Role of The Endothelium In Vascular Calcification

描述（由申请方提供）：血管钙化是血管疾病的常见并发症，与发病率和死亡率增加相关。它被认为是一个积极的过程，涉及多能细胞的骨软骨分化。先前的报告表明，内皮细胞（EC）在先天性软组织钙化疾病中提供钙化细胞，由异常骨形态发生蛋白（BMP）信号传导触发。基质Gla蛋白（MGP）是血管钙化的抑制剂，并且是血管钙化的拮抗剂。 BMP 2/4。MGP基因敲除小鼠显示出血管BMP信号的强烈激活和广泛的主动脉钙化。Ins 2-秋田/+小鼠，一种1型糖尿病模型，由于BMP和BMP受体的诱导，表现出类似的发现。初步数据显示，BMP激活导致正常EC中出现干细胞特征。事实上，EC谱系标志物与MGP缺失型动脉瘤中的成骨和多能干细胞标志物共染色，并且体外人主动脉EC中的MGP耗尽导致细胞响应于骨诱导因子而经历成骨分化。此外，Ins 2-秋田/+小鼠显示多种多能性相关标志物的主动脉表达增加。因此，正常的内皮细胞可能有助于骨祖细胞血管钙化时，受到增加的BMP活性。该提案的科学目的是确定内皮细胞在MGP缺乏症和糖尿病血管钙化中的作用。我们假设，正常内皮有助于多能细胞血管钙化，由于异常的BMP活性诱导干细胞特性和随后的骨生成的能力。具体目标1将在体外和体内确定MGP缺乏或高血糖背景下EC的多能性和骨软骨形成潜力。具体目标2将在两种血管钙化模型（MGP-/-小鼠和糖尿病Ins 2Akita/+小鼠）中使用线迹追踪确定EC是否为钙化病变贡献细胞。对于谱系追踪，将使用目前可用的Tie 2-GFP或Tie 2- Cre; R262 R-eGFP转基因小鼠。具体目标3将使用EC和SMC特异性MGP敲除小鼠确定EC和SMC特异性MGP损失在血管钙化发展中的相对贡献。如果成功的话，内皮可能成为治疗获得性血管疾病钙化的重要靶点。

DESCRIPTION (provided by applicant): Vascular calcification is a frequent complication of vascular disease associated with increased morbidity and mortality. It is recognized to be an active process involving osteochondrogenic differentiation in multipotent cells. Previous reports have suggested that endothelial cells (ECs) contribute calcifying cells in congenital disorders with soft tissue calcification, triggered by abnormal bone morphogenetic protein (BMP) signaling. Matrix Gla protein (MGP) is an inhibitor of vascular calcification, and an antagonist to BMP2/4. MGP null mice show strong activation of vascular BMP signaling and extensive aortic calcification. Ins2-Akita/+ mice, a model of type 1 diabetes, exhibit similar findings due to induction of BMPs and BMP receptors. Preliminary data show that BMP activation causes stem cell characteristics to emerge in normal ECs. Indeed, EC lineage markers co- stain with osteogenic and pluripotent stem cell markers in MGP null aortas, and MGP-depletion in human aortic ECs in vitro causes the cells to undergo osteogenic differentiation in response to osteoinductive factors. In addition, Ins2-Akita/+ mice exhibit increased aortic expression of multiple pluripotency-associated markers. Thus, normal ECs may contribute osteoprogenitor cells to vascular calcification when subjected to increased BMP activity. The scientific purpose of this proposal is to define the role of the endothelium in vascular calcification in MGP deficiency and diabetes. We hypothesize that normal endothelium contributes multipotent cells to vascular calcification due to the capacity of abnormal BMP activity to induce stem cell characteristics and subsequent osteogenesis. Specific Aim 1 will define multipotency and osteochondrogenic potential in ECs in the setting of MGP deficiency or hyperglycemia in vitro and in vivo. Specific Aim 2 will determine if ECs contribute cells to calcific lesions using lineag tracing in two models of vascular calcification, the MGP-/- mouse and the diabetic Ins2Akita/+ mouse. For lineage tracing, the currently available Tie2-GFP or Tie2- Cre;R262R-eGFP transgenic mice will be used. Specific Aim 3 will determine the relative contributions of EC- and SMC-specific loss of MGP in the development of vascular calcification using EC- and SMC-specific MGP knockout mice. If successful, the endothelium may emerge an important target to treat calcification in acquired vascular disease.