Endothelial dysfunction programme within EVGN (for specialists)

The Endothelial dysfunction area (area1) consists of 6 workpackages (WP).

The specific objectives of workpackages WP1, WP2, WP3 and WP4 are:

1. To elucidate the mechanisms involved in the regulation of eNOS activity under physiological conditions, particularly concentrating on hemodynamic forces (fluid shear stress), and the role of tyrosine phosphorylation of the enzyme on its enzyme activity and intracellular localization.
2. To determine the mechanisms of regulation of NO production in vascular tissue by DDAH, the enzyme that metabolises asymmetric methylarginines, including ADMA, endogenous inhibitor of NO release.
3. To develop novel tools specifically targeted to endothelial cells to detect and to scavenge oxygen-derived free radicals, and validate a standard HPLC-method to quantify superoxide anion formation using dihydro-ethidium for tissues and cells.
4. To define a role of microparticles in the induction and activation of the vascular NADPH oxidases.
5. To assess the effects of selective inhibitors of the soluble epoxide hydrolase (an enzyme which metabolises EETs) and sulfaphenazole (a selective inhibitor of CYP 2C9) on the flow-dependent and bradykinin-induced relaxation of porcine coronary arteries.
6. To study kallikrein gene expression and enzyme activity in mouse arteries and heart, and the regulation of kallikrein and other endothelial components of the kallikrein-kinin system (ACE, the angiotensin AT2 receptor and the B2 kinin receptor) by flow and angiotensins in arteries of normal mice, as well as in mice deficient in kallikrein, ACE, or AT receptors.
7. To identify the factors able to activate PKC or the Erk1/2 pathway responsible for ACE promoter activation in endothelial cells or in other ACE secreting cells, and study the molecular mechanisms underlying the endothelial dysfunction observed in kallikrein deficient mice.
8. To investigate the role of the kallikrein, B2 receptor and ACE genes in coronary circulation and myocardial ischemia and reperfusion, as well as in survival and remodelling after cardiac necrosis and apoptosis using genetically modified mice with kallikrein, ACE, or B2R gene inactivation, and mice with ACE gene duplication.
9. To identify target genes potentially involved in the modulation of the protective/deleterious effect of ACE gene by using gene titration studies, reproducing human genetic variability, coupled to functional genomic studies.
10. To study the ACE signalling pathway in a bid to identify angiotensin II- and bradykinin-independent effects of ACE inhibitors. As the phosphorylation of a specific serine residue (Ser1270) is implicated in ACE signalling, we will develop endothelial cell lines that over-express either wild-type ACE or S1270A-mutant ACE.
11. To study the KKS genes in human disease. We have developed immunological methods for quantification of human kallikrein and its molecular variant using antibodies to synthetic polymorph epitopes. This polymorphism will be studied in coronary insufficiency and diabetic complications.

Specific clinically-oriented objectives will also be addressed:
12. To determine genotypes of candidate genes (including eNOS, COX-2, PAI-1, p22phox, E-selectin, Gelatinase B, thrombospondin, IL-6, CD14) likely to affect endothelial function.
13. To assess the effects of sulfaphenazole (a selective inhibitor of CYP 2C9) on the flow-dependent and acetylcholine-induced vasodilatation of coronary and peripheral arteries in 40 patients with established coronary artery disease. In addition, we will determine the effects of the CYP 2C9 inhibitor on any alterations in inflammatory and oxidative stress reflecting serum markers.
14. To assess the link between polymorphisms in CYP4A11 and coronary artery disease.
15. To study the KKS genes in human diseases. A common missense polymorphism of the kallikrein gene (R53H), present (at the heterozygous state) in 5-7% of Caucasians, induces a major reduction in kallikrein activity. We will assess the effect of this loss of function polymorphism on urinary and immunoreactive plasma kallikrein. We will study in young volunteer subjects carrying the 53H allele the consequences of partial genetic deficiency in kallikrein activity on arterial morphology and function.

The workpackage WP6 is oriented towards the understanding of the effects and mechanisms of action of microparticles that are shed from the membrane of apoptotic (or activated) cells. In close coooperation with WP1-4, its specific objectives are:
16. To evaluate by DNA microarray the effects of apoptotic microparticles on endothelial cells and vascular smooth muscle cells, and to compare the effect of microparticles produced in vitro from differentially activated cells with those isolated from atherosclerotic plaque and those of circulating microparticles from patients with CAD, diabetes, hypertension, or cardiovascular and non-cardiovascular inflammatory conditions.
17. To determine the specific protein profile on circulating vs. in vitro obtained microparticles by proteomic analysis, and to examine whether apoptotic bodies carry already known circulating proinflammatory mediators (for example: HSP, CRP), which could mediate their effect on vascular cells.
18. To determine by HPLC-MS and by the presence of specific phospholipid epitopes the lipid profile of the different sources of microparticles and the possible contribution of oxidized lipids and other particle contents to their effects on vascular cells.
19. To isolate apoptotic bodies from human atherosclerotic plaque and determine their cause procoagulant (tissue factor expression or transfer), pro-angiogenic (VEGF expression) or pro-inflammatory activities on vascular smooth muscle and endothelial cells.