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Millions of individuals worldwide, across all age groups, suffer from the widespread health issue of gastric ulcers. In many experiments, cilostazol (Cls), a phosphodiesterase-3 inhibitor, was recently shown to have anti-ulcer activity. Notably, Cls increases the expression and transcriptional activity of PPAR-γ in vitro and in vivo. This study aimed to evaluate the protective effect of Cls against ethanol-induced gastric ulcers and clarify the possible underlying mechanisms with an emphasis on the role of PPAR-γ. Male albino rats were treated with ethanol to induce gastric ulcers, or they were pretreated with Cls, omeprazole (Omp), GW9662, or Cls + GW9662 for 14 consecutive days before receiving ethanol. Cls protects against ethanol-induced gastric ulcers. Cls treatment significantly reduced ethanol-induced upregulation of the pro-inflammatory markers (IL-1β, IL-6, TNF-α, and NF-κB), MDA (a marker of lipid peroxidation), and caspase-3 and cleaved caspase-3 (apoptotic markers). On the other hand, Cls treatment counteracted ethanol-induced downregulation of PPAR-γ, pErk-1, HO-1 and GSH (antioxidant markers), PECAM-1 and NO (healing markers), and Bcl-2 (antiapoptotic marker). However, when combined with GW9662, a potent antagonist of PPAR-γ, Cls loses its effects. In conclusion, these results suggest that PPAR-γ and pErk-1 are essential for Cls's protective effects against ethanol-induced gastric ulcers., (© 2024. The Author(s).)

Acute Respiratory Distress Syndrome (ARDS) is a severe lung condition with a high mortality rate for which there are no effective therapeutics. The failure of the alveolar-capillary barrier, composed of lung endothelial (EC) and alveolar epithelial (AEC) cells, is a critical factor leading to excessive inflammation and edema characteristic of acute lung injury (ALI) pathophysiology. Phosphodiesterases (PDE) are enzymes well-recognized for their roles in regulating endothelial permeability and inflammation. Although PDE inhibitors are used as therapeutics for inflammatory diseases like COPD (chronic obstructive pulmonary disease), their efficacy in treating ARDS has not yet been established. In this study, we investigated the effects of ensifentrine, an FDA-approved novel dual PDE 3/4 inhibitor, on lung endothelial and epithelial dysfunction caused by methicillin-resistant S. aureus (MRSA), a pathogen involved in bacterial ARDS. Human primary lung endothelial cells and alveolar epithelial cell lines (A549 and immortalized AEC) were treated with heat-killed MRSA, and their responses were assessed in the presence or absence of ensifentrine. Ensifentrine given either pre- or post-exposure attenuated MRSA-induced increased lung endothelial permeability. VE-cadherin junctions, which serve to stabilize the EC barrier, were disrupted by MRSA; however, ensifentrine effectively prevented this disruption. Pre-treatment with ensifentrine protected against MRSA-induced EC pro-inflammatory signaling by inhibiting the expression of VCAM-1, ICAM-1, and by reducing the IL-6 and IL-8 release. In AEC, MRSA caused the upregulation of ICAM-1, the activation of NF-kB, and the production of IL-8, all of which were inhibited by ensifentrine. These results indicate that the dual inhibition of phosphodiesterases 3 and 4 by ensifentrine is barrier protective and attenuates MRSA-induced inflammation in both lung endothelial and epithelial cells. The PDE3/4 inhibitor ensifentrine may represent a promising novel strategy for the treatment of MRSA-induced ARDS.

Current treatments for chronic obstructive pulmonary disease (COPD) are relatively limited and cannot meet the needs of all patients. Ensifentrine (development code RPL554), a representative drug of cyclic nucleotide phosphodiesterase 3/4 (PDE 3/4) inhibitors, has shown promising developments in the treatment of COPD in recent years, which need to be summarized. This article reviews the mechanism and clinical research progress of ensifentrine, focusing on its chemical structure, pharmacokinetics, pathophysiological mechanism, efficacy, and safety. Additionally, we provide clinical application suggestions and future research prospects.

Ensifentrine, an inhaled, selective phosphodiesterase (PDE) 3 and PDE4 inhibitor, is being developed by Verona Pharma plc for the treatment of respiratory diseases, including chronic obstructive pulmonary disease (COPD). In June 2024, ensifentrine (OHTUVAYRE™) inhalation suspension was approved for the maintenance treatment of COPD in adult patients in the USA. This article summarizes the milestones in the development of ensifentrine leading to this first approval for the maintenance treatment of COPD., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Cilostazol is a phosphodiesterase III inhibitor characterized by poor solubility. This limitation can be overcome by using a drug carrier capable of delivering the drug to the target site. Cyclodextrins are essential as drug carriers because of their outstanding complexation abilities and their capacity to improve drug bioavailability. This study comprises two stages: The first involves verifying different cyclodextrins and their complexation abilities towards cilostazol. This was accomplished using molecular docking simulations (MDS) and density functional theory (DFT). Both techniques indicate that the largest Sulfobutyl Ether-β-Cyclodextrin forms the most stable complex with cilostazol. Additionally, other important parameters of the complex are described, including binding sites, dominant interactions, and thermodynamic parameters such as complexation enthalpy, Gibbs free energy, and Gibbs free energy of solvation. The second stage involves a binding study between cilostazol and Phosphodiesterse3 (PDE3). This study was conducted using molecular docking simulations, and the most important energetic parameters are detailed. This is the first such report, and we believe that the results of our predictions will pave the way for future drug development efforts using cyclodextrin-cilostazol complexes as potential therapeutics.

Background: Intimal hyperplasia is the response to endothelial injury. Platelet-derived growth factor is released early and favors the formation of intimal hyperplasia. Although multiple treatments, from open surgery to endovascular techniques, have been used they remain controversial. There is currently interest in developing pharmacological strategies to address this pathology. Local vascular inflammation induced by vessel barotrauma generates intimal hyperplasia due to mechanical stress over the venous endothelium. Cilostazol is a selective phosphodiesterase type 3 (PDE3) selective inhibitor with a regulatory effect over intimal hyperplasia. The objective was to investigate cilostazol's role in inhibiting smooth muscle cell proliferation due to changes in the expression and release of PDGF-BB isoform and the effect on developing IH using an experimental model of vascular barotrauma (balloon-induced injury model)., Methods: We included 12 New Zealand rabbits. The balloon-induced injury model (BIIM) and experimental group cilostazol (20 mg/kg/day) included 6 rabbits each. Contralateral veins from 6 rabbits used in BIIM model has been taken as control group. We measured and compared the expression of PDGF-BB and the development of IH. A pathologist board chooses a PDGFRα antibody to localized its expression by immunohistochemistry analysis. Subsequently, using an automated immunohistochemical staining machine, the PDGFR expression was evaluated using a Zeiss Primo Star 4 light microscope., Results: The measurement obtained in the intimal layer was: 126.12 μm2 in the CG, 232 μm2 in the BIIM group, and 178 μm2 in the EG. A statistically significant difference was observed. Baseline serum concentrations of PDGF-BB in the BIIM group were 0.22 pg/mL. At 12 h 0.42 pg/mL, and 0.17 pg/mL at seven days. In the experimental group, the basal levels were 0.33 pg/mL. With the use of cilostazol, a lower peak was obtained at 12 h (0.08 pg/mL). This difference was statistically significant., Conclusions: Cilostazol induced a significant reduction of IH caused by barotrauma in the venous endothelium, which correlates with decrease in the PDGF-BB in serum. This could be attributed to the pharmacologic effect on PDGFR expression., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Background and Aim: Safe radical hepatectomy is important for patients with colorectal liver metastases complicated by sinusoidal obstruction syndrome (SOS) after oxaliplatin-based chemotherapy. This study aimed to investigate the impact of preoperative administration of cilostazol (CZ), an oral selective phosphodiesterase III inhibitor, on hepatectomy in rat SOS model., Material and Methods: Rats were divided into NL (normal liver), SOS (monocrotaline [MCT]-treated), and SOS + CZ (MCT + CZ-treated) groups. MCT or CZ was administered orally, and a 30% partial hepatectomy was performed 48 h after MCT administration. Postoperative survival rates were evaluated (n = 9, for each). Other rats were sacrificed on postoperative days (POD) 1 and 3 and evaluated histologically, immunohistochemically, biochemically, and using transmission electron microscopy (TEM), focusing particularly on SOS findings, liver damage, and liver sinusoidal endothelial cell (LSEC) injury., Results: The cumulative 10-day postoperative survival rate was significantly higher in the SOS + CZ group than in the SOS group (88.9% vs 33.3%, P = 0.001). Total SOS scores were significantly lower in the SOS + CZ group than in the SOS group on both POD 1 and 3. Serum biochemistry and immunohistochemistry showed that CZ reduced liver damage after hepatectomy. TEM revealed that LSECs were significantly preserved morphologically in the SOS + CZ group than in the SOS group on POD 1 (86.1 ± 8.2% vs 63.8 ± 9.3%, P = 0.003)., Conclusion: Preoperative CZ administration reduced liver injury by protecting LSECs and improved the prognosis after hepatectomy in rats with SOS., (© 2024 Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.)

Olprinone (OLP) is a selective inhibitor of phosphodiesterase III and is used clinically in patients with heart failure and those undergoing cardiac surgery; however, little is known about the effects of OLP on hepatoprotection. The purpose of this study aimed to determine whether OLP has protective effects in in vivo and in vitro rat models of endotoxin-induced liver injury after hepatectomy and to clarify the mechanisms of action of OLP. In the in vivo model, rats underwent 70% partial hepatectomy and lipopolysaccharide treatment (PH/LPS). OLP administration increased survival by 85.7% and decreased tumor necrosis factor-α, C-X-C motif chemokine ligand 1, and inducible nitric oxide synthase (iNOS) mRNA expression in the livers of rats treated with PH/LPS. OLP also suppressed nuclear translocation and/or DNA binding ability of nuclear factor kappa B (NF-κB). Pathological liver damage induced by PH/LPS was alleviated and neutrophil infiltration was reduced by OLP. Primary cultured rat hepatocytes treated with the pro-inflammatory cytokine interleukin-1β (IL-1β) were used as a model of in vitro liver injury. Co-treatment with OLP inhibited dose-dependently IL-1β-stimulated iNOS induction and NF-κB activation. Our results demonstrate that OLP may partially inhibit the induction of several inflammatory mediators through the suppression of NF-κB and thus prevent liver injury induced by endotoxin after liver resection.

Fracture healing in the aged is associated with a reduced healing capacity, which often results in delayed healing or non-union formation. Many factors may contribute to this deterioration of bone regeneration, including a reduced 'angiogenic trauma response'. The phosphodiesterase-3 (PDE-3) inhibitor cilostazol has been shown to exert pro-angiogenic and pro-osteogenic effects in preclinical studies. Therefore, we herein analyzed in a stable closed femoral fracture model whether this compound also promotes fracture healing in aged mice. Forty-two aged CD-1 mice (age: 16-18 months) were daily treated with 30 mg/kg body weight cilostazol ( n = 21) or vehicle (control, n = 21) by oral gavage. At 2 and 5 weeks after fracture, the femora were analyzed by X-ray, biomechanics, micro-computed tomography (µCT), histology, immunohistochemistry, and Western blotting. These analyses revealed a significantly increased bending stiffness at 2 weeks (2.2 ± 0.4 vs. 4.3 ± 0.7 N/mm) and an enhanced bone formation at 5 weeks (4.4 ± 0.7 vs. 9.1 ± 0.7 mm 3 ) in cilostazol-treated mice when compared to controls. This was associated with a higher number of newly formed CD31-positive microvessels (3.3 ± 0.9 vs. 5.5 ± 0.7 microvessels/HPF) as well as an elevated expression of phosphoinositide-3-kinase (PI3K) (3.6 ± 0.8 vs. 17.4 ± 5.5-pixel intensity × 10 4 ) and runt-related transcription factor (RUNX)2 (6.4 ± 1.2 vs. 18.2 ± 2.7-pixel intensity × 10 4 ) within the callus tissue. These findings indicate that cilostazol accelerates fracture healing in aged mice by stimulating angiogenesis and the expression of PI3K and RUNX2. Hence, cilostazol may represent a promising compound to promote bone regeneration in geriatric patients.

The mechanisms of pathogenesis of acute kidney injury (AKI) in snakebites is multifactorial and involves hemodynamic disturbances, with release of free radical causing cytotoxic effects. The phosphodiesterase-3 (PDE3) inhibitor, Cilostazol, has been reported to provide protection against renal oxidative stress., Objective: We evaluated the protective effects of cilostazol against Bothrops alternatus snake venom (BaV)-induced nephrotoxicity., Methods: Wistar rat kidneys (n = 6, 260-300 g) were isolated and perfused with Krebs-Henseleit solution containing 6 g/100 mL of bovine serum albumin. After 30 min, the kidneys were perfused with BaV to a final concentration of 1 and 3 μg/mL, and subsequently evaluated for perfusion pressure (PP), renal vascular resistance (RVR), urinary flow (UF), glomerular filtration rate (GFR), and percentage of electrolyte tubular sodium and chloride transport (%TNa + , %TCl - ). Oxidative stress and renal histological analyses were performed., Results: BaV caused a reduction in all the evaluated renal parameters (PP, RVR, GFR, UF, %TNa + , and %TCl - ). Although only the effects on PP and UF were reversed with cilostazol treatment, the decrease in the malondialdehyde levels, without changes in glutathione levels, further reduced the venom-induced renal tissue changes., Conclusion: Our data suggest that PDE3 is involved in BaV-induced nephrotoxicity, as cilostazol administration significantly ameliorated these effects., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier Ltd.)

Atherosclerosis is a multifactorial chronic disease associated with pro-inflammatory and pro-oxidative cardiovascular states. Cilostazol, a selective phosphodiesterase 3 inhibitor (PDE3), is clinically used in the treatment of intermittent claudication and secondary prevention of cerebral infarction. The aim of this study was to evaluate the cardioprotective effects of cilostazol and the molecular mechanisms involved in hypercholesterolemic rats. Male Wistar rats were divided into four groups: control group (C) and control + cilostazol group (C+CILO), that were fed a standard chow diet, and hypercholesterolemic diet group (HCD) and HCD + cilostazol (HCD+CILO) that were fed a hypercholesterolemic diet. Cilostazol treatment started after 30 days for C+CILO and HCD+CILO groups. Animals were administered cilostazol once a day for 15 days. Subsequently, serum and left ventricles were extracted for evaluation of lipid profile, inflammatory, and oxidative biomarkers. The HCD group displayed increased serum lipid levels, inflammatory cytokines production, and cardiac NF-kB protein expression and decreased cardiac Nrf2-mediated antioxidant activity. Conversely, the cilostazol treatment improved all these cardiac deleterious effects, inhibiting NF-kB activation and subsequently decreasing inflammatory mediators, reestablishing the antioxidant properties through Nrf2-mediated pathway, including increased SOD, GPx, and catalase expression. Taken together, our results indicated that cilostazol protects hypercholesterolemia-induced cardiac damage by molecular mechanisms targeting the crosstalk between Nrf2 induction and NF-kB inhibition in the heart., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Cilostazol, a phosphodiesterase III inhibitor, has vasodilating and antiplatelet properties with a low rate of bleeding complications. It has been used over the past 25 years for improving intermittent claudication in patients with peripheral artery disease (PAD). Cilostazol also has demonstrated efficacy in patients undergoing percutaneous revascularization procedures for both PAD and coronary artery disease. In addition to its antithrombotic and vasodilating actions, cilostazol also inhibits vascular smooth muscle cell proliferation via phosphodiesterase III inhibition, thus mitigating restenosis. Accumulated evidence has shown that cilostazol, due to its "pleiotropic" effects, is a useful, albeit underutilized, agent for both coronary artery disease and PAD. It is also potentially useful after ischemic stroke and is an alternative in those who are allergic or intolerant to classical antithrombotic agents (eg, aspirin or clopidogrel). These issues are herein reviewed together with the pharmacology and pharmacodynamics of cilostazol. Large studies and meta-analyses are presented and evaluated. Current guidelines are also discussed, and the spectrum of cilostazol's actions and therapeutic applications are illustrated., (© 2021, The American College of Clinical Pharmacology.)

Aims: Cilostazol (CLS) has shown antidepressant effect in cardiovascular patients, post-stroke depression, and animal models through its neurotrophic and antiinflammatory activities. Consequently, we aimed to investigate its safety and efficacy in patients with MDD by conducting double-blind, randomized, placebo-controlled pilot study., Methods: 80 participants with MDD (DSM-IV criteria) and Hamilton Depression Rating Scale (HDRS) score >20 were treated with CLS 50 mg or placebo twice daily plus escitalopram (ESC) 20 mg once daily for six weeks. Patients were evaluated by HDRS scores (weeks 0, 2, 4, and 6). Serum levels of CREB1, BDNF, 5-HT, TNF-α, NF- κB, and FAM19A5 were assessed pre- and post-treatment., Results: Co-administration of CLS had markedly decreased HDRS score at all-time points compared to the placebo group (p < 0.001). Early improvement, response, and remission rates after 6 weeks were significantly higher in the CLS group (90%, 90%, 80%, respectively) than in the placebo group (25%, 65%, 50% respectively) (p < 0.001). Moreover, the CLS group was superior to the placebo group in modulation of the measured neurotrophic and inflammatory biomarkers., Conclusion: CLS is safe and effective short-term adjunctive therapy in patients with MDD with no other comorbid conditions. Trial registration ID:NCT04069819., (© 2021 The Authors. CNS Neuroscience & Therapeutics published by John Wiley & Sons Ltd.)

Objective: platelets possess not only haemostatic but also inflammatory properties, which combined are thought to play a detrimental role in thromboinflammatory diseases such as acute coronary syndromes and stroke. Phosphodiesterase (PDE) 3 and -5 inhibitors have demonstrated efficacy in secondary prevention of arterial thrombosis, partially mediated by their antiplatelet action. Yet it is unclear whether such inhibitors also affect platelets' inflammatory functions. Here, we aimed to examine the effect of the PDE3A inhibitor cilostazol and the PDE5 inhibitor tadalafil on platelet function in various aspects of thromboinflammation. Approach and results: cilostazol, but not tadalafil, delayed ex vivo platelet-dependent fibrin formation under whole blood flow over type I collagen at 1000 s -1 . Similar results were obtained with blood from Pde3a deficient mice, indicating that cilostazol effects are mediated via PDE3A. Interestingly, cilostazol specifically reduced the release of phosphatidylserine-positive extracellular vesicles (EVs) from human platelets while not affecting total EV release. Both cilostazol and tadalafil reduced the interaction of human platelets with inflamed endothelium under arterial flow and the release of the chemokines CCL5 and CXCL4 from platelets. Moreover, cilostazol, but not tadalafil, reduced monocyte recruitment and platelet-monocyte interaction in vitro., Conclusions: this study demonstrated yet unrecognised roles for platelet PDE3A and platelet PDE5 in platelet procoagulant and proinflammatory responses.

Levosimendan is a distinctive inodilator combing calcium sensitization, phosphodiesterase inhibition and vasodilating properties through the opening of adenosine triphosphate-dependent potassium channels. It was first approved in Sweden in 2000 for the short-term treatment of acutely decompensated severe chronic heart failure when conventional therapy is not sufficient, and in cases where inotropic support is considered appropriate. After more than 20 years, clinical applications have considerably expanded across critical care and emergency medicine, and levosimendan is now under investigation in different cardiac settings (eg, septic shock, pulmonary hypertension) and for non-cardiac applications (eg, amyotrophic lateral sclerosis). This narrative review outlines key milestones in levosimendan history, by addressing regulatory issues, pharmacological peculiarities and clinical aspects (efficacy and safety) of a drug that did not receive great attention in the heart failure guidelines. A brief outlook to the ongoing clinical trials is also offered., Competing Interests: Professor Emanuel Raschi reports personal fees from Novartis for Consultancy, outside the submitted work. Prof. Dr. Luciano Potena reports personal fees from Novartis, personal fees from Biotest, personal fees from AstraZeneca, personal fees from Abbott, personal fees from Sandoz, outside the submitted work. The author reports no other conflicts of interest relevant to this work., (© 2021 Conti et al.)

Objective: Elevated intracellular cAMP concentrations potentiate insulin secretion from pancreatic β cells. Phosphodiesterase 3B (PDE3B) is highly expressed in these cells and plays a role in the regulation of insulin secretion., Materials and Methods: In this study, effects of amrinone, an inhibitor of PDE3B on insulin release from isolated pancreatic islets, were determined., Results: Exposure of islets to amrinone for 15, 30 and 90 min markedly increased secretion induced by 6.7 mM glucose. Amrinone enhanced also secretion stimulated by 6.7 mM glucose and DB-cAMP, an activator of PKA. It was also demonstrated that amrinone potentiated insulin secretion induced by 6.7 mM glucose in the combination with PMA (activator of PKC) or acetylcholine. However, the insulin-secretory response to glucose and glibenclamide was unchanged by amrinone., Conclusions: These results indicate that amrinone is capable of increasing insulin secretion; however, its action is restricted.

The need of circumventing life-threatening cardiovascular disorders (CVDs) and pulmonary hypertension (PHT) worldwide prompts researchers to develop effective therapeutic agents. Crucial role of cyclic nucleotide phosphodiesterase-5 (PDE5A) and cyclic nucleotide phosphodiesterase-3 (PDE3A) in cardiovascular signaling makes them potential drug targets for the treatment of CVDs and PHT. In this study, one-drug-multiple-target strategy has been employed to screen inhibitors exhibiting dual specificity through Phase-generated and statistically validated e-pharmacophore models of PDE5A and PDE3A. An extensive CoCoCo database of 7 million compounds with ∼150,000,000 conformations was virtually screened by sequential e-pharmacophore mapping followed by Lipinski Rule of Five (RO5) evaluation and hierarchical docking simulations. Finally, docked hits were subjected to rigorous MMGBSA analysis to estimate the relative spatial affinity of the drug-like compounds. The hits (354 and 366 ligands against PDE5A and PDE3A, respectively) were further optimized through 2D clustering followed by a comprehensive 2D and 3D interaction analysis. Five structurally diverse hits mapped equally well with the e-pharmacophore models and showed promising inhibitory interactions with conserved four catalytic features of PDE5A and PDE3A, thus exhibiting dual specificity. Proposed lead compounds exhibited the lowest MMGBSA binding energies and were found to be in agreement with Lipinski Rule of Five (RO5) and ADME/Tox criteria as compared to sildenafil. The proposed dual inhibitors could thus provide promising outcomes for the discovery of dual as well as multipotent drug like compounds after lead optimization and primary therapeutic interventions.

Ischemic preconditioning and postconditioning are strong measures preserving the heart against ischemia-reperfusion injury in experimental setting but are too invasive and impractical for clinical routine. The cardioprotective effects of ischemic preconditioning and postconditioning can be imitated pharmacologically, for example, with the phosphodiesterase inhibitors sildenafil and milrinone. We hypothesize that sildenafil-induced preconditioning is concentration dependent and further that a combined treatment of "nonprotective" versus "protective" concentrations of sildenafil and milrinone leads to a significant infarct size reduction. Experiments were performed on isolated hearts of male Wistar rats, randomized into 12 groups, mounted onto a Langendorff system, and perfused with Krebs-Henseleit buffer. All hearts underwent 33 minutes ischemia and 60 minutes of reperfusion. For determination of a concentration-dependent effect of sildenafil, hearts were perfused with increasing concentrations of sildenafil (0.1-1 µM) over 10 minutes before ischemia. In a second series of experiments, hearts were treated with 0.3 µM sildenafil or 1 µM milrinone as the "protective" concentrations. A higher concentration of respective drugs did not further reduce infarct size. In addition, a combination of "protective" and "nonprotective" concentrations of sildenafil and milrinone was applied. Sildenafil and milrinone in lower concentrations led to significant infarct size reduction, whereas combining both substances in cardioprotective concentrations did not enhance this effect. Sildenafil in a concentration of 0.3 µM induces myocardial protection. Furthermore, treatment with sildenafil and milrinone in lower concentrations had an equally strong cardioprotective effect regarding infarct size reduction compared with the administration of "protective" concentrations.

Background: Phosphodiesterase enzymes play a pivotal role in the pathogenesis of ischemia/reperfusion (IR). We examined the role of milrinone (MIL), a phosphodiesterase 3 inhibitor, on remote injury of the heart and lung after abdominal aortic cross-clamping., Design: Experimental study., Methods: Twenty-one Wistar rats were divided into 3 groups: (1) control (C, n = 7), underwent laparotomy and exploration of abdominal aorta only; (2) IR (n = 7), normal saline was applied intraperitoneally (i.p) before IR induced by clamping of the abdominal aorta for 1 hr and then allowing reperfusion for 1 hr; and (3) MIL + IR (n = 7), MIL was given (0.5 mg/kg, i.p) before IR. After sacrification, the lungs and hearts were taken out for analyses and the tissue malondialdehyde (MDA) and glutathione (GSH) were studied. All tissues were examined under light microscopy and transmission electron microscopy (TEM). Expressions of caveolin (Cav)-1 in the lung and Cav-1 and Cav-3 in the heart were examined immunohistochemically., Results: The MIL + IR group had significantly a lower magnitude of oxidative stress than the IR group both in the lung and heart (lung: P = 0.03 for MDA and 0.001 for GSH and heart: P = 0.002 for MDA and 0.000 for GSH). In light microscopy, the MIL + IR group had statistically a lower total injury score than the IR group for both the lung and heart tissue (P = 0.03 and P = 0.04, respectively). In TEM, regression of mitochondrial degeneration and lamellar bodies in type II pneumocytes in the lungs and obvious improvements in disruption at the intercalated discs and mitochondrial degeneration in the hearts in the MIL + IR group were detected compared with the IR group. The expression of both Cav-1 and Cav-3 in the MIL + IR group was improved compared with the IR group (P = 0.03 for both)., Conclusions: MIL attenuates remote injury of heart and lung in lower body IR by inhibiting oxidative stress. Moreover, Cav-1 and Cav-3 might have a potential role in MIL-induced cardioprotection., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Acute pancreatitis (AP) is an inflammatory disorder with a high mortality rate. Cilostazol is a selective phosphodiesterase-3 inhibitor drug that is commonly used as an antiplatelet, antithrombotic, and vasodilator drug. It exhibits antioxidant, anti-inflammatory, and anti-apoptotic activities, but its effect on AP has not been fully elucidated yet. The present study aimed to investigate the effects of cilostazol on L-arginine-induced AP and the possible protective mechanisms. A rat model of AP was established by a single i.p. injection of 3-g/kg L-arginine on day 13 of the experiment. The treated groups received a single daily oral dose of either 100 or 300 mg/kg/day for 14 consecutive days. Rats with AP showed histopathological changes of pancreatic tissue injury together with increased serum amylase enzyme activity and decreased serum insulin, pancreatic adiponectin, and cGMP levels. Moreover, AP rats showed increased pancreatic inflammatory biomarker (TNF-α, VCAM-1, and MPO) levels with decreased anti-inflammatory IL-10 levels. In addition, oxidative stress biomarkers (MDA and NO) were increased in AP with decreased antioxidant SOD activity and GSH level. Moreover, HO-1 immunostaining was increased in the AP group. Cilostazol pretreatment reversed the histopathological change; decreased the amylase activity and the levels of TNF-α, VCAM-1, and MPO; and increased the levels of insulin, adiponectin, cGMP, cAMP, and IL-10. Moreover, cilostazol decreased MDA and NO but increased SOD and GSH. Lastly, cilostazol increased the HO-1 immunostaining more than in the AP group. These data suggest that cilostazol protects against L-arginine-induced AP, which may be related to an increase in cGMP, cAMP, and upregulation of HO-1 with subsequent anti-inflammatory and antioxidant properties.

Phosphodiesterases (PDEs) are essential enzymes for the regulation of pathways mediated by cyclic adenosine monophosphate (cAMP). Secondary plant compounds like anthocyanins (ACs) can inhibit PDE activity and, consequently, may be beneficial for lipid metabolism. This study investigated 18 AC-rich juice extracts and pure reference compounds from red fruits for potential inhibitory effects on PDE 3B activity. Extracts were obtained through adsorption on Amberlite ® XAD 7 resin. Based on this screening, the chokeberry, blueberry, pomegranate, and cranberry extracts were active, with half maximal inhibitory concentrations (IC 50 ) ranging from 163 ± 3 µg/mL to 180 ± 3 µg/mL. The ACs in these extracts, peonidin-3-glucoside and cyanidin-3-arabinoside, were the most active single compounds (IC 50 = 56 ± 20 µg/mL, 108 ± 6 µg/mL). All extracts comprised high amounts of phenolic compounds, as determined by the Folin-Ciocalteu assay, ranging from 39.8 ± 1.5 to 73.5 ± 4.8 g gallic acid equivalents (GAE)/100 g extract. Pomegranate and chokeberry extracts exhibited the largest amounts of polyphenols (72.3 ± 0.7 g GAE/100 g, 70.6 ± 4.1 g GAE/100 g, respectively). Overall, our results showed that fruit juice extracts and their ACs can inhibit PDE activity. Any potential health benefits in vivo will be investigated in the future.

The promotion of apoptosis in tumor cells is a popular strategy for developing anti-cancer drugs. Here, we demonstrate that the plant indole alkaloid natural product nauclefine induces apoptosis of diverse cancer cells via a PDE3A-SLFN12 dependent death pathway. Nauclefine binds PDE3A but does not inhibit the PDE3A's phosphodiesterase activity, thus representing a previously unknown type of PDE3A modulator that can initiate apoptosis without affecting PDE3A's canonical function. We demonstrate that PDE3A's H840, Q975, Q1001, and F1004 residues-as well as I105 in SLFN12-are essential for nauclefine-induced PDE3A-SLFN12 interaction and cell death. Extending these molecular insights, we show in vivo that nauclefine inhibits tumor xenograft growth, doing so in a PDE3A- and SLFN12-dependent manner. Thus, beyond demonstrating potent cytotoxic effects of an alkaloid natural product, our study illustrates a potentially side-effect-reducing strategy for targeting PDE3A for anti-cancer therapeutics without affecting its phosphodiesterase activity.

Cilostazol, a phosphodiesterase 3 inhibitor, reduces the amyloid-beta (Aβ) burden in mouse models of Alzheimer disease by as yet unidentified mechanisms. In the present study, we examined the possibility that cilostazol ameliorates lysosomal dysfunction. Astrocytes treated with bafilomycin A1 (BafA1) exhibited markedly reduced DND-189 and acridine orange (AO) fluorescence, indicating reduced lysosomal acidity. In both cases, BafA1-induced alkalization was reversed by addition of cilostazol, dibutyryl cAMP or forskolin. All three agents significantly increased free zinc levels in lysosomes, and addition of the zinc chelator TPEN abrogated lysosomal reacidification. These treatments did not raise free zinc levels or reverse BafA1-mediated lysosomal alkalization in metallothionein 3 (Mt3)-null astrocytes, indicating that the increases in zinc in astrocytes were derived mainly from Mt3. Lastly, in FITC-Aβ-treated astrocytes, cilostazol reversed lysosomal alkalization, increased cathepsin D activity, and reduced Aβ accumulation in astrocytes. Cilostazol also reduced mHtt aggregate formation in GFP-mHttQ74-expressing astrocytes. Collectively, our results present the novel finding that cAMP/PKA can overcome the v-ATPase blocking effect of BafA1 in a zinc- and Mt3-dependent manner.

This study aimed to investigate whether a selective phosphodiesterase-3 (PDE3) inhibitor olprinone can positively influence the inflammation, apoptosis, and respiratory parameters in animals with acute respiratory distress syndrome (ARDS) model induced by repetitive saline lung lavage. Adult rabbits were divided into 3 groups: ARDS without therapy (ARDS), ARDS treated with olprinone i.v. (1 mg/kg; ARDS/PDE3), and healthy ventilated controls (Control), and were oxygen-ventilated for the following 4 h. Dynamic lung-thorax compliance (Cdyn), mean airway pressure (MAP), arterial oxygen saturation (SaO 2 ), alveolar-arterial gradient (AAG), ratio between partial pressure of oxygen in arterial blood to a fraction of inspired oxygen (PaO 2 /FiO 2 ), oxygenation index (OI), and ventilation efficiency index (VEI) were evaluated every hour. Post mortem , inflammatory and oxidative markers (interleukin (IL)-6, IL-1β, a receptor for advanced glycation end products (RAGE), IL-10, total antioxidant capacity (TAC), 3-nitrotyrosine (3NT), and malondialdehyde (MDA) and apoptosis (apoptotic index and caspase-3) were assessed in the lung tissue. Treatment with olprinone reduced the release of inflammatory mediators and markers of oxidative damage decreased apoptosis of epithelial cells and improved respiratory parameters. The results indicate a future potential of PDE3 inhibitors also in the therapy of ARDS.

Over 2,000 mutations have been reported in the cystic fibrosis transmembrane conductance regulator ( cftr ) gene, many of which cause disease but are rare and have no effective treatment. Thus, there is an unmet need for new, mutation-agnostic therapies for cystic fibrosis (CF). Phosphodiesterase (PDE) inhibitors are one such class of therapeutics that have been shown to elevate intracellular cAMP levels and stimulate CFTR-dependent anion secretion in human airway epithelia; however, the number of people with CF that could be helped by PDE inhibitors remains to be determined. Here we used Fisher rat thyroid (FRT) cells stably transduced with rare human CFTR mutants and studied their responsiveness to the dual phosphodiesterase 3/4 inhibitor RPL554 (Verona Pharma). Through its inhibitory effect on PDE4D, we find that RPL554 can elevate intracellular cAMP leading to a potentiation of forskolin-stimulated current mediated by R334W, T338I, G551D, and S549R mutants of CFTR when used alone or in combination with CFTR modulators. We also were able to reproduce these effects of RPL554 on G551D-CFTR when it was expressed in primary human bronchial epithelial cells, indicating that RPL554 would have stimulatory effects on rare CFTR mutants in human airways and validating FRT cells as a model for PDE inhibitor studies. Furthermore, we provide biochemical evidence that VX-809 causes surprisingly robust correction of several class III and IV CFTR mutants. Together, our findings further support the therapeutic potential of RPL554 for patients with CF with class III/IV mutations and emphasize the potential of PDEs as potential drug targets that could benefit patients with CF.

A simple, quick, easy and cheap tandem mass spectrometry (MS/MS) method for the determination of adenosine monophosphate (AMP) and cyclic adenosine monophosphate (cAMP) has been newly developed. This novel MS/MS method was applied for the evaluation of the inhibitory effect of a novel 2-oxo-1,2-dihydropyridine-3-carbonitrile derivative, also named DF492, on PDE3 enzyme activity in comparison to its parent drug milrinone. Molecule DF492, with an IC 50 of 409.5 nM, showed an inhibition of PDE3 greater than milrinone (IC 50 = 703.1 nM). To explain the inhibitory potential of DF492, molecular docking studies toward the human PDE3A were carried out with the aim of predicting the binding mode of DF492. The presence of different bulkier decorating fragments in DF492 was pursued to shift affinity of this novel molecule toward PDE3A compared to milrinone in accordance with both the theoretical and experimental results. The described mass spectrometric approach could have a wider potential use in kinetic and biomedical studies and could be applied for the determination of other phosphodiesterase inhibitor molecules.

Considerable evidence suggests that as breast cancer progresses, genetic and epigenetic mechanisms contribute to the emergence of self-renewing cells (CSC), which may also arise as a consequence of metastasis. Although the molecular pathways that trigger stemness and metastasis are known, key molecular and mechanistic gaps in our understanding of these processes remain unclear. Here, we first screened the inflammation-associated stemness gene phosphodiesterase 3A (PDE3A) using a medium-throughput siRNA library, which was overexpressed in breast tumors and significantly correlated with clinical progression. PDE3A induced the inflammatory nuclear factor NFκB signaling pathway by suppressing cAMP/PKA, which promotes the expression of the stem cell marker OCT4. In addition, PDE3A also promoted the translocation of CCDC88A from the cytoplasm to nuclei, thereby boosting the invasion-metastasis cascade in breast cancer. Most importantly, the PDE3A-selective inhibitor cilostazol dramatically suppressed breast tumor growth and reduced metastasis to the lungs in xenograft breast cancer models, with minimum toxicity. Taken together, we show that PDE3A could predispose patients with breast cancer to metastases by acting as a mediator of cancer stemness. PDE3A is a potential therapeutic target for advanced breast cancer., (©2019 American Association for Cancer Research.)

Aims: The present study aims to define maturation, yield, health, and ease of collection of murine immature oocytes recovered using the conventional method or from mice treated with cilostazol., Main Methods: The conventional method included the superovulation of mice and the recovery of germinal vesicle (GV) or metaphase (MI) oocytes from preovulatory follicles. The cilostazol method included the oral treatment of superovulated mice with 7.5 mg cilostazol once or twice to result in the ovulation of MI or GV oocytes, respectively., Key Findings: The cilostazol method resulted in >95% of GV or MI oocytes with a diameter range of 60-90 μm or 50.1-70 μm in comparison to <60.0% of GV or MI oocytes resulting from the conventional method, respectively (P < 0.0001). The cilostazol method resulted in GV oocytes having higher levels of co-occurrence of peripheral cortical granules (CG) and chromatin configuration of surrounded nucleolus and MI oocytes having higher levels of co-occurrence of normally organized spindles/chromosomes and peripheral CG with free domains than did the conventional method (P < 0.001). The cilostazol method was more time and labor efficient and resulted in higher oocyte yields of normal morphology than did the conventional method (P < 0.01)., Significance: The presented method provides not only oocytes with uniform size and synchronized developmental maturation but also a technique of oocyte collection that is efficient and resourceful. It is possible that not all immature oocytes resulting from the conventional method are from preovulatory follicles nor have been developed adequately and consequently ovulated as opposed to the presented method., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Introduction: This study aimed to investigate the dose-response and pharmacology of a range of single doses of nebulised ensifentrine (RPL554), an inhaled dual phosphodiesterase (PDE) 3/4 inhibitor in patients with asthma., Methods: In this randomised, placebo-controlled, double-blind crossover study, patients received single nebulised doses of ensifentrine 0.4, 1.5, 6 and 24 mg, salbutamol 2.5 and 7.5 mg, and placebo. Eligible patients were adults with asthma, pre-bronchodilator forced expiratory volume in 1 s (FEV 1 ) 60-90% predicted and ≥1.5 L, with post-salbutamol FEV 1 increase ≥15%. The co-primary objectives were peak and average FEV 1 over 12 h for ensifentrine vs placebo and salbutamol. Secondary endpoints included: peak and average systolic and diastolic blood pressure, pulse rate and ECG heart rate; and safety and tolerability (adverse events [AEs], and serum potassium). ClinicalTrials.gov: NCT02427165., Results: A total of 29 patients were randomised, with 25 (89%) completing the study. For the two co-primary endpoints there was a clear ensifentrine dose-response relationship, with all treatments superior to placebo (p < 0.001). There was no relationship between the ensifentrine dose and AE incidence or blood pressure. Ensifentrine 0.4, 1.5 and 6 mg had significantly lower effects than both salbutamol doses on pulse and heart rates. Ensifentrine did not impact potassium, whereas there was a was a dose-related reduction for salbutamol. Inhalation of ensifentrine resulted in a dose-related increase in plasma exposure., Conclusions: Single-dose ensifentrine demonstrated dose-dependent bronchodilation, and was as effective as a therapeutic dose of nebulised salbutamol. All ensifentrine doses were similarly well tolerated, and did not show the characteristic β 2 -agonist systemic adverse effects., (Copyright © 2019. Published by Elsevier Ltd.)

Recent reports show that protein kinase A (PKA), but not exchange protein activated by cAMP (EPAC), acts in a cell autonomous manner to constitutively reduce the angiogenic sprouting capacity of murine and human endothelial cells. Specificity in the cellular actions of individual cAMP-effectors can be achieved when a cyclic nucleotide phosphodiesterase (PDE) enzyme acts locally to control the "pool" of cAMP that activates the cAMP-effector. Here, we examined whether PDEs coordinate the actions of PKA during endothelial cell sprouting. Inhibiting each of the cAMP-hydrolyzing PDEs expressed in human endothelial cells revealed that phosphodiesterase 3 (PDE3) inhibition with cilostamide reduced angiogenic sprouting in vitro, while inhibitors of PDE2 and PDE4 family enzymes had no such effect. Identifying a critical role for PDE3B in the anti-angiogenic effects of cilostamide, silencing this PDE3 variant, but not PDE3A, markedly impaired sprouting. Importantly, using both in vitro and ex vivo models of angiogenesis, we show the hypo-sprouting phenotype induced by PDE3 inhibition or PDE3B silencing was reversed by PKA inhibition. Examination of the individual cellular events required for sprouting revealed that PDE3B and PKA each regulated angiogenic sprouting by controlling the invasive capacity of endothelial cells, more specifically, by regulating podosome rosette biogenesis and matrix degradation. In support of the idea that PDE3B acts to inhibit angiogenic sprouting by limiting PKA-mediated reductions in active cdc42, the effects of PDE3B and/or PKA on angiogenic sprouting were negated in cells with reduced cdc42 expression or activity. Since PDE3B and PKA were co-localized in a perinuclear region in human ECs, could be co-immunoprecipitated from lysates of these cells, and silencing PDE3B activated the perinuclear pool of PKA in these cells, we conclude that PDE3B-mediated hydrolysis of cAMP acts to limit the anti-angiogenic potential of PKA in ECs., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Introduction : A compound that simultaneously inhibits PDE3 and PDE4 should increase airway caliber by relaxing the smooth muscle and, simultaneously, suppress airway inflammatory responses. Ensifentrine (RPL554) is considered a PDE3/4 inhibitor, although its affinity for PDE3 is 3,440 times higher than that for PDE4, that is under clinical development for the treatment of asthma and COPD and, potentially, cystic fibrosis. Areas covered : We analyze the development of this molecule from its basic pharmacology to the present clinical Phase II studies. Expert opinion : Ensifentrine is an interesting drug but there is a lack of solid studies that still does not allow us to correctly allocate this molecule in the current COPD and even asthma therapeutic armamentarium. Furthermore, apparently ensifentrine has not yet entered Phase III clinical development and, in any case, there is no reliable evidence of its ability to elicit an anti-inflammatory activity in patients with COPD or asthma. Therefore, the real anti-inflammatory profile of ensifentrine must be clarified with new studies of basic pharmacology and adequate clinical studies specifically designed. However, at present the most intriguing perspective is linked to its possible use in the treatment of cystic fibrosis, also considering the lack of valid therapeutic options for this disease.

Background: Increasing evidence suggests that high glucose (HG) causes abnormalities in endothelial and vascular smooth muscle cell function (VSMC) and contributes to atherosclerosis. Receptor for advanced glycation end-products (RAGE) has been linked to the pathogenesis of both the macrovascular and microvascular complications of diabetes. Cilostazol is used to treat diabetic vasculopathy by ameliorating HG-induced vascular dysfunction., Objectives: In this study, we investigated whether the cilostazol suppression of HG-induced VSMC dysfunction is through RAGE signaling and its possible regulation mechanism., Method: We investigated the effect of HG and cilostazol on RAGE signaling in A7r5 rat VSMCs. Aortic tissues of streptozotocin (STZ) diabetic mice were also collected., Results: Aortic tissue samples from the diabetic mice exhibited a significantly decreased RAGE expression after cilostazol treatment. HG increased RAGE, focal adhesion kinase (FAK), matrix metalloproteinase-2 (MMP-2), intercellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expressions, and was accompanied with increased reactive oxygen species (ROS), cell proliferation, adhesion and migration. Cilostazol significantly reversed HG-induced RAGE, ROS, downstream gene expressions and cell functions. RAGE knockdown significantly reversed the expressions of HG-induced vasculopathy related gene expressions and cell functions. Cilostazol with RAGE knockdown had additive effects on downstream ERK/NF-κB signaling pathways, gene expressions and cell functions of A7r5 rat VSMCs in HG culture., Conclusions: Both in vitro and in vivo experimental diabetes models showed novel signal transduction of cilostazol-mediated protection against HG-related VSMC dysfunction, and highlighted the involvement of RAGE signaling and downstream pathways.

Aims: Cyclic AMP phosphodiesterases (PDEs) are important modulators of the cardiac response to β-adrenergic receptor (β-AR) stimulation. PDE3 is classically considered as the major cardiac PDE in large mammals and human, while PDE4 is preponderant in rodents. However, it remains unclear whether PDE4 also plays a functional role in large mammals. Our purpose was to understand the role of PDE4 in cAMP hydrolysis and excitation-contraction coupling (ECC) in the pig heart, a relevant pre-clinical model., Methods and Results: Real-time cAMP variations were measured in isolated adult pig right ventricular myocytes (APVMs) using a Förster resonance energy transfer (FRET) biosensor. ECC was investigated in APVMs loaded with Fura-2 and paced at 1 Hz allowing simultaneous measurement of intracellular Ca 2+ and sarcomere shortening. The expression of the different PDE4 subfamilies was assessed by Western blot in pig right ventricles and APVMs. Similarly to PDE3 inhibition with cilostamide (Cil), PDE4 inhibition with Ro 20-1724 (Ro) increased cAMP levels and inotropy under basal conditions. PDE4 inhibition enhanced the effects of the non-selective β-AR agonist isoprenaline (Iso) and the effects of Cil, and increased spontaneous diastolic Ca 2+ waves (SCWs) in these conditions. PDE3A, PDE4A, PDE4B and PDE4D subfamilies are expressed in pig ventricles. In APVMs isolated from a porcine model of repaired tetralogy of Fallot which leads to right ventricular failure, PDE4 inhibition also exerts inotropic and pro-arrhythmic effects., Conclusions: Our results show that PDE4 controls ECC in APVMs and suggest that PDE4 inhibitors exert inotropic and pro-arrhythmic effects upon PDE3 inhibition or β-AR stimulation in our pre-clinical model. Thus, PDE4 inhibitors should be used with caution in clinics as they may lead to arrhythmogenic events upon stress., (Copyright © 2019. Published by Elsevier Ltd.)

Serotonin (5-hydroxy-tryptamine, 5-HT) exerted concentration-dependent positive inotropic effects or positive chronotropic effects in transgenic (TG) mice which overexpress the human 5-HT 4a receptor in the heart but not in littermate wild-type (WT) mice. These positive inotropic effects and positive chronotropic effects are thought to be mediated by cyclic adenosine 3',5'-monophosphate (cAMP) in TG cardiomyocytes. To determine whether these effects are antagonized by endogenous phosphodiesterases (PDEs), the inotropic and chronotropic effects of 5-HT were tested in the additional presence of the PDE inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine hydrochloride (EHNA) (1 μM, a PDE2 inhibitor) or cilostamide (1 μM, a PDE3 inhibitor), rolipram (0.1 μM and 1 μM, a PDE4 inhibitor), and their combinations. For comparison, 3-isobutyl-1-methylxanthine (IBMX), an unspecific PDE inhibitor, was investigated. The use of 10 μM IBMX, the combination of rolipram (1 μM) and EHNA (1 μM), and the combination of rolipram (0.1 μM) and cilostamide (1 μM) each increased the potency of 5-HT to elevate the force of contraction in TG mice, but not the potency of 5-HT to increase the beating rate in TG mice. This indicates that PDE4 and PDE2 regulate the inotropic but not the chronotropic effects of 5-HT in TG mice. In contrast, cilostamide (1 μM) alone, EHNA (1 μM) alone, or in combination decreased the potency of 5-HT to increase force of contraction in TG mice. In summary, our present data suggest that the positive chronotropic effect of 5-HT in TG mice does not involve PDE activities, whereas the positive inotropic effect of 5-HT and the basal force in TG mice are diminished by endogenous activity of PDE4. Phosphorylation of PDE4, when PDE2 or PDE3 is inhibited, might enhance the activity of PDE4.

Objective: The aim of this study was to investigate the potential protective and therapeutic effects of milrinone, a specific phosphodiesterase (PDE) III inhibitor, on acoustic trauma-induced cochlear injury and apoptosis., Methods: A total number of 30 healthy Wistar albino rats were evenly divided into five groups as follows: group 1 was assigned as control group; group 2 and 3 were assigned as low-dosage groups (0.25 mg/kg) in which milrinone was administered 1 h before acoustic trauma (AT) and 2 h after AT, respectively; group 4 and 5 were assigned as high-dosage groups (0.50 mg/kg) in which the drug was administered 1 h before AT and 2 h after AT, respectively. Except control group, all treatment groups received a single dosage of milrinone for 5 days. Distortion product otoacoustic emissions (DPOAE) measurements were recorded before AT as well as at second and fifth post-traumatic days. At the end of fifth day, all rats were sacrificed and the cochlea of the rats was removed for histopathological evaluation. In addition, the groups were compared in terms of apoptotic index via caspase-3 staining., Results: In terms of signal-to-noise ratio (SNR), there was no statistically significant difference among the groups following AT (p > 0.05). After 5 days of milrinone treatment, the best SNR values were found in group 5, though all groups did not statistically differ (p > 0.05). In histopathological evaluation, vacuolization, inflammation, and edema scores in all treatment groups were statistically lower than those of the control group (p < 0.05). In group 2 and 4 where the drug was administered before AT, the inflammation and apoptosis index was lower than those of group 3 and 5 where the drug was administered after AT (p < 0.0001)., Conclusion: We reveal that milrinone has a protective effect on cochlear damage in the experimental acoustic model of rats. This protective effect was more apparent following the pre-traumatic milrinone administration, and is associated with its effect on decreasing inflammation and apoptosis. Based on DPOAE measurements following AT, especially in the group 5 (high-dosage group), milrinone may also have a therapeutic effect.

As the population of older individuals grows worldwide, researchers have increasingly focused their attention on identifying key molecular targets of age-related cognitive impairments, with the aim of developing possible therapeutic interventions. Two such molecules are the intracellular cyclic nucleotides, cAMP and cGMP. These second messengers mediate fundamental aspects of brain function relevant to memory, learning, and cognitive function. Consequently, phosphodiesterases (PDEs), which hydrolyze cAMP and cGMP, are promising targets for the development of cognition-enhancing drugs. Inhibitors that target PDEs work by elevating intracellular cAMP. In this review, we provide an overview of different PDE inhibitors, and then we focus on pharmacological and physiological effects of PDE3 inhibitors in the CNS and peripheral tissues. Finally, we discuss findings from experimental and preliminary clinical studies and the potential beneficial effects of the PDE3 inhibitor cilostazol on age-related cognitive impairments. In the innovation pipeline of pharmaceutical development, the antiplatelet agent cilostazol has come into the spotlight as a novel treatment for mild cognitive impairment. Overall, the repurposing of cilostazol may represent a potentially promising way to treat mild cognitive impairment, Alzheimer's disease, and vascular dementia. In this review, we present a brief summary of cAMP signaling and different PDE inhibitors, followed by a discussion of the pharmacological and physiological role of PDE3 inhibitors. In this context, we discuss the repurposing of a PDE3 inhibitor, cilostazol, as a potential treatment for age-related cognitive impairment based on recent research.

Background: The phosphodiesterase 3A (PDE3A) gene encodes a PDE that regulates cardiac myocyte cyclic adenosine monophosphate (cAMP) levels and myocardial contractile function. PDE3 inhibitors (PDE3i) are used for short-term treatment of refractory heart failure (HF), but do not produce uniform long-term benefit., Objectives: The authors tested the hypothesis that drug target genetic variation could explain clinical response heterogeneity to PDE3i in HF., Methods: PDE3A promoter studies were performed in a cloned luciferase construct. In human left ventricular (LV) preparations, mRNA expression was measured by reverse transcription polymerase chain reaction, and PDE3 enzyme activity by cAMP-hydrolysis., Results: The authors identified a 29-nucleotide (nt) insertion (INS)/deletion (DEL) polymorphism in the human PDE3A gene promoter beginning 2,214 nt upstream from the PDE3A1 translation start site. Transcription factor ATF3 binds to the INS and represses cAMP-dependent promoter activity. In explanted failing LVs that were homozygous for PDE3A DEL and had been treated with PDE3i pre-cardiac transplantation, PDE3A1 mRNA abundance and microsomal PDE3 enzyme activity were increased by 1.7-fold to 1.8-fold (p < 0.05) compared with DEL homozygotes not receiving PDE3i. The basis for the selective up-regulation in PDE3A gene expression in DEL homozygotes treated with PDE3i was a cAMP response element enhancer 61 nt downstream from the INS, which was repressed by INS. The DEL homozygous genotype frequency was also enriched in patients with HF., Conclusions: A 29-nt INS/DEL polymorphism in the PDE3A promoter regulates cAMP-induced PDE3A gene expression in patients treated with PDE3i. This molecular mechanism may explain response heterogeneity to this drug class, and may inform a pharmacogenetic strategy for a more effective use of PDE3i in HF., (Copyright © 2019 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Background and Purpose- Acetylsalicylic acid and clopidogrel are the 2 main antithrombotic drugs for secondary prevention in patients with ischemic stroke (IS) without indication for anticoagulation. Because of their limited efficacy and potential side effects, novel antiplatelet agents are urgently needed. Cilostazol, a specific phosphodiesterase (PDE)-3 inhibitor, protected from IS in clinical studies comprising mainly Asian populations. Nevertheless, the detailed mechanistic role of PDE-3 inhibitors in IS pathophysiology is hardly understood. In this project, we analyzed the efficacy and pathophysiologic mechanisms of a novel and only recently described PDE-3 inhibitor (substance V) in a mouse model of focal cerebral ischemia. Methods- Focal cerebral ischemia was induced by transient middle cerebral artery occlusion in 6- to 8-week-old male C57Bl/6 wild-type mice receiving substance V or vehicle 1 hour after ischemia induction. Infarct volumes and functional outcomes were assessed between day 1 and day 7, and findings were validated by magnetic resonance imaging. Blood-brain barrier damage, as well as the extent of local inflammatory response and cell death, was determined. Results- Inhibition of PDE-3 by pharmacological blockade with substance V significantly reduced infarct volumes and improved neurological outcome on day 1 and 7 after experimental cerebral ischemia. Reduced blood-brain barrier damage, attenuated brain tissue inflammation, and decreased local cell death could be identified as potential mechanisms. PDE-3 inhibitor treatment did neither increase the number of intracerebral hemorrhages nor affect platelet function. Conclusions- The novel PDE-3 inhibitor substance V protected mice from IS independent from platelet function. Pharmaceutical inactivation of PDE-3 might become a promising therapeutic approach to combat IS via inhibition of thromboinflammatory mechanisms and stabilization of the blood-brain barrier.

We investigated the effects of an injectable pimobendan solution (0.15 mg/kg) on cardiac function in healthy dogs. Fifteen dogs were divided into placebo, intravenous pimobendan injection, and subcutaneous pimobendan injection groups. In the placebo, the heart rate, systolic and end-diastolic left ventricular pressure (LVPs and LVEDP), and peak positive (max dP/dt) and negative (min dP/dt) first derivatives of the left ventricular pressure did not change for 60 min. After the intravenous pimobendan injection, LVEDP decreased significantly within 5 min, while the max dP/dt increased, and the effects continued until 60 min. In comparison, there were no hemodynamic changes after the subcutaneous pimobendan injection. This study demonstrates that injectable pimobendan induced a rapid inotropic effect and decreased the LVEDP in dogs.

Background: Levosimendan is a calcium sensitizer and phosphodiesterase inhibitor that has potent antioxidant and anti-inflammatory activities., Objectives: The aim of the current study is to investigate the potential protective effect of levosimendan on learning and memory impairment induced by diabetes., Methods: Adult Wister rats were randomly divided into four groups (n=15 rats/group): control, levosimendan, streptozotocin (STZ) induced diabetes, and levosimendan-STZ diabetes. Upon confirmation of the success of the STZ diabetic model, intraperitoneal levosimendan (100µg/kg/week) was administrated to the assigned groups for 4 weeks. Then, the radial arm water maze was used to evaluate spatial learning and memory. Oxidative stress biomarkers and brain-derived neurotrophic factor were evaluated in hippocampal tissues., Results: The results showed that Diabetes Mellitus (DM) impaired both short- and long- term memory (P<0.01), while levosimendan protected the animals from memory impairment. In addition, levosimendan prevented DM-induced reduction in the hippocampal levels of superoxide dismutase and glutathione peroxidase (P<0.05). Moreover, the administration of levosimendan prevented DM-induced increases in hippocampal thiobarbituric acid reactive substances level (P<0.05). Furthermore, levosimendan restored the ratio of reduced/oxidized glutathione (GSH/GSSG) in DM rats to that observed in the control group (P<0.05)., Conclusions: In summary, DM induced learning and memory impairment, and treatment with levosimendan impeded this impairment probably through preventing alterations in the antioxidant system in the hippocampus., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Aims: Regulation of vascular tone by 3',5'-cyclic adenosine monophosphate (cAMP) involves many effectors including the large conductance, Ca2+-activated, K+ (BKCa) channels. In arteries, cAMP is mainly hydrolyzed by type 3 and 4 phosphodiesterases (PDE3, PDE4). Here, we examined the specific contribution of BKCa channels to tone regulation by these PDEs in rat coronary arteries, and how this is altered in heart failure (HF)., Methods and Results: Concomitant application of PDE3 (cilostamide) and PDE4 (Ro-20-1724) inhibitors increased BKCa unitary channel activity in isolated myocytes from rat coronary arteries. Myography was conducted in isolated, U46619-contracted coronary arteries. Cilostamide (Cil) or Ro-20-1724 induced a vasorelaxation that was greatly reduced by iberiotoxin (IBTX), a BKCa channel blocker. Ro-20-1724 and Cil potentiated the relaxation induced by the β-adrenergic agonist isoprenaline (ISO) or the adenylyl cyclase activator L-858051 (L85). IBTX abolished the effect of PDE inhibitors on ISO but did not on L85. In coronary arteries from rats with HF induced by aortic stenosis, contractility and response to acetylcholine were dramatically reduced compared with arteries from sham rats, but relaxation to PDE inhibitors was retained. Interestingly, however, IBTX had no effect on Ro-20-1724- and Cil-induced vasorelaxations in HF. Expression of the BKCa channel α-subunit, of a 98 kDa PDE3A and of a 80 kDa PDE4D were lower in HF compared with sham coronary arteries, while that of a 70 kDa PDE4B was increased. Proximity ligation assays demonstrated that PDE3 and PDE4 were localized in the vicinity of the channel., Conclusion: BKCa channels mediate the relaxation of coronary artery induced by PDE3 and PDE4 inhibition. This is achieved by co-localization of both PDEs with BKCa channels, enabling tight control of cAMP available for channel opening. Contribution of the channel is prominent at rest and on β-adrenergic stimulation. This coupling is lost in HF.

Background: The calcium sensitizer levosimendan is increasingly used to improve hemodynamics in patients with acutely decompensated heart failure. By binding to cardiac troponin C the conformation of the calcium-troponin C complex is stabilized, which leads to acceleration of actin-myosin crossbrigde formation and increased force generating capacity of muscle fibers. Besides indications in cardiac failure, beneficial effects of levosimendan in skeletal muscle disorders are currently evaluated. The aim of this study was to investigate differential effects of levosimendan on skeletal muscle of pigs with and without susceptibility to malignant hyperthermia (MH) in order to identify possible risks of this emerging drug for patients with predisposition to MH., Methods: Muscle bundles of 17 pigs (9 MH susceptible (MHS); 8 MH non-susceptible (MHN)) were excised under general anesthesia and examined in the tissue bath with increasing concentrations of levosimendan (0.065; 0.125; 0.5; 1.0; 10 and 50 μg/ml). Baseline tension and twitch force were monitored continuously. Data are presented as median and interquartile range. Statistical evaluation was performed using D'Agostino & Pearson test for normal distribution and student's t test and 2-way ANOVA for differences between the groups. P < 0.05 was considered significant., Results: There were no differences between the groups concerning length, weight, initial twitch force and pre-drug resting tension of the investigated muscle strips. After an initial decrease in both groups, twitch amplitude was significantly higher in MHN (- 3.0 [- 5.2-0.2] mN) compared to MHS (- 7.5 [- 10.8- -4.5] mN) (p = 0.0034) muscle at an applied levosimendan concentration of 50 μg/ml. A marked increase in resting tension was detected following levosimendan incubation with 50 μg/ml in MHS muscle bundles (3.3 [0.9-6.1] mN) compared to MHN (- 0.7 [- 1.3-0.0] mN) (p < 0.0001)., Conclusions: This in vitro investigation revealed the development of significant contractures in muscle bundles of MHS pigs after incubation with levosimendan. However, the effect appeared only at supra-therapeutic concentrations and further research is needed to determine the impact of levosimendan on MHS individuals in vivo.

Ensifentrine (RPL554), an inhaled 'bifunctional' dual phosphodiesterase 3/4 inhibitor that exhibits both bronchodilator and anti-inflammatory activities, provides a new option in the treatment of chronic obstructive pulmonary disease (COPD) and other inflammatory airway diseases that are under clinical development. Ensifentrine appears to be initially under development for the treatment of COPD although it is not yet clear whether it should be understood as an add-on therapy in patients for the treatment of acute exacerbations of COPD or for the regular maintenance treatment of patients either alone, or on top of existing drug classes.

Bovine growing oocytes with a diameter of 105-115 μm from early antral follicles (1.2-1.8 mm) are able to resume meiosis, but lack the competence to mature to metaphase II. To confer full maturation competence onto the oocytes, culture systems which can support their growth and prevent their meiotic resumption during culture are needed. In this study, we cultured growing oocytes for 5 days to examine the effects of different phosphodiesterase (PDE) inhibitors on meiotic arrest and acquisition of full maturation competence of growing oocytes, and their gap junctional communication with cumulus cells. Growing oocyte-cumulus complexes (OCCs) were cultured with 3-isobutyl-1-methylxanthine (IBMX; broad-spectrum PDE inhibitor), rolipram (PDE4D inhibitor), cilostamide and milrinone (PDE3A inhibitors). The mean diameters of oocytes increased similarly in all groups. IBMX, cilostamide and milrinone induced antrum formation by OCCs and maintained meiotic arrest of oocytes during culture, whereas rolipram neither promoted antrum formation nor maintained oocyte meiotic arrest. Gap junctional communication between oocytes and cumulus cells was maintained by IBMX and cilostamide, but not by rolipram as judged by the transfer of injected lucifer yellow dye from oocytes to cumulus cells. In subsequent in vitro maturation, oocytes grown with IBMX, cilostamide and milrinone showed full maturation competence. These results suggest that PDE3A inhibition maintains the meiotic arrest of bovine growing oocytes and sustains their gap junctional communication with cumulus cells for 5 days, thereby contributing to their acquisition of full maturation competence., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Cilostazol, a phosphodiesterase 3 (PDE3) inhibitor, increases the intracellular level of cyclic adenosine monophosphate to cause vasodilation. Topical application of cilostazol is reported to improve local blood flow and enhance wound healing; however, its effect on human hair follicles is unknown., Objective: The purpose of this study was to determine the effect of cilostazol on hair growth., Methods: We investigated the expression of PDE3 in human dermal papilla cells (DPCs), outer root sheath cells (ORSCs), and hair follicles. The effects of cilostazol on DPC and ORSC proliferation were evaluated using BrdU and WST-1 assays. The expression of various growth factors in DPCs was investigated by growth factor antibody array. Additionally, hair shaft elongation was measured using ex vivo hair follicle organ cultures, and anagen induction was evaluated in C57BL/6 mice. Finally, the effects of cilostazol on vessel formation and activation of the mitogen-activated protein kinase pathway were evaluated., Results: We confirmed high mRNA and protein expression of PDE3 in human DPCs. Cilostazol not only enhanced the proliferation of human DPCs but also regulated the secretion of several growth factors responsible for hair growth. Furthermore, it promoted hair shaft elongation ex vivo, with increased proliferation of matrix keratinocytes. Cilostazol also accelerated anagen induction by stimulating vessel formation and upregulating the levels of phosphorylated extracellular signal-regulated kinase, c-Jun N-terminal kinase, and P38 after its topical application in C57BL/6 mice., Conclusion: Our results show that cilostazol promotes hair growth and may serve as a therapeutic agent for the treatment of alopecia., (Copyright © 2018 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.)

Background and Purpose: cAMP is a central second messenger that broadly regulates cell function and can underpin pathophysiology. In chronic obstructive pulmonary disease, a lung disease primarily provoked by cigarette smoke (CS), the activation of cAMP-dependent pathways, via inhibition of hydrolyzing PDEs, is a major therapeutic strategy. Mechanisms that disrupt cAMP signalling in airway cells, in particular regulation of endogenous PDEs, are poorly understood., Experimental Approach: We used a novel Förster resonance energy transfer (FRET) based cAMP biosensor in mice in vivo, ex vivo precision cut lung slices (PCLS) and in human cell models, in vitro, to track the effects of CS exposure., Key Results: Under fenoterol stimulation, FRET responses to cilostamide were significantly increased in in vivo, ex vivo PCLS exposed to CS and in human airway smooth muscle cells exposed to CS extract. FRET signals to rolipram were only increased in the in vivo CS model. Under basal conditions, FRET responses to cilostamide and rolipram were significantly increased in in vivo, ex vivo PCLS exposed to CS. Elevated FRET signals to rolipram correlated with a protein up-regulation of PDE4 subtypes. In ex vivo PCLS exposed to CS extract, rolipram reversed down-regulation of ciliary beating frequency, whereas only cilostamide significantly increased airway relaxation of methacholine pre-contracted airways., Conclusion and Implications: Exposure to CS, in vitro or in vivo, up-regulated expression and activity of both PDE3 and PDE4, which affected real-time cAMP dynamics. These mechanisms determine the availability of cAMP and can contribute to CS-induced pulmonary pathophysiology., (© 2018 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Alzheimer disease (AD) is the most common type of dementia, and is currently incurable. The efficacy of existing treatments for AD such as acetylcholinesterase inhibitors is limited to symptom improvement. Research on disease-modifying therapies (DMTs) has conventionally focused on amelioration of CNS pathogenesis. Two neuropathological changes correlate strongly with AD, the appearance of neurofibrillary tangles containing the microtubule-associated protein tau and extracellular amyloid deposits containing amyloid β-protein (Aβ). The aggregation of Aβ is believed to be the key pathogenic event in AD, with oligomeric assemblies thought to be the most neurotoxic form. Inhibitors of oligomer formation, therefore, could be valuable therapeutics for AD patients. The clinical phosphodiesterase type-3 inhibitor cilostazol (CSZ) was recently found to suppress the progression of cognitive decline in patients with stable AD receiving acetylcholinesterase inhibitors. Here we examined the effects of CSZ on in vitro aggregations of Aβ 1-40 and Aβ 1-42 including oligomerization, using the thioflavin T assay, photo-induced cross-linking of unmodified proteins, and electron microscopy. CSZ (25-100 μM) inhibited Aβ aggregation, especially oligomer formation. Considering that CSZ might be a key molecule for DMTs of AD, it cannot be ruled out that the low concentration of CSZ achievable in patient dosing may display some ant-oligomeric activity in synergy with its known therapeutic effects., (Copyright © 2018 Elsevier B.V. All rights reserved.)

It is currently unknown whether levosimendan can improve clinical outcomes in patients undergoing cardiac surgery. This meta-analysis aimed to assess the effect of levosimendan on mortality and the duration of intensive care unit (ICU) and hospital stay in adult patients undergoing cardiac surgery. A comprehensive search for eligible articles was conducted in PubMed, OVID and Cochrane databases of clinical trials and the Web of Science from database inception to August 2017. Stata/SE 11.0 was used to calculate the pooled odds ratio for postoperative mortality and the pooled standardized mean difference (SMD) for the duration of ICU stay and hospital stay. A total of 30 randomized controlled trials were included in the final analysis; the pooled results indicated that perioperative administration of levosimendan was associated with a reduction in postoperative mortality [5.8% vs 8.5%; odds ratio 0.66, 95% confidence interval 0.50-0.86, P = 0.002; I2 = 17.1%; 25 trials; 3239 patients] and length of ICU stay (SMD -0.32, 95% CI -0.58 to 0.06, P = 0.017; I2 = 88.0%; 23 trials; 2536 patients) compared with the control group but not in length of hospital stay (SMD -0.41, 95% CI -0.89 to 0.07, P = 0.094; I2 = 95.9%; 18 trials; 2047 patients). A subanalysis was conducted for trials published after 2015, and it suggested that levosimendan could not reduce the postoperative mortality (odds ratio = 0.91, 95% CI 0.63-1.31, P = 0.626; I2 = 0.9%), length of ICU stay (SMD -0.03, 95% CI -0.32 to 0.27, P = 0.850; I2 = 81.2%) or length of hospital stay (SMD 0.06, 95%CI -0.43 to 0.54, P = 0.821; I2 = 91.3%). To summarize, the evidence from studies published in the last 3 years indicated that perioperative administration of levosimendan was not associated with better clinical outcomes in adult patients undergoing cardiac surgery.

Background: Spontaneous firing of sinoatrial node cells (SANCs) is regulated by cAMP-mediated, PKA (protein kinase A)-dependent (cAMP/PKA) local subsarcolemmal Ca 2+ releases (LCRs) from RyRs (ryanodine receptors). LCRs occur during diastolic depolarization and activate an inward Na + /Ca 2+ exchange current that accelerates diastolic depolarization rate prompting the next action potential. PDEs (phosphodiesterases) regulate cAMP-mediated signaling; PDE3/PDE4 represent major PDE activities in SANC, but how they modulate LCRs and basal spontaneous SANC firing remains unknown., Methods: Real-time polymerase chain reaction, Western blot, immunostaining, cellular perforated patch clamping, and confocal microscopy were used to elucidate mechanisms of PDE-dependent regulation of cardiac pacemaking., Results: PDE3A, PDE4B, and PDE4D were the major PDE subtypes expressed in rabbit SANC, and PDE3A was colocalized with α-actinin, PDE4D, SERCA (sarcoplasmic reticulum Ca 2+ ATP-ase), and PLB (phospholamban) in Z-lines. Inhibition of PDE3 (cilostamide) or PDE4 (rolipram) alone increased spontaneous SANC firing by ≈20% ( P <0.05) and ≈5% ( P >0.05), respectively, but concurrent PDE3+PDE4 inhibition increased spontaneous firing by ≈45% ( P <0.01), indicating synergistic effect. Inhibition of PDE3 or PDE4 alone increased L-type Ca 2+ current (I Ca,L ) by ≈60% ( P <0.01) or ≈5% ( P >0.05), respectively, and PLB phosphorylation by ≈20% ( P >0.05) each, but dual PDE3+PDE4 inhibition increased I Ca,L by ≈100% ( P <0.01) and PLB phosphorylation by ≈110% ( P <0.05). Dual PDE3+PDE4 inhibition increased the LCR number and size ( P <0.01) and reduced the SR (sarcoplasmic reticulum) Ca 2+ refilling time ( P <0.01) and the LCR period (time from action potential-induced Ca 2+ transient to subsequent LCR; P <0.01), leading to decrease in spontaneous SANC cycle length ( P <0.01). When RyRs were disabled by ryanodine and LCRs ceased, dual PDE3+PDE4 inhibition failed to increase spontaneous SANC firing., Conclusions: Basal cardiac pacemaker function is regulated by concurrent PDE3+PDE4 activation which operates in a synergistic manner via decrease in cAMP/PKA phosphorylation, suppression of LCR parameters, and prolongation of the LCR period and spontaneous SANC cycle length., (© 2018 American Heart Association, Inc.)