Agnes Csiszár

Aging-Induced Dysregulation of Dicer1-Dependent MicroRNA Expression Impairs Angiogenic Capacity of Rat Cerebromicrovascular Endothelial Cells

Age-related impairment of angiogenesis is likely to play a central role in cerebromicrovascular rarefaction and development of vascular cognitive impairment, but the underlying mechanisms remain elusive. To test the hypothesis that dysregulation of Dicer1 (ribonuclease III, a key enzyme of the microRNA [miRNA] machinery) impairs endothelial angiogenic capacity in aging, primary cerebromicrovascular endothelial cells (CMVECs) were isolated from young (3 months old) and aged (24 months old) Fischer 344 × Brown Norway rats. We found an age-related downregulation of Dicer1 expression both in CMVECs and in small cerebral vessels isolated from aged rats. In aged CMVECs, Dicer1 expression was increased by treatment with polyethylene glycol-catalase. Compared with young cells, aged CMVECs exhibited altered miRNA expression profile, which was associated with impaired proliferation, adhesion to vitronectin, collagen and fibronectin, cellular migration (measured by a wound-healing assay using electric cell-substrate impedance sensing technology), and impaired ability to form capillary-like structures. Overexpression of Dicer1 in aged CMVECs partially restored miRNA expression profile and significantly improved angiogenic processes. In young CMVECs, downregulation of Dicer1 (siRNA) resulted in altered miRNA expression profile associated with impaired proliferation, adhesion, migration, and tube formation, mimicking the aging phenotype. Collectively, we found that Dicer1 is essential for normal endothelial angiogenic processes, suggesting that age-related dysregulation of Dicer1-dependent miRNA expression may be a potential mechanism underlying impaired angiogenesis and cerebromicrovascular rarefaction in aging.

Novel Fusogenic Liposomes for Fluorescent Cell Labeling and Membrane Modification

Efficient delivery of biomolecules into membranes of living cells as well as cell surface modifications are major biotechnological challenges. Here, novel liposome systems based on neutral and cationic lipids in combination with lipids modified by aromatic groups are introduced for such applications. The fusion efficiency of these liposome systems was tested on single cells in culture like HEK293, myofibroblasts, cortical neurons, human macrophages, smooth muscle cells, and even on tissue. Fusogenic liposomes enabled highly efficient incorporation of molecules into mammalian cell membranes within 1 to 30 min at fully unchanged cell growth conditions and did not affect cell behavior. We hypothesize that membrane fusions were induced in all cases by the interaction of the positively charged lipids and the delocalized electron system of the aromatic group generating local dipoles and membrane instabilities. Selected applications ranging from basic research to biotechnology are envisaged here.

Fluorescent Lipids: Functional Parts of Fusogenic Liposomes and Tools for Cell Membrane Labeling and Visualization

In this paper a rapid and highly efficient method for controlled incorporation of fluorescent lipids into living mammalian cells is introduced. Here, the fluorescent molecules have two consecutive functions: First, they trigger rapid membrane fusion between cellular plasma membranes and the lipid bilayers of their carrier particles, so called fusogenic liposomes, and second, after insertion into cellular membranes these molecules enable fluorescence imaging of cell membranes and membrane traffic processes. We tested the fluorescent derivatives of the following essential membrane lipids for membrane fusion: Ceramide, sphingomyelin, phosphocholine, phosphatidylinositol-bisphosphate, ganglioside, cholesterol, and cholesteryl ester. Our results show that all probed lipids could more efficiently be incorporated into the plasma membrane of living cells than by using other methods. Moreover, labeling occurred in a gentle manner under classical cell culture conditions reducing cellular stress responses. Staining procedures were monitored by fluorescence microscopy and it was observed that sphingolipids and cholesterol containing free hydroxyl groups exhibit a decreased distribution velocity as well as a longer persistence in the plasma membrane compared to lipids without hydroxyl groups like phospholipids or other artificial lipid analogs. After membrane staining, the fluorescent molecules were sorted into membranes of cell organelles according to their chemical properties and biological functions without any influence of the delivery system.

Mechanical Properties of Bare and Protein-Coated Giant Unilamellar Phospholipid Vesicles. A Comparative Study of Micropipet Aspiration and Atomic Force Microscopy

In this study, protein-coated giant phospholipid vesicles were used to model cell plasma membranes coated by surface protein layers that increase membrane stiffness under mechanical or osmotic stress. These changed mechanical properties like bending stiffness, membrane area compressibility modulus, and effective Youngs modulus were determined by micropipet aspiration, while bending stiffness, effective Youngs modulus, and effective spring constant of vesicles were analyzed by AFM. The experimental setups, the applied models, and the results using both methods were compared here. As demonstrated before, we found that bare vesicles were best probed by micropipet aspiration due to its high sensitivity. The mechanical properties of vesicles with protein surface layers were, however, better determined by AFM because it enables very local deformations of the membrane with barely any structural damage to the protein layer. Mechanical properties of different species of coating proteins, here streptavidin and avidin, could be clearly distinguished using this technique.

Plasma membrane functionalization using highly fusogenic immune activator liposomes.

Cell surface functionalization and target molecule incorporation into living cell membranes without functional damage represent major biotechnological challenges. One possible way to achieve these goals is to induce cell membrane fusion with an artificial membrane containing molecules equipped with reactive groups or ligands. In this work we developed a carrier system to incorporate lipopolysaccharide (LPS), an immune cell activating molecule from Gram-negative bacteria, into mammalian membranes. LPS is not present in untreated mammalian cells which hence are not detectable by the immune system. Here, we demonstrate the successful incorporation of LPS into fusogenic liposomes (FLs) and subsequent incorporation into mammalian plasma membranes using these FLs. Additionally, the presence of LPS in cell membranes was probed by the addition of non-activated macrophages. A high concentration of LPS in the plasma membrane of immortalized fibroblasts activated the immune cells, which in turn started to eliminate LPS-exhibiting cells. Our method for cellular membrane functionalization is a promising tool for biomedical applications and could provide the basis for specific cell targeting approaches.

Fusogenic Liposomes as Nanocarriers for the Delivery of Intracellular Proteins

Direct delivery of proteins and peptides into living mammalian cells has been accomplished using phospholipid liposomes as carrier particles. Such liposomes are usually taken up via endocytosis where the main part of their cargo is degraded in lysosomes before reaching its destination. Here, fusogenic liposomes, a newly developed molecular carrier system, were used for protein delivery. When such liposomes were loaded with water-soluble proteins and brought into contact with mammalian cells, the liposomal membrane efficiently fused with the cellular plasma membrane delivering the liposomal content to the cytoplasm without degradation. To explore the key factors of proteofection processes, the complex formation of fusogenic liposomes and proteins of interest and the size and zeta potential of the formed fusogenic proteoliposoms were monitored. Intracellular protein delivery was analyzed using fluorescence microscopy and flow cytometry. Proteins such as EGFP, Dendra2, and R-phycoerythrin or peptides such as LifeAct-FITC and NTF2-AlexaFluor488 were successfully incorporated into mammalian cells with high efficiency. Moreover, correct functionality and faithful transport to binding sites were also proven for the imported proteins.

Double-Shell Giant Vesicles Mimicking Gram-Negative Cell Wall Behavior during Dehydration

A biomimetic system modeling the behavior of Gram-negative bacteria under hyperosmotic stress was developed. To this end, we introduced a two-step electroswelling procedure for encapsulation of giant unilamellar vesicles by an additional membrane. Both membranes of the resulting double-shell vesicles (DSVs) were fluid. Additionally, the outer membrane was rigidified by a monolayer of streptavidin forming a two-dimensional crystal. For strong attachment of this protein layer, the outer membrane contained biotinylated lipids. This reinforced protein-lipid compound membrane served to model the assembly of the murein wall and outer membrane of Gram-negative bacteria. We characterized DSVs by confocal laser scanning microscopy. Furthermore, DSVs were exposed to hyperosmotic media (osmotic difference 0-1100 mosm/L), and the resulting shapes were analyzed. DSVs coated with streptavidin were much less deformed or destroyed by osmotic stress than bare DSVs or DSVs coated with noncrystalline avidin. Osmotically stressed DSVs coated with streptavidin displayed weak wrinkling of the outer membrane and formed small daughter vesicles of the inner membrane. Both features and the toughness against hyperosmotic stress are well described characteristics of Gram-negative bacteria.

Biotin-conjugated fusogenic liposomes for high-quality cell purification

Purification of defined cell populations from mixed primary cell sources is essential for many biomedical and biotechnological applications but often very difficult to accomplish due to missing specific surface markers. In this study, we developed a new approach for efficient cell population separation based on the specific membrane fusion characteristics of distinct cell types upon treatment with fusogenic liposomes. When such liposomes are conjugated with biotin, specific cell populations can be efficiently surface functionalized by biotin after liposomal treatment while other populations remain unlabeled. Due to the high affinity of biotin for avidin-like proteins, biotin functionalized cells are ideal targets for conjugation of e.g. avidin tagged magnetic beads, fluorophores or antibodies with bioanalytical relevance. Here, based on the differential biotinylation of distinct cell populations high quality separation of cardiac fibroblasts from myocytes, and cerebromicrovascular endothelial cells from fibroblasts was successfully established.

A bioanalytical assay to distinguish cellular uptake routes for liposomes.

Lipid‐based nanoparticles are frequently used for drug or DNA delivery into mammalian cells. However it is difficult to determine whether such particles are taken up via endocytosis or fusion to the plasma membrane. Here, we propose a simple and reliable analytical method to do so based on the unique spectral properties of the fluorescent tracer BODIPY FL. At high local concentrations, this dye displays an additional red‐shifted emission peak that is absent at low concentrations. In dye‐loaded liposomes taken up by endocytosis, the local dye concentration did not significantly change upon internalization. Accordingly, unchanged fluorescence spectra were detected. When cells were incubated with liposomes able to fuse with the plasma membrane of mammalian cells, a reduction of local dye concentration and much weaker emission in the red‐shifted peak were observed. The ratio of intensities in both fluorescence channels was shown to be a reliable indicator of the cellular uptake mechanism.

A variational approach to vesicle membrane reconstruction from fluorescence imaging

Biological applications like vesicle membrane analysis involve the precise segmentation of 3D structures in noisy volumetric data, obtained by techniques like magnetic resonance imaging (MRI) or laser scanning microscopy (LSM). Dealing with such data is a challenging task and requires robust and accurate segmentation methods. In this article, we propose a novel energy model for 3D segmentation fusing various cues like regional intensity subdivision, edge alignment and orientation information. The uniqueness of the approach consists in the definition of a new anisotropic regularizer, which accounts for the unbalanced slicing of the measured volume data, and the generalization of an efficient numerical scheme for solving the arising minimization problem, based on linearization and fixed-point iteration. We show how the proposed energy model can be optimized globally by making use of recent continuous convex relaxation techniques. The accuracy and robustness of the presented approach are demonstrated by evaluating it on multiple real data sets and comparing it to alternative segmentation methods based on level sets. Although the proposed model is designed with focus on the particular application at hand, it is general enough to be applied to a variety of different segmentation tasks.