Cyclic nucleotide phosphodiesterase regulation in vascular calcification

项目摘要 动脉钙化是由于羟磷灰石钙晶体在血管壁中的沉积而引起的。极有 在慢性肾病（CKD）和糖尿病患者以及外周动脉疾病患者中普遍存在 疾病（PAD）。当位于动脉中膜时，钙化与增加的 心血管疾病发病率和死亡率。钙化是一个高度调节的过程，由一系列 内源性刺激剂和抑制剂。升高的磷酸盐水平可以诱导成骨转化， 血管平滑肌细胞（SMC）。这些细胞释放细胞外基质降解酶，包括 导致钙化的基质金属蛋白酶（MMPs）。尽管取得了重大进展， 对钙化仍然知之甚少，并且还没有开发出预防或治疗钙化的可用药物。 出于这个原因，识别新的分子靶点并开发靶向它们的疗法的过程是非常重要的。 对于改善血管患者的预后至关重要。第二信使环核苷酸cAMP和 cGMP在多种由不同的环磷酸腺苷调控的人类疾病中发挥重要的调节作用， 核苷酸PDE同工酶已被证明是治疗人类疾病的理想和可行的药物靶标， 疾病然而，PDE在动脉钙化中的功能和调节仍然未知。这 该建议是基于我们的初步数据，表明磷酸二酯酶10A（PDE 10A）是最高的 在啮齿类动物钙化模型中所有PDE基因中诱导同种型。PDE 10A也显著增加， 体外钙化的VSMC、体内钙化的动脉和来自PAD患者的钙化的人胫动脉。 PDE 10A的敲除和抑制显著减弱磷酸盐诱导的VSMC成骨细胞增殖， PDE 10A的缺乏在体外减少了动脉的转化和钙化，而PDE 10A的缺乏在体内减少了动脉的钙化。使用 生物信息学分析和功能丧失策略，我们已经表明，MMP家族成员3（MMP- 3，基质溶解素-1）可以在钙化培养基中培养的VSMCs中被PDE 10A调节。我们进一步 初步的机制结果表明，PDE 10A的敲低和抑制都阻断了p38 MAPK 在钙化过程中激活VSMC。我们还发现，抑制p38 MAPK可减弱 钙化条件下MMP-3的上调。在这个项目中，我们提出PDE 10A介导动脉 通过调节p38 MAPK-MMP-3信号通路来钙化。在这一系列的实验中，我们将建立角色 PDE 10A在动脉钙化中的作用，并为PDE 10A抑制策略的潜在用途提供了见解， 减少CKD和PAD患者的钙化。我们的目的是1）研究PDE 10A在 血管SMC成骨转化和动脉钙化，2）检查 PDE 10A抑制动脉钙化，和3）评估p38-MAPK-MMP-3信号传导在动脉钙化中的意义。 PDE 10A介导的血管钙化。

PROJECT SUMMARY Arterial calcification results from the deposition of calcium hydroxyapatite crystals in the vessel wall. It is highly prevalent in patients with chronic kidney disease (CKD) and diabetes as well as those with peripheral artery disease (PAD). When located in the arterial media, calcification is strongly associated with increased cardiovascular morbidity and mortality. Calcification is a highly regulated process controlled by a series of endogenous stimulators and inhibitors. Elevated phosphate levels can induce osteogenic transformation of vascular smooth muscle cells (SMCs). These cells release extracellular matrix-degrading enzymes including the matrix metalloproteinases (MMPs) that contribute to calcification. Despite significant progress, arterial calcification continues to be poorly understood and no useable drugs to prevent or treat it have been developed. For this reason, the process of identifying novel molecular targets, and developing therapies to target them are critical for improving outcomes in our vascular patients. The second messenger cyclic nucleotides cAMP and cGMP play important regulatory roles in a variety of human diseases that are controlled by distinct cyclic nucleotide PDE isozymes that have proven to be ideal and feasible drug targets for the treatment of human diseases. The function and regulation of PDEs in arterial calcification, however, remains unknown. This proposal is based on our preliminary data showing that phosphodiesterase 1 0A (PDE10A) is the most highly induced isoform among all PDE genes in a rodent calcification model. PDE10A is also markedly increased in calcifying VSMCs in vitro, calcified arteries in vivo, and calcified human tibial arteries from patients with PAD. Knockdown and inhibition of PDE10A significantly attenuate phosphate-induced VSMC osteogenic transformation and calcification in vitro, and deficiency of PDE10A reduces arterial calcification in vivo. Using bioinformatics analyses and a loss-of-function strategy, we have shown that the MMP family member 3 (MMP- 3, stromelysin-1) can be regulated by PDE10A in VSMCs cultured in a calcification medium. Our further preliminary mechanistic results have shown that both knockdown and inhibition of PDE1 0A block p38 MAPK activation in VSMCs during calcification. We have additionally found that inhibition of p38 MAPK attenuates MMP-3 upregulation under calcifying conditions. In this project, we propose that PDE10A mediates arterial calcification by regulating p38 MAPK-MMP-3 signaling. In this series of experiments, we will establish the role of PDE10A in arterial calcification and provide insights into the potential use of PDE10A inhibition strategies to reduce calcification in patients with CKD and PAD. Our aims are to 1) investigate the actions of PDE10A in vascular SMC osteogenic transformation and arterial calcification, 2) examine the therapeutic potential of PDE10A inhibition in arterial calcification, and 3) assess the significance of p38-MAPK-MMP-3 signaling in PDE10A-mediated vascular calcification.