The counter regulatory axis of the renin angiotensin system in the brain and ischaemic stroke: insight from preclinical stroke studies and therapeutic potential

Stroke is the 2nd leading cause of death worldwide and the leading cause of physical disability and cognitive issues. Although we have made progress in certain aspects of stroke treatment, the consequences remain substantial and new treatments are needed. Hypertension has long been recognised as a major risk factor for stroke, both haemorrhagic and ischaemic. The renin angiotensin system (RAS) plays a key role in blood pressure regulation and this, plus local expression and signalling of RAS in the brain, both support the potential for targeting this axis therapeutically in the setting of stroke. While historically, focus has been on suppressing classical RAS signalling through the angiotensin type 1 receptor (AT1R), the identification of a counter-regulatory axis of the RAS signalling via the angiotensin type 2 receptor (AT2R) and Mas receptor has renewed interest in targeting the RAS. This review describes RAS signalling in the brain and the potential of targeting the Mas receptor and AT2R in preclinical models of ischaemic stroke. The animal and experimental models, and the route and timing of intervention, are considered from a translational perspective

Unscheduled changes in pre-clinical stroke model housing contributes to variance in physiological and behavioural data outcomes: a post hoc analysis

Ischaemic stroke presents a significant problem worldwide with no neuroprotective drugs available. Many of the failures in the search for neuroprotectants are attributed to failure to translate from pre-clinical models to humans, which has been combatted with rigorous pre-clinical stroke research guidelines. Here, we present post hoc analysis of a pre-clinical stroke trial, conducted using intraluminal filament transient middle cerebral artery occlusion in the stroke-prone spontaneously hypertensive rat, whereby unscheduled changes were implemented in the animal housing facility. These changes severely impacted body weight post-stroke resulting in a change from the typical body weight of 90.6% of pre-surgery weight post-stroke, to on average 80.5% of pre-surgery weight post-stroke. The changes also appeared to impact post-stroke blood pressure, with an increase from 215.4 to 240.3 mmHg between housing groups, and functional outcome post-stroke, with a 38% increased latency to contact in the sticky label test. These data highlight the importance of tightly controlled housing conditions when using physiological or behavioural measurements as a primary outcome

Experimental and theoretical investigation of drotaverine binding to bovine serum albumin

This study was motivated by the need to provide more insight into the possible mechanism of the intermolecular interactions between antispasmodic drug drotaverine and one of the serum albumins (BSA), with the aim to indicate the most probable sites of these interactions. For this purpose both experimental (spectrofluorometric titration at various temperatures) and theoretical (molecular mechanics) methods have been applied. The obtained results clearly showed that drotaverine quenched BSA fluorescence, and the most probable mechanism is static quenching. The negative value of the theoretically predicted binding free Gibbs energy (-23.8 kJ/mol) confirmed the existence of the intermolecular interactions involving drotaverine and one tryptophan within BSA protein and was well agreed with the experimentally determined value of -25.2 kJ/mol

Altered extracellular vesicle microrna expression in ischemic stroke and small vessel disease

Active transport of microRNAs (miRNA) in extracellular vesicles (EV) occurs in disease. Circulating EV-packaged miRNAs in the serum of stroke patients were compared to stroke mimics with matched cardio- and cerebrovascular risk factors, with corroboration of results in a pre-clinical model. An unbiased miRNA microarray was performed in stroke vs stroke mimic patients (n=39). Results were validated (n=173 patients) by real-time quantitative polymerase chain reaction. miRNA expression was quantified in total serum/EV (n=5-7) of naïve adult spontaneously hypertensive stroke-prone rats (SHRSP), their normotensive reference strain (Wistar Kyoto, WKY) and in circulating EV (n=3), peri-infarct brain (n=6) or EV derived from this region (n=3) in SHRSP following transient middle cerebral artery occlusion (tMCAO). Circulating EV concentration did not differ between stroke and stroke mimic patients. The microarray identified many altered EV-packaged miRNAs: levels of miRNA-17-5p, -20b-5p and -93-5p (miRNA-17 family members) and miRNA-27b-3p were significantly (p≤0.05) increased in stroke vs stroke mimic patients. Patients with small vessel disease (SVD) consistently had the highest miRNA levels. Circulating EV concentration was unaltered between naïve SHRSP and WKY but levels of miRNA-17-5p and -93-5p were significantly increased in SHRSP. tMCAO in SHRSP did not further alter circulating EV miRNA-17 family member expression and nor did it change total miRNA-17 family levels in peri-infarct brain tissue or in EV isolated from this region at 24hrs post-tMCAO. Changes in EV packaged miRNA expression was validated in patients with stroke, particularly those with SVD, and corroborated pre-clinically. Together, altered circulating EV levels of miRNA-17 family members may reflect the chronic sequelae underlying cerebrovascular SVD rather than the acute ischemic stroke itself

Modulating the counter-regulatory renin angiotensin system axis in experimental ischaemic stroke

Stroke is a leading cause of death and disability worldwide, yet treatment options are limited. Stroke can be caused by a ruptured cerebral blood vessel, known as haemorrhagic stroke, or more commonly by the blockage of a blood vessel within the brain, known as ischaemic stroke. Starvation of the brain tissue of oxygen and glucose during ischaemia results in a pathophysiological cascade of damage consisting of ionic dysregulation, excitotoxicity, oxidative stress and inflammation. Recanalization of the vessel, either pharmacologically using a clot busting drug or by mechanical clot removal known as thrombectomy, paradoxically results in further injury by similar mechanisms, known as reperfusion injury. Different aspects of stroke pathophysiology have been previously targeted as neuroprotective strategies, but none have proven effective in improving patient outcomes following stroke. Efforts by the preclinical stroke community to improve methodological rigor in experimental stroke research have been well implemented and will hopefully aid in the translation of novel neuroprotectants for stroke care. Targeting the renin angiotensin system (RAS) has been successful clinically for modulating hypertension, the major modifiable risk factor for stroke. In this system, the biologically active peptide, angiotensin II (Ang II) is produced through the actions of the enzymes renin and angiotensin converting enzyme (ACE), and acts upon the Ang II type 1 receptor (AT1R) to produce an increase in blood pressure, but also local tissue specific effects such as cardiac hypertrophy, fibrosis and neuronal cell death. A counter-regulatory axis of this system exists, consisting of an alternative enzyme, ACE2, which produces the peptides, Ang-(1-9) and Ang-(1-7), and alternative receptors, the Ang II type 2 receptor (AT2R) and Mas receptor (MasR). These peptides act upon these receptors, respectively, producing antagonistic effects to AT1R signalling. More recently, studies have demonstrated the neuroprotective potential of the counter-regulatory RAS, in particular agonism of AT2R. Ang-(1-9) is a novel peptide of the counter-regulatory RAS which acts upon the AT2R but, to date, this peptide has not been investigated in the setting of ischaemic stroke. The primary aim of this thesis was to investigate the therapeutic potential of Ang-(1-9) in a comorbid experimental stroke model, with secondary aims of investigating modulation of the RAS within the brain of this animal following stroke, and establishment of an in vitro model to study Ang-(1-9) effects on oxygen-glucose deprivation (OGD). In Chapter 3, the expression of RAS mRNA was confirmed in the brain of stroke prone spontaneously hypertensive rats (SHRSP). This animal was used throughout these studies as a comorbid animal model displaying chronic hypertension and resulting end-organ damage because hypertension is the key modifiable risk factor for ischaemic stroke. It was hypothesised that SHRSP would have different RAS gene expression to the normotensive Wistar Kyoto strain (WKY) and that brain RAS gene expression would be altered by salt-loading. Results, using reverse transcription quantitative PCR (RT-qPCR), demonstrated similar baseline RAS gene expression in the cortical tissue between the two strains but salt-loading in the SHRSP induced an downregulation of AT1R and upregulation of MasR compared to WKY, suggesting a differential response of RAS and counter-regulatory RAS receptor expression to this stressor between the two strains. It was also hypothesised that RAS gene expression would be altered in the SHRSP brain following experimental stroke. A transient middle cerebral artery occlusion (tMCAO) filament model of stroke was utilised without reperfusion or with 2-24 hr reperfusion in order to assess gene expression changes longitudinally post-stroke. Results demonstrated that genes for the components of the classical RAS, ACE and AT1R, were downregulated 24 hr following tMCAO in the SHRSP brain in line with previous reports in other animal models. Components of the counter-regulatory RAS axis, ACE2, AT2R and MasR, were also downregulated 24 hr following tMCAO in the SHRSP brain which contradicts previous studies in healthy mice or rats. Interestingly, however, during the MCA occlusion period, AT2R was significantly upregulated in line with previous reports of AT2R expression following permanent MCAO. Together, these results suggest that SHRSP may display altered RAS expression in response to insult compared to healthy animals but give reassurance for the ability to target AT2R in SHRSP due to its acute upregulation. A further interesting finding of this chapter was that AT2R expression varies greatly across the SHRSP brain following sham surgeries, with higher expression in the lower remainder region and in the “peri-infarct” region compared to cortical “infarct” tissue. In Chapter 4, it was hypothesised that Ang-(1-9) would have a beneficial effect on outcome in the SHRSP following tMCAO when it was delivered prior to stroke via an adeno-associated viral (AAV9) vector (AAV9-Ang-(1-9)) injected directly into the brain at striatal and cortical sites. AAV9 mediated transgene expression was first confirmed near the injection sites at the appropriate time-point for stroke induction, and further tMCAO surgeries were performed for competency training and to provide preliminary data for sample size calculations. Due to higher than expected mortality rate, and early terminations due to animals reaching the severity limit of the procedure, the study was terminated early with under-powered group sizes. Despite the early termination, animals receiving stereotactic AAV9-Ang-(1-9) prior to tMCAO showed trends towards functional improvement, measured by neurological scoring, the tapered beam test and sticky label test, compared to animals receiving burrhole surgery only or injections of control virus AAV9-eGFP (enhanced green fluorescent protein) but infarct volumes were similar across all groups. AAV9-Ang-(1-9) also had no effect on systemic blood pressure but tMCAO surgery induced an overall increase in systemic blood pressure across groups. Despite previous reports of AT2R agonism inducing upregulation of brain derived neurotrophic factor (BDNF), AAV9-Ang-(1-9) did not result in greater expression of BDNF following tMCAO in the SHRSP brain, indeed a trend towards reduced expression was observed. Similarly, despite previous reports of anti-fibrotic vascular effects of Ang-(1-9), AAV9-Ang-(1-9) did not affect vascular collagen deposition in the SHRSP brain following stroke. Both BDNF expression and vascular collagen deposition, however, were increased in the ipsilateral stroke hemisphere compared to the contralateral hemisphere. Overall, the results of this chapter demonstrated promising potential of treating ischaemic stroke with Ang-(1-9) and the use of AAV9 mediated transgene delivery, however due to limitations within the study further conclusive investigations are required to assess Ang-(1-9) in stroke. Furthermore, during this in vivo study, unanticipated changes in the animal unit facilities were implemented. These were explored in further detail in Chapter 5 which presents the observation of differences in post-stroke weight loss and functional outcome when animals were grouped depending on the bedding or cage type used, suggesting the importance of tight regulation of environmental variables in preclinical stroke studies. Finally, in Chapter 6, an in vitro model of ischaemia-reperfusion injury was established using OGD in primary neuronal cultures (PNC) cultured from fetal SHRSP brain tissue. PNC were used due to lack of RAS gene expression and adenoviral transduction observed in rat brain cell lines. It was hypothesised that the OGD PNC model could be used to demonstrate neuroprotective effects of Ang-(1-9) and would allow for mechanistic insight. Optimisation of the OGD protocol proved difficult with no rescue effect of OGD induced loss in cell viability or OGD induced cell death observed by previously published neuroprotective compounds or with Ang-(1-9) peptide or adenoviral mediated Ang-(1-9) expression. Interestingly, under normal culturing conditions, Ang-(1-9) and compound 21 (C21) induced a reduction in BDNF or vascular endothelial growth factor (VEGF) expression in PNC, despite previous reports of C21, the AT2R agonist, inducing upregulation of these molecules. This suggests that SHRSP PNC may respond differently to AT2R agonism depending on the model and disease setting. In summary, the findings presented in this thesis demonstrate encouraging potential for the use of Ang-(1-9) as a treatment for ischaemic stroke. However, this also highlights the difficulties encountered in preclinical stroke research and the need for further optimisation of these models for further investigation of Ang-(1-9) or other drug