Salome Smit

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Proteomics investigation into cardiac endothelial cells using the Orbitrap at the Proteomics facility of the University of Stellenbosch. Central Analytical Facility University of Stellenbosch. Salome Smit. Overview Proteomics analysis of cardiac endothelial cells - PowerPoint PPT Presentation
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Functional consequences of the inhibition of Plasmodial S-adenosylmethionine decarboxylase as a key regulator of polyamine metabolism

Proteomics investigation into cardiac endothelial cells using the Orbitrap at the Proteomics facility of the University of Stellenbosch

Salome SmitCentral Analytical Facility University of Stellenbosch

1Overview

Proteomics analysis of cardiac endothelial cellsSILAC experiment with HIV-1Tat proteinSummary 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 = CMECs3CMECsIn 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. 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.

TNF-: RELEASE, BINDING AND EFFECTSTNF-R1TNF-R2APOPTOSISINFLAMMATORY RESPONSE / PRO-SURVIVALInflammation; Tissue Injury (eg Ischaemia); Aging; Cardiovascular Risk Factors (Obesity; DM); Heart FailureTNF- ReleaseANTI-APOPTOSIS / ANTI-NECROSISVascular endothelial cells are the PRIMARY targets of circulating TNF- (Pober 2004); Express both TNF-R1 and TNF-R2 (Madge 2001)6What is the effect of TNF- on CMEC?

What would be a novel and optimal method to study these cells? To gain the most informationMETHODS: LARGE-SCALE PROTEOMICS: LTQ Orbitrap Velos MS (Thermo Scientific): Central Analytical Facility, FHS, Univ Stellenbosch

SDS-PAGEIN-GEL TRYPSINISATIONNANO LIQUID CHROMATOGRAPHYMASS SPECTOMETRYPROTEIN 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:

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