MSX2-Wnt Signaling in Cardiovascular Calcification

描述（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、瓣膜力学异常和炎症、慢性肾功能不全的常见后果。钙化性血管病变曾经被认为是良性的，但其有害的临床后果现在变得越来越清楚；它是导致CVA、Ml和PVD的病理生理学的一个关键组成部分——通过钙化血管病变的解剖和程度预示着截肢和心血管死亡率。治愈或完全逆转大血管钙化（MVC）的能力是尚未满足的临床需求。需要更好地理解MVC的开始和进展。成骨和炎症基因调控程序在MVC中被激活，通过与控制骨生理的机制重叠，涉及不同的外膜、内侧、内膜和瓣膜组织。我们已经证明了一个促骨程序——一个“前馈”BMP2-Msx2-Wnt信号级联——在糖尿病和血脂异常的主动脉肌成纤维细胞中被激活。相比之下，PTH/PTHrP受体激动剂（如特立帕肽）抑制血管肌成纤维细胞钙化和主动脉Msx2-Wnt基因表达。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Aim 1中，我们使用LDLR-/-。TOPGAL+报告小鼠（在Wnt响应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表达和来自外膜、内侧和瓣膜肌成纤维细胞的骨祖细胞募集联系起来。我们还测试了在CMV-Msx2转基因小鼠中观察到的主动脉钙化和Wnt表达增强是否激活了主动脉LacZ表达。原发性主动脉肌成纤维细胞的共培养研究将测试Msx2激活的旁分泌Writ信号是否被PTH抑制（1-34）。在Aim 2中，我们使用TOPGAL小鼠，在体内测试PTH（1-34）是否抑制典型Wnt信号。在Aim 3中，我们绘制了Msx2启动子蛋白- dna相互作用，将转录抑制传递给肌成纤维细胞中的PTH(1-34)，强调了介导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.