Ventilation and Perfusion at the Alveolar Level: Insights From Lung Intravital Microscopy

Intravital microscopy (IVM) offers unique possibilities for the observation of biological processes and disease related mechanisms in vivo. Especially for anatomically complex and dynamic organs such as the lung and its main functional unit, the alveolus, IVM provides exclusive advantages in terms of spatial and temporal resolution. By the use of lung windows, which have advanced and improved over time, direct access to the lung surface is provided. In this review we will discuss two main topics, namely alveolar dynamics and perfusion from the perspective of IVM-based studies. Of special interest are unanswered questions regarding alveolar dynamics such as: What are physiologic alveolar dynamics? How do these dynamics change under pathologic conditions and how do those changes contribute to ventilator-induced lung injury? How can alveolar dynamics be targeted in a beneficial way? With respect to alveolar perfusion IVM has propelled our understanding of the pulmonary microcirculation and its perfusion, as well as pulmonary vasoreactivity, permeability and immunological aspects. Whereas the general mechanism behind these processes are understood, we still lack a proper understanding of the complex, multidimensional interplay between alveolar ventilation and microvascular perfusion, capillary recruitment, or vascular immune responses under physiologic and pathologic conditions. These are only part of the unanswered questions and problems, which we still have to overcome. IVM as the tool of choice might allow us to answer part of these questions within the next years or decades. As every method, IVM has advantages as well as limitations, which have to be taken into account for data analysis and interpretation, which will be addressed in this review

On Top of the Alveolar Epithelium: Surfactant and the Glycocalyx

Gas exchange in the lung takes place via the air-blood barrier in the septal walls of alveoli. The tissue elements that oxygen molecules have to cross are the alveolar epithelium, the interstitium and the capillary endothelium. The epithelium that lines the alveolar surface is covered by a thin and continuous liquid lining layer. Pulmonary surfactant acts at this air-liquid interface. By virtue of its biophysical and immunomodulatory functions, surfactant keeps alveoli open, dry and clean. What needs to be added to this picture is the glycocalyx of the alveolar epithelium. Here, we briefly review what is known about this glycocalyx and how it can be visualized using electron microscopy. The application of colloidal thorium dioxide as a staining agent reveals differences in the staining pattern between type I and type II alveolar epithelial cells and shows close associations of the glycocalyx with intraalveolar surfactant subtypes such as tubular myelin. These morphological findings indicate that specific spatial interactions between components of the surfactant system and those of the alveolar epithelial glycocalyx exist which may contribute to the maintenance of alveolar homeostasis, in particular to alveolar micromechanics, to the functional integrity of the air-blood barrier, to the regulation of the thickness and viscosity of the alveolar lining layer, and to the defence against inhaled pathogens. Exploring the alveolar epithelial glycocalyx in conjunction with the surfactant system opens novel physiological perspectives of potential clinical relevance for future research

Drug delivery to atherosclerotic plaques using superparamagnetic iron oxide nanoparticles

Introduction: Magnetic drug targeting utilizes superparamagnetic iron oxide nanoparticles (SPIONs) to accumulate drugs in specified vasculature regions. Methods: We produced SPIONs conjugated with dexamethasone phosphate (SPION-DEXA). The efficacy of magnetic drug targeting was investigated in a rabbit model of atherosclerosis induced by balloon injury and high cholesterol diet. Results: In vitro, SPION-DEXA were well-tolerated by endothelial cells. SPION-DEXA were internalized by human peripheral blood mononuclear cells and induced CD163 expression comparable with the free drug. In vivo, magnetic targeting of SPIONs to abdominal aorta was confirmed by histology. Upon vascular injury followed by high-cholesterol diet, early administration of SPION-DEXA enhanced the inflammatory burden in the plaques. Increased macrophage content and larger intima–media thickness were observed in animals treated with SPION-DEXA compared with controls. In advanced atherosclerosis, no beneficial effect of local glucocorticoid therapy was detectable. Conclusion: Magnetic drug targeting represents an efficient platform to deliver drugs to diseased arteries in vivo. However, targeting of vascular injury in the lipid-rich environment using dexamethasone-conjugated SPIONs may cause accelerated inflammatory response

A novel human artery model to assess the magnetic accumulation of SPIONs under flow conditions

Magnetic targeting utilises the properties of superparamagnetic iron oxide nanoparticles (SPIONs) to accumulate particles in specified vasculature regions under an external magnetic field. As the behaviour of circulating particles varies depending on nanoparticle characteristics, magnetic field strength and flow dynamics, we established an improved ex vivo model in order to estimate the magnetic capture of SPIONs in physiological-like settings. We describe here a new, easy to handle ex vivo model of human umbilical artery. Using this model, the magnetic targeting of different types of SPIONs under various external magnetic field gradients and flow conditions was investigated by atomic emission spectroscopy and histology. Among tested particles, SPION-1 with lauric acid shell had the largest capacity to accumulate at the specific artery segment. SPION-2 (lauric acid/albumin-coated) were also successfully targeted, although the observed peak in the iron content under the tip of the magnet was smaller than for SPION-1. In contrast, we did not achieve magnetic accumulation of dextran-coated SPION-3. Taken together, the umbilical artery model constitutes a time- and cost-efficient, 3R-compliant tool to assess magnetic targeting of SPIONs under flow. Our results further imply the possibility of an efficient in vivo targeting of certain types of SPIONs to superficial arteries

Dextran-coated superparamagnetic iron oxide nanoparticles for magnetic resonance imaging: evaluation of size-dependent imaging properties, storage stability and safety

Background: Rising criticism of currently available contrast agents for magnetic resonance imaging, either due to their side effects or limited possibilities in terms of functional imaging, evoked the need for safer and more versatile agents. We previously demonstrated the suitability of novel dextran-coated superparamagnetic iron oxide nanoparticles (SPIONDex) for biomedical applications in terms of safety and biocompatibility. Methods: In the present study, we investigated the size-dependent cross-linking process of these particles as well as the size dependency of their imaging properties. For the latter purpose, we adopted a simple and easy-to-perform experiment to estimate the relaxivity of the particles. Furthermore, we performed an extensive analysis of the particles’ storage stability under different temperature conditions, showing their superb stability and the lack of any signs of agglomeration or sedimentation during a 12 week period. Results: Independent of their size, SPIONDex displayed no irritation potential in a chick chorioallantoic membrane assay. Cell uptake studies of ultra-small (30 nm) SPIONDex confirmed their internalization by macrophages, but not by non-phagocytic cells. Additionally, complement activation-related pseudoallergy (CARPA) experiments in pigs treated with ultra-small SPIONDex indicated the absence of hypersensitivity reactions. Conclusion: These results emphasize the exceptional safety of SPIONDex, setting them apart from the existing SPION-based contrast agents and making them a very promising candidate for further clinical development

Hypoxia-Mediated Impairment of the Mitochondrial Respiratory Chain Inhibits the Bactericidal Activity of Macrophages

In infected tissues oxygen tensions are low. As innate immune cells have to operate under these conditions, we analyzed the ability of macrophages (M phi) to kill Escherichia coli or Staphylococcus aureus in a hypoxic microenvironment. Oxygen restriction did not promote intracellular bacterial growth but did impair the bactericidal activity of the host cells against both pathogens. This correlated with a decreased production of reactive oxygen intermediates (ROI) and reactive nitrogen intermediates. Experiments with phagocyte NADPH oxidase (PHOX) and inducible NO synthase (NOS2) double-deficient M phi revealed that in E. coli- or S. aureus-infected cells the reduced antibacterial activity during hypoxia was either entirely or partially independent of the diminished PHOX and NOS2 activity. Hypoxia impaired the mitochondrial activity of infected M phi. Inhibition of the mitochondrial respiratory chain activity during normoxia (using rotenone or antimycin A) completely or partially mimicked the defective antibacterial activity observed in hypoxic E. coli-or S. aureus-infected wild-type M phi, respectively. Accordingly, inhibition of the respiratory chain of S. aureus-infected, normoxic PHOX-/- NOS2(-/-) M phi further raised the bacterial burden of the cells, which reached the level measured in hypoxic PHOX-/- NOS2(-/-) M phi cultures. Our data demonstrate that the reduced killing of S. aureus or E. coli during hypoxia is not simply due to a lack of PHOX and NOS2 activity but partially or completely results from an impaired mitochondrial antibacterial effector function. Since pharmacological inhibition of the respiratory chain raised the generation of ROI but nevertheless phenocopied the effect of hypoxia, ROI can be excluded as the mechanism underlying the antimicrobial activity of mitochondria

Non-immunogenic dextran-coated superparamagnetic iron oxide nanoparticles: a biocompatible, size-tunable contrast agent for magnetic resonance imaging

Iron oxide-based contrast agents have been in clinical use for magnetic resonance imaging (MRI) of lymph nodes, liver, intestines, and the cardiovascular system. Superparamagnetic iron oxide nanoparticles (SPIONs) have high potential as a contrast agent for MRI, but no intravenous iron oxide-containing agents are currently approved for clinical imaging. The aim of our work was to analyze the hemocompatibility and immuno-safety of a new type of dextran-coated SPIONs (SPIONdex) and to characterize these nanoparticles with ultra-high-field MRI. Key parameters related to nanoparticle hemocompatibility and immuno-safety were investigated in vitro and ex vivo. To address concerns associated with hypersensitivity reactions to injectable nanoparticulate agents, we analyzed complement activation-related pseudoallergy (CARPA) upon intravenous administration of SPIONdex in a pig model. Furthermore, the size-tunability of SPIONdex and the effects of size reduction on their biocompatibility were investigated. In vitro, SPIONdex did not induce hemolysis, complement or platelet activation, plasma coagulation, or leukocyte procoagulant activity, and had no relevant effect on endothelial cell viability or endothelial–monocytic cell interactions. Furthermore, SPIONdex did not induce CARPA even upon intravenous administration of 5 mg Fe/kg in pigs. Upon SPIONdex administration in mice, decreased liver signal intensity was observed after 15 minutes and was still detectable 24 h later. In addition, by changing synthesis parameters, a reduction in particle size <30 nm was achieved, without affecting their hemo- and biocompatibility. Our findings suggest that due to their excellent biocompatibility, safety upon intravenous administration and size-tunability, SPIONdex particles may represent a suitable candidate for a new-generation MRI contrast agent

Comparative analysis of nanosystems' effects on human endothelial and monocytic cell functions.

International audienceThe objective of our work was to investigate the effects of different types of nanoparticles on endothelial (HUVEC) and monocytic cell functions. We prepared and tested 14 different nanosystems comprising liposomes, lipid nanoparticles, polymer, and iron oxide nanoparticles. Some of the tested nanosystems contained targeting, therapeutic, or contrast agent(s). The effect of particles (0-400 mu g/mL) on endothelial-monocytic cell interactions in response to TNF-alpha was investigated using an arterial bifurcation model and dynamic monocyte adhesion assay. Spontaneous HUVEC migration (0-100 mu g/mL nanoparticles) and chemotaxis of monocytic cells towards MCP-1 in presence of particles (0-400 mu g/mL) were determined using a barrier assay and a modified Boyden chamber assay, respectively. Lipid nanoparticles dose-dependently reduced monocytic cell chemotaxis and adhesion to activated HUVECs. Liposomal nanoparticles had little effect on cell migration, but one formulation induced monocytic cell recruitment by HUVECs under non-uniform shear stress by about 50%. Fucoidan-coated polymer nanoparticles (25-50 mu g/mL) inhibited HUVEC migration and monocytic cell chemotaxis, and had a suppressive effect on monocytic cell recruitment under non-uniform shear stress. No significant effects of iron oxide nanoparticles on monocytic cell recruitment were observed except lauric acid and human albumin-coated particles which increased endothelial-monocytic interactions by 60-70%. Some of the iron oxide nanoparticles inhibited HUVEC migration and monocytic cell chemotaxis. These nanoparticle-induced effects are of importance for vascular cell biology and function and must be considered before the potential clinical use of some of the analyzed nanosystems in cardiovascular applications