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.

Pyrrolidine dithiocarbamate as an effector and trigger in ischemia-reperfusion injury in adipocutaneous flaps in rats.

BACKGROUND: Immune and inflammatory responses and the regulation of cellular events seem to be major factors in ischemia-reperfusion (I/R) injury. The inhibition of nuclear transcription factor κB (NF-κB) by pyrrolidine dithiocarbamate (PDTC) shows beneficial effects in animal models in various diseases and tissues with respect to I/R injury. If these results from other tissues could be transferred to adipocutaneous flaps in rats, a new approach to pharmacologic preconditioning could be defined for tissue transfer, and PDTC could be a solution as a trigger and effector to ameliorate the aftermath of the I/R injury. METHODS: Fifty-six male WISTAR rats were divided into four experimental groups. An epigastric adipocutaneous flap was raised, and the average flap necrosis was assessed for all groups on the fifth postoperative day using planimetric software. RESULTS: The control group had a significantly lower flap necrosis than the ischemic control group (p < .05). The PDTC nonischemic group had a significantly lower flap necrosis than the ischemic control group (p  =  .005). The ischemic PDTC group had a 10% reduction in flap necrosis that was not significant. CONCLUSION: Our data show that a significant reduction in flap necrosis can be achieved by intravenous application of PDTC.

Reduction of oedema formation after preconditioning with dopamine in an isolated rat lung model is mediated by adrenergic receptors.

BACKGROUND: Donor treatment with dopamine (DA) is an effective modality to improve organ quality by reduction of hypothermic, ischemic and reperfusion (I/R) injury. It is unknown by which mechanism DA reduces oedema formation and inflammation. Therefore we tested the first time in an isolated rat lung model if dopaminergic or adrenergic receptors are involved. MATERIAL/METHODS: Rats were treated for 1 hr with NaCl, DA or simultaneously with DA alpha- beta- D1- or D2-receptor blockers. Thereafter lungs were explanted, flushed with Perfadex-solution and stored at 4°C. Peak inspiratory pressure (PIP), pulmonary arterial pressure (PAP) and lung weight were measured during reperfusion of 3 hrs. Inflammatory mediators and the expression of adhesion molecules were measured after perfusion. RESULTS: Up to 6 hours of hypothermia did not influence oedema formation or PIP and PAP during reperfusion time. However, hypothermia after 8 hrs significantly increased PIP, PAP and pulmonary oedema in NaCl, alpha- and beta-blocker treated lungs, but significantly not in DA, D1- or D2-blocker treated lungs. Perfusion and ventilation alone induced a strong upregulation of cytokine-induced neutrophil chemoattractant-1 and adhesion molecules in untreated, alpha- and beta-blocker treated lungs, while in DA, D1- and D2-blocker treated lungs significant lower levels were found. CONCLUSIONS: Our study suggests that dopamine mediated protective effects on I/R damage and inflammation in donor lungs are most likely mediated via adrenergic receptors. These findings are highly relevant because new strategies for organ preservation are necessary in terms of long donation waiting lists.

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.

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.

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.

The additional detrimental effects of cold preservation on transplantation-associated injury in kidneys from living and brain-dead donor rats.

BACKGROUND: Brain death and cold preservation are major alloantigen-independent risk factors for transplantation outcome. The present study was conducted to assess the influence of these factors on transplantation-associated injury independently or in combination. METHODS: Brain death was induced in F344 rats. Renal grafts were harvested after 6 hr and either directly transplanted in unilateral nephrectomized Lewis recipient or subjected to 24 hr of cold preservation in University of Wisconsin solution before implantation. Allografts obtained from living donor rats were also subjected to cold preservation or not. DNA damage was assessed before implantation by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling staining. Ten days after transplantation, renal histology was performed according to Banff '97 classification. The expressions of cytokines and adhesion molecules were analyzed by quantitative polymerase chain reaction. RESULTS: Cold preservation significantly increased the number of terminal deoxynucleotide transferase-mediated dUTP nick-end labeling positive cells in renal allografts. Ten days after transplantation, histology revealed a higher degree of tubulitis and vasculitis scores when the grafts were subjected to cold storage. Vasculitis was aggravated when the graft was obtained from brain death (BD) donors. BD, but not cold preservation alone, was associated with papillary necrosis. This was more frequently observed after cold preservation. Immunohistology showed an increase in MHC class II+ cells after cold preservation. The combination of BD and cold preservation revealed a higher degree of VEGF and IL-10 expression. CONCLUSIONS: Our Study emphasizes that cold ischemia time should be limited when renal allografts from brain-dead donors are transplanted.

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 Ca(2+) influx and mitochondrial Ca(2+) 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 Ca(2+) underlie cold preservation injury. Dopamine improves the redox balance, prevents intracellular Ca(2+) accumulation and delays ATP depletion.

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 microg/min/kg) or vehicle (NaCl). Thereafter lungs were explanted, flushed with Perfadex solution and stored at 4 degrees 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< or =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.

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.

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.

Clinical review: immunomodulatory effects of dopamine in general inflammation.

Large quantitaties of inflammatory mediators are released during the course of endotoxaemia. These mediators in turn can stimulate the sympathetic nervous system (SNS) to release catecholamines, which ultimately regulate inflammation-associated impairment in tissue perfusion, myocardial impairment and vasodilatation. Treatment of sepsis is based on surgical and/or antibiotic therapy, appropriate fluid management and application of vasoactive catecholamines. With respect to the latter, discussions on the vasopressor of choice are still ongoing. Over the past decade dopamine has been considered the 'first line' vasopressor and is frequently used to improve organ perfusion and blood pressure. However, there is a growing body of evidence that dopamine has deleterious side effects; therefore, its clinical relevance seems to be more and more questionable. Nevertheless, it has not been convincingly demonstrated that other catecholamines are superior to dopamine in this respect. Apart from its haemodynamic action, dopamine can modulate immune responses by influencing the cytokine network. This leads to inhibition of expression of adhesion molecules, inhibition of cytokine and chemokine production, inhibition of neutrophil chemotaxis and disturbed T-cell proliferation. In the present review we summarize our knowledge of the immunomodulatory effects of dopamine, with an emphasis on the mechanisms by which these effects are mediated.