The Mechanism of Uremia Induced Vascular Calcification

描述(由申请人提供)： CKD患者血管钙化增多，其发病机制与正常骨形成相似。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)去分化的影响与磷的作用是相加的。一旦VSMC成为成骨细胞，它们就会分泌与细胞外基质(ECM)相互作用的基质小泡(MV)。在我们目前的VA Merit审查期间，我们已经对MV的含量进行了表征，并证明了增加Annexin-II和减少Fetuin-A含量会增强ECM的矿化能力。此外，这些MV的形成需要钙离子(细胞外到细胞内)的内流，并依赖于参与细胞骨架结构和组织的信号通路。转谷氨酰胺酶2(TG2)是一种钙依赖酶，与细胞膜上的SS1整合素和纤维连接蛋白是共受体，参与细胞骨架和细胞外基质之间的通讯，可以使细胞外基质蛋白发生交联，从而改变细胞内钙离子。我们的初步数据表明，与正常大鼠相比，CKD大鼠VSMC中TG2活性增加，在人和大鼠动脉钙化区域高表达，并在MV中表达。我们的假说是：CKD中谷氨酰胺转氨酶(TG2)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，加速了血管钙化。在目标1中，我们将验证这样一种假设，即CKD中TG2活性增加导致血管平滑肌细胞去分化，形成钙化基质囊泡(MV)和细胞外基质交联。与正常动物相比，我们将确定CKD患者VSMC中TG2活性的阻断在a)细胞分化为成骨细胞样表型，b)ECM蛋白质合成和TG交联，c)细胞钙化和d)MV钙化以及e)VSMC/MV和ECM之间的相互作用方面的影响。在目标2中，我们将检验这一假说，即CKD中细胞外钙的增加激活了膜和胞浆中的TG2，通过改变MV的组成以更有利于钙化的含量来增强VSMC/MV与ECM的相互作用。最后，我们将确定抑制CKD中增加的TG2活性是否可以防止体内的动脉钙化。我们将确定口服半胱胺(一种广泛的甘油三酯抑制剂)是否可以抑制自然发生的CKD-MBD(慢性肾脏疾病-矿物性骨骼疾病)大鼠模型的主动脉血管钙化。在初步的短期剂量发现研究后，我们将在20至34周内治疗CKD大鼠，并通过测定胸主动脉钙化和TG2活性来评估疗效，通过MicroCT测定骨钙含量和骨小梁交联度来评估安全性。这些研究将提供所需的体外和体内临床前研究，以确定半胱胺的应用是否可以通过抑制转谷氨酰胺酶活性来阻止CKD血管钙化的进展。半胱胺的衍生物已被批准用于人类其他用途。

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.