Grietje Beck

Increased circulating endothelial progenitor cells in septic patients: Correlation with survival

Objective:Endothelial damage and detachment of endothelial cells are known to occur in septic patients. Thus, recruitment of circulating endothelial progenitor cells (cEPCs) to these lesions might have a beneficial effect on the clinical course in septic patients. Therefore, we were interested in whether EPCs, detected by flow cytometry, are increasingly mobilized during sepsis and if this mobilization is associated with clinical outcome. Design:Prospective, nonrandomized study. Setting:Intensive care unit of a university hospital. Patients:Patients with (n = 32) and without (n = 15) sepsis and healthy volunteers (n = 15). Interventions:Peripheral blood mononuclear cells were isolated by Ficoll density gradient centrifugation, and cEPCs were characterized by three-color fluorescence flow cytometry using antibodies against CD133, CD34, and vascular endothelial growth factor receptor-2. Serum concentrations of vascular endothelial growth factor, granulocyte macrophage-colony stimulating factor, and erythropoietin were determined by enzyme-linked immunosorbent assay. Severity of sepsis was assessed according to Acute Physiology and Chronic Health Evaluation II scoring. Measurements and Main Results:In septic patients, the number of cEPCs was significantly higher than in nonseptic intensive care unit patients (p < .05) and healthy controls (p < .02). Nonsurvivors (n = 8), defined as death within 28 days after onset of sepsis, had significantly lower numbers of cEPCs than survivors (n = 24) (p < .0001). The number of cEPCs was correlated with survival in septic patients. Serum vascular endothelial growth factor concentrations were significantly higher in septic patients compared with nonseptic intensive care unit patients and healthy controls (p < .01) and correlated with the cEPC numbers (p < .0001). Similar findings were observed for granulocyte macrophage-colony stimulating factor and erythropoietin. Conclusions:Our data suggest that cEPC enumeration in peripheral blood of septic patients might be a valuable marker to assess the clinical outcome in these patients.

Increased Levels of Circulating Endothelial Progenitor Cells in Patients With Moyamoya Disease

Background and Purpose— Chronic cerebral ischemia leads to higher risk for strokes attributable to insufficient collateralization, resulting from inadequate capacity for arteriogenesis and angiogenesis. Patients with Moyamoya disease (MMD) have similar transient ischemic attack frequencies compared to patients with chronic cerebral ischemia with other etiologies, but a strong capacity for arteriogenesis and angiogenesis. The mechanisms involved in the upregulation of the arteriogenesis and angiogenesis in MMD still remain unknown. In the present study we investigated if circulating endothelial progenitor cells are increasingly mobilized during MMD. Methods— Twenty MMD patients, 8 patients with atherosclerotic cerebrovascular disease, and 15 healthy individuals were included in this study. Peripheral blood mononuclear cells were isolated by Ficoll density gradient centrifugation and circulating endothelial progenitor cells were characterized by triple staining using antibodies against CD133, CD34, and vascular endothelial growth factor receptor-2. Serum concentrations of vascular endothelial growth factor and granulocyte-macrophage colony-stimulating factor were determined by enzyme-linked immunosorbent assay. Results— In MMD patients the number of circulating endothelial progenitor cells was significantly higher than in atherosclerotic cerebrovascular disease patients (P<0.002) and healthy controls (P<0.0001). Serum vascular endothelial growth factor concentrations in MMD patients and in atherosclerotic cerebrovascular disease patients were significantly higher compared to those in healthy controls (P<0.0001). Similar findings were observed for granulocyte-macrophage colony-stimulating factor. An inverse correlation between circulating endothelial progenitor cell numbers and serum levels of vascular endothelial growth factor (r=−0.53; P<0,02) was found in the MMD group. Conclusion— Our results show increased circulating endothelial progenitor cell numbers in MMD, which may play a role in the increased arteriogenesis and angiogenesis in MMD. Moreover, our results suggest that increased circulating endothelial progenitor cell mobilization in MMD may not be entirely mediated by vascular endothelial growth factor or granulocyte-macrophage colony-stimulating factor.

Prevention of Cold‐Preservation Injury of Cultured Endothelial Cells by Catecholamines and Related Compounds

The present study was conducted to dissect the underlying mechanisms by which catecholamines protect cells against preservation injury.

Modulation of chemokine production and expression of adhesion molecules in renal tubular epithelial and endothelial cells by catecholamines.

Background. The present study was conducted to investigate whether catecholamines influence the production of chemokines and adhesion molecules in proximal tubular epithelial cells (PTECs) and endothelial cells. Methods. PTECs and human umbilical vein endothelial cells (HUVECs) were stimulated with various concentrations of dopamine (DA), adrenaline (AD), or noradrenaline (NA), and the production of interleukin (IL)-8, ENA-78, and Gro-&agr; was assessed by ELISA. The influence of catecholamine pretreatment on tumor necrosis factor (TNF)-&agr;–mediated production of these chemokines and the expression of adhesion molecules was also tested. Results. In PTECs, DA inhibited the production of all three chemokines in a dose-dependent fashion. Although inhibition in ENA-78 and Gro-&agr; production was also found in HUVECs, IL-8 production was up-regulated in these cells. Increased IL-8 secretion was predominantly observed at the apical site of the cells. In AD or NA stimulated cells, the production of Gro-&agr; was increased in PTECs and decreased in HUVECs. Down-regulation in IL-8 production was also observed after AD but not after NA stimulation of both cell types. Interestingly, TNF-&agr;–mediated up-regulation in intercellular adhesion molecule 1, vascular cell adhesion molecule (VCAM), and E-selectin was delayed in DA-pretreated HUVECs but not in PTECs. The influence of DA, but not AD or NA, on chemokine production was completely prevented by the addition of N-acetylcysteine. Conclusions. This study demonstrates that catecholamines differentially influence chemokine production and indicates that DA may have anti-inflammatory properties because it delays the expression of adhesion molecules and inhibits the production of chemokines in PTECs and endothelial cells under basal and inflammatory conditions.

Secreted phospholipases A2 induce the expression of chemokines in microvascular endothelium

Acute respiratory distress syndrome (ARDS) is characterized by alterations in microvascular permeability. In ARDS secreted phospholipase A(2) (sPLA(2)) IB and IIA are found to be highly upregulated. In this study, we therefore investigated the influence of exogenously added sPLA(2)-IB and sPLA(2)-IIA on the production of chemokines and adhesion molecules in lung microvascular endothelial cells (LMVEC). Treatment of LMVEC with sPLA(2)s resulted in a significant increase in the production of chemokines and adhesion molecules due to an increased expression of their mRNA and in an enhanced release of oleic acid. The upregulation of chemokines and adhesion molecules by LPS was stronger in the presence of sPLA(2). Activation of NF-kappaB occurred upon stimulation with sPLA(2). Moreover the MAPkinase pERK seems to be involved since a specific pERK inhibitor, e.g., U0126, but not a p38Kinase inhibitor, e.g., SB203580 prevented sPLA(2)-induced chemokine upregulation. Our data therefore suggest that LMVEC are a highly sensitive target for the direct action of extracellular sPLA(2)s.

Hypothermia-induced loss of endothelial barrier function is restored after dopamine pretreatment: role of p42/p44 activation.

Background. Donor dopamine usage is associated with improved immediate graft function after renal transplantation. Although prolonged cold preservation results in an increased vascular permeability, the present study was conducted to examine in vitro and in vivo if dopamine pretreatment influences endothelial barrier function under such conditions. Methods. To assess cold preservation injury in vitro and in vivo, cultured human umbilical vein endothelial cells (HUVEC) and Lewis donor rats were pretreated with dopamine or isotonic saline prior to cold storage. Injury was determined by lactate dehydrogenase (LDH) release, histology, and functional analysis. Results. In vitro cold storage resulted in intercellular gap formation in both untreated and dopamine pretreated HUVEC. In the latter monolayer integrity was completely restored upon rewarming and paracellular transport of fluorescein isothiocyanate-dextran was significantly reduced. In dopamine treated HUVEC, intercellular gap formation was independent of cell death and was associated with redistribution of junctional proteins and condensation of cytoskeleton proteins. In untreated HUVEC proteolysis and cell death were clearly evident after hypothermia. Closing of intercellular gaps was dependent on p42/p44 activation. Regeneration of adenosine triphosphate was only observed in dopamine pretreated cells. Only in dopamine treated Lewis renal allografts subjected to cold storage, activation of p42/p44 occurred upon rewarming. These grafts had a better renal function and displayed less inflammatory cells five days after transplantation. Conclusion. Our study demonstrates beneficial effects of dopamine treatment on cold storage induced endothelial barrier disturbances. This may contribute to the positive effects of catecholamines on immediate graft function of renal allografts in men.

Endothelial progenitor cells in regeneration after acute lung injury: do they play a role?

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are common disorders in patients requiring critical care. The clinical management of these disorders is difficult and unrewarding, and thus they are among the most common causes of death in intensive care units. The activation and damage of pulmonary endothelium comprise the hallmark of ALI/ARDS. Therefore, the recruitment of circulating endothelial progenitor cells (EPCs) to these lesions may exert a beneficial effect on the clinical course of ALI/ARDS. Consequently, cell-based therapies using stem cells to regenerate lung tissue have emerged as potential novel treatment strategies. Although initial studies suggested implantations of exogenously administered bone marrow-derived progenitor cells into damaged vessel walls, recent evidence indicates that this is rather a rare occurrence with uncertain physiologic significance. In the past few years, different populations of progenitor cells were identified, with different functional capacities. This review (1) highlights the different populations of EPCs identified or administered in different models of ALI/ARDS, (2) reports on whether beneficial effects of EPCs could be demonstrated, and (3) puts the conflicting results of different studies into perspective.

N-octanoyl dopamine, a non-hemodyanic dopamine derivative, for cell protection during hypothermic organ preservation.

Background Although donor dopamine treatment reduces the requirement for post transplantation dialysis in renal transplant recipients, implementation of dopamine in donor management is hampered by its hemodynamic side-effects. Therefore novel dopamine derivatives lacking any hemodynamic actions and yet are more efficacious in protecting tissue from cold preservation injury are warranted. We hypothesized that variation of the molecular structure would yield more efficacious compounds avoid of any hemodynamic effects. Methodology/Principal Findings To this end, we assessed protection against cold preservation injury in HUVEC by the attenuation of lactate dehydrogenase (LDH) release. Modification of dopamine by an alkanoyl group increased cellular uptake and significantly improved efficacy of protection. Further variation revealed that only compounds bearing two hydroxy groups in ortho or para position at the benzene nucleus, i.e. strong reductants, were protective. However, other reducing agents like N-acetyl cysteine and ascorbate, or NADPH oxidase inhibition did not prevent cellular injury following cold storage. Unlike dopamine, a prototypic novel compound caused no hemodynamic side-effects. Conclusions/Significance In conclusion, we demonstrate that protection against cold preservation injury by catecholamines is exclusively governed by strong reducing capacity and sufficient lipophilicity. The novel dopamine derivatives might be of clinical relevance in donor pre-conditioning as they are completely devoid of hemodynamic action, their increased cellular uptake would reduce time of treatment and therefore also may have a potential use for non-heart beating donors.

Modulation of IFN-gamma-induced immunogenicity by phosphatidylethanolamine-linked hyaluronic acid.

BACKGROUND The present study was conducted to examine the possibility of modulating interferon (IFN-gamma)-induced immunogenicity by a novel compound that is composed of a PLA2 inhibitor linked to hyaluronic acid (HYPE). METHODS HYPE was tested for its effect on IFN-gamma-induced expression of MHC class I, class II, and intercellular adhesion molecule (ICAM-1) in cultured endothelial and renal proximal tubular cells by flow cytometric analysis (FACS) as well as its ability to influence T cell activation in mixed lymphocyte reaction (MLR) or after mitogen stimulation. RESULTS In FACS, a profound inhibition in MHC class I and ICAM-1 staining was observed in stimulated or unstimulated cells that were incubated with HYPE. This was not due to down-regulation of antigen expression and only occurred when monoclonal antibodies, but not when polyclonal antibodies, were used. HYPE inhibited the induction of MHC class II in both cell types after IFN-gamma stimulation in a dose-dependent manner. Moreover, the induction of class II transactivator (CIITA) was completely inhibited under these conditions, most likely because it blocked the binding of IFN-gamma to the cell membrane. Addition of HYPE to MLR inhibited the proliferation of T cells and the secretion of interleukin (IL)-2, IFN-gamma, and IL-10. This was not observed when HYPE was added together with anti-CD3 or phytohemagglutinin (PHA). CONCLUSION Our study provides experimental evidence that HYPE has immunosuppressive features. This makes the compound an interesting candidate as an immunosuppressive drug, not only in organ transplantation, but also in diseases where IFN-gamma is overexpressed.

Amelioration of endotoxin-induced sepsis in rats by membrane anchored lipid conjugates.

ObjectiveIn the pathogenesis of septic shock, caused by either bacterial toxins or trauma, increased production of multiple proinflammatory mediators, such as phospholipase A2 (PLA2), cytokines, and chemokines, is known to be of major importance. The present study was undertaken to investigate the influence of a newly designed extracellular PLA2 inhibitor (ExPLI) on synthesis of proinflammatory mediators and mortality rate in a rat sepsis model. DesignProspective, randomized animal study. SettingExperimental laboratory. SubjectsMale Wistar-rats weighing 200–300 g. InterventionsMortality was induced by intraperitoneal bolus administration of lipopolysaccharide 15 mg/kg in 22 rats that were pretreated with NaCl or ExPLI (150 mg/kg). Furthermore, nine rats received a sublethal bolus of lipopolysaccharide (7.5 mg/kg) and nine rats received lipotechoic acid (8 mg/kg) simultaneously with or after ExPLI administration. Blood samples were collected from these rats, and cytokine concentrations were assessed by enzyme-linked immunosorbent assay. Lung and kidney were removed for RNA isolation and immunohistological analysis. Measurements and Main ResultsExPLI treatment significantly reduced lipopolysaccharide-induced mortality of rats (90.9 and 36.4%, p < .05). Up-regulation of tumor necrosis factor-&agr; and interleukin-6 production in serum after endotoxin treatment was significantly inhibited when ExPLIs were administered at the time of or before sepsis induction by using lipopolysaccharide or lipotechoic acid (p < .01). Similarly, messenger RNA expression of secreted PLA2-IIA, interleukin-1, or inducible nitric oxide synthase and the expression of intercellular adhesion molecule-1 were significantly down-regulated in lung and kidney of ExPLI-treated rats, as demonstrated by RNase protection assay, reverse transcription-polymerase chain reaction, or immunohistochemistry. ConclusionsExPLIs may be considered as potentially effective compounds to prevent the production of inflammatory mediators and to improve mortality rate in septic patients.