Neysan Rafat

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.

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.

Heterogeneity in lipopolysaccharide responsiveness of endothelial cells identified by gene expression profiling: role of transcription factors

Interindividual differences of endothelial cells in response to endotoxins might contribute to the diversity in clinical outcome among septic patients. The present study was conducted to test the hypothesis that endothelial cells (EC) with high and low proinflammatory potential exist and to dissect the molecular basis underlying this phenomenon. Thirty human umbilical vein endothelial cell (HUVEC) lines were stimulated for 24 h with lipopolysaccharide (LPS) and screened for interleukin (IL)‐8 production. Based on IL‐8 production five low and five high producers, tentatively called types I and II responders, respectively, were selected for genome‐wide gene expression profiling. From the 74 genes that were modulated by LPS in all type II responders, 33 genes were not influenced in type I responders. Among the 41 genes that were increased in both responders, 17 were expressed significantly stronger in type II responders. Apart from IL‐8, significant differences in the expression of proinflammatory related genes between types I and II responders were found for adhesion molecules [intercellular adhesion molecule (ICAM‐1), E‐selectin)], chemokines [monocyte chemoattractant protein (MCP‐1), granulocyte chemotactic protein (GCP‐2)], cytokines (IL‐6) and the transcription factor CCAAT/enhancer binding protein‐delta (C/EBP‐δ). Type I responders also displayed a low response towards tumour necrosis factor (TNF)‐α. In general, maximal activation of nuclear factor (NF)‐κB was achieved in type I responders at higher concentrations of LPS compared to type II responders. In the present study we demonstrate that LPS‐mediated gene expression differs quantitatively and qualitatively in types I and II responders. Our results suggest a pivotal role for common transcription factors as a low inflammatory response was also observed after TNF‐α stimulation. Further studies are required to elucidate the relevance of these findings in terms of clinical outcome in septic patients.

Donor dopamine treatment limits pulmonary oedema and inflammation in lung allografts subjected to prolonged hypothermia

Background. Endothelial barrier dysfunction severely compromises organ function after reperfusion. Because dopamine pretreatment improves hypothermia mediated barrier dysfunction, we tested the hypothesis that dopamine treatment of lung allografts positively affects tissue damage associated with hypothermic preservation and reperfusion. Methods. Rats were treated for 1 hr with dopamine (5 &mgr;g/min/kg) or vehicle (NaCl). Thereafter lungs were explanted, flushed with Perfadex solution and stored at 4°C for different time periods. Peak inspiratory pressure (PIP), pulmonary arterial pressure (PAP), and lung weight were measured online during reperfusion. Inflammatory mediators in the perfusate and the expression of adhesion molecules in situ were measured after perfusion. Results. Lungs could tolerate a cold ischemia time of up to 6 hr with stable PIP, PAP, and no edema formation upon reperfusion. Cold ischemia time above 6 hr significantly increased PIP, PAP, and pulmonary edema in untreated but not in dopamine treated lungs (P≤0.001 dopamine treated vs. untreated). Perfusion and ventilation alone induced a strong up-regulation of cytokine-induced neutrophil chemoattractant-1 and adhesion molecules in untreated lungs, whereas in dopamine treated lungs significantly lower levels were found. Dopamine treatment also inhibited tissue damage associated with hypothermic preservation as measured by nicotinamide adenine dinucleotide staining. Conclusion. Our study suggests that donor dopamine treatment is a highly effective modality to maintain organ quality of lung allograft. These findings are of high clinical relevance because prevention of tissue damage might reduce complications associated with lung transplantation and hence improve graft survival in lung transplant recipients.

The role of endothelial progenitor cells in sepsis

In sepsis and septic shock a series of immunological events are initiated that alter endothelial function in the macrocirculation and microcirculation. Endothelial swelling, deformation and apoptosis with detachment from the vasculature occur and endothelial cells (EC) appear in the circulation. Simultaneous to these pathological processes, reconstitution of the endothelial layer is initiated which can occur via migration and proliferation of surrounding mature ECs. However, terminally differentiated ECs have a low proliferative potential, hence their capacity to substitute damaged endothelium is limited. Therefore, adequate vascular repair requires additional support. Many studies have now convincingly demonstrated that vascular maintenance, repair, angiogenesis and neovascularization are partly mediated by recruitment of endothelial progenitor cells (EPCs) from the basal membrane. However, it seems that EPCs play a pivotal role not only in re-endothelialization after vascular damage, but also after severe inflammation. Recently, evidence was found that EPCs are increasingly mobilized during sepsis and that this mobilization is associated with clinical outcome. In septic patients the number of EPCs was significantly higher than in controls and was correlated with survival and the concentration of cytokines. In summary EPCs may exert an important function as an endogenous repair mechanism to maintain the integrity of the endothelial layer by replacing denuded parts of the microcirculation or by stimulation of EC proliferation. Therefore, EPC enumeration seems to be a valuable prognostic and diagnostic marker for the outcome in these patients and the induction of enhanced EPC mobilization a therapeutic option.

Die Rolle endothelialer Vorläuferzellen in der Sepsis

In sepsis and septic shock a series of immunological events are initiated that alter endothelial function in the macrocirculation and microcirculation. Endothelial swelling, deformation and apoptosis with detachment from the vasculature occur and endothelial cells (EC) appear in the circulation. Simultaneous to these pathological processes, reconstitution of the endothelial layer is initiated which can occur via migration and proliferation of surrounding mature ECs. However, terminally differentiated ECs have a low proliferative potential, hence their capacity to substitute damaged endothelium is limited. Therefore, adequate vascular repair requires additional support. Many studies have now convincingly demonstrated that vascular maintenance, repair, angiogenesis and neovascularization are partly mediated by recruitment of endothelial progenitor cells (EPCs) from the basal membrane. However, it seems that EPCs play a pivotal role not only in re-endothelialization after vascular damage, but also after severe inflammation. Recently, evidence was found that EPCs are increasingly mobilized during sepsis and that this mobilization is associated with clinical outcome. In septic patients the number of EPCs was significantly higher than in controls and was correlated with survival and the concentration of cytokines. In summary EPCs may exert an important function as an endogenous repair mechanism to maintain the integrity of the endothelial layer by replacing denuded parts of the microcirculation or by stimulation of EC proliferation. Therefore, EPC enumeration seems to be a valuable prognostic and diagnostic marker for the outcome in these patients and the induction of enhanced EPC mobilization a therapeutic option.

Effekte von Dopamin auf die zelluläre und humorale Immunantwort von Patienten mit Sepsis

ZusammenfassungZahlreiche In-vitro- und In-vivo-Studien belegen, dass Dopamin neben seinen hämodynamischen Effekten eine Reihe immunmodulatorischer Wirkungen induziert. Dopamin reduziert die Synthese proinflammatorischer und induziert die Synthese antiinflammatorischer Mediatoren. Dopamin hemmt die Synthese neurohypophysärer Hormone und hemmt die Zellproliferation sowie die Thrombozytenaggregation. Es reduziert die Phagozytoseaktivität neutrophiler Granulozyten und induziert Apoptose. Bei hohen Dopaminserumkonzentrationen, wie sie bei einer Sepsis durch vermehrte endogene Synthese, zusätzliche exogene Applikation und verringerte Clearance erreicht werden, könnten diese Effekte zu relevanten Veränderungen pathophysiologischer Abläufe führen. Um die Bedeutung von Dopamin für die zelluläre und humorale Immunantwort von Patienten mit Sepsis hervorzuheben, sind in dieser Übersicht die speziellen Wirkungen von Dopamin zusammengefasst und die zugrunde liegenden Mechanismen dargestellt.AbstractIn vitro and in vivo studies have demonstrated that apart from its hemodynamic action dopamine can modulate immune responses. Dopamine reduces the synthesis of proinflammatory and induces the synthesis of anti-inflammatory mediators. Dopamine inhibits neurohormone synthesis, lymphocyte proliferation and platelet aggregation. It reduces the phagocytic activity of neutrophils and induces apoptosis. Particularly with regard to sepsis, where high serum dopamine levels are reached by enhanced endogeneous production, exogeneous application and impaired clearance, this immunomodulation may have a clinical impact. This review summarizes dopamine-mediated immunomodulating effects to advance the knowledge regarding dopamine as an immune regulator under septic conditions.In vitro and in vivo studies have demonstrated that apart from its hemodynamic action dopamine can modulate immune responses. Dopamine reduces the synthesis of proinflammatory and induces the synthesis of anti-inflammatory mediators. Dopamine inhibits neurohormone synthesis, lymphocyte proliferation and platelet aggregation. It reduces the phagocytic activity of neutrophils and induces apoptosis. Particularly with regard to sepsis, where high serum dopamine levels are reached by enhanced endogenous production, exogenous application and impaired clearance, this immunomodulation may have a clinical impact. This review summarizes dopamine-mediated immunomodulating effects to advance the knowledge regarding dopamine as an immune regulator under septic conditions.

Therapeutic Effects of Bone Marrow-derived Progenitor Cells in Lipopolysaccharide-induced Acute Respiratory Distress Syndrome

Objective: Endotoxin-induced acute respiratory distress syndrome (ARDS) is characterized by diffuse dysfunction of the microvasculature including increased permeability with oedema formation and apoptosis or necrosis of endothelial- and epithelial cells. Concomitantly, an increased concentration of circulating endothelial progenitor cells (EPC) was found in septic patients, which seem to be involved in pulmonary regeneration. The number of circulating EPC correlated inversely to disease severity and mortality. Since bone marrow-derived endothelial progenitor cells (BMDPC) were found homing to damaged lung tissue, a reparative process seems to be initiated right after the initiation of vessel damage or degeneration. In the present study we investigated the potential of BMDPC as a treatment strategy in lipopolysaccharide (LPS)-induced ARDS. Methods: Male Wistar rats received lipopolysaccharide (LPS) (25 μg/kg) systemically and directly after LPS injection, the animals were administered 1x106 suspension of CD133+-cells dissolved in 1 ml of sodium chloride 0.9% or only 1 ml sodium chloride 0.9% for the control group. Mortality, macroscopic changes in lung tissue, disease symptoms, blood gas analyses, serum cytokine concentration, wet/dry weight and long-term results were analyzed. Results: Rats treated with BMDPC showed a significantly improved pulmonary gas exchange, an inhibition of proinflammatory cytokine synthesis, an improved clinical course and a reduced mortality (p<0.024) compared to rats treated with LPS alone. Conclusions: These findings suggest that the application of exogenous BMDPC can reduce the severity of septic organ damage. Cell therapy with BMDPC might therefore become a novel option in ARDS therapy.