title: Assessing the role of extracellular vesicles in renin-angiotensin system signalling in cardiomyocyte hypertrophy
Creators: name: Downie, Laura S.
Publisher: name: University of Glasgow
Year: 2019
Subjects: subject: cardiovascular disease, cardiac hypertrophy, extracellular vesicles, renin-angiotensin system, cell signalling.

description: Cardiovascular disease (CVD) involving the heart or blood vessels is the most common cause of death and disability worldwide. Following development of chronic hypertension, the left ventricular mass of the heart increases adaptively due to increased work load which further increases the risk of adverse cardiac events such as heart failure. Cardiomyocyte hypertrophy is associated with classical renin-angiotensin system (RAS) signalling where increased angiotensin II (Ang II) signalling via the angiotensin II type 1 receptor (AT1R) exacerbates the hypertrophic response and blockade of Ang II signalling via angiotensin receptor blockers (ARBs) inhibits the hypertrophic response. Experimental models of CVD, including cardiac hypertrophy, have shown that stimulation of the counter-regulatory axis of the RAS, in particular stimulation of the ACE2/Ang-(1-7)/Mas signalling arm, provides cardioprotective effects. Although there is growing evidence for the importance of Ang-(1-7) in cardioprotection, delivery of this peptide to the heart as a therapeutic proves difficult owing to its short half life and thus quick degradation in the circulation. Recently, extracellular vesicles (EVs) have become an area of interest in terms of therapeutic delivery vehicles in a variety of disease settings. Due to their phospholipid bilayer, EVs remain stable and are not immediately degraded in the circulation, however organ-targeting is still a major challenge. As there is a need for new therapeutic delivery strategies for Ang-(1-7), the primary aim of this thesis was to investigate the potential role of EVs in RAS signalling and to determine whether functional Ang-(1-7) could be delivered via EVs in both in vitro and in vivo models of Ang II-induced cardiomyocyte hypertrophy. Initially, in vitro investigations were carried out in the H9c2 model of Ang II-induced cardiomyocyte hypertrophy. EVs were obtained from the conditioned media of H9c2 cardiomyocytes +/- Ang II stimulation and successfully characterised in terms of size, protein concentration, and concentration of particles released though no significant differences were found upon Ang II stimulation. EVs obtained from cardiac fibroblasts [neonatal rat cardiac fibroblasts (NRCF)] +/- Ang II stimulation were also characterised and found to be significantly larger than those released from H9c2 cardiomyocytes. Next, the functional effect of the EVs was determined using the H9c2 model of cardiomyocyte hypertrophy. It was found that 1 hr post-treatment with EVs, the EVs could be found associated with the H9c2 cell nucleus. EVs released from FAM-labelled Ang II treated H9c2 cells were shown to co-localise at the recipient H9c2 cell nucleus along with the FAM-Ang II signal. The evidence for Ang II association with EVs was further demonstrated by ELISA, where it was found that Ang II levels were significantly increased in EVs released from Ang II stimulated H9c2 cells vs control cells. Treatment of recipient H9c2 cells with Ang II EVs induced a hypertrophic response in these cells, where a significant increase in cell size was observed along with increased gene expression of the hypertrophy marker, brain natriuretic peptide (BNP). It was found that proteinase K efficiently digests soluble Ang II peptide and that treatment of EVs with proteinase K before addition to recipient cells did not abolish the hypertrophic effect. The hypertrophic response observed by treatment with Ang II EVs could be blocked by pre-treating parental cells with losartan but not by pre-treatment with PD123,319. It was found that EVs derived from Ang II-stimulated NRCF cells could also induce a hypertrophic response in recipient H9c2 cardiomyocytes, and that Ang II was detectable within the EVs. As the Ang II EVs were shown to have a functional effect in terms of hypertrophy in recipient cells, parental cells were next treated with Ang-(1-7) before EV isolation. It was found that EVs derived from Ang-(1-7) treated H9c2 cardiomyocytes were able to significantly inhibit Ang II-induced hypertrophy in recipient cells, where the Ang II-induced increase in cell size was absent upon co-treatment with Ang-(1-7)-EVs. As functional Ang II was found at low levels within EVs derived from Ang II-stimulated cells, it was of interest to determine whether Ang II levels could be increased by exogenous loading via electroporation, and if Ang-(1-7) could also be exogenously loaded into EVs. Optimisation of electroporation with Ang II showed significantly higher levels present in Ang II electroporated EVs compared with naïve electroporated EVs. Similarly, it was found that electroporation of EVs in the presence of Ang-(1-7) produced significantly higher levels of Ang-(1-7) within EVs compared to naïve electroporated EVs. These Ang-(1-7)-loaded EVs inhibited Ang II-induced increase in cell size in recipient H9c2 cardiomyocytes. Next, EVs were derived from the serum of normotensive WKY rats and hypertensive SHRSP rats and characterised. WKY EVs were found to be significantly larger than SHRSP EVs and exhibited a significantly higher protein concentration. WKY EVs were electroporated in the presence of Ang-(1-7) and then found to inhibit Ang II-induced hypertrophy in H9c2 cardiomyocytes. Interestingly, the inhibitory effect was also found after treatment with naïve electroporated EVs, an effect presumed to be due to the detectable levels of endogenous Ang-(1-7). The protective effect of Ang-(1-7) electroporated serum-derived EVs was next investigated in vivo utilising an Ang II-infusion rat model. This involved two 30 μg bolus injections of electroporated EVs into the circulation of WKY rats infused with 200 ng/kg/min Ang II over a 2 week period. It was found that Ang II infusion did not significantly increase blood pressure, percentage fibrosis or cardiomyocyte hypertrophy compared with vehicle infusion. As a result, the protective effects of Ang-(1-7) EV bolus injection could not be established in this study. Finally, human serum EV populations were analysed between healthy control subjects and coronary artery bypass patients to determine potential differences in size, concentration of particles released, Ang II content and Ang-(1-7) content under normal and CVD conditions. It was found that EVs derived from the serum of patients with coronary artery disease were significantly smaller in size than those from healthy control subjects and there were significantly lower numbers of particles released. It was also found that patient EVs contained significantly higher levels of Ang II compared with healthy controls, however Ang-(1-7) levels did not significantly differ between the two groups. Overall, these studies provide evidence that different cells of the heart produce different populations of EVs. Additionally, these EVs are able to transport functional effector peptides associated with the RAS, including Ang II and Ang-(1-7), in an in vitro model of cardiomyocyte hypertrophy. Finally, for the first time, electroporation was used to load the cardioprotective peptide, Ang-(1-7), into EVs for delivery in an in vivo Ang II infusion model with promising effects valuable for future therapeutic studies.