Mechanistically, aldosterone inhibits the formation of bone marrow-derived progenitor cells by attenuating VEGFR-2, but effects on circulating EPCs were not investigated.48 Aldosterone also impairs vascular reactivity by decreasing glucose-6-phosphate dehydrogenase (G6PD) expression and activity in mature endothelial cells.18 We did AA26-9 not find changes in G6PD expression in monocytic EPCs by aldosterone treatment, suggesting that this mechanism is not involved in aldosterone-mediated EPC dysfunction (data not shown). endothelial progenitor cell This was done essentially as described.11,31,32 AA26-9 A considerable number of different EPC subtypes exist and the nomenclature is rather heterogeneous (see33 for review). We therefore here describe the different subtypes of cells we used in our studies and also mention this in the Results section; in brief, we used the following cells for our analyses, which have been extensively described and characterized in the literature: monocytic EPCs,11,34 CD34+/KDR+ cells (human study),35 sca1/flk1 cells (mouse study),11 early outgrowth (colony-forming units, CFU assays or Hill colonies)31 and late-outgrowth EPCs.32 Note, that alternatively to the term monocytic EPC also early EPC, angiogenic progenitor cells, or circulating angiogenic cells33 are used in the literature. All of the employed EPC subtypes expressed the MR (see Supplementary material online, and data not shown). For morphology and characteristics of late-outgrowth EPCs, see Supplementary material online, (or scrambled controls using the Stealth? Select RNAi Kit (Invitrogen, Germany; oligonucleotide concentration 150 nM, 48 h). FITC-labelled scrambled siRNA (control-FITC block-it fluorescent Oligo #2013, Invitrogen, Germany) was used as a transfection control. Transfection rate was 90% (data not shown). Forty-eight hours after transfection, expression was monitored by RT-PCR and western blot analysis (see above-mentioned section). Measurement of reactive oxygen species Intracellular reactive oxygen species (ROS) was determined using dihydroethidium (DHE) AA26-9 as described.12 After 20 min of incubation with DHE (2.5 M) at 37C and 5% CO2 in a humidified atmosphere, EPCs were evaluated using fluorescence microscopy. Signal intensity of cells and background was determined in at least four randomly chosen regions. Aldosterone level Plasma aldosterone levels were measured by commercially available radioimmunoassays (mice: DiaSorin GmbH, Dietzenbach, Germany; humans: DPC Biermann, Bad Nauheim, Germany). Mouse studies The study conforms to the published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). To test whether hyperaldosteronism would alter EPC biology, neovascularization capacity and endothelial function = 13)= 10) 0.05. Results Expression and functional importance of mineralocorticoid receptor activation for endothelial progenitor cells Monocytic (early) EPCs expressed the MR (NR3C2) both at the gene (and assays. Aldosterone treatment impaired formation of endothelial CFU and of UEA-1+/dil-acLDL+ cells from cultured peripheral blood mononuclear cells, indicating inhibition of EPC differentiation and functional impairment (and and Supplementary material online, and Supplementary material online, 0.05, **,## 0.01, ***,### 0.005. = 3C8 per group. Aldosterone increases oxidative stress in monocytic (early) endothelial progenitor cells in a protein kinase Mouse monoclonal to CD68. The CD68 antigen is a 37kD transmembrane protein that is posttranslationally glycosylated to give a protein of 87115kD. CD68 is specifically expressed by tissue macrophages, Langerhans cells and at low levels by dendritic cells. It could play a role in phagocytic activities of tissue macrophages, both in intracellular lysosomal metabolism and extracellular cellcell and cellpathogen interactions. It binds to tissue and organspecific lectins or selectins, allowing homing of macrophage subsets to particular sites. Rapid recirculation of CD68 from endosomes and lysosomes to the plasma membrane may allow macrophages to crawl over selectin bearing substrates or other cells. A-dependent manner Endothelial progenitor cell function is impaired by ROS.6,12,31 We tested whether aldosterone would alter ROS formation in monocytic (early) and late-outgrowth EPC. Treatment of EPC with aldosterone led to an up to four-fold increase in intracellular ROS production, which was blocked by pre-treatment of EPC with eplerenone or siRNA-mediated silencing of the MR (see and and Supplementary material online, 0.05, ***,### 0.005. = 3C8 per group. Aldosterone impairs monocytic (early) endothelial progenitor cell function, vascularization capacity, and endothelial function findings of aldosterone-mediated EPC dysfunction were operative and = n.s.), but an increase of intracellular ROS levels in response to aldosterone infusion (and on number and function of circulating sca-1+/flk-1+ EPC. Here, we co-treated the aldosterone-infused mice additionally with the beta-blocker metoprolol, which prevented blood pressure increase (see Supplementary material online, on intracellular oxidative stress and function of endothelial progenitor cells (EPCs). (experiments: aldosterone or vehicle was delivered by implanted osmotic minipumps continuously for 2 weeks at a dose rate of 50 g/kg/day. After 7 days matrigel plugs were implanted. After 14 days EPC function and number were determined, as well as vascularization of the implanted matrigel plug and sprouting capacity of explanted aortic rings. (on oxidative stress (oxidated dihydroethidium) in monocytic (early) EPCs ( 0.05, **,##= 4C6 individual experiments or animals per group. We then evaluated the consequences of hyperaldosteronism on endothelial function. Compared with controls, aldosterone-infused mice demonstrated a significant impairment of endothelium-dependent vasodilatation, whereas treatment with eplerenone alleviated this effect (CD31 staining of invading capillaries in matrigel plugs explanted 2 weeks after continuous aldosterone- or placebo infusion to mice. ( 0.05, **,##= 5C6 individual experiments or animals AA26-9 per group. In addition to effects of aldosterone on large vessels, we.