Phosphodiesterase Type 5 Inhibition Reverts Prostate Fibroblast-to-Myofibroblast Trans-Differentiation

Abstract Phosphodiesterase type 5 (PDE5) inhibitors have been demonstrated to improve lower urinary tract symptoms secondary to benign prostatic hyperplasia (BPH). Because BPH is primarily driven by fibroblast-to-myofibroblast trans-differentiation, this study aimed to evaluate the potential of the PDE5 inhibitor vardenafil to inhibit and reverse trans-differentation of primary human prostatic stromal cells (PrSC). Vardenafil, sodium nitroprusside, lentiviral-delivered short hairpin RNA-mediated PDE5 knockdown, sodium orthovanadate, and inhibitors of MAPK kinase, protein kinase G, Ras homolog family member (Rho) A, RhoA/Rho kinase, phosphatidylinositol 3 kinase and protein kinase B (AKT) were applied to PrSC treated with basic fibroblast growth factor (fibroblasts) or TGFβ1 (myofibroblasts) in vitro, in chicken chorioallantoic membrane xenografts in vivo, and to prostatic organoids ex vivo. Fibroblast-to-myofibroblast trans-differentiation was monitored by smooth muscle cell actin and IGF binding protein 3 mRNA and protein levels. Vardenafil significantly attenuated TGFβ1-induced PrSC trans-differentiation in vitro and in chorioallantoic membrane xenografts. Enhancement of nitric oxide/cyclic guanosine monophosphate signaling by vardenafil, sodium nitroprusside, or PDE5 knockdown reduced smooth muscle cell actin and IGF binding protein 3 mRNA and protein levels and restored fibroblast-like morphology in trans-differentiated myofibroblast. This reversal of trans-differentiation was not affected by MAPK kinase, protein kinase G, RhoA, or RhoA/Rho kinase inhibition, but vardenafil attenuated phospho-AKT levels in myofibroblasts. Consistently, phosphatidylinositol 3 kinase or AKT inhibition induced reversal of trans-differentiation, whereas the tyrosine phosphatase inhibitor sodium orthovanadate abrogated the effect of vardenafil. Treatment of prostatic organoids with vardenafil ex vivo reduced expression of myofibroblast markers, indicating reverse remodeling of stroma towards a desired higher fibroblast/myofibroblast ratio. Thus, enhancement of the nitric oxide/cyclic guanosine monophosphate signaling pathway by vardenafil attenuates and reverts fibroblast-to-myofibroblast trans-differentiation, hypothesizing that BPH patients might benefit from long-term therapy with PDE5 inhibitors.

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Preparation and Evaluation of Sildenafil Rapidly Disintegrating Tablets

International Journal of Pharmaceutical Sciences and Nanotechnology ◽

10.37285/ijpsn.2012.5.1.4 ◽

2012 ◽

Vol 5 (1) ◽

pp. 1617-1620

Author(s):

Adel M Aly ◽

A A Mohammed

Keyword(s):

Oral Cavity ◽

Cyclic Guanosine Monophosphate ◽

Rapid Onset ◽

Guanosine Monophosphate ◽

Direct Compression ◽

Onset Of Action ◽

Disintegration Time ◽

Maximum Serum ◽

Phosphodiesterase Type

Sildenafil citrate is an oral therapy for erectile dysfunction (ED). Sildenafil, a selective inhibitor of cyclic guanosine monophosphate-specific phosphodiesterase type 5 (PDE5), has been reported to be effective in men with ED associated with diabetes and prostate cancer, and in psychogenic ED. The main objective of this study is to prepare more effective, rapidly disintegrating and rapid onset of action sildenafil oral tablets. Sildenafil tablets were prepared using the newly introduced Pharmaburst® as a direct compression vehicle in comparison with the well-known excipients, namely mannitol, anhydrous lactose and primojel. The formula containing Pharmaburst® showed the most rapidly-disintegrating effect (15 sec) compared to the other formulations. Thus, Pharmaburst® can be utilized as an effective direct compression vehicle as well as a superdisintegrant with very rapid disintegration time in vitro and in the oral cavity. The rapidly-disintegrating sildenafil tablets showed maximum serum concentration within only two minutes (Cmax of 0.76 µg) by applying the tablets to the oral cavity of rabbits, whereas, the conventional sildenafil tablets have a comparatively lower Cmax (0.56µg) through about 45 minutes.

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Abstract 923: Dissociation Between Superoxide Accumulation And Nitroglycerin Induced Tolerance

Circulation ◽

10.1161/circ.116.suppl_16.ii_181-c ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

Vamsi K Addanki ◽

Pei-Suen Tsou ◽

Ho-Leung Fung

Keyword(s):

Nadph Oxidase ◽

Ex Vivo ◽

Dry Weight ◽

Causative Factor ◽

Cyclic Guanosine Monophosphate ◽

Guanosine Monophosphate ◽

Protein Accumulation ◽

Induced Tolerance

We hypothesize that superoxide (SO) accumulation is not a critical causative factor in inducing nitroglycerin (NTG) tolerance. Using p47phox−/− and gp91−/− mice vs. their respective wild-type (WT) controls, we showed that aorta from mice null of these critical NADPH oxidase subunits exhibited similar vascular tolerance after NTG dosing (20 mg/kg sc, tid for 3 days), as indicated by their ex vivo pEC 50 and cyclic guanosine monophosphate (cGMP, pmol/mg protein) accumulation upon NTG challenge. In vitro aorta SO production (cpm/mg dry weight) was enhanced by NTG incubation both in p47phox null and WT mice (Table 1 ). Pre-exposure of isolated mice aorta to 100 microM NTG for 1 hr resulted in vascular tolerance toward NTG and increased SO accumulation. Oxypurinol (Oxy, 1mM) reduced SO but failed to attenuate vascular tolerance (Table 2 ). In LLC-PK1 cells, pre-exposure to NTG (1 microM for 4 hours) resulted in increased SO accumulation and reduced cGMP response to 3.16 microM NTG vs. vehicle control. Exposure to 1 microM angiotensin II increased SO but did not reduce cGMP response. Taken together, these results indicate that in vivo vascular NTG tolerance in mice does not require the presence of the p47phox and gp91phox subunits of NADPH oxidase, and that increased SO accumulation may be a consequence, rather than a cause, of NTG tolerance. Table 1 Table 2

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The vasodilatory effect of sulfur dioxide via SGC/cGMP/PKG pathway in association with sulfhydryl-dependent dimerization

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00101.2015 ◽

2016 ◽

Vol 310 (11) ◽

pp. R1073-R1080 ◽

Cited By ~ 7

Author(s):

Qiuyu Yao ◽

Yaqian Huang ◽

Angie Dong Liu ◽

Mingzhu Zhu ◽

Jia Liu ◽

...

Keyword(s):

Sulfur Dioxide ◽

Soluble Guanylate Cyclase ◽

Hydrolytic Enzyme ◽

Cyclic Guanosine Monophosphate ◽

Guanosine Monophosphate ◽

Dependent Manner ◽

Protein Levels ◽

Phosphodiesterase Type ◽

Dose Dependent

The present study was designed to explore the role of soluble guanylate cyclase (sGC)/cyclic guanosine monophosphate (cGMP)/PKG pathway in sulfur dioxide (SO2)-induced vasodilation. We showed that SO2 induced a concentration-dependent relaxation of phenylephrine (PE)-precontracted rat aortic rings in association with an increase in cGMP concentration, whereas l-aspartic acid β-hydroxamate (HDX), an inhibitor of SO2 synthase, contracted rings in a dose-dependent manner. Pretreatment of aortic rings with the sGC inhibitor ODQ (30 μM) attenuated the vasodilatory effects of SO2, suggesting the involvement of cGMP pathway in SO2-induced vasodilation. Mechanistically, SO2 upregulated the protein levels of sGC and PKG dimers, while HDX inhibited it, indicating SO2 could promote cGMP synthesis through sGC activation. Furthermore, the dimerization of sGC and PKG and vasodilation induced by SO2 in precontracted rings were significantly prevented by thiol reductants dithiothreitol (DTT). In addition, SO2 reduced the activity of phosphodiesterase type 5 (PDE5), a cGMP-specific hydrolytic enzyme, implying that SO2 elevated cGMP concentration by inhibiting its hydrolysis. Hence, SO2 exerted its vasodilatory effects at least partly by promoting disulfide-dependent dimerization of sGC and PKG, resulting in an activated sGC/cGMP/PKG pathway in blood vessels. These findings revealed a new mode of action and mechanisms by which SO2 regulated the vascular tone.

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Novel enhancers of guanylyl cyclase-A activity via allosteric modulation

10.1101/2021.12.31.474340 ◽

2022 ◽

Author(s):

Henriette Andresen ◽

Cristina Pérez-Ternero ◽

Jerid Robinson ◽

Deborah M Dickey ◽

Adrian J Hobbs ◽

...

Keyword(s):

Small Molecules ◽

Natriuretic Peptide ◽

Guanylyl Cyclase ◽

Drug Target ◽

Ex Vivo ◽

Cyclic Guanosine Monophosphate ◽

Guanosine Monophosphate ◽

Allosteric Binding Sites ◽

Small Molecule Modulators

Natriuretic peptide receptor (NPR)-A (also known as NPR-A, NPR1 or guanylyl cyclase-A, GC-A) is an attractive but challenging target to activate with small molecules. GC-A is activated by endogenous atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP), and this activation leads to the production of cyclic guanosine monophosphate (cGMP). This system plays an important role in the regulation of cardiovascular and renal homeostasis. However, utilization of this receptor as a drug target has so far been limited to peptides, even though small molecule modulators allow oral administration and longer half-life. We have identified small molecular allosteric enhancers of GC-A, which strengthened ANP or BNP activation in various in vitro and ex vivo systems. These compounds do not mediate their actions through previously described allosteric binding sites or via known mechanisms of action. In addition, their selectivity and activity are dependent on only one amino acid in GC-A. Our findings show that there is a novel allosteric binding site on GC-A, which can be targeted by small molecules that increase the signaling effects of ANP and BNP.

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Polydatin Attenuates Carbachol-induced Contraction of Guinea Pig Gallbladder

Current Topics in Nutraceutical Research ◽

10.37290/ctnr2641-452x.18:34-38 ◽

2019 ◽

Vol 18 (1) ◽

pp. 34-38

Author(s):

Chen Lei ◽

Pan Xiang ◽

Shen Yonggang ◽

Song Kai ◽

Zhong Xingguo ◽

...

Keyword(s):

Protein Kinase ◽

Guinea Pig ◽

Smooth Muscles ◽

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

Cyclic Guanosine Monophosphate ◽

Guanosine Monophosphate ◽

Dependent Manner ◽

Underlying Mechanisms ◽

Dose Dependent

The aim of this study was to determine whether polydatin, a glucoside of resveratrol isolated from the root of Polygonum cuspidatum, warranted development as a potential therapeutic for ameliorating the pain originating from gallbladder spasm disorders and the underlying mechanisms. Guinea pig gallbladder smooth muscles were treated with polydatin and specific inhibitors to explore the mechanisms underpinning polydatin-induced relaxation of carbachol-precontracted guinea pig gallbladder. Our results shown that polydatin relaxed carbachol-induced contraction in a dose-dependent manner through the nitric oxide/cyclic guanosine monophosphate/protein kinase G and the cyclic adenosine monophosphate/protein kinase A signaling pathways as well as the myosin light chain kinase and potassium channels. Our findings suggested that there was value in further exploring the potential therapeutic use of polydatin in gallbladder spasm disorders.

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The cGMP-Dependent Protein Kinase 2 Contributes to Cone Photoreceptor Degeneration in the Cnga3-Deficient Mouse Model of Achromatopsia

International Journal of Molecular Sciences ◽

10.3390/ijms22010052 ◽

2020 ◽

Vol 22 (1) ◽

pp. 52

Author(s):

Mirja Koch ◽

Constanze Scheel ◽

Hongwei Ma ◽

Fan Yang ◽

Michael Stadlmeier ◽

...

Keyword(s):

Protein Kinase ◽

Mouse Model ◽

Cyclic Guanosine Monophosphate ◽

Guanosine Monophosphate ◽

Cone Photoreceptor ◽

Photoreceptor Degeneration ◽

Dependent Protein Kinase ◽

Genetic Ablation ◽

Cgmp Dependent Protein Kinase ◽

Dependent Protein

Mutations in the CNGA3 gene, which encodes the A subunit of the cyclic guanosine monophosphate (cGMP)-gated cation channel in cone photoreceptor outer segments, cause total colour blindness, also referred to as achromatopsia. Cones lacking this channel protein are non-functional, accumulate high levels of the second messenger cGMP and degenerate over time after induction of ER stress. The cell death mechanisms that lead to loss of affected cones are only partially understood. Here, we explored the disease mechanisms in the Cnga3 knockout (KO) mouse model of achromatopsia. We found that another important effector of cGMP, the cGMP-dependent protein kinase 2 (Prkg2) is crucially involved in cGMP cytotoxicity of cones in Cnga3 KO mice. Virus-mediated knockdown or genetic ablation of Prkg2 in Cnga3 KO mice counteracted degeneration and preserved the number of cones. Analysis of markers of endoplasmic reticulum stress and unfolded protein response confirmed that induction of these processes in Cnga3 KO cones also depends on Prkg2. In conclusion, we identified Prkg2 as a novel key mediator of cone photoreceptor degeneration in achromatopsia. Our data suggest that this cGMP mediator could be a novel pharmacological target for future neuroprotective therapies.

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Current Modulation of Guanylate Cyclase Pathway Activity—Mechanism and Clinical Implications

Molecules ◽

10.3390/molecules26113418 ◽

2021 ◽

Vol 26 (11) ◽

pp. 3418

Author(s):

Grzegorz Grześk ◽

Alicja Nowaczyk

Keyword(s):

Guanylate Cyclase ◽

Natriuretic Peptides ◽

Soluble Guanylate Cyclase ◽

Cyclic Guanosine Monophosphate ◽

Guanosine Monophosphate ◽

Pharmacological Intervention ◽

First Line ◽

Phosphodiesterase Type ◽

Activity Mechanism

For years, guanylate cyclase seemed to be homogenic and tissue nonspecific enzyme; however, in the last few years, in light of preclinical and clinical trials, it became an interesting target for pharmacological intervention. There are several possible options leading to an increase in cyclic guanosine monophosphate concentrations. The first one is related to the uses of analogues of natriuretic peptides. The second is related to increasing levels of natriuretic peptides by the inhibition of degradation. The third leads to an increase in cyclic guanosine monophosphate concentration by the inhibition of its degradation by the inhibition of phosphodiesterase type 5. The last option involves increasing the concentration of cyclic guanosine monophosphate by the additional direct activation of soluble guanylate cyclase. Treatment based on the modulation of guanylate cyclase function is one of the most promising technologies in pharmacology. Pharmacological intervention is stable, effective and safe. Especially interesting is the role of stimulators and activators of soluble guanylate cyclase, which are able to increase the enzymatic activity to generate cyclic guanosine monophosphate independently of nitric oxide. Moreover, most of these agents are effective in chronic treatment in heart failure patients and pulmonary hypertension, and have potential to be a first line option.

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IGFBP-1 expression is reduced in human type 2 diabetic glomeruli and modulates β1-integrin/FAK signalling in human podocytes

Diabetologia ◽

10.1007/s00125-021-05427-1 ◽

2021 ◽

Cited By ~ 1

Author(s):

Abigail C. Lay ◽

Lorna J. Hale ◽

Holly Stowell-Connolly ◽

Robert J. P. Pope ◽

Viji Nair ◽

...

Keyword(s):

Ex Vivo ◽

Igf Binding Proteins ◽

Impedance Sensing ◽

Human Type ◽

Early Stages ◽

Igf Binding ◽

Phosphoinositide 3 Kinase ◽

End Stage

AbstractAims/hypothesisPodocyte loss or injury is one of the earliest features observed in the pathogenesis of diabetic kidney disease (DKD), which is the leading cause of end-stage renal failure worldwide. Dysfunction in the IGF axis, including in IGF binding proteins (IGFBPs), is associated with DKD, particularly in the early stages of disease progression. The aim of this study was to investigate the potential roles of IGFBPs in the development of type 2 DKD, focusing on podocytes.MethodsIGFBPexpression was analysed in the Pima DKD cohort, alongside data from the Nephroseq database, and in ex vivo human glomeruli. Conditionally immortalised human podocytes and glomerular endothelial cells were studied in vitro, where IGFBP-1 expression was analysed using quantitative PCR and ELISAs. Cell responses to IGFBPs were investigated using migration, cell survival and adhesion assays; electrical cell-substrate impedance sensing; western blotting; and high-content automated imaging.ResultsData from the Pima DKD cohort and from the Nephroseq database demonstrated a significant reduction in glomerularIGFBP-1in the early stages of human type 2 DKD. In the glomerulus, IGFBP-1 was predominantly expressed in podocytes and controlled by phosphoinositide 3-kinase (PI3K)–forkhead box O1 (FoxO1) activity. In vitro,IGFBP-1 signalled to podocytes via β1-integrins, resulting in increased phosphorylation of focal-adhesion kinase (FAK), increasing podocyte motility, adhesion, electrical resistance across the adhesive cell layer and cell viability.Conclusions/interpretationThis work identifies a novel role for IGFBP-1 in the regulation of podocyte function and that the glomerular expression ofIGFBP-1is reduced in the early stages of type 2 DKD, via reduced FoxO1 activity. Thus, we hypothesise that strategies to maintain glomerular IGFBP-1 levels may be beneficial in maintaining podocyte function early in DKD.Graphical abstract

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Release of cyclic guanosine monophosphate evaluated as a diagnostic tool in cardiac diseases

Clinical Chemistry ◽

10.1093/clinchem/37.2.186 ◽

1991 ◽

Vol 37 (2) ◽

pp. 186-190 ◽

Cited By ~ 39

Author(s):

Karl-P Vorderwinkler ◽

Eilka Artner-Dworzak ◽

Gab Jakob ◽

Johanne Mair ◽

Franz Diensti ◽

...

Keyword(s):

Cyclic Guanosine Monophosphate ◽

Guanosine Monophosphate ◽

Cardiac Diseases ◽

Blood Samples ◽

Cutoff Value ◽

Intracellular Cgmp ◽

Glycerol Trinitrate ◽

Clinical Measurements ◽

And Storage

Abstract Concentrations of atrial natriuretic peptide (ANP) are increased in plasma of patients with impaired cardiac and renal function. The second messenger of ANP, cyclic guanosine monophosphate (cGMP), is released into the plasma specifically upon stimulation of cells with ANP. Although nitrates can also activate intracellular cGMP synthesis, we detected no increase in plasma cGMP concentrations after infusions of glycerol trinitrate. Because immunoreactive ANP is highly susceptible to degradation and nonspecific influences in blood samples, determinations of ANP require immediate centrifugation and storage of plasma at -20 degrees C. In contrast, we found that cGMP is stable for five days in vitro in blood samples containing EDTA. In 147 healthy blood donors, the upper cutoff value for plasma cGMP was 6.60 nmol/L, not significantly different (P greater than 0.05) from that for 222 patients with disorders other than cardiovascular and renal. In 69 patients with manifest congestive heart failure (NYHA stages II-IV), 65 had increased cGMP values. Using the above cutoff value for cGMP gave diagnostic sensitivity of 94.2% and specificity of 93.7%. Plasma cGMP may thus provide an alternative for routine clinical measurements of ANP in cardiac diseases in the absence of renal disorders.

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