Magnetic microbubble mediated vascular gene transfer for prevention of endothelial dysregulation during insulin resistance
Final Report Abstract
Gene targeting of the vasculature offers numerous therapeutic possibilities for patho-physiological conditions involving the vasculature, such as ischemia, tumour growth or vascular dysfunction upon inflammatory or insulin resistant conditions. By associating therapeutic lentiviral particles or plasmid DNA to magnetic microbubbles and their trapping and rupture at a specific site using external magnetic fields and ultra sound the side effects of systemic gene therapy are reduced and gene transfer efficiencies enhanced. However, optimization of this technique is still needed. The tyrosine phosphatase SHP-2 was shown in a previous study to be critical for growth factor induced angiogenesis in vitro and in vivo and influences endothelial inflammatory responses making it an interesting target to study in endothelial cells. Therefore, the aim of this study was to 1) establish and optimize a vascular site directed gene transfer technique using magnetic microbubbles to be applied for the study of SHP-2 function in the vasculature in vivo and 2) investigate whether SHP-2 is involved in patho-physiological endothelial processes, such as angiogenesis (first funding period) and the development of a pro-adhesive endothelium (second funding period) upon hypoxia and inflammatory conditions observed during insulin resistance and if modulation of SHP-2 activity can be used to restrict endothelial dysfunction and its consequences on angiogenesis and vascular inflammation. We successfully generated and characterized magnetic microbubbles associated to pDNA, oligonucleotides (AS-ODN, siRNA) and lentiviral particles. Using an external magnetic field and ultrasound we targeted magnetic microbubbles to the vascular wall in the mice dorsal skin fold chamber model. Using these MMB we achieved site specific reporter gene expression in vessels in vivo. Whereas treatment with MMB carrying pDNA did not result in any gene delivery to other vascular beds but at the target site, lentiviral MMB caused gene transfer at a second site, the spleen. Thus great efforts were invested to solve this problem and the use of different MMB as well as higher MNP amounts enhanced targeted site specific gene transfer although gene delivery to the spleen remained. Due to these technological difficulties we were unable to use the MMB-technique to study the function of SHP-2 in vivo. Different approaches to diminish expression in the spleen upon LV-MMB application are being tested. We found SHP-2 to positively influence hypoxic angiogenesis by stabilizing the hypoxia transcription factor HIF-1α in endothelial cells. Furthermore, the angiogenic response during insulin resistant conditions in vitro could be improved by over-expression of a constitutively active SHP-2 construct. Using external application of LV-MNP complexes in combination with a magnetic field we are now able to study the impact of SHP-2 on wound healing in vivo. In addition, SHP-2 was shown to control endothelial inflammatory signaling and adhesion molecule upregulation. Inhibition of SHP-2 activity in vivo resulted in enhanced neutrophil adhesion to the vascular wall, neutrophil transmigration as well as vascular leakage in the mouse cremaster muscle. Using the LV-MMB technique on aortas ex vivo, we were able to prevent the induction of a pro-adhesive phenotype in inflammatory conditions by over-expression of a constitutive active SHP-2 construct. Thus, targeted site directed gene delivery in vivo using MMB is possible and has great potential towards therapeutic use as well as being a powerful research tool.
Publications
- ARNO regulates VEGF-dependent tissue responses by stabilizing endothelial VEGFR-2 surface expression. Cardiovasc Res 2012; 93:111-9
Mannell HK, Pircher J, Chaudhry DI, Alig SK, Koch EG, Mettler R, Pohl U, Krötz F
(See online at https://doi.org/10.1093/cvr/cvr265) - Magnetic stents retain nanoparticlebound antirestenotic drugs transported by lipid microbubbles. Pharm Res. 2012;29:1295-1307
Räthel T, Mannell H, Pircher J, Gleich B, Pohl U, Krötz F
(See online at https://doi.org/10.1007/s11095-011-0643-y) - Site directed vascular gene delivery in vivo by ultrasonic destruction of magnetic nanoparticle coated microbubbles. Nanomedicine. 2012;8:1309-1318
Mannell H, Pircher J, Fochler F, Stampnik Y, Rathel T, Gleich B, Plank C, Mykhaylyk O, Dahmani C, Wornle M, Ribeiro A, Pohl U, Krötz F
(See online at https://doi.org/10.1016/j.nano.2012.03.007) - Targeted endothelial gene delivery by ultrasonic destruction of magnetic microbubbles carrying lentiviral vectors. Pharm Res. 2012;29:1282-1294
Mannell H, Pircher J, Rathel T, Schilberg K, Zimmermann K, Pfeifer A, Mykhaylyk O, Gleich B, Pohl U, Krötz F
(See online at https://doi.org/10.1007/s11095-012-0678-8) - The tyrosine phosphatase shp-1 regulates hypoxia inducible factor-1alpha (hif-1alpha) protein levels in endothelial cells under hypoxia. PLoS One. 2015;10 (3)
Alig SK, Stampnik Y, Pircher J, Rotter R, Gaitzsch E, Ribeiro A, Wornle M, Krötz F, Mannell H
(See online at https://doi.org/10.1371/journal.pone.0121113)
