The role of Angiotensin-(1-7) and Angiotensin-(1-9) in vascular remodelling

McKinney, Clare Adele (2014) The role of Angiotensin-(1-7) and Angiotensin-(1-9) in vascular remodelling. PhD thesis, University of Glasgow.

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Vascular remodelling is an adaptive process that allows vessels to respond to changes in haemodynamic conditions, however this process also underlies the pathogenesis of atherosclerosis, vein graft failure following coronary artery bypass graft (CABG) surgery and restenosis following stent deployment to an atherosclerotic vessel. Injury to the vessel wall causes denudation of the endothelial cell (EC) layer and the resultant pathological vascular remodelling involves growth and migration of vascular smooth muscle cells (VSMC) and degradation and reorganisation of the extracellular matrix (ECM). Regeneration of the endothelial layer, known as re endothelialisation, is essential for healing of injured vessels and therefore therapies that specifically target VSMC growth and migration, without preventing re-endothelialisation are optimal in these pathologies. Dysregulation of the renin angiotensin system (RAS) is one of the key contributing factors to remodelling of the vasculature, with the majority of the pathological processes involved, being mediated by angiotensin II (Ang II) signalling at the angiotensin type I receptor (AT1R). A counter regulatory axis of the RAS has been identified, centred around the enzymatic actions of angiotensin converting enzyme 2 (ACE2), and the resultant production of Angiotensin-(1-9) [Ang-(1-9)] and Angiotensin-(1-7) [Ang-(1-7)] from Ang I and Ang II respectively. This axis counter-regulates the actions of Ang II via the AT1R, thereby providing a vasculoprotective role. Ang-(1-7) acts via the receptor Mas and inhibits Ang II induced VSMC cell migration, proliferation, and vascular remodelling in vivo. Comparatively, less is known about the actions of Ang-(1-9), however it has been identified as a functional ligand for the angiotensin type 2 receptor (AT2R), inhibiting cardiac hypertrophy in vitro and cardiac fibrosis in vivo. However, the role of Ang-(1-9) in the vasculature is unexplored. Therefore, the main aim of this thesis was to investigate the interaction of Ang II and the counter-regulatory peptides Ang-(1-7) and Ang-(1-9) in the vasculature using primary human VSMC and EC, and to provide a direct comparison of Ang-(1-7) and Ang-(1-9) in order to further understand their signal transduction pathways.
First, a model of VSMC proliferation was established in VSMC isolated from human saphenous vein tissue (HSVSMC). Here it was demonstrated that while Ang II had no effect on HSVSMC proliferation, foetal calf serum (FCS) induced HSVSMC proliferation in a concentration dependent manner. Ang-(1-7) and Ang-(1-9) blocked FCS induced proliferation of HSVSMC via Mas or the AT2R, respectively. Ang II-induced HSVSMC migration via the AT1R, and was inhibited by both Ang-(1-7) and Ang-(1-9) via Mas and the AT2R, respectively. Further investigation into the functional interplay of Ang II, Ang-(1-7) and Ang-(1-9) in HSVSMC migration identified alterations in extracellular signal-related kinase 1/2 (ERK1/2) activity and matrix metalloproteinase 2 (MMP2) and MMP9 expression as potential mechanisms contributing to the observed results. Additionally, Ang II has recently been demonstrated to regulate expression of the microRNA-132/-212 (miR-132/-212) cluster in rat aortic VSMC, thereby regulating a number of target genes involved in VSMC migration. This pathway was assessed in HSVSMC and it was found that Ang II-mediated HSVSMC migration was associated with an increase in miR-132 but not miR-212 expression, and a decrease in phosphatase and tensin homologue (PTEN) expression, a miR-132 target, at the messenger RNA level. These changes were found to be via the AT1R and were inhibited by Ang-(1-7) and Ang-(1-9). However, PTEN protein levels were unchanged and no changes were observed in key proteins involved in the downstream signalling pathways of PTEN, such as Akt and monocyte chemoattractant protein 1 ( MCP-1). The role of miRNA-132 in Ang II induced HSVSMC migration was further investigated through the use of a miR-132 inhibitor and downregulation of DICER, a key enzyme involved in miRNA biogenesis. Here it was found that miR-132 or regulation of an alternative miRNA via DICER is not essential for Ang II induced HSVSMC migration. However, inhibition of miR-132 or DICER enhanced basal migration of unstimulated HSVSMC.
Next, the effect of the RAS peptides, particularly Ang-(1-9), on EC growth, migration and function was assessed. Ang II, Ang-(1-7) or Ang-(1-9) have no effect on growth or migration of EC isolated from human saphenous veins (HSVEC). A direct effect of Ang-(1-9) on nitric oxide (NO) release from HSVEC and Chinese hamster ovary (CHO) cells expressing the AT2R was demonstrated. Although in cell culture Ang-(1-9) induced NO release in an AT2R sensitive manner, it was found that in vessels from AT2R knockout (AT2R-/-) mice the biological effect of Ang-(1-9) was maintained and promoted vasodilation of both aortic and mesenteric artery rings. Furthermore, Ang-(1-9)-induced relaxation of AT2R-/- aortic rings, but not mesenteric artery rings, was blocked by A779, suggesting that in large vessels Ang-(1-9) may mediate is vasodilatory effects via conversion to Ang-(1-7) and signalling via Mas, while in resistance vessels Ang-(1-9) promotes vasodilation through an alternative mechanism.
The observation that Ang-(1-7) and Ang-(1-9) block HSVSMC, but not HSVEC, proliferation and migration, identified these peptides as potential therapeutic agents in vascular injury. A carotid artery wire injury model in mice was established, where injury to the carotid artery using a synthetic nylon fibre induced significant injury to the vessel, manifesting in the production of a large neointimal area at 28 days post injury. To assess the effects of Ang-(1-7) and Ang-(1-9), the peptides were delivered via subcutaneously via osmotic minipump. It was found that Ang-(1-7) infusion reduced neointimal formation and neointimal/media (NI/MA) ratio in comparison to control vessels via Mas. Similarly, Ang-(1-9) reduced neointimal formation and NI/MA ratio via the AT2R, as the AT2R antagonist PD123,319, but not the Mas antagonist A779,blocked the effects of Ang-(1-9), indicating that this was via a direct effect of Ang-(1-9), as opposed to conversion to Ang-(1-7) and signalling via Mas.
An interesting finding from the in vivo study was that a large proportion of vessels from animals co-infused with Ang-(1-7) and A779, or Ang-(1-9) and PD123,319 developed more complex lesions with increased vessel remodelling and neovascularisation, largely within the media, in comparison to all other groups. Analysis of the composition of these complex lesions revealed that they were composed of disorganised ECM and were highly cellular, containing a large number of VSMC, macrophages and proliferating cells. Re-endothelialisation had occurred on the lumenal lining of these vessels and neovascularisation of the complex lesion was observed.
In summary the data from this thesis demonstrates for the first time a direct biological role for Ang-(1-9) in the vasculature through inhibition of HSVSMC migration and proliferation, and increase NO bioavailability from HSVEC in vitro and reduced neointimal formation in an in vivo mouse model of vascular injury. Furthermore, this study provides a direct comparison of Ang-(1-9) and Ang-(1-7) in the vasculature and while the end biological effects are similar, they act via different receptors, the AT2R or Mas, respectively, and differences exist in their signal transduction mechanisms. These findings highlight the potential of Ang-(1-9) and Ang-(1-7) as therapeutic agents in the setting of vascular remodelling.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Angiotensin-(1-9), Angiotensin-(1-7), vascular remodelling
Subjects: R Medicine > RC Internal medicine
Colleges/Schools: College of Medical Veterinary and Life Sciences > Institute of Cardiovascular and Medical Sciences
Supervisor's Name: Nicklin, Dr. Stuart A. and Milligan, Prof. Graeme
Date of Award: 2014
Depositing User: Dr Clare McKinney
Unique ID: glathesis:2014-5752
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 11 Nov 2014 12:09
Last Modified: 13 Nov 2017 09:56
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