Role of The Endothelium In Vascular Calcification

描述(申请人提供)：血管钙化是血管疾病的一种常见并发症，与发病率和死亡率增加有关。它被认为是一个活跃的过程，涉及多潜能细胞的成骨分化。以往的报道表明，在由骨形态发生蛋白(BMP)信号异常触发的先天性软组织钙化疾病中，内皮细胞(ECs)参与了钙化细胞的形成。基质GLA蛋白(MGP)是血管钙化的抑制因子，也是血管钙化的拮抗剂。 BMP2/4。MGP基因缺失的小鼠表现出血管BMP信号的强烈激活和广泛的主动脉钙化。在1型糖尿病模型2-Akita/+小鼠中，由于BMP和BMP受体的诱导，也表现出类似的结果。初步数据显示，BMP激活导致正常内皮细胞出现干细胞特征。事实上，在MGP缺失的主动脉中，EC谱系标记与成骨和多能干细胞标记共染色，而体外培养的人主动脉内皮细胞中MGP缺失会导致细胞在成骨诱导因子的作用下进行成骨分化。此外，Ins2-Akita/+小鼠表现出多个多潜能相关标志物的主动脉表达增加。因此，当BMP活性增加时，正常内皮细胞可能有助于骨祖细胞促进血管钙化。这项建议的科学目的是确定内皮细胞在MGP缺乏症和糖尿病患者血管钙化中的作用。我们假设，由于异常的BMP活性诱导干细胞特征和随后的成骨能力，正常的内皮细胞在血管钙化中贡献了多潜能细胞。具体目标1将确定在体外和体内MGP缺乏或高血糖的情况下内皮细胞的多能性和成骨软骨潜能。具体目标2将在两种血管钙化模型-MGP-/-小鼠和糖尿病Ins2Akita/+小鼠中使用Lineag示踪法确定ECs是否对钙化病变起作用。对于谱系追踪，将使用目前可用的Tie2-GFP或Tie2-Cre；R262R-EGFP转基因小鼠。具体目标3将确定EC和SMC特异性MGP缺失在EC和SMC特异性MGP基因敲除小鼠血管钙化发展中的相对贡献。如果成功，内皮细胞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.