Delivering angiogenic transcription factor genes through myeloid-derived suppressor cells to treat ischemic stroke.

Introduction:: Primary treatments of ischemic stroke aim to break up the blood clots causing the stroke, with some treatments requiring highly invasive surgery (1). However, these treatments do not aim to recover the lost tissue within the penumbra. Thus, there is a need for alternative treatment, such as the use of cell-based therapies.

Previously, our lab found that transfecting fibroblasts with a cocktail of plasmid DNA for transcription factors Etv2, Fli1, and Foxc2 (EFF) can drive angiogenesis at the infarct when injected intracranially (2). While these results showed the potential of pro-endothelial gene therapies, intracranial administration is highly invasive.

To overcome this, one alternative is to use myeloid-derived suppressor cells (MDSCs). MDSCs are immature myeloid cells that can infiltrate diseased tissue from systemic circulation in chronic inflammatory conditions via endocrine signaling, whereby chemokine production recruits these MDSCs to the diseased site (3-5). These MDSCs are also able to exert some levels of angiogenesis due to their ability to secrete pro-angiogenic growth factors (6).

Thus, we targeted MDSCs to transfect with our EFF cocktail and deploy in ischemic stroke. Additionally, we looked at the role that extracellular vesicles (EVs) can play in our therapy. EVs have been found to mediate intracellular communication by transmitting bioactive compounds to targeted tissues (7). Thus, we predict that these EVs could play a role in transmitting our EFF cargo.


Materials and Methods:: MDSCs were transfected with the EFF cocktail via bulk electroporation. To study the transfection dynamics of MDSCs with our target plasmid DNA, qPCR analysis was performed on MDSCs at 24-,48-, and 72-hours post-transfection. The levels of EFF expressed in EVs secreted by the transfected MDSCs were also measured at the same collection points.

To study the biodistribution of the transfected cells in-vivo, mice underwent a middle cerebral artery occlusion (MCAo) surgery to induce a large striatal infarct. Transfected MDSCs were then isolated and labelled with a membrane dye and injected retro-orbitally four hours post-surgery due to increased blood-brain-barrier permeability. After 24 hours, the mice were sacrificed with their organs imaged under an in-vivo imaging system (IVIS) with the brain samples being sectioned and analyzed via fluorescent microscopy.

To study the bioactivity of the transfected MDSCs’ secretome, conditioned media derived from the transfected cells was collected 24 hours post-transfection. Endothelial cells that were embedded in Matrigel in a 2D culture were then exposed to the conditioned media in a tube formation assay. Images were taken of the endothelial cells periodically up to 7.5 hours post-exposure. Analysis of the images was performed using the Angiogenesis Analyzer plugin on ImageJ, emphasizing specific features of the network that indicate network integrity and proliferation, such as number of meshes, mesh size, number of branches, and master junction density.


Results, Conclusions, and Discussions:: qPCR results showed significant upregulation of EFF compared to sham in MDSCs and their secreted EVs at 24-,48-, and 72-hours post-transfection, indicating successful transfection and EV loading of EFF. In tracking these cells’ biodistribution, IVIS results show a high level of fluorescence in the ipsilateral striatum and liver of mice having undergone MCAo versus wild-type mice, indicating our transfected MDSCs can migrate to the infarct when injected systemically. To more closely study the biodistribution within the brain, IHC shows a significant amount of fluorescently labelled cells in the ipsilateral stratum as compared to the contralateral of the stroke-affected mice, confirming ipsilateral hemisphere sequestration of MDSCs. These results show that the MDSCs can reach the infarct even though there are occluded blood vessels blocking specific passage to the site.

Tube formation assay results show a significant increase in the number of meshes, average mesh size, number of branches, and master junction density of the media derived from EFF transfected MDSCs compared to our sham and negative control groups, suggesting greater network proliferation and integrity. This indicates the secretome of transfected MDSCs confers some pro-angiogenic effects. Significant increases in these measures show that the EFF group was able to form a more robust endothelial network as compared to the sham and negative control. With the increase in the number of branches for the EFF group, this shows there is more potential for outgrowth growth of the network, indicating the potential for more proliferation. Lastly, having a greater master junction density indicates that there may be more support to provide a stable foundation upon which meshes can grow.

Our results show that transfecting MDSCs with our EFF plasmid cocktail via bulk electroporation can upregulate the expression of these three transcription factors over several different time points. We were additionally able to show that our therapy can confer angiogenic effects in-vitro and migrate through systemic circulation reaching the infarcted hemisphere in stroke-affected mice. In total, these results establish a less-invasive method for deploying pro-angiogenic gene therapies to treat ischemic stroke. Future studies include assessing the long-term motor and cognitive recovery in these stroke-affected mice.


Acknowledgements (Optional): :

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