MSX2-WNT SIGNALING IN CARDIOVASCULAR CALCIFICATION

描述(申请人提供)：动脉硬化是以动脉壁增厚和硬化为特征的慢性疾病状态，失去弹性。动脉粥样硬化、Monckeberg氏内侧钙化硬化和小动脉硬化是动脉硬化的三种组织病理学类型。随着年龄的增长，糖耐量受损，糖尿病和高血压，管道动脉变得越来越动脉硬化，失去顺应性，以顺利的远端组织灌流。血管几何形状的改变，以及纤维化、交联化和矿化引起的血管基质材料性质的变化，都会损害这种温克塞尔生理学。动脉硬化--即内侧钙化性硬化症--已成为导致2型糖尿病(T2 DM)患者截肢风险的一个重要因素。更好地了解控制动脉纤维化、钙化和顺应性的信号通路将导致减轻动脉硬化性疾病负担的新策略。最近的数据表明，Wnt/2-catenin信号和Wnt7/LRP6相互作用在血管钙化和组织纤维化--肿瘤坏死因子、骨形态发生蛋白2和MSX2激活的成骨矿化的下游--中起重要作用。因此，这项提议的具体目的是：目的1：以SM22-CRE(+)、CTNNB1(FLOX/+)、LDLR(-/-)小鼠为研究模型，建立细胞自主的血管平滑肌细胞(VSMC)2-连环蛋白在糖尿病动脉硬化血管钙化和管壁纤维化中的作用。我们测试了VSMC2-catenin信号的遗传下调是否改变了糖尿病动脉硬化的启动和进展，包括饮食诱导的LDLR-/-小鼠LE血流量的减少。目的2：以糖尿病SM22-Cre(+)、LRP6(fl/fl)、LDLR(-/-)小鼠为模型，研究VSMC LRP6的表达在介导MSX2-Wnt信号在心血管钙化中的促钙化作用中的作用。LRP6通过2-连环蛋白和NFATc介导的转录，在培养中介导WNT7依赖的成骨和纤维化信号。为此，我们在体内确定了VSMC LRP6在体内对糖尿病动脉硬化血管钙化启动的细胞自主作用，以及对饮食诱导的主动脉成骨基因程序激活的影响。这些目标的结果将为降低VSMC2-连环蛋白水平和LRP6信号作为一种潜在的治疗策略来改善血管钙化从而降低II型糖尿病患者的下肢截肢风险提供生理学基础和验证。 与公共卫生相关：糖尿病患者的动脉硬化会增加心脏病发作和足部截肢的风险。这在一定程度上是通过代谢和炎症信号发生的，这些信号在大动脉中诱导骨样钙化。我们已经确定了两种蛋白质，LRP6和2-连环蛋白，在糖尿病小鼠的动脉钙化、纤维化和硬化过程中被激活。我们测试这些蛋白质的调节是否减少了动脉硬化，减少了心脏工作负荷，并改善了流向足部的血流量。

DESCRIPTION (provided by applicant): Arteriosclerosis is the chronic disease state characterized by thickening and hardening of arterial walls with loss of elasticity. Atherosclerosis, Monckeberg's medial calcific sclerosis, and arteriolosclerosis are the three histopathologic types of arteriosclerosis. With advancing age, impaired glucose tolerance, diabetes, and hypertension, the conduit arteries become increasingly arteriosclerotic, losing compliance necessary for smooth distal tissue perfusion. This Windkessel physiology is impaired by changes in vascular geometry, and by changes in vascular matrix material properties arising from fibrosis, cross-linking and mineralization. Arteriosclerosis- viz., medial calcific sclerosis - has emerged as a particularly important contributor to lower extremity (LE) amputation risk in type II diabetes (T2DM). A better understanding of signaling pathways that control arterial fibrosis, calcification, and compliance will lead to new strategies for diminishing arteriosclerotic disease burden. Recent data identify Wnt/ 2 -catenin signaling and Wnt7 /LRP6 interactions as important in vascular calcification and tissue fibrosis -- down-stream of TNF-, BMP2-, and Msx2- activated osteogenic mineralization. Thus, the specific aims of this proposal are: Aim 1: "To establish the contributions of cell-autonomous vascular smooth muscle cell (VSMC) 2-catenin actions to vascular calcification and mural fibrosis in diabetic arteriosclerosis, using SM22- Cre(+);Ctnnb1(flox/+);LDLR(-/-) mice as a model for study." We test whether genetic down- regulation of VSMC 2-catenin signaling alters initiation and progression of diabetic arteriosclerosis, including diet-induced reductions in LE blood flow in the LDLR-/- mouse. Aim 2: "To examine the role of VSMC LRP6 expression in mediating the pro-calcific actions of Msx2-Wnt signaling during cardiovascular calcification, using diabetic SM22- Cre(+);LRP6(fl/fl);LDLR(-/-) mice as a model for study." LRP6 mediates Wnt7-dependent osteogenic and fibrotic signals in culture via 2-catenin and NFATc-mediated transcription. In this aim, we determine in vivo the cell-autonomous roles of VSMC LRP6 to the initiation of vascular calcification in diabetic arteriosclerosis in vivo, and the impact upon diet-induced activation of aortic osteogenic gene programs. The outcomes of these aims will provide physiological rationale and validation for reducing VSMC 2-catenin levels and LRP6 signaling as a potential therapeutic strategy to ameliorate vascular calcification -- and thus reduce lower extremity amputation risk in type II diabetes PUBLIC HEALTH RELEVANCE: Arterial hardening in diabetes increases the risk for heart attacks & foot amputation. This occurs in part via metabolic & inflammatory signals that induce bone-like calcification in large arteries. We have identified that two proteins, LRP6 and 2-catenin, are activated with calcification, fibrosis, and hardening of the arteries in diabetic mice. We test whether regulation of these proteins reduces arterial hardening, decreases cardiac workload, and improves blood flow to the foot.