1. Proteomics investigation into cardiac endothelial cells using the Orbitrap at the Proteomics facility of the University of Stellenbosch Salome Smit Central Analytical Facility University of Stellenbosch

2. Overview 1.Proteomics analysis of cardiac endothelial cells 2.SILAC experiment with HIV-1Tat protein 3.Summary

3. Vascular endothelium long thought to be a mere selectively permeable barrier between the circulation and sub-endothelial tissues, is now known to be a master regulator of vascular homeostasis, Controlling functions such as vasomotor activity, thrombosis, inflammation and redox balance When endothelial function becomes compromised as observed in cardiovascular risk conditions such as diabetes mellitus, vascular homeostasis is lost resulting in increased oxidative stress, a loss of nitric oxide (NO) bioavailability, increased endothelial cell expression of pro-inflammatory vascular adhesion molecules and increased endothelial permeability. These pathophysiological changes underlie the phenomena of endothelial activation and endothelial dysfunction, of which the latter in particular is regarded as the early forerunner of atherosclerosis. All slides for CMEC work courtesy of Prof Hans Strijdom, University of Stellenbosch CARDIAC MICROVASCULAR ENDOTHELIAL CELLS = CMECs

4. CMECs In the heart, the myocardial capillaries (leading to ischaemic heart disease) are made up of cardiac microvascular endothelial cells (CMECs). CMECs show distinct structural and functional adaptations compared to other endothelial cell phenotypes in view of their location in the myocardium where they are closely associated with surrounding cardiomyocytes. There is intimate CMEC-cardiomyocyte arrangement cardiomyocytes are regarded as the primary cellular recipients of paracrine messengers secreted by CMECs, such as NO and endothelin-1.

5. CMECs CMECs: PIVOTAL ROLE IN BOTH MYOCARDIAL FUNCTION AND INJURY Optimal diffusion of oxygen and nutrients Reciprocal signalling with cardiomyocytes Regulate cardiomyocyte growth and development Regulation of cardiomyocyte contractile function & rhythmicity Therefore, CMECs are now recognized as important regulators of myocardial function.

6. TNF-α: RELEASE, BINDING AND EFFECTS TNF-R1TNF-R2 APOPTOSIS INFLAMMATORY RESPONSE / PRO-SURVIVAL Inflammation; Tissue Injury (eg Ischaemia); Aging; Cardiovascular Risk Factors (Obesity; DM); Heart Failure ↑TNF-α Release ANTI-APOPTOSIS / ANTI-NECROSIS Vascular endothelial cells are the PRIMARY targets of circulating TNF-α ( Pober 2004 ); Express both TNF-R1 and TNF-R2 ( Madge 2001 )

7. What is the effect of TNF-α on CMEC? What would be a novel and optimal method to study these cells? To gain the most information

8. METHODS: LARGE-SCALE PROTEOMICS: SDS-PAGE IN-GEL TRYPSINISATION NANO LIQUID CHROMATOGRAPHY MASS SPECTOMETRY PROTEIN ID Relatively few papers measure large-scale protein expression and regulation in vascular endothelial cells of any type (“only” 350 since 2001): Surprising! (Richardson 2010) Pubmed search: <5 papers reported on any form of proteomic analysis performed on CMECs

9. ControlTNF-α Control Down 226 TNF-α Up 269 Shared: 1214 Sieve™: 1511 proteins ControlTNF-α Control Down 77 TNF-α Up 143 Shared: 1056 Maxquant™: 1102 proteins TNF-α: 5ng / ml ; 24h PROTEIN REGULATION:

10. UP REGULATED AND TNF-α ONLY: DAVID Bioinformatics Resources® 16 proteins

11. UP REGULATED AND TNF-α ONLY: DAVID Bioinformatics Resources® MITOCHONDRIAL PROTEINS: ATP Synthase subunits (TNF only); Acetyltransferase component of Pyruvate dehydrogenase (  3-fold); Carnitine-Acylcarnitine carrier protein (  3-fold); Acyl CoA dehydrogenase (  5-fold); Isocitrate dehydrogenase (only TNF); ADP/ATP translocase 2 (  7.6-fold); Cytochrome C1 (  2-fold); Electron transfer flavoprotein (  2-fold); VDAC-1 (  2-fold); Cytochrome C1 (  2-fold); Cytochrome C oxidase (TNF only); Glycerol-3-phosphate dehydrogenase (TNF only) 51 proteins

12. UP REGULATED AND TNF-α ONLY: DAVID Bioinformatics Resources® 13 proteins

13. UP REGULATED AND TNF-α ONLY: Function, pathway, processP-value Nucleic Acid Metabolism0.000004 Protein Synthesis0.00006 Protein Trafficking0.00009 EIF-2 Signalling0.000015 Glutathione Metabolism0.0004 Interleukin Signalling0.0007 Oxidative Stress4x10 -7 Mitochondrial Dysfunction0.008 Ingenuity ® Systems

14. DOWN REGULATED AND CONTROL ONLY: DAVID Bioinformatics Resources® 27 proteins CYTOSKELETON PROTEINS: ADP Ribosylation Factor (  5-fold); Actin, alpha-1 (  4-fold); Actin, gamma-1 (control only); Alpha actinin-4 (  4-fold); Cofilin-1 (  5.5-fold); Gelsolin (  43-fold); Tubulin, beta 2 (  31-fold); R-ras (  52-fold)

15. DOWN REGULATED AND CONTROL ONLY: Function, pathway, processP-value Cellular Assembly & Organisation1.7 x 10 -9 Cellular Function & Maintenance1.7 x 10 -9 Cell Morphology1.7 x 10 -7 Cellular Growth & Proliferation3.8 x 10 -7 Integrin Signalling2.4 x 10 -10 Caveolar-mediated Endocytosis4.5 x 10 -9 Clathrin-mediated Endocytosis3.3 x 10 -8 Actin Cytoskeleton Signalling2.7 x 10 -7 Ingenuity ® Systems

16. EVIDENCE OF TNF-α SIGNALLING: TRADD TNF-R1 and Death Associated Protein (TRADD): Expressed only in TNF-α stimulated cells NF-κB: Expressed only in TNF-α stimulated cells Proteomics: Complement C4 (  2.2-fold); ICAM-1 (only TNF-α); MHC Class 1 (  1.6-fold); IL-1 (TNF-α only) Inflammatory / Immune Protein Expression: IκB-α β -tubulin Control0.5ng/ml5ng/ml20ng/ml IκB EXPRESSION

17. eNOS-NO Pathway:  eNOS: 27%  eNOS: 63%  NO: 44%  NO: 33%  NO: 23%

18. NOS-NO BIOSYNTHESIS PATHWAYS: ControlTNF-α 5ng/ml p-PKB t-PKB PKB/Akt REGULATION AND ACTIVATION PROTEOMICS: Heat shock protein 90-α (  5.8-fold) Heat shock protein 90-β (  42-fold) HEAT SHOCK PROTEIN 90 EXPRESSION HSP90 β- tubulin ControlTNF 0.5ng TNF 20ng NO PRODUCTION

19. Oxidative Stress:  ROS: 63% of studies  Nitrosative stress: 25% of studies  NADPH-oxidase: 25% of studies ROS included: Superoxide (50%), mito- ROS (25%) and H 2 O 2 (13%)

20. PROTEOMIC DATA SUGGEST  ANTI-OXIDANT PROTEINS AND OXIDATVE STRESS RESPONSE: PROTEOMICS: Park-7 (  2-fold ) SOD [Mn], mitochondrial (  2-fold) Thioredoxin (  3-fold) Glutathione-s-transferase (only in TNF) Glutathione peroxidase, GPX4 (only TNF) Peroxiredoxin (  2-fold) Function, pathway, processP-value Glutathione Metabolism0.0004 Oxidative Stress4x10 -7 DAVID Bioinformatics Resources® Ingenuity ® Systems

21. Control0.5ng/ml5ng/ml p22-phox β -tubulin P22-PHOX EXPRESSION OXIDATIVE STRESS PARAMETERS: 20ng/ml

22. MITOCHONDRIAL ROS PRODUCTION: Control: + MitoSox TM 20 µm TNF-α: + MitoSox TM 20 µm FACS confirmed results Thus proteomic results confirmed

23. Apoptosis / Cell Death:  Apoptosis: 50% of studies  Apoptosis: 38% of studies  Necrosis: 13% of studies

24. APOPTOSIS / CELL DEATH: PROTEOMICS: Bid (TNF only) RACK-1 (  2.7-fold) PEA-15 (inhibits TNF-R1-mediated Caspase 8 activity) (  6.3-fold) VDAC-1 (  1.6-fold) BOK (TNF only) Metadherin (anti-apoptotic) (TNF only) Gelsolin (anti-apoptotic) (  43-fold)

25. Take home message???? Vascular endothelial cells neglected in proteomics CMEC basically no thorough study This is novel and important study to gain knowledge into cardiovascular disease Increase in oxidative stress due to TNF-α – proteins increase to counteract eNOS – decreased HSP90 Cells undergo apoptosis – increase in apoptotic proteins and increase in some anti-apoptotic proteins – therefore cells are fighting back Due to increase in apoptotic proteins and hence increase in cell death the proteins involved in may be the result of cytoskeleton organisation which is decreased.

26. Quantitative proteomic analysis of HIV-1 Tat apoptosis in SH-SY5Y neuroblastoma cells Putuma P. Gqamana 1 ǂ, Tariq Ganief 1 ǂ, Salome Smit 2, Shaun Garnett 1, Andrew Nel 1, and Jonathan Blackburn 1┴. Quantitative proteomic analysis of HIV-1 Tat induced apoptosis in SH-SY5Y neuroblastoma cells. Manuscript in preparation.

27. Tat associated with neural cell death and probable agent of HIV associated dementia 2849 proteins were identified from SILAC treated cells which were either phospho- enriched or phospho-depleted (therefore reduced complexity of sample) 17 up regulated and 72 down regulated proteins identified from SILAC Dysregulation of proteins identified associated with several neurodegenerative disorders Cell adhesion proteins down regulated – associated with apoptosis Proteins identified may also have role in weakening of immune response From results: Tat neurotoxicity may activate early signalling via tyrosine phosphorylation receptors and cause mitochondrial and oxidative stress leading to apoptosis. This will form basis of future biomarker discovery for HIV associated dementia

28. Recent successes with Orbitrap 28 Orbitrap Velos MS Neuroblastoma cells7539 M. smegmatis3271 M. Bovis2368 P. falciparum1681 CMEC1663 V. Cholera1411 Urine biomarkers1500 Sample from another MS unit 546 vs 24 Single human peptide in Arabidopsis

29. Acknowledgements 29 Thanks to: Prof Hans Strijdom – US – CMEC And students Amanda, Mashudu and Corli Dr Putuma Gqamana – UCT – SILAC proteomics, neuroblastoma cells Dr Brandy Gqamana-Young – UCT – Urine proteomics Mae Newton-Foot and Zhou Fang – US – M. Smegmatis Louise Vos – US – M. Bovis Dr Martella du Preez and Lisa Schaeffer – CSIR – V. Cholera Dr Cobus Zwiegelaar – Azargen – human peptide

30. Proteomics Laboratory Senior Analyst: Dr Salome Smit Office: 021 938 9632 Fax nr : 086 690 7602 email: salomiesmit@sun.ac.zasalomiesmit@sun.ac.za Universiteit van Stellenbosch Besoek / Visit: www.sun.ac.za/safwww.sun.ac.za/saf www.facebook.com/pages/CAF-Proteomics-lab-University-of- Stellenbosch/278646975539969