Phosphodiesterase 4B Expression Research Articles

Expression Pattern of PDE4B, PDE4D, and SFRP5 Markers in Colorectal Cancer.

Background and Objectives: Colorectal cancer (CRC) is the most frequently diagnosed malignant disease of the gastrointestinal system, and new diagnostic and prognostic markers are needed to elucidate the complete tumor profile. Materials and Methods: We used CRC tumor tissues (Dukes' A-D) and adjacent noncancerous tissues of 43 patients. Immunohistochemistry was used to examine the expression of phosphodiesterase 4B (PDE4B), phosphodiesterase 4D (PDE4D), and secreted frizzled related protein 5 (SFRP5) markers. We also analyzed the expression levels of PDE4B, PDE4D, and SFRP5 in CRC tissues compared to control tissues using RNA-sequencing data from the UCSC Xena browser. Results: In CRC stages, the distribution of PDE4B-positive cells varied, with differing percentages between epithelium and lamina propria. Statistically significant differences were found in the number of PDE4B-positive epithelial cells between healthy controls and all CRC stages, as well as between different CRC stages. Similarly, significant differences were observed in the number of PDE4B-positive cells in the lamina propria between healthy controls and all CRC stages, as well as between different CRC stages. CRC stage Dukes' C exhibited a significantly higher number of PDE4B-positive cells in the lamina propria compared to CRC stage Dukes' B. Significant differences were noted in the number of PDE4D-positive epithelial cells between healthy controls and CRC stages Dukes' A, B, and D, as well as between CRC stage Dukes' C and stages A, B, and D. CRC stage Dukes' A had significantly more PDE4D-positive cells in the lamina propria compared to stage D. Significant differences were also observed in the number of SFRP5-positive cells in the lamina propria between healthy controls and all CRC stages, as well as between CRC stages Dukes' A and D. While the expression of PDE4D varied across CRC stages, the expression of SFRP5 remained consistently strong in both epithelium and lamina propria, with significant differences noted mainly in the lamina propria. The expression levels of PDE4B, PDE4D, and SFRP5 reveal significant differences in the expression of these genes between CRC patients and healthy controls, with notable implications for patient prognosis. Namely, our results demonstrate that PDE4B, PDE4D, and SFRP5 are significantly under-expressed in CRC tissues compared to control tissues. The Kaplan-Meier survival analysis and the log-rank (Mantel-Cox) test revealed distinct prognostic implications where patients with lower expression levels of SFRP5 exhibited significantly longer overall survival. The data align with our immunohistochemical results and might suggest a potential tumor-suppressive role for these genes in CRC. Conclusions: Considering significantly lower gene expression, aligned with our immunohistochemical data in tumor tissue in comparison to the control tissue, as well as the significantly poorer survival rate in the case of its higher expression, we can hypothesize that SFRP5 is the most promising biomarker for CRC out of the observed proteins. These findings suggest alterations in PDE4B, PDE4D, and SFRP5 expression during CRC progression, as well as between different stages of CRC, with potential implications for understanding the molecular mechanisms involved in CRC development and progression.

Zoledronic Acid Accelerates ER Stress-Mediated Inflammation by Increasing PDE4B Expression in Bisphosphonate-Related Osteonecrosis of the Jaw.

Long-term administration of bisphosphonates can lead to a significant side effect known as bisphosphonate-related osteonecrosis of the jaw (BRONJ). Although macrophage-mediated inflammation has been established as an important factor in BRONJ, the underlying mechanism remains elusive. In the current study, the roles of endoplasmic reticulum (ER) stress in zoledronic acid (ZOL)-induced inflammation were analyzed in macrophages, and the regulatory mechanism of ER stress activation was next investigated. An in vitro model of BRONJ was established by treating RAW264.7 cells with ZOL. The activation of ER stress was analyzed by western blotting and transmission electron microscopy, and inflammation was assessed by quantitative real-time PCR and enzyme-linked immunosorbent assay. ER stress was significantly activated in ZOL-treated macrophages, and inhibition of ER stress by TUDCA, an ER stress inhibitor, suppressed ZOL-induced inflammation in macrophages. Mechanistically, phosphodiesterase 4B (PDE4B) was significantly increased in ZOL-treated macrophages. Forced expression of PDE4B promoted ER stress and inflammation, whereas PDE4B knockdown decreased ZOL-induced ER stress and inflammation in macrophages. More importantly, PDE4B inhibitor could improve ZOL-induced BRONJ in vivo. These data suggest that ZOL accelerates ER stress-mediated inflammation in BRONJ by increasing PDE4B expression. PDE4B inhibition may represent a potential therapeutic strategy for BRONJ. Subsequent research should concentrate on formulating medications that selectively target PDE4B, thereby mitigating the risk of BRONJ in patients undergoing bisphosphonate treatment.

Fear memory regulation by the cAMP signaling pathway as an index of reexperiencing symptoms in posttraumatic stress disorder

Posttraumatic stress disorder (PTSD) is a psychiatric disorder associated with traumatic memory, yet its etiology remains unclear. Reexperiencing symptoms are specific to PTSD compared to other anxiety-related disorders. Importantly, reexperiencing can be mimicked by retrieval-related events of fear memory in animal models of traumatic memory. Recent studies revealed candidate PTSD-associated genes that were related to the cyclic adenosine monophosphate (cAMP) signaling pathway. Here, we demonstrate the tight linkage between facilitated cAMP signaling and PTSD by analyzing loss- and gain-of-cAMP signaling effects on fear memory in mice and the transcriptomes of fear memory-activated mice and female PTSD patients with reexperiencing symptoms. Pharmacological and optogenetic upregulation or downregulation of cAMP signaling transduction enhanced or impaired, respectively, the retrieval and subsequent maintenance of fear memory in mice. In line with these observations, integrative mouse and human transcriptome analysis revealed the reduced mRNA expression of phosphodiesterase 4B (PDE4B), an enzyme that degrades cAMP, in the peripheral blood of PTSD patients showing more severe reexperiencing symptoms and the mouse hippocampus after fear memory retrieval. Importantly, more severe reexperiencing symptoms and lower PDE4B mRNA levels were correlated with decreased DNA methylation of a locus within PDE4B, suggesting the involvement of methylation in the mechanism of PTSD. These findings raise the possibility that the facilitation of cAMP signaling mediating the downregulation of PDE4B expression enhances traumatic memory, thereby playing a key role in the reexperiencing symptoms of PTSD patients as a functional index of these symptoms.

PDE4B promotes the progression of gastric cancer via the PI3K/AKT/MYC pathway and immune infiltration.

Phosphodiesterase 4B (PDE4B) is a key enzyme involved in regulating intracellular cyclic adenosine monophosphate levels and plays a significant role in the diagnosis, classification, treatment, and prognosis of various cancers. However, the role of PDE4B in gastric cancer (GC) remains unclear. We used the GEPIA2 (Gene Expression Profiling Interactive Analysis 2) database to analyze the differential expression level of PDE4B across tumor samples and verified our findings via qPCR and immunohistochemical analysis. We also analyzed the correlation between PDE4B expression levels and clinical pathological parameters, and prognosis, in the database. The effects of PDE4B on GC proliferation, migration, and invasion were evaluated through in vitro and in vivo experiments. Enrichment analysis was performed using bioinformatic tools, and results were validated by western blot analysis. The correlation between PDE4B expression and immune cell infiltration was investigated using bioinformatics tools. PDE4B is highly expressed in GC and is significantly associated with deep infiltration, distant metastasis, tumor, node, metastasis (TNM) stage, and preoperative CA199 levels. Over-expression of PDE4B promotes proliferation, clonal formation, migration, and invasion of GC cells and is associated with poor prognosis. PDE4B promotes the infiltration of immune cells into the tumor microenvironment (TME) and the phosphorylation of PI3K/AKT pathway, increasing MYC expression. PDE4B can serve as an independent prognostic biomarker for GC. We found that PDE4B can promote immune cell infiltration of the TME and mediate malignancy in gastric cancer through the PI3K/AKT/MYC pathway.

Resistance to Prostaglandin E2 Promotes Monocyte Activation During Chronic HIV Infection.

Monocyte activation is a driver of inflammation in the course of chronic HIV infection. Prostaglandin E2 (PGE2) is known to mediate anti-inflammatory effects, notably the inhibition of tumor necrosis factor- (TNF-) production by monocytes. We aim to investigate the effects of PGE2 on activation of monocytes in chronic HIV infection and the mechanisms through which PGE2 modulates their inflammatory signature. We recruited a group of people with HIV (PWH) and matched healthy uninfected persons. We compared plasma levels of PGE2, monocyte activation, and sensitivity of monocytes to the inhibitory actions mediated by PGE2. We found increased plasma levels of PGE2 in PWH, and an activated phenotype in circulating monocytes, compared with uninfected individuals. Monocytes from PWH showed a significant resistance to the inhibitory actions mediated by PGE2; the concentration of PGE2 able to inhibit 50 of the production of TNF- by lipopolysaccharide-stimulated monocytes was 10 times higher in PWH compared with uninfected controls. Furthermore, the expression of phosphodiesterase 4B, a negative regulator of PGE2 activity, was significantly increased in monocytes from PWH. Resistance to the inhibitory actions mediated by PGE2 could account, at least in part, for the inflammatory profile of circulating monocytes in PWH.

Phosphodiesterase 4B activation exacerbates pulmonary hypertension induced by intermittent hypoxia by regulating mitochondrial injury and cAMP/PKA/p-CREB/PGC-1α signaling

Proliferation of smooth muscle cells, oxidative stress, and pulmonary vasoconstriction resulting from intermittent hypoxia (IH) facilitate pulmonary hypertension (PH) in patients with obstructive sleep apnea. The role of Phosphodiesterase 4 B (PDE4B) in PH has not yet been established. Herein, we investigated whether PDE4B inhibition ameliorates experimental PH by modulating cAMP signaling. We performed an integrative analysis of PDE4B expression in Gene Expression Omnibus datasets, experimental IH-induced rat PH samples, and IH-induced pulmonary arterial smooth muscle cells (PASMCs). PDE4B expression was modulated using siRNA in vitro and a specific adeno-associated virus serotype 1 in vivo. In the databases of mouse models of IH-induced and sustained hypoxia-induced PH and in a rat model of six weeks of IH, the expression of PDE4B was up-regulated. Inhibition of PDE4B attenuated IH-induced pulmonary vascular remodeling and right ventricular hypertrophy. Our results also showed that PDE4B deficiency inhibited IH-induced proliferation of PASMCs with less mitochondrial reactive oxygen species and mitochondrial damage. Meanwhile, IH induced an increase in ATF4, which positively regulated the expression of PDE4B through transcription, and inhibition of ATF4 exerted effects similar to those of PDE4B inhibition. Mechanistically, downregulating the expression of PDE4B resulted in the activation of the cAMP/PKA/p-CREB/PGC-1α pathway in PASMCs after IH. Taken together, our present study provides evidence that inhibition of PDE4B attenuates IH-induced PH by regulating cAMP signaling.

Black carrot extract protects against hepatic injury through epigenetic modifications.

We studied the epigenetic regulation of how black carrot extract (BCE) protects against ethanol-induced hepatic damage. We have shown that the butanol-extracted fraction of BCE (BCE-BuOH) increased intracellular cyclic adenosine monophosphate (cAMP) levels by suppressing the expression of phosphodiesterase 4b (PDE4b); however, the detailed mechanism remains to be elucidated. We focused on changes in histone modifications involved in the suppression of pde4 expression. The methylation level of histone H3 lysine 9 (H3K9), which regulates gene expression of PDE4b, decreased after treatment with 100 mM ethanol but was significantly increased by treatment with 400 μg/ml BCE-BuOH. In contrast, ethanol induced an increase in H3K9 acetylation. However, treatment with BCE-BuOH inhibited the increase in acetylation through an increase in Sirtuin 1 (Sirt1), a histone deacetylase. Furthermore, BCE-BuOH treatment increased the level of methionine adenosyltransferase (MAT) 2a mRNA and increased intracellular S-adenosylmethionine. The present results indicate that BCE-BuOH is useful for protection against alcohol-induced hepatic injury. PRACTICAL APPLICATIONS: We have reported that black carrot extract (BCE) suppressed liver steatosis and liver fibrosis on a rat alcoholic liver disease model. The results from this study have shown that BCE regulated the alcoholic-induced hepatic injury at the level of epigenetic modifications. These results suggested that BCE is useful for protection against alcoholic-induced hepatic injury.

Transcription factor JDP2 activates PDE4B to participate in hypoxia/reoxygenation-induced H9c2 cell injury.

Myocardial ischemia/reperfusion (I/R) injury is a clinical challenge in the treatment of acute myocardial infarction (AMI). Phosphodiesterase 4B (PDE4B) expression is upregulated in AMI tissues. Thus, the present study aimed to investigate the role of PDE4B in myocardial I/R injury. H9c2 cardiomyocytes were subjected to hypoxia/reoxygenation (H/R) to establish an in vitro myocardial I/R model. PDE4B expression was detected via reverse transcription-quantitative PCR (RT-qPCR) and western blotting before and after transfection with PDE4B interference plasmids in H/R-stimulated H9c2 cells. Cell viability and cytotoxicity were assessed using the Cell Counting Kit-8 and lactate dehydrogenase assays, respectively. Furthermore, oxidative stress was assessed using malondialdehyde, superoxide dismutase and glutathione/glutathione oxidized ratio detection kits. Cell apoptosis was detected via a TUNEL assay and western blotting. c-Jun dimerization protein 2 (JDP2) expression was also detected via RT-qPCR and western blotting. The dual luciferase reporter and chromatin immunoprecipitation assays were performed to verify the interaction between JDP2 and PDE4B. Following co-transfection with PDE4B interference plasmid and JDP2 overexpression plasmid, cell viability, cytotoxicity, oxidative stress and cell apoptosis were assessed. The results demonstrated that PDE4B knockdown reversed H/R-induced loss of viability and cytotoxicity of H9c2 cells. H/R-induced oxidative stress and cardiomyocyte apoptosis were also alleviated by PDE4B knockdown. In addition, the transcription factor JDP2 was expressed at high levels in H/R-stimulated H9c2 cells, and JDP2 overexpression upregulated PDE4B expression. Notably, JDP2 overexpression partly reversed the ameliorative effect of PDE4B knockdown on H/R-induced H9c2 injury. Taken together, the results of the present study suggested that JDP2-activated PDE4B contributed to H/R-induced H9c2 cell injury.

PDE4B Is a Homeostatic Regulator of Cyclic AMP in Dendritic Cells.

Chronic decreases in the second messenger cyclic AMP (cAMP) occur in numerous settings, but how cells compensate for such decreases is unknown. We have used a unique system—murine dendritic cells (DCs) with a DC-selective depletion of the heterotrimeric GTP binding protein Gαs—to address this issue. These mice spontaneously develop Th2-allergic asthma and their DCs have persistently lower cAMP levels. We found that phosphodiesterase 4B (PDE4B) is the primary phosphodiesterase expressed in DCs and that its expression is preferentially decreased in Gαs-depleted DCs. PDE4B expression is dynamic, falling and rising in a protein kinase A-dependent manner with decreased and increased cAMP concentrations, respectively. Treatment of DCs that drive enhanced Th2 immunity with a PDE4B inhibitor ameliorated DC-induced helper T cell response. We conclude that PDE4B is a homeostatic regulator of cellular cAMP concentrations in DCs and may be a target for treating Th2-allergic asthma and other settings with low cellular cAMP concentrations.

PDE4B Proposed as a High Myopia Susceptibility Gene in Chinese Population.

Myopia is the most common cause of refractive error worldwide. High myopia is a severe type of myopia, which usually accompanies pathological changes in the fundus. To identify high myopia susceptibility genes, DNA-pooling based genome-wide association analysis was used to search for a correlation between single nucleotide polymorphisms and high myopia in a Han Chinese cohort (cases vs. controls in discovery stage: 507 vs. 294; replication stage 1: 991 vs. 1,025; replication stage 2: 1,021 vs. 52,708). Three variants (rs10889602T/G, rs2193015T/C, rs9676191A/C) were identified as being significantly associated with high myopia in the discovery, and replication stage. rs10889602T/G is located at the third intron of phosphodiesterase 4B (PDE4B), whose functional assays were performed by comparing the effects of rs10889602T/T deletion of this risk allele on PDE4B and COL1A1 gene and protein expression levels in the rs10889602T/Tdel/del, rs10889602T/Tdel/wt, and normal control A549 cell lines. The declines in the PDE4B and COL1A1 gene expression levels were larger in the rs10889602T/T deleted A549 cells than in the normal control A549 cells (one-way ANOVA, p < 0.001). The knockdown of PDE4B by siRNA in human scleral fibroblasts led to downregulation of COL1A1. This correspondence between the declines in rs10889602 of the PDE4B gene, PDE4B knockdown, and COL1A1 protein expression levels suggest that PDE4B may be a novel high myopia susceptibility gene, which regulates myopia progression through controlling scleral collagen I expression levels. More studies are needed to determine if there is a correlation between PDE4B and high myopia in other larger sample sized cohorts.

Phosphodiesterase 4B is required for NLRP3 inflammasome activation by positive feedback with Nrf2 in the early phase of LPS- induced acute lung injury

Acute lung injury (ALI) is associated with overproduction of inflammatory mediators in lung tissue. Previous studies have revealed that inflammation induces activation of phosphodiesterase 4B (PDE4B) accompanied by the production of inflammatory mediators, but the detailed mechanism remains unclear. Here, we focused on the NOD-, LRR- and pyrin domain-containing protein 3(NLRP3) inflammasome complexes to study the crosstalk between PDE4B and NF-E2-related factor 2 (Nrf2). We used global knockout PDE4B or Nrf2 mice to prepare LPS induced acute lung injury model by intratracheally administration, and LPS primed bone marrow-derived macrophages (BMDMs), following overexpression of PDE4B or Nrf2, luciferase activity analysis, and chIP-qPCR analyses. We found that deficiency of PDE4B could potently attenuate the lung histopathological changes, suppress the secretion of pro-inflammatory mediators such as tumor necrosis factor α (TNF-α), interleukin (IL)-1β, IL-6, IL-18, and cleaved caspase-1, 8, and GSDMD accompanied with defective activation of the ROS/Nrf2/NLRP3. Meanwhile deficiency of Nrf2 showed the similar results. Furtherly, overexpression by PDE4B or Nrf2 plasmid transfection in MH-S cells could enhance the Nrf2 or PDE4B expression. Luciferase analysis suggested that Nrf2 activated PDE4B promoter activity, while PDE4B could increase Nrf2 substrate ARE activity in MH-S cells in dose dependent manners. ChIP-qPCR analyses showed that Nrf2 bound to the PDE4B promoter region at ̴ 1532 to ̴1199 position in macrophages. Altogether, deficiency of PDE4B inhibit the inflammasome activation and pyroptosis in LPS stimulated lung injury model and macrophages by regulating ROS/Nrf2/NLRP3 activation. The study provides new insight that PDE4B is required for NLRP3 inflammasome activation by positive feedback with Nrf2.

Evidence for reprogramming of monocytes into reparative alveolar macrophages in vivo by targeting PDE4b.

Increased lung vascular permeability and neutrophilic inflammation are hallmarks of acute lung injury. Alveolar macrophages (AMϕ), the predominant sentinel cell type in the airspace, die in massive numbers while fending off pathogens. Recent studies indicate that the AMϕ pool is replenished by airspace-recruited monocytes, but the mechanisms instructing the conversion of recruited monocytes into reparative AMϕ remain elusive. Cyclic AMP (cAMP) is a vascular barrier protective and immunosuppressive second messenger in the lung. Here, we subjected mice expressing GFP under the control of the Lysozyme-M promoter (LysM-GFP mice) to the LPS model of rapidly resolving lung injury to address the impact of mechanisms determining cAMP levels in AMϕ and regulation of mobilization of the reparative AMϕ-pool. RNA-seq analysis of flow-sorted Mϕ identified phosphodiesterase 4b (PDE4b) as the top LPS-responsive cAMP-regulating gene. We observed that PDE4b expression markedly increased at the time of peak injury (4 h) and then decreased to below the basal level during the resolution phase (24 h). Activation of transcription factor NFATc2 was required for the transcription of PDE4b in Mϕ. Inhibition of PDE4 activity at the time of peak injury, using intratracheal rolipram, increased cAMP levels, augmented the reparative AMϕ pool, and resolved lung injury. This response was not seen following conditional depletion of monocytes, thus establishing airspace-recruited PDE4b-sensitive monocytes as the source of reparative AMϕ. Interestingly, adoptive transfer of rolipram-educated AMϕ into injured mice resolved lung edema. We propose suppression of PDE4b as an effective approach to promote reparative AMϕ generation from monocytes for lung repair.

The Potential Regulatory Network of Glutamate Metabolic Pathway Disturbance in Chinese Han Withdrawal Methamphetamine Abusers.

ObjectsTo explore the long-term influence of methamphetamine abuse on metabolomics character, with gas chromatography-mass spectrometry (GS-MS) technology, and the potential regulatory network using the bioinformatics method.MethodsForty withdrawal methamphetamine abusers (WMA) were recruited from Shanghai Gaojing Forced Isolation Detoxification Institute. Forty healthy controls (HC) were recruited from society. GS-MS technology was used to detect metabolic products in serum. A bioinformatics method was used to build a regulatory network. Q-PCR was used to detect the candidate gene expressions, and ELISA was used to detect the regulatory enzyme expressions.ResultsFour pathways were significantly changed in the MA compared to the HC: (1) the arginine synthesis pathway, (2) alanine, aspartic acid and glutamate metabolic pathway, (3) cysteine and methionine metabolic pathway, and (4) the ascorbate and aldarate pathway (enrichment analysis p < 0.05, Impactor factor > 0.2). When focusing on the ‘Alanine, aspartate, and glutamate metabolism’ pathway, a regulatory network was established, and the expression of candidate regulatory genes and enzymes was verified. It was found that the expression of DLG2 (Discs large MAGUK scaffold protein 2), PLA2G4 (Phospholipase A2 group IVE), PDE4D (Phosphodiesterase 4D), PDE4B (Phosphodiesterase 4B), and EPHB2 (Ephrin type-B receptor 2) were significantly different between the two groups (p < 0.05), However, after adjusting for age and BMI, only DLG2, PLA2G4, and EPHB2 remained significant (p < 0.05). The expression of enzymes was not significantly different (p > 0.05).ConclusionMethamphetamine abuse influences the metabolic process in the long term, and DLG2, PLA2G4, and EPHB2 may regulate the glutamate metabolism pathway.

Long non-coding RNA nuclear enriched abundant transcript 1 (NEAT1) sponges microRNA-124-3p to up-regulate phosphodiesterase 4B (PDE4B) to accelerate the progression of Parkinson’s disease

ABSTRACT Reportedly, long non-coding RNA (lncRNA) are crucial modulators in neurodegenerative diseases. Herein, we investigated the role of lncRNA nuclear enriched abundant transcript 1 (NEAT1) in Parkinson’s disease (PD). In-vitro PD model was established based on SH-SY5Y cells treated with 1-methyl-4-phenylpyridinium (MPP+). NEAT1, microRNA (miR) −124-3p and phosphodiesterase 4B (PDE4B) expression levels were examined by qRT-PCR. CCK-8 assay and LDH release assay were adopted to delve into the cell viability and cytotoxicity, respectively. Besides, western blot was utilized to determine mTOR, p-mTOR and PDE4B expression levels. ELISA was executed to detect the levels of tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β) and interleukin 6 (IL-6). Dual-luciferase reporter assay and RIP assay were used to probe the relationship between miR-124-3p and NEAT1 or PDE4B. We demonstrated that, in SH-SY5Y cells treated with MPP+, NEAT1 and PDE4B expression levels were raised, while miR-124-3p expression was repressed; NEAT1 depletion or miR-124-3p overexpression increased the cell viability and suppressed cell injury. Besides, miR-124-3p was confirmed as the direct target of NEAT1, and its down-regulation counteracted the impact of NEAT1 depletion on SH-SY5Y cells. PDE4B was as the downstream target of miR-124-3p, and its overexpression weakens the impact of miR-124-3p on SH-SY5Y cells. Additionally, NEAT1 decoyed miR-124-3p to modulate PDE4B expression. Collectively, in MPP+-induced SH-SY5Y cells, NEAT1 depletion increases cell viability, represses cytotoxicity and reduces inflammatory response by regulating miR-124-3p and PDE4B expression levels, suggesting that NEAT1 may be a promising target for treating PD.

Low concentration of Bupivacaine ameliorates painful diabetic neuropathy by mediating miR-23a/PDE4B axis in microglia

Bupivacaine (Bup) has a certain research basis in pain-related diseases, but it has not been studied in painful diabetic neuropathy. In this study, we investigated the role of Bupivacaine in painful diabetic neuropathy. Mouse model with painful diabetic neuropathy was established, and then treated with different concentrations of Bupivacaine. The blood glucose level in the tail vein and the changes in body weight was measured. The mechanical allodynia, thermal hyperalgesia and thermal allodynia was assessed by pain behavioral tests. Microglia were treated with high glucose (HG) and different concentrations of Bupivacaine. The levels of inflammatory cytokines were detected by using Enzyme-linked immunosorbent assays. Dual luciferase reporter assay explored the relationship between miR-23a and phosphodiesterase 4 B (PDE4B). The results displayed that Bupivacaine ameliorated the mechanical allodynia, thermal hyperalgesia, and thermal allodynia in mice with painful diabetic neuropathy, and is more effective at low concentration. Moreover, low concentration of Bupivacaine inhibited inflammation and promoted miR-23a expression in mice with painful diabetic neuropathy and in microglia induced by HIGH GLUCOSE. Overexpression of miR-23a reduced the levels of inflammatory cytokines by down-regulating PDE4B expression. Knockdown of miR-23a reversed the inhibition effect of Bupivacaine on microglial inflammation. These results revealed that low concentration of Bupivacaine inhibited microglial inflammation through down-regulating PDE4B via miR-23a, thereby attenuated painful diabetic neuropathy.

Cardiac Overexpression of PDE4B Blunts β-Adrenergic Response and Maladaptive Remodeling in Heart Failure.

The cyclic AMP (adenosine monophosphate; cAMP)-hydrolyzing protein PDE4B (phosphodiesterase 4B) is a key negative regulator of cardiac β-adrenergic receptor stimulation. PDE4B deficiency leads to abnormal Ca2+ handling and PDE4B is decreased in pressure overload hypertrophy, suggesting that increasing PDE4B in the heart is beneficial in heart failure. We measured PDE4B expression in human cardiac tissues and developed 2 transgenic mouse lines with cardiomyocyte-specific overexpression of PDE4B and an adeno-associated virus serotype 9 encoding PDE4B. Myocardial structure and function were evaluated by echocardiography, ECG, and in Langendorff-perfused hearts. Also, cAMP and PKA (cAMP dependent protein kinase) activity were monitored by Förster resonance energy transfer, L-type Ca2+ current by whole-cell patch-clamp, and cardiomyocyte shortening and Ca2+ transients with an Ionoptix system. Heart failure was induced by 2 weeks infusion of isoproterenol or transverse aortic constriction. Cardiac remodeling was evaluated by serial echocardiography, morphometric analysis, and histology. PDE4B protein was decreased in human failing hearts. The first PDE4B-transgenic mouse line (TG15) had a ≈15-fold increase in cardiac cAMP-PDE activity and a ≈30% decrease in cAMP content and fractional shortening associated with a mild cardiac hypertrophy that resorbed with age. Basal ex vivo myocardial function was unchanged, but β-adrenergic receptor stimulation of cardiac inotropy, cAMP, PKA, L-type Ca2+ current, Ca2+ transients, and cell contraction were blunted. Endurance capacity and life expectancy were normal. Moreover, these mice were protected from systolic dysfunction, hypertrophy, lung congestion, and fibrosis induced by chronic isoproterenol treatment. In the second PDE4B-transgenic mouse line (TG50), markedly higher PDE4B overexpression, resulting in a ≈50-fold increase in cardiac cAMP-PDE activity caused a ≈50% decrease in fractional shortening, hypertrophy, dilatation, and premature death. In contrast, mice injected with adeno-associated virus serotype 9 encoding PDE4B (1012 viral particles/mouse) had a ≈50% increase in cardiac cAMP-PDE activity, which did not modify basal cardiac function but efficiently prevented systolic dysfunction, apoptosis, and fibrosis, while attenuating hypertrophy induced by chronic isoproterenol infusion. Similarly, adeno-associated virus serotype 9 encoding PDE4B slowed contractile deterioration, attenuated hypertrophy and lung congestion, and prevented apoptosis and fibrotic remodeling in transverse aortic constriction. Our results indicate that a moderate increase in PDE4B is cardioprotective and suggest that cardiac gene therapy with PDE4B might constitute a new promising approach to treat heart failure.

Combination of Phosphodiesterase 4 (PDE4) Inhibitor Roflumilast and R-CHOP As First-Line Therapy for High-Risk Diffuse Large B Cell Lymphoma Patients

R-CHOP remains the standard therapy for diffuse large B cell lymphoma (DLBCL), even though long-term disease free survival is achieved in only ~60-70% of the patients. One approach to improve on this cure rate is to implement rationally devised therapeutic strategies that capitalize on a better understanding of lymphoma biology, and on robust pre-clinical data. Still, in many instances clinical translation is limited by the difficulty in inhibiting key lymphomagenic proteins (e.g., MYC), or by subpar clinical responses and/or toxicity associated with the use of inhibitors to well-validated targets (e.g., SYK, BTK, PI3K, VEGF, etc.). Using genome-wide studies, we identified high expression of phosphodiesterase 4B (PDE4B) in fatal DLBCLs (Nat Med. PMID: 11786909). PDE4 hydroxylases cyclic adenosine monophosphate (cAMP) thus terminating its inhibitory signals and promoting B lymphocytes survival. Using in vitro and in vivo models, we showed that PDE4 inhibition augments cAMP signals and suppresses PI3K-AKT activity in DLBCL (Blood, PMID: 15331441). We mapped these events to SYK inhibition downstream of the B cell receptor (BCR) (Blood, PMID: 19369227). PDE4 inhibitors also suppress the pro-angiogenic lymphoma microenvironment by directly inhibiting the endothelium, and by decreasing VEGF production/secretion by the lymphoma cells, as we showed in a mouse engineered to develop lymphoma in a Pde4b KO background (Leukemia, PMID: 26503641). Earlier, we built on these pre-clinical data to test the safety and pharmacodynamics of the PDE4 inhibitor roflumilast (FDA-approved for COPD) in patients with relapsed/refractory mature B cell malignancies. In that first-in-cancer drug-repurposing study (NCT01888952), we found that roflumilast was well tolerated, clinically active, and that it suppressed PI3K activity, which correlated with the degree of clinical response (Clin. Cancer Res PMID: 27542768). Aberrant BCR signaling and angiogenesis are integral to the pathogenesis of DLBCL. Yet, combination of either ibrutinib or bevacizumab with R-CHOP was not beneficial in this disease. Thus, considering the pleiotropic anti-lymphoma properties of PDE4 inhibitors (seeBloodPMID: 27756749), which includes BCR suppression and anti-angiogenesis, we opened a trial to test the combination of roflumilast with R-CHOP in treatment naïve DLBCLs (NCT03458546). This is a single center, phase Ib, open label, non-randomized single arm study of roflumilast + R-CHOP (RR-CHOP) aimed at untreated high-risk DLBCL patients. Key eligibility criteria include: non-GCB DLBCL (Han's algorithm), NCCN-IPI risk score of ≥ 2, Ann Arbor stage II-IV; key exclusion criteria include active CNS involvement and documented history of severe depression (a specific concern related to the use of roflumilast). Patients will receive R-CHOP every 21 days for 6 cycles, and roflumilast PO daily (500 mcg) throughout the 18-week treatment period. Given the anti-angiogenic properties of PDE4 inhibitors that we documented pre-clinically, and the prohibitive cardiotoxicity of bevacizumab + R-CHOP combination, we will monitor serum levels of troponin and B-type natriuretic peptide, as biomarkers for cardiac toxicity. In addition, PBMC, plasma and urine samples will be collected for measurement SYK/PI3K/AKT activity and VEGF levels at baseline and before cycles 3 and 6. Exome sequencing of germline and tumor DNA is to be performed too. Adverse events will be assessed and documented according to the CTCAE version 4.03 criteria. Eligible patients will be included in the intent-to-treat analysis. The primary objective of this study is to test safety and tolerability of RR-CHOP; the secondary objectives are to preliminarily assess anti-tumor efficacy of the combination and the biomarker potential of SYK/PI3K/AKT activity and VEGF levels. Given its exploratory nature, this study was designed for a cohort of 10 patients, in which no statistical assessment of response or sample size calculation was included. To date, we have met ~50% of the target accrual, with half of those patients being of Hispanics ethnicity. If RR-CHOP is proven safe, the next step will be to initiate a randomized trial that compares RR-CHOP to R-CHOP in untreated DLBCL patients, which should conclusively validate (or refute) our extensive, biologically-informed, pre-clinically data on the benefits of PDE4 inhibitors for the treatment of mature B cell malignancies. Disclosures No relevant conflicts of interest to declare. OffLabel Disclosure: roflumilast - PDE4 inhibitor, FDA-approved for COPD patients.

Correlation Between Phosphodiesterase 4B Expression and Ejection Fraction Following Coronary Artery Occlusion-Induced Heart Failure

Correlation Between Phosphodiesterase 4B Expression and Ejection Fraction Following Coronary Artery Occlusion-Induced Heart Failure

Dexamethasone Inhibits Synergistic Induction of PDE4B Expression by Roflumilast and Bacterium NTHi.

Phosphodiesterase 4B (PDE4B) plays an important role in inflammation. Recently we have reported that roflumilast as a PDE4-selective inhibitor, synergizes with nontypeable Haemophilus influenzae (NTHi) to up-regulate PDE4B expression in vitro and in vivo. Clinical evidence and our previous results suggest that synergistic induction of PDE4B could be counterproductive for suppressing inflammation or may contribute to tolerance to roflumilast. We thus investigated if dexamethasone inhibits the synergistic induction of PDE4B by roflumilast and NTHi as well as inflammation. Here, dexamethasone markedly suppressed the synergistic induction of PDE4B in human lung epithelial cells and in vivo. We also found that dexamethasone further suppressed NTHi-induced inflammatory response in vitro and in vivo. Moreover, Compound A, as a dissociating non-steroidal glucocorticoid receptor (GR) ligand, inhibited the synergistic induction of PDE4B, thereby suggesting the requirement of dexamethasone-mediated GR activation in the suppression of PDE4B expression. Taken together, our data suggest that dexamethasone may help attenuate inflammation and tolerance through suppressing the PDE4B expression in chronic obstructive pulmonary disease (COPD) patients using roflumilast.

Altered expression of microRNA-23a in psoriatic arthritis modulates synovial fibroblast pro-inflammatory mechanisms via phosphodiesterase 4B

ObjectivesTo investigate the functional role of miR-23a in synovial fibroblasts (SFC) activation in psoriatic arthritis (PsA). MethodsDifferential expression of the miR-23a-27a-24-2 cluster was identified by real-time quantitative PCR in PsA synovial tissue and peripheral blood mononuclear cells (PBMC) compared to osteoarthritis (OA) and correlated with disease activity. For regulation experiments, PsA synovial fibroblasts (SFC) were cultured with Toll-like receptor (TLR) ligands and pro-inflammatory cytokines. PsA SFC were transfected with a miR-23a inhibitor to assess the functional effect on migration, invasion and expression of pro-inflammatory meditators. The direct interaction between miR-23a and predicted target mRNA, phosphodiesterase 4B (PDE4B), was examined by luciferase reporter gene assay, with the expression and regulation confirmed by RT-PCR and western blot. A PDE4 inhibitor was used to analyse the function of PDE4B signalling in both miR-23a and Poly(I:C)-induced PsA SFC activation. ResultsSynovial tissue expression of miR-23a was lower in PsA compared to OA and correlated inversely with disease activity and synovitis. TLR activation via Poly(I:C) and LPS, but not Pam3CSK4, significantly decreased miR-23a expression, with no significant effect observed in reponse to stimulation with pro-inflammatory cytokines. Decreased miR-23a expression enhanced PsA SFC migration, invasion and secretion of IL-6, IL-8, MCP-1, RANTES and VEGF. We identified PDE4B as a direct target of miR-23a and demonstrated enhanced mRNA and protein expression of PDE4B in anti-miR-23a transfected PsA SFC. Poly(I:C) and/or miR-23a-induced migration and enhanced cytokine expression was suppressed by the blockade of PDE4 signalling. ConclusionsIn PsA, dysregulated miR-23a expression contributes to synovial inflammation through enhanced SFC activation, via PDE4B signalling, and identifies a novel anti-inflammatory mechanism of PDE4 blockade.