MicroRNAs and extracellular vesicles in pre-clinical and clinical stroke studies

Stroke is a leading cause of death and disability worldwide and remains a largely unmet clinical need. Despite decades of pre-clinical stroke research there are only two licensed interventions: intravenous delivery of thrombolytic recombinant tissue plasminogen activator (rt-PA) within 4.5 hours of stroke or mechanical thrombectomy. However, the number of patients eligible to receive either treatment is limited. An alternative intervention is needed, one which directly address specific aspects of stroke pathophysiology. Through their ability to alter the expression of multiple genes involved in stroke pathophysiology microRNAs (miRNA or miR) offer a novel therapeutic intervention. miRNA expression is altered both in experimental stroke and in patients with stroke. It was initially hypothesised that modulation of specific dysregulated miRNAs would be therapeutically beneficial in pre-clinical experimental ischaemic stroke. Recently, active transport of miRNAs in extracellular vesicles (EV), such as exosomes, has been demonstrated pre-clinically between cells in atherosclerosis and cardiac hypertrophy disease settings. It was therefore hypothesised that miRNAs packaged in exosomes would differ between patients with stroke and patients without stroke, raising the potential for novel exosomal miRNAs to be used as biomarkers or therapeutic agents for modulation. In Chapter 3, investigations were carried out to test the hypotheses that modulation of either miR-494 or miR-21 would be therapeutically beneficial in pre-clinical in vitro models of stroke. While miR-494 expression was unchanged in brain tissue of spontaneously hypertensive stroke prone rats (SHRSP) harvested at either 24 or 72 hours following transient middle cerebral artery occlusion (tMCAO) its expression was successfully up-regulated in B50 neuronal and GPNT cerebral endothelial cell lines following delivery of miR-494 mimic in combination with siPORT, a lipid based transfection reagent. mRNA expression of putative miR-494 target genes (PTEN, MMP2 and MMP9) was investigated post-miR-494 modulation but there was no obvious change in their expression. Modulation of miR-494 expression did not appear to be therapeutically beneficial (or detrimental) when assessed by a cell survival assay (MTS). As the balance of evidence did not indicate that miR-494 would be a suitable target for modulation in experimental stroke subsequent similar experiments investigated the therapeutic potential of miR-21. Its expression was significantly increased in SHRSP brain tissue at 72 hours following ischaemic stroke. miR-21 expression was successfully increased in cerebral endothelial cells following delivery of miR-21 mimics (with siPORT). mRNA expression of putative target genes (PDCD4 and PTEN) was unchanged following miR-21 modulation and cell survival (assessed by MTS assay) was unaffected. Subsequent experiments looked at vessel reactivity of aortae taken from miR-21+/- and miR-21-/- mice in comparison to wild type (WT) mice. Treatment of vessels with L-NAME to block endogenous nitric oxide (NO) bioavailability resulted in unopposed contraction to U46619 in WT mice while there was no change in contraction in miR-21-/- mice aortae, consistent with reduced basal NO bioavailability, and a detrimental phenotype associated with the loss of miR-21 expression. As the data generated in this study were primarily neutral and gave no indication that either miR-494 or miR-21 would be therapeutically beneficial in the setting of ischaemic stroke, subsequent studies focussed on investigating exosomal miRNA in ischaemic stroke. In Chapter 4, exosomal miRNA expression was profiled in blood samples from stroke patients and subsequently in pre-clinical rodent stroke models. Patients with suspected stroke were recruited and a blood sample taken at 48 hours post-stroke. All participants gave full informed consent and the study was approved by the Scotland A Research Ethics Committee. Exosomes were isolated from 200 μL serum before RNA was extracted. A miRNA microarray was performed (OpenArray™ platform) on samples from 39 patients. Validation of results was performed by real-time quantitative polymerase chain reaction using samples from 173 patients to determine the expression levels of specific miRNAs. Microarray experiments identified 26 exosomal miRNAs that were significantly dysregulated between stroke and non-stroke patients or between specific TOAST subtypes and non-stroke controls. Of these, changes in 13 miRNAs were validated in the larger cohort and levels of 9 miRNAs (-27b, -93, -20b, -17, -199a, -30a, let-7e, -218 and -223) were found to be significantly increased in definitively diagnosed stroke patients as compared to non-stroke patients. Differences in exosomal miRNA expression were observed between TOAST subtypes with small vessel disease patients consistently having the highest levels of these miRNAs. miRNA expression did not correlate with baseline or day 7 NIHSS score, although there was a trend towards patients with better functional outcome post-stroke (as assessed by modified Rankin Score at 1 month) having a higher level of some exosomal miRNAs. Subsequently total and exosomal miR-17 family (miRNAs -17, -93 and -20b) expression was investigated in pre-clinical models of hypertension and stroke. Total circulating miR-17 expression was unchanged between the serum of normotensive WKY and hypertensive SHRSP rats, whilst exosomal miR-17 expression was significantly increased in SHRSP vs. WKY. miR-17 family expression was unchanged in peri-infarct brain tissue of SHRSP at both 24 and 72 hours post-tMCAO. Experiments profiling total and exosomal circulating miR-17 family expression in serum of SHRSP post tMCAO or permanent MCAO revealed that expression was variable and changes observed were not significant. Cellular expression of miR-17 family miRNAs was unchanged following hypoxic challenge in neuronal, glial and cerebral endothelial cell lines and exosomal miRNA expression was highly variable, with no changes detected as significant. This study both identified and validated (for the first time) changes in exosome packaged miRNA expression in patients with stroke across differing stroke subtypes. The pre-clinical experimental findings corroborate the human data and support a functional role for these findings. In Chapter 5 exosomal packaged miR-17 family miRNAs were delivered in pre-clinical models of ischaemic stroke (both in vivo and in vitro) to test the hypothesis that they would be therapeutically beneficial following in vitro hypoxic challenge or in vivo experimental stroke. Bioinformatics analysis highlighted a number of important target genes implicated in stroke pathophysiology for each miRNA including genes involved in the regulation of the cellular response to stress, apoptosis and angiogenesis. miRNAs were artificially loaded (by electroporation) into EVs harvested from SHRSP brain. While miRNA loaded EVs did not successfully modulate miRNA expression either in vivo or in vitro it is believed that this is a result of technical issues with the loading of the miRNAs into the EVs. This study should be repeated when miRNAs have been successfully loaded into EVs, as these experiments remain of interest. In summary, the findings presented in this thesis confirm that packaging of miRNAs into exosomes is significantly dysregulated in stroke patients and that as a result the circulating exosomal miRNA profile is altered. This will direct future studies looking into paracrine signalling in the setting of stroke and the modulation of specific miRNAs as a novel therapy in the setting of experimental stroke

MicroRNAs in Aldosterone Production and Action

The secretion of aldosterone by the adrenal cortex is a tightly regulated process. Loss of this control can result in severe hypertension and end-organ damage, so detailed understanding of the various mechanisms by which the body regulates aldosterone biosynthesis is key. The emergence of microRNAs (miRNAs) as negative regulators of numerous physiological processes has naturally led to their study in the context of aldosterone production. We summarise several studies that have demonstrated a significant role for microRNAs in aldosterone biosynthesis and action, thereby presenting a possible therapeutic role in the treatment of common forms of hypertension such as primary aldosteronism. Furthermore, the presence of microRNAs in the circulation offers the prospect of accessible and informative biomarkers that may simplify the currently protracted and technically difficult diagnosis of such conditions

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

Circulating microRNAs as diagnostic markers in primary aldosteronism

Primary aldosteronism (PA) is a common and highly treatable condition, usually resulting from adrenocortical tumorous growth or hyperplasia. PA is currently underdiagnosed owing to its complex and protracted diagnostic procedures. A simplified biomarker-based test would be highly valuable in reducing cardiovascular morbidity and mortality. Circulating microRNAs are emerging as potential biomarkers for a number of conditions due to their stability and accessibility. PA is known to alter microRNA expression in adrenocortical tissue; if these changes or their effects are mirrored in the circulating miRNA profile, then this could be exploited by a diagnostic test. However, the reproducibility of studies to identify biomarker-circulating microRNAs has proved difficult for other conditions due to a series of technical challenges. Therefore, any studies seeking to definitively identify circulating microRNA biomarkers of PA must address this in their design. To this end, we are currently conducting the circulating microRNA arm of the ongoing ENS@T-HT study. In this review article, we present evidence to support the utility of circulating microRNAs as PA biomarkers, describe the practical challenges to this approach and, using ENS@T-HT as an example, discuss how these might be overcome

Positive impact of pre-stroke surgery on survival following transient focal ischemia in hypertensive rats

We describe a positive influence of pre-stroke surgery on recovery and survival in a commonly used experimental stroke model. Two groups of male, stroke-prone spontaneously hypertensive rats (SHRSPs) underwent transient middle cerebral artery occlusion (tMCAO). Group 1 underwent the procedure without any prior intervention whilst group 2 had an additional general anaesthetic 6 days prior to tMCAO for a cranial burrhole and durotomy. Post-stroke recovery was assessed using a 32 point neurological deficit score and tapered beam walk and infarct volume determined from haematoxylin–eosin stained sections. In group 2 survival was 92% (n = 12) versus 67% in group 1 (n = 18). In addition, post-tMCAO associated weight loss was significantly reduced in group 2. There was no significant difference between the two groups in experimental outcomes: infarct volume (Group 1 317 ± 18.6 mm<sup>3</sup> versus Group 2 332 ± 20.4 mm<sup>3</sup>), and serial (day 0–14 post-tMCAO) neurological deficit scores and tapered-beam walk test. Drilling a cranial burrhole under general anaesthesia prior to tMCAO in SHRSP reduced mortality and gave rise to infarct volumes and neurological deficits similar to those recorded in surviving Group 1 animals. This methodological refinement has significant implications for animal welfare and group sizes required for intervention studies

Galanin-immunoreactivity identifies a distinct population of inhibitory interneurons in laminae I-III of the rat spinal cord

Background: Inhibitory interneurons constitute 30-40% of neurons in laminae I-III and have an important anti-nociceptive role. However, because of the difficulty in classifying them we know little about their organisation. Previous studies have identified 3 non-overlapping groups of inhibitory interneuron, which contain neuropeptide Y (NPY), neuronal nitric oxide synthase (nNOS) or parvalbumin, and have shown that these differ in postsynaptic targets. Some inhibitory interneurons contain galanin and the first aim of this study was to determine whether these form a different population from those containing NPY, nNOS or parvalbumin. We also estimated the proportion of neurons and GABAergic axons that contain galanin in laminae I-III. Results: Galanin cells were concentrated in laminae I-IIo, with few in laminae IIi-III. Galanin showed minimal co-localisation with NPY, nNOS or parvalbumin in laminae I-II, but most galanin-containing cells in lamina III were nNOS-positive. Galanin cells constituted similar to 7%, 3% and 2% of all neurons in laminae I, II and III, and we estimate that this corresponds to 26%, 10% and 5% of the GABAergic neurons in these laminae. However, galanin was only found in similar to 6% of GABAergic boutons in laminae I-IIo, and similar to 1% of those in laminae IIi-III. Conclusions: These results show that galanin, NPY, nNOS and parvalbumin can be used to define four distinct neurochemical populations of inhibitory interneurons. Together with results of a recent study, they suggest that the galanin and NPY populations account for around half of the inhibitory interneurons in lamina I and a quarter of those in lamina I

Protease-activated receptor 2 activation induces behavioural changes associated with depression-like behaviour through microglial-independent modulation of inflammatory cytokines

Rationale: Major depressive disorder (MDD) is a leading cause of disability worldwide but currently prescribed treatments do not adequately ameliorate the disorder in a significant portion of patients. Hence, a better appreciation of its aetiology may lead to the development of novel therapies. Objectives: In the present study, we have built on our previous findings indicating a role for protease-activated receptor-2 (PAR2) in sickness behaviour to determine whether the PAR2 activator, AC264613, induces behavioural changes similar to those observed in depression-like behaviour. Methods: AC264613-induced behavioural changes were examined using the open field test (OFT), sucrose preference test (SPT), elevated plus maze (EPM), and novel object recognition test (NOR). Whole-cell patch clamping was used to investigate the effects of PAR2 activation in the lateral habenula with peripheral and central cytokine levels determined using ELISA and quantitative PCR. Results: Using a blood–brain barrier (BBB) permeable PAR2 activator, we reveal that AC-264613 (AC) injection leads to reduced locomotor activity and sucrose preference in mice but is without effect in anxiety and memory-related tasks. In addition, we show that AC injection leads to elevated blood sera IL-6 levels and altered cytokine mRNA expression within the brain. However, neither microglia nor peripheral lymphocytes are the source of these altered cytokine profiles. Conclusions: These data reveal that PAR2 activation results in behavioural changes often associated with depression-like behaviour and an inflammatory profile that resembles that seen in patients with MDD and therefore PAR2 may be a target for novel antidepressant therapies

Machine learning for classification of hypertension subtypes using multi-omics: a multi-centre, retrospective, data-driven study

Background: Arterial hypertension is a major cardiovascular risk factor. Identification of secondary hypertension in its various forms is key to preventing and targeting treatment of cardiovascular complications. Simplified diagnostic tests are urgently required to distinguish primary and secondary hypertension to address the current underdiagnosis of the latter. Methods: This study uses Machine Learning (ML) to classify subtypes of endocrine hypertension (EHT) in a large cohort of hypertensive patients using multidimensional omics analysis of plasma and urine samples. We measured 409 multi-omics (MOmics) features including plasma miRNAs (PmiRNA: 173), plasma catechol O-methylated metabolites (PMetas: 4), plasma steroids (PSteroids: 16), urinary steroid metabolites (USteroids: 27), and plasma small metabolites (PSmallMB: 189) in primary hypertension (PHT) patients, EHT patients with either primary aldosteronism (PA), pheochromocytoma/functional paraganglioma (PPGL) or Cushing syndrome (CS) and normotensive volunteers (NV). Biomarker discovery involved selection of disease combination, outlier handling, feature reduction, 8 ML classifiers, class balancing and consideration of different age- and sex-based scenarios. Classifications were evaluated using balanced accuracy, sensitivity, specificity, AUC, F1, and Kappa score. Findings: Complete clinical and biological datasets were generated from 307 subjects (PA=113, PPGL=88, CS=41 and PHT=112). The random forest classifier provided ∼92% balanced accuracy (∼11% improvement on the best mono-omics classifier), with 96% specificity and 0.95 AUC to distinguish one of the four conditions in multi-class ALL-ALL comparisons (PPGL vs PA vs CS vs PHT) on an unseen test set, using 57 MOmics features. For discrimination of EHT (PA + PPGL + CS) vs PHT, the simple logistic classifier achieved 0.96 AUC with 90% sensitivity, and ∼86% specificity, using 37 MOmics features. One PmiRNA (hsa-miR-15a-5p) and two PSmallMB (C9 and PC ae C38:1) features were found to be most discriminating for all disease combinations. Overall, the MOmics-based classifiers were able to provide better classification performance in comparison to mono-omics classifiers. Interpretation: We have developed a ML pipeline to distinguish different EHT subtypes from PHT using multi-omics data. This innovative approach to stratification is an advancement towards the development of a diagnostic tool for EHT patients, significantly increasing testing throughput and accelerating administration of appropriate treatment. Funding: European Union's Horizon 2020 Research and Innovation Programme under Grant Agreement No. 633983, Clinical Research Priority Program of the University of Zurich for the CRPP HYRENE (to Z.E. and F.B.), and Deutsche Forschungsgemeinschaft (CRC/Transregio 205/1)

MicroRNAs in Aldosterone Production and Action

The secretion of aldosterone by the adrenal cortex is a tightly regulated process. Loss of this control can result in severe hypertension and end-organ damage, so detailed understanding of the various mechanisms by which the body regulates aldosterone biosynthesis is key. The emergence of microRNAs (miRNAs) as negative regulators of numerous physiological processes has naturally led to their study in the context of aldosterone production. We summarise several studies that have demonstrated a significant role for microRNAs in aldosterone biosynthesis and action, thereby presenting a possible therapeutic role in the treatment of common forms of hypertension such as primary aldosteronism. Furthermore, the presence of microRNAs in the circulation offers the prospect of accessible and informative biomarkers that may simplify the currently protracted and technically difficult diagnosis of such conditions.</p

Preclinical validation of the therapeutic potential of Glasgow Oxygen Level Dependent (GOLD) technology: A theranostic for acute stroke

In acute stroke patients, penumbral tissue is non-functioning but potentially salvageable within a time window of variable duration and represents target tissue for rescue. Reperfusion by thrombolysis and/or thrombectomy can rescue penumbra and improve stroke outcomes, but these treatments are currently available to a minority of patients. In addition to the utility of Glasgow Oxygen Level Dependent (GOLD) as an MRI contrast capable of detecting penumbra, its constituent perfluorocarbon (PFC) oxygen carrier, combined with normobaric hyperoxia, also represents a potential acute stroke treatment through improved oxygen delivery to penumbra. Preclinical studies were designed to test the efficacy of an intravenous oxygen carrier, the perfluorocarbon emulsion Oxycyte® (O-PFC), combined with normobaric hyperoxia (50% O2) in both in vitro (neuronal cell culture) and in vivo rat models of ischaemic stroke. Outcome was assessed through the quantification of lipid peroxidation and oxidative stress levels, mortality, infarct volume, neurological scoring and sensorimotor tests of functional outcome in two in vivo models of stroke. Additionally, we investigated evidence for any positive or negative interactions with the thrombolytic recombinant tissue plasminogen activator (rt-PA) following embolus-induced stroke in rats. Treatment with intravenous O-PFC + normobaric hyperoxia (50% O2) provided evidence of reduced infarct size and improved functional recovery. It did not exacerbate oxidative stress and showed no adverse interactions with rt-PA. The positive results and lack of adverse effects support human trials of O-PFC + 50% O2 normobaric hyperoxia as a potential therapeutic approach. Combined with the diagnostic data presented in the preceding paper, O-PFC and normobaric hyperoxia is a potential theranostic for acute ischaemic stroke