Role of cAMP-phosphodiesterase 1C signaling in regulating growth factor receptor stability, vascular smooth muscle cell growth, migration, and neointimal hyperplasia.

RATIONALE: Neointimal hyperplasia characterized by abnormal accumulation of vascular smooth muscle cells (SMCs) is a hallmark of occlusive disorders such as atherosclerosis, postangioplasty restenosis, vein graft stenosis, and allograft vasculopathy. Cyclic nucleotides are vital in SMC proliferation and migration, which are regulated by cyclic nucleotide phosphodiesterases (PDEs). OBJECTIVE: Our goal is to understand the regulation and function of PDEs in SMC pathogenesis of vascular diseases. METHODS AND RESULTS: We performed screening for genes differentially expressed in normal contractile versus proliferating synthetic SMCs. We observed that PDE1C expression was low in contractile SMCs but drastically elevated in synthetic SMCs in vitro and in various mouse vascular injury models in vivo. In addition, PDE1C was highly induced in neointimal SMCs of human coronary arteries. More importantly, injury-induced neointimal formation was significantly attenuated by PDE1C deficiency or PDE1 inhibition in vivo. PDE1 inhibition suppressed vascular remodeling of human saphenous vein explants ex vivo. In cultured SMCs, PDE1C deficiency or PDE1 inhibition attenuated SMC proliferation and migration. Mechanistic studies revealed that PDE1C plays a critical role in regulating the stability of growth factor receptors, such as PDGF receptor ß (PDGFRß) known to be important in pathological vascular remodeling. PDE1C interacts with low-density lipoprotein receptor-related protein-1 and PDGFRß, thus regulating PDGFRß endocytosis and lysosome-dependent degradation in an low-density lipoprotein receptor-related protein-1-dependent manner. A transmembrane adenylyl cyclase cAMP-dependent protein kinase cascade modulated by PDE1C is critical in regulating PDGFRß degradation. CONCLUSIONS: These findings demonstrated that PDE1C is an important regulator of SMC proliferation, migration, and neointimal hyperplasia, in part through modulating endosome/lysosome-dependent PDGFRß protein degradation via low-density lipoprotein receptor-related protein-1.

Modulation of Polycystic Kidney Disease Severity by Phosphodiesterase 1 and 3 Subfamilies.

Aberrant intracellular calcium levels and increased cAMP signaling contribute to the development of polycystic kidney disease (PKD). cAMP can be hydrolyzed by various phosphodiesterases (PDEs). To examine the role of cAMP hydrolysis and the most relevant PDEs in the pathogenesis of PKD, we examined cyst development in Pde1- or Pde3-knockout mice on the Pkd2(-/WS25) background (WS25 is an unstable Pkd2 allele). These PDEs were selected because of their importance in cross-talk between calcium and cyclic nucleotide signaling (PDE1), control of cell proliferation and cystic fibrosis transmembrane conductance regulator (CFTR) -driven fluid secretion (PDE3), and response to vasopressin V2 receptor activation (both). In Pkd2(-/WS25) mice, knockout of Pde1a, Pde1c, or Pde3a but not of Pde1b or Pde3b aggravated the development of PKD and was associated with higher levels of protein kinase A-phosphorylated (Ser133) cAMP-responsive binding protein (P-CREB), activating transcription factor-1, and CREB-induced CRE modulator proteins in kidney nuclear preparations. Immunostaining also revealed higher expression of P-CREB in Pkd2(-/) (WS25);Pde1a(-/-), Pkd2(-) (/WS25);Pde1c(-/-), and Pkd2(-/) (WS25);Pde3a(-/-) kidneys. The cystogenic effect of desmopressin administration was markedly enhanced in Pkd2(-/WS25);Pde3a(-/-) mice, despite PDE3 accounting for only a small fraction of renal cAMP PDE activity. These observations show that calcium- and calmodulin-dependent PDEs (PDE1A and PDE1C) and PDE3A modulate the development of PKD, possibly through the regulation of compartmentalized cAMP pools that control cell proliferation and CFTR-driven fluid secretion. Treatments capable of increasing the expression or activity of these PDEs may, therefore, retard the development of PKD.

Role of phosphodiesterases in the shaping of sub-plasma-membrane cAMP oscillations and pulsatile insulin secretion.

Specificity and versatility in cyclic AMP (cAMP) signalling are governed by the spatial localisation and temporal dynamics of the signal. Phosphodiesterases (PDEs) are important for shaping cAMP signals by hydrolyzing the nucleotide. In pancreatic ß-cells, glucose triggers sub-plasma-membrane cAMP oscillations, which are important for insulin secretion, but the mechanisms underlying the oscillations are poorly understood. Here, we investigated the role of different PDEs in the generation of cAMP oscillations by monitoring the concentration of cAMP in the sub-plasma-membrane space ([cAMP](pm)) with ratiometric evanescent wave microscopy in MIN6 cells or mouse pancreatic ß-cells expressing a fluorescent translocation biosensor. The general PDE inhibitor IBMX increased [cAMP](pm), and whereas oscillations were frequently observed at 50 µM IBMX, 300 µM-1 mM of the inhibitor caused a stable increase in [cAMP](pm). The [cAMP](pm) was nevertheless markedly suppressed by the adenylyl cyclase inhibitor 2',5'-dideoxyadenosine, indicating IBMX-insensitive cAMP degradation. Among IBMX-sensitive PDEs, PDE3 was most important for maintaining a low basal level of [cAMP](pm) in unstimulated cells. After glucose induction of [cAMP](pm) oscillations, inhibitors of PDE1, PDE3 and PDE4 inhibitors the average cAMP level, often without disturbing the [cAMP](pm) rhythmicity. Knockdown of the IBMX-insensitive PDE8B by shRNA in MIN6 cells increased the basal level of [cAMP](pm) and prevented the [cAMP](pm)-lowering effect of 2',5'-dideoxyadenosine after exposure to IBMX. Moreover, PDE8B-knockdown cells showed reduced glucose-induced [cAMP](pm) oscillations and loss of the normal pulsatile pattern of insulin secretion. It is concluded that [cAMP](pm) oscillations in ß-cells are caused by periodic variations in cAMP generation, and that several PDEs, including PDE1, PDE3 and the IBMX-insensitive PDE8B, are required for shaping the sub-membrane cAMP signals and pulsatile insulin release.

Role of phosphodiesterases in adult-onset pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is characterized by increased mean pulmonary artery pressure (mPAP) due to vasoconstriction and structural changes in the small pulmonary arteries (PAs); proliferation of pulmonary artery smooth muscle cells (PASMCs) contributes to the remodeling. The abnormal pathophysiology in the pulmonary vasculature relates to decreased cyclic nucleotide levels in PASMCs. Phosphodiesterases (PDEs) catalyze the hydrolysis of cAMP and cGMP, thereby PDE inhibitors are effective in vasodilating the PA and decreasing PASMC proliferation. Experimental studies support the use of PDE3, PDE5, and PDE1 inhibitors in PAH. PDE5 inhibitors such as sildenafil are clinically approved to treat different forms of PAH and lower mPAP, increase functional capacity, and decrease right ventricular hypertrophy, without decreasing systemic arterial pressure. New evidence suggests that the combination of PDE inhibitors with other therapies for PAH may be beneficial in treating the disease. Furthermore, inhibiting PDEs in the heart and the inflammatory cells that infiltrate the PA may offer new targets to reduce right ventricular hypertrophy and inhibit inflammation that is associated with and contributes to the development of PAH. This chapter summarizes the advances in the area and the future of PDEs in PAH.

PDE1A inhibition elicits cGMP-dependent relaxation of rat mesenteric arteries.

BACKGROUND AND PURPOSE: PDE1, a subfamily of cyclic nucleotide PDEs consisting of three isoforms, PDE1A, PDE1B and PDE1C, has been implicated in the regulation of vascular tone. The PDE1 isoform(s) responsible for tone regulation is unknown. This study used isoform-preferring PDE1 inhibitors, Lu AF58027, Lu AF64196, Lu AF66896 and Lu AF67897, to investigate the relative contribution of PDE1 isoforms to regulation of vascular tone. EXPERIMENTAL APPROACH: In rat mesenteric arteries, expression and localization of Pde1 isoforms were determined by quantitative PCR and in situ hybridization, and physiological impact of PDE1 inhibition was evaluated by isometric tension recordings. KEY RESULTS: In rat mesenteric arteries, Pde1a mRNA expression was higher than Pde1b and Pde1c. In situ hybridization revealed localization of Pde1a to vascular smooth muscle cells (VSMCs) and only minor appearance of Pde1b and Pde1c. The potency of the PDE1 inhibitors at eliciting relaxation showed excellent correlation with their potency at inhibiting PDE1A. Thus, Lu AF58027 was the most potent at inhibiting PDE1A and was also the most potent at eliciting relaxation in mesenteric arteries. Inhibition of NOS with l-NAME, soluble GC with ODQ or PKG with Rp-8-Br-PET-cGMP all attenuated the inhibitory effect of PDE1 on relaxation, whereas PKA inhibition with H89 had no effect. CONCLUSIONS AND IMPLICATIONS: Pde1a is the dominant PDE1 isoform present in VSMCs, and relaxation mediated by PDE1A inhibition is predominantly driven by enhanced cGMP signalling. These results imply that isoform-selective PDE1 inhibitors are powerful investigative tools allowing examination of physiological and pathological roles of PDE1 isoforms.

Inhibition of phosphodiesterase-1 attenuates cold-induced pulmonary hypertension.

Chronic exposure to cold caused pulmonary arterial hypertension (cold-induced pulmonary hypertension [CIPH]) and increased phosphodiesterase-1C (PDE-1C) expression in pulmonary arteries (PAs) in rats. The purpose of this study is to investigate a hypothesis that inhibition of PDE-1 would decrease inflammatory infiltrates and superoxide production leading to attenuation of CIPH. Three groups of male rats were exposed to moderate cold (5±1°C) continuously, whereas 3 groups were maintained at room temperature (23.5±1°C, warm; 6 rats/group). After 8-week exposure to cold, 3 groups in each temperature condition received continuous intravenous infusion of 8-isobutyl-methylxanthine (8-IBMX) (PDE-1 inhibitor), apocynin (NADPH oxidase inhibitor) or vehicle, respectively, for 1 week. Cold exposure significantly increased right-ventricular systolic pressure compared with warm groups (33.8±3.2 versus 18.6±0.3 mm Hg), indicating that animals developed CIPH. Notably, treatment with 8-IBMX significantly attenuated the cold-induced increase in right ventricular pressure (23.5±1.8 mm Hg). Cold exposure also caused right-ventricular hypertrophy, whereas 8-IBMX reversed cold-induced right ventricular hypertrophy. Cold exposure increased PDE-1C protein expression, macrophage infiltration, NADPH oxidase activity, and superoxide production in PAs and resulted in PA remodeling. 8-IBMX abolished cold-induced upregulation of PDE-1C in PAs. Interestingly, inhibition of PDE-1 eliminated cold-induced macrophage infiltration, NADPH oxidase activation, and superoxide production in PAs and reversed PA remodeling. Inhibition of NADPH oxidase by apocynin abolished cold-induced superoxide production and attenuated CIPH and PA remodeling. In conclusion, inhibition of PDE-1 attenuated CIPH and reversed cold-induced PA remodeling by suppressing macrophage infiltration and superoxide production, suggesting that upregulation of PDE-1C expression may be involved in the pathogenesis of CIPH.

[Role of cyclic nucleotide phosphodiesterases in the cAMP compartmentation in cardiac cells]. / Rôle des phosphodiestérases des nucléotides cycliques dans la compartimentation subcellulaire de l'AMP cyclique des myocytes cardiaques.

In the light of the knowledge accumulated over the years, it becomes clear that intracellular cAMP is not uniformly distributed within cardiomyocytes and that cAMP compartmentation is required for adequate processing and targeting of the information generated at the membrane. Localized cAMP signals may be generated by interplay between discrete production sites and restricted diffusion within the cytoplasm. In addition to specialized membrane structures that may limit cAMP spreading, degradation of the second messenger by cyclic nucleotide phosphodiesterases (PDEs) appears critical for the formation of dynamic microdomains that confer specificity of the response to various hormones. This review summarizes the main findings that support the cAMP compartmentation hypothesis in cardiac cells, with a special emphasis on PDEs. The respective roles of the four main cardiac cAMP-PDE families (PDE1 to PDE4) in the organization of cAMP microdomains and hormonal specificity in cardiac cells are reviewed. The evidence that these PDEs are modified in heart failure is summarized, and the implication for the progression of the disease is discussed. Finally, the potential benefits that could be awaited from the manipulation of specific PDE subtypes in heart failure are presented.