Phosphodiesterase 5A Research Articles

Downregulation of miR-214 promotes dilated Cardiomyopathy Progression through PDE5A-Mediated cGMP regulation.

Dilated cardiomyopathy (DCM) is a myocardial disorder resulting in a substantial decline in cardiac function and potentially leading to heart failure. This research combines bioinformatics analysis with empirical validation to explore the roles and mechanisms of miR-214 in DCM. Using the DEseq2 R package, a total of 125 differentially expressed circulating miRNAs (DE c-miRNAs) and 784 DE genes (DEGs) were identified. Cross-analysis between target genes of DE c-miRNAs and DEGs identified 124 common genes, and protein-protein interaction analysis of common genes identified 11 hub genes. Twelve DE c-miRNAs were further verified by quantifying their levels in the serum of DCM patients and healthy individuals. miR-214 levels were significantly decreased in serum from DCM patients, positively correlated with left ventricular ejection fraction and left ventricular fractional shortening. Further analysis showed that miR-214 directly targets and negatively regulates phosphodiesterase 5A (PDE5A). Elevated PDE5A expression reduced cGMP levels; however, using sildenafil, a PDE5A inhibitor, reversed this effect, substantiating the regulatory mechanism of miR-214 on cGMP via PDE5A. These results provide new potential targets for the diagnosis and treatment of DCM.

MiR-30d Attenuates Pulmonary Arterial Hypertension via Targeting MTDH and PDE5A and Modulates the Beneficial Effect of Sildenafil.

Pulmonary arterial hypertension (PAH) is associated with aberrant pulmonary vascular smooth muscle cell (PASMC) function and vascular remodeling. MiR-30d plays an important role in the pathogenesis of several cardiovascular disorders. However, the function of miR-30d in PAH progression remained unknown. Our study shows that circulating miR-30d level is significantly reduced in the plasma from PAH patients. In miR-30d transgenic (TG) rats, overexpressing miR-30d attenuates monocrotaline (MCT)-induced pulmonary hypertension (PH) and pulmonary vascular remodeling. Increasing miR-30d also inhibits platelet-derived growth factor-bb (PDGF-bb)-induced proliferation and migration of human PASMC. Metadherin (MTDH) and phosphodiesterase 5A (PDE5A) are identified as direct target genes of miR-30d. Meanwhile, nuclear respiratory factor 1 (NRF1) acts as a positive upstream regulator of miR-30d. Using miR-30d knockout (KO) rats treated with sildenafil, a PDE5A inhibitor that is used in clinical PAH therapies, it is further found that suppressing miR-30d partially attenuates the beneficial effect of sildenafil against MCT-induced PH and vascular remodeling. The present study shows a protective effect of miR-30d against PAH and pulmonary vascular remodeling through targeting MTDH and PDE5A and reveals that miR-30d modulates the beneficial effect of sildenafil in treating PAH. MiR-30d should be a prospective target to treat PAH and pulmonary vascular remodeling.

Identification of Critical Genes Differentiating Stable and Unstable Atherosclerotic Plaques: A Bioinformatic and Computational Analysis.

Identification of biomarkers to distinguish between stable and unstable plaque formation would be very useful to predict plaque vulnerability. We downloaded microarray profiles of gene set enrichment (GSE) accession numbers including GSE71226 and GSE20680 (group A: containing healthy vs stable plaque samples) and GSE62646 and GSE34822 (group B: containing stable vs unstable plaque samples) from Gene expression omnibus (GEO) database. Differentially expressed genes were compared in both data sets of each group. Ten and 12 key genes were screened in groups A and B, respectively. Gene Ontology (GO) enrichment was applied by the plugin "BiNGO" (Biological networks gene ontology tool) of the Cytoscape. The key genes were mostly enriched in the biological process of positive regulation of the cellular process. The protein-protein interaction and co-expression network were analyzed by the STRING (search tool for the retrieval of interacting genes/proteins) and GeneMANIA (gene multiple association network integration algorithm) plugin of Cytoscape, respectively, which showed that Epidermal growth factor (EGF), Heparin-binding EGF like growth factor (HBEGF), and Matrix metalloproteinase 9 (MMP9) were at the core of the network. Further validation of key genes using two datasets showed that Phosphodiesterase 5A (PDE5A) and Protein S (PROS1) were decreased in unstable plaques, while Suppressor of cytokine signaling (SOCS3), HBEGF, and Leukocyte immunoglobulin-like receptor B4 (LILRB4) were increased. The present study used several datasets to identify key genes associated with stable and unstable atherosclerotic plaque.

Potential drug targets for tumors identified through Mendelian randomization analysis.

According to the latest cancer research data, there are a significant number of new cancer cases and a substantial mortality rate each year. Although a substantial number of clinical patients are treated with existing cancer drugs each year, the efficacy is unsatisfactory. The incidence is still high and the effectiveness of most cancer drugs remains unsatisfactory. Therefore, we evaluated the human proteins for their causal relationship to for cancer risk and therefore also their potential as drug targets. We used summary tumors data from the FinnGen and cis protein quantitative trait loci (cis-pQTL) data from a genome-wide association study, and employed Mendelian randomization (MR) to explore the association between potential drug targets and nine tumors, including breast, colorectal, lung, liver, bladder, prostate, kidney, head and neck, pancreatic caners. Furthermore, we conducted MR analysis on external cohort. Moreover, Bidirectional MR, Steiger filtering, and colocalization were employed to validate the main results. The DrugBank database was used to discover potential drugs of tumors. Under the threshold of False discovery rate (FDR) < 0.05, results showed that S100A16 was protective protein and S100A14 was risk protein for human epidermal growth factor receptor 2-positive (HER-positive) breast cancer, phosphodiesterase 5A (PDE5A) was risk protein for colorectal cancer, and melanoma inhibitory activity (MIA) was protective protein for non-small cell lung carcinoma (NSCLC). And there was no reverse causal association between them. Colocalization analysis showed that S100A14 (PP.H4.abf = 0.920) and S100A16 (PP.H4.abf = 0.932) shared causal variation with HER-positive breast cancer, and PDE5A (PP.H4.abf = 0.857) shared causal variation with colorectal cancer (CRC). The MR results of all pQTL of PDE5A and MIA were consistent with main results. In addition, the MR results of MIA and external outcome cohort were consistent with main results. In this study, genetic predictions indicate that circulating S100 calcium binding protein A14 (S100A14) and S100 calcium binding protein A16 (S100A16) are associated with increase and decrease in the risk of HER-positive breast cancer, respectively. Circulating PDE5A is associated with increased risk of CRC, while circulating MIA is associated with decreased risk of NSCLC. These findings suggest that four proteins may serve as biomarkers for cancer prevention and as potential drug targets that could be expected for approval.

Phosphodiesterase 5A regulates the vomeronasal pump in mice.

The vomeronasal organ (VNO) is a specialized chemoreceptive structure in many vertebrates that detects chemical stimuli, mostly pheromones, which often elicit innate behaviors such as mating and aggression. Previous studies in rodents have demonstrated that chemical stimuli are actively transported to the VNO via a blood vessel-based pumping mechanism, and this pumping mechanism is necessary for vomeronasal stimulation in behaving animals. However, the molecular mechanisms that regulate the vomeronasal pump remain mostly unknown. In this study, we observed a high level of expression of phosphodiesterase 5A (PDE5A) in the vomeronasal blood vessel of mice. We provided evidence to support the potential role of PDE5A in vomeronasal pump regulation. Local application of PDE5A inhibitors-sildenafil or tadalafil-to the vomeronasal organ (VNO) reduced stimulus delivery into the VNO, decreased the pheromone-induced activity of vomeronasal sensory neurons, and attenuated male-male aggressive behaviors. PDE5A is well known to play a role in regulating blood vessel tone in several organs. Our study advances our understanding of the molecular regulation of the vomeronasal pump.

Mex-3 RNA binding family member A (MEX3A)/circMPP6 complex promotes colorectal cancer progression by inhibiting autophagy

RNA-binding proteins (RBPs)-RNA networks have contributed to cancer development. Circular RNAs (circRNAs) are considered as protein recruiters; nevertheless, the patterns of circRNA-protein interactions in colorectal cancer (CRC) are still lacking. Processing bodies (PBs) formed through liquid-liquid phase separation (LLPS) are membrane-less organelles (MLOs) consisting of RBPs and RNA. Previous evidence suggests a connection between PBs dynamics and cancer progression. Despite the increasingly acknowledged crucial role of RBPs and RNA in the accumulation and maintenance of MLOs, there remains a lack of specific research on the interactions between PBs-related RBPs and circRNAs in CRC. Herein, we identify that MEX-3 RNA binding family member A (MEX3A), frequently upregulated in CRC tissues, predicts poorer patient survival. Elevated MEX3A accelerates malignance and inhibits autophagy of CRC cells. Importantly, MEX3A undergoes intrinsically disordered regions (IDRs)-dependent LLPS in the cytoplasm. Specifically, circMPP6 acts as a scaffold to facilitate the interaction between MEX3A and PBs proteins. The MEX3A/circMPP6 complex modulates PBs dynamic and promotes UPF-mediated phosphodiesterase 5A (PDE5A) mRNA degradation, consequently leading to the aggressive properties of CRC cells. Clinically, CRC patients exhibiting high MEX3A expression and low PDE5A expression have the poorest overall survival. Our findings reveal a collaboration between MEX3A and circMPP6 in the regulation of mRNA decay through triggering the PBs aggregation, which provides prognostic markers and/or therapeutic targets for CRC.

Icariin has the potential to induce the differentiation of bone marrow mesenchymal stem cells into brown fat cells via PDE5A inhibition

BackgroundBone marrow mesenchymal stem cells (BMMSCs) possess the ability of adipogenic differentiation. Icariin (ICA) is a prenylated flavonol glycoside with diverse pharmacological activities and has been reported to promote osteogenic differentiation of BMMSCs. Nevertheless, the effects of ICA on BMMSC adipogenic differentiation into brown fat cells are still unclear. This study aimed to explore the effects and mechanistic basis of ICA on the differentiation of BMMSCs into brown fat cells. MethodsOil Red-O staining assay was applied to detect the adipogenic differentiation of BMMSCs after induction. RT-qPCR and Western blot were conducted to detect the expression of lipogenic markers PPARγ and FABP4 as well as the brown fat biomarkers BMP7, PGC-1α, and UCP1 in BMMSCs. Moreover, phosphodiesterase-5A (PDE5A) expression in BMMSCs treated with ICA was measured by RT-qPCR and Western blot. ResultsICA promoted the adipogenic differentiation of BMMSCs and increased the expression levels of lipogenic markers PPARγ and FABP4 and the brown fat biomarkers BMP7, PGC-1α, and UCP1 during the adipogenic differentiation of BMMSCs. Furthermore, PDE5A was identified as a target of ICA, and its expression was reduced by ICA treatment. Moreover, PDE5A inhibition enhanced BMP7, PGC-1α, and UCP1 levels in BMMSCs. Additionally, overexpression of PDE5A notably reversed the effects of ICA in the differentiation of BMMSCs into brown fat cells. ConclusionICA induces the differentiation of BMMSCs into brown fat cells via PDE5A inhibition, highlighting the therapeutic value of ICA for treating obesity-related diseases.

RUNX1 Isoforms Regulate RUNX1 and Target-Genes Differentially in Platelets/Megakaryocytes: Association with Clinical Cardiovascular Events

RUNX1 is an essential transcription factor for hematopoiesis. Germline RUNX1 haplodeficiency is associated with thrombocytopenia, platelet dysfunction and predisposition to myeloid malignancies. Three major RUNX1 isoforms (A, B and C) are recognized and share a DNA binding RUNT domain; RUNX1A lacks the transactivation domain. Two distinct promoters regulate RUNX1 expression: distal P1 regulates RUNX1C; proximal P2 regulates RUNX1B and RUNX1A. Little is known regarding the differential effects of isoforms in RUNX1 autoregulation, target-gene regulation and association with clinical events. We performed studies in megakaryocytic HEL cells, HeLa cells (which do not express RUNX1) and in platelets of healthy subjects (n=74) and provide evidence for isoform-specific differential autoregulation and target-gene regulation by RUNX1. We show an association between expression of specific RUNX1-regulated genes in whole blood with cardiovascular events in patients. There are 5 RUNX1 consensus binding sites in P1 promoter and 1 in P2 promoter within ~1000 bp from ATG. Wild type RUNX1 P1 and P2 promoter regions were individually cloned into PGL4 luciferase promoter vector. ChIP studies using PMA-treated megakaryocytic HEL cells showed RUNX1 binding to chromatin regions encompassing the RUNX1 binding sites in both promoters. Mutations of 2 RUNX1 binding sites in intron region of P1 promoter reduced promoter activity; mutations of first 2 RUNX1 binding sites at the exon region of P1 promoter increased activity. Mutation of single RUNX1 binding site in P2 promoter increased promoter activity. Thus, RUNX1 binds to P1 and P2 promoters to regulate activities. To examine the RUNX1 autoregulation by individual isoforms, we co-transfected each isoform with P1 or P2 promoter vector in HeLa cells, which do not express endogenous RUNX1. In response to RUNX1B over-expression, both P1 and P2 promoters showed a dose-dependent decrease in promoter activity. RUNX1C over-expression increased P1 and P2 promoter activities. Thus, RUNX1B and RUNX1C regulate P1 and P2 promoters differentially. In HEL cells, which have endogenous RUNX1, RUNX1B overexpression decreased RUNX1C and RUNX1A mRNA/protein expression by ~50%. RUNX1C overexpression increased RUNX1B and RUNX1A mRNA/protein expression. Thus, RUNX1B decreases RUNX1C and RUNX1A expression; RUNX1C increases RUNX1B and RUNX1A. We studied the regulation by RUNX1 isoforms of target genes PCTP (phosphatidylcholine transfer protein), MYL9 (myosin light chain), PDE5A (phosphodiesterase 5A) and F13A (factor XIIIA) . In HeLa cells. RUNX1B overexpression increased PCTP, MYL9 and PDE5A (protein and mRNA); RUNX1C overexpression reduced PCTP and MYL9. In HEL cells RUNX1B increased PCTP, MYL9, PDE5A and F13A expression; RUNX1C reduced PCTP and MYL9 expression. To obtain evidence in vivo, we studied the relationships between RUNX1 isoforms and their relationships to RUNX1-target genes by RNAseq in leukocyte-poor platelets (74 healthy donors; ages 30-75 yrs). RUNX1B correlated negatively with RUNX1C (R= -0.413 and p=1.5e-06) and RUNX1A (R= -0.493 and p=0.00e+oo); RUNX1C correlated positively with RUNX1A (R=0.45, p&amp;lt;0.0001). RUNX1B correlated positively with PCTP, F131A, PDE5A and RAB1B, and negatively with MYL9 (Figure 1); no significant relationships were observed with RUNX1C or RUNX1A. To understand the relationships to cardiovascular events (MI or death), we explored whole blood gene expression using microarrays in 587 patients (2 cohorts, 190 and 397 patients) presenting to cardiac catherization for chest pain and followed for 3.8 years. In our previous studies RUNX1B transcripts were higher and RUNX1C lower in those with MI or death. We now found that higher expressions of F13A1(Figure 2) and RAB31, which have been shown by us to be RUNX1-targets, were associated with cardiovascular events. Conclusions: RUNX1 isoforms autoregulate RUNX1 expression differentially and regulate target genes in a differential manner, a pattern consistent with in vivo platelet gene expression. The relative expression of RUNX1B and RUNX1C isoforms and of their target genes associate with clinical cardiovascular events. RUNX1-isoform specific effects need to be considered in clinical scenarios where RUNX1 is involved and in its pharmacologic modulation.

No impact of polymorphism in the phosphodiesterase 5A gene in Cavalier King Charles Spaniels on pimobendan-induced inhibition of platelet aggregation response.

A variant in the canine phosphodiesterase (PDE) 5A gene (PDE5A:E90K) is associated with decreased concentrations of circulating cyclic guanosine monophosphate (cGMP) and response to PDE5 inhibitor treatment. Pimobendan is a PDE inhibitor recommended for medical treatment of certain stages of myxomatous mitral valve disease (MMVD) in dogs. PDE5A:E90K polymorphism attenuates the inhibitory effect of pimobendan on in vitro platelet aggregation and increases basal platelet aggregation in Cavalier King Charles Spaniels (CKCS). Selected clinical variables (MMVD severity, sex, age, hematocrit, platelet count in platelet-rich plasma [PRP], and echocardiographic left ventricular fractional shortening [LV FS]) will not show an association with results. Fifty-two privately owned CKCS with no or preclinical MMVD. Using blood samples, we prospectively assessed PDE5A genotype using Sanger sequencing and adenosine diphosphate-induced platelet aggregation response (area under the curve [AUC], maximal aggregation [MaxA], and velocity [Vel]) with and without pimobendan using light transmission aggregometry. Dogs also underwent echocardiography. Pimobendan inhibited platelet function as measured by AUC, MaxA, and Vel at a concentration of 10 μM (P < .0001) and Vel at 0.03 μM (P < .001). PDE5A:E90K polymorphism did not influence the inhibitory effect of pimobendan or basal platelet aggregation response. The PDE5A:E90K polymorphism did not influence in vitro basal platelet aggregation response or the inhibitory effect of pimobendan on platelet aggregation in CKCS. Dogs with the PDE5A:E90K polymorphism did not appear to have altered platelet function or response to pimobendan treatment.

MicroRNA-1 protects the endothelium in acute lung injury.

Acute lung injury (ALI) and its most severe form, acute respiratory distress syndrome (ARDS), cause severe endothelial dysfunction in the lung, and vascular endothelial growth factor (VEGF) is elevated in ARDS. We found that the levels of a VEGF-regulated microRNA, microRNA-1 (miR-1), were reduced in the lung endothelium after acute injury. Pulmonary endothelial cell-specific (EC-specific) overexpression of miR-1 protected the lung against cell death and barrier dysfunction in both murine and human models and increased the survival of mice after pneumonia-induced ALI. miR-1 had an intrinsic protective effect in pulmonary and other types of ECs; it inhibited apoptosis and necroptosis pathways and decreased capillary leak by protecting adherens and tight junctions. Comparative gene expression analysis and RISC recruitment assays identified miR-1 targets in the context of injury, including phosphodiesterase 5A (PDE5A), angiopoietin-2 (ANGPT2), CNKSR family member 3 (CNKSR3), and TNF-α-induced protein 2 (TNFAIP2). We validated miR-1-mediated regulation of ANGPT2 in both mouse and human ECs and found that in a 119-patient pneumonia cohort, miR-1 correlated inversely with ANGPT2. These findings illustrate a previously unknown role of miR-1 as a cytoprotective orchestrator of endothelial responses to acute injury with prognostic and therapeutic potential.

Identification of selective inhibitors for phosphodiesterase 5A using e-pharmacophore modelling and large-scale virtual screening-based structure guided drug discovery approaches

The inhibition of Phosphodiesterase 5A (PDEA5) has the potential to modulate pulmonary arterial hypertension and cardiovascular diseases. Exploring the cross-reactivity of clinically available PDE5A therapeutics with PDE6A is intriguing in order to develop highly selective PDE5A compounds in cardiovascular arena. In the current study, we leveraged e-pharmacophore based screening and molecular dynamics (MD) simulation to discover more selective PDE5A inhibitors as compared to the PDE6A catalytic domain. e-Pharmacophore based mapping of the CoCoCo database (7 million compounds: ∼ 150,000,000 conformers), followed by Glide docking, MM-GBSA, and protein–inhibitor interaction analysis, revealed 1536427, 4832637 and 6788240 as stable, tight binders of PDE5A instead of PDE6A. These compounds adhere to Lipinski Rule of Five (RO5) and ADME/Tox criteria. MD simulations analysis showed that 1536427 stays stable and tightly binds to catalytic (Q-region) core of PDE5A catalytic domain as compared to sildenafil. Pronounced inward motions of the hydrophobic (H-region) and Lid region indicate the closure of PDE5A-1536427 complex, whereas this region in PDE6A-1536427 is more open. Significant differences in the interactions, stability, and dynamics of 1536427 were observed in the catalytic domain of PDE6A, demonstrating less specificity for PDE6A in comparison to PDE5A. After lead optimization and therapeutic interventions, this proposed lead may emerge as a promising PDE5A selective inhibitor. Communicated by Ramaswamy H. Sarma

Skin cancer signal associated with phosphodiesterase inhibitors: gaining insight through the FDA pharmacovigilance database

ABSTRACT Background Panoramic views of post-marketing safety profiles, such as cancer signal, of phosphodiesterase 5A (PDE5A) inhibitors have yet to be fully evaluated. Research design and methods Data from the FDA Adverse Event Reporting System (FAERS) concerning the timeframe between January 1st, 2004 to 30 June 2022 was analyzed through a disproportionality study to understand the association between sildenafil, tadalafil, and vardenafil and cancer. This association was identified using the Reporting odds ratio (ROR) and Bayesian Confidence Propagation Neural Network (BCPNN) approaches. Results Sildenafil associated ROR values and IC025 ranged from 9.19 (95% CI 7.72–10.94, IC025 2.77) for metastatic malignant melanoma to 132.23 (95% CI 95.49–183.11, IC025 4.69) for malignant melanoma stage II. Tadalafil associated ROR and IC025 ranged from 6.79 (95% CI 5.41–8.54, IC025 2.27) for metastatic malignant melanoma to 180.17 (95% CI 130.11–249.50, IC025 4.89) for malignant melanoma stage II. Vardenafil associated ROR and IC025 ranged from 23.38 (95% CI 15.20–35.96, IC025 2.63) for metastatic malignant melanoma to 245.77 (95% CI 154.42–391.16, IC025 2.10) for malignant melanoma stage III. Conclusions This study supports the association between sildenafil, tadalafil, and vardenafil with skin cancer signal in erectile dysfunction (ED) patients.

Identification of novel changes in skeletal muscle proteome after sildenafil treatment

Insulin Resistance (IR) is associated with increased matrix proteins, including collagens, fibronectins, and laminins in the endomysium surrounding skeletal muscle. The increased ECM deposition in skeletal muscle IR results in ECM stiffening that decreases muscle integrity and cellular signaling. Recent evidence has shown that Sildenafil, a phosphodiesterase 5a inhibitor, improves insulin sensitivity in humans, and in high fat diet mice, reduces collagen deposition in the matrix surrounding skeletal muscle. In addition, data shows that a short-term treatment of Sildenafil for 7 days increases protein synthesis and decreases muscle fatigue in humans, which may mitigate the effects of IR. However, very few studies have examined the molecular mechanisms and metabolic signaling directly associated with Sildenafil’s influence on skeletal muscle. The aim of our study was to perform an unbiased proteomic analysis on Sildenafil’s effects on skeletal muscle in C2C12 myotubes. We hypothesized that Sildenafil would alter the abundance of proteins in metabolic pathways that are involved in communicating with the ECM. We performed a dose response (n = 3 for each dose; 0.5 μM, 1 μM, 2.5 μM, 5 μM, and 10 μM) and time response (n = 3 for each time; 30 min, 1 hour, 4 hour, 6 hour) curve. Proteomic analyses revealed that the highest number of proteins significantly changing was at 1 μM for 30 min (n = 423). Analysis of the significant proteins revealed 396 and 27 proteins that increased and decreased, respectively. KEGG analysis revealed that 20% of the total changing proteins (n = 83) were in metabolic pathways. The top 10 metabolic protein increasers, represented as a fold change of abundance compared to vehicle, were ASCL3 (1.27 ± 0.08), GT251 (1.12 ± 0.10), ODPA (1.15 ± 0.03), PCKGM (1.08 ± 0.04), PCY1A (1.23 ± 0.04), PGM1 (1.06 ± 0.04), PTG1S (1.21 ± 0.05), PYR1 (1.37 ± 0.09). SAHH (1.19 + 0.07), and THIM (1.13 ± 0.08). Of the 83 metabolic proteins, only 6 were decreasing, P5CR2 (0.78 ± 0.04), ALD1 (0.82 ± 0.11), P4K2A (0.67 ± 0.05), DGAT1 (0.27 ± 0.19), SCLY (0.77 ± 0.15), and PLCD3 (0.61 ± 0.16). The results from this preliminary study conclude that Sildenafil affects proteins involved in metabolic pathways. Further analysis will be performed to confirm the roles of these proteins in relation to ECM signaling/communication in these metabolic pathways. University of Arizona's Research, Innovation &amp; Impact Faculty Seed Grant This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Flavonoids and prenylhydroquinones from the prepared folium of Epimedium sagittatum Maxim. and their inhibition against phosphodiesterase5A

An effort to identify novel active substances of the prepared folium of Epimedium sagittatum Maxim. (PFES) that was an important herb for male erectile dysfunction (ED) was taken. At present, phosphodiesterase-5A (PDE5A) is the most important target of new drugs for the treatment of ED. Therefore, the inhibition ingredients in PFES were systematically screened for the first time in this study. Eleven compounds, including eight new flavonoids and three prenylhydroquinones were isolated: sagittatosides DN (1−11), and their structures were elucidated by spectra and chemical analyses. Among them, a novel prenylflavonoid with oxyethyl group (1) was obtained and three prenylhydroquinones (9–11) were firstly isolated from Epimedium. All compounds were analyzed for the inhibition against PDE5A by molecular docking, and they all showed significant binding affinity as same as sildenafil. Their inhibitory activities were verified, and the results showed compound 6 had significant inhibition against PDE5A1. The isolation of new flavonoids and prenylhydroquinones with inhibitory activities of PDE5A from PFES implied that this herb might be a good source for the treatment of ED agents finding.

Inhibition of phosphodiesterase 5A by tadalafil improves SIRT1 expression and activity in insulin-resistant podocytes

A decrease in intracellular levels of 3′,5′-cyclic guanosine monophosphate (cGMP) has been implicated in the progression of diabetic nephropathy. Hyperglycemia significantly inhibits cGMP-dependent pathway activity in the kidney, leading to glomerular damage and proteinuria. The enhancement of activity of this pathway that is associated with an elevation of cGMP levels may be achieved by inhibition of the cGMP specific phosphodiesterase 5A (PDE5A) using selective inhibitors, such as tadalafil. Hyperglycemia decreased the insulin responsiveness of podocytes and impaired podocyte function. These effects were associated with lower protein amounts and activity of the protein deacetylase sirtuin 1 (SIRT1) and a decrease in the phosphorylation of adenosine monophosphate-dependent protein kinase (AMPK). We found that PDE5A protein levels increased in hyperglycemia, and PDE5A downregulation improved the insulin responsiveness of podocytes with reestablished SIRT1 expression and activity. PDE5A inhibitors potentiate nitric oxide (NO)/cGMP signaling, and NO modulates the activity and expression of SIRT1. Therefore, we investigated the effects of tadalafil on SIRT1 and AMPK in the context of improving the insulin sensitivity in podocytes and podocyte function in hyperglycemia. Our study revealed that tadalafil restored SIRT1 expression and activity and activated AMPK by increasing its phosphorylation. Tadalafil also restored stimulating effect of insulin on glucose transport in podocytes with high glucose-induced insulin resistance. Additionally, tadalafil improved the function of podocytes that were exposed to high glucose concentrations. Our results display novel mechanisms involved in the pathogenesis of glomerulopathies in diabetes, which may contribute to the development of more effective treatment strategies for diabetic nephropathy.

Phosphodiesterase 5a Signalling in Skeletal Muscle Pathophysiology.

Phosphodiesterase 5A (PDE5A) is involved in cGMP hydrolysis, regulating many physiological processes. Increased activity of PDE5A has been found in several pathological conditions, and the pharmacological inhibition of PDE5 has been demonstrated to have several therapeutic applications. We have identified the presence of three different Pde5a isoforms in cardiomyocytes, and we have found that the expression of specific Pde5a isoforms may have a causal role in the onset of pathological responses in these cells. In our previous study, we demonstrated that PDE5A inhibition could ameliorate muscular dystrophy by acting at different levels, as assessed by the altered genomic response of muscular cells following treatment with the PDE5A inhibitor tadalafil. Thus, considering the importance of PDE5A in various pathophysiological conditions, we further investigated the regulation of this enzyme. Here, we analysed the expression of Pde5a isoforms in the pathophysiology of skeletal muscle. We found that skeletal muscle tissues and myogenic cells express Pde5a1 and Pde5a2 isoforms, and we observed an increased expression of Pde5a1 in damaged skeletal muscles, while Pde5a2 levels remained unchanged. We also cloned and characterized the promoters that control the transcription of Pde5a isoforms, investigating which of the transcription factors predicted by bioinformatics analysis could be involved in their modulation. In conclusion, we found an overexpression of Pde5a1 in compromised muscle and identified an involvement of MyoD and Runx1 in Pde5a1 transcriptional activity.

Impaired Platelet Function and Thrombus Formation in PDE5A-Deficient Mice.

Intracellular cyclic GMP (cGMP) inhibits platelet function. Platelet cGMP levels are controlled by phosphodiesterase 5A (PDE5A)-mediated degradation. However, the exact role of PDE5A in platelet function and thrombus formation remains poorly understood. In this study, we characterized the role of PDE5A in platelet activation and function. Platelets were isolated from wild type or PDE5A-/- mice to measure platelet aggregation, activation, phosphatidylserine exposure (annexin-V binding), reactive oxygen species (ROS) generation, platelet spreading as well as clot retraction. Cytosolic calcium mobilization was measured using Fluo-4 AM by a microplate reader. Western blot was used to measure the phosphorylation of VASP, ERK1/2, p38, JNK, and AKT. FeCl3-induced arterial thrombosis and venous thrombosis were assessed to evaluate the in vivo hemostatic function and thrombus formation. Additionally, in vitro thrombus formation was assessed in a microfluidic whole-blood perfusion assay. PDE5A-deficient mice presented significantly prolonged tail bleeding time and delayed arterial and venous thrombus formation. PDE5A deficiency significantly inhibited platelet aggregation, ATP release, P-selectin expression, and integrin aIIbb3 activation. In addition, an impaired spreading on collagen or fibrinogen and clot retraction was observed in PDE5A-deficient platelets. Moreover, PDE5A deficiency reduced phosphatidylserine exposure, calcium mobilization, ROS production, and increased intracellular cGMP level along with elevated VASP phosphorylation and reduced phosphorylation of ERK1/2, p38, JNK, and AKT. In conclusion, PDE5A modulates platelet activation and function and thrombus formation, indicating that therapeutically targeting it might be beneficial for the treatment of thrombotic diseases.

Shunxin decoction improves diastolic function in rats with heart failure with preserved ejection fraction induced by abdominal aorta constriction through cyclic guanosine monophosphate-dependent protein kinase Signaling Pathway.

To determine whether Shunxin decoction improves diastolic function in rats with heart failure with preserved ejection fraction (HFpEF) by regulating the cyclic guanosine monophosphate-dependent protein kinase (cGMP-PKG) signaling pathway. Except for control group 8 and sham surgery group 8, the remaining 32 male Sprague-Dawlay rats were developed into HFpEF rat models using the abdominal aorta constriction method. These rats in the HFpEF model were randomly divided into the model group, the Shunxin high-dose group, the Shunxin low-dose group, and the Qiliqiangxin capsule group. The three groups received high-dose Shunxin decoction, low-dose Shunxin decoction, and Qiliqiangxin capsule by gavage, respectively, for 14 d. After the intervention, the diastolic function of each rat was evaluated by testing E/A, heart index, hematoxylin-eosin staining, Masson, myocardial ultrastructure, and N-terminal pro-brain natriuretic peptide (NT-proBNP). The Bioinformatics Analysis Tool for Molecular Mechanism of Traditional Chinese Medicine (BATMAN-TCM) software was used to predict targets for which Shunxin decoction acts on the cGMP-PKG pathway. Natriuretic peptide receptor A (NPRA) and guanylate cyclase (GC) were detected by immunohistochemistry, and eNOS, phosphodiesterase 5A (PDE5A), and cGMP-dependent protein kinase 1(PKG I) were determined by Western blotting. Compared to the model group, the thickness of the interventricular septum at the end of diastole (IVSd) and the thickness of the posterior wall at the end of diastole (PWd) of the Shunxin decoction high-dose group, Shunxin decoction low-dose group, and Qiliqiangxin capsule group were all significantly reduced ( < 0.01). Furthermore, Shunxin decoction high-dose group E/A value was decreased ( < 0.01). Compared to the model group, the expression of NPRA and GC increased in the Shunxin decoction low-dose group and the Qiliqiangxin capsule group ( < 0.01). Compared to the model group, the expressions of eNOS and PKG I increased ( < 0.05) in the Shunxin decoction high-dose group. The expression of PDE5A expression decreased in the myocardium of the Shunxin decoction high-dose group, Shunxin decoction low-dose group, and Qiliqiangxin capsule group compared to the model group ( < 0.01). Shunxin decoction can improve diastolic function in rats with HFpEF. It increases the expression of NPRA, GC, and eNOS in the myocardial cell cGMP-PKG signaling pathway, upregulates cGMP expression, decreases PDE5A expression to reduce the cGMP degradation. Thus, the cGMP continually stimulates PKG I, reversing myocardial hypertrophy and improving myocardial compliance in HFpEF rats.

Cellular Redox Metabolism Is Modulated by the Distinct Localization of Cyclic Nucleotide Phosphodiesterase 5A Isoforms.

3′-5′ cyclic nucleotide phosphodiesterases (PDEs) are a family of evolutionarily conserved cAMP and/or cGMP hydrolyzing enzymes, components of transduction pathways regulating crucial aspects of cell life. Among them, cGMP-specific PDE5—being a regulator of vascular smooth muscle contraction—is the molecular target of several drugs used to treat erectile dysfunction and pulmonary hypertension. Production of full-length murine PDE5A isoforms in the milk-yeast Kluyveromyces lactis showed that the quaternary assembly of MmPDE5A1 is a mixture of dimers and tetramers, while MmPDE5A2 and MmPDE5A3 only assembled as dimers. We showed that the N-terminal peptide is responsible for the tetramer assembly of MmPDE5A1, while that of the MmPDE5A2 is responsible for its mitochondrial localization. Overexpression of the three isoforms alters at different levels the cAMP/cGMP equilibrium as well as the NAD(P)+/NAD(P)H balance and induces a metabolic switch from oxidative to fermentative. In particular, the mitochondrial localization of MmPDE5A2 unveiled the existence of a cAMP-cGMP signaling cascade in this organelle, for which we propose a metabolic model that could explain the role of PDE5 in some cardiomyopathies and some of the side effects of its inhibitors.

Protein tyrosine phosphatase PTPN22 negatively modulates platelet function and thrombus formation

AbstractProtein tyrosine phosphatase nonreceptor type 22 (PTPN22) is a protein tyrosine phosphatase that negatively regulates T-cell signaling. However, whether it is expressed and functions in platelets remains unknown. Here we investigated the expression and role of PTPN22 in platelet function. We reported PTPN22 expression in both human and mouse platelets. Using PTPN22−/− mice, we showed that PTPN22 deficiency significantly shortened tail-bleeding time and accelerated arterial thrombus formation without affecting venous thrombosis and the coagulation factors VIII and IX. Consistently, PTPN22-deficient platelets exhibited enhanced platelet aggregation, granule secretion, calcium mobilization, lamellipodia formation, spreading, and clot retraction. Quantitative phosphoproteomic analysis revealed the significant difference of phosphodiesterase 5A (PDE5A) phosphorylation in PTPN22-deficient platelets compared with wild-type platelets after collagen-related peptide stimulation, which was confirmed by increased PDE5A phosphorylation (Ser92) in collagen-related peptide–treated PTPN22-deficient platelets, concomitant with reduced level and vasodilator-stimulated phosphoprotein phosphorylation (Ser157/239). In addition, PTPN22 interacted with phosphorylated PDE5A (Ser92) and dephosphorylated it in activated platelets. Moreover, purified PTPN22 but not the mutant form (C227S) possesses intrinsic serine phosphatase activity. Furthermore, inhibition of PTPN22 enhanced human platelet aggregation, spreading, clot retraction, and increased PDE5A phosphorylation (Ser92). In conclusion, our study shows a novel role of PTPN22 in platelet function and arterial thrombosis, identifying new potential targets for future prevention of thrombotic or cardiovascular diseases.