Phosphodiesterase 4D Research Articles

An Empirical Quantitative Structure-Activity Relationship Equation Assists the Discovery of High-Affinity Phosphodiesterase 4D Inhibitors as Leads to PET Radioligands.

A positron emission tomography (PET) radioligand for imaging phosphodiesterase 4D (PDE4D) would benefit drug discovery and the investigation of neuropsychiatric disorders. The most promising radioligand to date, namely, [11C]T1650, has shown unstable quantification in humans. Structural elaboration of [11C]T1650 was therefore deemed necessary. High target affinity in the low nM range is usually required for successful PET radioligands. In our PDE4D PET radioligand development, we formulated and optimized an empirical equation (log[IC50 (nM)] = P1 + P2 + P3 + P4) that well described the relationship between binding affinity and empirically derived values (P1-P4) for the individual fragments in four subregions commonly composing each inhibitor (R2 = 0.988, n = 62). This equation was used to predict compounds that would have high inhibitory potency. Fourteen new compounds were obtained with IC50 of 0.3-10 nM. Finally, eight compounds were judged to be worthy of future radiolabeling and evaluation as PDE4D PET radioligands.

Circ_0101874 overexpression strengthens PDE4D expression by targeting miR-335-5p to promote neuronal injury in ischemic stroke

BackgroundIschemic stroke has been a public concern, while its pathogenesis is not fully understood. Increasing evidence suggests that circular RNAs (circRNAs) are involved in this disorder. The purpose of this study was to explore the role of circ_0101874 in ischemic stroke. MethodsThe in vivo model of ischemic stroke was established in mice with middle cerebral artery occlusion (MCAO) treatment. The in vitro model of ischemic stroke was established in SK-N-SH cells with oxygen-glucose deprivation (OGD) treatment. The expression of circ_0101874, miR-335-5p and phosphodiesterase 4D (PDE4D) mRNA was measured by quantitative real-time PCR (qPCR). The release of inflammatory factors was checked by ELISA. Cell viability, cell proliferation and cell apoptosis were detected using CCK-8 assay, EdU assay and flow cytometry assay, respectively. The protein levels of cyclinD1, cleaved-caspase-3 and PDE4D were detected by western blot. The interaction between miR-335-5p and circ_0101874 or PDE4D was validated by dual-luciferase reporter assay and RIP assay. ResultsCirc_0101874 was highly expressed in MCAO animal models and OGD-induced SK-N-SH cells. Circ_0101874 knockdown suppressed OGD-enhanced inflammation, cell apoptosis and oxidative stress and promoted OGD-inhibited cell viability and cell proliferation in SK-N-SH cells. Circ_0101874 directly bound to miR-335-5p, and miR-335-5p inhibition reversed the effects of circ_0101874 knockdown. PDE4D was a target gene of miR-335-5p, and PDE4D overexpression recovered OGD-promoted SK-N-SH cell injuries that were blocked by miR-335-5p enrichment. Circ_0101874 bound to miR-335-5p to enhance the expression of PDE4D. ConclusionCirc_0101874 knockdown alleviated OGD-induced neuronal cell injury by suppressing PDE4D via regulating miR-335-5p.

Phosphodiesterase 4D contributes to angiotensin II-induced abdominal aortic aneurysm through smooth muscle cell apoptosis

Abdominal aortic aneurysm (AAA) is a permanent expansion of the abdominal aorta that has a high mortality but limited treatment options. Phosphodiesterase (PDE) 4 family members are cAMP-specific hydrolyzing enzymes and have four isoforms (PDE4A-PDE4D). Several pan-PDE4 inhibitors are used clinically. However, the regulation and function of PDE4 in AAA remain largely unknown. Herein, we showed that PDE4D expression is upregulated in human and angiotensin II-induced mouse AAA tissues using RT-PCR, western blotting, and immunohistochemical staining. Furthermore, smooth muscle cell (SMC)-specific Pde4d knockout mice showed significantly reduced vascular destabilization and AAA development in an experimental AAA model. The PDE4 inhibitor rolipram also suppressed vascular pathogenesis and AAA formation in mice. In addition, PDE4D deficiency inhibited caspase 3 cleavage and SMC apoptosis in vivo and in vitro, as shown by bulk RNA-seq, western blotting, flow cytometry and TUNEL staining. Mechanistic studies revealed that PDE4D promotes apoptosis by suppressing the activation of cAMP-activated protein kinase A (PKA) instead of the exchange protein directly activated by cAMP (Epac). Additionally, the phosphorylation of BCL2-antagonist of cell death (Bad) was reversed by PDE4D siRNA in vitro, which indicates that PDE4D regulates SMC apoptosis via the cAMP-PKA-pBad axis. Overall, these findings indicate that PDE4D upregulation in SMCs plays a causative role in AAA development and suggest that pharmacological inhibition of PDE4 may represent a potential therapeutic strategy.

Roflumilast-Mediated Phosphodiesterase 4D Inhibition Reverses Diabetes-Associated Cardiac Dysfunction and Remodeling: Effects Beyond Glucose Lowering

Patients with type 2 diabetes have a substantial risk of developing cardiovascular disease. Phosphodiesterase 4 (PDE4) dysregulation is of pathophysiological importance in metabolic disorders. For determination of the role of PDE4 in diabetic cardiac dysfunction, mice fed with a high-fat diet (HFD) were treated by pharmacological inhibition of PDE4 or cardiac specific knocking down of PDE4D. Mice on HFD developed diabetes and cardiac dysfunction with increased cardiac PDE4D5 expression. PDE4 inhibitor roflumilast can reverse hyperglycemia and cardiac dysfunction, accompanied by the decrease of PDE4D expression and increase of muscle specific miRNA miR-1 level in hearts. Either cardiac specific PDE4D knockdown or miR-1 overexpression significantly reversed cardiac dysfunction in HFD mice, despite persistence of hyperglycemia. Findings of gain- and loss-of-function studies of PDE4D in cardiomyocytes indicated that inhibition of insulin-induced PDE4D protected cardiac hypertrophy by preserving miR-1 expression in cardiomyocytes through promoting cAMP-CREB-Sirt1 signaling-induced SERCA2a expression. We further revealed that insulin also induced PDE4D expression in cardiac fibroblasts, which causes cardiac fibrosis through TGF-β1 signaling-mediated miR-1 reduction. Importantly, the expression of PDE4D5 was increased in human failing hearts of individuals with diabetes. These studies elucidate a novel mechanism by which hyperinsulinemia-induced cardiac PDE4D expression contributes to diabetic cardiac remodeling through reducing the expression of miR-1 and upregulation of miR-1 target hypertrophy and fibrosis-associated genes. Our study suggests a therapeutic potential of PDE4 inhibitor roflumilast in preventing or treating cardiac dysfunction in diabetes in addition to lowering glucose.

Hemodynamic stress‐induced cardiac remodelling is not modulated by ablation of phosphodiesterase 4D interacting protein

Adrenergic stimulation in the heart activates the protein kinase A (PKA), which phosphorylates key proteins involved in intracellular Ca2+ handling. PKA is held in proximity to its substrates by protein scaffolds, the A kinase anchoring proteins (AKAPs). We have previously identified the transcript of phosphodiesterase 4D interacting protein (Pde4dip; also known as myomegalin), one of the sarcomeric AKAPs, as being differentially expressed following hemodynamic overload, a condition inducing hyperadrenergic state in the heart. Here, we addressed whether PDE4DIP is involved in the adverse cardiac remodelling following hemodynamic stress. Homozygous Pde4dip knockout (KO) mice, generated by CRISPR‐Cas9 technology, and wild‐type (WT) littermates were exposed to aortocaval shunt (shunt) or transthoracic aortic constriction (TAC) to induce hemodynamic volume overload (VO) or pressure overload (PO), respectively. The mortality, cardiac structure, function and pathological cardiac remodelling were followed up after hemodynamic injuries. The PDE4DIP protein level was markedly downregulated in volume‐overloaded‐ but upregulated in pressure‐overloaded‐WT hearts. Following shunt or TAC, mortality rates were comparably increased in both genotypes. Twelve weeks after shunt or TAC, Pde4dip‐KO animals showed a similar degree of cardiac hypertrophy, dilatation and dysfunction as WT mice. Cardiomyocyte hypertrophy, myocardial fibrosis, reactivation of cardiac stress genes and downregulation of ATPase, Ca2+ transporting, cardiac muscle, slow twitch 2 transcript did not differ between WT and Pde4dip‐KO hearts following shunt or TAC. In summary, despite a differential expression of PDE4DIP protein in remodelled WT hearts, Pde4dip deficiency does not modulate adverse cardiac remodelling after hemodynamic VO or PO.

Biomarker discovery for practice of precision medicine in hypopharyngeal cancer: a theranostic study on response prediction of the key therapeutic agents

BackgroundHypopharyngeal cancer is a relatively rare malignancy with poor prognosis. Current chemotherapeutic algorithm is still far from personalized medicine, and the identification of the truly active therapeutic biomarkers and/or targets is eagerly awaited.MethodsVenturing to focus on the conventional key chemotherapeutic drugs, we identified the most correlative genes (and/or proteins) with cellular sensitivity to docetaxel (TXT), cisplatin (CDDP) and 5-fluorouracil (5-FU) in the expression levels, through 3 steps approach: genome-wide screening, confirmation study on the quantified expression levels, and knock-down and transfection analyses of the candidates. The probable action pathways of selected genes were examined by Ingenuity Pathway Analysis using a large-scale database, The Cancer Genome Atlas.ResultsThe first genome-wide screening study derived 16 highly correlative genes with cellular drug sensitivity in 15 cell lines (|R| > 0.8, P < 0.01 for CDDP and 5-FU; |R| > 0.5, P < 0.05 for TXT). Among 10 genes the observed correlations were confirmed in the quantified gene expression levels, and finally knock-down and transfection analyses provided 4 molecules as the most potent predictive markers-AGR2 (anterior gradient 2 homolog gene), and PDE4D (phosphodiesterase 4D, cAMP-specific gene) for TXT; NINJ2 (nerve Injury-induced protein 2); CDC25B (cell division cycle 25 homolog B gene) for 5-FU- in both gene and protein expression levels. Overexpression of AGR2, PDE4D signified worse response to TXT, and the repressed expression sensitized TXT activity. Contrary to the findings, in the other 2 molecules, NINJ2 and CDC25, there observed opposite relationship to cellular drug response to the relevant drugs. IPA raised the potential that each selected molecule functionally interacts with main action pathway (and/or targets) of the relevant drug such as tubulin β chain genes for TXT, DNA replication pathway for CDDP, and DNA synthesis pathway and thymidylate synthetase gene for 5-FU.ConclusionWe newly propose 4 molecules -AGR2, PDE4D,NINJ2 and CDC25B) as the powerful exploratory markers for prediction of cellular response to 3 key chemotherapeutic drugs in hypopharyngeal cancers and also suggest their potentials to be the therapeutic targets, which could contribute to the development of precision medicine of the essential chemotherapy in hypopharyngeal patients. (339 words).

Circular RNA from phosphodiesterase 4D can attenuate chondrocyte apoptosis and matrix degradation under OA milieu induced by IL-1β via circPDE4D/miR-4306/SOX9 Cascade

Background Phosphodiesterase 4D (PDE4D) is a novel molecular therapeutic agent for human diseases, including Alzheimer’s disease, ischemic stroke, asthma, and cancers. Circular RNA from PDE4D (circPDE4D; ID hsa_circ_0072568) was one of the most downregulated circRNAs in OA patients. However, its precise role in OA-related chondrocytes was largely unknown. Methods Expressions of circPDE4D, microRNA (miR)-4306, and sex-determining region Y-box 9 (SOX9) were measured by quantitative real-time PCR; protein levels of SOX9 and proteins related to apoptosis and extracellular matrix (ECM) were detected by Western blotting. Cell apoptosis was assessed by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, 5-ethynyl-2′-deoxyuridine and Annexin V-fluorescein isothiocyanate apoptosis assays. MiR-4306 response elements were predicted by bioinformatics algorithm and identified using dual-luciferase reporter, RNA immunoprecipitation, and biotin-coupled miRNA capture assays. Results CircPDE4D was markedly downregulated in OA cartilages and interleukin (IL)-1β-stressed human normal chondrocytes (HNC). Ectopic expression of circPDE4D rescued cell viability, proliferation, and expressions of B-cell lymphoma/leukemia-2 (Bcl-2) and Collagen type II α1 in IL-1β-insulted HNC, and meanwhile declined apoptosis rate and levels of Bcl-2-associated X protein, cleaved caspase-3, cleaved poly (ADP-ribose) polymerase-1, matrix metalloproteinase-13, ADAM metallopeptidase with thrombospondin type 1 motif 5, IL-6, and IL-8. CircPDE4D and SOX9 were competing endogenous RNAs (ceRNAs) for miR-4306, and circPDE4D could positively regulate SOX9 expression via miR-4306. Conclusion CircPDE4D and miR-4306 were important regulators in regulating IL-1β-induced HNC apoptosis and matrix degradation via regulating the key transcription factor SOX9, suggesting a novel circPDE4D/miR-4306/SOX9 ceRNA pathway in OA-related chondrocyte dysfunction.

PDE4D binds and interacts with YAP to cooperatively promote HCC progression

The role of cAMP in the development of hepatocellular carcinoma (HCC) is controversial and the biological function of cAMP-hydrolysing enzyme phosphodiesterase 4D (PDE4D) in HCC remains unclear. In this study, we observed markedly higher PDE4D expression in HCC patients with poor survival. PDE4D bound to yes-associated protein (YAP), and PDE4D expression positively correlated with YAP expression in HCC. Overexpression of PDE4D increased YAP dephosphorylation and activity and promoted HCC cell growth in vitro and in vivo, which was attenuated by the YAP inhibitor verteporfin. In contrast, silencing PDE4D reduced YAP expression and HCC cell growth. Notably, forced expression of YAP promoted PDE4D and YAP target gene expression and cell growth, which were abrogated by the PDE4D inhibitor roflumilast. Mechanistically, silencing of YAP caused PDE4D downregulation and HCC cell apoptosis via extracellular signal-regulated kinase (ERK) activation. Roflumilast activated cAMP-PKA signaling and induced cAMP-PKA-dependent YAP phosphorylation at serine 127, resulting in YAP degradation and suppression of HCC growth, which were reversed by the PKA inhibitor PKI. Additionally, transfection of the YAP-S127A mutant reversed roflumilast-mediated suppression of YAP and cell growth. Taken together, our findings indicate that PDE4D binds to and interacts with YAP to promote HCC progression. Targeting the PDE4D-YAP interaction with roflumilast may be an effective strategy for HCC treatment.

Selective inhibition of phosphodiesterase 4D increases tau phosphorylation at Ser214 residue.

Tau is a protein that normally participates in the assembly and stability of microtubules. However, it can form intraneuronal hyperphosphorylated aggregates that are hallmarks of Alzheimer's disease and other neurodegenerative disorders known as tauopathies. Tau can be phosphorylated by multiple kinases at several sites. Among such kinases, the cAMP-dependent protein kinase A (PKA) phosphorylates tau at Ser214 (pTAU-S214), an event that was shown to reduce the pathological assembly of the protein. Given that the neuronal cAMP/PKA-activated cascade is involved in synaptic plasticity and memory, and that cAMP-enhancing strategies demonstrated promising therapeutic potential for the treatment of cognitive deficits, we investigated the impact of cAMP on pTAU-S214 in N2a cells and rat hippocampal slices. Our results confirm that the activation of adenylyl cyclase increases pTAU-S214 in both model systems and, more interestingly, this effect is mimicked by GEBR-7b, a phosphodiesterase 4D inhibitor with proven pro-cognitive efficacy in rodents.

Endosomal b2‐adrenergic receptor controls nuclear cAMP signal in learning and memory via sequestration of phosphodiesterase 4D

Learning and memory‐related behaviors depend on GPCR signaling to the nucleus to induce Immediate early gene (IEG) expression. However, the molecular mechanisms mediating this process remain unclear. We have previously identified a subpopulation of β2AR which is preferentially phosphorylated by GRK sites and internalized to the endosome upon stimulation. Here, we demonstrate that GRK phosphorylated endosomal β2Adrenergic Receptor (β2AR) indirectly facilitates nuclear cAMP signaling through sequestration of a PDE4D/β‐arrestin complex. Combining subcellular cAMP biosensors and transgenic mice, we found deleting GRK phosphorylation sites on β2AR (GRKD) blocked agonist‐induced receptor endocytosis and nuclear cAMP signaling in hippocampal neurons. This resulted in impaired PKA‐mediated IEGs expression in vitro,reduced learning induced IEG expression in vivo, and impaired long‐term memory in a Morris water maze. Mechanistically, while β2AR stimulation promoted β‐arrestin‐dependent recruitment of PDE4D5, inhibition of β‐arrestin‐PDE4D5 association alone prevented β2AR‐induced increases in nuclear cAMP signaling without affecting receptor endocytosis. Furthermore, direct PDE4 inhibition was sufficient to rescue the β2AR‐induced nuclear cAMP signal in GRKD neurons and ameliorate the long‐term memory deficits in GRKD mice. This work indicates that the sequestration of the β‐arrestin/PDE4D complex by the GRK‐phosphorylated β2AR critically controls the receptor‐induced nuclear cAMP signals. This constitutes a novel mechanism by which GPCR activation promotes nuclear cAMP signaling through endocytosis dependent relocalization of PDE4D isoforms, IEG expression, and learning and memory‐related behaviors.

Association of plaque enhancement on vessel wall MRI and the phosphodiesterase 4D variant with stroke recurrence in patients with symptomatic intracranial atherosclerosis.

Vessel wall MRI (VW-MRI) can be used to evaluate the nature of intracranial atherosclerosis (ICAS) plaque in vivo. Phosphodiesterase 4D (PDE4D) participates in stroke development. This study aims to explore the value of VW-MRI findings and the PDE4D genevariant in predicting stroke recurrence in patients with ICAS. We prospectively recruited 324 symptomatic ICAS patients. VW-MRI was performed to determine luminal and wall changes. PDE4D genesingle-nucleotide polymorphisms (SNPs)-namely, SNP32, SNP83, and SNP87-were determined by direct sequencing. The risk factors of stroke recurrence were analyzed using the multivariate Cox proportional hazards model. Of the 324 subjects, 97 (29.9%) experienced recurrent ischemic stroke during the follow-up period. A total of 254 patients (78.4%) showed plaque enhancement; 87 of these patients experienced stroke recurrence. The CT/CC genotype frequencies of PDE4D83 were significantly higher in participants with recurrent stroke than in patients without stroke recurrence (p = 0.019 and p < 0.001, respectively). However, the PDE4D32 and PDE4D87 variants were not correlated with recurrent stroke. Multivariate analysis showed that plaque enhancement from VW-MRI (HR 4.52, 95% CI 2.35-8.73, p < 0.001) and the PDE4D83 variant (HR 7.43, 95% CI 1.75-31.87, p = 0.005) were independently correlated with stroke recurrence. Kaplan-Meier curves showed significant differences in stroke recurrence rates between the plaque-enhanced group and the non-enhanced group (p < 0.001) and between the PDE4D83 variant carriers and noncarriers (p = 0.002). Plaque enhancement on VW-MRI and the presence of the PDE4D83 variant are associated with ischemic stroke recurrence in subjects with symptomatic ICAS.

Genetic determinants of intracranial large artery stenosis in the northern Manhattan study

BackgroundIntracranial stenosis is one of the most common causes of stroke worldwide. Several single nucleotide polymorphisms have been associated with intracranial atherosclerosis, which is inferred to be the most common underlying cause of intracranial large artery stenosis (ILAS). We previously reviewed known genetic variants related to ILAS in predominantly Asian cohorts, but their prevalence and role in ILAS among western multiethnic populations are uncertain. MethodsWe leveraged existing imaging and genetic data from the Northern Manhattan Study, a multiethnic prospective cohort study. Based on literature review, we selected adiponectin Q (ADIPOQ) rs2241767 and rs182052, ring finger protein 213 (RNF213) rs112735431, apolipoprotein E (APOE) rs429358, phosphodiesterase 4D (PDE4D) rs2910829, lipoprotein lipase (LPL) rs320, and aldosterone synthase (CYP11B2) rs1799998 variants as candidates to explore. We defined ILAS as luminal stenosis >50% in any intracranial large artery using time-of-flight magnetic resonance angiography (MRA). ResultsWe included 1109 participants (mean age 70 ± 9 years, 70% Hispanic, 60% women) in this study. ILAS was identified in 81 (7%) NOMAS participants. Logistic regression analyses adjusted for age, sex, principal components, and vascular risk factors showed ILAS prevalence associated with CYP11B2 rs1799998 under the dominant model (OR = 0.56, 95%CI: 0.35–0.89) and LPL rs320 heterozygote genotype (OR = 1.68, 95%CI: 1.05–2.71). The genotype distributions of ADIPOQ rs2241767 and rs182052, APOE rs429358 and CYP11B2 rs1799998 variants were significantly different among non-Hispanic white and Black, and Hispanic groups. When participants were further stratified by race/ethnicity, the estimates were consistent for CYP11B2 rs1799998 across race/ethnic groups but not for LPL rs320. ConclusionThe CYP11B2 rs1799998 variant may be a protective genetic factor for ILAS across race/ethnic groups, but the risk of ILAS associated with LPL rs320 varies by race/ethnic group. Further functional studies may help elucidate the role that these variants play in the pathophysiology of ILAS.

The relationship between phosphodiesterase 4D gene polymorphism and coronary heart disease.

Coronary atherosclerotic heart disease is one of the most common heart diseases that seriously endanger human health. The study found that intracellular second messenger CAMP plays an important role in inhibiting the proliferation and migration of vascular smooth muscle and the local inflammatory response at the damaged vessel. Phosphodiesterase 4D (PDE4D) can specifically degrade cAMP. The purpose of this article is to investigate the relationship between phosphodiesterase 4D gene polymorphism and coronary heart disease and the effect of phosphodiesterase 4D gene polymorphism on cardiovascular, using polymerase chain reaction-restriction fragment length polymorphism method (PCR-RFLP) was used to detect 50 patients with coronary heart disease (ACS group) and 100 patients who were diagnosed with coronary artery disease by coronary angiography at the same time as the control group (NC group). The results of the study showed that the frequencies of AA, AG, and GG genotypes in 150 samples were 25.67%, 54.66%, and 22.67%, respectively, which was consistent with Hard Weber's law (X = 2.186, P = 0.101). The distribution of GG genotype (18% vs. 27%), AA + AG genotype (85% vs. 74%), G (57% vs. 55%), A (43% vs. 45 %) There was no statistically significant difference in allele frequency (P <0.05). From this, it can be seen that the rs918592 polymorphism of the PDE4D gene is not associated with coronary heart disease.

Phosphodiesterase 4D promotes angiotensin II-induced hypertension in mice via smooth muscle cell contraction

Hypertension is a common chronic disease, which leads to cardio-cerebrovascular diseases, and its prevalence is increasing. The cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) pathway participates in multiple cardiovascular diseases. Phosphodiesterase (PDE) 4 has been shown to regulate PKA activity via cAMP specific hydrolysis. However, whether PDE4-cAMP-PKA pathway influences hypertension remains unknown. Herein, we reveal that PDE4D (one of PDE4 isoforms) expression is upregulated in the aortas of experimental hypertension induced by angiotensin II (Ang II). Furthermore, knockout of Pde4d in mouse smooth muscle cells (SMCs) attenuates Ang II-induced hypertension, arterial wall media thickening, vascular fibrosis and vasocontraction. Additionally, we find that PDE4D deficiency activates PKA-AMP-activated protein kinase (AMPK) signaling pathway to inhibit myosin phosphatase targeting subunit 1 (MYPT1)-myosin light chain (MLC) phosphorylation, relieving Ang II-induced SMC contraction in vitro and in vivo. Our results also indicate that rolipram, a PDE4 inhibitor, may be a potential drug for hypertension therapy.

MicroRNA-223-3p Protect Against Radiation-Induced Cardiac Toxicity by Alleviating Myocardial Oxidative Stress and Programmed Cell Death via Targeting the AMPK Pathway.

Objectives: Radiotherapy improves the survival rate of cancer patients, yet it also involves some inevitable complications. Radiation-induced heart disease (RIHD) is one of the most serious complications, especially the radiotherapy of thoracic tumors, which is characterized by cardiac oxidative stress disorder and programmed cell death. At present, there is no effective treatment strategy for RIHD; in addition, it cannot be reversed when it progresses. This study aims to explore the role and potential mechanism of microRNA-223-3p (miR-223-3p) in RIHD. Methods: Mice were injected with miR-223-3p mimic, inhibitor, or their respective controls in the tail vein and received a single dose of 20 Gy whole-heart irradiation (WHI) for 16 weeks after 3 days to construct a RIHD mouse model. To inhibit adenosine monophosphate activated protein kinase (AMPK) or phosphodiesterase 4D (PDE4D), compound C (CompC) and AAV9-shPDE4D were used. Results: WHI treatment significantly inhibited the expression of miR-223-3p in the hearts; furthermore, the levels of miR-223-3p decreased in a radiation time-dependent manner. miR-223-3p mimic significantly relieved, while miR-223-3p inhibitor aggravated apoptosis, oxidative damage, and cardiac dysfunction in RIHD mice. In addition, we found that miR-223-3p mimic improves WHI-induced myocardial injury by activating AMPK and that the inhibition of AMPK by CompC completely blocks these protective effects of miR-223-3p mimic. Further studies found that miR-223-3p lowers the protein levels of PDE4D and inhibiting PDE4D by AAV9-shPDE4D blocks the WHI-induced myocardial injury mediated by miR-223-3p inhibitor. Conclusion: miR-223-3p ameliorates WHI-induced RIHD through anti-oxidant and anti-programmed cell death mechanisms via activating AMPK by PDE4D regulation. miR-223-3p mimic exhibits potential value in the treatment of RIHD.

Insights into the potential of withanolides as Phosphodiesterase-4 (PDE4D) inhibitors

Medicinal herbs have been used as traditional medicines for centuries. The molecular mechanism of action of their bioactive molecules against various diseases or therapeutic targets is still being explored. Here, the active compounds (withanolides) of a well-known Indian medicinal herb, Ashwagandha (Withania somnifera), have been studied for their most potential therapeutic targets and their mechanism of action using ligand-based screening and receptor-based approaches. Ligand-based screening predicted the six top therapeutic targets, namely, Protein kinase C alpha (PRKCA), Protein kinase C delta (PRKCD), Protein kinase C epsilon (PRKCE), Androgenic Receptor (AR), Cycloxygenase-2 (PTGS-2) and Phosphodiesterase-4D (PDE4D). Further, when these predictions were validated using receptor-based studies, i.e. molecular docking, molecular dynamics simulation and free energy calculations, it was found that PDE4D was the most potent target for four withanolides, namely, Withaferin-A, 17-Hydroxywithaferin-A, 27-Hydroxywithanone and Withanolide-R. These compounds had a better binding affinity and similar interactions as that of an already known inhibitor (Zardaverine) of PDE4D. These results warrant further in-vitro and in-vivo investigations to examine their therapeutic potential as an inhibitor of PDE4D. Communicated by Ramaswamy H. Sarma

5-Hydroxymethylcytosine Signatures in Circulating Cell-Free DNA as Early Warning Biomarkers for COVID-19 Progression and Myocardial Injury

Background: The symptoms of coronavirus disease 2019 (COVID-19) range from moderate to critical conditions, leading to death in some patients, and the early warning indicators of the COVID-19 progression and the occurrence of its serious complications such as myocardial injury are limited. Methods: We carried out a multi-center, prospective cohort study in three hospitals in Wuhan. Genome-wide 5-hydroxymethylcytosine (5hmC) profiles in plasma cell-free DNA (cfDNA) was used to identify risk factors for COVID-19 pneumonia and develop a machine learning model using samples from 53 healthy volunteers, 66 patients with moderate COVID-19, 99 patients with severe COVID-19, and 38 patients with critical COVID-19. Results: Our warning model demonstrated that an area under the curve (AUC) for 5hmC warning moderate patients developed into severe status was 0.81 (95% CI 0.77–0.85) and for severe patients developed into critical status was 0.92 (95% CI 0.89–0.96). We further built a warning model on patients with and without myocardial injury with the AUC of 0.89 (95% CI 0.84–0.95). Conclusion: This is the first study showing the utility of 5hmC as an accurate early warning marker for disease progression and myocardial injury in patients with COVID-19. Our results show that phosphodiesterase 4D and ten-eleven translocation 2 may be important markers in the progression of COVID-19 disease.

FGF1 and insulin control lipolysis by convergent pathways

Inexorable increases in insulin resistance, lipolysis, and hepatic glucose production (HGP) are hallmarks of type 2 diabetes. Previously, we showed that peripheral delivery of exogenous fibroblast growth factor 1 (FGF1) has robust anti-diabetic effects mediated by the adipose FGF receptor (FGFR) 1. However, its mechanism of action is not known. Here, we report that FGF1 acutely lowers HGP by suppressing adipose lipolysis. On a molecular level, FGF1 inhibits the cAMP-protein kinase A axis by activating phosphodiesterase 4D (PDE4D), which separates it mechanistically from the inhibitory actions of insulin via PDE3B. We identify Ser44 as an FGF1-induced regulatory phosphorylation site in PDE4D that is modulated by the feed-fast cycle. These findings establish the FGF1/PDE4 pathway as an alternate regulator of the adipose-HGP axis and identify FGF1 as an unrecognized regulator of fatty acid homeostasis.

MicroRNA-137-3p Improves Nonalcoholic Fatty Liver Disease through Activating AMPKα.

Nonalcoholic fatty liver disease (NAFLD) is one of the most common chronic liver diseases worldwide and can develop to nonalcoholic steatohepatitis and later hepatic cirrhosis with a high prevalence to hepatocellular carcinoma. Oxidative stress and chronic hepatic inflammation are implicated in the pathogenesis of NAFLD. MicroRNA-137-3p (miR-137-3p) are associated with oxidative stress and inflammation; however, its role and mechanism in NAFLD remain unclear. Mice were fed with a high-fat diet (HFD) for 24 weeks to establish the NAFLD model. To overexpress or suppress hepatic miR-137-3p expression, mice were intraperitoneally injected with the agomir, antagomir, or respective controls of miR-137-3p at a dose of 100 mg/kg weekly for 6 consecutive weeks before the mice were sacrificed. To validate the involvement of AMP-activated protein kinase alpha (AMPKα) or cAMP-specific phosphodiesterase 4D (PDE4D), HFD mice were intraperitoneally injected with 20 mg/kg compound C or 0.5 mg/kg rolipram every other day for 8 consecutive weeks before the mice were sacrificed. Hepatic miR-137-3p expression was significantly decreased in mice upon HFD stimulation. miR-137-3p agomir alleviated, while miR-137-3p antagomir facilitated HFD-induced oxidative stress, inflammation, and hepatic dysfunction in mice. Mechanistically, we revealed that miR-137-3p is directly bound to the 3′-untranslated region of PDE4D and subsequently increased hepatic cAMP level and protein kinase A activity, thereby activating the downstream AMPKα pathway. In summary, miR-137-3p improves NAFLD through activating AMPKα and it is a promising therapeutic candidate to treat NAFLD.

MiR-139-5p has an antidepressant-like effect by targeting phosphodiesterase 4D to activate the cAMP/PKA/CREB signaling pathway.

BackgroundPhosphodiesterase 4D (PDE4D) inhibitor is commonly used to treat depression, but side effects seriously decrease its efficacy. PDE4D was a downstream target mRNA of miR-139-5p. Therefore, we examined the effects of hippocampal miR-139-5p gain- and loss-of-function on depression-like behaviors, the expression level of PDE4D, and hippocampus neurogenesis.MethodsBioinformatic analyses were carried out to to screen differential genes. Quantitative real-time polymerase chain reaction (qRT-PCR) and luciferase reporter assay were used to confirm the relationship between miR-139-5p and PDE4D. MiR-139-5p mimics, miR-139-5p inhibitor, or miR-NC were used to explore the function of miR-139-5p in HT-22 cells. We further explored the role of miR-139-5p in vivo using AAV-injection. Elisa, western blotting, and fluorescence in situ hybridization (FISH) were used to detect the expression of miR-139-5p and PDE4D in CRC tissues.ResultsHere, we showed that PDE4D messenger RNA (mRNA) was a direct target of microRNA (miR)-139-5p, which was downregulated in a chronic ultra-mild stress (CUMS)-induced depression mouse model. Moreover, in experiments in vitro, miR-139-5p mimic repressed PDE4D expression in HT-22 cells, but promoted phosphorylated cyclic-AMP response element-binding protein (p-CREB) and brain-derived neurotrophic factor (BDNF) expression. Interestingly, adeno-associated virus (AAV)-miR-139-5p downregulated susceptibility to stress-induced depression-like behaviors in mice. AAV-miR-139-5p suppressed PDE4D in mouse hippocampal cells, increasing expression level of cyclic adenosine monophosphate (cAMP), p-CREB, and BDNF, and stimulating mouse hippocampal neurogenesis.ConclusionsOur findings suggested that miR-139-5p acted like an antidepressant by targeting PDE4D, thereby regulating the cAMP/protein kinase A (PKA)/CREB/BDNF pathway to improve depression.