title: Exploring TGF-β superfamily-dependent regulation of smooth muscle cell phenotypes in the context of saphenous vein graft disease
Creators: name: Schwartze, Julian Tristan
Publisher: name: University of Glasgow
Year: 2021

description: Coronary artery bypass grafting (CABG) is a surgical technique utilised to bypass one or more stenosed coronary arteries to re-establish blood supply to cardiac tissue. The great saphenous vein of the leg remains the most used conduit for all non-left anterior descending (LAD) territories. Despite advances in surgical technique and optimised pharmacological treatment, approximately 50% of all saphenous vein grafts (SVGs) fail at 10 years following coronary implantation often requiring potentially life-threatening and costly re-interventions. Pathogenic drivers of SVG disease include early thrombosis, neointima formation (NF) and accelerated atherosclerosis. Together, these pathologies culminate in SVG obstruction, thromboembolism, aneurysm formation and haemorrhaging. The key driver of SVG disease is occluding NF which is defined as a continuous thickening of the inner most layer of the venous conduit which culminates in lumenal obstruction. Pre-clinical and clinical studies have demonstrated that dedifferentiated smooth muscle cells (SMCs) are the main contributor to NF. This process is termed SMC phenotype switching which entails a loss in contractile properties and instead a gain in migratory and proliferative capacity. Targeting SMC phenotype switch-driven NF with specific pharmacological compounds and gene therapy remain important therapeutic strategies to prolong SVG patency following CABG surgery. Transforming growth factor (TGF)-b1 and bone morphogenetic protein (BMP)-9 are both pleiotropic growth factors which are members of TGF-b superfamily. Whereas TGF-b1 may signal via the activin receptor-like kinase (ALK)5 or ALK1 receptors, BMP-9 mainly signals via the ALK1 receptor. Studies in small and large animal models have shown that TGF-b1 is up-regulated following vein grafting. In contrast, BMP-9 has not yet been investigated in the context of vascular injurydriven NF. To date, the role of TGF-b1 signalling in NF remains controversial. Our group has shown that TGF-b1/ALK1 signalling drives SMC migration and that NF in ligation-injured carotid arteries is blunted in heterozygous Smad1 knockout mice. Since BMP-9 signals via the ALK1 receptor, it may be speculated that this ligand acts as a pathogenic mediator of NF. Hence, the main aims of this PhD were to (i) determine the presence of BMP-9 and SMC phenotype switching in pre-implantation SVGs and murine vascular injury models, (ii) to evaluate ALK1 and ALK5-driven regulation of primary human saphenous vein (HSV) SMC phenotypes, (iii) to characterise single HSVSMC transcriptomes following BMP-9 and/or TGF-b1 treatment and to (iv) develop a replication-deficient human adenovirus serotype 5 vector (HAdV-5) expressing the activin/BMP receptor activin A receptor type IIa (ACVR2A). Immunohistochemistry (IHC) demonstrated that BMP-9 was present in human pre-implantation SVGs and murine carotid arteries. Moreover, intimal SVG SMCs displayed a decrease in myosin heavy chain (MYH)11 and a trend towards a decrease in calponin expression compared to medial SMCs, suggesting in vivo SMC phenotype switching. In addition, carotid artery wire injury in mice triggered a trend towards an increase in total vascular BMP-9 levels, which was paralleled by SMC phenotype switching, indicating a potential role for BMP-9 in NF. Together, these results prompted an observational multiple timepoint carotid artery ligation study in mice which aimed to link BMP-9 presence with vascular injury responses. Carotid ligation triggered intimal and medial SMC proliferation which was paralleled by a loss of SMC a-smooth muscle actin (SMA) indicating SMC phenotype switching. Due to the COVID-19 pandemic and subsequent lab closure, BMP-9 staining could not be completed. A crosslinking study revealed that recombinant human (rh) BMP-9 bound to ALK1, ALK2, bone morphogenetic protein receptor (BMPR)2, ACVR2A/B and endoglin on primary HSVSMCs. Functionally, BMP-9-treated HSVSMCs displayed a time-dependent increase in s-mothers against decapentaplegic (SMAD)1 phosphorylation paralleled by an increase in ID1 (encodes inhibitor of differentiation-1) mRNA expression levels indicating that BMP-9 activates the SMAD1 pathway in this cell type. The observation of in vivo SMC phenotype switching in pre-implantation SVGs prompted development and generation of a ligand-independent contractile differentiation protocol for primary HSVSMCs and primary human coronary artery (HCA) SMCs. This enabled investigation of functional ALK1 and ALK5 signalling during contractile SMC differentiation. In parallel, the direct, ligand-mediated effect of TGF-b1 and BMP-9 on primary HSVSMCs was studied. Immunoblot, quantitative real-time polymerase chain reaction (qRT-PCR) and BrdU proliferation assays demonstrated that smooth muscle differentiation supplement (SMDS)-treated HSVSMCs displayed an accumulation of contractile SMC markers which was paralleled by a decrease in proliferation. These observations indicated the presence of a contractile HSVSMC phenotype. ALK1 target gene ID1 mRNA expression levels were suppressed in SMDS-treated HSVSMCs indicating dampened ALK1 pathway activity during contractile differentiation. Moreover, SMDS treatment induced the expression of mRNA for the activin/BMP receptor ACVR2A in HSVSMCs suggesting a potential role for ACVR2A during contractile differentiation. Similar observations were made in HCASMCs. This prompted development and generation of a replication-deficient HAdV-5 expressing the ACVR2A transgene. HAdV-5-mediated delivery of ACVR2A to primary HSVSMCs was successful. However, increased ACVR2A expression had no effect on aSMA mRNA levels in 15% fetal calf serum (FCS)- or SMDS-treated HSVSMCs. Pharmacological ALK5 inhibition prevented contractile differentiation in SMDS-treated HSVSMCs. In contrast, pharmacological ALK1 inhibition had no effect on the induction of contractile gene expression. In line with these findings, TGF-b1 drove contractile gene expression in an ALK5-dependent manner and suppressed serum-induced HSVSMC proliferation and migration. BMP-9 had no effect on TGF-b1/ALK5-driven contractile gene expression. These findings led to an investigation into the impact of BMP-9 and/or TGF-b1 stimulation on angiotensin (Ang)II-dependent intracellular Ca2+ mobilisation, which served as an upstream surrogate marker of SMC contraction. Following development of a robust fluorescence-based Ca2+ handling protocol, it was observed that TGF-b1-treated HSVSMCs displayed an increase in AngII-driven intracellular Ca2+ mobilisation in an ALK5-dependent manner, indicating an increase in contractile potential. In contrast, BMP-9 blunted this effect indicating ALK5 antagonism and/or differential utilisation of intracellular Ca2+. Opposed to findings in primary HSVSMCs, TGF-b1 had no effect on AngIIdependent Ca2+ release in primary HCASMCs indicating differential regulation of Ca2+ handling in this cell type. Single cell (sc) region of interest (ROI) analysis revealed heterogeneous Ca2+ transient responses in HSVSMC populations independent of ligand treatment. However, TGF-b1-treated HSVSMC populations demonstrated a trend towards a reduction in non-responding cells and instead an increase in cells responding with higher fluorescence intensities indicating a more homogeneous intracellular Ca2+ release response. TGF-b1-induced homogeneity was reversed in the presence of BMP-9 paralleling whole ROI findings. Since primary HSVSMCs displayed heterogeneous Ca2+ transient responses, influenced by TGF-b1 and BMP-9, the next aim was to evaluate potential transcriptome heterogeneity in BMP-9 and/or TGF-b1-treated HSVSMCs using the 10x Chromium microfluidics-based scRNA sequencing (seq) platform. Combined uniform manifold approximation and projection (UMAP)/Louvain clustering uncovered transcriptional heterogeneity in untreated and ligand treated HSVSMCs potentially reflecting a heterogeneous composition of SMC sub-lineages within pre-implantation SVG media. Both BMP-9 and TGF-b1 drove target gene expression demonstrating functional ALK5 and ALK1/ALK2 pathway activation. Principal component analysis (PCA) showed greater spatial distances between TGF-b1 and BMP-9-treated HSVSMCs suggesting that TGF-b1 triggered greater transcriptional activation compared to BMP-9. Within BMP-9 treated HSVSMCs, combined RNA velocity and gene ontology (GO) analysis revealed a potential osteogenic HSVSMC sub-lineage. Finally, GO identified potentially contractile and pro-fibrotic sub-lineages within TGF-b1-treated HSVSMCs. In conclusion, the TGF-b1/ALK5 pathway positively regulates the contractile HSVSMC phenotype. In contrast, BMP-9 drives ID1 expression and blunts TGFb1/ALK5-driven AngII-dependent Ca2+ responses via ALK1 and/or ALK2 indicating partial ALK5 antagonism. Together, these data suggest that ALK5 agonism/ALK1 antagonism may protect HSVSMCs from phenotype switching and dampen NF in the context of SVG disease.