Effect of the hydrogen sulfide donor GYY4137 on platelet activation and microvascular thrombus formation in mice.

This study evaluates the effect of the H2S donor GYY4137 (GYY) on adhesion molecule expression, protein S-sulfhydration and morphology of platelets in vitro and on kinetics of microvascular thrombus formation in vivo. Using flowcytometry, untreated resting, TRAP-activated, or TRAP-activated and GYY-exposed human platelets were studied for expression of P-selectin, GPIb and GPIIb/IIIa as well as for fibrinogen binding. By means of electron microscopy, platelet morphology and intracellular granule numbers were assessed. Platelet shape change was studied using immunohistochemistry for P-selectin, NSF and F-actin by SR-SIM. Biotin switch assay served for the analysis of platelet protein S-sulfhydration by GYY. Using the FeCl3 and the light/dye model in dorsal skinfold chamber-equipped mice, the effect of GYY and its vehicle DMSO was studied on venular thrombus formation and tail-vein bleeding time. Soluble (s)P-selectin plasma concentrations were measured in GYY- or DMSO-treated animals. Exposure to GYY increased the S-sulfhydration of platelet proteins. GYY reduced dose-dependently the TRAP-induced adhesion molecule expression and attenuated the morphological signs of TRAP-associated platelet activation. In mice, GYY caused a significant prolongation of venular thrombus formation and tail-vein bleeding time. Application of an anti-P-selectin antibody in DMSO-exposed animals prolonged thrombosis formation comparably as GYY did. GYY reversed the TRAP-induced distribution of P-selectin at the plasma membrane of platelets. This indicates reduced exocytosis and shedding of P-selectin, which is supported by significantly lower sP-selectin concentrations in GYY- vs. DMSO-treated mice. H2S acts anti-thrombotic and seems to regulate thrombogenesis by interference with platelet activation and adhesion molecule-mediated aggregation.

Innovative strategy for microRNA delivery in human mesenchymal stem cells via magnetic nanoparticles.

Bone marrow derived human mesenchymal stem cells (hMSCs) show promising potential in regeneration of defective tissue. Recently, gene silencing strategies using microRNAs (miR) emerged with the aim to expand the therapeutic potential of hMSCs. However, researchers are still searching for effective miR delivery methods for clinical applications. Therefore, we aimed to develop a technique to efficiently deliver miR into hMSCs with the help of a magnetic non-viral vector based on cationic polymer polyethylenimine (PEI) bound to iron oxide magnetic nanoparticles (MNP). We tested different magnetic complex compositions and determined uptake efficiency and cytotoxicity by flow cytometry. Additionally, we monitored the release, processing and functionality of delivered miR-335 with confocal laser scanning microscopy, real-time PCR and live cell imaging, respectively. On this basis, we established parameters for construction of magnetic non-viral vectors with optimized uptake efficiency (~75%) and moderate cytotoxicity in hMSCs. Furthermore, we observed a better transfection performance of magnetic complexes compared to PEI complexes 72 h after transfection. We conclude that MNP-mediated transfection provides a long term effect beneficial for successful genetic modification of stem cells. Hence, our findings may become of great importance for future in vivo applications.

Spray- and laser-assisted biomaterial processing for fast and efficient autologous cell-plus-matrix tissue engineering.

At present, intensive investigation aims at the creation of optimal valvular prostheses. We introduced and tested the applicability and functionality of two advanced cell-plus-matrix seeding technologies, spray-assisted bioprocessing (SaBP) and laser-assisted bioprocessing (LaBP), for autologous tissue engineering (TE) of bioresorbable artificial grafts. For SaBP, human mesenchymal stem cells (HMSCs), umbilical cord vein endothelial cells (HUVECs) and fibrin were simultaneously spray-administered on poly(ε-caprolactone) (PCL) substrates. For LaBP, HUVECs and HMSCs were separately laser-printed in stripes, followed by fibrin sealing. Three-leaflet valves were manufactured following TE of electrospun PCL tissue equivalents. Grafts were monitored in vitro under static and dynamic conditions in bioreactors. SaBP and LaBP resulted in TE of grafts with homogeneous cell distribution and accurate cell pattern, respectively. The engineered valves demonstrated immediate sufficient performance, complete cell coating, proliferation, engraftment, HUVEC-mediated invasion, HMSC differentiation and extracellular matrix deposition. SaBP revealed higher efficiency, with at least 12-fold shorter processing time than the applied LaBP set-up. LaBP realized coating with higher cell density and minimal cell-scaffold distance. Fibrin and PCL stability remain issues for improvement. The introduced TE technologies resulted in complete valvular cell-plus-matrix coating, excellent engraftment and HMSCs differentiation. SaBP might have potential for intraoperative table-side TE considering the procedural duration and ease of implementation. LaBP might accelerate engraftment with precise patterns.

Magnetic Nanoparticle Based Nonviral MicroRNA Delivery into Freshly Isolated CD105(+) hMSCs.

Genetic modifications of bone marrow derived human mesenchymal stem cells (hMSCs) using microRNAs (miRs) may be used to improve their therapeutic potential and enable innovative strategies in tissue regeneration. However, most of the studies use cultured hMSCs, although these can lose their stem cell characteristics during expansion. Therefore, we aimed to develop a nonviral miR carrier based on polyethylenimine (PEI) bound to magnetic nanoparticles (MNPs) for efficient miR delivery in freshly isolated hMSCs. MNP based transfection is preferable for genetic modifications in vivo due to improved selectivity, safety of delivery, and reduced side effects. Thus, in this study different miR/PEI and miR/PEI/MNP complex formulations were tested in vitro for uptake efficiency and cytotoxicity with respect to the influence of an external magnetic field. Afterwards, optimized magnetic complexes were selected and compared to commercially available magnetic vectors (Magnetofectamine, CombiMag). We found that all tested transfection reagents had high miR uptake rates (yielded over 60%) and no significant cytotoxic effects. Our work may become crucial for virus-free introduction of therapeutic miRs as well as other nucleic acids in vivo. Moreover, in the field of targeted stem cell therapy nucleic acid delivery prior to transplantation may allowfor initial cell modulation in vitro.

Improved transfection in human mesenchymal stem cells: effective intracellular release of pDNA by magnetic polyplexes.

AIM: Magnetically guided transfection has been shown as a promising approach for the genetic modification of cells. We observed that polyethylenimine (PEI)-condensed pDNA, combined with magnetic nanoparticles (MNPs) via biotin-streptavidin interactions could provide higher transfection efficiency than pDNA/PEI alone, even without the application of a magnetic force. Therefore, we intended to investigate the beneficial properties of MNP-based transfection. MATERIALS & METHODS: We performed three-color fluorescent labeling of magnetic transfection complexes and traced them inside human mesenchymal stem cells over time using confocal microscopy in order to study pDNA release kinetics by colocalization studies. RESULTS: We demonstrated that MNP-combined pDNA/PEI complexes provide more rapid and efficient release of pDNA than pDNA/PEI alone, which could be explained by the retention of PEI on the surface of the MNPs due to strong biotin-streptavidin interactions. CONCLUSION: The process of pDNA liberation may significantly influence the efficiency of the transfection vector. Therefore, it should be carefully considered when creating novel gene delivery agents.

Analyzing migratory properties of human CD133(+) stem cells in vivo after intraoperative sternal bone marrow isolation.

Human bone marrow stem cell populations have been applied for cardiac regeneration purposes within different clinical settings in the recent past. The migratory capacity of applied stem cell populations towards injured tissue, after undergoing specific peri-interventional harvesting and isolation procedures, represents a key factor limiting therapeutic efficacy. We therefore aimed at analyzing the migratory capacity of human cluster of differentiation (CD) 133(+) bone marrow stem cells in vivo after intraoperative harvesting from the sternal bone marrow. Human CD133(+) bone marrow stem cells were isolated from the sternal bone marrow of patients undergoing cardiac surgery at our institution. Migratory capacity towards stromal cell-derived factor-1α (SDF-1α) gradients was tested in vitro and in vivo by intravital fluoresecence microscopy, utilizing the cremaster muscle model in severe combined immunodeficient (SCID) mice and analyzing CD133(+) cell interaction with the local endothelium. Furthermore, the role of a local inflammatory stimulus for CD133(+) cell interaction with the endothelium was studied. In order to describe endothelial response upon chemokine stimulation laser scanning microscopy of histological cremaster muscle samples was performed. SDF-1α alone was capable to induce relevant early CD133(+) cell interaction with the endothelium, indicated by the percentage of rolling CD133(+) cells (45.9±1.8% in "SDF-1" vs. 17.7±2.7% in "control," p<0.001) and the significantly reduced rolling velocity after SDF-1α treatment. Furthermore, SDF-1α induced firm endothelial adhesion of CD133(+) cells in vivo. Firm endothelial adhesion, however, was significantly enhanced by additional inflammatory stimulation with tumor necrosis factor-α (TNF-α) (27.9±4.3 cells/mm(2)in "SDF-1 + TNF" vs. 2.2±1.1 cells/mm(2) in "control," p<0.001). CD133(+) bone marrow stem cells exhibit sufficient in vivo homing towards SDF-1α gradients in an inflammatory microenvironment after undergoing standardized intraoperative harvesting and isolation from the sternal bone marrow.

Targeted delivery of human VEGF gene via complexes of magnetic nanoparticle-adenoviral vectors enhanced cardiac regeneration.

This study assessed the concept of whether delivery of magnetic nanobeads (MNBs)/adenoviral vectors (Ad)-encoded hVEGF gene (Ad(hVEGF)) could regenerate ischaemically damaged hearts in a rat acute myocardial infarction model under the control of an external magnetic field. Adenoviral vectors were conjugated to MNBs with the Sulfo-NHS-LC-Biotin linker. In vitro transduction efficacy of MNBs/Ad-encoded luciferase gene (Ad(luc)) was compared with Ad(luc) alone in human umbilical vein endothelial cells (HUVECs) under magnetic field stimulation. In vivo, in a rat acute myocardial infarction (AMI) model, MNBs/Ad(hVEGF) complexes were injected intravenously and an epicardial magnet was employed to attract the circulating MNBs/Ad(hVEGF) complexes. In vitro, compared with Ad(luc) alone, MNBs/Ad(luc) complexes had a 50-fold higher transduction efficiency under the magnetic field. In vivo, epicardial magnet effectively attracted MNBs/Ad(hVEGF) complexes and resulted in strong therapeutic gene expression in the ischemic zone of the infarcted heart. When compared to other MI-treated groups, the MI-M(+)/Ad(hVEGF) group significantly improved left ventricular function (p<0.05) assessed by pressure-volume loops after 4 weeks. Also the MI-M(+)/Ad(hVEGF) group exhibited higher capillary and arteriole density and lower collagen deposition than other MI-treated groups (p<0.05). Magnetic targeting enhances transduction efficiency and improves heart function. This novel method to improve gene therapy outcomes in AMI treatment offers the potential into clinical applications.

Magnetic targeting strategies in gene delivery.

Gene delivery is a process of the insertion of transgenes into cells with the purpose to obtain the expression of encoded protein. The therapeutic application of this process is termed gene therapy, which is becoming a promising instrument to treat genetic and acquired diseases. Although numerous methods of gene transfer have already been developed, including biological, physical and chemical approaches, the optimal strategy has to be discovered. Importantly, it should be effective, selective and safe to be translated to the clinic. Magnetic targeting has been demonstrated as an effective strategy to decrease side effects of gene transfer, while increasing the selectivity and efficiency of the applied vector. This article will focus on the latest progress in the development of different magnetic vectors, based on both viral and nonviral gene delivery agents. It will also include a description of magnetic targeting applications in stem cells and in vivo, which has gained interest in recent years due to the rapid development of technology.