Investigating Angiotensin II in cardiac remodelling and the counter-regulatory actions of Angiotensin-(1-9)

Cardiovascular diseases (CVDs) including, hypertension, coronary heart disease and heart failure are the leading cause of death worldwide. Hypertension, a chronic increase in blood pressure above 140/90 mmHg, is the single main contributor to deaths due to heart disease and stroke. In the heart, hypertension results in adaptive cardiac remodelling, including LV hypertrophy to normalize wall stress and maintain cardiac contractile function. However, chronic increases in BP results in the development of hypertensive heart disease (HHD). HHD describes the maladaptive changes during cardiac remodelling which result in reduced systolic and diastolic function and eventually heart failure. This includes ventricular dilation due to eccentric hypertrophy, cardiac fibrosis which stiffens the ventricular wall and microvascular rarefaction resulting in a decrease in coronary blood flow albeit an increase in energy demand. Chronic activation of the renin-angiotensin-system (RAS) with its effector peptide angiotensin (Ang)II plays a key role in the development of hypertension and the maladaptive changes in HHD. Ang II acts via the angiotensin type 1 receptor (AT1R) to mediate most of its pathological actions during HHD, including stimulation of cardiomyocyte hypertrophy, activation of cardiac fibroblasts and increased collagen deposition. The counter-regulatory axis of the RAS which is centred on the ACE2/Ang-(1-7)/Mas axis has been demonstrated to counteract the pathological actions of Ang II in the heart and vasculature. Ang-(1-7) via the Mas receptor prevents Ang II-induced cardiac hypertrophy and fibrosis and improves cardiac contractile function in animal models of HHD. In contrast, less is known about Ang-(1-9) although evidence has demonstrated that Ang-(1-9) also antagonises Ang II and is anti-hypertrophic and anti-fibrotic in animal models of acute cardiac remodelling. However, so far it is not well documented whether Ang-(1-9) can reverse established cardiac dysfunction and remodelling and whether it is beneficial when administered chronically. Therefore, the main aim of this thesis was to assess the effects of chronic Ang-(1-9) administration on cardiac structure and function in a model of Ang II-induced cardiac remodelling. Furthermore, this thesis aimed to investigate novel pathways contributing to the pathological remodelling in response to Ang II. First, a mouse model of chronic Ang II infusion was established and characterised by comparing the structural and functional effects of the infusion of a low and high dose of Ang II after 6 weeks. Echocardiographic measurements demonstrated that low dose Ang II infusion resulted in a gradual decline in cardiac function while a high dose of Ang II induced acute cardiac contractile dysfunction. Both doses equally induced the development of cardiac hypertrophy and cardiac fibrosis characterised by an increase in the deposition of collagen I and collagen III. Moreover, increases in gene expression of fibrotic and hypertrophic markers could be detected following high dose Ang II infusion over 6 weeks. Following this characterisation, the high dose infusion model was used to assess the effects of Ang-(1-9) on cardiac structural and functional remodelling in established disease. Initially, it was evaluated whether Ang-(1-9) can reverse Ang II-induced cardiac disease by administering Ang-(1-9) for 2-4 weeks following an initial 2 week infusion of a high dose of Ang II to induce cardiac contractile dysfunction. The infusion of Ang-(1-9) for 2 weeks was associated with a significant improvement of LV fractional shortening compared to Ang II infusion. However, after 4 weeks fractional shortening declined to Ang II levels. Despite the transient improvement in cardiac contractile function, Ang-(1-9) did not modulate blood pressure, LV hypertrophy or cardiac fibrosis. To further investigate the direct cardiac effects of Ang-(1-9), cardiac contractile performance in response to Ang-(1-9) was evaluated in the isolated Langendorff-perfused rat heart. Perfusion of Ang-(1-9) in the paced and spontaneously beating rat heart mediated a positive inotropic effect characterised by an increase in LV developed pressure, cardiac contractility and relaxation. This was in contrast to Ang II and Ang-(1-7). Furthermore, the positive inotropic effect to Ang-(1-9) was blocked by the AT1R antagonist losartan and the protein kinase A inhibitor H89. Next, endothelial-to-mesenchymal transition (EndMT) as a novel pathway that may contribute to Ang II-induced cardiac remodelling was assessed in Ang II-infused mice in vivo and in human coronary artery endothelial cells (HCAEC) in vitro. Infusion of Ang II to mice for 2-6 weeks resulted in a significant decrease in myocardial capillary density and this was associated with the occurrence of dual labelling of endothelial cells for endothelial and mesenchymal markers. In vitro stimulation of HCAEC with TGFβ and Ang II revealed that Ang II exacerbated TGF-induced gene expression of mesenchymal markers. This was not correlated with any changes in SMAD2 or ERK1/2 phosphorylation with co-stimulation of TGFβ and Ang II. However, superoxide production was significantly increased in HCAEC stimulated with Ang II but not TGFβ. Finally, the role of Ang II in microvesicle (MV)-mediated cardiomyocyte hypertrophy was investigated. MVs purified from neonatal rat cardiac fibroblasts were found to contain detectable Ang II and this was increased by stimulation of fibroblasts with Ang II. Treatment of cardiomyocytes with MVs derived from Ang II-stimulated fibroblasts induced cardiomyocyte hypertrophy which could be blocked by the AT1R antagonist losartan and an inhibitor of MV synthesis and release brefeldin A. Furthermore, Ang II was found to be present in MVs isolated from serum and plasma of Ang II-infused mice and SHRSP and WKY rats. Overall, the findings of this thesis demonstrate for the first time that the actions of Ang-(1-9) in cardiac pathology are dependent on its time of administration and that Ang-(1-9) can reverse Ang II-induced cardiac contractile dysfunction by acting as a positive inotrope. Furthermore, this thesis demonstrates evidence for an involvement of EndMT and MV signalling as novel pathways contributing to Ang II-induced cardiac fibrosis and hypertrophy, respectively. These findings provide incentive to further investigate the therapeutic potential of Ang-(1-9) in the treatment of cardiac contractile dysfunction in heart disease, establish the importance of novel pathways in Ang II-mediated cardiac remodelling and evaluate the significance of the presence of Ang II in plasma-derived MVs.

Warfare in the West Highlands and Isles of Scotland, c. 1544-1615

Warfare has long been associated with Scottish Highlanders and Islanders, especially in the period known in Gaelic tradition as ‘Linn nan Creach’ (the ‘Age of Forays’), which followed the forfeiture of the Lordship of the Isles in 1493. The sixteenth century in general is remembered as a particularly tumultuous time within the West Highlands and Isles, characterised by armed conflict on a seemingly unprecedented scale. Relatively little research has been conducted into the nature of warfare however, a gap filled by this thesis through its focus on a series of interconnected themes and in-depth case studies spanning the period c. 1544-1615. It challenges the idea that the sixteenth century and early seventeenth century was a time of endless bloodshed, and explores the rationale behind the distinctive mode of warfare practised in the West Highlands and Isles. The first part of the thesis traces the overall ‘Process of War’. Chapter 1 focuses on the mentality of the social elite in the West Highlands and Isles and demonstrates that warfare was not their raison d'être, but was tied inextricably to chiefs’ prime responsibility of protecting their lands and tenants. Chapter 2 assesses the causation of warfare and reveals that a recurrent catalyst for armed conflict was the assertion of rights to land and inheritance. There were other important causes however, including clan expectation, honour culture, punitive government policies, and the use of proxy warfare by prominent magnates. Chapter 3 takes a fresh approach to the military capacity of the region through analysis of armies and soldiers, and the final thematic chapter tackles the conduct of warfare in the West Highlands and Isles, with analysis of the tactics and strategy of militarised personnel. The second part of this thesis comprises five case studies: the Clanranald, 1544-77; the Colquhouns of Luss and the Lennox, 1592-1603; the MacLeods of Harris and MacDonalds of Sleat, 1594-1601; the Camerons, 1569-1614; and the ‘Islay Rising’, 1614-15. This thesis adopts a unique approach by contextualising the political background of warfare in order to instil a deeper understanding of why early modern Gaelic Scots resorted to bloodshed. Overall, this period was defined by a sharp rise in military activity, followed by an even sharper decline, a trajectory that will be evidenced vividly in the final case study on the ‘Islay Rising’. Although warfare was widespread, it was not unrestrained or continuous, and the traditional image of a region riven by perpetual bloodshed has been greatly exaggerated.

The standardisation of Scottish Gaelic orthography 1750-2007: a corpus approach

This thesis investigates the standardisation of Modern Scottish Gaelic orthography from the mid-eighteenth century to the twenty-first. It presents the results of the first corpus-based analysis of Modern Scottish Gaelic orthographic development combined with an analytic approach that places orthographic choices in their sociolinguistic context. The theoretical framework behind the analysis centres on discussion of how the language ideologies of the phonographic ideal, historicism, autonomy, vernacularism and the ideology of the standard itself have shaped orthographic conventions and debates. It argues that current spelling norms reflect an orthography that is the result of compromise, historical factors and pragmatic function. The research uses a digital corpus to examine how three particular features have been used over time: the dialect variation between <eu> and <ia>; variation in s + stop consonant clusters (sd/st, sg/sc, sb/sp); and the use of the grave and acute accents. Evidence is drawn from the Corpas na Gàidhlig electronic corpus created at the University of Glasgow: the sub-corpus used in this study includes 117 published texts representing a period of over 250 years from 1750 to 2007, and a total size of over four and a quarter million words. The results confirm a key period of reform between 1750 and the early nineteenth century, and thereafter a settled norm being established in the early nineteenth century. Since then, some variation has been acceptable although changes and reform of some features have centred on increasing uniformity and regularisation.

The Scottish Parliament in the 15th and 16th centuries

Earlier histories of the Scottish parliament have been somewhat constitutional in emphasis and have been exceedingly critical of what was understood to be parliament's subservience to the crown. Estimates by constitutional historians of the extreme weakness of parliament rested on an assessment of the constitutional system. The argument was that many of its features were not consistent with a reasonably strong parliament. Because the 'constitution' is apparently fragmented, with active roles played by bodies such as the lords of articles, the general council and the convention of estates, each apparently suggesting that parliament was inadequate, historians have sometimes failed to appreciate the positive role played by the estates in the conduct of national affairs. The thesis begins with a discussion of the reliability of the printed text of APS and proceeds to an examination of selected aspects of the work of parliament in a period from c 1424-c 1625. The belief of constitutional historians such as Rait that conditions In Scotland proved unfavourable to the interests and. effectiveness of parliament in the fifteenth and sixteenth centuries, is also examined. Chapter 1 concludes that APS is a less than reliable text, particularly for the reign of James I. Numerous statutes were excluded from the printed text and they are offered below for the first time. These statutes have been a useful addition to our understanding of the reign of James I. Chapter 2 analyses the motives behind the schemes for shire representation and concludes that neither constitutional theory nor political opportunism explains the support which James I and James VI gave to these measures. Both these monarchs were motivated by the realisation that their particular ambitions were dependent on winning the support of the estates whose ranks should include representatives from the shires. Chapter 3 examines the method of electing the lords of articles, the composition of this committee, and some aspects of its operation. The conclusion is that in the main the estates were the deciding force in the choice of the lords of articles. The committee's composition was more a reflection of a desire for a balance between representatives from north and south of the Forth and for the most important burghs and clergy to be selected than an attempt at electing government favourites. The articles did exercise a significant control over the items which came before parliament but this control was not absolute and applied to government as well as private legislation. Chapter 4 questions the traditional view that the general council and convention of estates were the same body. It is argued that they were two different institutions with different powers, but that they nevertheless worked within certain limits and were careful not to usurp the authority of parliament. Chapter 5 concedes that taxation was sometimes decided outside parliament; that the irregularity of taxation certainly weakened the bargaining power of the estates and that the latter did not appear to capitalise on these occasions when taxation was an issue. But the tendency was to ensure that, whether in or out of parliament, the decision to impose taxation was taken by a large number of each estate. The infrequency of taxation was a direct consequence of an unwillingness among the estates to agree to a regular taxation and their preference to ensure for the crown an alternative source of income. Moreover taxation was one issue, which more than any other, would be subject to contentious opposition by the estates, and could lead to the crown's defeat. Chapter 6 is concerned with ecclesiastical representation after the Reformation and the church's attitudes to the possibility of ministerial representation. Some ministers had doctrinal misgivings but the majority came to believe that the church's absence from parliament bad severely reduced. the influence of the church. That no agreement was forthcoming on a system of ministerial representation, particularly after 1597, is attributable to the estates' unwillingness to compromise and, not to the strength of opposition in the church. Chapter 7 examines the institutions which are sometime seen as 'rivals' of parliament and concludes that institutions such as the privy council were generally very careful in matters which needed the approval of parliament, and seemed aware of the greater authority of parliament. Chapter 8 which illustrates how parliament had the right to be consulted in all important matters of state, brings together the main points of the earlier chapters and offers further illustrations of the essential role which parliament played in the conduct of national affairs. Whether or not the system can be regarded as constitutionally sound, the estates in Scotland could observe parliament's day-to-day operation with some satisfaction. All in all, there is little convincing evidence that parliament was as weak as some historians would have us believe.