The Mechanism of Uremia Induced Vascular Calcification

描述（由申请人提供）： CKD患者的血管钙化增加，其发病机制与正常骨生成相似。我们以前已经证明，尿毒症是一个主要的危险因素，体外研究表明，尿毒症对血管平滑肌细胞（VSMC）去分化的影响是加性的磷。一旦VSMC变成成骨细胞样，它们分泌与细胞外基质（ECM）相互作用的基质囊泡（MV）。在我们目前的VA Merit审查中，我们已经表征了MV的含量，并证明增加膜联蛋白-II和减少胎球蛋白-A含量导致ECM矿化能力增加。此外，这些MV的形成需要钙的流入（细胞外到细胞内），并依赖于参与细胞骨架结构和组织的信号转导途径。转氨酶2（TG 2）是钙依赖性酶，其是细胞膜上与β 1整联蛋白和纤连蛋白的共受体，因此参与细胞骨架和ECM之间的通讯，可以交联ECM蛋白，并且可以改变细胞内钙。我们的初步数据表明，与正常大鼠相比，在CKD的VSMC中TG 2活性增加，在人和大鼠动脉的钙化区域中高度表达，并且在MV中表达。我们的假设是：CKD中转氨酶（TG 2）活性增加通过诱导促进血管平滑肌细胞-细胞外基质相互作用的促钙化环境加速血管钙化。在目的1中，我们将检验以下假设：CKD中TG 2活性增加诱导血管平滑肌细胞去分化，伴随钙化基质囊泡（MV）形成和细胞外基质交联。我们将确定与正常动物相比，阻断CKD的VSMC中TG 2活性对a）细胞分化成成骨细胞样表型，B）ECM蛋白合成和ECM的TG交联，c）细胞钙化和d）MV钙化潜力和e）VSMC/MV与ECM之间的相互作用的影响。在目标2中，我们将测试CKD中细胞外钙增加激活膜和细胞溶质TG 2以通过将MV的组成改变为更促钙化的含量来增强VSMC/MV与ECM的相互作用的假设。最后，我们将确定在CKD中抑制增加的TG 2活性是否可以预防体内动脉钙化。我们将确定口服胱胺（一种广泛的TG抑制剂）是否可以抑制自然发生的CKD-MBD（慢性肾脏疾病-矿物质骨病）大鼠模型中的主动脉血管钙化。在最初的短期剂量探索研究之后，我们将用和不用胱胺治疗CKD大鼠20至34周，并通过测定胸主动脉的钙化和TG 2活性来评估疗效，通过microCT测定骨钙含量和小梁交联来评估安全性。这些研究将提供所需的临床前体外和体内研究，以确定给予胱胺（其衍生物已被批准用于人类的其他用途）是否可以通过抑制转氨酶活性来阻止CKD中血管钙化的进展。

DESCRIPTION (provided by applicant): Patients with CKD have increased vascular calcification and the pathogenesis parallels normal bone osteogenesis. We have previously demonstrated that uremia is a major risk factor and in vitro studies show that the effect of uremia on vascular smooth muscle cells (VSMC) de-differentiation is additive to that of phosphorus. Once the VSMC become osteoblast like, they secrete matrix vesicles (MV) that interact with the extracellular matrix (ECM). During our current VA Merit review, we have characterized the content of the MV and demonstrated that increased annexin-II and decreased fetuin-A content leads to increased ability to mineralize ECM. Furthermore, the formation of these MV requires an influx of calcium (extracellular to intracellular) and was dependent on signaling pathways involved in cytoskeleton structure and organization. Transglutaminase 2 (TG2) is a calcium dependent enzyme that is a co-receptor with ss1 integrins and fibronectin on the cell membrane, and therefore involved in the communication between the cytoskeleton and the ECM, can cross link ECM proteins, and can alter intracellular calcium. Our preliminary data demonstrates that TG2 activity is increased in VSMC from CKD compared to Normal rats, is highly expressed in calcifying areas of both human and rat arteries, and is expressed in MV. Our Hypothesis is: Increased transglutaminase (TG2) activity in CKD accelerates vascular calcification by inducing a pro-calcifying environment facilitating vascular smooth muscle cell-extracellular matrix interaction. In Aim 1, we will test the hypothesis that increased TG2 activity in CKD induces vascular smooth muscle cell de-differentiation with calcifying matrix vesicle (MV) formation and extracellular matrix cross-linking. We will determine the effect of blockade of TG2 activity in VSMC from CKD, compared to Normal animals on a) cellular differentiation to an osteoblast like phenotype, b) ECM protein synthesis and TG cross-linking of the ECM, c) cellular calcification and d) MV calcification potential and e) the interaction between VSMC/MV and the ECM. In Aim 2 we will test the hypothesis that increased extracellular calcium in CKD activates both membrane and cytosolic TG2 to enhance the interaction of VSMC/MV with the ECM by altering the composition of MV to a more pro-calcifying content. Finally, we will determine if inhibition of the increased TG2 activity in CKD can prevent arterial calcification in vivo. We will determine if oral administration of cystamine, a broad TG inhibitor, can inhibit aorta vascular calcification in a naturally occurring rat model of CKD-MBD (Chronic Kidney Disease-Mineral Bone Disorder). After initial short term dose finding studies, we will treat CKD rats with and without cystamine from 20 to 34 weeks and assess efficacy by determining calcification and TG2 activity of the thoracic aorta, and safety by determining bone calcium content and trabecular cross-linking by microCT. These studies will provide the needed pre-clinical in vitro and in vivo studies to determine if the administration of cystamine, a derivative of which is approved for other uses in humans, may halt progression of vascular calcification in CKD through its inhibition of transglutaminase activity.