PDE4 inhibitor, roflumilast protects cardiomyocytes against NO-induced apoptosis via activation of PKA and Epac dual pathways

Myocyte apoptosis plays an important role in myocardial infarction and cAMP is crucial in the regulation of myocyte apoptosis. Phosphodiesterase-4 (PDE4) inhibitor blocks the hydrolysis of cAMP via inhibition of PDE4 and is attractive candidate for novel anti-inflammatory drugs. However, its function in cardiovascular diseases and cardiomyocyte apoptosis is unclear. Therefore, we investigated whether roflumilast, a PDE4 inhibitor, exerts protective effect against NO-induced apoptosis in both of H9c2 cells and neonatal rat cardiomyocytes (NRCMs), focusing on cAMP downstream molecules such as protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac). According to our data, intracellular cAMP was increased by roflumilast treatment in H9c2 cells and NRCMs. Roflumilast inhibited SNP-induced apoptosis and this effect was reversed by PKA specific inhibitor H-89 and KT-5720. In addition, PKA specific activator N(6)-benzoyladenosine 3',5-cyclic monophosphate (N(6)Bz-cAMP) mimicked the effects of roflumilast. CREB phosphorylation by roflumilast was also inhibited by H-89, indicating that roflumilast protects SNP-induced apoptosis via PKA-dependent pathway. Roflumilast increased Epac1/GTP-Rap1 and the protective effect was abolished by Epac1 siRNA transfection, demonstrating that Epac signaling was also involved in this protective response. In support, Epac specific activator 8-(4-chlrorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (8CPT-2Me-cAMP) protected SNP-induced apoptosis. PI3K/Akt inhibitor LY294002 blocked roflumilast-induced Akt phosphorylation and protective effect. Furthermore, inhibition of Epac1 with siRNA had no effect on roflumilast-induced CREB phosphorylation, whereas inhibited Akt phosphorylation, implicating that Akt phosphorylation was regulated by Epac pathway. In addition, it was also observed that rolipram and cilomilast exert similar effects as roflumilast. In summary, our data indicate that roflumilast protects NO-induced apoptosis via both cAMP-PKA/CREB and Epac/Akt-dependent pathway. Our study suggests a possibility of PDE4 inhibitor roflumilast as a potential therapeutic agent against myocardial ischemia/reperfusion (I/R) injury.     

Reduction of serum FABP4 level by sitagliptin,a DPP-4 inhibitor,in patients with type 2 diabetes mellitus

Fatty acid binding protein 4 (FABP4), also known as adipocyte FABP or aP2, is secreted from adipocytes in association with lipolysis as a novel adipokine, and elevated serum FABP4 level is associated with obesity, insulin resistance, and atherosclerosis. However, little is known about the modulation of serum FABP4 level by therapeutic drugs. Sitagliptin (50 mg/day), a dipeptidyl peptidase 4 (DPP-4) inhibitor that increases glucagon-like peptide 1 (GLP-1), was administered to patients with type 2 diabetes (n = 24) for 12 weeks. Treatment with sitagliptin decreased serum FABP4 concentration by 19.7% (17.8 ± 1.8 vs. 14.3 ± 1.5 ng/ml, P < 0.001) and hemoglobin A1c without significant changes in adiposity or lipid variables. In 3T3-L1 adipocytes, sitagliptin or exendin-4, a GLP-1 receptor agonist, had no effect on short-term (2 h) secretion of FABP4. However, gene expression and long-term (24 h) secretion of FABP4 were significantly reduced by sitagliptin, which was not mimicked by exendin-4. Treatment with recombinant DPP-4 increased gene expression and long-term secretion of FABP4, and the effects were cancelled by sitagliptin. Furthermore, knockdown of DPP-4 in 3T3-L1 adipocytes decreased gene expression and long-term secretion of FABP4. In conclusion, sitagliptin decreases serum FABP4 level, at least in part, via reduction in the expression and consecutive secretion of FABP4 in adipocytes by direct inhibition of DPP-4.     

COPD patients with chronic bronchitis and higher sputum eosinophil counts show increased type-2 and PDE4 gene expression in sputum

Chronic obstructive pulmonary disease (COPD) patients with higher eosinophil counts are associated with increased clinical response to phosphodiesterase-4-inhibitors (PDE4i). However, the underlying inflammatory mechanisms associated with this increased response is not yet elucidated. This post hoc analysis focused on sputum gene expression in patients with chronic bronchitis who underwent 32-day treatment with two doses of the inhaled PDE4i CHF6001 (tanimilast) or placebo on top of triple therapy. Biological characterization and treatment effects were assessed between patients with different sputum eosinophil levels (eosinophil^high ≥ 3%; eosinophil^low < 3%) at baseline (primary samples) or at the end of the treatment of the placebo arm (validation samples). Forty-one genes were differentially expressed in primary samples (p-adjusted for false discovery rate < 0.05); all up-regulated in eosinophil^high patients and functionally enriched for type-2 and PDE4 inflammatory processes. Eleven out of nineteen genes having immune system biological processes annotations including IL5RA, ALOX15, IL1RL1, CLC, GATA1 and PDE4D were replicated using validation samples. The expression of a number of these inflammatory mediators was reduced by tanimilast treatment, with greater effects observed in eosinophil^high patients. These findings suggest that type-2 and PDE4 overexpression in COPD patients with higher sputum eosinophil counts contribute to the differential clinical response to PDE4i observed in previous clinical trials.     

Three-dimensional structures of PDE4D in complex with roliprams and implication on inhibitor selectivity

Selective inhibitors against the 11 families of cyclic nucleotide phosphodiesterases (PDEs) are used to treat various human diseases. How the inhibitors selectively bind the conserved PDE catalytic domains is unknown. The crystal structures of the PDE4D2 catalytic domain in complex with (R)- or (R,S)-rolipram suggest that inhibitor selectivity is determined by the chemical nature of amino acids and subtle conformational changes of the binding pockets. The conformational states of Gln369 in PDE4D2 may play a key role in inhibitor recognition. The corresponding Y329S mutation in PDE7 may lead to loss of the hydrogen bonds between rolipram and Gln369 and is thus a possible reason explaining PDE7's insensitivity to rolipram inhibition. Docking of the PDE5 inhibitor sildenafil into the PDE4 catalytic pocket further helps understand inhibitor selectivity.     

Signaling from beta1- and beta2-adrenergic receptors is defined by differential interactions with PDE4

Beta1- and beta2-adrenergic receptors (betaARs) are highly homologous, yet they play clearly distinct roles in cardiac physiology and pathology. Myocyte contraction, for instance, is readily stimulated by beta1AR but not beta2AR signaling, and chronic stimulation of the two receptors has opposing effects on myocyte apoptosis and cell survival. Differences in the assembly of macromolecular signaling complexes may explain the distinct biological outcomes. Here, we demonstrate that beta1AR forms a signaling complex with a cAMP-specific phosphodiesterase (PDE) in a manner inherently different from a beta2AR/beta-arrestin/PDE complex reported previously. The beta1AR binds a PDE variant, PDE4D8, in a direct manner, and occupancy of the receptor by an agonist causes dissociation of this complex. Conversely, agonist binding to the beta2AR is a prerequisite for the recruitment of a complex consisting of beta-arrestin and the PDE4D variant, PDE4D5, to the receptor. We propose that the distinct modes of interaction with PDEs result in divergent cAMP signals in the vicinity of the two receptors, thus, providing an additional layer of complexity to enforce the specificity of beta1- and beta2-adrenoceptor signaling.     

Purification and characterization of the human PDE4A catalytic domain (PDE4A330-723) expressed in Sf9 cells

The human PDE4A catalytic domain (PDE4A330-723) expressed in Sf9 cells was found to be heavily phosphorylated on both serines of the conserved SPS motif by mass spectrometric analysis. The purified protein exists as a tetramer at a concentration approximately 1 mg/ml from light scattering measurement and has a Km of 2 microM in hydrolyzing cAMP. In comparison, a partially purified PDE4A330-723 expressed in Escherichia coli has an apparent Km of 10 microM. The EC50 values for the Mg2+- or Co2+-mediated cAMP hydrolysis between the two enzymes differed by less than twofold. In addition, both enzymes exhibit similar sensitivities toward inhibition by a diverse set of inhibitors. Together with the fact that its adjacent peptide was covalently labeled by an electrophilic cAMP analogue, these results support that the SPS motif is not part of but is positioned near the active site. An efficient purification protocol that provides a highly purified PDE4A catalytic domain suitable for crystallization study is described.     

CD4+ T cell responses elicited by different subsets of human skin migratory dendritic cells

Skin dendritic cells (DC) are professional APC critical for initiation and control of adaptive immunity. In the present work we have analyzed the CD4+ T cell stimulatory function of different subsets of DC that migrate spontaneously from human skin explants, including CD1a+CD14- Langerhans' cells (LC), CD1a-CD14- dermal DC (DDC), and CD1a-CD14+ LC precursors. Skin migratory DC consisted of APC at different stages of maturation-activation that produced IL-10, TGF-beta1, IL-23p19, and IL-12p40, but did not release IL-12p70 even after exposure to DC1-driving stimuli. LC and DDC migrated as mature/activated APC able to stimulate allogeneic naive CD4+ T cells and to induce memory Th1 cells in the absence of IL-12p70. The potent CD4+ T cell stimulatory function of LC and DDC correlated with their high levels of expression of MHC class II, adhesion, and costimulatory molecules. The Th1-biasing function of LC and DDC depended on their ability to produce IL-23. By contrast, CD1a-CD14+ LC precursors migrated as immature-semimature APC and were weak stimulators of allogeneic naive CD4+ T cells. However, and opposite of a potential tolerogenic role of immature DC, the T cell allostimulatory and Th1-biasing function of CD14+ LC precursors increased significantly by augmenting their cell number, prolonging the time of interaction with responding T cells, or addition of recombinant human IL-23 in MLC. The data presented in this study provide insight into the function of the complex network of skin-resident DC that migrate out of the epidermis and dermis after cutaneous immunizations, pathogen infections, or allograft transplantation.     

Analysis of the polymorphic variants of the PDE4D gene in patients with acute stroke in the Moldavian population

The risk of the ischemic stroke is mediated by both environmental and genetic factors. Recent studies of DeCode group identified the risk of polymorphisms for ischemic stroke in the phosphodiesterase 4D gene (PDE4D). The goal of this study was to explore the role of two variants of the gene encoding PDE4D [SNP41 (rs152312) and SNP87 (rs2910829)] in the Moldavian patients with ischemic stroke and in control. No significant association with ischemic stroke was observed with SNP41 and 87.     

Discovery of potent,selective, and orally bioavailable PDE5 inhibitor: Methyl-4-(3-chloro-4-methoxybenzylamino)-8-(2-hydroxyethyl)-7-methoxyquinazolin-6-ylmethylcarbamate (CKD 533)

In a continuing effort to discover novel PDE5 inhibitors, we have successfully found quinazolines with 4-benzylamino substitution as potent and selective PDE5 inhibitors. Initial lead compound (1) was found to be easily metabolized when incubated with human liver microsomes mainly through C6 amide hydrolysis. Blocking of this metabolic hot spot led to discovery of 10 (CKD533) which is highly potent, selective and orally efficacious in conscious rabbit model for erectile dysfunction and now is undergoing preclinical toxicology study.     

T cell antigen receptor expression by subsets of Ly-2-L3T4- (CD8-CD4-) thymocytes

The V beta 8-specific mAb F23.1 and KJ16 were used as fluorescent stains to test for TCR expression on the surface of subpopulations of early, CD4-CD8- (L3T4-Ly-2-) thymocytes from adult CBA mice. A surprisingly high proportion (27%) of Ly-2-L3T4- thymocytes were strongly F23.1 and KJ16 positive. No positive cells were detected among Ly-2-L3T4- thymocytes from V beta 8-negative SJL mice. In contrast to the adult thymus, Ly-2-L3T4- cells from embryonic CBA thymus lacked F23.1-positive cells. Subsets of adult CBA Ly-2-L3T4- thymocytes were separated to determine which expressed V beta 8. The major subset, Ly-1 low B2A2-M1/69+Thy-1+Pgp-1-, representing a phenotype similar to embryonic Ly-2-L3T4- thymocytes and the phenotype commonly isolated from adult thymocytes as Ly-1 "dull," lacked cells strongly positive for F23.1. In contrast, a series of subsets of adult CBA Ly-2-L3T4- thymocytes which were B2A2-M1/69- and Pgp-1+ all included strongly F23.1-positive cells. A minor subset, negative for most markers except Pgp-1 and presumed on the basis of this phenotype and some reconstitution studies to include the earliest intrathymic precursors, contained 28% F23.1-positive cells. However, no F.23.1-positive cells were detected in equivalent "prethymic" populations from bone marrow or from athymic mouse spleen. The subsets of Ly-2-L3T4- thymocytes which were Ly-1 high, B2A2-M1/69-, and Pgp-1+ all contained about 70% F23.1-positive cells, indicating a V beta 8 usage much higher than the mature T cell average. These results indicate that a series of distinct developmental events have occurred within these CD4-CD8- thymocytes previously considered as a single group of early precursor cells, and that some aspects of repertoire selection may be occurring amongst thymocytes which lack CD4 or CD8.     

Inhibition of phosphodiesterase activity, airway inflammation and hyperresponsiveness by PDE4 inhibitor and glucocorticoid in a murine model of allergic asthma

Phosphodiesterase 4 (PDE4) isozyme plays important roles in inflammatory and immunomodulatory cells. In this study, piclamilast, a selective PDE4 inhibitor, was used to investigate the role of PDE4 in respiratory function and inflammation in a murine asthma model. Sensitized mice were challenged with aerosolized ovalbumin for 7 days, piclamilast (1, 3 and 10 mg/kg) and dexamethasone (2 mg/kg) were orally administered once daily during the period of challenge. Twenty-four hours after the last challenge, airway hyperresponsiveness to methacholine was determined by whole-body plethysmography, airway inflammation and mucus secretion by histomorphometry, pulmonary cAMP-PDE activity by HPLC, cytokine levels in bronchoalveolar lavage fluid and their mRNA expression in lung by ELISA and RT-PCR, respectively. In control mice, significant induction of cAMP-PDE activity was parallel to the increases of hyperresponsiveness, inflammatory cells, cytokine levels, mRNA expression as well as goblet cell hyperplasia. However, piclamilast dose-dependently and significantly improved airway resistance and dynamic compliance, and the maximal effect was similar to that of dexamethasone. Piclamilast treatment dose-dependently and significantly prevented the increase in inflammatory cell number and goblet cell hyperplasia, as well as production of cytokines, including eotaxin, TNFalpha and IL-4. Piclamilast exerted a weaker inhibitory effect than dexamethasone on eosinophils and neutrophils, had no effect on lymphocyte accumulation. Moreover, piclamilast inhibited up-regulation of cAMP-PDE activity and cytokine mRNA expression; the maximal inhibition of cAMP-PDE was greater than that exerted by dexamethasone, and was similar to dexamethasone on cytokine mRNA expression. This study suggests that inhibition of PDE4 by piclamilast robustly improves the pulmonary function, airway inflammation and goblet cell hyperplasia in murine allergenic asthma.     

Attenuation of the activity of the cAMP-specific phosphodiesterase PDE4A5 by interaction with the immunophilin XAP2

The cyclic AMP-specific phosphodiesterase (PDE4) isoform PDE4A5 interacted with the immunophilin XAP2 in a yeast two-hybrid assay. The interaction was confirmed in biochemical pull-down analyses. The interaction was specific, in that PDE4A5 did not interact with the closely related immunophilins AIPL1, FKBP51, or FKBP52. XAP2 also did not interact with other PDE4A isoforms or typical isoforms from the three other PDE4 subfamilies. Functionally, XAP2 reversibly inhibited the enzymatic activity of PDE4A5, increased the sensitivity of PDE4A5 to inhibition by the prototypical PDE4 inhibitor 4-[3-(cyclopentyloxy)-4-methoxyphenyl]-2-pyrrolidinone (rolipram) and attenuated the ability of cAMP-dependent protein kinase to phosphorylate PDE4A5 in intact cells. XAP2 maximally inhibited PDE4A5 by approximately 60%, with an IC50 of 120 nm, and reduced the IC50 for rolipram from 390 nm to 70-90 nm. Co-expression of XAP2 and PDE4A5 in COS7 cells showed that they could be co-immunoprecipitated and also reduced both the enzymatic activity of PDE4A5 and its IC50 for rolipram. Native XAP2 and PDE4A5 could be co-immunoprecipitated from the brain. The isolated COOH-terminal half of XAP2 (amino acids 170-330), containing its tetratricopeptide repeat domain, but not the isolated NH2-terminal half (amino acids 1-169), containing the immunophilin homology region, similarly reduced PDE4A5 activity and its IC50 for rolipram. Mutation of Arg271 to alanine, in the XAP2 tetratricopeptide repeat region, attenuated its ability to both interact with PDE4A5 in two-hybrid assays and to inhibit PDE4A5 activity. Either the deletion of a specific portion of the unique amino-terminal region or specific mutations in the regulatory UCR2 domain of PDE4A5 attenuated its ability be inhibited by XAP2. We suggest that XAP2 functionally interacts with PDE4A5 in cells.     

Computational investigation of the dynamic control of cAMP signaling by PDE4 isoform types

Cyclic adenosine monophosphate (cAMP) is a generic signaling molecule that, through precise control of its signaling dynamics, exerts distinct cellular effects. Consequently, aberrant cAMP signaling can have detrimental effects. Phosphodiesterase 4 (PDE4) enzymes profoundly control cAMP signaling and comprise different isoform types wherein enzymatic activity is modulated by differential feedback mechanisms. Because these feedback dynamics are non-linear and occur coincidentally, their effects are difficult to examine experimentally but can be well simulated computationally. Through understanding the role of PDE4 isoform types in regulating cAMP signaling, PDE4-targeted therapeutic strategies can be better specified. Here, we established a computational model to study how feedback mechanisms on different PDE4 isoform types lead to dynamic, isoform-specific control of cAMP signaling. Ordinary differential equations describing cAMP dynamics were implemented in the VirtualCell environment. Simulations indicated that long PDE4 isoforms exert the most profound control on oscillatory cAMP signaling, as opposed to the PDE4-mediated control of single cAMP input pulses. Moreover, elevating cAMP levels or decreasing PDE4 levels revealed different effects on downstream signaling. Together these results underline that cAMP signaling is distinctly regulated by different PDE4 isoform types and that this isoform specificity should be considered in both computational and experimental follow-up studies to better define PDE4 enzymes as therapeutic targets in diseases in which cAMP signaling is aberrant.     

Stereochemistry of the Enzymatic Reduction of 2-(4-Methoxybenzyl)-1-Cyclohexanone by Solanum Aviculare Cells in vitro

During the investigation of chemical properties of the dicyclic system of insect juvenile hormone analogues, biotransformation of 2-(4-methoxybenzyl)-1-cyclohexanone (1) by plant cell cultures was studied. Among other components, the cis-(1S, 2S)- and cis-(1R, 2R)-2-(4-methoxybenzyl)-1-cyclohexanol enantiomers 2a and 2b were found in the reaction mixture. Higher stereoselectivity of the biotransformation was observed for trans-(1S, 2R)-enantiomer 3a formation, which occurred in at least 60% of calculated enantiomeric excess.     

脉冲振荡测定COPD患者呼吸阻抗的临床意义

通过对40名COPD患者，30名健康者，以德国Master sereen PFT肺功能仪对其进行呼吸阻抗测定，并与常规肺功能比较，探讨脉冲振荡法在COPD患者中的应用。