THYMOSIN (beta)4 - A NOVEL REGULATOR OF LOW DENSITY LIPOPROTEIN RECEPTOR RELATED PROTEIN 1 (LRP1) IN VASCULAR DISEASE

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Cardiovascular diseases (CVD) are the leading cause of morbidity and mortality worldwide. Maintaining a stable medial layer of cells is essential to prevent fatal vascular diseases; however, endothelial damage triggers release of cytokines and growth factors, such as PDGFB and TGFb, that causes the phenotypic switching of smooth muscle cells (VSMC) from a quiescent, contractile state to an active, synthetic type, which display enhanced proliferation and migration.DThymosinb4 (Tb4) was identified as a key regulator of VSMC differentiation in the developing yolk sac, the coronary and the systemic vasculature. Tb4 is a ubiquitously expressed, small actin monomer binding protein. An interaction screen identified relevant Tb4 protein binding partners, of which, Low density lipoprotein receptor-related protein 1 (LRP1) and Basigin were prioritised for further investigation. GWAS studies have revealed that LRP1 variants associate with risk of aneurysm and, in animal studies, absence of LRP1 predisposes to abdominal aortic aneurysm and atherosclerosis. Studies suggest that Tb4 plays an important role to protect against vascular disease.In the embryo, Tb4 acts primarily in a paracrine manner, secreted from endothelial cells to induce differentiation of the surrounding mesoderm into smooth muscle. Therefore, studies will be performed to distinguish a cell-autonomous versus paracrine role in vascular protection. EC- or VSMC-specific Tb4 knockdown mice will be generated by crossing the Tb4shRNA line with PdgfbCreERT2 (EC) or Myh11CreERT2 (VSMC) lines, respectively, and induced with 4-hydroxy-tamoxifen. Transmission electron microscopy (TEM) will be used to understand morphological changes in VSMC. Changes in the levels of contractile and synthetic markers at the mRNA level will be measured by single-cell RNA sequencing and QPCR and, at the protein level, by immunohistochemistry. Changes in the two major ECM components produced by VSMC, elastin and collagen, will be analyzed immunohistochemically. Evidence of interaction with the LRP1 pathway will be sought by comparing readouts PDGF-B and TGFfo62 signalling pathways (phosphorylation of LRP-1, PDGFRfo62, and ERK in sectioned vessels and expression of downstream genes, Pai-1 and c-myc, by qRT-PCR). Medial VSMCs will be examined to determine hyperplasia/proliferation using EdU.Exploring Basigin as a putative target of Tb4 in vascular protection: The transmembrane glycoprotein Basigin is upregulated in atherosclerosis, potently induces extracellular matrix metalloproteinases (MMPs), and is required for interleukin (IL)-18-induced VSMC migration. We hypothesise that Tb4 sustains baseline suppression of Basigin, to maintain vascular stability by limiting induction of IL-18 and MMPs. The mechanisms by which Tb4-Basigin control IL-18 expression and MMP activation will be explored in VSMCs and in Tb4KO mice, using loss-/gain- of function approaches and insight from RNAseq.Exploring the therapeutic potential of Tb4 in vascular protection: Exogenous TFO624 or vehicle will be infused into mice using osmotic mini pump in AAA and atherosclerotic mouse models to evaluate the potential of Tb4 to protect arteries against disease and, in atherosclerosis, to assess potential for disease regression after initiation of plaque formation.The project will provide training in several MRC strategic skill priority areas: Whole organ (in vivo): The proposed research uses well-established murine models of aortic disease. The student will be trained to perform surgical implantation of osmotic mini pumps for sustained release of Angiotensin II; an atherosclerosis (hypercholesterolaemia) model will also be used. Monitoring of disease progression will be performed by regular blood sampling and ultrasound imaging of the aorta. Quantitative skills: A transcriptomic analysis will be performed to gain insight into the processes disrupted in aortas of TB4KO mice. Bioinformatics training will be provided.