Age Impairs Soluble Guanylyl Cyclase Function in Mouse Mesenteric Arteries

Endothelial dysfunction (ED) comes with age, even without overt vessel damage such as that which occurs in atherosclerosis and diabetic vasculopathy. We hypothesized that aging would affect the downstream signalling of the endothelial nitric oxide (NO) system in the vascular smooth muscle (VSM). With this in mind, resistance mesenteric arteries were isolated from 13-week (juvenile) and 40-week-old (aged) mice and tested under isometric conditions using wire myography. Acetylcholine (ACh)-induced relaxation was reduced in aged as compared to juvenile vessels. Pretreatment with L-NAME, which inhibits nitrix oxide synthases (NOS), decreased ACh-mediated vasorelaxation, whereby differences in vasorelaxation between groups disappeared. Endothelium-independent vasorelaxation by the NO donor sodium nitroprusside (SNP) was similar in both groups; however, SNP bolus application (10−6 mol L−1) as well as soluble guanylyl cyclase (sGC) activation by runcaciguat (10−6 mol L−1) caused faster responses in juvenile vessels. This was accompanied by higher cGMP concentrations and a stronger response to the PDE5 inhibitor sildenafil in juvenile vessels. Mesenteric arteries and aortas did not reveal apparent histological differences between groups (van Gieson staining). The mRNA expression of the α1 and α2 subunits of sGC was lower in aged animals, as was PDE5 mRNA expression. In conclusion, vasorelaxation is compromised at an early age in mice even in the absence of histopathological alterations. Vascular smooth muscle sGC is a key element in aged vessel dysfunction.

Suppression of Proliferation of Human Glioblastoma Cells by Combined Phosphodiesterase and Multidrug Resistance-Associated Protein 1 Inhibition

The paucity of currently available therapies for glioblastoma multiforme requires novel approaches to the treatment of this brain tumour. Disrupting cyclic nucleotide-signalling through phosphodiesterase (PDE) inhibition may be a promising way of suppressing glioblastoma growth. Here, we examined the effects of 28 PDE inhibitors, covering all the major PDE classes, on the proliferation of the human U87MG, A172 and T98G glioblastoma cells. The PDE10A inhibitors PF-2545920, PQ10 and papaverine, the PDE3/4 inhibitor trequinsin and the putative PDE5 inhibitor MY-5445 potently decreased glioblastoma cell proliferation. The synergistic suppression of glioblastoma cell proliferation was achieved by combining PF-2545920 and MY-5445. Furthermore, a co-incubation with drugs that block the activity of the multidrug resistance-associated protein 1 (MRP1) augmented these effects. In particular, a combination comprising the MRP1 inhibitor reversan, PF-2545920 and MY-5445, all at low micromolar concentrations, afforded nearly complete inhibition of glioblastoma cell growth. Thus, the potent suppression of glioblastoma cell viability may be achieved by combining MRP1 inhibitors with PDE inhibitors at a lower toxicity than that of the standard chemotherapeutic agents, thereby providing a new combination therapy for this challenging malignancy.

Inhibition of Phosphodiesterase 3A by Cilostazol Dampens Proinflammatory Platelet Functions

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.

Design of a randomised, double-blind, crossover, placebo-controlled trial of effects of sildenafil on cerebrovascular function in small vessel disease: Oxford haemodynamic adaptation to reduce pulsatility trial (OxHARP)

Background Cerebral small vessel disease (SVD) is associated with increased cerebrovascular pulsatility, endothelial dysfunction, and impaired vascular reactivity. Vasodilating phosphodiesterase inhibitors may improve cardiovascular pulsatility and reactivity, and potentially reduce progression of SVD. Hypothesis: Sildenafil, a PDE5 inhibitor, will reduce cerebrovascular pulsatility and increase cerebrovascular reactivity compared to placebo, and is non-inferior to cilostazol, a PDE3 inhibitor. Methods OxHARP is a randomised, double-blind, crossover trial of sildenafil 50 mg thrice daily, cilostazol 100 mg twice daily and placebo in 75 patients with mild to moderate small vessel disease and a previous lacunar or cryptogenic stroke or TIA. Participants undergo a physiological assessment at baseline and on each treatment, including transcranial Doppler ultrasound (TCD, DWL DopplerBox) to assess cerebrovascular pulsatility and reactivity to 4–6% carbon dioxide. In up to 60 patients, cerebrovascular pulsatility, perfusion and reactivity will also be assessed by MRI. Outcome measures The primary outcome is difference in middle cerebral artery pulsatility (Gosling’s Pulsatility Index, PI) after 3 weeks of sildenafil versus placebo. Secondary outcomes including non-inferiority of sildenafil vs cilostazol in effects on PI, percentage increase in MCA blood flow velocity and BOLD-fMRI response during inhalation of 4–6% carbon dioxide. Discussion Reduction in cerebral pulsatility and increased cerebrovascular reactivity during treatment with sildenafil would indicate potential benefit to prevent progression of SVD, suggesting a need for trials with clinical outcomes. Trial Registration OxHARP is registered with ClinicalTrials.org, NCT03855332

Phenotypical Effect of Phosphodiesterase 5 (PDE5) Inhibitor on Behavioral Activities of Fruit Fly Drosophila melanogaster

Phosphodiesterase 5 (PDE5) inhibitor is a class of drugs currently used to treat erectile dysfunction. Physiologically, inhibition of PDE5 may lead to vasodilation, blood flow increment, and penile erection. However, PDE5 inhibitors have been reported not only to modify the function of the male reproductive organ but also to influence other physiological systems. To explore the effect of PDE5 inhibitor on metazoan physiological systems, a fruit fly (Drosophila melanogaster) model organism is used since the catalytic domain of fruit fly PDE5/6 shares a high similarity of amino acid sequence (58%) with the PDE5 of humans. This study aimed to investigate whether the effect of PDE5 inhibition by sildenafil is phenotypically observable as changes in the behavioral states. Two behavioral phenotypes of D. melanogaster, negative geotaxis, and ethanol sensitivity, were used as test parameters in this explorative study. The results demonstrated that sildenafil had a significant effect on reducing locomotor activity, as reflected by negative geotaxis assay, but it had no influence on the fruit fly sensitivity to ethanol. Taken together, our results suggested that PDE5 inhibition might impair the physiological condition of the metazoan species. Also, an explorative study using D. melanogaster might offer valuable insight as a model organism in the discovery and repurposing approach of PDE5 inhibitor.

Pancreatic fat cells of humans with type 2 diabetes display reduced adipogenic and lipolytic activity

Obesity, especially visceral fat accumulation, increases the risk of type 2 diabetes (T2D). The purpose of this study was to investigate the impact of T2D on the pancreatic fat depot. Pancreatic fat pads from 17 partial pancreatectomized patients (PPP) were collected, pancreatic preadipocytes isolated and in vitro differentiated. Patients were grouped using HbA1c into normal glucose tolerant (NGT), prediabetic (PD) and T2D. Transcriptome profiles of preadipocytes and adipocytes were assessed by RNAseq. Insulin sensitivity was estimated by quantifying AKT phosphorylation on western blots. Lipogenic capacity was assessed with oil red O staining, lipolytic activity via fatty acid release. Secreted factors were measured using ELISA. Comparative transcriptome analysis of preadipocytes and adipocytes indicates defective upregulation of genes governing adipogenesis (NR1H3), lipogenesis (FASN, SCD, ELOVL6, FADS1) and lipolysis (LIPE) during differentiation of cells from T2D-PPP. In addition, the ratio of leptin/adiponectin mRNA was higher in T2D than in NGT-PPP. Preadipocytes and adipocytes of NGT-PPP were more insulin sensitive than T2D-PPP cells in regard to AKT phosphorylation. Triglyceride accumulation was similar in NGT and T2D adipocytes. Despite a high expression of the receptors NPR1 and NPR2 in NGT and T2D adipocytes, lipolysis was stimulated by ANP 1.74-fold in NGT cells only. This stimulation was further increased by the PDE5 inhibitor dipyridamole (3.09-fold). Dipyridamole and forskolin increased lipolysis receptor-independently 1.88-fold and 1.48-fold, respectively, solely in NGT cells. In conclusion, the metabolic status persistently affects differentiation and lipolysis of pancreatic adipocytes. These alterations could aggravate the development of T2D.

Effect of sildenafil citrate on prediabetic and diabetic albino rats treated with metformin

Diabetes Miletus (DM) is a global epidemic disease. It is estimated that there are already 415 million adults aged 20–79 years diabetics worldwide. Sildenafil citrate is a phosphodiesterase type 5 (PDE5) inhibitor, which increases cyclic guanosine monophosphate (cGMP) and metformin (MET) is a biguanide used for the treatment of type 2 diabetes which increases peripheral insulin sensitivity. Aim: This study aims to assess the effect of sildenafil citrate and metformin on lipid profile and glycemic control in diabetic and prediabetic albino rats. Materials and methods: Adult male albino rats are used and divided into nine groups each group consists of 10 rats, diabetes is induced by feeding a high-fat diet (HFD) for an initial period of 2 weeks followed by a single intraperitoneal injection of (35 mg/kg) Streptozotocin. Prediabetes is induced by feeding (HFD) and glucose in water for a period of 2 weeks. Sildenafil was given in a dose of (5 &10 mg/kg/day orally for 4 weeks), metformin was given in a dose of (50 &100 mg/kg/day orally for 4 weeks) using oral gavages to normal healthy rats, diabetic and prediabetic rats. Blood samples were collected after 4 weeks of treatment in all experimental groups. Results: Combined administration of sildenafil and metformin on diabetic rats improving hyperglycemia, oxidative stress, and hyperlipidemia induced by streptozotocin than the administration of metformin or sildenafil alone. Conclusion: Sildenafil has beneficial effects against some diabetic complications. The present study showed that sildenafil with metformin has beneficial effects against diabetic complications.