Andreas Schmiedl

No Evidence of Transdifferentiation of Human Endothelial Progenitor Cells Into Cardiomyocytes After Coculture With Neonatal Rat Cardiomyocytes

Background— Recent studies have suggested the differentiation of human endothelial progenitor cells (huEPCs) isolated from peripheral blood into cardiomyocytes. This study investigates whether, when cocultured, neonatal rat cardiomyocytes (NRCMs) can induce transdifferentiation of huEPCs into cardiomyocytes. Methods and Results— Coculture experiments with 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine (DiI)–labeled huEPCs and NRCMs have been performed. Cocultures have been analyzed by means of flow cytometry, 3D confocal laser microscopy, species-specific reverse transcriptase–polymerase chain reaction for the expression of human cardiac marker genes, and electron microscopy. Although fluorescence-activated cell sorting (FACS) analysis and conventional wide-field fluorescence microscopy suggested the existence of DiIpos cardiomyocytes in cocultures, no convincing evidence of cardiac differentiation of huEPCs has been obtained. Apparently, DiIpos cardiomyocytes were identified as necrotic NRCMs or NRCM-derived vesicles with high levels of autofluorescence or, alternatively, as NRCMs lying on top of or below labeled huEPCs or huEPC fragments. Accordingly, no expression of human Nkx2.5, GATA-4, or cardiac troponin I was detected. Conclusions— No convincing evidence of transdifferentiation of huEPCs into cardiomyocytes was obtained. Although we cannot exclude that recent contrary data may be due to slightly different culture protocols, our study has revealed that recently applied standard analysis tools including FACS and wide-field fluorescence microscopy are not sufficient to demonstrate transdifferentiation in coculture settings and can lead to misinterpretation of the data obtained solely with these methods.

Regulation of Expression and Function of Dipeptidyl Peptidase 4 (DP4), DP8/9, and DP10 in Allergic Responses of the Lung in Rats

The expression of dipeptidyl peptidase 4 (DP4, CD26) affects T-cell recruitment to lungs in an experimental rat asthma model. Furthermore, the gene of the structural homologous DP10 represents a susceptibility locus for asthma in humans, and the functional homologous DP8/9 are expressed in human leukocytes. Thus, although several mechanisms may account for a role of DP4-like peptidases in asthma, detailed information on their anatomical sites of expression and function in lungs is lacking. Therefore, bronchi and lung parenchyma were evaluated using immunohistochemistry and histochemical/enzymatic activity assays, as well as quantitative real-time PCR for this family of peptidases in naïve and asthmatic rat lungs derived from wild-type F344 and DP4-deficient F344 rat strains. Surprisingly, results show not only that the induction of experimental asthma increases DP4 enzymatic activity in the bronchoalveolar lavage fluid and parenchyma, but also that DP8/9 enzymatic activity is regulated and, as well as the expression of DP10, primarily found in the bronchial epithelium of the airways. This is the first report showing a differential and site-specific DP4-like expression and function in the lungs, suggesting a pathophysiologically significant role in asthma.

The effect of titanium dioxide nanoparticles on pulmonary surfactant function and ultrastructure

BackgroundPulmonary surfactant reduces surface tension and is present at the air-liquid interface in the alveoli where inhaled nanoparticles preferentially deposit. We investigated the effect of titanium dioxide (TiO2) nanosized particles (NSP) and microsized particles (MSP) on biophysical surfactant function after direct particle contact and after surface area cycling in vitro. In addition, TiO2 effects on surfactant ultrastructure were visualized.MethodsA natural porcine surfactant preparation was incubated with increasing concentrations (50-500 μg/ml) of TiO2 NSP or MSP, respectively. Biophysical surfactant function was measured in a pulsating bubble surfactometer before and after surface area cycling. Furthermore, surfactant ultrastructure was evaluated with a transmission electron microscope.ResultsTiO2 NSP, but not MSP, induced a surfactant dysfunction. For TiO2 NSP, adsorption surface tension (γads) increased in a dose-dependent manner from 28.2 ± 2.3 mN/m to 33.2 ± 2.3 mN/m (p < 0.01), and surface tension at minimum bubble size (γmin) slightly increased from 4.8 ± 0.5 mN/m up to 8.4 ± 1.3 mN/m (p < 0.01) at high TiO2 NSP concentrations. Presence of NSP during surface area cycling caused large and significant increases in both γads (63.6 ± 0.4 mN/m) and γmin (21.1 ± 0.4 mN/m). Interestingly, TiO2 NSP induced aberrations in the surfactant ultrastructure. Lamellar body like structures were deformed and decreased in size. In addition, unilamellar vesicles were formed. Particle aggregates were found between single lamellae.ConclusionTiO2 nanosized particles can alter the structure and function of pulmonary surfactant. Particle size and surface area respectively play a critical role for the biophysical surfactant response in the lung.

Inhibition of CD26/Dipeptidyl Peptidase IV Enhances CCL11/Eotaxin-Mediated Recruitment of Eosinophils In Vivo

Chemokines mediate the recruitment of leukocytes to the sites of inflammation. N-terminal truncation of chemokines by the protease dipeptidyl peptidase IV (DPPIV) potentially restricts their activity during inflammatory processes such as allergic reactions, but direct evidence in vivo is very rare. After demonstrating that N-terminal truncation of the chemokine CCL11/eotaxin by DPPIV results in a loss of CCR3-mediated intracellular calcium mobilization and CCR3 internalization in human eosinophils, we focused on the in vivo role of CCL11 and provide direct evidence for specific kinetic and rate-determining effects by DPPIV-like enzymatic activity on CCL11-mediated responses of eosinophils. Namely, it is demonstrated that i.v. administration of CCL11 in wild-type F344 rats leads to mobilization of eosinophils into the blood, peaking at 30 min. This mobilization is significantly increased in DPPIV-deficient F344 rats. Intradermal administration of CCL11 is followed by a dose-dependent recruitment of eosinophils into the skin and is significantly more effective in DPPIV-deficient F344 mutants as well as after pharmacological inhibition of DPPIV. Interestingly, CCL11 application leads to an up-regulation of DPPIV, which is not associated with negative feedback inhibition via DPPIV-cleaved CCL11(3–74). These findings demonstrate regulatory effects of DPPIV for the recruitment of eosinophils. Furthermore, they illustrate that inhibitors of DPPIV have the potential to interfere with chemokine-mediated effects in vivo including but not limited to allergy.

Simvastatin attenuates ventilator-induced lung injury in mice

IntroductionMechanical ventilation (MV) is a life saving intervention in acute respiratory failure without alternative. However, particularly in pre-injured lungs, even protective ventilation strategies may evoke ventilator-induced lung injury (VILI), which is characterized by pulmonary inflammation and vascular leakage. Adjuvant pharmacologic strategies in addition to lung protective ventilation to attenuate VILI are lacking. Simvastatin exhibited anti-inflammatory and endothelial barrier stabilizing properties in vitro and in vivo.MethodsMice were ventilated (12 ml/kg; six hours) and subjected to simvastatin (20 mg/kg) or sham treatment. Pulmonary microvascular leakage, oxygenation, pulmonary and systemic neutrophil and monocyte counts and cytokine release in lung and blood plasma were assessed. Further, lung tissue was analyzed by electron microscopy.ResultsMechanical ventilation induced VILI, displayed by increased pulmonary microvascular leakage and endothelial injury, pulmonary recruitment of neutrophils and Gr-1high monocytes, and by liberation of inflammatory cytokines in the lungs. Further, VILI associated systemic inflammation characterized by blood leukocytosis and elevated plasma cytokines was observed. Simvastatin treatment limited pulmonary endothelial injury, attenuated pulmonary hyperpermeability, prevented the recruitment of leukocytes to the lung, reduced pulmonary cytokine levels and improved oxygenation in mechanically ventilated mice.ConclusionsHigh-dose simvastatin attenuated VILI in mice by reducing MV-induced pulmonary inflammation and hyperpermeability.

The surface to volume ratio of mitochondria, a suitable parameter for evaluating mitochondrial swelling : correlations during the course of myocardial global ischaemia

Cellular changes occuring in the left ventricular myocardium during ischaemia after different methods of cardiac arrest have been evaluated by morphological and morphometric parameters: volume densities of mitochondria (VVMi), sarcoplasm (VVSp), myofibrils (VVMf), surface densities of mitochondria (SVMi). The surface to volume ratio of mitochondria (SVratioMi) has been used as an independent parameter of mitochondrial swelling. Since ischaemic swelling of myocardial cells increases the volume of the reference space and ischaemic swelling of mitochondria decreases the free sarcoplasm, VVMi and VVSp cannot be considered as reliable indicators of the degree of oedema. SVMi/VVMf remains nearly constant after different forms of cardiac arrest, demonstrating the integrity of mitochondrial outer membranes. The inverse linear ratio between SVratioMi and the mean mitochondrial volume indicates that the increase in mitochondrial volume is achieved by surface smoothing. Loss of matrix structure and fragmentation of cristae occur at an SVratioMi of about 5.8, cristolysis at 5.5 to 5.6 and amorphous matrix densities at an SVratioMi of less than 5.5 μm2/μm3. The SVratioMi is a suitable parameter for evaluating mitochondrial swelling both at the onset and during global myocardial ischaemia, independent of the method of cardiac arrest used. It serves as an indicator of the state of structural preservation of mitochondria during ischaemia.

Distribution of surfactant proteins in type II pneumocytes of newborn, 14-day old, and adult rats: an immunoelectron microscopic and stereological study

Surfactant proteins (SP) have an important impact on the function of the pulmonary surfactant. In contrast to humans, rat lungs are immature at birth. Alveolarization starts on postnatal day 4. Little is known about the distribution of SP during postnatal alveolarization. By immunoelectron microscopy, we studied the distribution of SP-A, SP-D, SP-B, and precursors of SP-C in type II pneumocytes before, near the end and after alveolarization and in mature lungs. We determined the subcellular volume fractions and the relative labeling index to obtain information about preferential labeling of compartments and non-randomness of labeling. Independently of alveolarization, the overall cellular distribution of SP was non-random. A preferential labeling for SP-A and SP-D was found in small vesicles and multivesicular bodies (mvb). SP-B and precursors of SP-C were localized in mvb and lamellar bodies (lb). There are no postnatal changes in labeling for all three SP in these compartments. Labeling intensity for SP-B in lb increased in close correlation with a significant increase in the volume fractions of lb during alveolarization. Our results support the concept that postnatal alveolarization in rat lungs is associated with significant increases in the SP-B content in lb and volume fraction of lb in type II pneumocytes. The postnatal compartment-specific distribution of SP-A, precursors of SP-C and SP-D does not change.

Cathepsin G and Neutrophil Elastase Contribute to Lung-Protective Immunity against Mycobacterial Infections in Mice

The neutrophil serine proteases cathepsin G (CG) and neutrophil elastase (NE) are involved in immune-regulatory processes and exert antibacterial activity against various pathogens. To date, their role and their therapeutic potential in pulmonary host defense against mycobacterial infections are poorly defined. In this work, we studied the roles of CG and NE in the pulmonary resistance against Mycobacterium bovis bacillus Calmette-Guérin (BCG). CG-deficient mice and even more pronounced CG/NE-deficient mice showed significantly impaired pathogen elimination to infection with M. bovis BCG in comparison to wild-type mice. Moreover, granuloma formation was more pronounced in M. bovis BCG-infected CG/NE-deficient mice in comparison to CG-deficient and wild-type mice. A close examination of professional phagocyte subsets revealed that exclusively neutrophils shuttled CG and NE into the bronchoalveolar space of M. bovis BCG-infected mice. Accordingly, chimeric wild-type mice with a CG/NE-deficient hematopoietic system displayed significantly increased lung bacterial loads in response to M. bovis BCG infection. Therapeutically applied human CG/NE encapsulated in liposomes colocalized with mycobacteria in alveolar macrophages, as assessed by laser scanning and electron microscopy. Importantly, therapy with CG/NE-loaded liposomes significantly reduced mycobacterial loads in the lungs of mice. Together, neutrophil-derived CG and NE critically contribute to deceleration of pathogen replication during the early phase of antimycobacterial responses. In addition, to our knowledge, we show for the first time that liposomal encapsulated CG/NE exhibit therapeutic potential against pulmonary mycobacterial infections. These findings may be relevant for novel adjuvant approaches in the treatment of tuberculosis in humans.

Postnatal maturation of tendon, cruciate ligament, meniscus and articular cartilage: a histological study in sheep.

Orthopaedic basic science data on immature skeletons are rare in the literature. Since the number of knee injuries in young humans is steadily increasing, studies on immature animals such as sheep, which can be used as model systems are becoming more and more important. However, no baseline data are available on physiologic and morphologic changes during growth in the relevant tissues. In the present study, histomorphometric changes in the tendon of the musculus flexor digitalis superficialis, the cranial cruciate ligament, the medial meniscus and the articular cartilage of the medial femoral condyle were identified in sheep between the ages of 1 and 40 weeks postnatally. Profound changes in tissue composition during growth could be observed. A high cellularity in the early postnatal period decreases to a constant lower level after 18 weeks. Similar changes during postnatal growth could be observed for blood vessel density. Also, staining of alpha-smooth muscle actin (SMA) and vascular endothelial growth factor (VEGF) steadily decreased. In contrast, the number of components of extracellular matrix steadily increased in all tissues. The age of 18 weeks seems to be a threshold after which the tissue composition of the observed structures remains constant in this species.

Human Umbilical Cord Blood Mononuclear Cells in a Double-Hit Model of Bronchopulmonary Dysplasia in Neonatal Mice

Background Bronchopulmonary dysplasia (BPD) presents a major threat of very preterm birth and treatment options are still limited. Stem cells from different sources have been used successfully in experimental BPD, induced by postnatal hyperoxia. Objectives We investigated the effect of umbilical cord blood mononuclear cells (MNCs) in a new double-hit mouse model of BPD. Methods For the double-hit, date mated mice were subjected to hypoxia and thereafter the offspring was exposed to hyperoxia. Human umbilical cord blood MNCs were given intraperitoneally by day P7. As outcome variables were defined: physical development (auxology), lung structure (histomorphometry), expression of markers for lung maturation and inflammation on mRNA and protein level. Pre- and postnatal normoxic pups and sham treated double-hit pups served as control groups. Results Compared to normoxic controls, sham treated double-hit animals showed impaired physical and lung development with reduced alveolarization and increased thickness of septa. Electron microscopy revealed reduced volume density of lamellar bodies. Pulmonary expression of mRNA for surfactant proteins B and C, Mtor and Crabp1 was reduced. Expression of Igf1 was increased. Treatment with umbilical cord blood MNCs normalized thickness of septa and mRNA expression of Mtor to levels of normoxic controls. Tgfb3 mRNA expression and pro-inflammatory IL-1β protein concentration were decreased. Conclusion The results of our study demonstrate the therapeutic potential of umbilical cord blood MNCs in a new double-hit model of BPD in newborn mice. We found improved lung structure and effects on molecular level. Further studies are needed to address the role of systemic administration of MNCs in experimental BPD.