C. Hanusch

Hypothermic injury: the mitochondrial calcium, ATP and ROS love-hate triangle out of balance.

Background/Aims: Catecholamines prevent hypothermic cell death which accounts for severe tissue damage and impaired allograft function after prolonged organ preservation. Here, we identified cellular processes which govern hypothermia-mediated cell death in endothelial cells and how they are influenced by dopamine. Methods: Lactate dehydrogenase assay, intracellular ATP, reactive oxygen species and reduced thio-group measurement, intracellular calcium measurement and mitochondrial calcium staining were performed in the study. Results: Intracellular ATP was almost completely depleted within 12 hrs of hypothermic preservation in untreated human umbilical vein endothelial cells (HUVEC), while dopamine pre-treatment significantly delayed ATP depletion. 4 hrs after hypothermia a redox imbalance was observed in untreated cells, which increased with the duration of hypothermia. The redox imbalance was primarily caused by depletion of SH reduction equivalents and was significantly inhibited by dopamine. In addition, hypothermia-induced Ca2+ influx and mitochondrial Ca2+ accumulation were both prevented by dopamine. The protective effect of dopamine was abrogated by ionomycin and sodium azide and partly by oligomycin and CCCP. Conclusions: Our data demonstrated that loss of intracellular ATP, generation of a redox imbalance and accumulation of intracellular Ca2+ underlie cold preservation injury. Dopamine improves the redox balance, prevents intracellular Ca2+ accumulation and delays ATP depletion.

Circulating endothelial progenitor cells are increased in human lung cancer and correlate with stage of disease

AIM Recent studies suggest that circulating endothelial progenitor cells (cEPCs) are recruited to the angiogenic vascular system of non-small-cell lung cancer (NSCLC) and correlate with clinical behaviour. Consequently, the level of cEPCs has been proposed as a novel biomarker for disease progression in NSCLC. The role of cEPCs for the vascularisation of small-cell lung cancer (SCLC) is still unknown. Therefore, this study aims to examine the level of cEPCs as well as the level of EPC mobilising mediators in the blood of lung cancer patients and the correlation with tumour stage and disease progression. METHODS In this study, 36 patients with biopsy-proven lung cancer (32 NSCLC, 4 SCLC) and 15 healthy individuals were recruited. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient centrifugation, and cEPCs were characterised by triple staining using antibodies against CD133, CD34 and vascular endothelial growth factor-receptor (VEGFR)-2. Serum concentrations of VEGF were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS In lung cancer patients, the number of cEPCs was significantly higher than in healthy controls (p=0.000). Regarding tumour stage, NSCLC patients with UICC III-IV had significantly higher EPC counts than UICC I-IIB patients (p=0.044). Serum VEGF concentrations in lung cancer patients were significantly higher than in healthy controls (p=0.000) and correlated with cEPC numbers for all the groups (r=0.42, p=0.003). Follow-up data (n=20) revealed significantly higher cEPC numbers in lung cancer patients with tumour progression than in patients without evidence or progression of disease. The relative change in cEPC numbers between pre- and post-treatment assessment was significantly correlated to disease progression (p=0.000, log rank test) and the combined end points of progression and/or death (p=0.003, log rank test). CONCLUSION Our results show increased cEPCs numbers in lung cancer patients, which may play a role in the vascularisation of lung cancer. Moreover, our results suggest an association of cEPC numbers with tumour stage and progression.

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.

Pre-ischaemic conditioning of the pulmonary endothelium by immunotargeting of catalase via angiotensin-converting-enzyme antibodies

BACKGROUND Alleviation of oxidative stress via targeted delivery of catalase to the pulmonary endothelium by conjugation of angiotensin-converting-enzyme (ACE) monoclonal antibodies attenuates lung injury in an in vivo model of warm lung ischaemia and reperfusion. This study evaluates treatment of lung allografts with conjugates of anti-ACE antibody with catalase (9B9-CAT) in the setting of hypothermic preservation and reports the effect on ischaemia/reperfusion injury in this model. METHODS Rats were injected 1h prior to lung harvesting with mouse immunoglobulin G (IgG) (negative controls), catalase only (CAT) or anti-ACE mAb 9B9 conjugated with catalase (9B9-CAT). Lungs were flushed with low-potassium dextran (LPD) solution, excised and stored at 4 degrees C for 4 and 8h. Grafts were isolated and directly reperfused at 37 degrees C for up to 180 min. Peak inspiratory pressure (PIP), pulmonary arterial pressure (PAP) and lung weight were measured during reperfusion. Anti-oxidative capacity and catalase activity were measured in frozen lung tissue and inflammatory parameters were detected during reperfusion in perfusate solution. RESULTS Cold ischaemia of 8h significantly increased lung weight gain, PIP and PAP in non-immune mouse IgG and CAT-treated lungs than in 9B9-CAT-treated lungs (p<0.005). Significantly higher catalase activity and anti-oxidative status were found in the lung tissue of animals conditioned with 9B9-CAT after 4 and 8h of cold storage than in animals treated with catalase (CAT) alone or in animals treated with non-immune mouse IgG (p<0.01). CONCLUSION These results validate immunotargeting by anti-ACE mAb conjugated with catalase as a prospective and specific strategy to augment anti-oxidative defence of the pulmonary endothelium during lung transplantation. Vascular immunotargeting of anti-oxidative enzymes could limit reactive oxygen species mediated ischaemia-reperfusion (I/R) injury of the lung and has the potential to become a promising modality for extension of the viability of banked transplantation tissue.

Hypothermic preservation of lung allograft inhibits cytokine-induced chemoattractant-1, endothelial leucocyte adhesion molecule, vascular cell adhesion molecule-1 and intracellular adhesion molecule-1 expression

Organ dysfunction is a major clinical problem after lung transplantation. Prolonged cold ischaemia and reperfusion injury are believed to play a central role in this complication. The influence of cold preservation on subsequent warm reperfusion was studied in an isolated, ventilated and perfused rat lung. Rat lungs were flushed with cold Perfadex‐solution and stored at 4°C for different time periods. Thereafter lungs were perfused and ventilated for up to 3 h. Physiological parameters, production of inflammatory mediators and leucocyte infiltration were measured before and after perfusion. Lungs subjected to a cold ischaemia time of up to 6 h showed stable physiological conditions when perfused for 3 h. However, cold‐ischaemia time beyond 6 h resulted in profound tissue oedema, thereby impairing ventilation and perfusion. Warm reperfusion and ventilation per se induced a strong inflammatory response, as demonstrated by a significant up‐regulation of chemokines and adhesion molecules (cytokine‐induced chemoattractant‐1, intracellular adhesion molecule and endothelial leucocyte adhesion molecule), accompanied by enhanced leucocyte infiltration. Although the up‐regulation of inflammatory mediators was blunted in lungs that were subjected to cold ischaemia, this did not influence leucocyte infiltration. In fact, cold ischaemia time correlated with leucocyte sequestration. Although cold preservation inhibits the expression of inflammatory mediators it does not affect leucocyte sequestration during warm reperfusion. Cold preservation might cause impairment of the endothelial barrier function, as evidenced by tissue oedema and profound leucocyte infiltration.

Intraoperative lung edema monitoring by microwave reflectometry

Microwave reflectometry might be a suitable tool for the thoracic surgeon to monitor edema formation of the lung during lung surgery. A new setup of microwave reflectometry for lung water measurements was developed and tested for clinical application. Three lung models were used for the microwave reflectometry tests: 1) the model of an ex vivo isolated perfused rat lung to investigate lung edema formation during ischemia-reperfusion (n=6), 2) the in situ lung of a human patient to demonstrate the feasibility of lung water monitoring during a surgical operation, 3) the model of an ex vivo isolated perfused human lung to investigate edema formation during postischemic reperfusion and to investigate the changes in water content in the region of a tumor. During human lung operation, significant changes in water content occurred in different lung areas. During isolated perfusion, a significant increase in lung water was measured in models 1) and 3) (P=0.03). Water content of tumor tissue was higher than in the surrounding healthy lung tissue. Microwave reflectometry offers a non-invasive approach to monitor lung edema formation in experimental models and during thoracic surgery.

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.

Immunotargeting of the Pulmonary Endothelium via Angiotensin-Converting-Enzyme in Isolated Ventilated and Perfused Human Lung

Vascular immunotargeting of catalase via angiotensin-converting-enzyme (ACE) attenuated lung ischemia reperfusion injury in the rat. As this might be a promising modality for extension of the viability of human lung grafts for transplantation we tested the hypothesis whether anti-ACE antibodies are suitable for human lung protection within the model of isolated perfused and ventilated human lung resections. Right after surgery for lung cancer, human lung specimens were isolated, ventilated and perfused under physiological conditions with 500 μg of either mouse monoclonal antibodies (mAb) to human ACE (9B9, I2H5, 3G8) or non-immune mouse IgG (as a negative control) followed by wash-out perfusion. Perfusion pressure, pH and lung weight gain were measured before and during perfusion. After mAb perfusion and wash-out perfusion period lung tissue was tested for the uptake of mAbs by immunohistochemistry and by enzyme-capture technique. Furthermore, antibody concentration and ACE shedding were measured within the perfusate. We found that ACE activity in tumor and normal lung tissue did not differ between the groups perfused with different mAbs. However, ACE activity in normal lung tissue (17.0 ± 6.0 U/g) was significantly higher compared to tumor tissue (6.0 ± 3.0; p < 0.01). Absolute retaining of mAbs was with 1.3 ± 1.1% of injected dose per gram of tissue in normal lung tissue, 0.7 ± 0.7% of injected dose per gram of tumor tissue and was significantly higher compared to non-immune mouse IgG (0.1 ± 0.1%/g; p < 0.01). Anti-ACE mAbs concentration in the perfusate dropped significantly to 47 ± 11% (p < 0.001) at 40 min of perfusion. No significant difference between different anti-ACE mAbs in the depletion from perfusate has been observed. mAb 9B9 showed the most intense immunostaining (i.e., most significant lung uptake) after each experiment in normal and tumor lung tissue compared to mAbs i2H5 and 3G8 (p < 0.01). These results validate the possibility of immunotargeting of pulmonary endothelium in the human lung tissue by anti-ACE mAbs under in vivo conditions. Furthermore, the model might be useful to investigate targeted therapies in lung cancer without side effects for the patient.

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.