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。它在慢性肾病（CKD）和糖尿病患者以及外周动脉疾病（PAD）患者中非常普遍。当位于动脉中层时，钙化与心血管发病率和死亡率的增加密切相关。钙化是一个受一系列内源性刺激物和抑制物控制的高度调节的过程。磷酸盐水平升高可诱导血管平滑肌细胞（SMC）的成骨转化。这些细胞释放细胞外基质降解酶，包括有助于钙化的基质金属蛋白酶（MMPs）。尽管取得了重大进展，动脉钙化仍然知之甚少，也没有开发出可用的药物来预防或治疗它。出于这个原因，识别新的分子靶点并开发针对它们的治疗方法对于改善我们血管患者的结局至关重要。第二信使环核苷酸cAMP和cGMP在由不同的环核苷酸PDE同工酶控制的多种人类疾病中发挥重要的调节作用，这些环核苷酸PDE同工酶已被证明是用于治疗人类疾病的理想且可行的药物靶标。然而，PDE在动脉钙化中的功能和调节仍然未知。这个建议是基于我们的初步数据显示，磷酸二酯酶10A（PDE 10A）是最高度诱导异构体之间的所有PDE基因在啮齿类动物钙化模型。PDE 10A在体外钙化的VSMC、体内钙化的动脉和来自PAD患者的钙化的人胫动脉中也显著增加。PDE 10A的敲低和抑制显著减弱磷酸盐诱导的VSMC体外成骨转化和钙化，PDE 10A的缺乏减少体内动脉钙化。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）可以调节PDE 10A在钙化培养基中培养的VSMCs。我们进一步的初步机制研究结果表明，PDE 10A的敲低和抑制都能阻断钙化过程中VSMC中p38 MAPK的激活。我们还发现，抑制p38 MAPK减弱了钙化条件下MMP-3的上调。在这个项目中，我们提出PDE 10A通过调节p38 MAPK-MMP-3信号转导介导动脉钙化。在这一系列实验中，我们将确定PDE 10A在动脉钙化中的作用，并深入了解PDE 10A抑制策略在减少CKD和PAD患者钙化中的潜在用途。我们的目的是1）研究PDE 10A在血管SMC成骨转化和动脉钙化中的作用，2）检查PDE 10A抑制在动脉钙化中的治疗潜力，和3）评估p38-MAPK-MMP-3信号传导在PDE 10A介导的血管钙化中的意义。

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.