Nitric oxide (NO) plays a significant role in controlling the physiology and pathophysiology of the body, including the endothelial antiplatelet function and therefore, antithrombogenic property of ...the blood vessels. This property of NO can be exploited to prevent thrombus formation on artificial surfaces like extracorporeal membrane oxygenators, which when come into contact with blood lead to protein adsorption and thereby platelet activation causing thrombus formation. However, NO is extremely reactive and has a very short biological half‐life in blood, so only endogenous generation of NO from the blood contacting material can result into a stable and kinetically controllable local delivery of NO. In this regards, highly hydrophilic bioactive nanogels are presented which can endogenously generate NO in blood plasma from endogenous NO‐donors thereby maintaining a physiological NO flux. It is shown that NO releasing nanogels could initiate cGMP‐dependent protein kinase signaling followed by phosphorylation of vasodilator‐stimulated phosphoprotein in platelets. This prevents platelet activation and aggregation even in presence of highly potent platelet activators like thrombin, adenosine 5′‐diphosphate, and U46619 (thromboxane A2 mimetic).
Highly hydrophilic bioactive nanogels are designed to endogeneously generate nitric oxide in blood plasma from endogenous nitric oxide‐donors to maintain a physiological nitic oxide flux. The nanogels could initiated cyclic guanosine monophosphate‐dependent protein kinase signaling followed by phosphorylation of vasodilator‐stimulated phosphoproteinin platelets. This prevented platelet activation and aggregation even in presence of highly potent platelet activators.
Hereditary retinal degeneration (RD) is often associated with excessive cGMP signalling in photoreceptors. Previous research has shown that inhibition of cGMP-dependent protein kinase G (PKG) can ...reduce photoreceptor loss in two different RD animal models. In this study, we identified a PKG inhibitor, the cGMP analogue CN238, which preserved photoreceptor viability and functionality in rd1 and rd10 mutant mice. Surprisingly, in explanted retinae, CN238 also protected retinal ganglion cells from axotomy-induced retrograde degeneration and preserved their functionality. Furthermore, kinase activity-dependent protein phosphorylation of the PKG target Kv1.6 was reduced in CN238-treated rd10 retinal explants. Ca2+-imaging on rd10 acute retinal explants revealed delayed retinal ganglion cell repolarization with CN238 treatment, suggesting a PKG-dependent modulation of Kv1-channels. Together, these results highlight the strong neuroprotective capacity of PKG inhibitors for both photoreceptors and retinal ganglion cells, illustrating their broad potential for the treatment of retinal diseases and possibly neurodegenerative diseases in general.
Abstract
Aims
Sodium-glucose-cotransporter-2 inhibitors showed favourable cardiovascular outcomes, but the underlying mechanisms are still elusive. This study investigated the mechanisms of ...empagliflozin in human and murine heart failure with preserved ejection fraction (HFpEF).
Methods and results
The acute mechanisms of empagliflozin were investigated in human myocardium from patients with HFpEF and murine ZDF obese rats, which were treated in vivo. As shown with immunoblots and ELISA, empagliflozin significantly suppressed increased levels of ICAM-1, VCAM-1, TNF-α, and IL-6 in human and murine HFpEF myocardium and attenuated pathological oxidative parameters (H2O2, 3-nitrotyrosine, GSH, lipid peroxide) in both cardiomyocyte cytosol and mitochondria in addition to improved endothelial vasorelaxation. In HFpEF, we found higher oxidative stress-dependent activation of eNOS leading to PKGIα oxidation. Interestingly, immunofluorescence imaging and electron microscopy revealed that oxidized PKG1α in HFpEF appeared as dimers/polymers localized to the outer-membrane of the cardiomyocyte. Empagliflozin reduced oxidative stress/eNOS-dependent PKGIα oxidation and polymerization resulting in a higher fraction of PKGIα monomers, which translocated back to the cytosol. Consequently, diminished NO levels, sGC activity, cGMP concentration, and PKGIα activity in HFpEF increased upon empagliflozin leading to improved phosphorylation of myofilament proteins. In skinned HFpEF cardiomyocytes, empagliflozin improved cardiomyocyte stiffness in an anti-oxidative/PKGIα-dependent manner. Monovariate linear regression analysis confirmed the correlation of oxidative stress and PKGIα polymerization with increased cardiomyocyte stiffness and diastolic dysfunction of the HFpEF patients.
Conclusion
Empagliflozin reduces inflammatory and oxidative stress in HFpEF and thereby improves the NO–sGC–cGMP–cascade and PKGIα activity via reduced PKGIα oxidation and polymerization leading to less pathological cardiomyocyte stiffness.
•Cyclic nucleotides are important for neuroplasticity and memory function.•Altered cyclic nucleotide signaling is implicated in memory dysfunction.•Cyclic nucleotides gradients are mainly regulated ...by phosphodiesterases.•Cyclic nucleotide signaling is spatiotemporally defined.•New tools provide novel ways to measure and manipulate cyclic nucleotides.
A plethora of studies indicate the important role of cAMP and cGMP cascades in neuronal plasticity and memory function. As a result, altered cyclic nucleotide signaling has been implicated in the pathophysiology of mnemonic dysfunction encountered in several diseases. In the present review we provide a wide overview of studies regarding the involvement of cyclic nucleotides, as well as their upstream and downstream molecules, in physiological and pathological mnemonic processes. Next, we discuss the regulation of the intracellular concentration of cyclic nucleotides via phosphodiesterases, the enzymes that degrade cAMP and/or cGMP, and via A-kinase-anchoring proteins that refine signal compartmentalization of cAMP signaling. We also provide an overview of the available data pointing to the existence of specific time windows in cyclic nucleotide signaling during neuroplasticity and memory formation and the significance to target these specific time phases for improving memory formation. Finally, we highlight the importance of emerging imaging tools like Förster resonance energy transfer imaging and optogenetics in detecting, measuring and manipulating the action of cyclic nucleotide signaling cascades.
Platelets are circulating sentinels of vascular integrity and are activated, inhibited, or modulated by multiple hormones, vasoactive substances or drugs. Endothelium- or drug-derived NO strongly ...inhibits platelet activation via activation of the soluble guanylate cyclase (sGC) and cGMP elevation, often in synergy with cAMP-elevation by prostacyclin. However, the molecular mechanisms and diversity of cGMP effects in platelets are poorly understood and sometimes controversial. Recently, we established the quantitative human platelet proteome, the iloprost/prostacyclin/cAMP/protein kinase A (PKA)-regulated phosphoproteome, and the interactions of the ADP- and iloprost/prostacyclin-affected phosphoproteome. We also showed that the sGC stimulator riociguat is in vitro a highly specific inhibitor, via cGMP, of various functions of human platelets. Here, we review the regulatory role of the cGMP/protein kinase G (PKG) system in human platelet function, and our current approaches to establish and analyze the phosphoproteome after selective stimulation of the sGC/cGMP pathway by NO donors and riociguat. Present data indicate an extensive and diverse NO/riociguat/cGMP phosphoproteome, which has to be compared with the cAMP phosphoproteome. In particular, sGC/cGMP-regulated phosphorylation of many membrane proteins, G-proteins and their regulators, signaling molecules, protein kinases, and proteins involved in Ca2+ regulation, suggests that the sGC/cGMP system targets multiple signaling networks rather than a limited number of PKG substrate proteins.
•Direct sGC stimulators (NO donors, riociguat) inhibit platelet function at multiple sites.•NO/riociguat affect more than 200 phosphoproteins (cGMP phosphoproteome) in human platelets.•Multiple signaling networks are identified as targets of the sGC/cGMP system in human platelets.
Long noncoding RNAs (lncRNAs) play a crucial role in several malignances, involving nasopharyngeal carcinoma (NPC), a heterogeneous disease. This study investigated mechanism of serine/arginine ...repetitive matrix protein 2‐alternative splicing (SRRM2‐AS) in NPC cell proliferation, differentiation, and angiogenesis. Initially, differentially expressed lncRNAs were screened out via microarray analysis. Vascular endothelial growth factor (VEGF) protein positive rate and microvessel density (MVD) were determined in NPC and adjacent tissues. NPC CNE‐2 cells were treated with a series of vector and small interfering RNA to explore the effect of SRRM2‐AS in NPC. The target relationship between myosin light chain kinase (MYLK) and SRRM2‐AS was verified. Levels of SRRM2‐AS, MYLK, cGMP, PKG, VEGF, PCNA, Ki‐67, B‐cell lymphoma‐2 (Bcl‐2), Bcl‐2‐associated X protein (Bax), and Caspase 3 were determined after transfection. Finally, the effect of SRRM2‐AS on cell proliferation, colony formation, angiogenesis, cell cycle, and apoptosis in NPC was evaluated. SRRM2‐AS was highly expressed and MYLK was poorly expressed in NPC tissues. VEGF protein positive rate and MVD were elevated in NPC tissues. MYLK was confirmed to be a target gene of SRRM2‐AS. Silencing of SRRM2‐AS elevated levels of MYLK, cGMP, PKG, Bax, and Caspase 3, but decreased levels of VEGF, PCNA, Ki‐67, and Bcl‐2. Especially, silencing of SRRM2‐AS suppressed cell proliferation, colony formation and angiogenesis, blocked cell cycle, and enhanced cell apoptosis in NPC. Our results suggested that silencing of SRRM2‐AS protected against angiogenesis of NPC cells by upregulating MYLK and activating the cGMP‐PKG signaling pathway, which provides a new target for NPC treatment.
Ischemic stroke is one of the leading causes of mortality worldwide. The available treatments are not effective. Phosphodiesterase 9A (PDE9A) is an intracellular cyclic guanosine monophosphate (cGMP) ...hydrolase considered to be a promising therapeutic target for brain diseases. This study explored neuroprotective effects and the underlying mechanism of LW33, a novel PDE9A inhibitor, on ischemic stroke in vitro and in vivo.
A middle cerebral artery occlusion (MCAO) model was established in adult male Sprague-Dawley rats and an oxygen-glucose deprivation/reoxygenation (OGD/R) model was established in human SH-SY5Y cells to mimic ischemia-reperfusion injury in vitro.
LW33 increased cell viability, reduced lactate dehydrogenase activity, and OGD/R-induced apoptosis of SH-SY5Y cells. The protective effects of LW33 against stroke occurred in the recovery phase. LW33 administration significantly reduced cerebral infarction volume in MCAO rats, without causing significant deformation or necrosis of neurons in the cortex. LW33 also improved learning and cognitive dysfunction and reduced other pathological changes in MCAO rats in the recovery period. Moreover, LW33 stimulated the cGMP/PKG/CREB pathway and up-regulated the expression of the apoptosis-related proteins, and this effect was reversed by KT5823 treatment.
LW33 inhibited cell apoptosis and promoted neuronal repair to alleviate OGD/R and MCAO induced pathological alterations via the cGMP/PKG/CREB pathway, indicating that LW33 may be a promising therapeutic target for ischemic stroke.
Display omitted
•Protein kinase G (PKG) is a major receptor of cGMP.•The contributions of the 6-oxo and 2-NH2 cGMP substituents to PKG binding are highly non-additive.•Such non-additivity stems ...primarily from mutual protein–ligand conformational selection.•The 6-oxo substituent controls conformational selection at the level of the protein.•The 2-NH2 substituent controls conformational selection at the level of the unbound ligand.
Protein kinase G (PKG) is a major receptor of cGMP, and controls signaling pathways distinct from those regulated by cAMP. However, the contributions of the two substituents that differentiate cGMP from cAMP (i.e. 6-oxo and 2-NH2) to the cGMP-versus-cAMP selectivity of PKG remain unclear. Here, using NMR to map how binding affinity and dynamics of the protein and ligand vary along a ligand double-substitution cycle, we show that the contributions of the two substituents to binding affinity are surprisingly non-additive. Such non-additivity stems primarily from mutual protein–ligand conformational selection, whereby not only does the ligand select for a preferred protein conformation upon binding, but also, the protein selects for a preferred ligand conformation. The 6-oxo substituent mainly controls the conformational equilibrium of the bound protein, while the 2-NH2 substituent primarily controls the conformational equilibrium of the unbound ligand (i.e. syn versus anti). Therefore, understanding the conformational dynamics of both the protein and ligand is essential to explain the cGMP-versus-cAMP selectivity of PKG.
The cellular mechanisms underlying hereditary photoreceptor degeneration are still poorly understood, a problem that is exacerbated by the enormous genetic heterogeneity of this disease group. ...However, the last decade has yielded a wealth of new knowledge on degenerative pathways and their diversity. Notably, a central role of cGMP-signalling has surfaced for photoreceptor cell death triggered by a subset of disease-causing mutations.
In this review, we examine key aspects relevant for photoreceptor degeneration of hereditary origin. The topics covered include energy metabolism, epigenetics, protein quality control, as well as cGMP- and Ca2+-signalling, and how the related molecular and metabolic processes may trigger photoreceptor demise. We compare and integrate evidence on different cell death mechanisms that have been associated with photoreceptor degeneration, including apoptosis, necrosis, necroptosis, and PARthanatos. A special focus is then put on the mechanisms of cGMP-dependent cell death and how exceedingly high photoreceptor cGMP levels may cause activation of Ca2+-dependent calpain-type proteases, histone deacetylases and poly-ADP-ribose polymerase. An evaluation of the available literature reveals that a large group of patients suffering from hereditary photoreceptor degeneration carry mutations that are likely to trigger cGMP-dependent cell death, making this pathway a prime target for future therapy development.
Finally, an outlook is given into technological and methodological developments that will with time likely contribute to a comprehensive overview over the entire metabolic complexity of photoreceptor cell death. Building on such developments, new imaging technology and novel biomarkers may be used to develop clinical test strategies, that fully consider the genetic heterogeneity of hereditary retinal degenerations, in order to facilitate clinical testing of novel treatment approaches.
PDF
