Cytokine profiling in anti neutrophil cytoplasmic antibody-associated vasculitis: a cross-sectional cohort study.

ANCA-associated vasculitides (AAV) are severe diseases, potentially affecting lungs, kidney, and other organs. Nevertheless, risk profiling remains difficult. Aim of the current study was to analyze serological characteristics in AAV. The principal goal was to identify diagnostic markers that potentially allow a more sophisticated risk profiling in AAV. AAV subjects were recruited and evaluated for disease activity, disease stage, medication, and laboratory findings. Serum concentrations of the following parameters were measured: IL-1ß, IL-6, IL-17 A, IL-17 F, IL-21, IL-22, IL-23, TNF-α, sCD40L, IL-4, IL-10, IL-25, IL-31, IL-33, and INF-γ. A total number of 62 AAV subjects was included in the study (39 females; 23 males). Forty-five subjects were PR3+, 17 subjects showed ANCA specificity for MPO. The majority of all cytokines fell under the lower detection limit of the assay. Serum IL-10 was higher in both, AAV and SSc as compared to controls; it was also higher in early systemic AAV. Serum IL-33 was elevated in AAV and SSc; in AAV, higher levels were found in non-necrotizing GN and RTX untreated subjects. Serum CD40L was raised in AAV as well; higher concentrations were also found in PR3+ and MPO+ patients and early systemic, generalized, and refractory AAV. IL-10 may potentially serve as a marker of early systemic AAV. IL-33 may help to identify subjects with a higher risk for necrotizing GN in AAV.

Bone morphogenetic protein-5 and early endothelial outgrowth cells (eEOCs) in acute ischemic kidney injury (AKI) and 5/6-chronic kidney disease.

Early endothelial outgrowth cells (eEOCs) reproducibly have been shown to act protectively in acute ischemic kidney injury (AKI) and chronic kidney injury. Bone morphogenetic protein-5 (BMP-5) acted antifibrotically in human hypertensive nephropathy. The aim of the current study was to analyze effects of BMP-5 treatment in an eEOC-based therapy of murine AKI and 5/6-nephrectomy. Male C57/Bl6N mice were either subjected to unilateral renal artery clamping postuninephrectomy or to 5/6-nephrectomy. Untreated or BMP-5-pretreated murine eEOCs were injected into recipient animals at the time of reperfusion (AKI) or at 2 and 5 days after 5/6-nephrectomy. Analysis of renal function and morphology was performed at 48 h and at 6 wk (AKI) or at 8 wk (5/6 model). Cellular consequences of eEOC treatment were evaluated using different in vitro assays. AKI was mitigated significantly by injecting BMP-5-pretreated eEOCs. Renal function was improved at 48 h [corrected] after cell therapy. In 5/6-nephrectomy, the cells failed to act renoprotectively, [corrected] but proteinuria was reduced after administering untreated eEOCs." Next, the original version read as "BMP-5 acts as a potent eEOC agonist in murine AKI in the short [corrected] term. Cell effects in 5/6-nephrectomy are heterogenous, but untreated cells act antifibrotically [corrected] without any impact on EnMT.

Angiopoietin-1 treated early endothelial outgrowth cells (eEOCs) are activated in vitro and reduce renal damage in murine acute ischemic kidney injury (iAKI).

BACKGROUND: Acute kidney injury (AKI) severely worsens prognosis of hospitalized patients. Early Endothelial Outgrowth Cells act protective in murine acute ischemic renal failure and renoprotective actions of eEOCs have been documented to increase after cell pretreatment with 8-O-cAMP and Melatonin. Angiopoietin-1 is critically involved in maintaining vascular integrity and regeneration. Aim of the study was to analyze the consequences of eEOC treatment with Ang-1 in murine AKI. METHODS: After 40 minutes of unilateral renal artery clamping with contralateral nephrectomy, male C57/Bl6N mice were injected with either untreated or pretreated (Ang-1) syngeneic murine eEOCs. Two days later serum creatinine levels and morphology were evaluated. Cultured, Ang-1 treated murine eEOCs were analyzed for production/release of proangiogenic and proinflammatory mediators, migratory activity, and cell survival, respectively. RESULTS: Angiopoietin-1 pretreatment of eEOCs significantly reduced serum creatinine in cell-injected mice. In vitro analysis showed increased migration of Ang-1 treated eEOCs and supernatant from Ang-1 treated eEOCs stimulated migration of cultured mature endothelial cells. In addition, Ang-1 reduced percentages of Annexin V+/PI+ eEOCs. Intrarenal numbers of eEOCs remained unaffected by Ang-1 and eEOCs did not produce more or less proangiogenic/proinflammatory mediators after being stimulated with Ang-1. CONCLUSIONS: Angiopoietin-1 pretreatment of eEOCs increases the cells' renoprotective competence in ischemic AKI. Thus, the armentarium of eEOC agonists in AKI is increasingly being expanded and the treatment of AKI with eEOCs becomes a promising future option.

Endothelial progenitor cells (EPC) in sepsis with acute renal dysfunction (ARD).

INTRODUCTION: Sepsis is characterized by systemic microvascular dysfunction. Endothelial progenitor cells (EPCs) are critically involved in maintaining vascular homeostasis under both physiological and pathological conditions. The aim of the present study was to analyze the endothelial progenitor cell system in patients suffering from sepsis with acute renal dysfunction. METHODS: Patients with newly diagnosed sepsis were recruited from the ICU in a nonrandomized prospective manner. Blood samples were obtained within the first 12 hours after the diagnosis of sepsis. For quantifying endothelial progenitor cells (EPCs), CD133+/Flk-1+ cells were enumerated by cytometric analysis. Analysis of EPC proliferation was performed by a colony-forming units (CFU) assay. Blood concentrations of proangiogenic mediators were measured by ELISA. Acute renal dysfunction was diagnosed according to the Acute Kidney Injury Network (AKIN) criteria. Depending on the overall mean creatinine concentration during the stay at the ICU, patients were either assigned to a 'normal creatinine group' or to a 'high creatinine group'. Survival rates, frequency of dialysis, the simplified acute physiology score (SAPS) II scores, and different laboratory parameters were collected/used for further clinical characterization RESULTS: Circulating EPCs were significantly higher in all sepsis patients included in the study as opposed to healthy controls. Patients within the 'high creatinine group' showed an even more pronounced EPC increase. In contrast, EPC proliferation was severely affected in sepsis. Neither total circulating EPCs nor EPC proliferation differed between patients requiring dialysis and patients without renal replacement therapy. Cell numbers and cell proliferation also did not differ between surviving patients and patients with sepsis-related death. Serum levels of vascular endothelial growth factor (VEGF), stromal derived factor-1 (SDF-1), and Angiopoietin-2 were higher in sepsis than in healthy controls. Sepsis patients within the 'high creatinine group' showed significantly higher mean serum levels of uric acid. CONCLUSIONS: Sepsis significantly affects the endothelial progenitor cell system, as reflected by increased EPC numbers, increased concentrations of proangiogenic mediators, and reduced proliferative capacity of the cells. This occurs independently from the frequency of dialysis and from patient survival. Increased serum levels of uric acid are possibly responsible for stronger EPC mobilization in sepsis patients with higher average creatinine levels.

Enhancing the chondrogenic potential of chondrogenic progenitor cells by deleting RAB5C.

Osteoarthritis (OA) is the most prevalent chronic joint disease that affects a large proportion of the elderly population. Chondrogenic progenitor cells (CPCs) reside in late-stage OA cartilage tissue, producing a fibrocartilaginous extracellular matrix; these cells can be manipulated in vitro to deposit proteins of healthy articular cartilage. CPCs are under the control of SOX9 and RUNX2. In our earlier studies, we showed that a knockdown of RUNX2 enhanced the chondrogenic potential of CPCs. Here we demonstrate that CPCs carrying a knockout of RAB5C, a protein involved in endosomal trafficking, exhibited elevated expression of multiple chondrogenic markers, including the SOX trio, and increased COL2 deposition, whereas no changes in COL1 deposition were observed. We report RAB5C as an attractive target for future therapeutic approaches designed to increase the COL2 content in the diseased joint.

LDL lipid apheresis rapidly increases peripheral endothelial progenitor cell competence.

BACKGROUND AND AIM: Endothelial progenitor cells (EPCs) have been shown to promote neovascularization under physiologic and pathologic conditions. Statins have been documented to increase the total number of circulating EPCs in long-term treated patients. Lipid apheresis is used to treat patient with refractory hyperlipidemia. The aim of our study was to evaluate whether lipid apheresis is associated with EPC mobilization. METHODS: Thirteen patients with refractory hyperlipidemia (analysis at the beginning and at the end of a single lipid apheresis treatment) and 10 healthy controls were included into the study. For quantifying total peripheral EPCs, CD133+/Flk-1+ myelo-monocytic blood cells were enumerated by flow cytometry. The proliferative potential of EPCs was evaluated by a "colony-forming unit" assay. In some patients, EPC eNOS expression was evaluated before and after treatment. RESULTS: Circulating EPCs and the cells' proliferative activity were lower in hyperlipidemia patients as compared to controls (0.14 +/- 0.07 vs. 0.6 +/- 0.14, P = 0.01, and 13.9 +/- 4.9 vs. 45.6 +/- 8.1, P = 0.0007). Lipid apheresis treatment was not associated with an increase in total EPCs. The cells' proliferative activity was strongly stimulated by lipid apheresis as reflected by an increase in the number of EPC colonies (13.9 +/- 4.9 to 34.1 +/- 7.3, P = 0.035). Analysis of EPC eNOS expression revealed a threefold increase in the cellular expression intensity after lipid apheresis. CONCLUSIONS: Patients with refractory hyperlipidemia exhibit lower peripheral EPC numbers and a lower proliferative activity of circulating EPCs than healthy controls. A single lipid apheresis treatment significantly stimulates EPC proliferation, it furthermore increases cellular eNOS. In summary, these results show that lipid apheresis mediates beneficial effects on the EPC system as an essential element in the process of vascular repair in the human organism.

Acute Kidney Injury-Associated Systemic Inflammation Is Aggravated in Insulin-Dependent Diabetes Mellitus.

BACKGROUND: Acute kidney injury (AKI) significantly worsens the prognosis of hospitalized patients. Diabetes mellitus (DM) affects a growing number of individuals in the western world. DM subjects are at a higher risk for acquiring AKI during the stay at the hospital. The current study intended to quantify serum levels of specific immunomodulatory cytokines in diabetic mice suffering from AKI. METHODS: DM was induced in male C57/Bl6N mice by systemic injections of beta cell-toxic streptozotocin. Animals underwent bilateral renal ischemia (45 min) 6 weeks later. RESULTS: Post-ischemic diabetic mice showed significantly differing serum concentrations of the majority of all analytes as compared to untreated controls and non-diabetic (post-ischemic) animals. CONCLUSIONS: Together, our data suggest DM-associated immune activation in AKI. One may suppose that inadequate stimulation of the humoral/cellular immune response potentially contributes to the higher ischemia susceptibility of the organ in DM.

Humoral and Cellular Patterns of Early Endothelial Progenitor Cells in Relation to the Cardiovascular Risk in Axial Spondylarthritis.

BACKGROUND: Spondylarthritis (SpA) significantly affects sacroiliac, intervertebral and peripheral joints. Patients with SpA suffer from increased cardiovascular risk (CVR). The endothelial progenitor cell (EPC) system critically perpetuates vascular repair. The aim of the study was to evaluate circulating EPCs in axial (ax)SpA with special attention on parameters of disease activity and CVR. METHODS: Disease activity and functional impairment were quantified in 50 axSpA patients by using standardized parameters (Bath ankylosing spondylitis disease activity index (BASDAI), C-reactive protein (CRP), finger-floor distance (FFD) and Ott' sign). Circulating EPCs and EPC regeneration were analyzed (fluorescence-activated cell sorting (FACS) and colony-forming unit (CFU) assay). Serum vasomodulatory mediators were quantified by enzyme-linked immunosorbent assay (ELISA). RESULTS: EPC colony numbers were lower in axSpA as compared to controls. Females displayed more colonies than males. In addition, fewer colonies were observed in smokers, in patients with a BASDAI of below 4 and in hypertension. Circulating CD133+/KDR+ cells did not differ between the groups. Follow-up analysis (33 months later) did not show any differences in gender, colony formation, CD133+/KDR+ cells or serum levels of vasomodulatory mediators if related to the categories of BASDAI, Ott' sign or FFD. CONCLUSIONS: EPC colony formation is significantly affected in axSpA with particularly low levels in males. EPC-related parameters do not allow predicting disease activity-related or functional parameters nor are they useful for CVR assessment in SpA.

Endothelial autophagy and Endothelial-to-Mesenchymal Transition (EndoMT) in eEPC treatment of ischemic AKI.

BACKGROUND: Autophagy enables cells to digest endogenous/exogenous waste products, thus potentially prolonging the cellular lifespan. Early endothelial progenitor cells (eEPCs) protect mice from ischemic acute kidney injury (AKI). The mid-term prognosis in AKI critically depends on vascular rarefication and interstitial fibrosis with the latter partly being induced by mesenchymal transdifferentiation of endothelial cells (EndoMT). This study aimed to determine the impact of eEPC preconditioning with different autophagy inducing agents [suberoylanilide hydroxamic acid (SAHA)/temsirolimus] in ischemic AKI. METHODS: Male C57/Bl6 N mice were subjected to bilateral renal ischemia (40 min). Animals were injected with either untreated, or SAHA- or temsirolimus-pretreated syngeneic murine eEPCs at the time of reperfusion. Mice were analyzed 48 h and 4 weeks later. In addition, cultured eEPCs were treated with transforming growth factor (TGF)-ß ± SAHA, autophagy (perinuclear LC3-II), and stress-induced premature senescence (SIPS-senescence-associated ß-galactosidase, SA-ß-Gal), and were evaluated 96 h later. RESULTS: Cultured eEPCs showed reduced perinuclear density of LC3-II + vesicles and elevated levels of SA-ß-Gal after treatment with TGF-ß alone, indicating impaired autophagy and aggravated SIPS. These effects were completely abrogated by SAHA. Systemic administration of either SAHA or tems pretreated eEPCs resulted in elevated intrarenal endothelial p62 at 48 h and 4 weeks, indicating stimulated endothelial autophagy. This effect was most pronounced after injection of SAHA-treated eEPCs. At 4 weeks endothelial expression of mesenchymal alpha-smooth muscle actin (αSMA) was reduced in animals receiving untreated and SAHA-pretreated cells. In addition, SAHA-treated cells reduced fibrosis at week 4. Tems in contrast aggravated EndoMT. Postischemic renal function declined after renal ischemia and remained unaffected in all experimental cell treatment groups. CONCLUSION: In ischemic AKI, intrarenal endothelial autophagy may be stabilized by systemic administration of pharmacologically preconditioned eEPCs. Early EPCs can reduce postischemic EndoMT and fibrosis in the mid-term. Autophagy induction in eEPCs either increases or decreases the mesenchymal properties of intrarenal endothelial cells, depending on the substance being used. Thus, endothelial autophagy induction in ischemic AKI, mediated by eEPCs is not a renoprotective event per se.

Dynamics of pulse wave velocity and vascular augmentation index in association with endothelial progenitor cells in SLE.

Patients with SLE display a significantly higher cardiovascular risk (CVR). Pulse wave velocity (PWV) has meanwhile been established as a reliable parameter of end-organ damage. Endothelial progenitor cells (EPCs) are critically involved in vascular repair under both physiological and pathological conditions. The aim of the study was to analyse PWV and the Vascular Augmentation Index (VAI) and EPC numbers/regeneration in a well-defined German SLE cohort. Thirty patients were included. Only two individuals displayed a PWV of above 10 m/s. There was no correlation between PWV percentiles and disease activity as reflected by the SLE Disease Activity Index. Neither EPC colonies nor percentages of circulating EPCs (CD133+/KDR+) correlated with PWV/VAI in a positive or negative manner. Thus, it can be questioned whether pulse wave analysis and/or EPC proliferation and circulating cell numbers are truly useful for CVR assessment in SLE.

Peroxide release into saliva from five different home bleaching systems in vivo.

PURPOSE: This study determined hydrogen peroxide release into the oral cavity during use of different home bleaching products and compared them with accepted safe levels. METHODS: Determination of peroxide in saliva was performed with peroxidase, phenol and 4-aminoantipyrin in a photometric method. Upper jaw incisors were bleached with individual trays charged with 350 mg Opalescence 10%, Opalescence 15% (OP) and Vivastyle (V). Additionally, Whitestrips (WS) designed for upper or lower jaw were used. All systems were adopted by five subjects for 30 minutes on different days. Whole saliva was collected at 2-minute intervals during the first 10 minutes of bleaching and every 5 minutes thereafter. RESULTS: Highest release of peroxide was found for all products in the saliva sample collected initially after application of the bleaching agent. Total amount of peroxide released into saliva during 30-minute bleaching period was 0.78+/-0.45 for Opalescence 10% and 1.52+/-0.44 mg for Opalescence 15%. Significantly more peroxide was released from Vivastyle (2.67+/-1.03 mg) and from Whitestrips (upper: 3.25+/-5,65, lower: 2.09+/-0.34 mg). A significantly smaller fraction of the charged peroxides was released into saliva from individual trays than from Whitestrips during the 30-minute use time. From the peroxide loaded in the trays or strips the following fractions were released during the application period: Opalescence 10% (6.4+/-3.7%), Opalescence 15% (8+/-2.4%), Vivastyle (18.6+/-8.5%), upper Whitestrips (30.4+/-4.9), lower Whitestrips (27.4+/-4.4%). In terms of amount/kg body weight the bleaching systems led to a single exposure of 0.013-0.056 mg/kg which is distinctly less than the maximum safe daily dose of 0.26 mg/kg/day if calculated for a small person (58 kg/128 lbs).

Determination of peroxides in saliva--kinetics of peroxide release into saliva during home-bleaching with Whitestrips and Vivastyle.

Aim of the study was to determine peroxides in saliva, released during bleaching procedures. Upper incisors of five subjects were bleached with Whitestrips (5% H2O2) and Vivastyle (10% carbamide peroxide, tray charged with 225mg) for 30min, each on different days. Saliva was collected before and during the whole period of bleaching at different intervals. The amount of peroxide in the salivary samples was assessed with peroxidase, phenol and 4-aminoantipyrin in a photometric assay. Additionally the amount of peroxides in the bleaching material was determined before and after the bleaching, so that the peroxide release into saliva could be balanced. The amount of peroxides released into saliva was related to the bleaching system and only partially influenced by the individual salivary flow rate. Bleaching with Vivastyle led to lower release of peroxides into saliva compared to Whitestrips (Vivastyle: 0.8+/-0.17mg; Whitestrips: 1.5+/-0.84mg). Salivary flow rate was not correlated to release of peroxides from the bleaching products. It can be concluded that the enzymatic method adopting 4-aminoantipyrin and peroxidase is valid for the determination of peroxides in saliva. Furthermore distinctly more peroxides are released into the oral cavity from Whitestrips than from trays charged with Vivastyle .

Immobilisation and activity of human alpha-amylase in the acquired enamel pellicle.

UNLABELLED: Amylase is an important salivary component and structural element of the acquired enamel pellicle. Aim of the study was to establish a method for precise and direct determination of pellicle bound amylase activity in order to analyse kinetics and activity of the immobilised enzyme. Six bovine enamel slabs (5mm diameter) were fixed on individual maxillary trays and worn by five subjects for different times (3, 30 and 120 min) on buccal and palatal sites on different days. Slabs were removed from the trays and rinsed with aqua dest. Afterwards, pellicle bound amylase activity was determined directly with a photometric method using 2-chloro-4-nitrophenyl-4-O-beta-D-galactopyranosylmaltotriosid (GalG2CNP) as substrate yielding the coloured product chloronitrophenolate (CNP). All investigated pellicles exhibited immobilised amylase activity. Mean activity was 1.39 +/- 187 mU/cm(2) (n=87, range 0.14-11.5 mU/cm(2)). Product formation of CNP by immobilised amylase was linear over time. Pellicle bound amylase showed a Michaelis type kinetic (Km = 3.3 x 10(-3) M). Immobilised activity on buccal surfaces ranged between 0.25 and 11.1 mU/cm(2) (palatal slabs: 0.14-3.06 mU/cm(2)). Thirty minutes pellicles formed on buccal sites exhibited significantly higher immobilised amylase activity (2.85 +/- 3.65 mU/cm(2)) than palatal ones (0.63 +/- 0.32 mU/cm(2)). Amylase activity showed great intraindividual variability when comparing same positions on different days. CONCLUSION: Pellicle bound amylase activity can be determined directly with GalG2CNP and shows a Michaelis Menten kinetic. Enzyme activity of the amylase immobilised in the in situ pellicle reveals great intra- and interindividual differences.

Fibrate treatment of eEOCs in murine AKI.

BACKGROUND: Early endothelial outgrowth cells (eEOCs) protect mice from acute kidney injury (AKI). Peroxisome proliferator-activated receptor-alpha (PPAR-α) has been shown to mediate renoprotective effects under different experimental conditions. The aim of the study was to investigate consequences of fibrate treatment of murine eEOCs in a cell-based therapeutic approach to AKI. METHODS: Male C57/Bl6N mice, subjected to unilateral renal ischemia (40 min) post-uninephrectomy, were systemically injected with 0.5 × 10(6) untreated or fenofibrate (FF 1, 5, 10 or 50 µm)/clofibrate (CF 1 mm) pretreated syngeneic murine eEOCs. Renal function and morphology were analyzed 48 h later. Cellular consequences of eEOC treatment with fibrates (FF 1, 5, 10, 50 µm, CF 1 mm) were evaluated using different in vitro assays (direct cell migration, apoptosis/necrosis, ELISA studies). RESULTS: Administration of untreated eEOCs did not protect mice from AKI. Injection of eEOCs treated with CF (1 mm) or FF 50 µm did not result in any protection from ischemia-induced renal dysfunction. In vitro analysis showed reduced cellular secretion of vasoprotective vascular endothelial growth factor (VEGF), an effect that was more pronounced with CF; FF increased percentages of apoptotic/necrotic eEOCs, and both substances failed to stimulate migration of cultured cells. With lower FF concentrations (1, 5, 10 µm) cell survival was increased and 10 µm FF stimulated VEGF secretion. In vivo administration of FF-treated eEOCs (10 µm) also did not result in any renoprotective effect. CONCLUSION: PPAR-α activation using fibrates does not stimulate renoprotective effects of syngeneic murine eEOCs in ischemic AKI, although lower fibrate concentrations significantly activate eEOCs in vitro.

Endothelial progenitor cells in systemic lupus erythematosus.

BACKGROUND AND AIM: Systemic lupus erythematosus (SLE) is an autoimmune-mediated disease, characterized by inflammation of small arteries and arterioles. Patients with SLE suffer from a 17-fold higher risk for developing atherosclerosis than healthy individuals. Endothelial progenitor cells (EPCs) have been shown to be critically involved in microvascular repair under both physiological and pathological conditions. The aim of the present study was to analyze EPC regeneration and mobilization in SLE patients with variable disease activity and undergoing different treatment regimens. METHODS: Forty-eight patients with SLE were analyzed. Healthy, age- and sex-matched individuals served as controls. Total circulating EPCs were enumerated by FACS analysis, and regenerative activity of the cells was analyzed by a colony-forming assay. Vasomodulatory mediators were quantified by ELISA. RESULTS: SLE patients did not show lower or higher percentages of total circulating EPCs, but they displayed significantly lower colony numbers as compared with healthy controls, indicating impaired EPC regeneration and mobilization. Low and high disease activity were associated with decreased EPC regeneration, while moderate disease activity was not. Hypertension and, to some extent, renal involvement were associated with reduced colony formation. Patients not receiving hydroxychloroquine (HCQ) treatment and those undergoing glucocorticoid therapy showed impaired EPC regeneration as well. CONCLUSIONS: SLE patients suffer from both defective regeneration and mobilization of EPCs. Such an impairment of the EPC system, as one key regulatory element in the process of vasorepair, could potentially promote microvascular damage in SLE. Long-term glucocorticoid therapy may further suppress the EPC system, while HCQ may prevent regeneration of the cells.

Angiopoietin-2 modulates eEOC-mediated renoprotection in AKI in a dose-dependent manner.

BACKGROUND: Early endothelial outgrowth cells (eEOCs) significantly protect mice from acute kidney injury (AKI). Angiopoietin-2 (Ang-2) has been shown to be critically involved in vascular repair and homeostasis. The aim of this study was to investigate consequences of Ang-2 treatment of syngeneic murine eEOCs in a cell-based therapeutic approach for AKI. METHODS: Male 8- to 12-week-old C57/Bl6N mice, subjected to unilateral renal ischemia (40 minutes) postuninephrectomy were systemically injected with 0.5 × 10(6) untreated or Ang-2-pretreated syngeneic murine eEOCs. Renal function and morphology were analyzed 48 hours later. Cellular consequences of eEOC treatment with Ang-2 were evaluated using different in vitro assays (direct and indirect migration, apoptosis/necrosis, ELISA studies). RESULTS: Administration of untreated eEOCs did not protect mice from AKI. Ang-2 dose-dependently modulated cell effects in AKI. While incubating the cells at a concentration of 200 ng/mL (1 hour) did not have any effect on renal function, doubling the concentration (400 ng/mL) resulted in significant renoprotection of cell-injected mice. With 800 ng/mL, cell injection dramatically worsened renal function of treated animals. In vitro analysis showed significantly accelerated migration of cultured mature endothelial cells after incubation with supernatant from Ang-2-treated eEOCs (200 and 400 ng/mL). These effects were most pronounced with 400 mg/mL. In addition, Ang-2 promoted survival of eEOCs. Cellular releases of VEGF and IL-6 were decreased by Ang-2, while TGF-ß levels in the medium of Ang-2-stimulated eEOCs were not different from those in untreated cells. CONCLUSION: Ang-2 acts as modulator of eEOCs in AKI. The migration analysis indicates that the Ang-2 significantly alters indirect (paracrine) activity of eEOCs, thus promoting renoprotection in a dose-dependent manner.

Impairment and Differential Expression of PR3 and MPO on Peripheral Myelomonocytic Cells with Endothelial Properties in Granulomatosis with Polyangiitis.

Background. Granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA) are autoimmune-mediated diseases characterized by vasculitic inflammation of respiratory tract and kidneys. Clinical observations indicated a strong association between disease activity and serum levels of certain types of autoantibodies (antineutrophil cytoplasm antibodies with cytoplasmic [cANCA in GPA] or perinuclear [pAN CA in MPA] immunofluorescence). Pathologically, both diseases are characterized by severe microvascular endothelial cell damage. Early endothelial outgrowth cells (eEOCs) have been shown to be critically involved in neovascularization under both physiological and pathological condition. Objectives. The principal aims of our study were (i) to analyze the regenerative activity of the eEOC system and (ii) to determine mPR3 and MPO expression in myelo monocytic cells with endothelial characteristics in GPA and MPA patients. Methods. In 27 GPA and 10 MPA patients, regenerative activity blood-derived eEOCs were analyzed using a culture-forming assay. Flk-1(+), CD133(+)/Flk-1(+), mPR3(+), and Flk-1(+)/mPR3(+) myelomonocytic cells were quantified by FACS analysis. Serum levels of Angiopoietin-1 and TNF-α were measured by ELISA. Results. We found reduced eEOC regeneration, accompanied by lower serum levels of Angiopoietin-1 in GPA patients as compared to healthy controls. In addition, the total numbers of Flk-1(+) myelomonocytic cells in the peripheral circulation were decreased. Membrane PR3 expression was significantly higher in total as well as in Flk-1(+) myelomonocytic cells. Expression of MPO was not different between the groups. Conclusions. These data suggest impairment of the eEOC system and a possible role for PR3 in this process in patients suffering from GPA.