Phosphodiesterases 2, 3 and 4 can decrease cardiac effects of H2-histamine-receptor activation in isolated atria of transgenic mice.

Histamine exerts cAMP-dependent positive inotropic effects (PIE) and positive chronotropic effects (PCE) on isolated left and right atria, respectively, of transgenic mice which overexpress the human H2-receptor in the heart (=H2-TG). To determine whether these effects are antagonized by phosphodiesterases (PDEs), contractile studies were done in isolated left and right atrial preparations of H2-TG. The contractile effects of histamine were tested in the additional presence of the PDE-inhibitorserythro-9-(2-hydroxy-3-nonyl)adenine hydrochloride (EHNA, 1 µM, PDE2-inhibitor) or cilostamide (1 µM, PDE3-inhibitor), rolipram (10 µM, a PDE4-inhibitor), and their combinations. Cilostamide (1 µM) and EHNA (1 µM), rolipram (1 µM), and EHNA (1 µM) and the combination of rolipram (0.1 µM) and cilostamide (1 µM) each increased the potency of histamine to elevate the force of contraction (FOC) in H2-TG. Cilostamide (1 µM) and rolipram (10 µM) alone increased and EHNA (1 µM) decreased alone, and their combination increased the potency of histamine to increase the FOC in H2-TG indicating that PDE3 and PDE4 regulate the inotropic effects of histamine in H2-TG. The PDE inhibitors (EHNA, cilostamide, rolipram) alone did not alter the potency of histamine to increase the heart beat in H2-TG whereas a combination of rolipram, cilostamide, and EHNA, or of rolipram and EHNA increased the potency of histamine to act on the beating rate. In summary, the data suggest that the PCE of histamine in H2-TG atrium involves PDE 2 and 4 activities, whereas the PIE of histamine are diminished by activity of PDE 3 and 4.

Docking and quantitative structure-activity relationship of bi-cyclic heteroaromatic pyridazinone and pyrazolone derivatives as phosphodiesterase 3A (PDE3A) inhibitors.

PDE3s belong to the phosphodiesterases family, where the PDE3A isoform is the major subtype in platelets involved in the cAMP regulation pathway of platelet aggregation. PDE3A inhibitors have been designed as potential antiplatelet agents. In this work, a homology model of PDE3A was developed and used to obtain the binding modes of bicyclic heteroaromatic pyridazinones and pyrazolones. Most of the studied compounds adopted similar orientations within the PDE3A active site, establishing hydrogen bonds with catalytic amino acids. Besides, the structure-activity relationship of the studied inhibitors was described by using a field-based 3D-QSAR method. Different structure alignment strategies were employed, including template-based and docking-based alignments. Adequate correlation models were obtained according to internal and external validations. In general, QSAR models revealed that steric and hydrophobic fields describe the different inhibitory activities of the compounds, where the hydrogen bond donor and acceptor fields have minor contributions. It should be stressed that structural elements of PDE3A inhibitors are reported here, through descriptions of their binding interactions and their differential affinities. In this sense, the present results could be useful in the future design of more specific and potent PDE3A inhibitors that may be used for the treatment of cardiovascular diseases.

B cell-derived IL-4 acts on podocytes to induce proteinuria and foot process effacement.

The efficacy of B cell depletion therapies in diseases such as nephrotic syndrome and rheumatoid arthritis suggests a broader role in B cells in human disease than previously recognized. In some of these diseases, such as the minimal change disease subtype of nephrotic syndrome, pathogenic antibodies and immune complexes are not involved. We hypothesized that B cells, activated in the kidney, might produce cytokines capable of directly inducing cell injury and proteinuria. To directly test our hypothesis, we targeted a model antigen to the kidney glomerulus and showed that transfer of antigen-specific B cells could induce glomerular injury and proteinuria. This effect was mediated by IL-4, as transfer of IL-4-deficient B cells did not induce proteinuria. Overexpression of IL-4 in mice was sufficient to induce kidney injury and proteinuria and could be attenuated by JAK kinase inhibitors. Since IL-4 is a specific activator of STAT6, we analyzed kidney biopsies and demonstrated STAT6 activation in up to 1 of 3 of minimal change disease patients, suggesting IL-4 or IL-13 exposure in these patients. These data suggest that the role of B cells in nephrotic syndrome could be mediated by cytokines.

Novel approaches to targeting PDE3 in cardiovascular disease.

Inhibitors of PDE3, a family of dual-specificity cyclic nucleotide phosphodiesterases, are used clinically to increase cardiac contractility by raising intracellular cAMP content in cardiac myocytes and to reduce vascular resistance by increasing intracellular cGMP content in vascular smooth muscle myocytes. When used in the treatment of patients with heart failure, PDE3 inhibitors are effective in the acute setting but increase sudden cardiac death with long-term administration, possibly reflecting pro-apoptotic and pro-hypertrophic consequences of increased cAMP-mediated signaling in cardiac myocytes. cAMP-mediated signaling in cardiac myocytes is highly compartmentalized, and different phosphodiesterases, by controlling cAMP content in functionally discrete intracellular microcompartments, regulate different cAMP-mediated pathways. Four variants/isoforms of PDE3 (PDE3A1, PDE3A2, PDE3A3, and PDE3B) are expressed in cardiac myocytes, and new experimental results have demonstrated that these isoforms, which are differentially localized intracellularly through unique protein-protein interactions, control different physiologic responses. While the catalytic regions of these isoforms may be too similar to allow the catalytic activity of each isoform to be selectively inhibited, targeting their unique protein-protein interactions may allow desired responses to be elicited without the adverse consequences that limit the usefulness of existing PDE3 inhibitors.

Dual PDE3/4 and PDE4 inhibitors: novel treatments for COPD and other inflammatory airway diseases.

Selective phosphodiesterase (PDE) 4 and dual PDE3/4 inhibitors have attracted considerable interest as potential therapeutic agents for the treatment of respiratory diseases, largely by virtue of their anti-inflammatory (PDE4) and bifunctional bronchodilator/anti-inflammatory (PDE3/4) effects. Many of these agents have, however, failed in early development for various reasons, including dose-limiting side effects when administered orally and lack of sufficient activity when inhaled. Indeed, only one selective PDE4 inhibitor, the orally active roflumilast-n-oxide, has to date received marketing authorization. The majority of the compounds that have failed were, however, orally administered and non-selective for either PDE3 (A,B) or PDE4 (A,B,C,D) subtypes. Developing an inhaled dual PDE3/4 inhibitor that is rapidly cleared from the systemic circulation, potentially with subtype specificity, may represent one strategy to improve the therapeutic index and also exhibit enhanced efficacy versus inhibition of either PDE3 or PDE4 alone, given the potential positive interactions with regard to anti-inflammatory and bronchodilator effects that have been observed pre-clinically with dual inhibition of PDE3 and PDE4 compared with inhibition of either isozyme alone. This MiniReview will summarize recent clinical data obtained with PDE inhibitors and the potential for these drugs to treat COPD and other inflammatory airways diseases such as asthma and cystic fibrosis.

Regulation of ecto-apyrase CD39 (ENTPD1) expression by phosphodiesterase III (PDE3).

The ectoenzyme CD39 suppresses thrombosis and inflammation by suppressing ATP and ADP to AMP. However, mechanisms of CD39 transcriptional and post-translational regulation are not well known. Here we show that CD39 levels are modulated by inhibition of phosphodiesterase 3 (PDE3). RAW macrophages and human umbilical vein endothelial cells (HUVECs) were treated with the PDE3 inhibitors cilostazol and milrinone, then analyzed using qRT-PCR, immunoprecipitation/Western blot, immunofluorescent staining, radio-thin-layer chromatography, a malachite green assay, and ELISA. HUVECs expressed elevated CD39 protein (2-fold [P<0.05] for cilostazol and 2.5-fold [P<0.01] for milrinone), while macrophage CD39 mRNA and protein were both elevated after PDE3 inhibition. HUVEC ATPase activity increased by 25% with cilostazol and milrinone treatment (P<0.05 and P<0.01, respectively), as did ADPase activity (47% and 61%, P<0.001). There was also a dose-dependent elevation of soluble CD39 after treatment with 8-Br-cAMP, with maximal elevation of 60% more CD39 present compared to controls (1 mM, P<0.001). Protein harvested after 8-Br-cAMP treatment showed that ubiquitination of CD39 was decreased by 43% compared to controls. A DMSO or PBS vehicle control was included for each experiment based on solubility of cilostazol, milrinone, and 8-Br-cAMP. These results indicate that PDE3 inhibition regulates endothelial CD39 at a post-translational level.

Functional regulatory T cells produced by inhibiting cyclic nucleotide phosphodiesterase type 3 prevent allograft rejection.

Regulatory T cells (T(regs)) manipulated ex vivo have potential as cellular therapeutics in autoimmunity and transplantation. Although it is possible to expand naturally occurring T(regs), an attractive alternative possibility, particularly suited to solid organ and bone marrow transplantation, is the stimulation of total T cell populations with defined allogeneic antigen-presenting cells (APCs) under conditions that lead to the generation or expansion of donor-reactive, adaptive T(regs). Here we demonstrate that stimulation of mouse CD4(+) T cells by immature allogeneic dendritic cells combined with pharmacological inhibition of phosphodiesterase 3 (PDE) resulted in a functional enrichment of Foxp3(+) T cells. Without further manipulation or selection, the resultant population delayed skin allograft rejection mediated by polyclonal CD4(+) effectors or donor-reactive CD8(+) T cell receptor transgenic T cells and inhibited both effector cell proliferation and T cell priming for interferon-γ production. Notably, PDE inhibition also enhanced the enrichment of human Foxp3(+) CD4(+) T cells driven by allogeneic APCs. These cells inhibited T cell proliferation in a standard in vitro mixed lymphocyte assay and, moreover, attenuated the development of vasculopathy mediated by autologous peripheral blood mononuclear cells in a functionally relevant humanized mouse transplant model. These data establish a method for the ex vivo generation of graft-reactive, functional mouse and human T(regs) that uses a clinically approved agent, making pharmacological PDE inhibition a potential strategy for T(reg)-based therapies.

Cilostazol and atherogenic dyslipidemia: a clinically relevant effect?

INTRODUCTION: Cilostazol is a reversible, selective inhibitor of PDE3A able to significantly improve walking distance in patients with intermittent claudication. However, beyond its antiplatelet and vasodilator properties, cilostazol seems to have significant effects on atherogenic dyslipidemia. AREAS COVERED: The effects of cilostazol on plasma lipids, lipoproteins, apolipoproteins and postprandial lipemia are reviewed. A literature search (using Medline and Scopus) was performed up to 24 October 2010. The authors also manually reviewed the references of selected articles for any pertinent material. EXPERT OPINION: Cilostazol is able to significantly lower plasma triglyceride levels, with a concomitant increase in high-density lipoprotein (HDL) cholesterol concentrations. Additional effects on pro-atherogenic lipoproteins and apolipoproteins include those on remnant-like particles, HDL subclasses, apolipoprotein B and postprandial lipemia. Cilostazol can improve the pro-atherogenic lipid profile in patients with peripheral arterial disease or type 2 diabetes. Further studies are needed to establish whether cilostazol treatment exerts clinically relevant effects on atherogenic dyslipidemia in high-risk patients.

Suppressing apoptosis with milrinone simulating extracorporeal circulation: a pilot study.

BACKGROUND: After cardioplegia, ischemia/reperfusion injury can induce apoptosis. The aim of this study was to evaluate our ex vivo microperfusion model on human myocardium during simulated cardioplegia (cp) and reperfusion (rep). In addition, the aim was to verify the anti-apoptotic properties of the phosphodiesterase 3 inhibitor milrinone. METHODS: Cardiac biopsies were retrieved from the right auricle of patients undergoing elective CABG prior to induction of cardiopulmonary bypass. Biopsies were exposed to ex vivo conditions with varying periods of cp/rep (30/10, 60/20, 120/40 min). Group I consisted of untreated controls (n=15), Group II of treated controls who had cp/rep (n=15) while Group III had cp/rep+milrinone (n=15). For the detection of apoptosis, anti-activated caspase-3 and PARP-1 cleavage immunostaining were used. RESULTS: The percentage of apoptotic cardiomyocytes in Group I was significantly (P<0.05) lower compared to Group II, revealing a time-dependent increase. In Group III with milrinone treatment, apoptosis was significantly suppressed (P<0.05). CONCLUSIONS: Milrinone significantly suppressed apoptosis in our ex vivo setting. This finding warrants further study aiming to evaluate the potential beneficial effects of milrinone on the suppression of ischemia/reperfusion injury in a clinical setting.

Partial reversal of experimental pulmonary hypertension by phosphodiesterase-3/4 inhibition.

Phosphodiesterase (PDE) inhibitors are currently under investigation for the therapy of pulmonary hypertension. The present study was designed to investigate chronic effects of oral pumafentrine, a mixed selective PDE-3/4 inhibitor, in monocrotaline (MCT)-induced pulmonary hypertension in rats. Treatment with pumafentrine (10 mg.kg(-1) daily) from week 4 to 6 after a single injection of MCT (60 mg.kg(-1)) partially reversed pulmonary hypertension and right heart hypertrophy in rats. In addition, small pulmonary arterial muscularisation, media hypertrophy and decrease in lumen area were largely reversed. Inhibition of smooth muscle proliferation under pumafentrine was demonstrated in vivo as was a pro-apoptotic effect of pumafentrine on vascular cells. Moreover, pumafentrine dose-dependently increased cyclic adenosine monophosphate levels and inhibited proliferation of cultured pulmonary arterial smooth muscle cells. In conclusion, oral pumafentrine partially reverses monocrotaline-induced pulmonary hypertension, lung vascular remodelling and right heart hypertrophy in rats.