Circulating bone marrow-derived endothelial progenitor cells (EPCs) promote vascular reparative processes and neoangiogenesis, and their number in peripheral blood correlates with endothelial function and cardiovascular risk. We tested the hypothesis that the cytokine erythropoietin (EPO) stimulates EPCs in humans. We studied 11 patients with renal anemia and 4 healthy subjects who received standard doses of recombinant human EPO (rhEPO). Treatment with rhEPO caused a significant mobilization of CD34 ؉ /CD45 ؉ circulating progenitor cells in peripheral blood (measured by flow cytometry), and increased the number of functionally active EPCs (measured by in vitro assay) in patients (week 2, 312% ؎ 31%; week 8, 308% ؎ 40%; both P < .01 versus baseline) as well as in healthy subjects (week 8, 194% ؎ 15%; P < .05 versus baseline). IntroductionStem cell therapy emerges as a promising approach in cardiovascular medicine. Current research focuses on bone marrow-derived endothelial progenitor cells (EPCs), which promote vascular reparative processes. [1][2][3] EPCs are considered to originate from CD34-positive (CD34 ϩ ) stem cells. 3 These cells differentiate via separate pathways into erythrocytes, thrombocytes, various lineages of leukocytes, and also endothelial cells. EPCs are found mainly in the bone marrow, but may also circulate in the vasculature where they home and incorporate into sites of active neovascularization. 1,[4][5][6][7] In experimental studies, increased neovascularization by these cells improves cardiac function after myocardial ischemia. [8][9][10] In patients with myocardial infarction, the clinical outcome is strongly correlated to the number of mobilized EPCs from the bone marrow. 11 Thus, the search for substances that modulate the number and/or function of EPCs is a matter of considerable interest. For example, vascular endothelial growth factor (VEGF) has been shown to regulate EPC proliferation and differentiation. 12 Erythropoietin (EPO) is a cytokine stimulating erythrocyte differentiation. It is produced mainly in the renal interstitium in response to hypoxic stimuli. Currently the main indication for use of recombinant human EPO (rhEPO) is treatment of anemia due to EPO deficiency in patients with chronic renal failure. EPO also appears to have direct biologic effects on endothelial cells. 13,14 Furthermore, both VEGF and EPO share important activities with respect to neoangiogenesis. 12,15 The main target of both cytokines seems to be the vasculature. 16 Thus, EPO could affect EPC proliferation and differentiation as well.We tested the hypothesis that EPO modulates the number of functionally active EPCs in humans. For this purpose, we assessed circulating CD34 ϩ cells in whole blood using flow cytometry, and the number of functionally active EPCs in an in vitro assay during 8 weeks of treatment with standard rhEPO doses in 11 patients with renal anemia and in 4 healthy subjects. Patients and methods Study participants and protocolThe study protocols were approved by the Hannover Medic...

Abstract-The number of circulating endothelial progenitor cells (EPCs) correlates with endothelial dysfunction and cardiovascular risk in humans. We explored whether angiotensin II receptor antagonist therapy affects the number of regenerative EPCs in patients with type 2 diabetes. In a prospective double-blind parallel group study, we randomly treated 18 type 2 diabetics with olmesartan (40 mg) or placebo for 12 weeks. We analyzed circulating CD34 Key Words: receptors, angiotensin II Ⅲ endothelium Ⅲ blood vessels Ⅲ cardiovascular diseases I n experimental studies using different animal models of cardiovascular injury and repair, bone marrow-derived endothelial progenitor cells (EPCs) have been shown to be responsible for endothelial and hence vascular repair. [1][2][3][4][5][6] Circulating EPCs incorporate into sites of active neovascularization, where they orchestrate re-endothelialization of damaged vessel walls, also by secreting a large number of cytokines that attract and govern cells that are indispensable in the process of vascular repair. 7,8 Human data are even more intriguing, because in patients with coronary artery disease the number of EPCs correlates with the number of cardiovascular risk factors, and this correlation exists even in apparently healthy subjects without manifest atherosclerosis. 9,10 In the latter population, the number of EPCs also significantly correlated with the degree of endothelial dysfunction. 10 Moreover, in patients with clinical conditions known to be associated with increased cardiovascular risk such as type 2 diabetes mellitus or renal failure, the number and/or function of EPCs is significantly altered. [11][12][13] Theoretically, EPCs can be expanded in vitro for therapeutic use, 5,14 but currently this procedure is laborious and expensive. Experimental work and studies in humans have revealed that the number of functionally active EPCs can be increased by pharmacological intervention, however, eg, administration of statins 15,16 and recombinant human EPO (rHuEPO) or its analogue darbepoetin. 12,17,18 This finding could be of therapeutic relevance, because persistent stimulation of EPCs by targeted pharmacological intervention could, at least theoretically, repair endothelial injury and progression of atherosclerotic vascular disease in patients at risk. We have therefore explored the effect of angiotensin II subtype 1-receptor antagonist therapy on EPCs in patients with type 2 diabetes mellitus in a prospective double-blind parallel group study. We verified the results in a second open study. Patients and MethodsThe studies were approved by the Hannover Medical School Ethics Committee. We have obtained written informed consent from all participants. In a prospective double-blind parallel group trial, 18 patients with type 2 diabetes mellitus randomly received placebo (nϭ9; age, 60.4Ϯ2.6 years; body mass index, 29.4Ϯ1.2 kg/m 2 ) or 40 mg of the angiotensin II receptor antagonist olmesartan (nϭ9; 61.7Ϯ2.4 years; 30.0Ϯ1.6 kg/m 2 ) for 12 weeks. They were normotensive, ie...

EMPs are elevated in active adult AAV. EMP levels correlate with disease activity and might serve as a marker of endothelial activation and damage. Differential detection of endothelial, platelet- and leucocyte-derived MPs may provide more insight in to the pathogenesis of AAV.

Rationale Atherosclerosis is a major cause of death in patients with chronic kidney disease. Chronic inflammation of the arterial wall including invasion, proliferation and differentiation of leukocytes is important in atherosclerotic lesion development. How atherosclerotic inflammation is altered in renal impairment is incompletely understood. Objective This study analyzed leukocytes of the atherosclerotic aorta in mice with impaired and normal renal function and studied a mechanism for the alteration in aortic myeloid leukocytes. Methods and Results Unilateral nephrectomy significantly decreased glomerular filtration rate and increased atherosclerotic lesion size and aortic leukocyte numbers in two murine atherosclerosis models, Apolipoprotein E (Apoe−/−) and LDL receptor (LDLr−/−) deficient mice. The number of aortic myeloid cells increased significantly. They took up less oxidized LDL, while CD11c expression, interaction with T cells and aortic T cell proliferation were significantly enhanced in renal impairment. In human PBMC cultures, chronic kidney disease serum decreased lipid uptake and increased HLAII expression. Supplementation with Interleukin (IL)-17A similarly increased HLAII and CD11c expression and impaired oxLDL uptake. IL-17A expression was increased in atherosclerotic mice with renal impairment. Ablation of IL-17A in LDLr−/− mice by lethal irradiation and reconstitution with Il17a−/− bone marrow abolished the effect of renal impairment on aortic CD11b+ myeloid cell accumulation, CD11c expression and cell proliferation. Atherosclerotic lesion size was decreased to levels observed in normal kidney function. Conclusions Kidney function modifies arterial myeloid cell accumulation and phenotype in atherosclerosis. Our results suggest a central role for IL-17A in aggravation of vascular inflammation and atherosclerosis in renal impairment.

Interleukin (IL)-17A signaling via Interleukin 17 receptor A (Il17ra) contributes to the inflammatory host response by inducing recruitment of innate immune cells, but also plays a role in homeostatic neutrophilic granulocyte regulation. Monocytes, the other main innate immune cell, have a longer life span and can pursue multiple differentiation pathways towards tissue macrophages. Monocytes are divided into two subpopulations by expression of the Ly6C/Gr1 surface marker in mice. We here investigated the role of Il17ra in monocyte homeostasis and macrophage generation. In Il17ra-/- and in mixed bone marrow chimeric wt/Il17ra-/- mice, the concentrations of circulating Il17ra-/-Gr1low monocytes were significantly decreased compared to wt cells. Pulmonary, splenic and resident peritoneal Il17ra-/- macrophages were significantly fewer than of wt origin. Bone marrow progenitor and monocyte numbers were equal, but the proportion of Il17ra-/-Gr1low monocytes was already decreased at bone marrow level. After monocyte depletion, initial Gr1high and Gr1low monocyte regeneration of Il17ra-/- and wt cells was very similar. However, Il17ra-/-Gr1low counts were not sustained. After labeling with either fluorescent beads or BrdU, Il17ra-/-Gr1high monocyte transition to Gr1low cells was not detectable unlike wt cells. Monocyte recruitment in acute peritonitis, which is known to be largely due to Gr1high cell migration, was unaffected in an identical environment. Unilateral ureteral obstruction induces a less acute inflammatory and fibrotic kidney injury. Compared to wt cells in the same environment, Il17ra-/- macrophage accumulation in the kidney was decreased. In the absence of Il17ra on all myeloid cells, renal fibrosis was significantly attenuated. Our data show that Il17ra modulates Gr1low monocyte counts and suggest defective Gr1high to Gr1low monocyte transition as an underlying mechanism. Lack of Il17ra altered homeostatic tissue macrophage formation and diminished renal inflammation and fibrosis. Il17ra appears to be a novel modulator of monocyte phenotype and possible therapeutic target in renal fibrosis.

The clinical importance of microparticles resulting from vesiculation of platelets and other blood cells is increasingly recognized, although no standardized method exists for their measurement. Only a few studies have examined the analytical and preanalytical steps and variables affecting microparticle detection. We focused our analysis on microparticle detection by flow cytometry. The goal of our study was to analyze the effects of different centrifugation protocols looking at different durations of high and low centrifugation speeds. We also analyzed the effect of filtration of buffer and long-term freezing on microparticle quantification, as well as the role of Annexin V in the detection of microparticles. Absolute and platelet-derived microparticles were 10- to 15-fold higher using initial lower centrifugation speeds at 1500 × g compared with protocols using centrifugation speeds at 5000 × g (P < 0.01). A clear separation between true events and background noise was only achieved using higher centrifugation speeds. Filtration of buffer with a 0.2 μm filter reduced a significant amount of background noise. Storing samples for microparticle detection at −80°C decreased microparticle levels at days 28, 42, and 56 (P < 0.05 for all comparisons with fresh samples). We believe that staining with Annexin V is necessary to distinguish true events from cell debris or precipitates. Buffers should be filtered and fresh samples should be analyzed, or storage periods will have to be standardized. Higher centrifugation speeds should be used to minimize contamination by smaller size platelets.

IntroductionApoptosis is essential to maintain homeostasis in multicellular organisms and apoptotic cells are rapidly and effectively cleared by professional phagocytes before they undergo secondary necrosis and release their noxious cytoplasmic content into the environment. 1 Ineffective clearance of apoptotic cells contributes to disease pathogenesis. 2,3 This may be true especially for autoimmune diseases such as systemic lupus erythematosus (SLE) because apoptotic cells are thought to be a potential source of autoantigens and disturbed clearance of apoptotic corpses may initiate and drive autoimmunity. [4][5][6] This concept of acquired autoimmunity has also been confirmed in several animal models that showed disturbed engulfment of apoptotic cells. [7][8][9][10] Vascular endothelial cells (ECs) serve as a crucial barrier between tissues and the circulation. They secrete a variety of substances, regulate coagulation, and participate in the immune response. 11 Under physiologic conditions circulating ECs (CECs) are almost not traceable, whereas in a variety of vascular diseases such as myocardial infarction, small-vessel vasculitis, transplantation, or cancer high numbers of CECs are detectable in the peripheral circulation. [12][13][14][15][16][17] Consequentially, accumulation of CECs may affect the homeostasis of the vessel wall by interfering with the EC layer both in the vicinity of endothelial lesions or even distant from the site of injury.Clearance of dying cells or cellular debris has been mostly ascribed to professional phagocytes, for example, antigenpresenting macrophages, neutrophils, or dendritic cells, but it is conceivable that other cell types such as epithelial cells or ECs may also partake in this process. 18,19 ECs are not professional phagocytes although specific subpopulations, such as liver ECs or cells from high endothelial venules, are able to phagocytose apoptotic cells. 20,21 Phagocytosis of circulating endothelial debris by healthy endothelium thus appears to be an intriguing concept.The role of endothelial-or platelet-derived microparticles in the circulation is the subject of several recently published studies but few, if any, data, shed light on the impact of apoptotic endothelial corpses on the adjacent endothelium. [22][23][24][25][26][27][28][29][30][31] The aim of the present study was to establish an in vitro model to study the interaction of apoptotic or necrotic ECs with a healthy EC layer. We here demonstrate that microvascular ECs, when exposed to apoptotic cells, react with the release of proinflammatory chemotactic cytokines and that this response triggers enhanced adhesion of primary neutrophils and macrophages. Patients, materials, and methodsThe study was approved by the Hannover Medical School Ethics Committee and conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from all patients included in this study. EC cultureHuman umbilical vein endothelial cells (HUVECs) were isolated from umbilical cords by exposure of the vein...

Reduced kidney function increases the risk for atherosclerosis and cardiovascular death. Leukocytes in the arterial wall contribute to atherosclerotic plaque formation. We investigated the role of fractalkine receptor CX3CR1 in atherosclerotic inflammation in renal impairment.