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

心血管疾病（CVD）是全球发病率和死亡率的主要原因。维持稳定的细胞中层对于预防致命的血管疾病至关重要;然而，内皮损伤触发细胞因子和生长因子如PDGFB和TGFb的释放，这导致平滑肌细胞（VSMC）从静止的收缩状态到活性的合成类型的表型转换，胸腺素b4（Tb 4）是卵黄囊、冠状动脉和全身血管中VSMC分化的关键调节因子。Tb 4是一种广泛表达的小肌动蛋白单体结合蛋白。相互作用筛选确定了相关的Tb 4蛋白结合伴侣，其中，低密度脂蛋白受体相关蛋白1（LRP 1）和Basigin优先进行进一步研究。GWAS研究表明，LRP 1变异与动脉瘤的风险有关，在动物研究中，LRP 1的缺乏易患腹主动脉瘤和动脉粥样硬化。研究表明，Tb 4在预防血管疾病中起重要作用，在胚胎中，Tb 4主要以旁分泌方式发挥作用，由内皮细胞分泌，诱导周围中胚层分化为平滑肌。因此，将进行研究以区分细胞自主与旁分泌在血管保护中的作用。EC或VSMC特异性Tb 4敲低小鼠将通过分别将Tb 4shRNA系与PdgfbCreERT 2（EC）或Myh 11 CreERT 2（VSMC）系杂交产生，并用4-羟基-他莫昔芬诱导。透射电子显微镜（TEM）将用于了解VSMC的形态学变化。将通过单细胞RNA测序和QPCR在mRNA水平上测量收缩和合成标志物水平的变化，并通过免疫组织化学在蛋白质水平上测量。VSMC产生的两种主要ECM成分弹性蛋白和胶原蛋白的变化将进行化学分析。将通过比较PDGF-B和TGF β 62信号传导通路的读数（通过qRT-PCR检测切片血管中LRP-1、PDGFR β 62和ERK的磷酸化以及下游基因派-1和c-myc的表达）来寻找与LRP 1通路相互作用的证据。将使用EdU.Exploring Basigin作为Tb 4在血管保护中的假定靶点：跨膜糖蛋白Basigin在动脉粥样硬化中上调，有效地诱导细胞外基质金属蛋白酶（MMP），并且是白细胞介素（IL）-18诱导的VSMC迁移所需的。我们假设Tb 4维持Basigin的基线抑制，以通过限制IL-18和MMP的诱导来维持血管稳定性。Tb 4-Basigin控制IL-18表达和MMP活化的机制将在VSMC和Tb 4KO小鼠中使用功能丧失/获得方法和来自RNAseq的见解进行探索。在AAA和动脉粥样硬化小鼠模型中，使用渗透性微型泵将外源性TFO 624或溶媒输注到小鼠体内，以评价Tb 4保护动脉的潜力该项目将提供MRC几个战略技能优先领域的培训：整个器官（体内）：拟议的研究使用成熟的主动脉疾病小鼠模型。学生将接受培训，以进行渗透微型泵的手术植入，持续释放血管紧张素II;动脉粥样硬化（高胆固醇血症）模型也将被使用。将通过定期采血和主动脉超声成像监测疾病进展。定量技能：将进行转录组学分析，以深入了解TB 4KO小鼠睾丸中被破坏的过程。将提供生物信息学培训。

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.