Local anti-angiogenic therapy by magnet-assisted downregulation of SHP2 phosphatase
Graphical abstract
Introduction
Angiogenesis, the formation of new blood vessels from existing ones, is controlled by pro- and anti-angiogenic growth factors. This regulation is important during development but it is also a promising target for treatment strategies in disease. In ischemic diseases, such as myocardial infarction or peripheral arterial disease, the stimulation of vessel formation is beneficial to enhance oxygen supply to organs and limbs [1]. In contrast, several cancer therapies aim at the reduction of vessel growth to control tumor progression [2]. Importantly, the modulation of vascularization is only required at distinct locations. Thus, the systemic application of vascular modulators can result in a lack of effect at the site of interest or even systemic adverse effects. Therefore, several strategies for the local modulation of angiogenesis have been developed. To increase angiogenesis in wound healing, electrical stimulation was administered close to the wound, which enhanced the release of pro-angiogenic growth factors [3]. In other studies angiogenesis was stimulated directly by the local injection of vasoactive growth factor proteins [4], mRNAs [5] or endothelial progenitor cells. For the local inhibition of angiogenesis, implants coated with an angiogenesis inhibitor were applied [6]. Moreover, RGD peptides enabled the tumor endothelium-specific expression of the vascular endothelial growth factor (VEGF) scavenger sFlt1, which resulted in diminished tumor vascularization and growth in mice [7]. Local delivery of compounds can also be achieved by the application of nanoparticles. The use of nanoparticles is advantageous because they have high payloads, they increase the half-life of the compounds in the blood and they reduce toxicity. In particular, iron-based magnetic nanoparticles (MNPs) have the advantage that they can be guided to a specific target by magnetic fields [8]. MNPs have been applied to carry drugs, viral vectors, plasmids or even cells to a specific destination [9]. However, there is only very sparse data on the use of MNPs to modulate angiogenesis [10,11]. To test new (anti-)angiogenic agents, different 2D and 3D assays are currently employed but most of them do not cover all steps of angiogenesis [12] and it is unclear if the results can be transferred to the in vivo situation. Embryonic stem cells (ESCs) are a promising option because they fully recapitulate spatio-temporal events of vasculo- and angiogenesis in an organotypic environment of all 3 germ layers [13].
Therefore, in the current study we have developed a strategy for the testing of site-specific lentivirus-based gene transfer on vessel formation in ESCs using MNPs and magnetic fields. Thereby, we could demonstrate that local shRNA-based downregulation of the Src homology 2-containing tyrosine phosphatase (SHP2) effectively reduces vascularization in ESCs in vitro, which was confirmed by a wound assay in mouse in vivo. Thus, MNP-assisted lentiviral transduction using RNAi technology enables local inhibition of angiogenesis in ESCs vitro and re-vascularization in mouse in vivo.
Section snippets
Cultivation of embryonic stem cells (ESCs)
Native murine wildtype (WT) D3 ESCs were obtained from the American Type Culture Collection (ATCC, Manassas, USA). Transgenic murine D3 flt1/eGFP ESCs were generated as previously described [14]. Undifferentiated ESC lines were cultured under sterile conditions at 37āÆĀ°C in a humidified atmosphere with 5% CO2 on irradiated and neomycin-resistant feeder cells (Merck Millipore, Darmstadt, Germany). Basal cultivation medium (Dulbecco's Modified Eagle Medium (DMEM; Life technologies, Darmstadt,
Global lentiviral SHP2 knockdown reduces angiogenesis in flt1/eGFP ESCs
To establish a targeted anti-angiogenic therapeutic approach, we have applied an in vitro model based on differentiating ESCs. This resembles 3D vascular growth similar to the in vivo situation. We used transgenic murine ESCs expressing the enhanced green fluorescent protein (eGFP) under control of the endothelium-specific flt1 promoter (flt1/eGFP ESCs) [14]. The cells were differentiated into the mesodermal lineage using embryoid bodies (EBs) and the mass culture method (Fig. S1 a). During
Discussion
Local anti-angiogenic therapy is a promising approach for various diseases with uncontrolled vessel formation such as vascular malformations, tumor growth or retinopathies. In our present study we have developed a strategy for the site-specific inhibition of angiogenesis in the ESC system in vitro, which could be recapitulated under in vivo conditions.
For local gene transfer, we utilized complexes of lentiviral vectors and MNPs. Lentiviral vectors enable highly efficient gene delivery because
Conclusion
Our study demonstrates that local RNAi-based knockdown of SHP2 using MNPs and magnetic fields is an innovative strategy to site-specifically diminish vessel formation in the ESC system in vitro and the dorsal skinfold chamber in mouse in vivo.
Author contributions
S.R. performed in vitro ESC experiments, analyzed the data and contributed to the writing of the manuscript, Y.H. and H.M. performed and analyzed in vivo experiments, A.H. and B.G. calculated magnetic fields, O.M. synthesized and characterized SO-Mag5 particles, A.P. designed and provided lentiviral vectors, D.W. designed the study, analyzed data and wrote the paper.
Competing financial interest
None.
Acknowledgements
We thank Rainer Quaas (Chemicell GmbH) for providing data on nano-screenMag particles. The work was supported by funding to the junior research group āMagnetic nanoparticles (MNPs)āendothelial cell replacement in injured vesselsā by the Ministry of Innovation, Science, Research and Technology of the State of North Rhine-Westphalia (DW) and the DFG Research Unit FOR 917 Nanoguide (AP, BG, HM).
References (35)
- et al.
Biocompatible, purified VEGF-A mRNA improves cardiac function after intracardiac injection 1 week post-myocardial infarction in swine
Mol Ther Methods Clin Dev
(2018) - et al.
Anti-angiogenic inhibition of tumor growth by systemic delivery of PEI-g-PEG-RGD/pCMV-sFlt-1 complexes in tumor-bearing mice
J. Control. Release
(2006) - et al.
Embryonic stem cell-derived embryoid bodies development in collagen gels recapitulates sprouting angiogenesis
Lab. Investig.
(2001) - et al.
Endostatin influences endothelial morphology via the activated ERK1/2-kinase endothelial morphology and signal transduction
Microvasc. Res.
(2006) - et al.
HIF-1alpha dependent wound healing angiogenesis in vivo can be controlled by site-specific lentiviral magnetic targeting of SHP-2
Mol. Ther.
(2017) - et al.
Improvement of vascular function by magnetic nanoparticle-assisted circumferential gene transfer into the native endothelium
J. Control. Release
(2016) - et al.
Improved heart repair upon myocardial infarction: combination of magnetic nanoparticles and tailored magnets strongly increases engraftment of myocytes
Biomaterials
(2018) - et al.
Promoting blood vessel growth in ischemic diseases: challenges in translating preclinical potential into clinical success
Dis. Model. Mech.
(2013) - et al.
The role of angiogenesis in cancer treatment
Biomedicines
(2017) - et al.
Angiogenesis is induced and wound size is reduced by electrical stimulation in an acute wound healing model in human skin
PLoS One
(2015)
Growth factor-induced therapeutic angiogenesis in the heart: protein therapy
Cardiovasc. Res.
Local inhibition of angiogenesis by halofuginone coated silicone materials
J. Mater. Sci. Mater. Med.
Magnetic nanoparticles: from design and synthesis to real world applications
Nanomaterials (Basel)
Magnetic nanoparticles: novel options for vascular repair?
Nanomedicine (London)
Highly effective antiangiogenesis via magnetic mesoporous silica-based siRNA vehicle targeting the VEGF gene for orthotopic ovarian cancer therapy
Int. J. Nanomedicine
A novel targeted angiogenesis technique using VEGF conjugated magnetic nanoparticles and in-vitro endothelial barrier crossing
BMC Cardiovasc. Disord.
Development of a one-step embryonic stem cell-based assay for the screening of sprouting angiogenesis
BMC Biotechnol.
Cited by (10)
The Cell-Specific Role of SHP2 in Regulating Bone Homeostasis and Regeneration Niches
2023, International Journal of Molecular SciencesInhibition of Vascular Growth by Modulation of the Anandamide/Fatty Acid Amide Hydrolase Axis
2021, Arteriosclerosis, Thrombosis, and Vascular BiologyTargeting the SHP2 phosphatase promotes vascular damage and inhibition of tumor growth
2021, EMBO Molecular MedicineThe Potential Application of Magnetic Nanoparticles for Liver Fibrosis Theranostics
2021, Frontiers in ChemistryShp2 inhibition benefits epidermal growth factor receptor-mutated non-small cell lung cancer therapy
2021, Mini-Reviews in Medicinal Chemistry