Greetje Vande Velde

Magnetoliposomes as magnetic resonance imaging contrast agents

Among the wide variety in iron oxide nanoparticles which are routinely used as magnetic resonance imaging (MRI) contrast agents, magnetoliposomes (MLs) take up a special place. In the present work, the two main types (large and small MLs) are defined and their specific features are commented. For both types of MLs, the flexibility of the lipid coating allows for efficient functionalization, enabling bimodal imaging (e.g., MRI and fluorescence) or the use of MLs as theranostics. These features are especially true for large MLs, where several magnetite cores are encapsulated within a single large liposome, which were found to be highly efficient theranostic agents. By carefully fine-tuning the number of magnetite cores and attaching Gd(3+) -complexes onto the liposomal surface, the large MLs can be efficiently optimized for dynamic MRI. A special type of MLs, biogenic MLs, can also be efficiently used in this regard, with potential applications in cancer treatment and imaging. Small MLs, where the lipid bilayer is immediately attached onto a solid magnetite core, give a very high r2 /r1 ratio. The flexibility of the lipid bilayer allows the incorporation of poly(ethylene glycol)-lipid conjugates to increase blood circulation times and be used as bone marrow contrast agents. Cationic lipids can also be incorporated, leading to high cell uptake and associated strong contrast generation in MRI of implanted cells. Unique for these small MLs is the high resistance the particles exhibit against intracellular degradation compared with dextran- or citrate-coated particles. Additionally, intracellular clustering of the iron oxide cores enhances negative contrast generation and enables longer tracking of labeled cells in time.

Quantification of Lung Fibrosis and Emphysema in Mice Using Automated Micro-Computed Tomography

Background In vivo high-resolution micro-computed tomography allows for longitudinal image-based measurements in animal models of lung disease. The combination of repetitive high resolution imaging with fully automated quantitative image analysis in mouse models of lung fibrosis lung benefits preclinical research. This study aimed to develop and validate such an automated micro-computed tomography analysis algorithm for quantification of aerated lung volume in mice; an indicator of pulmonary fibrosis and emphysema severity. Methodology Mice received an intratracheal instillation of bleomycin (n = 8), elastase (0.25U elastase n = 9, 0.5U elastase n = 8) or saline control (n = 6 for fibrosis, n = 5 for emphysema). A subset of mice was scanned without intervention, to evaluate potential radiation-induced toxicity (n = 4). Some bleomycin-instilled mice were treated with imatinib for proof of concept (n = 8). Mice were scanned weekly, until four weeks after induction, when they underwent pulmonary function testing, lung histology and collagen quantification. Aerated lung volumes were calculated with our automated algorithm. Principal Findings Our automated image-based aerated lung volume quantification method is reproducible with low intra-subject variability. Bleomycin-treated mice had significantly lower scan-derived aerated lung volumes, compared to controls. Aerated lung volume correlated with the histopathological fibrosis score and total lung collagen content. Inversely, a dose-dependent increase in lung volume was observed in elastase-treated mice. Serial scanning of individual mice is feasible and visualized dynamic disease progression. No radiation-induced toxicity was observed. Three-dimensional images provided critical topographical information. Conclusions We report on a high resolution in vivo micro-computed tomography image analysis algorithm that runs fully automated and allows quantification of aerated lung volume in mice. This method is reproducible with low inherent measurement variability. We show that it is a reliable quantitative tool to investigate experimental lung fibrosis and emphysema in mice. Its non-invasive nature has the unique benefit to allow dynamic 4D evaluation of disease processes and therapeutic interventions.

Quantitative evaluation of MRI-based tracking of ferritin-labeled endogenous neural stem cell progeny in rodent brain

Endogenous neural stem cells have the potential to facilitate therapy for various neurodegenerative brain disorders. To increase our understanding of neural stem and progenitor cell biology in healthy and diseased brain, methods to label and visualize stem cells and their progeny in vivo are indispensable. Iron oxide particle based cell-labeling approaches enable cell tracking by MRI with high resolution and good soft tissue contrast in the brain. However, in addition to important concerns about unspecific labeling and low labeling efficiency, the dilution effect upon cell division is a major drawback for longitudinal follow-up of highly proliferating neural progenitor cells with MRI. Stable viral vector-mediated marking of endogenous stem cells and their progeny with a reporter gene for MRI could overcome these limitations. We stably and efficiently labeled endogenous neural stem/progenitor cells in the subventricular zone in situ by injecting a lentiviral vector expressing ferritin, a reporter for MRI. We developed an image analysis pipeline to quantify MRI signal changes at the level of the olfactory bulb as a result of migration of ferritin-labeled neuroblasts along the rostral migratory stream. We were able to detect ferritin-labeled endogenous neural stem cell progeny into the olfactory bulb of individual animals with ex vivo MRI at 30 weeks post injection, but could not demonstrate reliable in vivo detection and longitudinal tracking of neuroblast migration to the OB in individual animals. Therefore, although LV-mediated labeling of endogenous neural stem and progenitor cells resulted in efficient and stable ferritin-labeling of stem cell progeny in the OB, even with quantitative image analysis, sensitivity remains a limitation for in vivo applications.

MRI visualization of endogenous neural progenitor cell migration along the RMS in the adult mouse brain: validation of various MPIO labeling strategies.

The adult rodent brain contains neural progenitor cells (NPCs), generated in the subventricular zone (SVZ), which migrate along the rostral migratory stream (RMS) towards the olfactory bulb (OB) where they differentiate into neurons. The aim of this study was to visualize endogenous NPC migration along the RMS with magnetic resonance imaging (MRI) in adult healthy mice. We evaluated various in situ (in vivo) labeling approaches using micron-sized iron oxide particles (MPIOs) on their efficiency to label endogenous NPCs. In situ labeling and visualization of migrating NPCs were analyzed by a longitudinal MRI study and validated with histology. Here, we visualized endogenous NPC migration in the mouse brain by in vivo MRI and demonstrated accumulation of MPIO-labeled NPCs in the OB over time with ex vivo MRI. Furthermore, we investigated the influence of in situ injection of MPIOs on adult neurogenesis. Quantitative analysis of bromodeoxyuridine labeled cells revealed altered proliferation in the SVZ and NPC migration after in situ MPIO injection. From the labeling strategies presented in this report, intraventricular injection of a small number of MPIOs combined with the transfection agent poly-l-lysine hydrobromide was the best method as labeling of the NPCs was successful and proliferation in the SVZ was only marginally affected. While MRI visualization of endogenous NPC migration can provide insight into aberrant NPC migration in disease models, this work emphasizes the importance to carefully explore the impact on adult neurogenesis when new in situ labeling strategies are developed.

Towards non-invasive monitoring of pathogen-host interactions during Candida albicans biofilm formation using in vivo bioluminescence.

Candida albicans is a major human fungal pathogen causing mucosal and deep tissue infections of which the majority is associated with biofilm formation on medical implants. Biofilms have a huge impact on public health, as fungal biofilms are highly resistant against most antimycotics. Animal models of biofilm formation are indispensable for improving our understanding of biofilm development inside the host, their antifungal resistance and their interaction with the host immune defence system. In currently used models, evaluation of biofilm development or the efficacy of antifungal treatment is limited to ex vivo analyses, requiring host sacrifice, which excludes longitudinal monitoring of dynamic processes during biofilm formation in the live host. In this study, we have demonstrated for the first time that non‐invasive, dynamic imaging and quantification of in vitro and in vivo C. albicans biofilm formation including morphogenesis from the yeast to hyphae state is feasible by using growth‐phase dependent bioluminescent C. albicans strains in a subcutaneous catheter model in rodents. We have shown the defect in biofilm formation of a bioluminescent bcr1 mutant strain. This approach has immediate applications for the screening and validation ofantimycotics under in vivo conditions, for studying host–biofilm interactions in different transgenic mouse models and for testing the virulence of luminescent C. albicans mutants, hereby contributing to a better understanding of the pathogenesis of biofilm‐associated yeast infections.

MRI assessment of blood outgrowth endothelial cell homing using cationic magnetoliposomes.

The use of contrast material to stimulate magnetic resonance imaging (MRI) of migrating cells has become an important area of research. In the present study, cationic magnetoliposomes (MLs) were used to magnetically label human blood outgrowth endothelial cells (BOECs) and follow their homing by magnetic resonance imaging (MRI). The biodistribution and functional integration capacity of BOECs, which have shown extensive promise as gene delivery vehicles, have thus far only rarely been investigated. MLs were avidly internalized by BOECs giving clear MRI contrast in phantom studies and the magnetic labeling did not affect cell proliferation, viability, morphology or homeostasis and elicited only minor reactive oxygen species levels. Intravenous injection of labeled BOECs was compared with injection of free MLs and unlabeled BOECs, resulting in homing of BOECs toward the liver and spleen, which was confirmed by histology. The MLs used offer great potential for cellular tracking studies by MRI when low levels of widely distributed cells are present. In particular, the use of these MLs will allow to evaluate the efficacy of new methods to enhance BOEC homing and integration to optimize their use as efficient vehicles for gene therapy.

Bioluminescence imaging of stroke-induced endogenous neural stem cell response

Brain injury following stroke affects neurogenesis in the adult mammalian brain. However, a complete understanding of the origin and fate of the endogenous neural stem cells (eNSCs) in vivo is missing. Tools and technology that allow non-invasive imaging and tracking of eNSCs in living animals will help to overcome this hurdle. In this study, we aimed to monitor eNSCs in a photothrombotic (PT) stroke model using in vivo bioluminescence imaging (BLI). In a first strategy, inducible transgenic mice expressing firefly luciferase (Fluc) in the eNSCs were generated. In animals that received stroke, an increased BLI signal originating from the infarct region was observed. However, due to histological limitations, the identity and exact origin of cells contributing to the increased BLI signal could not be revealed. To overcome this limitation, we developed an alternative strategy employing stereotactic injection of conditional lentiviral vectors (Cre-Flex LVs) encoding Fluc and eGFP in the subventricular zone (SVZ) of Nestin-Cre transgenic mice, thereby specifically labeling the eNSCs. Upon induction of stroke, increased eNSC proliferation resulted in a significant increase in BLI signal between 2days and 2weeks after stroke, decreasing after 3months. Additionally, the BLI signal relocalized from the SVZ towards the infarct region during the 2weeks following stroke. Histological analysis at 90days post stroke showed that in the peri-infarct area, 36% of labeled eNSC progeny differentiated into astrocytes, while 21% differentiated into mature neurons. In conclusion, we developed and validated a novel imaging technique that unequivocally demonstrates that nestin(+) eNSCs originating from the SVZ respond to stroke injury by increased proliferation, migration towards the infarct region and differentiation into both astrocytes and neurons. In addition, this new approach allows non-invasive and specific monitoring of eNSCs over time, opening perspectives for preclinical evaluation of candidate stroke therapeutics.

Magnetic resonance imaging for noninvasive assessment of lung fibrosis onset and progression: cross-validation and comparison of different magnetic resonance imaging protocols with micro-computed tomography and histology in the bleomycin-induced mouse model.

ObjectivesBleomycin instillation is frequently used to model lung fibrosis, although the onset and severity of pathology varies highly between mice. This makes non–invasive fibrosis detection and quantification essential to obtain a comprehensive analysis of the disease course and to validate novel therapies. Magnetic resonance imaging (MRI) of lung disease progression and therapy may provide such a sensitive in vivo readout of lung fibrosis, bypassing radiotoxicity concerns (when using micro-CT [&mgr;CT]) and elaborate invasive end point measurements (histology). We aimed to optimize and evaluate 3 different lung MRI contrast and acquisition methods to visualize disease onset and progression in the bleomycin-induced mouse model of lung fibrosis using a small-animal MRI scanner. For validation, we compared the MRI results with established &mgr;CT and histological measures of lung fibrosis. Materials and MethodsFree-breathing bleomycin-instilled and control mice were scanned in vivo with respiration-triggered conventional, ultrashort echo time and self-gated MRI pulse sequences (9.4 T) and &mgr;CT at baseline and weekly at days 7, 14, 21, and 28 after bleomycin instillation. After the last imaging time point, the mice were killed and the lungs were isolated for criterion standard histological analysis of lung fibrosis and quantification of lung collagen content for validation of the imaging results. The agreement between quantitative MRI and &mgr;CT data and standard measurements was analyzed by linear regression. ResultsAll 3 MRI protocols were able to visualize and quantify lung pathology onset and progression in individual bleomycin-instilled mice. In vivo MRI results were in excellent agreement with in vivo &mgr;CT and criterion standard histological measures of lung fibrosis. Ultrashort echo time MRI appeared particularly useful for detecting early disease; self-gated MRI, for improved breathing motion handling. DiscussionMagnetic resonance imaging sensitively visualizes and quantifies lung fibrosis in vivo, which makes it a noninvasive, translatable, safe, and potentially more versatile alternative to invasive methods or &mgr;CT, thereby stimulating pathogenesis and preclinical research.

Transplacental sildenafil rescues lung abnormalities in the rabbit model of diaphragmatic hernia

Introduction The management of congenital diaphragmatic hernia (DH) would benefit from an antenatal medical therapy, which addresses both lung hypoplasia and persistent pulmonary hypertension. We aimed at evaluating the pulmonary effects of sildenafil in the fetal rabbit model for DH. Methods We performed a dose-finding study to achieve therapeutic fetal plasmatic concentrations without toxicity following maternal sildenafil administration. Subsequently, DH fetuses were randomly exposed to transplacental placebo or sildenafil 10 mg/kg/day from gestational day 24 until examination at term (day 30). Efficacy measures were ipsilateral pulmonary vascular and airway morphometry, micro-CT-based branching analysis, Doppler flow in the main pulmonary artery and postnatal lung mechanics. Results Fetal sildenafil plasmatic concentration was above the minimal therapeutic level for at least 22 h/day without maternal and fetal side effects. The placebo-exposed DH fetuses had increased wall thickness in peripheral pulmonary vessels and significantly less fifth-order vessels compared with controls (CTR). Sildenafil-exposed DH fetuses, instead, had a medial and adventitial thickness in peripheral pulmonary vessels in the normal range and normal vascular branching. Fetal pulmonary artery Doppler showed a reduction of pulmonary vascular resistances both in DH and in CTR fetuses treated by sildenafil compared with the placebo-treated ones. Sildenafil also reversed the mean terminal bronchiolar density to normal and improved lung mechanics, yet without measurable impact on lung-to-bodyweight ratio. Conclusions In the rabbit model for DH, antenatal sildenafil rescues vascular branching and architecture, reduces pulmonary vascular resistances and also improves airway morphometry and respiratory mechanics.

De novo design of a biologically active amyloid

Aggregation by design Amyloid aggregation is driven by short sequences within proteins that self-assemble into characteristic amyloid structures. About 30 human proteins are implicated in amyloid-associated diseases, but many more contain short sequences that are potentially amyloidogenic. Gallardo et al. designed a peptide based on an amyloidogenic sequence in the vascular endothelial growth factor receptor VEGFR2. The peptide induced VEGFR2 to form aggregates with features characteristic of amyloids. Amyloids were toxic only in cells that required VEGFR2 activity, suggesting that the toxicity was due to loss of function of VEGFR2, rather than to inherent toxicity of the aggregates. The peptide inhibited VEGFR2-dependent tumor growth in a mouse tumor model. Science, this issue p. 10.1126/science.aah4949 A designed peptide drives a protein that does not usually aggregate to form amyloids. INTRODUCTION It has been shown that most proteins possess amyloidogenic segments. However, only about 30 human proteins are known to be involved in amyloid-associated pathologies, and it is still not clear what determines amyloid toxicity in these diseases. We investigated whether an endogenously expressed protein that contains sequences with known amyloidogenic segments, but is not known to aggregate either under normal or pathological conditions, can be induced to do so by seeding it with a peptide comprising the protein’s own amyloidogenic fragment. We chose to target the protein vascular endothelial growth factor receptor 2 (VEGFR2) because it has well-characterized biological function and so could provide a model system with which to investigate the relationship between protein loss of function and amyloid toxicity in different cellular contexts. RATIONALE The capacity of the amyloid conformation of disease proteins to catalyze their own amyloid conversion demonstrates the sequence specificity of amyloid assembly. Because the core of amyloids consists of short amyloidogenic sequence fragments, we hypothesized that a short amyloidogenic protein sequence of VEGFR2, a protein normally not associated with protein aggregation, should be able to interact with and specifically induce the aggregation of VEGFR2, resulting in its functional knockdown. We used TANGO, an algorithm that predicts aggregation-prone sequences, to identify potential amyloidogenic fragments in VEGFR2. We synthesized these fragments as a tandem repeat in a peptide framework in which each unit is flanked by charged residues and coupled by a short peptide linker. The thinking behind this design was that the tandem repeats would promote the formation of diffusable soluble oligomeric aggregates, whereas the charged residues would kinetically stabilize these oligomers and reduce the rate of insoluble fibril formation. RESULTS By screening for loss of function, we identified one peptide, termed “vascin,” that was highly potent at inhibiting VEGFR2. This sequence was derived from the translocation signaling sequence of VEGFR2. We found that vascin is an amyloidogenic peptide that readily forms small β-structured oligomers, ranging from dimers to nonamers, that slowly convert to amyloid fibrils. When added to cell culture medium, these oligomers are efficiently absorbed by the cell, where they interact with and promote the aggregation and partial degradation of nascent VEGFR2. Vascin aggregation does not induce the aggregation of known disease amyloids. Neither do vascin oligomers affect the function of the related EGF receptor or the surface translocation of other receptors. We found vascin only to be toxic to cells that are dependent on VEGFR2 function, suggesting that toxicity is due to loss of VEGFR2 function and not to vascin aggregation or vascin-induced VEGFR2 aggregation. Consistent with this, we found that vascin is active in vivo and could reduce tumor growth in a VEGFR2-sensitive subcutaneous B16 melanoma syngenic tumor model in mice but is not intrinsically toxic to other tissues. CONCLUSION We found that a short amyloidogenic protein fragment can induce the aggregation of a protein normally not associated with amyloidosis in a manner that recapitulates key biophysical and biochemical characteristics of natural amyloids. In addition, we found that amyloid toxicity is observed only in cells that both express VEGFR2 and are dependent on VEGFR2 activity for survival. Thus, rather than being generic, amyloid toxicity here appears to be both protein-specific and conditional on a requirement for VEGFR2 protein function. A synthetic amyloid peptide induces aggregation. We designed vascin, a synthetic amyloid peptide based on an amyloidogenic fragment of the signal peptide of VEGFR2. Vascin forms prefibrillar oligomers that penetrate mammalian cells and interacts with the nascent VEGFR2 protein, resulting in its aggregation and functional knockdown. [Composition includes parts of an image from iStock.com/luismmolina.] Most human proteins possess amyloidogenic segments, but only about 30 are associated with amyloid-associated pathologies, and it remains unclear what determines amyloid toxicity. We designed vascin, a synthetic amyloid peptide, based on an amyloidogenic fragment of vascular endothelial growth factor receptor 2 (VEGFR2), a protein that is not associated to amyloidosis. Vascin recapitulates key biophysical and biochemical characteristics of natural amyloids, penetrates cells, and seeds the aggregation of VEGFR2 through direct interaction. We found that amyloid toxicity is observed only in cells that both express VEGFR2 and are dependent on VEGFR2 activity for survival. Thus, amyloid toxicity here appears to be both protein-specific and conditional—determined by VEGFR2 loss of function in a biological context in which target protein function is essential.