Vascular tone & cAMP phosphodiesterase in angioplasty

描述（由申请人提供）：在大鼠主动脉球囊导管去内皮化（BAL）后，平滑肌细胞（SMC）增殖和迁移增强导致内膜增厚。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依赖性蛋白激酶（PKA），可以减少血管成形术后SMC的增殖/迁移和病变。除了SMC生长的变化，收缩力也可能受到损伤的影响。总体假设：BAL导致SMC和血管壁相关炎症细胞中特异性cAMP PDEs的表达和活性增强，从而影响SMC环核苷酸水平、蛋白质磷酸化和收缩性。该项目将首先确定在bal损伤的大鼠主动脉或生长因子刺激的大鼠主动脉SMC中，哪些高亲和力、camp选择性的PDE3和PDE4亚型上调，而随后的目标是评估PDE上调对camp依赖性磷酸化、血管收缩性和细胞位置的影响。目的1A：测定体内BAL后PDE3A/3B和PDE4A/4B/4D基因蛋白表达的时间过程。主动脉内侧SMC的初步数据显示，BAL与PDE4B mRNA的双相增加有关，PDE3B mRNA的双相增加较少，或者其他基因没有变化（PDE3A, 4D）。目的：测定血清、PDGF-BB或bFGF刺激下RASMC中PDE4B、PDE4D和PDE3A蛋白剪接变异体的时间过程。目的2A：确定PDE抑制剂增强PKA活性的时间过程，或PKA依赖于SMC中血管扩张剂敏感底物VASP的磷酸化。细胞内cAMP的这些指标将用于确定抑制BAL中过表达的PDE3/4是否恢复或增强β激动剂和福斯克林依赖的激活。目的2B：确定体外PDE3/4上调对VASP磷酸化和PKA活性的影响。目的3：在损伤后24小时和1-2周，用各种血管扩张剂加/减PDE抑制剂来描述BAL主动脉的收缩性。目的4：应用免疫组织化学和原位杂交技术，在BAL术后24小时和7-14天检测PDE在主动脉细胞表达的特异性。PDE3/4的增加预计会减少药物产生的血管松弛，从而增加cAMP和cGMP水平。PDE过表达有利于血管痉挛，可能影响血管壁重塑。特异性PDEs的上调代表了对损伤的重要反应，可能作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