title: Dissecting transforming growth factor-beta signalling pathways in the context of acute vascular injury
Creators: name: Low, Emma Louise
Publisher: name: University of Glasgow
Year: 2018

description: Coronary artery bypass grafting (CABG) is a mainstay in the treatment of coronary heart disease (CHD), a leading cause of premature death in the UK. However, fewer than 60 % of saphenous vein grafts remain patent in the long-term due to the formation of a hyperplastic, occlusive neointima within the grafted vessel. Excessive vascular smooth muscle cell (VSMC) proliferation, migration and extracellular matrix (ECM) synthesis are key events in the pathogenesis of vein graft intimal hyperplasia (IH), and subsequent necrotic core formation and intraplaque haemorrhage further accelerate the process of vein graft failure by forming unstable atherosclerotic lesions. Early IH therefore represents an important target for therapeutic interventions aimed at improving clinical outcomes after CABG. The pleiotropic cytokine transforming growth factor-beta (TGFβ) is highly expressed in restenotic vessels from CHD patients and is acutely upregulated following vein graft implantation in large and small animal models of vein graft disease. To date, most studies investigating the role of TGFβ in IH have used global approaches to target TGFβ, or have focused on the canonical activin receptor-like kinase 5 (ALK5), Smad2/3-mediated pathway. However, TGFβ elicits a diverse range of cellular responses by activating several distinct signalling pathways, and in certain cell types can also signal via ALK1, activating a separate set of receptor-regulated Smad proteins (R-Smads; Smad1/5) that can antagonise ALK5 signalling. Importantly, the role of ALK1 in the pathogenesis of vein graft IH remains unclear and studies have yet to conclusively show whether TGFβ is able to signal via ALK1 in VSMCs. Moreover, in vivo studies indicate that the three mammalian TGFβ isoforms have discrete biological functions, especially during wound healing, a process similar to IH involving cell migration, proliferation and ECM formation. Therefore, the principal aim of this thesis was to dissect the roles of ALK5- and ALK1-mediated signalling in VSMCs during vein graft IH. In addition, this thesis aimed to evaluate whether TGFβ1, TGFβ2 and TGFβ3 differentially regulate VSMC behaviour in the context of vein graft IH. Initially, the expression of the TGFβ isoforms, ALK receptors and R-Smads in VSMCs during the development of IH was evaluated in both small and large animal models of arterial injury and vein graft disease. IHC analysis of wire-injured mouse carotid arteries 14 days post-injury revealed that TGFβ1, TGFβ3, ALK5 and ALK1 were expressed in αSMA+ intimal and medial VSMCs. Interestingly, while nuclear localisation of phosphorylated Smad2/3 (pSmad2/3) within αSMA+ VSMCs was observed in both non-injured and injured vessels, nuclear pSmad1/5 was only detected within VSMCs following vascular injury. IHC analysis of TGFβ signalling components in diseased pre-implantation human saphenous vein (HSV) with pre-existing IH showed that TGFβ1, TGFβ3, TβRII (TGFβ type II receptor), ALK5 and ALK1 were expressed in αSMA+ VSMCs within both the intima and media. Importantly, dual staining for TβRII and ALK5 or ALK1 showed strong co-localisation between ALK5/ALK1 and TβRII. Both pSmad2/3 and pSmad1/5 were localised to the nuclei of intimal αSMA+ VSMCs, suggesting that both ALK5 and ALK1 signalling pathways may be active in these cells. Confocal microscopy analysis of three failed vein graft specimens obtained from patients undergoing cardiac transplantation revealed abundant nuclear-localised pSmad2/3 and pSmad1/5 in αSMA+ intimal VSMCs. These data suggest that both the ALK5 and ALK1 pathways may be activated in VSMCs during the development of IH. Having localised TGFβ1, TGFβ3, TβRII, ALK5, ALK1, pSmad2/3 and pSmad1/5 to intimal vein graft SMCs, subsequent mechanistic characterisation of TGFβ signalling via ALK5/ALK1 was performed in SMC outgrowth cultures from pre-implantation HSV segments from CABG patients (HSVSMC). Affinity labelling and crosslinking studies using 125I-TGFβ1 revealed binding of TGFβ1 to ALK5, ALK1 and TβRII, as well as the accessory receptors endoglin and betaglycan in HSVSMC. qRT-PCR confirmed the expression of these receptors at the RNA level, while immunoblotting revealed that treatment with all three TGFβ isoforms could induce a rapid increase in pSmad2 as well as pSmad1/5. Immunocytochemistry demonstrated the nuclear localisation of both pSmad2/3 and pSmad1/5 signalling complexes following stimulation of HSVSMC with TGFβ1, while qRT-PCR evaluation of ALK5 and ALK1 target genes (SERPINE1 and ID1, respectively) confirmed the transcriptional activation of both ALK signalling pathways by all three TGFβ isoforms. Importantly, pharmacological inhibition of ALK5 or ALK1 (using SB525334 or K02288, respectively) or siRNA-mediated knockdown of ALK5 or ALK1 in TGFβ-stimulated HSVSMC, reduced the expression of pSmad2 and pSmad1/5, respectively, confirming that TGFβ can bind to and signal through both ALK5 and ALK1 in HSVSMC. Functional assays performed in HSVSMCs indicated that TGFβ1, TGFβ2 and TGFβ3 regulate VSMC proliferation and migration in a similar manner in vitro. To gain insight into how the ALK5 and ALK1 TGFβ signalling pathways regulate VSMC proliferation, migration and apoptosis, functional assays were performed in HSVSMC treated with TGFβ1 ± SB525334 or K02288. Pharmacological inhibition of ALK5 or ALK1 did not significantly alter HSVSMC proliferation in response to TGFβ1. Interestingly, TGFβ1-mediated HSVSMC migration was significantly attenuated in the presence of ALK1 small molecule inhibitor, K02288, whereas inhibition of ALK5 signalling by SB525334 had no significant effect on HSVSMC migration. TGFβ1 protected from hydrogen peroxide-induced HSVSMC apoptosis and inhibition of ALK5 or ALK1 signalling reversed this effect. These studies indicate that TGFβ signalling via ALK5 and ALK1 differentially regulates HSVSMC migration, but not proliferation or apoptosis. Data output from the Human TGFβ/BMP RT2 Profiler PCR Arrays suggested that several TGFβ signalling pathway genes were differentially expressed following rTGFβ treatment in HSVSMCs, whereby some TGFβ isoform-specific effects on gene expression were observed. However, following validation, no TGFβ isoform-specific effects on gene expression were detected. Whole genome expression profiling of migrating HSVSMCs treated with TGFβ1 ± SB525334 or K02288 was performed in order to compare the gene expression profiles directly regulated by ALK5 and ALK1. In total, the expression of 3,235 genes was modulated by TGFβ1 treatment compared with non-stimulated HSVSMCs, approximately half of which appeared to be co-ordinately dysregulated following ALK5 and ALK1 inhibition. Two groups of putative ALK5- and ALK1-specific transcriptional targets were chosen for more detailed evaluation and validation. qRT-PCR validation in HSVSMC confirmed fibroblast growth factor 2 (FGF2) and Mal, T-cell differentiation protein like (MALL) as ALK5-specific target genes, and fatty acid desaturase 1 (FADS1), H1 histone family member 0 (H1F0) and scavenger receptor class A member 3 (SCARA3) as ALK1-specific target genes. Together, this data indicates that TGFβ regulates HSVSMC behaviour during the pathogenesis of vein graft IH by activating distinct, ALK receptor-specific transcriptional networks. Overall, the findings from this thesis indicate that the ALK1/Smad1/5 TGFβ signalling pathway is activated following vascular injury and induces specific transcriptional changes to promote VSMC migration. Moreover, these studies indicate that TGFβ1, TGFβ2 and TGFβ3 regulate VSMC behaviour in a similar manner in vitro and all isoforms appear to have equivalent effects on the induction of established ALK5 and ALK1 target genes. Together, these findings highlight the potential of targeting non-canonical TGFβ signalling pathways in the setting of vein graft failure.