Cyclic nucleotide phosphodiesterase regulation in vascular calcification

项目总结 动脉钙化是由于钙化的羟基磷灰石晶体沉积在血管壁。它在慢性肾脏疾病(CKD)和糖尿病患者以及外周动脉疾病(PAD)患者中高度流行。当钙化位于动脉中层时，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和抑制因子控制的高度调控的过程。磷酸盐水平升高可诱导血管平滑肌细胞(SMCs)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，但对动脉钙化的了解仍然很少，也没有开发出可用于预防或治疗它的药物。因此，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的血管患者的预后至关重要。第二信使环核苷酸cAMP和cGMP在多种人类疾病中起着重要的调节作用，这些疾病由不同的环核苷酸PDE同工酶控制，已被证明是治疗人类疾病的理想和可行的药物靶点。然而，PDE在动脉钙化中的作用和调控仍不清楚。这一建议是基于我们的初步数据显示，在啮齿动物钙化模型中，磷酸二酯酶10A(PDE10A)是所有PDE基因中诱导程度最高的异构体。PDE10A在体外钙化的VSMCs、体内钙化的动脉和来自PAD患者的钙化的人胫骨动脉中也显著增加。在体外，PDE10A的基因敲除和抑制可显著减轻磷酸盐诱导的VSMC成骨转化和钙化，在体内，PDE10A的缺失可减少动脉钙化。利用生物信息学分析和功能丧失策略，我们已经证明，在钙化介质中培养的VSMC中，基质金属蛋白酶家族成员3(基质分解素-1)可以被PDE10A调节。我们进一步的机制结果表明，在钙化过程中，PDE10A的敲除和抑制都阻断了VSMC中p38MAPK的激活。此外，我们还发现，在钙化条件下，抑制p38MAPK可以减弱基质金属蛋白酶-3的上调。在本项目中，我们认为PDE10A通过调节p38MAPK-MMP3信号来介导动脉钙化。在这一系列实验中，我们将确定PDE10A在动脉钙化中的作用，并为PDE10A抑制策略在CKD和PAD患者中减少钙化的潜在应用提供见解。我们的目的是1)研究PDE10A在血管SMC成骨转化和动脉钙化中的作用，2)检测PDE10A抑制动脉钙化的治疗潜力，3)评估p38-MAPK-MMP-3信号在PDE10A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.