: 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 1 A: 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 2 A: 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 2 B: 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
