cGMP-dependent Protein Kinase Research Articles

Atrial natriuretic peptide (ANP) modulates stress-induced autophagy in endothelial cells

Atrial natriuretic peptide (ANP), a cardiac hormone involved in the regulation of water/sodium balance and blood pressure, is also secreted by endothelial cells, where it exerts protective effects in response to stress. Autophagy is an intracellular self-renewal process involved in the degradation of dysfunctional cytoplasmic elements. ANP was recently reported to act as an extracellular regulator of cardiac autophagy. However, its role in the regulation of endothelial autophagy has never been investigated. Here, we tested the effects of ANP in the regulation of autophagy in human umbilical vein endothelial cells (HUVECs). We found that ANP rapidly increases autophagy and autophagic flux at physiological concentrations through its predominant pathway, mediated by natriuretic peptide receptor type A (NPR-A) and protein kinase G (PKG). We further observed that ANP is rapidly secreted by HUVEC under stress conditions, where it mediates stress-induced autophagy through autocrine and paracrine mechanisms. Finally, we found that the protective effects of ANP in response to high-salt loading or tumor necrosis factor (TNF)-α are blunted by concomitant inhibition of autophagy. Overall, our results suggest that ANP acts as an endogenous autophagy activator in endothelial cells. The autophagy mechanism mediates the protective endothelial effects exerted by ANP.

Signaling Paradigms of H2S-Induced Vasodilation: A Comprehensive Review

Hydrogen sulfide (H2S), a gas traditionally considered toxic, is now recognized as a vital endogenous signaling molecule with a complex physiology. This comprehensive study encompasses a systematic literature review that explores the intricate mechanisms underlying H2S-induced vasodilation. The vasodilatory effects of H2S are primarily mediated by activating ATP-sensitive potassium (K_ATP) channels, leading to membrane hyperpolarization and subsequent relaxation of vascular smooth muscle cells (VSMCs). Additionally, H2S inhibits L-type calcium channels, reducing calcium influx and diminishing VSMC contraction. Beyond ion channel modulation, H2S profoundly impacts cyclic nucleotide signaling pathways. It stimulates soluble guanylyl cyclase (sGC), increasing the production of cyclic guanosine monophosphate (cGMP). Elevated cGMP levels activate protein kinase G (PKG), which phosphorylates downstream targets like vasodilator-stimulated phosphoprotein (VASP) and promotes smooth muscle relaxation. The synergy between H2S and nitric oxide (NO) signaling further amplifies vasodilation. H2S enhances NO bioavailability by inhibiting its degradation and stimulating endothelial nitric oxide synthase (eNOS) activity, increasing cGMP levels and potent vasodilatory responses. Protein sulfhydration, a post-translational modification, plays a crucial role in cell signaling. H2S S-sulfurates oxidized cysteine residues, while polysulfides (H2Sn) are responsible for S-sulfurating reduced cysteine residues. Sulfhydration of key proteins like K_ATP channels and sGC enhances their activity, contributing to the overall vasodilatory effect. Furthermore, H2S interaction with endothelium-derived hyperpolarizing factor (EDHF) pathways adds another layer to its vasodilatory mechanism. By enhancing EDHF activity, H2S facilitates the hyperpolarization and relaxation of VSMCs through gap junctions between endothelial cells and VSMCs. Recent findings suggest that H2S can also modulate transient receptor potential (TRP) channels, particularly TRPV4 channels, in endothelial cells. Activating these channels by H2S promotes calcium entry, stimulating the production of vasodilatory agents like NO and prostacyclin, thereby regulating vascular tone. The comprehensive understanding of H2S-induced vasodilation mechanisms highlights its therapeutic potential. The multifaceted approach of H2S in modulating vascular tone presents a promising strategy for developing novel treatments for hypertension, ischemic conditions, and other vascular disorders. The interaction of H2S with ion channels, cyclic nucleotide signaling, NO pathways, ROS (Reactive Oxygen Species) scavenging, protein sulfhydration, and EDHF underscores its complexity and therapeutic relevance. In conclusion, the intricate signaling paradigms of H2S-induced vasodilation offer valuable insights into its physiological role and therapeutic potential, promising innovative approaches for managing various vascular diseases through the modulation of vascular tone.

Bifidobacterium pseudolongum‐Derived Bile Acid from Dietary Carvacrol and Thymol Supplementation Attenuates Colitis via cGMP‐PKG‐mTORC1 Pathway

AbstractCarvacrol and thymol (CAT) have been widely recognized for their antimicrobial and anti‐inflammatory properties, yet their specific effects on colitis and the mechanisms involved remain insufficiently understood. This study establishes a causative link between CAT administration and colitis mitigation, primarily through the enhancement of Bifidobacterium pseudolongum abundance in the colon. This increase promotes the production of secondary bile acids, particularly hyodeoxycholic acid (HDCA) and 12‐ketodeoxycholic acid (12‐KCAC), which exert anti‐inflammatory effects. Notably, CAT does not alleviate colitis symptoms in germ‐free mice, indicating the necessity of gut microbiota. This research uncovers a novel regulatory mechanism where HDCA and 12‐KCAC inhibit colonic inflammation by reducing the expression of transmembrane guanylate cyclase 1A in the colonic epithelium. This downregulation elevates intracellular Ca2+ and cGMP levels, activating protein kinase G (PKG). Activated PKG subsequently suppresses the mTOR signaling pathway, thereby ameliorating dextran sulfate sodium (DSS)‐induced colonic damage. These findings highlight potential metabolites and therapeutic targets for preventing and treating colitis. Bifidobacterium pseudolongum, HDCA, and 12‐KCAC emerge as promising candidates for therapeutic interventions in colitis and related disorders characterized by impaired tight junction function.

Endothelial estrogen - myocardial cGMP axis critically determines angiogenesis and cardiac performance during pressure-overload.

Estrogen exerts beneficial cardiovascular effects by binding to specific receptors on various cells to activate nuclear and non-nuclear actions. Estrogen receptor α (ERα) non-nuclear signaling confers protection against heart failure remodeling, involving myocardial cyclic guanosine monophosphate (cGMP) - cGMP-dependent protein kinase G (PKG) activation; however, its tissue-specific role remains elusive. Herein, we examined the cell type-specific role of ERα non-nuclear signaling in estrogen-conferred protection against heart failure. We first assessed the tissue-specific impacts of ERα in estrogen's cardiac benefits, utilizing endothelial ERα deletion (ERαf/f/Tie2Cre+) and myocyte ERα deletion (ERαf/f/αMHCCre+) female mice. Female mice were ovariectomized and the effect of estradiol (E2) was assessed in hearts exposed to 3week pressure-overload (TAC). E2 failed to improve cardiac function in ERαf/f/Tie2Cre+ TAC hearts but provided benefits in ERαf/f/αMHCCre+ TAC hearts, indicating that endothelial ERα is essential. We next assessed the role of non-nuclear signaling in endothelial cells, employing animals with endothelial-specific inactivation of ERα non-nuclear signaling (ERαKI/KI/Tie2Cre+). Female OVX mice were supplemented with E2 and subjected to 3-week TAC. ERαKI/KI/Tie2Cre+ TAC hearts revealed exacerbated cardiac dysfunction and reduced myocardial PKG activity as compared to littermate TAC hearts, which was associated with attenuated myocardial induction of vascular endothelial growth factor (VEGF) and angiogenesis as assessed with CD31-stained capillary density. This phenotype of ERαKI/KI/Tie2Cre+ was rescued by myocardial PKG activation from chronic treatment with soluble guanylate cyclase (sGC) stimulator. We performed co-culture experiments to determine endothelial-cardiomyocyte interactions. VEGF induction by E2 in cardiac myocytes required co-existence of intact endothelial ERα signaling in a NOS-dependent manner. On the other hand, VEGF was induced in myocytes directly with an sGC stimulator in the absence of endothelial cells. An endothelial estrogen - myocardial cGMP axis stimulates angiogenic response and improves cardiac performance during pressure-overload.

Novel Celecoxib Derivative, RF26, Blocks Colon Cancer Cell Growth by Inhibiting PDE5, Activating cGMP/PKG Signaling, and Suppressing β-catenin-dependent Transcription.

Previous studies have reported that the cGMP-specific PDE5 isozyme is overexpressed in colon adenomas and adenocarcinomas and essential for colon cancer cell proliferation, while PDE5 selective inhibitors (e.g., sildenafil) have been reported to have cancer chemopreventive activity. This study aimed to determine the anticancer activity of a novel PDE5 inhibitor, RF26, using colorectal cancer (CRC) cells and the role of PDE5 in CRC tumor growth in vivo. The objective of this study was to characterize the anticancer activity of a novel celecoxib derivative, RF26, in CRC cells previously reported to lack COX-2 inhibition but have potent PDE5 inhibitory activity. Anticancer activity of RF26 was studied using human CRC cell lines. Its effects on intracellular cGMP levels, cGMP-dependent protein kinase (PKG) activity, β-catenin levels, TCF/LEF transcriptional activity, cell cycle distribution, and apoptosis were measured. CRISPR/cas9 PDE5 knockout techniques were used to determine if PDE5 mediates the anticancer activity of RF26 and validate PDE5 as a cancer target. RF26 was appreciably more potent than celecoxib and sildenafil to suppress CRC cell growth and was effective at concentrations that increased intracellular cGMP levels and activated PKG signaling. RF26 suppressed β-catenin levels and TCF/LEF transcriptional activity and induced G1 cell cycle arrest and apoptosis within the same concentration range. CRISPR/cas9 PDE5 knockout CRC cells displayed reduced sensitivity to RF26, proliferated slower than parental cells, and failed to establish tumors in mice. Further evaluation of RF26 for the prevention or treatment of cancer and studying the role of PDE5 in tumorigenesis are warranted.

Salidroside enhances NO bioavailability and modulates arginine metabolism to alleviate pulmonary arterial hypertension

BackgroundSalidroside (SAL), derived from Rhodiola, shows protective effects in pulmonary arterial hypertension (PAH) models, but its mechanisms are not fully elucidated.ObjectivesInvestigate the therapeutic effects and the mechanism of SAL on PAH.MethodsMonocrotaline was used to establish a PAH rat model. SAL’s impact on oxidative stress and inflammatory responses in lung tissues was analyzed using immunohistochemistry, ELISA, and Western blot. Untargeted metabolomics explored SAL’s metabolic regulatory mechanisms.ResultsSAL significantly reduced mean pulmonary artery pressure, right ventricular hypertrophy, collagen deposition, and fibrosis in the PAH rats. It enhanced antioxidant enzyme levels, reduced inflammatory cytokines, and improved NO bioavailability by upregulating endothelial nitric oxide synthase (eNOS), soluble guanylate cyclase (sGC), cyclic guanosine monophosphate (cGMP), and protein kinase G (PKG) and decreases the expression of endothelin-1 (ET-1). Metabolomics indicated SAL restored metabolic balance in PAH rats, particularly in arginine metabolism.ConclusionsSAL alleviates PAH by modulating arginine metabolism, enhancing NO synthesis, and improving pulmonary vascular remodeling.

The Rpfor gene modulates the locomotory activity and host-seeking behaviour of Rhodnius prolixus.

The molecular bases of animal behaviour are intricate due to the pleiotropic nature of behaviour-modulating genes, which are often expressed across multiple tissues. The foraging gene (for) encodes a cGMP-dependent protein kinase (PKG), pivotal in regulating downstream target proteins through phosphorylation. In insects, for has been implicated in various behavioural contexts and physiological processes regarding searching for food. Rhodnius prolixus, a hematophagous bug that transmits Trypanosoma cruzi, the causative agent of Chagas disease, exhibits specific activity patterns associated with its hematophagous behaviour. Our previous work demonstrated a correlation between locomotor activity profiles and the expression of Rpfor, suggesting its involvement in modulating triatomine locomotion. In this study, we investigated the impact of Rpfor knockdown on locomotory activity, host-seeking behaviour, feeding performance and lipid metabolism in R. prolixus nymphs. Using RNA interference, we achieved a significant reduction of Rpfor expression in both the brain and fat body of R. prolixus nymphs. Knocked-down nymphs exhibited diminished non-oriented locomotory activity compared with controls, without altering the characteristic bimodal pattern of activity. Additionally, they displayed an increased tendency to approach a host, suggesting a role for Rpfor in modulating host-seeking behaviour. Feeding performance and lipid metabolism remained unaffected by Rpfor knockdown. Our findings underscore the multifaceted role of Rpfor in modulating locomotor activity and host-seeking behaviour in R. prolixus nymphs, shedding light on the molecular mechanisms underlying their hematophagous behaviour and potential implications for disease transmission. Further research is necessary to elucidate the intricate interplay between Rpfor expression, behaviour and physiological processes in triatomine bugs.

Abstract We109: AAV9-Mediated Overexpression of Mixed Lineage Kinase 3 After Transaortic Constriction Improves Left Ventricular Function

Introduction: Mixed-lineage kinase 3 (MLK3) is a cGMP-dependent protein kinase 1α (PKG1α) substrate, preserving left ventricular (LV) function without affecting blood pressure (BP). MLK3 kinase-dependent effects maintain LV function, while kinase-independent signalling regulates BP. Thus, enhancing MLK3 kinase activity may improve LV function in heart failure (HF) while circumventing PKG1-induced hypotension. No MLK3-activation strategies exist currently, thus hindering progress in MLK3 research and potential therapeutics. Hypothesis: Augmenting MLK3 after pressure overload by transaortic constriction (TAC) would prevent LV hypertrophy and dysfunction in mice. Goal: To test the effect of adeno-associated virus 9 (AAV9)-mediated MLK3 overexpression on LV function, when administered in the setting of pre-existing LV hypertrophy and dysfunction. Methods: Male mice 12 weeks of age underwent 26G TAC or sham surgery. At day 5 post-TAC we administered by retro-orbital injection AAV9 encoding MLK3 or TdTomato negative control, each under control of the CM-specific cTNT promoter (AAV-MLK3: n=12 TAC, 7 sham; AAV-TdTomato: n=10 Tac, 8 sham. At day 28 post-AAV9 injection, we measured cardiac structure and function by echocardiography, invasive LV hemodynamics, and organ weights. Results: Echocardiography 5 days post-TAC confirmed TAC-induced LV hypertrophy and reduced systolic function prior to AAV9 treatment. At day 28, AAV-MLK3-treated TAC mice demonstrated improved LV ejection fraction and fractional shortening compared with TAC-AAV-Td controls, indicating improved systolic function. MLK3-treated TAC mice maintained higher LV systolic pressure post-TAC vs. Td controls, further indicating improved cardiac performance. Diastolic indices, including ratio of mitral E/A inflow velocities, tau, and LV relaxation rate were also improved in AAV-MLK3 TAC versus AAV-Td controls. AAV9-MLK3 administration had no effect on LV wall thickness or on LV or whole heart mass normalized to tibia length. MLK3 expression was increased 3-fold in AAV9-MLK3 TAC LVs compared with AAV9-Td controls. Conclusion: AAV9-mediated MLK3 overexpression after the onset of LV hypertrophy and dysfunction improves LV function in mice.

The regulatory effects of arginine on ovary development in the Chinese mitten crab Eriocheir sinensis

This study aimed to investigate the effects of arginine (Arg) on the vitellogenesis and ovarian maturation of Chinese mitten crab. Six isonitrogenous and isolipidic diets (1.83, 2.52, 3.26, 3.96, 4.81 and 5.52 % Arg) were fed to adult female Chinese mitten crab (60.05 ± 4.33 g). The feeding period lasted for eleven weeks. The results showed that gonadosomatic index of crabs increased with increasing dietary Arg concentrations from 1.83 % to 2.52 % and significantly decreased from 2.52 % to 5.52 %. The quadratic regression analysis based on gonadosomatic index estimated that the optimal Arg requirement for adult female crabs was 2.60 %. Dietary supplementation with 2.52 % Arg significantly increased the crude protein concentration in the ovary. A dose of 2.52 % Arg significantly decreased the vitellogenesis-inhibiting hormone (VIH) content in the hemolymph. Arg (2.52 %) significantly upregulated the Vg and protein kinase A (PKA) gene expressions and increased the cyclic adenosine monophosphate (cAMP) concentration in the hepatopancreas. Moreover, dietary supplementation with 2.52 % Arg significantly decreased the cyclic guanosine monophosphate (cGMP) concentration and downregulated protein kinase G (PKG) and p38 mitogen-activated protein kinase (p38 MAPK) gene expression. Arg at 2.52 % significantly increased hemolymph Vg content and the gene expression of the vitellogenin receptor (VgR). In conclusion, Arg at 2.52 % improved vitellogenesis and ovarian maturation by reducing the VIH content and regulating Vg expression via the cGMP/cAMP pathway.

Endothelial TRPV4 channel mediates the vasodilation induced by Tanshinone IIA

Tanshinone IIA (TSA), the main lipo-soluble component from the dried rhizome of Salvia miltiorrhiza, has been shown to induce vasodilation. However, the underlying mechanisms remains unclear. This study aimed to investigate the effect of TSA on the vasodilation of small resistant arteries ex vivo. Vascular myography revealed that endothelial denudation reduced significantly the vasodilatory effect of TSA. Blocking transient receptor potential vanilloid 4 (TRPV4) channels prevented TSA-induced vasodilation. Whole-cell patch-clamp analysis revealed that the current passing through TRPV4 channels increased after TSA treatment in endothelial cells (ECs). This was attributed to reduced TRPV4 protein degradation along with its increased expression. The TRPV4 inhibitor HC-067047 lowed nitric oxide (NO) production and TSA-induced expression of endothelial nitric oxide synthase (eNOS). Moreover, it increased the production of cyclic guanosine monophosphate (cGMP) and protein kinase G (PKG). The present results indicate that TSA induces endothelium-dependent vasodilation, which is mediated by the TRPV4-NO-PKG signaling pathway. These findings highlight the potential of TSA, a compound known in traditional Chinese medicine as Danshen (Salvia miltiorrhiza), for future cardiovascular therapeutic strategies.

The clinical significance and anti-tumor role of PRKG1 in bladder cancer.

cGMP-dependent protein kinase 1 (PRKG1) has shown to be associated with some tumorigenesis, while the role of PRKG1 in bladder cancer is unclear. To investigate the biological and clinical significance of PRKG1 in bladder cancer, we detected the expression of PRKG1 and explored the function of PRKG1 in bladder cancer cells. The PRKG1 transcripts data was downloaded from The Cancer Genome Atlas (TCGA) database, and immunohistochemistry staining was conducted on formalin-fixed paraffin-embedded (FFPE) sample tissues. Relationship between clinical characteristics of patients and expression of PRKG1 was analyzed in FFPE samples, TCGA database, and GSE19423 dataset. PRKG1 was over-expressed, and cell proliferation, migration, invasion, apoptosis, and spheroidizing ability were then detected. Chemosensitivity to cisplatin was detected with cell viability, and half-maximal drug inhibitory concentration (IC50) was calculated. In addition, the relation between PRKG1 expression and the infiltration level of tumor immune cells in tumor microenvironment were analyzed. The results showed expression of PRKG1 was lower in bladder cancer, compared with normal tissues both at protein and transcript levels. Lower PRKG1 expression was related to higher tumor grade, T stage, and muscle invasion, also predicted worse overall survival and recurrence free survival in patients treated with Bacillus Calmette-Guerin (BCG) intravesical immunotherapy. Analysis of tumor immune cells infiltration showed lower PRKG1 was associated with non-inflamed tumor microenvironment. The present study firstly identified the anti-tumor role and tumor immune regulatory role of PRKG1, also found loss of PRKG1 could be used as a prognosis factor. The present study provided a potential biomarker and therapy target to bladder cancer.

In silico exploration of potential PRKG1 Inhibitors: A comprehensive study using MTIOPEN Screening, molecular Docking, and MD simulation

The enzyme protein kinase G1 (PRKG1) plays a crucial role in cellular signaling pathways such as smooth muscle relaxation, neuronal signaling, and platelet aggregation. The dysregulation of PRKG1 leads to different diseases, making it a promising drug target. In this study, we employed a comprehensive in silico strategy to explore potential inhibitors of PRKG1 by using the crystal structure of the PRKG1 protein. The active site of PRKG1 protein was parameterized within a three-dimensional grid box. The 100 hit compounds identified during virtual screening were docked to the prepared PRKG1 receptor to predict binding affinities. The top ten compounds were chosen, and their binding affinities ranged from −10.734 to −10.398 kcal/mol. Finally, a 200 ns long simulation was run to confirm the stability of the protein–ligand complexes at the binding sites of the two top compounds against the PRKG1 receptor. All these findings suggest that the selected compounds can serve as potential compounds to inhibit the PRKG1 function in biological assays.

NONO2P, a novel nitric oxide donor, causes vasorelaxation through NO/sGC/PKG pathway, K+ channels opening and SERCA activation

Background & aimsThe treatment of cardiovascular diseases (CVD) could greatly benefit from using nitric oxide (NO) donors. This study aimed to investigate the mechanisms of action of NONO2P that contribute to the observed responses in the mesenteric artery. The hypothesis was that NONO2P would have similar pharmacological actions to sodium nitroprusside (SNP) and NO. MethodsMale Wistar rats were euthanized to isolate the superior mesenteric artery for isometric tension recordings. NO levels were measured using the DAF-FM/DA dye, and cyclic guanosine monophosphate (cGMP) levels were determined using a cGMP-ELISA Kit. ResultsNONO2P presented a similar maximum efficacy to SNP. The free radical of NO (NO•) scavengers (PTIO; 100 μM and hydroxocobalamin; 30 μM) and nitroxyl anion (NO−) scavenger (L-cysteine; 3 mM) decreased relaxations promoted by NONO2P. The presence of the specific soluble guanylyl cyclase (sGC) inhibitor (ODQ; 10 μM) nearly abolished the vasorelaxation. The cGMP-dependent protein kinase (PKG) inhibition (KT5823; 1 μM) attenuated the NONO2P relaxant effect. The vasorelaxant response was significantly attenuated by blocking inward rectifying K+ channels (Kir), voltage-operated K+ channels (KV), and large conductance Ca2+-activated K+ channels (BKCa). NONO2P-induced relaxation was attenuated by cyclopiazonic acid (10 μM), indicating that sarcoplasmic reticulum Ca2+-ATPase (SERCA) activation is involved in this relaxation. Moreover, NONO2P increased NO levels in endothelial cells and cGMP production. ConclusionsNONO2P induces vasorelaxation with the same magnitude as SNP, releasing NO• and NO−. Its vasorelaxant effect involves sGC, PKG, K+ channels opening, and SERCA activation, suggesting its potential as a therapeutic option for CVD.

Activation loop phosphorylation and cGMP saturation of PKG regulate egress of malaria parasites.

The cGMP-dependent protein kinase (PKG) is the sole cGMP sensor in malaria parasites, acting as an essential signalling hub to govern key developmental processes throughout the parasite life cycle. Despite the importance of PKG in the clinically relevant asexual blood stages, many aspects of malarial PKG regulation, including the importance of phosphorylation, remain poorly understood. Here we use genetic and biochemical approaches to show that reduced cGMP binding to cyclic nucleotide binding domain B does not affect in vitro kinase activity but prevents parasite egress. Similarly, we show that phosphorylation of a key threonine residue (T695) in the activation loop is dispensable for kinase activity in vitro but is essential for in vivo PKG function, with loss of T695 phosphorylation leading to aberrant phosphorylation events across the parasite proteome and changes to the substrate specificity of PKG. Our findings indicate that Plasmodium PKG is uniquely regulated to transduce signals crucial for malaria parasite development.

Naringenin modulates the NO‑cGMP‑PKG signaling pathway by binding to AKT to enhance osteogenic differentiation in hPDLSCs.

Naringenin (NAR) is a prominent flavanone that has been recognized for its capacity to promote the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs). The present study aimed to explore how NAR promotes the osteogenic differentiation of hPDLSCs and to assess its efficacy in repairing alveolar bone defects. For this purpose, a protein‑protein interaction network of NAR action was established by mRNA sequencing and network pharmacological analysis. Gene and protein expression levels were evaluated by reverse transcription‑quantitative and western blotting. Alizarin red and alkaline phosphatase staining were also employed to observe the osteogenic capacity of hPDLSCs, and immunofluorescence was used to examine the co‑localization of NAR molecular probes and AKT in cells. The repair of mandibular defects was assessed by micro‑computed tomography (micro‑CT), Masson staining and immunofluorescence. Additionally, computer simulation docking software was utilized to determine the binding affinity of NAR to the target protein, AKT. The results demonstrated that activation of the nitric oxide (NO)‑cyclic guanosine monophosphate (cGMP)‑protein kinase G (PKG) signaling pathway could promote the osteogenic differentiation of hPDLSCs. Inhibition of AKT, endothelial nitric oxide synthase and soluble guanylate cyclase individually attenuated the ability of NAR to promote the osteogenic differentiation of hPDLSCs. Micro‑CT and Masson staining revealed that the NAR gavage group exhibited more new bone formation at the defect site. Immunofluorescence assays confirmed the upregulated expression of Runt‑related transcription factor 2 and osteopontin in the NAR gavage group. In conclusion, the results of the present study suggested that NAR promotes the osteogenic differentiation of hPDLSCs by activating the NO‑cGMP‑PKG signaling pathway through its binding to AKT.

(007) TESTOSTERONE POSITIVELY REGULATES ADENOSINE-INDUCED RELAXATION IN THE VAGINA: AN EXPERIMENTAL STUDY IN RATS

Abstract Introduction It has been previously demonstrated that testosterone (T) has a pivotal role in controlling vaginal relaxation through the related molecular machinery, i.e., improving the nitric oxide (NO)/protein kinase G (PKG)/cyclic guanosine monophosphate (cGMP) vaginal signaling and maintaining the integrity of the muscular wall. Adenosine (ADO) is a purinergic neuromodulator that, in the male, shows a positive effect on penile erection by controlling smooth muscle relaxation and local blood flow increase. However, the role, if any, of T on the ADO-induced relaxation signaling is yet to be investigated. Objective The aim of this study was to evaluate the effect of sex steroids on ADO-mediated vaginal relaxation using a validated animal model of ovariectomized (OVX) and sex steroid supplemented Sprague-Dawley rats. Methods Subgroups of OVX rats were treated with 17β-estradiol (E2; 10 mg/kg/die), testosterone (T; 30 mg/kg/week), or testosterone and letrozole (T+L, 2.5 mg/kg/die) for 6 weeks. The experimental groups were compared with a group of intact rats. In vitro contractility studies were performed on noradrenaline (NA) pre-contracted distal vaginal strips, isolated from each experimental group, stimulated with increasing doses of ADO (1 μM - 1 mM), with or without NO-synthase inhibitor L-NAME (100 μM) pretreatment, or treated with ADORA2A selective agonist CGS21680 (10 Pm-100 μM). Messenger ribonucleic acid (mRNA) isolated from vaginal tissue was analyzed by semi-quantitative Reverse Transcriptase-Polymerase Chain Reaction. Results The results showed that OVX resulted in a decreased responsiveness to ADO, completely restored by T supplementation, with or without letrozole. In contrast, E2 did not affect the hyporesponsiveness to ADO in OVX rats. Likewise, ADO induced dose-dependent relaxation was significantly reduced by in vitro L-NAME (100 μM) only in the OVX animals treated with T or T+L. Accordingly, vagina expressed all ADO receptors (ADORA1, ADORA2A, ADORA2B, ADORA3) mRNAs, with pro-relaxant ADORA2A (ADO receptor coupled to adenylyl cyclase activation) being significantly modulated by hormonal treatments: OVX significantly reduced ADORA2A mRNA expression, an effect counteracted by either T or T+L administration, but not by E2. These data are supported by the observed T-induced upregulation of cyclic adenosine monophosphate (cAMP)-dependent genes, where T, but not E2, treatment upregulated the mRNA expression of the ubiquitous isoform of ADCY (ADCY4), protein kinase A (PKA) catalytic subunit b (PKAcb), PKA regulatory subunit 2b (PKAr2b) and cAMP responsive element modulator (CREM). Finally, as further demonstration of the functional activity of ADO signaling in distal vagina, stimulation by increasing doses of CGS21680, a selective agonist of ADORA2A receptor, enhanced the relaxant effect induced by T and T+L treatments but not by E2. Conclusions Our data demonstrated for the first time a novel ADO-mediated relaxant mechanism in vagina and suggested that T positively modulates the responsiveness to ADO stimulation, thus confirming this process as a major player in the relaxant mechanism regulation of muscle contractility in vagina. Disclosure No.

C-type natriuretic peptide/cGMP/FoxO3 signaling attenuates hyperproliferation of pericytes from patients with pulmonary arterial hypertension

Pericyte dysfunction, with excessive migration, hyperproliferation, and differentiation into smooth muscle-like cells contributes to vascular remodeling in Pulmonary Arterial Hypertension (PAH). Augmented expression and action of growth factors trigger these pathological changes. Endogenous factors opposing such alterations are barely known. Here, we examine whether and how the endothelial hormone C-type natriuretic peptide (CNP), signaling through the cyclic guanosine monophosphate (cGMP) -producing guanylyl cyclase B (GC-B) receptor, attenuates the pericyte dysfunction observed in PAH. The results demonstrate that CNP/GC-B/cGMP signaling is preserved in lung pericytes from patients with PAH and prevents their growth factor-induced proliferation, migration, and transdifferentiation. The anti-proliferative effect of CNP is mediated by cGMP-dependent protein kinase I and inhibition of the Phosphoinositide 3-kinase (PI3K)/AKT pathway, ultimately leading to the nuclear stabilization and activation of the Forkhead Box O 3 (FoxO3) transcription factor. Augmentation of the CNP/GC-B/cGMP/FoxO3 signaling pathway might be a target for novel therapeutics in the field of PAH.

Nitric oxide releasing nanofiber stimulates revascularization in response to ischemia via cGMP-dependent protein kinase.

Nitric oxide (NO) promotes angiogenesis via various mechanisms; however, the effective transmission of NO in ischemic diseases is unclear. Herein, we tested whether NO-releasing nanofibers modulate therapeutic angiogenesis in an animal hindlimb ischemia model. Male wild-type C57BL/6 mice with surgically-induced hindlimb ischemia were treated with NO-releasing 3-methylaminopropyltrimethoxysilane (MAP3)-derived or control (i.e., non-NO-releasing) nanofibers, by applying them to the wound for 20 min, three times every two days. The amount of NO from the nanofiber into tissues was assessed by NO fluorometric assay. The activity of cGMP-dependent protein kinase (PKG) was determined by western blot analysis. Perfusion ratios were measured 2, 4, and 14 days after inducing ischemia using laser doppler imaging. On day 4, Immunohistochemistry (IHC) with F4/80 and gelatin zymography were performed. IHC with CD31 was performed on day 14. To determine the angiogenic potential of NO-releasing nanofibers, aorta-ring explants were treated with MAP3 or control fiber for 20 min, and the sprout lengths were examined after 6 days. As per either LDPI (Laser doppler perfusion image) ratio or CD31 capillary density measurement, angiogenesis in the ischemic hindlimb was improved in the MAP3 nanofiber group; further, the total nitrate/nitrite concentration in the adduct muscle increased. The number of macrophage infiltrations and matrix metalloproteinase-9 (MMP-9) activity decreased. Vasodilator-stimulated phosphoprotein (VASP), one of the major substrates for PKG, increased phosphorylation in the MAP3 group. MAP3 nanofiber or NO donor SNAP (s-nitroso-n-acetyl penicillamine)-treated aortic explants showed enhanced sprouting in an ex vivo aortic ring assay, which was partially abrogated by KT5823, a potent inhibitor of PKG. These findings suggest that the novel NO-releasing nanofiber, MAP3 activates PKG and promotes therapeutic angiogenesis in response to hindlimb ischemia.

Isopsoralen Improves Glucocorticoid-induced Osteoporosis by Regulating Purine Metabolism and Promoting cGMP/PKG Pathway-mediated Osteoblast Differentiation.

The effects of Isopsoralen (ISO) in promoting osteoblast differentiation and inhibiting osteoclast formation are well-established, but the mechanism underlying ISO's improvement of Glucocorticoid- Induced Osteoporosis (GIOP) by regulating metabolism remains unclear. This study aims to elucidate the mechanism of ISO treatment for GIOP through non-targeted metabolomics based on ISO's efficacy in GIOP. Initially, we established a GIOP female mouse model and assessed ISO's therapeutic effects using micro-CT detection, biomechanical testing, serum calcium (Ca), and phosphorus (P) level detection, along with histological analyses using hematoxylin and eosin (HE), Masson, and tartrate-resistant acidic phosphatase (TRAP) staining. Subsequently, non-targeted metabolomics was employed to investigate ISO's impact on serum metabolites in GIOP mice. RT-qPCR and Western blot analyses were conducted to measure the levels of enzymes associated with these metabolites. Building on the metabolomic results, we explored the effects of ISO on the cyclic Guanosine Monophosphate (cGMP)/Protein Kinase G (PKG) pathway and its role in mediating osteoblast differentiation. Our findings demonstrate that ISO intervention effectively enhances the bone microarchitecture and strength of GIOP mice. It mitigates pathological damage, such as structural damage in bone trabeculae, reduced collagen fibers, and increased osteoclasts, while improving serum Ca and P levels in GIOP mice. Non-- targeted metabolomics revealed purine metabolism as a common pathway between the Control and GIOP groups, as well as between the ISO high-dose (ISOH) group and the GIOP group. ISO intervention upregulated inosine and adenosine levels, downregulated guanosine monophosphate levels, increased Adenosine Deaminase (ADA) expression, and decreased cGMP-specific 3',5'-cyclic phosphodiesterase (PDE5) expression. Additionally, ISO intervention elevated serum cGMP levels, upregulated PKGI and PKGII expression in bone tissues, as well as the expression of Runt-related transcription factor 2 (Runx2) and Osterix, and increased serum Alkaline Phosphatase (ALP) activity. In summary, ISO was able to enhance the bone microstructure and bone strength of GIOP mice and improve their Ca, P, and ALP levels, which may be related to ISO's regulation of purine metabolism and promotion of osteoblast differentiation mediated by the cGMP/PKG pathway. This suggests that ISO is a potential drug for treating GIOP. However, further research is still needed to explore the specific targets and clinical applications of ISO.

Cardiomyocyte Specific MLK3 Deletion Opposes LV Dysfunction and Cardiac Fetal Gene Re-Expression

Study Objective: To determine the cardiac myocyte (CM) and blood pressure-independent effects of Mixed lineage kinase 3 (MLK3) in the regulation of left ventricular (LV) function at baseline and in response to pathological stress. Background: MLK3 functions as a direct substrate effector of cGMP-dependent protein kinase 1α (PKG1α). Whole-body MLK3 deletion in mice increases cardiac dysfunction and remodeling after pressure overload and prevents the therapeutic effect of cGMP augmentation on LV function. However, MLK3 deletion also causes hypertension, which may confound these findings. Hypothesis: MLK3 opposes LV dysfunction through a blood pressure-independent function in the cardiac myocyte (CM). Methods: We generated mice harboring LoxP sites flanking the MAP3K11 gene encoding MLK3 (MLK3fl/fl) and crossed them with αMHC-Cre transgenic mice, enabling constitutive postnatal cardiac myocyte-specific MLK3 deletion (CMKO). Male and female 3- and 6-month-old MLK3 CMKO and control MLK3 intact (MLK3fl/fl Cre-) littermates were studied in the basal state. We also performed transaortic constriction (TAC) or sham surgery for 4 weeks on 3-month-old male mice. We assessed LV structure and function by organ mass, echocardiography, pressure volume loop analysis, qPCR, and immunoblot. In basal and TAC studies, we used age-matched αMHC-Cre x MLK3+/+ (non-floxed) mice as additional controls. Results: MLK3 gene excision in CMs was confirmed by PCR. By 6 months of age, MLK3 CMKO mice displayed: progressive reduction in LV ejection fraction; increase in LV internal diameter; and LV hypertrophy, indicating LV dysfunction and pathological remodeling, compared with littermate controls or with αMHC-Cre x MLK3+/+ non-floxed mice. This effect was more prominent in females than in males. LV fetal gene expression did not differ between genotypes at 3 months. At 6 months of age, however, the fetal genes nppa, RCAN, and CTGF were significantly upregulated in both male and female MLK3 CMKO mice, compared with MLK3 intact, indicating pathological remodeling. After 4-week TAC, MLK3 CMKO LVs developed more severe reduction in LV systolic function compared with MLK3 intact littermates as assayed by echocardiographic measures of LV ejection fraction; and invasive hemodynamic indices of preload recruitable stroke work, maximum LV pressure and LV developed pressure. The MLK3 CMKO LVs also demonstrated a more eccentric remodeling phenotype in which LV mass/tibia length, LV end diastolic and end systolic diameters increased more severely in CMKO mice, with corresponding reduced LV wall thicknesses. Finally, lung mass/tibia length was elevated in the MLK3 CMKO TAC mice compared to MLK3 intact TAC littermates, indicating overt heart failure. Conclusion: (1) MLK3 functions as a tonic inhibitor of LV dysfunction and pathological remodeling through a role in the CM, possibly through sex-specific mechanisms. (2) Intact MLK3 is required for normal LV compensation and concentric remodeling in response to pressure overload. These findings have important clinical implications, as drugs which activate PKG1 in heart failure improve outcomes but are limited by excess hypotension. As a myocardial PKG1 substrate which opposes pathological LV remodeling through blood pressure-independent mechanisms, MLK3 may represent a candidate therapeutic target for heart failure. NIH 1R01HL162919. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.