title: Extracellular vesicle mediated delivery of angiotensin (1-9) after ischaemic stroke
Creators: name: Rooney, Rebecca Audrey
Publisher: name: University of Glasgow
Year: 2024

description: Stroke remains a leading cause of global disability and mortality. For those who experience a stroke, interventions such as mechanical thrombectomy, or more commonly, rtPA are used to enable total or partial reperfusion that can save lives and reduce the degree of disability. With advancements in medicine, 80% of stroke incidences are preventable, and hopefully one day, with further advancements in pioneering medical science, the degree of disability and morbidity from stroke will be minimal. Recently, extracellular vesicles (EVs) have been studied for their role in disease, including stroke, due to their cell signalling potential and internalised cargo. In doing so, EVs are now understood to be excellent delivery vectors, whereby beneficial microRNAs, compounds or peptides may be encased within EVs that can be used to treat various diseases. Activation of the receptors within the counter-regulatory axis of the Renin Angiotensin Aldosterone system (RAAS), such as the Angiotensin II Type II receptor, AT2R, can lead to vasodilation, inhibition of inflammation, prevention of cell death and stimulation of cell growth, all of which are crucial pathways in the brain following stroke. Several preclinical and clinical stroke studies have highlighted the potential benefit of targeting RAAS to improve post-stroke outcomes. By eliciting the activation of AT2R by its novel functional ligand Angiotensin- (1-9) [Ang-(1-9)], these protective downstream pathways can be activated. It was therefore hypothesised that loading EVs with Ang-(1-9) would activate neuroprotective mechanisms in a comorbid model of experimental stroke. Consequently, this research would establish the therapeutic potential of Ang-(1-9) in ischaemic stroke, along with assessing the ability of EVs to successfully deliver potentially therapeutic, internalised cargo.

In chapter 3, published peer-reviewed data was systematically reviewed to summarise whether RAAS targeting interventions improved post-stroke functional outcomes, including neurological outcomes, infarct volumes and the expression of peptides or cognate receptors, in experimental stroke models. From the available literature there were 414 hits from which a total of 27 publications met the inclusion criteria and were included in further data analysis. Of the studies that assessed the effects of RAAS targeting interventions on neurological outcomes, 31 out of 42 studies demonstrated that RAAS targeting interventions improved post-stroke neurological outcomes. These positive findings were also reflected in infarct volume, whereby, of the studies that assessed the effects of RAAS targeting interventions on infarct volume, 12 out of 16 studies found that RAAS targeting interventions reduced infarct size following stroke. RAAS targeting interventions also led to differential expression of genes coding for cognate receptors and key enzymes in the RAAS. These findings put into perspective the beneficial effects of activating the counter regulatory axis of the RAAS and inhibiting the classical axis of the RAAS, on post-stroke outcomes.

In chapter 4, it was hypothesised that AT2R deficiency in C57BL/6 mice would influence the characteristics of circulating EVs. EVs were isolated from plasma samples of male and female wildtype C57BL/6 mice or AT2R deficient mice and were characterised using conventional nanoparticle tracking analysis (NTA), protein quantification and nano flow cytometry. AT2R deficiency in female mice led to a significantly increased concentration of EVs compared to wildtype female mice, indicative that AT2R deficiency can directly impact EV characteristics. A sex-based difference was identified in AT2R deficient mice, whereby EV particle size was significantly greater in AT2R deficient females compared to AT2R deficient males. EV protein content was similar between groups. To understand if knocking down AT2R affected EV surface markers, and potentially the cellular origin of EVs, plasma derived EVs were analysed for the presence of platelet marker CD41 and endothelial cell marker CD31 using nano flow cytometry. Again, sex-based differences were identified in the AT2R deficient mice, whereby it was found that EVs from AT2R deficient female mice possessed significantly greater levels of CD41 and CD31 compared to EVs derived from AT2R deficient male mice. From current literature, miRNAs were identified that were linked with the RAAS. Two miRNAs, miRNA-132 and -146a, were expressed within EVs isolated from plasma of wildtype mice or AT2R deficient C57BL/6 mice, however the expression of these miRNAs was similar between groups. Overall, it was determined that AT2R deficiency led to differences in EV concentration compared to wildtype mice and that AT2R deficiency led to sex-based difference in EV size and surface marker expression.

In chapter 5, EV isolation methods were compared to determine whether isolation method influenced EV concentration, size and protein content. Size exclusion chromatography (SEC) was the method that provided the greatest EV concentration from SHRSP plasma samples and was used for further downstream experiments. EVs were loaded with peptides or miRNAs by electroporation which led to a significant reduction in EV concentration. SEC isolation of EVs following electroporation led to the greatest EV concentration compared to other isolation techniques. Over and above NTA characterisation and in line with standardised characterisation guidelines, SEC isolated EVs were found to possess common EV biomarkers, as determined by western blot, and structure, as determined by transmission electron microscopy. EVs and their loaded cargo were then assessed in preclinical cell culture models of stroke. Ang-(1-9) peptide and Ang-(1-9) electroporated EVs did not affect the viability of immortalised B50 rat neuronal cells or SHRSP rat primary neuronal cells, suggesting their safety for use in animal models. EVs were electroporated with fluorescently-labelled Ang-(1-9) peptides or lipophilic dyes, as it may have provided information into the location of EVs and potentially the delivery site of internalised cargo in further in vivo studies. Treatment of immortalised B50 rat neuronal cells and GPNT rat cerebral endothelial cells with 5-FAM-Ang-(1-9) or electroporated EVs did not produce fluorescence, however primary neuronal cells treated with 5-FAMAng-(1-9) peptide demonstrated abundant fluorescence. Lipophilic labelling of EVs led to unreproducible red fluorescence in GPNT cerebral endothelial cells and primary neuronal cells. Ultimately loading of EVs with 5-FAM-Ang-(1-9) or labelling with lipophilic dye was unreproducible and could not be carried forward to in vivo studies.

In chapter 6 intranasal administration of EVs and EV brain targeting ability was assessed. EVs were loaded with exogenous Cel-miR-39 and administered intranasally to SHRSP rats. The expression of Cel-miR-39 EV cargo was identified throughout the brain, demonstrating the effectiveness of intranasal EV administration and the subsequent delivery of internal EV cargo. To target EVs to the brain, EVs were linked to Rabies virus glycoprotein (RVG) peptide using a CP05 linker previously reported to conjugate to CD63 tetraspanin on the surface of EVs. Primary neuronal cells expressed the receptor for RVG, CHRNB2, at greater levels than immortalised B50 rat neuronal cells, and as such primary neuronal cells were used in further targeting investigations. SEC isolated EVs were also shown to possess CD63 tetraspanin required for RVG-CP05 targeting conjugation. Nontargeted, Cel-miR-39 loaded EVs, and RVG-targeted, Cel-miR-39 loaded EVs at various concentrations were compared for their ability to deliver miRNA cargo throughout SHRSP rat brain following intranasal administration. Overall RVG targeting did not improve EV miRNA cargo delivery to the brain using the intranasal EV administration method, and as such RVG targeting of EVs was not used in further in vivo studies.

In the final results chapter, chapter 7, the effects of occlusion and reperfusion duration on post-stroke gene expression within brain tissue was determined. Genes related to the RAAS, such as ACE, ACE2 and Mas1, and growth, such as VEGFA and VEGFB, were assessed whereby occlusion duration led to differential expression of RAAS-related genes. The subsequent in vivo study aimed to assess the effects of intranasally administered Ang- (1-9) electroporated EVs on functional outcomes following tMCAO. In this stage the difficulty of in vivo research was highlighted and some functional outcomes that were originally planned could not be conducted due to animals exceeding humane endpoints as a result of tMCAO surgery. Nevertheless, the effects of Ang-(1-9) EVs on gene expression within brain tissue of SHRSPs in the acute phase following tMCAO was determined, whereby Ang-(1-9) EVs led to differential expression of genes related to RAAS, growth and inflammation.

The findings of this thesis highlight the possible benefit of RAAS targeting interventions, including Ang-(1-9), and how EVs may be used as a delivery vector to facilitate the use of RAAS targeting interventions in ischaemic stroke. Some aspects of this preclinical study were challenging; however, the data demonstrates the potential application of EVs in disease treatment. Further study must be carried out to conclusively determine the likely benefits of Ang-(1-9) in ischaemic stroke.