Kai Nowak

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.

Mechanical Properties of Mesh Materials Used for Hernia Repair and Soft Tissue Augmentation

Background Hernia repair is the most common surgical procedure in the world. Augmentation with synthetic meshes has gained importance in recent decades. Most of the published work about hernia meshes focuses on the surgical technique, outcome in terms of mortality and morbidity and the recurrence rate. Appropriate biomechanical and engineering terminology is frequently absent. Meshes are under continuous development but there is little knowledge in the public domain about their mechanical properties. In the presented experimental study we investigated the mechanical properties of several widely available meshes according to German Industrial Standards (DIN ISO). Methodology/Principal Findings Six different meshes were assessed considering longitudinal and transverse direction in a uni-axial tensile test. Based on the force/displacement curve, the maximum force, breaking strain, and stiffness were computed. According to the maximum force the values were assigned to the groups weak and strong to determine a base for comparison. We discovered differences in the maximum force (11.1±6.4 to 100.9±9.4 N/cm), stiffness (0.3±0.1 to 4.6±0.5 N/mm), and breaking strain (150±6% to 340±20%) considering the direction of tension. Conclusions/Significance The measured stiffness and breaking strength vary widely among available mesh materials for hernia repair, and most of the materials show significant anisotropy in their mechanical behavior. Considering the forces present in the abdominal wall, our results suggest that some meshes should be implanted in an appropriate orientation, and that information regarding the directionality of their mechanical properties should be provided by the manufacturers.

Oncological outcome of primary non-metastatic soft tissue sarcoma treated by neoadjuvant isolated limb perfusion and tumor resection

Isolated limb perfusion (ILP) is an effective limb salvage strategy in patients with advanced soft tissue sarcoma (STS) where surgery alone would result in significant functional morbidity or mandate an amputation. Most previous reports of patients undergoing ILP focus on limb salvage rates rather than local and distant relapse rates. Here, we report the oncological outcome of sarcoma patients treated by ILP and 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.

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.

Pure bronchoplastic resections of the bronchus without pulmonary resection for endobronchial carcinoid tumours

OBJECTIVES Bronchopulmonary carcinoid tumours are relatively uncommon primary lung neoplasms. A small proportion of these lesions are predominantly endobronchial and do not extend beyond the bronchial wall. Endoscopic resection can be performed, but carries around a one in three risk of local recurrence and, therefore, mandates long-term surveillance. An alternative is complete surgical resection via bronchoplastic resection. We present our experience of surgical resection in patients with endobronchial carcinoids. METHODS From 2000 to 2010, 13 patients (age 45±16 years, 10 males) underwent pure bronchoplastic resection, including systematic nodal dissection, for endobronchial carcinoid tumours, without the resection of lung parenchyma. RESULTS There was no significant operative morbidity or mortality. This is a retrospective review of a consecutive case series. The last follow-up for all patients was obtained in 2011. The mean maximum tumour size was 18±8 mm. No lymph node invasion was observed. The median follow-up was 6.3±3.3 years, with no regional recurrence. In 1 case, a tumourlet was identified at 5 years in the contralateral airway and viewed as a metachronous new lesion. CONCLUSIONS Bronchial sleeve resection is a safe procedure for suitably located endobronchial carcinoid tumours. Endoscopic resection should be reserved for patients who decline, or are unfit, for surgery.

Heparins that block VEGF-A-mediated von Willebrand factor fiber generation are potent inhibitors of hematogenous but not lymphatic metastasis

Von Willebrand factor (VWF) serves as a nidus for platelet aggregation and thrombosis. We hypothesize that VWF fibers contribute to the development of venous thromboembolism (VTE) and to metastasis formation. Here, we show that vascular and lymphatic endothelial cells (ECs) express VWF in vitro and release VWF fibers after activation by tumor cell supernatants. In contrast, an ex vivo analysis of primary mouse tumors revealed the presence of VWF fibers in the blood microvasculature but not in lymphatic vessels. Unlike the anticoagulant Fondaparinux, an inhibitor of thrombin generation, the low-molecular-weight heparin (LMWH) Tinzaparin inhibited VWF fiber formation and vessel occlusion in tumor vessels by blocking thrombin-induced EC activation and vascular endothelial growth factor-A (VEGF-A)-mediated VWF release. Intradermal tumor cell inoculation in VWF- and ADAMTS13-deficient mice did not alter lymph node metastases compared with wild type animals. Interestingly, multiple tumor-free distal organs exhibited hallmarks of malignancy-related VTE, including luminal VWF fibers, platelet-rich thrombi and vessel occlusions. Furthermore, ADAMTS13 deficiency, characterized by prolonged intraluminal VWF network lifetimes, resulted in a severely increased number of metastatic foci in an experimental model of hematogenous lung seeding. Treatment with Tinzaparin inhibited tumor-induced release of VWF multimers, impeded platelet aggregation and decreased lung metastasis. Thus, our data strongly suggest a critical role of luminal VWF fibers in determining the occurrence of thrombosis and cancer metastasis. Moreover, the findings highlight LMWHs as therapeutic strategy to treat thrombotic complications while executing anti-metastatic activities.

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.

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.

Monitoring water content of rat lung tissuein vivo using microwave reflectometry

Measurement of lung water is an important diagnostic means of assessing pulmonary oedema. Water content affects the dielectric spectrum at microwave frequencies, but quantification is still a problem. A new lung tissue model is presented that allows the calculation of water content from dielectric permitivity. The dielectric permitivity of lung tissue was measured by microwave reflectometry using a noninvasine surface probe. During perfusion of rat lungs (n=22) with blood, injury was induced by interruption of the blood supply for a duration between 0 (control) and 2h. Water content was assessed from dielectric permitivity using a new mixture formula and was also determined by drying and weighing. The mixture formula allows for the dielectric polarisation of water, dry matter and air in the tissue. A linear correlation was found between total water content determined from dielectric permitivity and that from drying and weighing (y=1.001x, R2=0.8). Lung injury showed an increase in total water content from 80.9±1.2% (control) to 84.1±0.9% (p<0.01). The analysis of dielectric permitivity data at microwave frequencies with the new tissue model is sensitive enough to detect water accumulation produced by lung injury and it can be used to monitor total water content without tissue destruction.