Vascular tone & cAMP phosphodiesterase in angioplasty

描述(由申请人提供)：大鼠主动脉球囊导管脱内皮化(BAL)后，增强的平滑肌细胞(SMC)增殖和迁移是导致内膜增厚的原因。最近的研究表明，通过特异性抑制cAMP磷酸二酯酶PDE3和/或PDE4来激活cAMP依赖的蛋白激酶(PKA)可以减少血管成形术后SMC的增殖/迁移和损伤。除了SMC生长的变化外，损伤还可能影响收缩能力。总体假设：BAL导致SMC和血管壁相关炎症细胞中特定cAMP PDE的表达和活性增加，进而影响SMC的环核苷酸水平、蛋白磷酸化和收缩。该项目将首先确定哪些高亲和力、cAMP选择性的PDE3和PDE4亚型在BAL损伤的大鼠主动脉或生长因子刺激的大鼠主动脉SMC中上调，而随后的目标是评估PDE上调对cAMP依赖的磷酸化、血管收缩和细胞定位的影响。目的：研究BAL后PDE3A/3B和PDE4A/4B/4D基因蛋白表达的时程变化。主动脉中段SMC的先导数据显示，BAL与PDE4B基因的双相增加有关，并且较小(PDE3B)或其他基因(PDE3A、4D)没有变化。目的：研究PDE4B、PDE4D和PDE3A蛋白剪接变异体在血清、PDGF-BB和bFGF刺激的RASMC中的时程变化。目的：确定PDE抑制剂增强SMC中PKA活性或依赖于PKA的血管扩张剂敏感底物Vasp磷酸化的时程。这些细胞内cAMP指数将被用来确定抑制BAL中过表达的PDE3/4是否恢复或增强β-激动剂和Forsklin依赖的激活。目的2B：研究PDE3/4在体外上调对Vasp磷酸化和PKA活性的影响。目的3：观察不同血管扩张剂加/减PDE抑制剂对BAL损伤后24小时和1-2周的主动脉收缩性能的影响。目的：用免疫组织化学和原位杂交法鉴定BAL后24小时和7~14天PDE表达的细胞特异性。据预测，PDE3/4的增加会降低药物产生的血管松弛，从而增加cAMP和cGMP水平。PDE的过度表达有利于血管痉挛，这可能会影响血管壁重塑。特定PDE的上调代表了对损伤的重要反应，可能成为再狭窄和高血压的治疗靶点

DESCRIPTION (provided by the applicant): Enhanced smooth muscle cell (SMC) proliferation and migration account for intimal thickening, which follows balloon catheter de-endothelialization (BAL) of rat aorta. Recent studies show activation of cAMP-dependent protein kinase (PKA) by specific inhibition of cAMP phosphodiesterases PDE3 and/or PDE4 reduces SMC proliferation/ migration and lesions after angioplasty. In addition to changes in SMC growth, contractility may be affected by injury. Overall hypothesis: BAL leads to enhanced expression and activity of specific cAMP PDEs in both SMC and vessel wall-associated inflammatory cells, which then affects SMC cyclic nucleotide levels, protein phosphorylation and contractility. The project will first identify which high affinity, cAMP-selective PDE3 and PDE4 isoforms are upregulated in the BAL-injured rat aorta or growth factor-stimulated rat aortic SMC, while later aims assess the impact of PDE upregulation on cAMP-dependent phosphorylation, vessel contractility, and cellular locale. Aim 1A: Determine the time course of protein expression for PDE3A/3B and PDE4A/4B/4D genes following BAL in vivo. Pilot data for aortic medial SMC, show, that BAL is associated with biphasic increases in PDE4B mRNA, and smaller (PDE3B) or, no changes in other genes (PDE3A, 4D). Aim lB: To determine the time course of PDE4B, PDE4D and PDE3A protein splice variants in RASMC stimulated with serum, PDGF-BB or bFGF. Aim 2A: Determine the time course for PDE inhibitor enhancement of PKA activity, or PKA-dependent phosphorylation of a vasodilator-sensitive substrate VASP in SMC. These indices of intracellular cAMP will be used to determine if inhibition of overexpressed PDE3/4 in BAL restores or enhances beta-agonist and forskolin-dependent activation. Aim 2B: Determine the impact of PDE3/4 upregulation in vitro on VASP phosphorylation and PKA activity. Aim 3: Characterize contractility of the BAL aorta at 24 hr and 1-2 weeks after injury with various vasodilators plus/minus PDE inhibitors. Aim 4: Identify at 24 hr and 7-14 days after BAL the aortic cellular specificity of PDE expression by immunohistochemistry and in situ hybridization. The increase in PDE3/4 is predicted to reduce vasorelaxation produced by agents, which increase cAMP and cGMP levels. PDE overexpression favors vasospasm, which may affect vessel wall remodeling. Upregulation of specific PDEs represents an important response to injury that may serve as a therapeutic target in restenosis and hypertension