Investigating the role of angiotensin-(1-9) in neointimal formation

Summary During coronary artery bypass graft (CABG) procedures, the patient’s autologous saphenous vein (SV) is grafted either side of an occlusion in the coronary artery. This allows oxygenated blood to bypass the narrowed section thus reperfusing the myocardium and relieving symptoms of coronary heart disease. Damage to the SV endothelium can promote the adherence of platelets and the formation of an occlusive thrombus that leads to early vein graft failure. Where thrombosis is limited, local inflammation and endothelial cell dysfunction can promote the inward migration of medial vascular smooth muscle cells (VSMC) . Migrated VSMCs, now residing in the intima, proliferate extensively forming an occlusive lesion known as the neointima. Neointimal VSMCs exhibit a synthetic phenotype and excrete components of extracellular matrix. Accelerated atherosclerosis can then lead to the total occlusion of the conduit resulting in myocardial infarction or requirement for further surgery. Approximately 40% of vein grafts fail over a period of ten years. The need to develop new therapies to prevent late vein graft failure (VGF) is therefore urgent. The renin angiotensin system (RAS) provides vital modulation of blood pressure and vascular tone through the octapeptide angiotensin II (Ang II). Excessive Ang II signalling through the angiotensin II type I receptor (AT1R) promotes pathophysiological processes important in the development and progression of vein graft disease, including but not limited to inflammation, VSMC migration and fibrosis. A non-canonical axis of the RAS exists to counterbalance the effects of Ang II. Angiotensin-converting enzyme-related carboxypeptidase 2 (ACE2) degrades Ang II to angiotensin-(1-7) [Ang-(1-7)] and angiotensin I (Ang I) to angiotensin-(1-9) [Ang-(1-9)]. Here, the effect of subcutaneous infusion of soluble Ang-(1-9) peptide was investigated in a mouse model of neointima formation induced by carotid artery ligation. In this model, subcutaneous delivery of Ang-(1-9) decreased circulating concentrations of the proinflammatory cytokine monocyte chemotactic protein-1 (MCP-1) (an effector of Ang II with a known role in vein graft remodelling) 14 days after ligation but not at the earlier 7-day time point. Infusion of Ang-(1-9) significantly attenuated VSMC proliferation as determined by immunofluorescence assessment of arterial proliferating cell nuclear antigen (PCNA) expression. However, Ang-(1-9) infusion did not inhibit neointima formation as determined by histological staining and morphometric analysis. Next, VSMC were isolated from surplus sections of human SV donated by CABG patients. Platelet derived growth factor beta chain dimer (PDGF-BB) is a potent mitogen and plays a key role in the development of vein graft disease. Here, PDGF-BB induced both directional and chemotactic HSVSMC migration. In both migration models (scratch and Boyden chamber), PDGF-BB induced migration was attenuated by pre-incubation with Ang-(1-9). PDGF-BB also promoted potent pro-proliferative effects on HSVSMCs as determined by incorporation of a thymidine analogue (bromodeoxyuridine). At micromolar concentrations, Ang-(1-9) blocked the pro-proliferative effects of PDGF-BB. Downstream effects of PDGF-BB and Ang-(1-9) were assessed via an antibody based protein array. At an acute time-point, PDGF-BB induced activation of the mitogen-activated protein kinase (MAPK) pathway as determined by phosphorylation of extracellular signal–regulated kinases (ERK1/2). Likewise, PDGF-BB elicited activation of the phosphoinositide 3-kinase (PI3K) pathway as detected by phosphorylation of protein kinase B (Akt) and proline rich Akt residue 40 (PRAS40). Preincubation with Ang-(1-9) selectively inhibited PDGF-BB induced ERK1/2 phosphorylation. This is in agreement with previously reported effects of the AT2R. The MEKK inhibitor U0126 was then employed to demonstrate that selective inhibition of ERK1/2 prevented PDGF-BB induced HSVSMC proliferation. Therefore, it is suggested here that Ang-(1-9) confers therapeutic effects in VSMC in vitro and in vivo and could be a putative target for application in CABG. CABG surgeries are ideally suited to viral gene therapy owing to the opportunity to treat the vein ex vivo in the operating theatre before implantation. Therefore, local overexpression of Ang-(1-9) is achievable and may be beneficial in the setting of vein graft failure following CABG. Here, the effects of an adenoviral vector encoding an intracellular cleaved and secreted Ang-(1-9) peptide [RAdAng-(1-9)] was characterised. Direct transduction with RAdAng-(1-9) significantly inhibited HSVSMC migration induced by Ang II. Unexpectedly, RAdAng-(1-9) conferred pro-proliferative effects on HSVSMC and HSV endothelial cells (HSVEC). The hepatoma cell line HepG2 were then transduced with RAdAng-(1-9) and the effect of the conditioned culture media on recipient HSVSMC proliferation assessed. This aimed to mimic the subsequently performed in vivo model where the vector is delivered systemically resulting in liver transgene expression. HepG2 cells transduced with RAdAng-(1-9) expressed and secreted the Ang-(1-9) fusion protein as detected by immunoblotting of the conditioned culture media. Conditioned media collected from RAdAng-(1-9) but not RAdControl transduced HepG2 cells inhibited serum induced HSVSMC proliferation. Subsequently, an in vivo proof of concept gene transfer study was conducted. Intravascular delivery of a control vector (AdLacZ) led to strong liver transgene expression (three days after infusion) and did not affect neointima formation following wire injury. Importantly, RAdAng-(1–9) administered intravenously 48 hours before wire injury surgery significantly inhibited neointima formation 28 days after endothelial denudation, whereas administration of saline or RAdControl had no effect. These data elucidate novel therapeutic effects of Ang-(1-9) in vitro and in vivo. This is the first evidence that Ang-(1-9) can inhibit the effects of PDGF-BB in HSVSMC. Furthermore, this is the first evidence that gene therapy with Ang-(1-9) can be used to inhibit neointima formation in vivo . Taken together Ang-(1-9) may be a potential candidate for use in CABG. Further investigations should investigate how best to deliver this peptide and fully understand its mechanisms of action