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.

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.

Protective Effects of Epigallocatechin Gallate for Male Sexual Dysfunction in Streptozotocin-Induced Diabetic Rats.

Sexual dysfunction is a common problem for men with diabetes. Epigallocatechin gallate (EGCG) is known to ameliorate erectile function in aging rats. However, there has not yet been a report to evaluate its effects on diabetic male rat sexual behavior in the literature. In this study, we investigated the effects of EGCG on male sexual behavior in diabetic rats. Diabetic rats were induced by a single intraperitoneal injection of 65 mg/kg of streptozotocin. After streptozotocin injection for one week, animals were then orally treated with 40 mg/kg of EGCG or vehicle. Copulatory behavior and fasting blood glucose levels were recorded before treatment, as well as 7 and 14 days after treatment. Serum LH, testosterone, and PDE5a levels were measured by EIA assay after the last behavioral test. Data showed that diabetic rats who had diminished sexual functions demonstrated significantly increased latencies in mount, intromission, and ejaculation, as well as significant decreases in frequencies of intromission and ejaculation, compared to non-diabetic controls, indicating sexual function recovery. Lower blood glucose levels were also found in diabetic rats after EGCG treatment. Additionally, the lower LH and higher PDE5a levels in diabetic rats than controls were also noted. The findings declared that EGCG had a protective effect on male sexual behavior in diabetic rats.

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.

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.

Characterisation of pomegranate juice effects on human corpus cavernosum.

Pomegranate (POM) juice may benefit the erectile process, but the scientific evidence is lacking. This study evaluates the molecular characterisation and confirmation of POM's action on human corpus cavernosum (HCC) obtained from patients (n = 16) undergoing penile prosthesis implantation. After phenylephrine contraction, the relaxant effects of POM with various inhibitors in the presence and absence of palmitic acid (PA)-induced acute oxidative stress were investigated. Electrical field stimulation (EFS)- and acetylcholine (ACh)-induced relaxation were performed using organ bath preparation. Expression of neuronal nitric oxide synthase (nNOS), endothelial (eNOS), phosphodiesterase (PDE)-5A and cGMP levels were assessed in cells from ex vivo organ cultures of HCC, using RT-PCR, ELISA and immunohistochemistry techniques. POM induced marked relaxation of HCC (maximum response: 97.0 ± 3.1%) and reversed the PA-induced decrease of EFS (20 Hz). nNOS transcription was increased by 7-fold in POM-treated cells without influencing eNOS and PDE5A expressions. We conclude that POM induced marked relaxation of HCC via: (i) nNOS stimulation, and (ii) downstream relaxation stimulated by nNOS and cGMP and bypassing the NO and PDE5. This action provides a rationale for the therapeutic or preventative use of POM in men with erectile dysfunction who do not respond well to PDE5 inhibitors.

Role of rare germline copy number variation in melanoma-prone patients.

AIM: This work evaluates a possible causative role for germline copy number variants (CNVs) in melanoma predisposition. PATIENTS & METHODS: A total of 41 melanoma-prone Brazilian patients were investigated for CNVs using 850K single nucleotide polymorphism arrays. RESULTS: Ten rare CNVs were identified in nine patients, comprising 54 known genes, mostly related to cancer. In silico analyses revealed gene enrichment for cellular development and growth, and proliferation, highlighting five genes directly associated with the melanoma phenotype (ANGPT1, IDH1, PDE5A, HIST1H1B and GCNT2). CONCLUSION: Patients harboring rare CNVs exhibited a decreased age of disease onset, in addition to an overall higher skin cancer predisposition. Our findings suggest that rare CNVs contribute to melanoma susceptibility, and should be taken into account when investigating cancer risk factors.

Low androgen induced penile maldevelopment involves altered gene expression of biomarkers of smooth muscle differentiation and a key enzyme regulating cavernous smooth muscle cell tone.

PURPOSE: We determined the effects of low androgens in the neonatal period on biomarkers of smooth muscle cell differentiation, Myh11 and Acta2, and on Pde5A expression in the penis. MATERIALS AND METHODS: One-day-old pups were treated daily with the gonadotropin-releasing hormone antagonist antide with or without dihydrotestosterone for 1 to 6 days. Tissues were collected at age day 7 and at adulthood at age 120 days. Penes were examined by quantitative reverse transcriptase-polymerase chain reaction, Western blot and immunohistochemistry. Testes were assayed for the intratesticular testosterone and steroidogenic enzymes Cyp17α1 and StAR. RESULTS: Gonadotropin-releasing hormone antagonist exposure suppressed the neonatal testicular testosterone surge 70% to 80%. Quantitative reverse transcriptase-polymerase chain reaction revealed 80% to 90% reductions in Cyp17α1 and StAR protein, and 40% to 60% reductions in Myh11 and ACTA2 as a result of gonadotropin-releasing hormone antagonist compared to controls. Dihydrotestosterone co-administration mitigated these decreases. Western blot confirmed the Myh11 decrease at the protein level. Immunohistochemistry of Acta2 confirmed cavernous smooth muscle cell loss at the tissue level. Also, gonadotropin-releasing hormone antagonist exposure decreased Pde5a expression and dihydrotestosterone co-administration mitigated the decrease. Comparison of data between 2 parts of the penis body (corpora cavernosa and corpus spongiosum) showed that antagonist induced decreases in Myh11, Acta2 and Pde5a expression occurred only in the corpora cavernosa, implying that the latter is the target site of low androgen action. CONCLUSIONS: As evidenced by gonadotropin-releasing hormone antagonist induced suppression of the neonatal testosterone surge and reduced steroidogenesis, low androgens in the neonatal period altered gene expression of biomarkers of smooth muscle cell differentiation. This led to loss of cavernous smooth muscle cells and consequently to penile maldevelopment.

NO-releasing xanthine KMUP-1 bonded by simvastatin attenuates bleomycin-induced lung inflammation and delayed fibrosis.

BACKGROUND AND PURPOSE: Pulmonary fibrosis (PF) is a progressing lung injury initiated by pulmonary inflammation (PI). Bleomycin (BLM) is the most common pathogenesis of PF through early PI and extensive extracellular matrix deposition. This study is aimed to determine whether NO-releasing KMUP-1 inhibits PI and PF, and if so, the benefits of KMUP-1S resulted from simvastatin (SIM)-bonding to KMUP-1. EXPERIMENT APPROACH: C57BL/6 male mice were intra-tracheally administered BLM (4 U/kg) at day 0. KMUP-1 (1-5 mg/kg), KMUP-1S (2.5 mg/kg), SIM (5 mg/kg), Plus (KMUP-1 2.5 mg/kg + SIM 2.5 mg/kg), and clarithromycin (CAM, 10 mg/kg) were orally and daily administered for 7 and 28 days, respectively, to mice, sacrificed at day-7 and day-28 to isolate the lung tissues, for examining the inflammatory and fibrotic signaling and measuring the cell population and MMP-2/MMP-9 activity in broncholaveolar lavage fluid (BAL). KEY RESULTS: KMUP-1 and KUP-1S significantly decreased neutrophil counts in BAL fluid. Fibroblastic foci were histologically assessed by H&E and Masson's trichrome stain and treated with KMUP-1 and references. Lung tissues were determined the contents of collagen and the expressions of TGF-ß, α-SMA, HMGB1, CTGF, eNOS, p-eNOS, RhoA, Smad3, p-Smad3, MMP-2 and MMP-9 by Western blotting analyses, respectively. These changes areregulated by NO/cGMP and inhibited by various treatments. KMUP-1 and KMUP-1S predominantly prevented HMGB1/MMP-2 expression at day-7 and reduced TGF-ß/phosphorylated Smad3 and CTGF at day-28. CONCLUSIONS AND IMPLICATIONS: KMUP-1 and KMUP-S restore eNOS, inhibit iNOS/ROCKII/MMP-2/MMP-9, attenuate histologic collagen disposition and reduce BALF inflammatory cells, potentially useful for the treatment of BLM-lung PF.

Ent-labdane-type diterpene glycosides obtained from Rubus chingii Hu and their inhibitory effects on PDE5A activity.

In this study, 16 new ent-labdane-type diterpene glycosides, designated as goshonosides J1-J16 (1-16), along with nine previously known diterpene glycosides (17-25) were extracted from the fruits of Rubus chingii Hu. The structures of goshonosides J1-J16 were elucidated using various analytical techniques, such as nuclear magnetic resonance, electron capture detector ECD, high-resolution electrospray ionization mass spectrometry HREIMS, single-crystal X-ray diffraction, and hydrolysis. Furthermore, the isolates' efficacy in inhibiting the activity of phosphodiesterase type 5 A was evaluated. Goshonosides J1, J2, and G effectively inhibited the activity of the aforementioned enzyme (IC50 values: 6.15 ± 1.76, 3.27 ± 0.65, and 9.61 ± 2.36 µM, respectively). Our findings highlight the remarkable structural diversity of bioactive compounds in R. chingii Hu and offer insights into the use of this shrub.

OTUD1 promotes isoprenaline- and myocardial infarction-induced heart failure by targeting PDE5A in cardiomyocytes.

Heart failure represents a major cause of death worldwide. Recent research has emphasized the potential role of protein ubiquitination/deubiquitination protein modification in cardiac pathology. Here, we investigate the role of the ovarian tumor deubiquitinase 1 (OTUD1) in isoprenaline (ISO)- and myocardial infarction (MI)-induced heart failure and its molecular mechanism. OTUD1 protein levels were raised markedly in murine cardiomyocytes after MI and ISO treatment. OTUD1 deficiency attenuated myocardial hypertrophy and cardiac dysfunction induced by ISO infusion or MI operation. In vitro, OTUD1 knockdown in neonatal rat ventricular myocytes (NRVMs) attenuated ISO-induced injuries, while OTUD1 overexpression aggravated the pathological changes. Mechanistically, LC-MS/MS and Co-IP studies showed that OTUD1 bound directly to the GAF1 and PDEase domains of PDE5A. OTUD1 was found to reverse K48 ubiquitin chain in PDE5A through cysteine at position 320 of OTUD1, preventing its proteasomal degradation. PDE5A could inactivates the cGMP-PKG-SERCA2a signaling axis which dysregulate the calcium handling in cardiomyocytes, and leading to the cardiomyocyte injuries. In conclusion, OTUD1 promotes heart failure by deubiquitinating and stabilizing PDE5A in cardiomyocytes. These findings have identified PDE5A as a new target of OTUD1 and emphasize the potential of OTUD1 as a target for treating heart failure.

A label-free LC/MS-based enzymatic activity assay for the detection of PDE5A inhibitors.

Phosphodiesterase type 5 (PDE5), a cyclic nucleotide phosphodiesterase, controls the duration of the cyclic guanosine monophosphate (cGMP) signal by hydrolyzing cGMP to GMP. Inhibiting the activity of PDE5A has proven to be an effective strategy for treating pulmonary arterial hypertension and erectile dysfunction. Current enzymatic activity assay methods for PDE5A mainly use fluorescent or isotope-labeled substrates, which are expensive and inconvenient. Here, we developed an LC/MS-based enzymatic activity assay for PDE5A without labeling, which detects the enzymatic activity of PDE5A by quantifying the substrate cGMP and product GMP at a concentration of 100 nM. The accuracy of this method was verified by a fluorescently labeled substrate. Moreover, a new inhibitor of PDE5A was identified by this method and virtual screening. It inhibited PDE5A with an IC50 value of 870 nM. Overall, the proposed strategy provides a new method for screening PDE5A inhibitors.

Co-expression of soluble guanylyl cyclase subunits and PDE5A shRNA to elevate cellular cGMP level: A potential gene therapy for myocardial cell death.

BACKGROUND: Genetic manipulation on the NO-sGC-cGMP pathway has been rarely achieved, partially due to complexity of the soluble guanylyl cyclase (sGC) enzyme. OBJECTIVE: We aim to develop gene therapy directly targeting the pathway to circumvent cytotoxicity and tolerance after prolonged use of NO-donors and the insufficiency of PDE inhibitors. METHODS: In this study, we constructed lentivirus vectors expressing GUCY1A3 and GUCY1B3 genes, which encoded the α1 and ß1 subunits of soluble guanylyl cyclase (sGC), respectively, to enhance cGMP synthesis. We also constructed lentiviral vector harboring PDE5A shRNA to alleviate phosphodiesterase activity and cGMP degradation. RESULTS: Transductions of human HEK293 cells with the constructs were successful, as indicated by the fluorescent signal and altered gene expression produced by each vector. Overexpression of GUCY1A3 and GUCY1B3 resulted in increased sGC enzyme activity and elevated cGMP level in the cells. Expression of PDE5A shRNA resulted in decreased PDE5A expression and elevated cGMP level. Co-transduction of the three lentiviral vectors resulted in a more significant elevation of cGMP in HEK293 cells without obvious cytotoxicity. CONCLUSION: To the best of our knowledge, this is the first study to show that co-expression of exogenous subunits of the soluble guanylyl cyclase could form functional enzyme and increase cellular cGMP level in mammalian cells. Simultaneous expression of PDE5A shRNA could alleviate feedback up-regulation on PDE5A caused by cGMP elevation. Further studies are required to evaluate the effects of these constructs in vivo.

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

Background: Bone 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. Methods: Oil 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. Results: ICA 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. Conclusion: ICA induces the differentiation of BMMSCs into brown fat cells via PDE5A inhibition, highlighting the therapeutic value of ICA for treating obesity-related diseases.

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.

Sildenafil Citrate Downregulates PDE5A mRNA Expression in Women with Recurrent Pregnancy Loss without Altering Angiogenic Factors-A Preliminary Study.

In our previous study, we showed that sildenafil citrate (SC), a selective PDE5A blocker, modulated NK cell activity in patients with recurrent pregnancy loss, which correlated with positive pregnancy outcomes. It was found that NK cells had a pivotal role in decidualization, angiogenesis, spiral artery remodeling, and the regulation of trophoblast invasion. Thus, in the current study, we determined the effects of SC on angiogenic factor expression and production, as well as idNK cell activity in the presence of nitric synthase blocker L-NMMA. Methods: NK cells (CD56+) were isolated from the peripheral blood of 15 patients and 15 fertile women on MACS columns and cultured in transformation media containing IL-15, TGF-ß, and AZA-a methylation agent-for 7 days in hypoxia (94% N2, 1% O2, 5% CO2). Cultures were set up in four variants: (1) with SC, (2) without SC, (3) with NO, a synthase blocker, and (4) with SC and NO synthase blocker. NK cell activity was determined after 7 days of culturing as CD107a expression after an additional 4h of stimulation with K562 erythroleukemia cells. The expression of the PDE5A, VEGF-A, PIGF, IL-8, and RENBP genes was determined with quantitative real-time PCR (qRT-PCR) using TaqMan probes and ELISA was used to measure the concentrations of VEGF-A, PLGF, IL-8, Ang-I, Ang-II, IFN-γ proteins in culture supernatants after SC supplementation. Results: SC downregulated PDE5A expression and had no effect on other studied angiogenic factors. VEGF-A expression was increased in RPL patients compared with fertile women. Similarly, VEGF production was enhanced in RPL patients' supernatants and SC increased the concentration of PIGF in culture supernatants. SC did not affect the expression or concentration of other studied factors, nor idNK cell activity, regardless of NO synthase blockade.

Discovery of two novel hetero-tricyclic lead scaffolds as PDE5A inhibitor: virtual screening, molecular docking and pharmacophore modeling approach.

Phosphodiesterase 5A enzyme has been the upcoming and promising target in hypertension management. In this research, reported 270 bioactive natural products having antihypertensive potential were selected and docked against PDE5A using vLife MDS 4.6 software. Based on docking score, π-stacking, H-bond and ionic interactions with PDE5A, 82 tricyclic compounds were selected for further study. Protein residue Gln817A was associated in H-boding, Leu804A in ionic interaction whereas Val782A and Phe820A were associated in π-stacking interaction with ligand. In silico docking studies resulted in discovery of oxygen containing naphthofuran and nitrogen and oxygen containing pyrano quinolizine tricyclic lead scaffolds as novel PDE5A inhibitors. Additionally, developed pharmacophore model suggested that one centre of hydrogen bond acceptor, one aromatic centre and two aliphatic centres are minimum pharmacophoric features required in the molecule so as to show sildenafil like activity. The identified lead scaffolds would provide novel platform for drug discovery of bioactive natural products.

Myocardial Impact of NHE1 Regulation by Sildenafil.

The cardiac Na+/H+ exchanger (NHE1) is a membrane glycoprotein fundamental for proper cell functioning due its multiple housekeeping tasks, including regulation of intracellular pH, Na+ concentration, and cell volume. In the heart, hyperactivation of NHE1 has been linked to the development of different pathologies. Several studies in animal models that reproduce the deleterious effects of ischemia/reperfusion injury or cardiac hypertrophy have conclusively demonstrated that NHE1 inhibition provides cardioprotection. Unfortunately, NHE1 inhibitors failed to reproduce these effects in the clinical arena. The reasons for those discrepancies are not apparent yet. However, a reasonable clue to consider would be that drugs that completely abolish the exchanger activity, including that its essential housekeeping function may not be the best therapeutic approach. Therefore, interventions tending to specifically reduce its hyperactive state without affecting its basal activity emerge as a novel potential gold standard. In this regard, a promising goal seems to be the modulation of the phosphorylation state of the cytosolic tail of the exchanger. Recent own experiments demonstrated that Sildenafil, a phosphodiesterase 5A inhibitor drug that has been widely used for the treatment of erectile dysfunction is able to decrease NHE1 phosphorylation, and hence reduce its hyperactivity. In connection, growing evidence demonstrates cardioprotective properties of Sildenafil against different cardiac pathologies, with the distinctive characteristic of directly affecting cardiac tissue without altering blood pressure. This mini-review was aimed to focus on the regulation of NHE1 activity by Sildenafil. For this purpose, experimental data reporting Sildenafil effects in different animal models of heart disease will be discussed.

The NO-cGMP-PKG pathway in skeletal remodeling.

The nitric oxide (NO)-cyclic guanosine monophosphate (cGMP)-protein kinase G (PKG) pathway plays a critical role in skeletal homeostasis. Preclinical data using NO and its donors and genetically modified mice demonstrated that NO was required in bone remodeling and partly mediated the anabolic effects of mechanical stimuli and estrogen. However, the off-target effects and tachyphylaxis of NO limit its long-term use, and previous clinical trials using organic nitrates for osteoporosis have been disappointing. Among the other components in the downstream pathway, targeting cGMP-specific phosphodiesterase to promote the NO-cGMP-PKG signal is a viable option. There are growing in vitro and in vivo data that, among many other PDE families, PDE5A is highly expressed in skeletal tissue, and inhibiting PDE5A using currently available PDE5A inhibitors might increase the osteoanabolic signal and protect the skeleton. These preclinical data open the possibility of repurposing PDE5A inhibitors for treating osteoporosis. Further research is needed to address the primary target bone cell of PDE5A inhibition, the contribution of direct and indirect effects of PDE5A inhibition, and the pathophysiological changes in skeletal PDE5A expression in aging and hypogonadal animal models.

Identification of potential genes in upper tract urothelial carcinoma using next-generation sequencing with bioinformatics and in vitro analyses.

BACKGROUND: We aimed to identify prognostic biomarkers of upper tract urothelial carcinomas (UTUCs), including microRNAs (miRNAs) and genes which account for only 5% to 10% of all urothelial carcinomas (UCs). In Taiwan, this figure is markedly higher, where it can reach up to 30% of UC cases. MATERIALS AND METHODS: Using next-generation sequencing (NGS), we analyzed two pairs of renal pelvis tumors and adjacent normal urothelial tissues to screen miRNAs and messenger RNAs. By combining bioinformatics analysis from miRmap, Gene Expression Omnibus (GEO), and Oncomine and Ingenuity® Pathway Analysis databases, we identified candidate genes. To search for upstream miRNAs with exact target binding sites, we used miRmap, TargetScan, and miRDB to enforce evidence. Then, we clarified gene and protein expression through an in vitro study using western blot analysis and quantitative real-time reverse transcriptase-PCR. RESULTS: Interactions between selected target genes obtained using the NGS and miRmap methods were assessed through a Venn diagram analysis. Six potential genes, namely, PDE5A, RECK, ZEB2, NCALD, PLCXD3 and CYBRD1 showed significant differences. Further analysis of gene expression from the GEO dataset indicated lower expression of PDE5A, RECK, ZEB2, and CYBRD1 in bladder cancer tissue than in normal bladder mucosa, which indicated that PDE5A, RECK, ZEB2, and CYBRD1 may act as tumor suppressors in UTUC. In addition, we compared the expression of these genes in various UC cell lines (RT4, BFTC905, J82, T24, UMUC3, 5637, BFTC 909, UMUC14) and found decreased expression of PDE5A in muscle-invasive UC cells compared with the RT4 cell line. Furthermore, by using paired UTUC and normal tissues from 20 patients, lower PDE5A expression was also demonstrated in tumor specimens. CONCLUSIONS: Our findings suggest these candidate genes may play some roles in UTUC progression. We propose that these markers may be potential targets clarified by in vitro and in vivo experiments. PDE5A also potentially presents tumor suppressor genes, as identified by comparing the expression between normal and tumor specimens.