of the redox-dependent posttranslational modifications through which oxidants signal is S-glutathionylation, which is reversed by glutaredoxin (GRX) enzymes. I have previously demonstrated that S-glutathionylation and the level of GRX1 exert an important control over the activation of the pro-inflammatory transcription factor NF-B [1], which has been shown to be activated by cyclic stretch as well [2].During this short term research fellowship I will test the hypothesis that mechanical stretch exerted on lung epithelial cells or lung tissue slices results in attenuated protein S-glutathionylation and that this is related to the activation of the transcription factor NF-B and inflammatory cytokine and chemokine production. This will be tested by 1. determining that stretch induces oxidant production, NF-B activation and attenuated protein S-glutathionylation in lung epithelial cells and lung tissue slices and 2. examining whether oxidative stress and alterations in S-glutathionylation in response to stretch are linked to NF-B activation by treatment with antioxidants or genetically manipulating GRX1 levels before applying stretch to lung epithelial cells. For this research, two generations of Flexercell systems and stretch devices are available as well as in house expertise with ‘lung slice’ models. This newly acquired technology and methodology will be used in future research projects on structure-function relationships in the context of respiratory diseases in general with an emphasis on redox regulation of physiological and pathophysiological processes. For my personal development as an independent researcher at Maasticht University I aim to deepen translational research in this area, keeping to clinically relevant models. I regard the Leicester Institute for Lung Health as a centre of excellence and prof. Brightling as an outstanding mentor in this respect because of his expertise in clinical as well as basic research with a specific interest in integrating models of airway physiology at the organ level to alterations in tissue biomechanics and ultrastructure at the tissue level. Given the complementarity, it is my believe that this fellowship will therefore be a starting point for a long-term collaboration.

BackgroundHealthy lungs are a dynamic environment, where each breath causes expansion and contraction, both circumferential and longitudinal. Airway and alveolar epithelial cells are therefore continuously exposed to luminal shear stress generated by the airflow, as well as mechanical stretch. A number of respiratory diseases, including COPD are characterized by fibrosis and remodeling which impact on the forces generated upon inflation [3, 4]. Also the loss of alveolar attachments to airways in emphysema will alter the stretch pressure that the airway is subjected to and make the airways more prone to collapse [5]. Consequent dynamic hyperinflation as seen in an important proportion of patients with COPD upon exercise and even daily activities causes acute alterations in airway stretch pressures [6]. It is however important to consider that these processes that influence airway dynamics are not uniformly distributed throughout the anatomy of the lungs. Moreover, traditional measurements of lung mechanics have been inferred from pressure, volume and flow relationships at the entrance to the airways. The aim of AirPROM FP7, coordinated by Prof. Brightling, is to develop an integrated multi-scale predictive computational model of the airway. One of the goals is to integrate models of large and small airway physiology at the organ level to alterations in tissue biomechanics and ultrastructure at the tissue level. This will lead to a better estimation of the physical properties of micro-airways. Through this effort the molecular effects of more accurate stretch pressures can be tested in vitro.

From the vasculature we know that hemodynamic forces play an important role in the regulation of structure, tone and response to agonists [7]. In the lung, research intomechanobiology has been mostly confined to mechanical ventilation and models of acute respiratory distress syndrome. From this research it is clear that mechanical stress causes excessive alveolar distention leading to injury and increased pulmonary vascular permeability as well as inflammation [8]. In vitro studies applying cyclic stretch toalveolar or bronchial epithelial cells causing their deformation have shown to triggeralterations in cytoskeletal organization [9], activation of kinases and the production of oxidants by mitochondria as well as NADPH-dependent oxidases (Nox) [10], amongst others. The oxidative stress related to stretch is thought to be a major cause of lung injury. However, oxidants do not only cause damage, but they also have the capacity to specifically induce or influence signal transduction. The best known example hereof is the release of endothelium-derived relaxing factor or nitric oxide that causes vessel relaxation [11]. One of the redox-dependent posttranslational protein modificationsthrough which oxidants signal is S-glutathionylation. This is the binding of the antioxidant molecule glutathione to cysteine animo acids, which occurs under physiological conditions and is enhanced by exposure to low levels of oxidants. S-glutathionylation is reversed by glutaredoxin (GRX) enzymes, with GRX1 occurring in the cytosol, and GRX2 in mitochondria and nucleus [12]. The function of numerous proteins has been shown to be affected by S-glutathionylation, including actin polymerization and matrix metalloproteases. In addition, I have previously demonstrated that S-glutathionylation and the level of GRX1 exert an important control over the activation of the pro-inflammatory transcription factor NF-B [1], which has been shown to be activated by cyclic stretch as well [2]. In brief, I show that H2O2 and Nox4 induce

S-glutathionylation of cysteine 179 of Inhibitory kappa B kinase (IKK) which consequently inhibits its kinase activity. Moreover, I found that endogenous levels of GRX1 determine IKK and NF-B activation, as for instance primary lung epithelial cells derived from GRX1 k.o. mice had a markedly attenuated response to LPS with respect to the production of the chemokines Kc and MIP2. The aim of this fellowship is to apply stretch pressures that are clinically relevant in vitroand ex vivo in order to investigate if the previously reported redox control over NF-B is deregulated by mechanical stress and thus contributes to inflammation and injury.

Hypothesis and aimsDuring this short term research fellowship I will test the hypothesis that mechanical stretch exerted on lung epithelial cells or lung tissue slices results in attenuatedprotein S-glutathionylation and that this is related to the activation of the transcription factor NF-B and inflammatory cytokine and chemokine production.

1. It will be determined whether stretch induces oxidant production, NF-B activation and attenuated protein S-glutathionylation in lung epithelial cells and lung tissue slices.

2. By treatment with antioxidants such as N-acetylcysteine or genetically manipulating GRX1 levels, it will be examined whether oxidative stress and alterations in S-glutathionylation in response to stretch are linked to NF-B activation.

WorkplanCell culture and stretch modelsThe non-malignant mouse alveolar epithelial type II cell line C10 will be used in most experiments, including in experiments with genetic manipulation of GRX1 levels through overexpression and siRNA knock down. Some experiments will in addition be performed using primary bronchial epithelial cells, either submerged or at air-liquid interface,obtained through bronchoscopy from healthy controls or patients with COPD. Cells will be seeded on flexible bottom BioFlex plates and grown until confluency. Next, the plates will be mounted onto the Flexercell Tension system (Flexercell International Corporation) and cells will be subjected to static or cyclic strains of various degrees and for various time frames to reflect physiological breathing cycles and pathological parameters. In addition to the cell culture model, tissue slices of human lung will be subjected to stretch and parameters detailed below will be analyzed microscopically.

Biochemical analysesOxidant production will be visualized and quantified using the fluorescent probe DCF and protein S-glutathionylation using the staining technique I developed [13]. Expression of GRX1, Nox4 and cytokines including Kc and MIP2 will be evaluated by QPCR, as well as protein levels by Western blotting or ELISA. NF-B activity will be assessed using commercially available kits, nuclear translocation by immunohistochemistry or in cells stably expressing an NF-B-luciferase reporter plasmid.

Timetable and feasibilityI have experience with all the biochemical assays to be used during this fellowship, as well as with the C10 line, including overexpression and knock-down of GRX1. The first month will be used to get acquainted with the Flexercell systems and establish appropriate stretch conditions in this cell line in association with the read-outs. In the second and third month, aim 2 will be conducted as will select experiments in primary human cells and tissue slices.

Host institute, guidance and expertiseThe Leicester Institute for Lung Health (ILH) was created to harmonise and synergise respiratory research and clinical development. This has greatly strengthened the ability to undertake high quality translational research in airway disease and respiratory infections. In 2009 the University reorganised biomedical research making respiratory science one of 3 priority areas. The respiratory science theme now has 150 researchers, including >40 clinical and non-clinical principal investigators with >£30M in external funding in the last 5 years (including MRC programme) and >200 original papers in the last decade (8 N Eng J Med, 5 Lancet) representing major breakthroughs in our understanding of respiratory disease.Professor Chris Brightling (CB) is a Wellcome Senior Clinical Fellow and runs a group of 25 researchers. He leads the Medical Research Council COPD Consortium, is coordinator for AirPROM (FP7), and co-coordinator for EvA (FP7). The focus of the group is translational research in airways disease. With co-PIs in Nottingham CB has been awarded £1M to develop primary cell ex vivo airway models and has a shared capability to explore mechanotransduction in these models. The groups have 2 generations of Flexercell systems and access to bespoke stretch devices as well as in house expertise with ‘lung slice’ models in collaboration with Dr Yassine Amrani. Full access and support to this capability and our clinical research facility will made available to Dr Reynaert.

Relevance to home institute Research into COPD is central at the department of Respiratory Medicine at Maastricht University and is widely recognized. The newly acquired techniques involving in vitroand ex vivo stretch models, as well as culture of primary human airway epithelial cells and tissue slices will be most valuable to the research conducted at our institute and will be applicable to other projects currently being performed, as well as those in the future. This will in addition greatly strengthen the translational component of our research.

Relevance to personal development and careerIn this project, some of the research and findings from my PhD projects, as well as tools I developed are placed into a new pathophysiological context, namely that of stretch and pulmonary biomechanics. These structure-function relations can be applied on most every pulmonary disease and I believe I bring into this field the unique concept or redox signaling. Until May of next year I have my own funding through a prestigious grant from the Netherlands Organization for Scientific Research, in which redox modulation of matrix breakdown is the central topic. Upon return to Maastricht, the newly acquired technologies with respect to stretch, primary epithelial cultures and lung tissue slices will

be applied to this as well as some of the other projects I am currently involved in (see c.v.). Furthermore, I see this fellowship as an opportunity to gain new expertise and expand my network of collaborations that will be useful for applying for follow-up funding and establishing an independent research position at Maastricht University.

References1. Reynaert, N. L., A. van der Vliet, A. S. Guala, T. McGovern, M. Hristova, C. Pantano, N. H. Heintz, J. Heim, Y. S. Ho, D. E. Matthews, E. F. Wouters, and Y. M. Janssen-Heininger. Dynamic redox control of NF-kappaB through glutaredoxin-regulated S-glutathionylation of inhibitory kappaB kinase beta. Proc Natl Acad Sci U S A 2006; 103(35):13086-13091.2. Liu, Y. Y., S. K. Liao, C. C. Huang, Y. H. Tsai, D. A. Quinn, and L. F. Li. Role for nuclear factor-kappaB in augmented lung injury because of interaction between hyperoxia and high stretch ventilation. Transl Res 2009; 154(5):228-240.3. Ebihara, T., N. Venkatesan, R. Tanaka, and M. S. Ludwig. Changes in extracellular matrix and tissue viscoelasticity in bleomycin-induced lung fibrosis. Temporal aspects. Am J Respir Crit Care Med 2000; 162(4 Pt 1):1569-1576.4. Faffe, D. S., and W. A. Zin. Lung parenchymal mechanics in health and disease. Physiol Rev 2009; 89(3):759-775.5. Milic-Emili, J. Does mechanical injury of the peripheral airways play a role in the genesis of COPD in smokers? Rev Mal Respir 2003; 20(6 Pt 1):833-840.6. O'Donnell, D. E., and P. Laveneziana. The clinical importance of dynamic lung hyperinflation in COPD. Copd 2006; 3(4):219-232.7. Birukov, K. G. Cyclic stretch, reactive oxygen species, and vascular remodeling. Antioxid Redox Signal 2009; 11(7):1651-1667.8. Tschumperlin, D. J., F. Boudreault, and F. Liu. Recent advances and new opportunities in lung mechanobiology. J Biomech 43(1):99-107.9. Papaiahgari, S., A. Yerrapureddy, P. M. Hassoun, J. G. Garcia, K. G. Birukov, and S. P. Reddy. EGFR-activated signaling and actin remodeling regulate cyclic stretch-induced NRF2-ARE activation. Am J Respir Cell Mol Biol 2007; 36(3):304-312.10. Chapman, K. E., S. E. Sinclair, D. Zhuang, A. Hassid, L. P. Desai, and C. M. Waters. Cyclic mechanical strain increases reactive oxygen species production in pulmonary epithelial cells. Am J Physiol Lung Cell Mol Physiol 2005; 289(5):L834-841.11. Ignarro, L. J., R. E. Byrns, G. M. Buga, and K. S. Wood. Endothelium-derived relaxing factor from pulmonary artery and vein possesses pharmacologic and chemical properties identical to those of nitric oxide radical. Circ Res 1987; 61(6):866-879.12. Shelton, M. D., P. B. Chock, and J. J. Mieyal. Glutaredoxin: role in reversible protein s-glutathionylation and regulation of redox signal transduction and protein translocation. Antioxid Redox Signal 2005; 7(3-4):348-366.13. Reynaert, N. L., K. Ckless, A. S. Guala, E. F. Wouters, A. van der Vliet, and Y. M. Janssen-Heininger. In situ detection of S-glutathionylated proteins following glutaredoxin-1 catalyzed cysteine derivatization. Biochim Biophys Acta 2006; 1760(3):180-187.

Curriculum vitae

-Personal detailsTitle(s), initial(s), first name, surname: Prof.dr. E.F.M. Wouters, EmielMale/female: MaleDate and place of birth: 16 May 1953, GeelNationality: BelgiumCivil status: Married, 3 children

-Master's (‘Doctoraal’)University: University LeuvenDate: 2 July 1978Main subject: Medicine

-DoctorateUniversity/College of Higher Education: University MaastrichtDate: 20 November 1987Supervisor (‘Promotor’): Prof.dr. L.H. GreveTitle of thesis: Bronchial response in COPD measured by forced oscillation

technique.

-Work experience since graduating• 1978-1980: training internal medicine St. Annadal hospital Maastricht• 1980-1981: training internal medicine Catholic University Hospital Leuven• 1981-1984: training chest physician University Hospital Maastricht• 1984-1987: assistant professor department of Pulmonology, University Hospital Maastricht• 1987-1992: associate professor department of Pulmonology, University Hospital Maastricht• 1992-to date: professor in Pulmonology and chairman department of Respiratory Medicine, University

Hospital Maastricht

-PhD thesis promotorPromoter or co-promotor of 36 PhD thesis since 1991 in chronological order:

Schols AMWJ, Wesseling GJ, Jorna THJM, Cuijpers CEJ, Ketelaars CAJ, Staal-van den Brekel AJ,Dentener MA, Baarends EM, Simons JPFHA, Korn SH, Lamers RJS, Wirnsberger RM, Pennings HJ,Cobben NAM, Koerts-de Lang E, Creutzberg EC, Langendijk JA, Engelen MPKJ, Pouw EM, HendriksJJE, Gosker H, Vernooy J, Hochsenbag M, Langen RCJ, Vermeeren M, Wagena E, Broekhuizen R,Chavannes N, Rutten E, Reynaert N, Willems D, Haegens A, Groenewegen K, Geraerts L, Franssen F,Mercken E

-International activities• European School of Respiratory Medicine• European Respiratory Society National Delegate: 1997-1999• European Respiratory Society Council member: 1997-1999• European Respiratory Society Chairman of the clinical assembly group 01.02 Rehabilitation and chronic

care: 1999-2004• Editor European Respiratory Monograph: 2002-2006• Member European Respiratory Society working group on Integrated Care: 2008-2011• Member Task Force ERS/ATS Pulmonary Rehabilitation guidelines: 2005-2006• Member Task Force ERS/ATS COPD guidelines: 2002-2004• Member Task Force ERS/ATS Outcome measures• Editorial board member of several medical journals• Member International Advisory Board of Multidisciplinary Respiratory Medicine (MRM): 2010• Member Advisory Board Scientific Research Europäisches Zentrum für Allergie und Asthma Davos

(EACD): 2005-2009• Member Scientific Advisory Board German Competence network Asthma and COPD: 2010• Visiting professorship University Vermont, Burlington (USA)

-Academic activities• Scientific advisory Board Dutch Asthma Foundation: 1997-1999 member; 1999-2000 chairman.• AZPO (Advisory Council on Research and Care Dutch Asthma Foundation): chairman 2000-2006.• NWO (Netherlands Organization for Scientific Research): member medical science.• IWC (Advisory Board Scientific research University Maastricht): chairman 1998-2000.• AWO (Advisory Board Scientific research University Hospital Maastricht): member 1998-2000.• COB (Advisory Board Scientific research University Maastricht and University Hospital Maastricht):

chairman 2000-2002.• NVALT (Dutch Association of Chest Physicians) working group Rehabilitation: chairman 1994-2003.• NVALT (Dutch Association of Chest Physicians) Scientific Advisory Board: member 1999-2007.• Programme coordinator Graduate School VLAG II (Food Technology, Agrobiotechnology, Nutrition &

Health Sciences.• Member of the board of the Research Institute NUTRIM (Nutrition and Toxicology Research Institute

Maastricht)• Royal Netherlands Academy of Arts and Sciences: Advisory Committee Multifactorial diseases in the

genomics centry: member 2003-2006.• Board member of the Dutch Respiratory Society.

-Management activities• Chairman of the Treatment and Care Unit Thorax in the University Hospital Maastricht. The treatment

and Care Unit Thorax is composed by the Department Cardiology, Cardiopulmonary Surgery andPulmonology (1998-2002).

• Director of the Centre for Chronic Diseases, University Hospital Maastricht (2003-to date).• Chairman Board of Directors CIRO Horn (2005-to date).

-Editoral board memberships• Editor International Journal of Respiratory Care• Member Editorial Board American Journal of Respiratory and Critical Care Medicine• Member Editorial Board CHEST• Member Editorial Board Respiration• Member Editorial Board Chronic Respiratory Disease• Member Editorial Board Pulmonary Medicine

-Funding over the past 5 years

CIROCO – AstraZeneca 400.000 euro, ECLIPSE study – GSK 2.000.000 euro, Wijerhorst Foundation3.300.000 euro, Wijerhorst Foundation 3.000.000 euro, Dutch Asthma Foundation – Vernooy 245.000 euro.

Emiel FM Wouters, selected publications

1. John R Hurst PhD FRCP1, Jørgen Vestbo FRCP MD*2, 3, Antonio Anzueto MD4, NicholasLocantore PhD5, Hana Müllerova PhD5, Ruth Tal-Singer PhD5, Bruce Miller PhD5, DavidLomas PhD FRCP6, Alvar Agusti FRCP MD PhD7, William MacNee MB ChB MD FRCP8, PeterCalverley MD9, Stephen Rennard MD10, Emiel FM Wouters MD PhD11 and Jadwiga AWedzicha MD FRCP1 for the Evaluation of COPD Longitudinally to Identify PredictiveSurrogate Endpoints (ECLIPSE) investigators. Frequency, Determinants and Impact ofExacerbations in a large cohort with Chronic Obstructive Pulmonary Disease. NEJM 2010 inpress.

2. Haegens A, Heeringa P, van Suylen RJ, Steele C, Aratani Y, O'Donoghue RJ, Mutsaers SE,Mossman BT, Wouters EF, Vernooy JH. Myeloperoxidase deficiency attenuateslipopolysaccharide-induced acute lung inflammation and subsequent cytokine andchemokine production. J Immunol 2009;182:7990-6.

3. Vernooy JH, Drummen NE, Van Suylen RJ, Cloots RH, Moller GM, Bracke KR, Zuyderduyn S,Dentener MA, Brusselle GG, Hiemstra PS, Wouters EF. Enhanced pulmonary leptinexpression in patients with severe COPD and asymptomatic smokers. Thorax 2009;64:26-32

4. Vernooy JH, Moller GM, V Suylen RJ, van Spijk MP, Cloots RH, Hoet PH, Pennings HJ,Wouters EF. Increased Granzyme A Expression In Type II Pneumocytes of Patients WithSevere COPD. Am J Respir Crit Care Med 2007;175:464–472.

5. Reynaert NL, van der Vliet A, Guala AS, McGovern T, Hristova M, Pantano C, Heintz NH,Heim J, Ho YS, Matthews DE, Wouters EF, Janssen-Heininger YM. Dynamic redox control ofNF-{kappa}B through glutaredoxin-regulated S-glutathionylation of inhibitory {kappa}Bkinase {beta}. Proc Natl Acad Sci USA 2006;103:13086-13091.

6. Mercken EM, Hageman GJ, Schols AM, Akkermans MA, Bast A, Wouters EF. Rehabilitationdecreases exercise-induced oxidative stress in chronic obstructive pulmonary disease. Am JRespir Crit Care Med 2005; 172:994-1001.

7. Wouters EF, Postma DS, Fokkens B, Hop WC, Prins J, Kuipers AF, Pasma HR, Hensing CA,Creutzberg EC. Withdrawal of fluticasone propionate from combinedsalmeterol/fluticasone treatment in patients with COPD causes immediate and sustaineddisease deterioration: a randomised controlled trial. Thorax 2005;60:480-7.

8. Langen RC, Van Der Velden JL, Schols AM, Kelders MC, Wouters EF, Janssen-Heininger YM.Tumor necrosis factor-alpha inhibits myogenic differentiation through MyoD proteindestabilization. FASEB J 2004; 18:227-237.

9. Wouters EF. Management of severe COPD. Lancet 2004;364:883-95.

10. Vernooy JH, Kucukaycan M, Jacobs JA, Chavannes NH, Buurman WA, Dentener MA,Wouters EF. Local and systemic inflammation in patients with chronic obstructivepulmonary disease. Soluble tumor necrosis factor receptors are increased in sputum. Am JRespir Crit Care Med 2002; 166: 1218-1224.

Professor Christopher E Brightling

Contact Details:University of Leicester, Institute for lung Health, Glenfield Hospital, Leicester, LE3 9QP Email:ceb@le.ac.uk Tel: 0116 256 3998

Qualifications:BSc (Hons) Pharmacology Imperial College London 1990, MBBS St Mary’s Hospital London1993, MRCP London 1996, PhD University of Leicester 2002, FCCP College of Chest PhysiciansUSA 2007

Current Post:Professor of Respiratory Medicine, Wellcome Senior Clinical Research Fellow, HonoraryConsultant, University of Leicester.

Area of expertise:Understanding the pathophysiology and improving the clinical management of airway disease, inparticular the utilisation of biomarkers in the management of airway disease and the interactionsbetween inflammatory and structural cells in asthma and chronic obstructive pulmonary disease.

Leadership in the field:CEB is a respiratory clinical scientist with an international reputation leading a group of over 20researchers. He is a member of the British Thoracic Society severe asthma registry steeringcommittee, the BTS/SIGN asthma guideline committee, the American & European ThoracicSocieties joint severe asthma guideline group, the World Health Organisation severe asthmastrategy group, the Food and Drug Administration & National Institute for Health ChronicObstructive Pulmonary Disease biomarker group, and the American Chronic Cough guidelinesteering committee.

Current external funding:AirPROM FP7 (coordinator 16M Euro pending final contract negotiation, ~2M Leicester), MRCCOPD Consortium (£6M CI ~1M Leicester), Wellcome Trust Senior Fellowship (£1.2M), MedicalResearch Council (£1.1M), NC3R (250K), Asthma UK (210K), National Institute for Health (Co-PI1.5M, £160K to CEB), Emphysema versus Airways FP7 (Co-PI £2.1M 180K to CEB),Biotechnology and Biological Sciences Research Council (£80K), AstraZeneca (£110K),GlaxoSmithKline (£370K), MedImmune (£190K), Roche (£350K).

Prestige Factors:AirPROM (FP7) coordinator, Lead for MRC COPD consortium, Local Lead for TherapeuticCapability Cluster, Associate Editor for Chest (2005-) & Open Respiratory Medicine (2008-),editorial board for American Journal of Respiratory and Critical Care Medicine (2010-) & ClinicalMedicine (2007-). Reviewer for 24 international journals and grant reviewer for Wellcome Trust,MRC, Asthma UK, British Lung Foundation, NH/MRC Australia, and Astma Fonds.

CEB is chief investigator for 3 current multi-centre multi-nation phase IIa/b studies for noveltherapies in asthma and COPD.

CEB has been awarded 9 postgraduate regional and national research prizes and his students andpost-doctoral fellows have been awarded 10 national and international awards.

Professor Christopher E BrightlingSelected Publications from last 2 years (from >125 publications in total, >4000citations, H index 34):1. Kaur D, Saunders R, Hollins F, Woodman L, Doe C, Siddiqui S, Bradding P,

Brightling CE. Mast cell fibroblastoid differentiation mediated by airway smoothmuscle in asthma J Immunol 2010 in press

2. Doe C, Bafadhel M, Siddiqui S, Desai D, Mistry V, Rugman P, McCormick M, MayR, Sleeman MA, Anderson IK, Brightling CE. Expression of the Th17-associatedcytokines interleukin (IL)-17A and F in asthma and chronic obstructive pulmonarydisease Chest. 2010 in press

3. Gupta S, Siddiqui S, Haldar P, Entwisle JJ, Mawby D, Wardlaw AJ, Bradding P,Pavord ID, Green RH, Brightling CE. Quantitative analysis of high-resolutioncomputed tomography scans in severe asthma subphenotypes Thorax.2010;65(9):775-81

4. Kulkarni NS, Hollins F, Sutcliffe A, Saunders R, Shah S, Siddiqui S, Gupta S, HaldarP, Green R, Pavord I, Wardlaw A, Brightling CE. Eosinophil protein in airwaymacrophages: a novel biomarker of eosinophilic inflammation in patients with asthmaJ Allergy Clin Immunol. 2010 Jul;126(1):61-9.e3.

5. Siddiqui S, Mistry V, Doe C, Stinson S, Foster M, Brightling C. Airway wallexpression of OX40/OX40L and interleukin-4 in asthma Chest. 2010;137(4):797-804.

6. Haldar P, Brightling C, Hargadon B, Gupta S, Monteiro W, Sousa A, Marshall R,Bradding P, Green R, Wardlaw AJ, Pavord ID. Mepolizumab and exacerbations ofrefractory eosiniophilic asthma New Engl J Med 2009; 360(10): 973-84

7. Saunders R, Siddiqui S, Kaur D, Doe C, Sutcliffe A, Hollins F, Bradding P, WardlawA, Brightling CE. Fibrocyte localization to the airway smooth muscle is a feature ofasthma J Allergy Clin Immunol 2009;123(2):376-84

8. Woodman L, Siddiqui S, Cruse G, Sutcliffe A, Saunders R, Kaur D, Bradding P,Brightling C Mast cells promote airway smooth muscle cell differentiation viaautocrine upregulation of TGF-β1 J Immunol. 2008;181(7):5001-7

9. Siddiqui S, Mistry V, Doe C, Roach K, Morgan A, Wardlaw A, Pavord I, Bradding P,Brightling CE. Airway hyper responsiveness is dissociated from airway wallstructural remodelling. J Allergy and Clin Immunol 2008; 122: 335-41

10. Saha SK, Berry M, Parker D, Siddiqui S, May R, Monk P, Bradding P, Wardlaw AJ,Pavord ID, Brightling CE Increased sputum and bronchial biopsy interleukin (IL)-13expression in severe asthma J Allergy Clin Immunol 2008; 121: 685-91