MSX2-Wnt Signaling in Cardiovascular Calcification

说明(申请人提供)：心血管钙化是衰老、糖尿病、高胆固醇血症、高血压、瓣膜机械和炎症异常以及慢性肾功能不全的常见后果。曾经被认为是良性的，钙化性血管病变的有害临床后果现在变得清晰起来；它是导致CVA、ML和PVD的病理生理学的关键组成部分--从钙性血管病变的解剖和范围来看，截肢和心血管死亡是先兆。治愈或基本上逆转大血管钙化(MVC)的能力代表着一种尚未满足的临床需求。需要对MVC的启动和发展有更好的理解。成骨和炎症基因调节程序在MVC中被激活--可变地涉及外膜、内膜、内膜和瓣膜组织--通过与控制骨生理的机制重叠的机制。我们已经证明，在糖尿病和血脂异常的情况下，在主动脉肌成纤维细胞中，一个促成骨程序--BMP2-MSX2-Wnt信号级联--被激活。相反，甲状旁腺素/甲状旁腺素受体激动剂(如特立帕帝)抑制血管成纤维细胞钙化和主动脉MSX2-Wnt基因的表达。我们现在研究肌成纤维细胞MSX2-Wnt信号在MVC中的作用和调控。在目标1中，我们使用LDLR-/-.TOPGAL+报告小鼠(Wnt反应TCF/Lef元件控制下的LacZ转基因)，将主动脉Wnt信号的空间激活与饮食诱导的MSX2-Wnt表达和外膜、内侧和瓣膜肌成纤维细胞的成骨细胞募集联系起来。我们还测试了我们在CMV-MSX2转基因小鼠中观察到的增强的主动脉钙化和Wnt表达是否激活了主动脉LacZ的表达。原代主动脉肌成纤维细胞的共培养研究将测试甲状旁腺素(1-34)是否抑制MSX2激活的旁分泌写入信号。在目标2中，我们利用TOPGAL小鼠，在体内测试PTH(1-34)是否抑制典型的Wnt信号。在目标3中，我们将MSX2启动子的蛋白质-DNA相互作用映射到肌成纤维细胞中的PTH(1-34)，强调介导BMP2和Wnt激活的元件。

DESCRIPTION (provided by applicant): Cardiovascular calcification is a common consequence of aging, diabetes, hypercholesterolemia, hypertension, abnormal valve mechanics & inflammation, & chronic renal insufficiency. Once thought benign, the deleterious clinical consequences of calcific vasculopathy are now becoming clear; it is a key component of pathophysiology leading to CVA, Ml, and PVD -- with amputation and cardiovascular mortality portended by the anatomy and extent of calcific vasculopathy. The ability to cure or substantially reverse macrovascular calcification (MVC) represents an unmet clinical need. A better understanding of MVC initiation and progression is required. Osteogenic & inflammatory gene regulatory programs are activated in MVC-- variably involving adventitial, medial, intimal, and valvular tissues -- via mechanisms overlapping those that control bone physiology. We've shown that a pro-osteogenic program -- a "feed-forward" BMP2-Msx2-Wnt signaling cascade -- is activated in aortic myofibroblasts by diabetes and dyslipidemia. By contrast, PTH/PTHrP receptor agonists (e.g., teriparatide) suppress vascular myofibroblast calcification and aortic Msx2-Wnt gene expression. We now study the role and regulation of myofibroblast Msx2-Wnt signaling in MVC. In Aim 1, we use LDLR-/-.TOPGAL+ reporter mice (LacZ transgene under control of Wnt-responsive TCF/LEF element) to relate spatial activation of aortic Wnt signaling to diet-induced Msx2-Wnt expression and recruitment of osteoprogenitors from adventitial, medial, & valvular myofibroblasts. We also test if the enhanced aortic calcification and Wnt expression we observe in CMV-Msx2 transgenic mice activates aortic LacZ expression. Co-culture studies of primary aortic myofibroblasts will test if Msx2--activated paracrine Writ signaling is inhibited by PTH (1-34). In Aim 2, using TOPGAL mice, we test if PTH(1-34) inhibits canonical Wnt signaling in vivo. In Aim 3, we map Msx2 promoter protein-DNA interactions that convey transcriptional suppression to PTH(1-34) in myofibroblasts, emphasizing elements that mediate BMP2 and Wnt activation.