Despoina Pantazi Chemist, MSc, PhD Staff of the UOI (IDAX)

1. UNIVERSITY OF IOANNINA / SCHOOL OF SCIENCES DEPARTMENT OF CHEMISTRY / LABORATORY OF BIOCHEMISTRY ΕΠΙΔΡΑΣΗ ΤΗΣ ΜΗΧΑΝΙΚΗΣ ΤΑΣΗΣ ΣΤΗ ΒΙΟΣΥΝΘΕΣΗ ΤΗΣ ΦΩΣΦΑΤΙΔΥΛΟΧΟΛΙΝΗΣΣE ΠΝΕΥΜΟΝΟΚΥΤΤΑΡΑ ΤΥΠΟΥ ΙΙ The Effect of Mechanical Stretch on Biosynthesis of Phosphatidylcholine (PC) on Alveolar Type II Cells (ATII) Despoina Pantazi Chemist, MSc, PhD Staff of the UOI (IDAX) http://www.chem.uoi.gr Laboratory of Biochemistry

2. Pulmonary surfactant • Synthesized by type II alveolar cells • Consists of surfactant specific proteins and phospholipids (dipalmitoylphosphatidylcholine, PC) • Reduces surface tension (prevents alveolar collapse during expiration) • Exhibits host-defense properties

3. ALVEOLAR TYPE II CELLS Surfactant is biosynthesized, organized and stored into various structures, such as lamellar bodies in alveolar type II (ΑΤΙΙ) cells. Under this form surfactant is secreted into the alveolar space through the formation of microvilli in the apical cell surface. Alveolar type II cell from human lung ATII

4. LB Golgi Υπόφαση SER Orgeig S. Conference proceedings, Australian Frontiers, 31 July to 1 August 2003, Canberra Australia.

5. EM of Lamellar Bodies and Tubular Myelin From J. Goerke, Pulmonary surfactant: functions and molecular composition Biochim. Biophys. Acta 1408 (1998)79-89.

6. Composition of human lung surfactant 68.0 % PC 10.0 % PG,5.0 % PE 4.0 % Sph 90 % Phospholipids 90 % Lipids 10 % Neutral Lipids Hydrophilic proteinsSP-A, SP-D (50 -49 %) 10 % Proteins HydrophobicSP-B, SP-C (1 %) Rooney S.A. 1985. Am. Rev. Respir. Dis., 131:439-460.

7. DPPC Structure DPPC, the major lipid component of surfactant, is responsible for the equilibration of pressure in the alveoli.

8. LUNG SURFACTANT HOMEOSTASIS • Surfactant homeostasis is critical for lung function and is maintained through the balanced actions of biosynthesis, secretion, recycling and decomposition of its constituents • Qualitative and quantitative alterations of surfactant composition are associated to acute respiratory distress syndrome (ARDS)

9. MECHANICAL VENTILATION • Mechanical ventilation is required for critically-ill patients • However, it is established that certain modes of ventilation provoque inflammation not only in injured, but even in non-injured lung

10. MECHANICAL FORCES • Lung is subjected to mechanical forces continuously during development, normal breathing and mechanical ventilation • The main forces are • Strain is more prominent in lung epithelium during breathing • Shear forces acts mainly on the vascular endothelium • Cell proliferation, differentiation, apoptosis, release of different inflammatory cytokines and factors are some of the stretch- induced responses in AT II cells Substantial evidence indicates that mechanical stress during respiration is the signal for lung surfactant secretion from AT-II cells

11. AIM To investigate the effect of mechanical stimulation onphosphatidylcholine biosynthesis within AT-II cells - A549-cell line was used as a model for AT-II cells - A mode of static stretch was applied

12. FLOW-CHART A549 CELLS 800xg, 10 min, 4 ºC Cell Homogenate Pellet Protein Enzyme activities Enzyme expression Lipid analysis Force 35 g/cm2 for 1 & 4 h Cell Count (~ 5% Deformation) Homogenisation Petriperm Plexiglass

13. RESULTS

14. EFFECT OF STRETCH ON PHOSPHATIDYLCHOLINE CONTENT PC * PC (% Difference from Control) CONTROL STRETCH 1h STRETCH 4h After stretch for 1 h PC was increased by 22.9 %. At 4h, PC declined to the control levels Control: unstretched cells; *: Statistical difference from the control

15. Enzyme activities that implicated in production of PC ? CPT PC PLA2 PC Lyso - - DPPC LPCAT

16. O-CO-R O-CO-R R΄-OC-O HO P-Cho ΟΗ ΕΝZYMIC ACTIVITIES (1) • Final step of the de novo formation PC CDP-choline CMP CPT Diacylglycerol(DAG) Phosphatidylcholine(PC) R: palmitate R’: unsaturated acyl chain

17. BIOSYNTHESIS OF PC: CPT 100 CPT CPT 80 * * 60 CPT Activity (% Difference from Control) 40 20 0 -20 CONTROL STRETCH 1h STRETCH 4h -40 CPT activity was increased at 47 ± 9 % (p=0.037) after stretch for 1 h while after stretch for 4 h the enzyme activity recurred to the control levels. Control: unstretched cells; *: Statistical difference from the control

18. (Lyso-PC) Phosphatidylcholine(PC) O-CO-R O-CO-R O-CO-R O-CO-R HO HO R-OC-O R΄-OC-O P-Cho PCho P-Cho PCho R: palmitate ΕΝZYMIC ACTIVITIES (2) 2. Remodelling of PC for DPPC formation A aiPLA2 Lyso-phosphatidylcholine (Lyso-PC) B LPCAT palmitate (DPPC)

19. D TOTAL ΕΝZYMIC ACTIVITIES: variations

20. RELATIVE GENE EXPRESSION

21. IN CONCLUSION • Static stretch of A549 cells causes a transient increase in total phosphatidylcholine levels • The concomitant increase in CPT justifies this alteration • The enrichment of stretched cells in DPPC can be attributed to the concerted actions of aiPLA2 and, in particular, of LPCAT in which the expression is definitively increased • Our data suggest that in A549 cells mild static mechanical stretch promotes the biosynthesis of PC

22. CONCLUSIONS - CLINICAL IMPLICATIONS Possible clinical implications of our findings • A low level of stretch can induce surfactant production which can act in a protective way • This phenomenon has a limited duration and the system equilibrates after 4 h. Probably, cyclic stretch could help in the further production • Our results are compatible with previous work where PC was reduced after prolonged ventilation

23. Future Studies - Perspectives Part I • Signaling and regulation of surfactant biosynthesis under different stretching conditions • Relevance of our findings with mechanical ventilation

24. Instrumentation of Cyclic Stretcher

25. Future Studies Part II • Lipidomics is the detailed analysis and global characterization of the structure and function of lipids (the lipidome) within a living system • The enrichment of the list of lipids with new structures from biological samples, and/or after the influence of various parameters probably will help to the understanding of mechanisms of lung diseases • Lipidomic and further proteomic analysis of biological samples (BAL, serum, etc) in the unit LC-LTQ-ORBITRAP-MSn in Department of Chemistry in UOI

26. LC- LTQ – ORBITRAP – MSn INSTRUMENTATION Solvent Platform Autosampler Pump • When the HPLC system is connected to an MS detector, the components exit the LC column and travel through a red tubing to the MS detector. The LTQ Orbitrap features a rapid scan rateand high mass accuracy. Enables faster, more sensitive and more reliable detection and identification of compounds in low levels in complex mixtures. It outstands mass accuracy, mass resolution and reliable high sensitivity MSn performance.

27. REFERENCES • Pantazi D., Nakos G., Kitsiouli E., Trangas T., Lekka M.E. 2011. Non-Injurious mechanical stretch induces phosphatidylcholine biosynthesis in alveolar type II cells. Under preparation. • Fisher, A.B., Dodia C. 1997. Role of acidic Ca2+ -independent phospholipase A2 in synthesis of lung dipalmitoylphosphatidylcholine. Am. J. Physiol. Lung Cell Mol. Physiol. 272:L238-L243. • Edwards Y.S. 2001. Stretch stimulation: its effect on alveolar type II cell function in the lung. Comp. Biochem. Physiol. 129:245-260. • Torday J.S., Rehan V.K. 2002. Stretch-stimulated surfactant synthesis is coordinated by the paracrine actions of PTHP and leptin. Am. J. Physiol. Lung Cell Mol. Physiol.283:L130-L135. • Fahy E., Subramaniam S., Murphy R.C., Nishijima M., Raetz C.R.H., Shimizu T., Spener F., van Meer G., Wakelam M.J.O., Dennis E.A. 2009. Update of the LIPID MAPS comprehensive classification system for lipids. J. Lipid Res., 50:S9-S14.

28. Acknowledgements • Professor M.E. Lekka • Professor G. Nakos • Assoc. Professor D. Galanopoulou • Assoc. Professor T. Trangas • E. Kitsiouli, PhD • T. Karkampounas, PhD • 7. Candidate MSc & PhD studentsof Laboratory of Biochemistry

29. ΣΑΣ ΕΥΧΑΡΙΣΤΩ ΓΙΑ ΤΟ ΧΡΟΝΟ ΣΑΣ