CircPDE5A-encoded novel regulator of the PI3K/AKT pathway inhibits esophageal squamous cell carcinoma progression by promoting USP14-mediated de-ubiquitination of PIK3IP1.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is a common gastrointestinal tumor and has become an important global health problem. The PI3K/AKT signaling pathway plays a key role in the development of ESCC. CircRNAs have been reported to be involved in the regulation of the PI3K/AKT pathway, but the underlying mechanisms are unclear. Therefore, this study aimed to identify protein-coding circRNAs and investigate their functions in ESCC. METHODS: Differential expression of circRNAs between ESCC tissues and adjacent normal tissues was identified using circRNA microarray analysis. Thereafter, LC-MS/MS was used to identify circPDE5A-encoded novel protein PDE5A-500aa. Molecular biological methods were used to explore the biological functions and regulatory mechanisms of circPDE5A and PDE5A-500aa in ESCC. Lastly, circRNA-loaded nanoplatforms were constructed to investigate the therapeutic translation value of circPDE5A. RESULTS: We found that circPDE5A expression was down-regulated in ESCC cells and tissues and that it was negatively associated with advanced clinicopathological stages and poorer prognosis in ESCC. Functionally, circPDE5A inhibited ESCC proliferation and metastasis in vitro and in vivo by encoding PDE5A-500aa, a key regulator of the PI3K/AKT signaling pathway in ESCC. Mechanistically, PDE5A-500aa interacted with PIK3IP1 and promoted USP14-mediated de-ubiquitination of the k48-linked polyubiquitin chain at its K198 residue, thereby attenuating the PI3K/AKT pathway in ESCC. In addition, Meo-PEG-S-S-PLGA-based reduction-responsive nanoplatforms loaded with circPDE5A and PDE5A-500aa plasmids were found to successfully inhibit the growth and metastasis of ESCC in vitro and in vivo. CONCLUSION: The novel protein PDE5A-500aa encoded by circPDE5A can act as an inhibitor of the PI3K/AKT signaling pathway to inhibit the progression of ESCC by promoting USP14-mediated de-ubiquitination of PIK3IP1 and may serve as a potential target for the development of therapeutic agents.

Hsa_circ_0092355 Accelerates Papillary Thyroid Cancer Progression by Regulating the miR-543/PDE5A Pathway.

CircRNAs have been found to participate in the progression of various tumors. In the present study, we aimed to clarify the role of hsa_circ_0092355 in papillary thyroid cancer (PTC) cell development. RT-qPCR was used to determine the expression of hsa_circ_0092355, miR-543, and PDE5A. PTC cell proliferation was ascertained via a cell colony formation assay and the CCK-8 test. Western blotting was performed to examine the expression levels of PDE5A and apoptosis-associated proteins (Bcl-2 and Bax) in PTC cells. A scratch wound assay was performed to measure the migration of PTC cells. A mouse xenograft test was performed to assess the effects of hsa_circ_0092355 in vivo. RIP and dual-luciferase reporter assays confirmed the association between miR-543 and hsa_circ_0092355 or PDE5A. Associations between miR-543, hsa_circ_0092355, and PDE5A were evaluated using Pearson's correlation coefficient. Upregulation of hsa_circ_0092355 was observed in PTC tissues. The hsa_circ_0092355 knockdown blocked the proliferation and migration of PTC cells and induced apoptosis. Moreover, hsa_circ_0092355 knockdown blocked PTC xenograft tumor growth in vivo. The miR-543 inhibitor could reverse the changes induced by hsa_circ_0092355 knockdown by hsa_circ_0092355 targeting miR-543. Furthermore, miR-543 suppresses PTC progression by downregulating PDE5A expression. Our findings suggest that the PTC tumor promoter hsa_circ_0092355 may promote carcinogenesis by controlling the miR-543/PDE5A pathway.

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

BACKGROUND: 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. HYPOTHESIS: 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. ANIMALS: Fifty-two privately owned CKCS with no or preclinical MMVD. METHODS: 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. RESULTS: 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. CONCLUSIONS AND CLINICAL IMPORTANCE: 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.

Plantain peels restore sexual performance, hormonal imbalance, and modulate nitric oxide production and key enzymes of penile function in paroxetine-sexually impaired male rats.

This study focused on the effect of unripe (UPP) and ripe (RPP) plantain peels' extracts (200 and 400 mg/kg) on sexual behavior, hormonal profiles [testosterone, follicle-stimulating hormone (FSH), and luteinizing hormone (LH)], and enzymes [acetylcholinesterase (AChE), phosphodiesterase-5' (PDE-5), arginase, angiotensin-I converting enzyme (ACE), ecto-5'neucleotidase, and adenosine deaminase (ADA)] in paroxetine (PAR)-induced penile dysfunction rats. From the result, arginase, PDE-5', ACE, ecto-5'-nucleotidase ADA, and AChE activities, sexual activities, hormonal profile, and NO level were reduced, while thiobarbituric acid reactive species (TBARS) level increased (p < .05) relative to normal control rats. However, treatment with UPP and URP reduced the activities of these enzymes, decreased TBARS levels, and increased hormones, and penile NO levels in PAR-induced rats. Thus, the use of UPP and RPP could be channeled towards the improvement of sexual performance in erectile dysfunction (ED) disorder. PRACTICAL APPLICATIONS: Plantain fruits are a tropical staple food crop commonly consumed at various stages of ripeness and cooking methods. However, its peels are regarded as a waste product with reported cases of environmental menace. Interestingly, plantain peel is being used as a major raw material for industrial applications in the agro-based industries and in folklore for the treatment of many human ailments due to its rich phytochemicals such as polyphenols, carotenoids, alkaloids, etc., which have been reported. A prelude study has also indicated its usefulness in ED management, but further pharmacological investigations are needed to proffer information on its effect in ED management and its anti-androgenic activity in male Wistar rats. The information from this study could be of pharmaceutical importance in designing natural remedies capable of improving penile rigidity, hormone profiles, and alteration of enzymes linked with ED.

Main active components of Si-Miao-Yong-An decoction (SMYAD) attenuate autophagy and apoptosis via the PDE5A-AKT and TLR4-NOX4 pathways in isoproterenol (ISO)-induced heart failure models.

Heart failure (HF), the main cause of death in patients with many cardiovascular diseases, has been reported to be closely related to the complicated pathogenesis of autophagy, apoptosis, and inflammation. Notably, Si-Miao-Yong-An decoction (SMYAD) is a traditional Chinese medicine (TCM) used to treat cardiovascular disease; however, the main active components and their relevant mechanisms remain to be discovered. Based on our previous ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS) results, we identified angoriside C (AC) and 3,5-dicaffeoylquinic acid (3,5-DiCQA) as the main active components of SMYAD. In vivo results showed that AC and 3,5-DiCQA effectively improved cardiac function, reduced the fibrotic area, and alleviated isoproterenol (ISO)-induced myocarditis in rats. Moreover, AC and 3,5-DiCQA inhibited ISO-induced autophagic cell death by inhibiting the PDE5A/AKT/mTOR/ULK1 pathway and inhibited ISO-induced apoptosis by inhibiting the TLR4/NOX4/BAX pathway. In addition, the autophagy inhibitor 3-MA was shown to reduce ISO-induced apoptosis, indicating that ISO-induced autophagic cell death leads to excess apoptosis. Taken together, the main active components AC and 3,5-DiCQA of SMYAD inhibit the excessive autophagic cell death and apoptosis induced by ISO by inhibiting the PDE5A-AKT and TLR4-NOX4 pathways, thereby reducing myocardial inflammation and improving heart function to alleviate and treat a rat ISO-induced heart failure model and cell heart failure models. More importantly, the main active components of SMYAD will provide new insights into a promising strategy that will promote the discovery of more main active components of SMYAD for therapeutic purposes in the future.

Tadalafil enhances the therapeutic efficacy of BET inhibitors in hepatocellular carcinoma through activating Hippo pathway.

Bromodomain and extraterminal (BET) proteins are promising therapeutic targets for hematological and solid tumors. However, BET inhibitor monotherapy did not show a significant therapeutic benefit for hepatocellular carcinoma (HCC) in preclinical trials. Here, we identified YAP/TAZ genes, as determinants for sensitivity to BET inhibitors. YAP/TAZ expression, especially TAZ, promote resistance to BET inhibitor. In addition, we analyzed that the mRNA level of PDE5 was positively correlated with YAP/TAZ based on TCGA database and demonstrated tadalafil, a PDE5 inhibitor, could block YAP/TAZ protein expression by activating Hippo pathway. Cotreatment with tadalafil and JQ-1 synergistically reduced YAP/TAZ protein expression, suppressed proliferation and induced G0-G1 arrest of cultured HCC cells. JQ-1 alone does not show significant benefits in a mouse model of HCC induced by c-Myc/N-Ras plasmids. In contrast, the combination, tadalafil and JQ-1, successfully suppressed tumor progression, enhanced antitumor immunity by improving the ratio of activated CD8 and extended the survival time of mice. Our data define the key role of YAP/TAZ in mediating resistance to BET inhibitor, described the PDE5/PKG/Hippo/YAP/TAZ axis and identified a common clinical drug that can be developed as an effective combined strategy to overcome BET inhibitor resistance in MYC/Ras-driven HCC.

LncRNA HOXA-AS3 Promotes the Progression of Pulmonary Arterial Hypertension through Mediation of miR-675-3p/PDE5A Axis.

Pulmonary arterial hypertension (PAH) seriously threatens the elder people. Long non-coding RNAs (lncRNAs) are involved in multiple diseases. However, the study of the lncRNAs in the occurrence of PAH is just beginning. For this, we sought to explore the biological function of lncRNA HOXA cluster antisense RNA 3 (HOXA-AS3) in PAH. Hypoxia (HYP) was used to mimic in vitro model of PAH. Gene and protein expressions in cells were detected by q-PCR and Western blotting, respectively. In addition, cell proliferation and viability were tested by CCK-8 and MTT assay. Cell apoptosis was measured by flow cytometry. Wound healing was used to detect cell migration. Furthermore, the connection of HOXA-AS3, miR-675-3p, and phosphodiesterase 5A (PDE5A) was verified by dual-luciferase report assay. HOXA-AS3 and PDE5A were upregulated in human pulmonary artery smooth muscle cells (HPASMCs) in the presence of HYP, while miR-675-3p was downregulated. Moreover, knockdown of HOXA-AS3 suppressed the growth and migration of HPASMCs, but induced the apoptosis. Overexpression of miR-675-3p achieved the same effect. MiR-675-3p inhibitor or overexpression of PDE5A notably reversed the inhibitory effect of HOXA-AS3 knockdown on PAH. Finally, HOXA-AS3 could sponge miR-675-3p, and PDE5A was directly targeted by miR-675-3p. HOXA-AS3 increased the development of PAH via regulation of miR-675-3p/PDE5 axis, which could be the potential biomarker for treatment of PAH.

Therapeutic Benefit of the Association of Lodenafil with Mesenchymal Stem Cells on Hypoxia-induced Pulmonary Hypertension in Rats.

Pulmonary arterial hypertension (PAH) is characterized by the remodeling of pulmonary arteries, with an increased pulmonary arterial pressure and right ventricle (RV) overload. This work investigated the benefit of the association of human umbilical cord mesenchymal stem cells (hMSCs) with lodenafil, a phosphodiesterase-5 inhibitor, in an animal model of PAH. Male Wistar rats were exposed to hypoxia (10% O2) for three weeks plus a weekly i.p. injection of a vascular endothelial growth factor receptor inhibitor (SU5416, 20 mg/kg, SuHx). After confirmation of PAH, animals received intravenous injection of 5.105 hMSCs or vehicle, followed by oral treatment with lodenafil carbonate (10 mg/kg/day) for 14 days. The ratio between pulmonary artery acceleration time and RV ejection time reduced from 0.42 ± 0.01 (control) to 0.24 ± 0.01 in the SuHx group, which was not altered by lodenafil alone but was recovered to 0.31 ± 0.01 when administered in association with hMSCs. RV afterload was confirmed in the SuHx group with an increased RV systolic pressure (mmHg) of 52.1 ± 8.8 normalized to 29.6 ± 2.2 after treatment with the association. Treatment with hMSCs + lodenafil reversed RV hypertrophy, fibrosis and interstitial cell infiltration in the SuHx group. Combined therapy of lodenafil and hMSCs may be a strategy for PAH treatment.

Sildenafil Potentiates the Therapeutic Efficacy of Docetaxel in Advanced Prostate Cancer by Stimulating NO-cGMP Signaling.

PURPOSE: Docetaxel plays an indispensable role in the management of advanced prostate cancer. However, more than half of patients do not respond to docetaxel, and those good responders frequently experience significant cumulative toxicity, which limits its dose duration and intensity. Hence, a second agent that could increase the initial efficacy of docetaxel and maintain tolerability at biologically effective doses may improve outcomes for patients. EXPERIMENTAL DESIGN: We determined phosphodiesterase 5 (PDE5) expression levels in human and genetically engineered mouse (GEM) prostate tissues and tumor-derived cell lines. Furthermore, we investigated the therapeutic benefits and underlying mechanism of PDE5 inhibitor sildenafil in combination with docetaxel using in vitro, Pten conditional knockout (cKO), derived tumoroid and xenograft prostate cancer models. RESULTS: PDE5 expression was higher in both human and mouse prostate tumors and cancer cell lines compared with normal tissues/cells. In GEM prostate-derived cell lines, PDE5 expression increased from normal prostate (wild-type) epithelial cells to androgen-dependent and castrated prostate-derived cell lines. The addition of physiologically achievable concentrations of sildenafil enhanced docetaxel-induced prostate cancer cell growth inhibition and apoptosis in vitro, reduced murine 3D tumoroid growth, and in vivo tumorigenicity as compared with docetaxel alone. Furthermore, sildenafil enhanced docetaxel-induced NO and cGMP levels thereby augmenting antitumor activity. CONCLUSIONS: Our results demonstrate that sildenafil's addition could sensitize docetaxel chemotherapy in prostate cancer cells at much lesser concentration than needed for inducing cell death. Thus, the combinatorial treatment of sildenafil and docetaxel may improve anticancer efficacy and reduce chemotherapy-induced side-effects among patients with advanced prostate cancer.

Discovery of Novel Agents on Spindle Assembly Checkpoint to Sensitize Vinorelbine-Induced Mitotic Cell Death Against Human Non-Small Cell Lung Cancers.

Non-small cell lung cancer (NSCLC) accounts about 80% of all lung cancers. More than two-thirds of NSCLC patients have inoperable, locally advanced or metastatic tumors. Non-toxic agents that synergistically potentiate cancer-killing activities of chemotherapeutic drugs are in high demand. YL-9 was a novel and non-cytotoxic compound with the structure related to sildenafil but showing much less activity against phosphodiesterase type 5 (PDE5). NCI-H460, an NSCLC cell line with low PDE5 expression, was used as the cell model. YL-9 synergistically potentiated vinorelbine-induced anti-proliferative and apoptotic effects in NCI-H460 cells. Vinorelbine induced tubulin acetylation and Bub1-related kinase (BUBR1) phosphorylation, a necessary component in spindle assembly checkpoint. These effects, as well as BUBR1 cleavage, were substantially enhanced in co-treatment with YL-9. Several mitotic arrest signals were enhanced under combinatory treatment of vinorelbine and YL-9, including an increase of mitotic spindle abnormalities, increased cyclin B1 expression, B-cell lymphoma 2 (Bcl-2) phosphorylation and increased phosphoproteins. Moreover, YL-9 also displayed synergistic activity in combining with vinorelbine to induce apoptosis in A549 cells which express PDE5. In conclusion. the data suggest that YL-9 is a novel agent that synergistically amplifies vinorelbine-induced NSCLC apoptosis through activation of spindle assembly checkpoint and increased mitotic arrest of the cell cycle. YL-9 shows the potential for further development in combinatory treatment against NSCLC.

Phosphodiesterase-5 inhibitors use and risk for mortality and metastases among male patients with colorectal cancer.

Phosphodiesterase-5 (PDE5) inhibitors are suggested to have anti-tumor effects and to inhibit surgery-induced immunosuppression. We aimed to explore whether post-diagnostic use of PDE5 inhibitors was associated with a better prognosis among male patients with colorectal cancer (CRC) and the role of open surgery in the association. Here we show that post-diagnostic use of PDE5 inhibitors is associated with a decreased risk of CRC-specific mortality (adjusted HR = 0.82, 95% CI 0.67-0.99) as well as a decreased risk of metastasis (adjusted HR = 0.85, 95% CI 0.74-0.98). Specifically, post-operative use of PDE5 inhibitors has a strong anti-cancer effect. The reduced risk of metastasis is mainly due to distant metastasis but not regional lymphatic metastasis. PDE5 inhibitors have the potential to be an adjuvant drug for patients with CRC to improve prognosis, especially those who have undergone open surgery.

Differential expression of caveolin-1 during pathogenesis of combined pulmonary fibrosis and emphysema: Effect of phosphodiesterase-5 inhibitor.

INTRODUCTION: Combined pulmonary fibrosis and emphysema (CPFE) is a relatively new entity within the spectrum of cigarette smoke induced lung disorders. Currently there is no consensus about its treatment. We hypothesized that caveolin-1 critically determines the parenchymal and vascular remodeling leading to the development of CPFE. We assessed the effect of therapeutic targeting of caveolin-1 in mesenchymal and endothelial cells by the phosphodiesterase-5 inhibitor, sildenafil. METHODS: Male Wistar rats (n = 168) were exposed to; room air (control); bleomycin (7 U/kg), bleomycin+sildenafil (50 mg/kg/day P.O.), cigarette smoke (CS) (4 Gold Flake 69 mm/day), CS + sildenafil, CS + bleomycin, CS + bleomycin+sildenafil. Animals were euthanized at 8, 9, 11, 12 weeks and lung histopathological changes, collagen deposition, ROS, Xanthine oxidase, caveolin-1 determined. RESULTS: Cigarette smoke causes progressive ROS accumulation, caveolin-1 up-regulation in alveolar epithelial cells, alveolar macrophages, peribronchiolar fibroblasts, endothelial and vascular smooth muscle cells, interstitial inflammation and emphysema. Sildenafil reduces oxidative stress, parenchymal caveolin-1 and attenuates emphysema caused by CS. Bleomycin increases lung ROS and downregulates caveolin-1 leading to fibroblast proliferation and fibrosis. Combined cigarette smoke and bleomycin exposure, results in differential caveolin-1 expression and heterogeneous parenchymal remodeling with alternating areas of emphysema and fibrosis. Increased caveolin-1 induces premature senescence of lung fibroblasts and emphysema. Decreased caveolin-1 is associated with propagation of EMT and fibrosis. Sildenafil attenuates the parenchymal remodeling however it is not effective in reducing VSMC hypertrophy in combined group. CONCLUSION: CPFE is characterized by heterogenous parenchymal remodeling and differential caveolin-1 expression. Sildenafil therapy attenuates parenchymal pathologies in CPFE. Additional therapy is however needed for attenuating VSMC remodeling.

A Multi-Biochemical and In Silico Study on Anti-Enzymatic Actions of Pyroglutamic Acid against PDE-5, ACE, and Urease Using Various Analytical Techniques: Unexplored Pharmacological Properties and Cytotoxicity Evaluation.

In the current study, pyroglutamic acid (pGlu), a natural amino acid derivative, has efficiently inhibited the catalytic activities of three important enzymes, namely: Human recombinant phosphodiesterase-5A1 (PDE5A1), human angiotensin-converting enzyme (ACE), and urease. These enzymes were reported to be associated with several important clinical conditions in humans. Radioactivity-based assay, spectrophotometric-based assay, and an Electrospray Ionization-Mass Spectrometry-based method were employed to ascertain the inhibitory actions of pGlu against PDE5A1, ACE, and urease, respectively. The results unveiled that pGlu potently suppressed the activity of PDE5A1 (half-maximal inhibitory concentration; IC50 = 5.23 µM) compared with that of standard drug sildenafil citrate (IC50 = 7.14 µM). Moreover, pGlu at a concentration of 20 µg/mL was found to efficiently inhibit human ACE with 98.2% inhibition compared with that of standard captopril (99.6%; 20 µg/mL). The urease-catalyzed reaction was also remarkably inactivated by pGlu and standard acetohydroxamic acid with IC50 values of 1.8 and 3.9 µM, respectively. Remarkably, the outcome of in vitro cytotoxicity assay did not reveal any significant cytotoxic properties of pGlu against human cervical carcinoma cells and normal human fetal lung fibroblast cells. In addition to in vitro assays, molecular docking analyses were performed to corroborate the outcomes of in vitro results with predicted structure-activity relationships. In conclusion, pGlu could be presented as a natural and multifunctional agent with promising applications in the treatment of some ailments connected with the above-mentioned anti-enzymatic properties.

Pharmacological and transcriptomic characterization of the nitric oxide pathway in aortic rings isolated from the tortoise Chelonoidis carbonaria.

In this study the nitric oxide (NO)-soluble guanylate cyclase (sGC) and phosphodiesterase-5 (PDE-5) pathway was characterized in tortoise Chelonoidis carbonaria aorta. Concentration response curves (CCR) to ATP, ADP, AMP, adenosine and histamine were performed in the presence and absence of L-NAME in aorta pre-contracted with ACh (3 µM). CCR to SNP, BAY 41-2272 (sGC stimulator), BAY 60-2770 (sGC activator) and tadalafil (PDE-5 inhibitor) were constructed in the presence and absence of ODQ (10 µM). ATP (pEC50 6.1 ±â€¯0.1), ADP (pEC50 6.0 ±â€¯0.2), AMP (pEC50 6.8 ±â€¯0.1) and histamine (pEC50 6.8 ±â€¯0.12) relaxed Chelonoidis aorta and the addition of L-NAME reduced their efficacy (p < .05). Adenosine effects (pEC50 6.6 ±â€¯0.1) were not changed in the presence of L-NAME. SNP (pEC50 7.5 ±â€¯0.7; Emax 102.2 ±â€¯2.5%), BAY 41-2272 (pEC50 7.3 ±â€¯0.2; Emax 130.3 ±â€¯10.2%), BAY 60-2770 (pEC50 11.4 ±â€¯0.1; Emax 130.3 ±â€¯6.5%) and tadalafil (pEC50 6.7 ±â€¯0.3; Emax 121.3 ±â€¯15.3%) relaxed Chelonoidis aorta. The addition of ODQ reduced the SNP and tadalafil maximum response (p < .05) and promoted 63 fold right shift on BAY 41-2272 curve. In contrast, no alteration was observed on BAY 60-2770 response. Transcriptomic analysis for eNOS and sGC were found in aorta and brain libraries with high homology when compared with human transcripts. The NO-sGC-PDE-5 is functionally present in Chelonoidis aorta with a functional and genomic similarity to mammalian vessels. Unlike most of mammalian vessels, ACh did not cause endothelium-dependent relaxation in Chelonoidis carbonaria aortic rings.

Metabolic role of cGMP in S. cerevisiae: the murine phosphodiesterase-5 activity affects yeast cell proliferation by altering the cAMP/cGMP equilibrium.

In higher eukaryotes, cAMP and cGMP are signal molecules of major transduction pathways while phosphodiesterases (PDE) are a superfamily of cAMP/cGMP hydrolysing enzymes, modulatory components of these routes. Saccharomyces cerevisiae harbours two genes for PDE: Pde2 is a high affinity cAMP-hydrolysing enzyme, while Pde1 can hydrolyse both cAMP and cGMP. To gain insight into the metabolic role of cGMP in the physiology of yeast, the murine Pde5a1 gene encoding a specific cGMP-hydrolysing enzyme, was expressed in S. cerevisiae pdeΔ strains. pde1Δ and pde2Δ PDE5A1-transformed strain displayed opposite growth-curve profiles; while PDE5A1 recovered the growth delay of pde1Δ, PDE5A1 reversed the growth profile of pde2Δ to that of the untransformed pde1Δ. Growth test analysis and the use of Adh2 and Adh1 as respiro-fermentative glycolytic flux markers confirmed that PDE5A1 altered the metabolism by acting on Pde1-Pde2/cyclic nucleotides content and also on the TORC1 nutrient-sensing cascade. cGMP is required during the log-phase of cell proliferation to adjust/modulate cAMP levels inside well-defined ranges. A model is presented proposing the role of cGMP in the cAMP/PKA pathway. The expression of the PDE5A1 cassette in other mutant strains might constitute the starting tool to define cGMP metabolic role in yeast nutrient signaling.

Upregulation of SERT and ADORA1 in broilers with acute right ventricular failure.

Pulmonary hypertension (PH), remains a challenging disease with a large impact on both humans and meat-type chickens. PH is characterized by the onset of idiopathic pulmonary arterial hypertension leading to right ventricular failure. In this experiment relative gene expression of adenosine A1 receptor (ADORA1), serotonin transporter (SERT), phosphodiesterase 5A (PDE5) and troponin T2 were compared in hearts from broilers with acute right ventricular failure and from healthy birds. There were major increases in adenosine A1 receptor (177%) and serotonin transporter (475%), and more modest but significant increases in PDE5 (146%) and troponin T2 (140%) gene expressions in broilers with right ventricular failure compared to healthy birds (P<0.01). This novel report shows that pulmonary hypertension related gene expression in broilers is similar to that in humans. This molecular similarity between PH in broilers and human patients suggests, first, that they will make a suitable animal model for study PH in humans, but also that the literature on PH in humans may be profitably applied to the study of PH in broilers.

Hydroalcoholic Extract of Levisticum officinale Increases cGMP Signaling Pathway by Down-Regulating PDE5 Expression and Induction of Apoptosis in MCF-7 and MDA-MB-468 Breast Cancer Cell Lines

Background: This study aimed to investigate Levisticum officinale hydroalcoholic extract (LOHE) effect on both cGMP signaling pathway and phosphodiesterase 5 (PDE5) gene expression pattern and to examine the role of LOHE in apoptosis induction of MCF-7 and MDA-MB-468 cell lines. Methods: The half maximal inhibitory concentration (IC50) of LOHE was examined in both cell lines using the MTT assay. Using IC50 values of LOHE on both cells, the type of cell death was detected by flowcytometric analysis. The values of PDE5 and cGMP were evaluated by real-time PCR and ELISA methods, respectively. Results: The IC50 values were measured as 150 µg/ml for MDA-MB-468 and 200 µg/ml for MCF-7. At 12 hour of treatment, a significant decrease in the PDE5 expression and maximum increase in the amount of intracellular cGMP were observed (p < 0.05). However, these effects were more noticeable in MDA-MB-468 triple-negative cells. Conclusion: Our data suggest that LOHE extract could be a potential source for new strategies towards targeting both PDE5 and cGMP signaling pathways.

Effect of Riociguat and Sildenafil on Right Heart Remodeling and Function in Pressure Overload Induced Model of Pulmonary Arterial Banding.

Pulmonary arterial hypertension (PAH) is a progressive disorder characterized by remodeling of the pulmonary vasculature and a rise in right ventricular (RV) afterload. The increased RV afterload leads to right ventricular failure (RVF) which is the reason for the high morbidity and mortality in PAH patients. The objective was to evaluate the therapeutic efficacy and antiremodeling potential of the phosphodiesterase type 5 (PDE5) inhibitor sildenafil and the soluble guanylate cyclase stimulator riociguat in a model of pressure overload RV hypertrophy induced by pulmonary artery banding (PAB). Mice subjected to PAB, one week after surgery, were treated with either sildenafil (100 mg/kg/d, n = 5), riociguat (30 mg/kg/d, n = 5), or vehicle (n = 5) for 14 days. RV function and remodeling were assessed by right heart catheterization, magnetic resonance imaging (MRI), and histomorphometry. Both sildenafil and riociguat prevented the deterioration of RV function, as determined by a decrease in RV dilation and restoration of the RV ejection fraction (EF). Although both compounds did not decrease right heart mass and cellular hypertrophy, riociguat prevented RV fibrosis induced by PAB. Both compounds diminished TGF-beta1 induced collagen synthesis of RV cardiac fibroblasts in vitro. Treatment with either riociguat or sildenafil prevented the progression of pressure overload-induced RVF, representing a novel therapeutic approach.

Mathematical Modelling of Nitric Oxide/Cyclic GMP/Cyclic AMP Signalling in Platelets.

Platelet activation contributes to normal haemostasis but also to pathologic conditions like stroke and cardiac infarction. Signalling by cGMP and cAMP inhibit platelet activation and are therefore attractive targets for thrombosis prevention. However, extensive cross-talk between the cGMP and cAMP signalling pathways in multiple tissues complicates the selective targeting of their activities. We have used mathematical modelling based on experimental data from the literature to quantify the steady state behaviour of nitric oxide (NO)/cGMP/cAMP signalling in platelets. The analysis provides an assessment of NO-induced cGMP synthesis and PKG activation as well as cGMP-mediated cAMP and PKA activation though modulation of phosphodiesterase (PDE2 and 3) activities. Both one- and two-compartment models of platelet cyclic nucleotide signalling are presented. The models provide new insight for understanding how NO signalling to cGMP and indirectly cAMP, can inhibit platelet shape-change, the initial step of platelet activation. Only the two-compartment models could account for the experimental observation that NO-mediated PKA activation can occur when the bulk platelet cAMP level is unchanged. The models revealed also a potential for hierarchical interplay between the different platelet phosphodiesterases. Specifically, the models predict, unexpectedly, a strong effect of pharmacological inhibitors of cGMP-specific PDE5 on the cGMP/cAMP cross-talk. This may explain the successful use of weak PDE5-inhibitors, such as dipyridamole, in anti-platelet therapy. In conclusion, increased NO signalling or PDE5 inhibition are attractive ways of increasing cGMP-cAMP cross-talk selectively in platelets.

Identification of murine phosphodiesterase 5A isoforms and their functional characterization in HL-1 cardiac cell line.

Phosphodiesterase 5A (PDE5A) specifically degrades the ubiquitous second messenger cGMP and experimental and clinical data highlight its important role in cardiac diseases. To address PDE5A role in cardiac physiology, three splice variants of the PDE5A were cloned for the first time from mouse cDNA library (mPde5a1, mPde5a2, and mPde5a3). The predicted amino acidic sequences of the three murine isoforms are different in the N-terminal regulatory domain. mPDE5A isoforms were transfected in HEK293T cells and they showed high affinity for cGMP and similar sensitivity to sildenafil inhibition. RT-PCR analysis showed that mPde5a1, mPde5a2, and mPde5a3 had differential tissue distribution. In the adult heart, mPde5a1 and mPde5a2 were expressed at different levels whereas mPde5a3 was undetectable. Overexpression of mPDE5As induced an increase of HL-1 number cells which progress into cell cycle. mPDE5A1 and mPDE5A3 overexpression increased the number of polyploid and binucleated cells, mPDE5A3 widened HL-1 areas, and modulated hypertrophic markers more efficiently respect to the other mPDE5A isoforms. Moreover, mPDE5A isoforms had differential subcellular localization: mPDE5A1 was mainly localized in the cytoplasm, mPDE5A2 and mPDE5A3 were also nuclear localized. These results demonstrate for the first time the existence of three PDE5A isoforms in mouse and highlight their potential role in the induction of hypertrophy.