Low-dose adrenomedullin-2/intermedin(8-47) reduces pulmonary ischemia/reperfusion injury.

Adrenomedullin-2/intermedin stabilizes the pulmonary microvascular barrier challenged by application of thrombin ex vivo and by experimental ventilation in vivo. Here, we test the hypothesis that adrenomedullin-2/intermedin(8-47) protects mouse lungs from ischemia/reperfusion injury in vivo. C57BL/6 mice were anesthetized, intubated, ventilated, and heparinized. Blood vessels and the main bronchus of the left lung were clamped for 90min. Thereafter, lungs were reperfused for 120min. Five min before clamping and before reperfusion, mice obtained intravenous injections of adrenomedullin-2/intermedin(8-47). After reperfusion, mice were sacrificed and bronchoalveolar lavage of the left and the right lung was performed separately. The integrity of the blood-air barrier was investigated by electron microscopy using stereological methods. In response to ischemia/reperfusion injury, intraalveolar leukocytes accumulated in the ischemic lung. Two applications of 10ng/kg body weight adrenomedullin-2/intermedin(8-47) dramatically reduced leukocyte infiltration to about 15% (p≤0.001). Also the proportion of the subpopulation of neutrophil granulocytes decreased (12% vs 5%, p=0.013). Electron microscopy revealed a protection of the blood-air barrier by adrenomedullin-2/intermedin(8-47). Adrenomedullin-2/intermedin(8-47) ameliorates early ischemia/reperfusion injury in mouse lungs by protecting the integrity of the blood-air barrier and by potently reducing leukocyte influx into the alveolar space. Adrenomedullin-2/intermedin(8-47) might be of therapeutic interest in lung transplantation and cardiopulmonary bypass.

Dry powder aerosolization of a recombinant surfactant protein-C-based surfactant for inhalative treatment of the acutely inflamed lung.

OBJECTIVE: Inhalative application of substantial amounts of pulmonary surfactant to the acutely inflamed lung represents a desirable therapeutic approach but was impossible under clinical conditions because of the technical limitations of currently available devices. We developed a new dry powder aerosolizer for administration of a recombinant surfactant protein-C-based surfactant, determined aerosol characteristics, and evaluated its use in animal models of acute lung injury. DESIGN: Laboratory experiment. SETTING: University laboratory. SUBJECTS: Rabbits and mice. INTERVENTIONS: The efficacy of an aerosol application of recombinant surfactant protein-C surfactant was assessed in three animal models of acute lung injury: in rabbits with acute lung injury caused by repetitive lavage with prolonged and injurious ventilation; in rabbits at day 4 after inhalative application of bleomycin; and in bleomycin-challenged, spontaneously breathing mice. MEASUREMENTS AND MAIN RESULTS: Analysis of aerosolizer characteristics revealed favorable properties making inhalative surfactant treatment in acute lung injury/acute respiratory distress syndrome possible. The generated aerosol had a mass median aerodynamic diameter of 1.6 microm, with 85% of all particles being smaller than 5 microm. The average mass of surfactant being aerosolized was approximately 800 mg/min, thus allowing delivery of large amounts of surfactant. Biochemical and biophysical surfactant properties remained unaltered after aerosolization. In both rabbit models aerosolization of approximately 500 mg recombinant surfactant protein-C surfactant resulted in a far-reaching restoration of gas exchange and compliance, with Pao2/Fio2 values approaching control values. In bleomycin-challenged, spontaneously breathing mice, surfactant aerosolization resulted in a restoration of compliance. CONCLUSIONS: The described dry powder aerosolizer may be applicable to surfactant therapy of acute lung injury/acute respiratory distress syndrome. This conclusion is based on four main factors. High doses comparable to those used for intratracheal instillation in humans can be generated within a relatively short time period, the device can be connected to the inspiratory limb of the ventilator circuit, the aerosolized surfactant material is biophysically fully active, and therapeutic efficacy was proven in three different animal models of acute lung injury/acute respiratory distress syndrome.

Monitoring of experimental rat lung transplants by high-resolution flat-panel volumetric computer tomography (fpVCT).

OBJECTIVE: Noninvasive assessment of experimental lung transplants with high resolution would be favorable to exclude technical failure and to follow up graft outcome in the living animal. Here we describe a flat-panel Volumetric Computed Tomography (fpVCT) technique using a prototype scanner. METHODS: Lung transplantation was performed in allogeneic as well as in corresponding syngeneic rat strain combinations. At different time points post-transplantation, fpVCT was performed. RESULTS: Lung transplants can be visualized in the living rat with high-spatial resolution. FpVCT allows a detailed analysis of the lung and the bronchi. Infiltrates developing during rejection episodes can be diagnosed and follow-up studies can easily be performed. CONCLUSIONS: With fpVCT it is possible to control the technical success of the surgical procedure. Graft rejection can be visualized individually in the living animal noninvasively, which is highly advantageous for studying the pathogenesis of chronic rejection or to monitor new therapies.

Treatment with keratinocyte growth factor does not improve lung allograft survival in the rat.

PURPOSE: Lung allografts are threatened by primary graft dysfunction, infections, and rejection. Novel therapies protecting pulmonary allografts are badly needed. Keratinocyte growth factor (KGF) protects the lung against a variety of injurious stimuli and exerts anti-inflammatory effects. The aim of the study was to test the potential of recombinant truncated KGF (DeltaN23-KGF, palifermin) to attenuate pulmonary allograft rejection. MATERIALS AND METHODS: Intratracheal instillation of 5 mg/kg DeltaN23-KGF was performed twice in donor rats on days 3 and 2 before explantation of the lung. In control animals, an equivalent volume of vehicle was instilled. Left lungs were transplanted in the fully allogeneic Dark Agouti to Lewis rat strain combination and in the less stringent Fischer 344 to Wistar Kyoto combination. Allograft recipients were additionally treated with DeltaN23-KGF post-transplantation. Graft outcome, leukocytic infiltration, and major histocompatibility complex (MHC) class II antigen expression was analyzed. RESULTS: In both rat strain combinations, DeltaN23-KGF treatment did not improve pulmonary allograft outcome. Graft infiltration by macrophages and T lymphocytes remained unchanged. In addition, we demonstrated that MHC class II antigens were more abundant in KGF-treated allografts compared to control-treated grafts, which probably results in an increased alloreactivity. CONCLUSION: In conclusion, intratracheal DeltaN23-KGF treatment is not effective to prevent acute pulmonary allograft rejection.

Time-dependent changes in pulmonary surfactant function and composition in acute respiratory distress syndrome due to pneumonia or aspiration.

BACKGROUND: Alterations to pulmonary surfactant composition have been encountered in the Acute Respiratory Distress Syndrome (ARDS). However, only few data are available regarding the time-course and duration of surfactant changes in ARDS patients, although this information may largely influence the optimum design of clinical trials addressing surfactant replacement therapy. We therefore examined the time-course of surfactant changes in 15 patients with direct ARDS (pneumonia, aspiration) over the first 8 days after onset of mechanical ventilation. METHODS: Three consecutive bronchoalveolar lavages (BAL) were performed shortly after intubation (T0), and four days (T1) and eight days (T2) after intubation. Fifteen healthy volunteers served as controls. Phospholipid-to-protein ratio in BAL fluids, phospholipid class profiles, phosphatidylcholine (PC) molecular species, surfactant proteins (SP)-A, -B, -C, -D, and relative content and surface tension properties of large surfactant aggregates (LA) were assessed. RESULTS: At T0, a severe and highly significant reduction in SP-A, SP-B and SP-C, the LA fraction, PC and phosphatidylglycerol (PG) percentages, and dipalmitoylation of PC (DPPC) was encountered. Surface activity of the LA fraction was greatly impaired. Over time, significant improvements were encountered especially in view of LA content, DPPC, PG and SP-A, but minimum surface tension of LA was not fully restored (15 mN/m at T2). A highly significant correlation was observed between PaO2/FiO2 and minimum surface tension (r = -0.83; p < 0.001), SP-C (r = 0.64; p < 0.001), and DPPC (r = 0.59; p = 0.003). Outcome analysis revealed that non-survivors had even more unfavourable surfactant properties as compared to survivors. CONCLUSION: We concluded that a profound impairment of pulmonary surfactant composition and function occurs in the very early stage of the disease and only gradually resolves over time. These observations may explain why former surfactant replacement studies with a short treatment duration failed to improve outcome and may help to establish optimal composition and duration of surfactant administration in future surfactant replacement studies in acute lung injury.

Compartment- and cell-specific expression of coagulation and fibrinolysis factors in the murine lung undergoing inhalational versus intravenous endotoxin application.

Intraalveolar and intravascular fibrin formation are typical hallmarks of acute inflammatory lung diseases, and may foster subsequent fibroproliferative events. We investigated the regulation and cellular sources of key coagulation and fibrinolysis factors in lungs undergoing compartmentalized challenge with endotoxin (LPS). BALB/c mice received 15 ng LPS either by intravenous injection or by inhalation. Quantitative gene expression analysis (real-time RT-PCR) was performed for tissue factor (TF), TF pathway inhibitor (TFPI), tissue-type plasminogen activator (t-PA), urokinase-type-PA (u-PA), PA inhibitor-1 (PAI-1), and PAI-2 in peripheral white blood cells (PBC) as well as in alveolar macrophages (AM), type-II pneumocytes (ATII), endothelial cells (EC) and smooth muscle cells (SMC), all obtained by laser microdissection. Neither route of LPS administration caused substantial protein leakage or leukocyte recruitment into the alveolar space. Compartmentalized upregulation of procoagulant and downregulation of fibrinolytic activities was, however, observed in response to both modes of LPS challenge. Intraalveolar endotoxin, in particular, caused strong upregulation of TF ( approximately 20-fold increase in gene expression) and PAI-2 (225-fold increase) in microdissected AM, upregulation of PAI-1 in microdissected ATII (300-fold increase) and EC (180-fold increase), upregulation of t-PA in EC (40-fold), and downregulation of u-PA in vascular smooth muscle cells. TFPI was largely unchanged in all cell types, and PBC showed no major gene regulatory response to inhaled endotoxin. We conclude that the lung possesses a cell-specific alveolar coagulation and fibrinolysis system, being independent of the vascular coagulation cascade and responding readily with enhanced procoagulant and anti-fibrinolytic activities to LPS challenge.