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Abstract-In response to biological and mechanical injury, or in vitro culturing, vascular smooth muscle cells (VSMCs) undergo phenotypic modulation from a differentiated "contractile" phenotype to a dedifferentiated "synthetic" one. This results in the capacity to proliferate, migrate, and produce extracellular matrix proteins, thus contributing to neointimal formation. Cyclic nucleotide phosphodiesterases (PDEs), by hydrolyzing cAMP or cGMP, are critical in the homeostasis of cyclic nucleotides that regulate VSMC growth. Here, we demonstrate that PDE1A, a Ca 2ϩ -calmodulin-stimulated PDE preferentially hydrolyzing cGMP, is predominantly cytoplasmic in medial "contractile" VSMCs but is nuclear in neointimal "synthetic" VSMCs. Using primary VSMCs, we show that cytoplasmic and nuclear PDE1A were associated with a contractile marker (SM-calponin) and a growth marker (Ki-67), respectively. This suggests that cytoplasmic PDE1A is associated with the "contractile" phenotype, whereas nuclear PDE1A is with the "synthetic" phenotype. To determine the role of nuclear PDE1A, we examined the effects loss-of-PDE1A function on subcultured VSMC growth and survival using PDE1A RNA interference and pharmacological inhibition. Reducing PDE1A function significantly attenuated VSMC growth by decreasing proliferation via G 1 arrest and inducing apoptosis. Inhibiting PDE1A also led to intracellular cGMP elevation, p27Kip1 upregulation, cyclin D1 downregulation, and p53 activation. We further demonstrated that in subcultured VSMCs redifferentiated by growth on collagen gels, cytoplasmic PDE1A regulates myosin light chain phosphorylation with little effect on apoptosis, whereas inhibiting nuclear PDE1A has the opposite effects. These suggest that nuclear PDE1A is important in VSMC growth and survival and may contribute to the neointima formation in atherosclerosis and restenosis. (Circ Res. 2006;98:777-784.) Key Words: PDE Ⅲ smooth muscle cell Ⅲ growth Ⅲ apoptosis Ⅲ vascular injury V ascular smooth muscle cells (VSMCs) in response to injury and hormonal stimuli exhibit phenotypic plasticity, changing from a differentiated (quiescent, contractile) phenotype to a dedifferentiated (active, synthetic) one. 1 This process was originally defined as "phenotypic modulation." 2 Under normal conditions, VSMCs residing in the media of vessels are quiescent with a very low turnover rate. 3,4 Quiescent VSMCs are fully differentiated cells that possess the "contractile" phenotype and function principally to maintain vascular tone. If the vessel is injured or cells are placed in tissue culture, VSMCs respond by changing from the "contractile" to the "synthetic" phenotype. 4 Synthetic VSMCs contribute to neointima formation by downregulating contractile proteins and acquiring the capacity to proliferate, migrate, and produce extracellular matrix proteins. 5 Therefore, phenotypic modulation of VSMCs plays a key role in the pathogenesis of cardiovascular disorders such as atherosclerosis, postangioplasty restenosis, bypass vein graft failure, and ca...

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Isoforms in the PDE1 family of cyclic nucleotide phosphodiesterases were recently found to comprise a significant portion of the cGMP-inhibited cAMP hydrolytic activity in human hearts. We examined the expression of PDE1 isoforms in human myocardium, characterized their catalytic activity, and quantified their contribution to cAMP hydrolytic and cGMP hydrolytic activity in subcellular fractions of this tissue. Western blotting with isoform-selective anti-PDE1 monoclonal antibodies showed PDE1C1 to be the principal isoform expressed in human myocardium. Immunohistochemical analysis showed that PDE1C1 is distributed along the Z-lines and M-lines of cardiac myocytes in a striated pattern that differs from that of the other major dual-specificity cyclic nucleotide phosphodiesterase in human myocardium, PDE3A. Most of the PDE1C1 activity was recovered in soluble fractions of human myocardium. It binds both cAMP and cGMP with K m values of ϳ1 M and hydrolyzes both substrates with similar catalytic rates. PDE1C1 activity in subcellular fractions was quantified using a new PDE1-selective inhibitor, IC295. At substrate concentrations of 0.1 M, PDE1C1 constitutes the great majority of cAMP hydrolytic and cGMP hydrolytic activity in soluble fractions and the majority of cGMP hydrolytic activity in microsomal fractions, whereas PDE3 constitutes the majority of cAMP hydrolytic activity in microsomal fractions. These results indicate that PDE1C1 is expressed at high levels in human cardiac myocytes with an intracellular distribution distinct from that of PDE3A and that it may have a role in the integration of cGMP-, cAMP-and Ca 2؉ -mediated signaling in these cells. PDE13 cyclic nucleotide phosphodiesterases are dual-specificity enzymes that bind and hydrolyze cAMP and cGMP in a mutually competitive manner (for review, see Ref. 1). Their activity is increased by their binding to Ca 2ϩ and calmodulin, a feature unique to the PDE1 family of cyclic nucleotide phosphodiesterases. Three PDE1 genes, PDE1A, PDE1B, and PDE1C, have been identified, and several protein isoforms are generated from these genes by alternative splicing. PDE1A and PDE1B isoforms have significantly higher affinities for cGMP than for cAMP, whereas PDE1C isoforms have similar affinities for both substrates (2-4). The modulation of the catalytic activity of PDE1 isoforms by Ca 2ϩ -dependent stimulation and their susceptibility to mutually competitive inhibition by cAMP and cGMP suggest that these enzymes may be points of interaction among several signaling pathways.Cyclic nucleotide phosphodiesterases are particularly important in cardiac muscle. In humans, alterations in cAMP metabolism and cAMP-mediated signaling leading to decreases in intracellular cAMP content and in the phosphorylation of some substrates of cAMP-dependent protein kinase are prominent features of the pathophysiology of heart failure, and inhibition of PDE3 cAMP hydrolytic activity has a major role in its treatment (5). Changes in cGMP-mediated signaling in cardiac disease have not been ch...