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Vascular smooth muscle cells (VSMCs) can switch from their contractile state to a synthetic phenotype resulting in high migratory and proliferative capacity and driving atherosclerotic lesion formation. The cysteine-rich LIM-only protein 4 (CRP4) reportedly modulates VSM-like transcriptional signatures, which are perturbed in VSMCs undergoing phenotypic switching. Thus, we hypothesized that CRP4 contributes to adverse VSMC behaviours and thereby to atherogenesis in vivo. The atherogenic properties of CRP4 were investigated in plaque-prone apolipoprotein E (ApoE) and CRP4 double-knockout (dKO) as well as ApoE-deficient CRP4 wildtype mice. dKO mice exhibited lower plaque numbers and lesion areas as well as a reduced content of α-smooth muscle actin positive cells in the lesion area, while lesion-associated cell proliferation was elevated in vessels lacking CRP4. Reduced plaque volumes in dKO correlated with significantly less intra-plaque oxidized low-density lipoprotein (oxLDL), presumably due to upregulation of the antioxidant factor peroxiredoxin-4 (PRDX4). This study identifies CRP4 as a novel pro-atherogenic factor that facilitates plaque oxLDL deposition and identifies the invasion of atherosclerotic lesions by VSMCs as important determinants of plaque vulnerability. Thus, targeting of VSMC CRP4 should be considered in plaque-stabilizing pharmacological strategies.

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.

ABSTRACT Silent information regulator 2-related enzyme 1 (SIRT1) is an aging-related protein activated with aging. Herein, we evaluated the role of SIRT1 in aging-related erectile dysfunction. The expression of SIRT1 was modulated in aged Sprague-Dawley rats following intragastric administration of resveratrol (Res; 5 mg kg−1), niacinamide (NAM; 500 mg kg−1) or Res (5 mg kg−1) + tadalafil (Tad; phosphodiesterase-5 [PDE5] inhibitor; 5 mg kg−1) for 8 weeks. Then, we determined erectile function by the ratio of intracavernosal pressure (ICP)/mean systemic arterial pressure (MAP). Cavernosal tissues were extracted to evaluate histological changes, cell apoptosis, nitric oxide (NO)/cyclic guanosine monophosphate (cGMP), the superoxide dismutase (SOD)/3,4-methylenedioxyamphetamine (MDA) level, and the expression of SIRT1, p53, and forkhead box O3 (FOXO3a) using immunohistochemistry, terminal deoxynucleotidyl transferase (TdT)-mediated 2'-deoxyuridine 5'-triphosphate (dUTP) nick-end labeling (TUNEL), enzyme-linked immunosorbent assays, and western blot analysis. Compared with the control, Res treatment significantly improved erectile function, reflected by an increased content of smooth muscle and endothelium, NO/cGMP and SOD activity, and reduced cell apoptosis and MDA levels. The effect of Res was improved by adding Tad. In addition, the protein expression of SIRT1 was increased in the Res group, accompanied by decreased p53 and FOXO3a levels. In addition, inhibition of SIRT1 by NAM treatment resulted in adverse results compared with Res treatment. SIRT1 activation ameliorated aging-related erectile dysfunction, supporting the potential of SIRT1 as a target for erectile dysfunction treatment.

It is widely accepted that disorders of the male (uro)genital tract, such as erectile dysfunction (ED) and benign diseases of the prostate (lower urinary tract symptomatology or benign prostatic hyperplasia), can be approached therapeutically by influencing the function of both the vascular and non-vascular smooth muscle of the penile erectile tissue or the transition zone/periurethral region of the prostate, respectively. As a result of the discovery of nitric oxide (NO) and cyclic guanosine monophosphate (GMP) as central mediators of penile smooth muscle relaxation, the use of drugs known to increase the local production of NO and/or elevate the intracellular level of the second messenger cyclic GMP have attracted broad attention in the treatment of ED of various etiologies. Specifically, the introduction of vasoactive drugs, including orally active inhibitors of the cyclic GMP-specific phosphodiesterase (PDE) 5, has offered great advantage in the pharmacotherapy of ED and other diseases of the genitourinary tract. These drugs have been proven efficacious with a fast on-set of action and an improved profile of side-effects. This review summarizes current strategies for the treatment of ED utilizing the application of vasoactive drugs via the oral, transurethral, topical, or self-injection route.

It has been well established that male erectile dysfunction is frequently associated with vascular diseases. The normal function of cavernous arteries is considered a prerequisite to maintain sufficient blood flow to the trabecular spaces in order to enable penile erection. Contractility of cavernous arteries is regulated by the peripheral autonomic nervous system and endogenous factors released from the endothelial cell layer. A significant increase of blood flow in the central cavernous arteries is the initial event leading to penile tumescence and rigidity. Besides the significance of the nitric oxide/cyclic guanosine monophosphate (cGMP)-mediated mechanisms, the cyclic AMP (cAMP) signalling pathway is also involved in the regulation of tone of the erectile tissue, and interactions between cGMP- and cAMP-mediated mechanisms have been demonstrated. The aim of the present study was to investigate by means of immunohistochemistry the presence of the phosphodiesterase (PDE) isoenzymes 3, 4 (cAMP-specific PDEs) and 5 (cGMP-specific PDE) in thin sections of human central cavernous arteries (HCA) and their functional significance in the mechanism of vessel tone regulation. Functional experiments were performed using circular segments of HCA and strip preparations of the human corpus cavernosum (HCC). Relaxant effects induced by the cumulative addition of the PDE inhibitors milrinone (PDE3 inhibitor), rolipram (PDE4 inhibitor) and sildenafil (PDE5 inhibitor; 0.01, 0.1, 1 and 10 M) were studied in preparations of HCA and HCC challenged by 1 M norepinephrine (NE). Moreover, immunohistochemistry was carried out in order to evaluate the expression of PDE3, PDE4 and PDE5 in thin sections of HCA. Milrinone, rolipram and sildenafil dose-dependently reversed the NE-induced tension of the isolated vascular segments and HCC strips with sildenafil being the most effective drug. Neither rolipram nor milrinone reached an EC50 value. Abundant immunoreactivities specific for PDE3, PDE4 and PDE5 were observed in the entire smooth musculature of the wall of HCA and resistance arteries. In addition, immunoreactivity for PDE4 was also detected in the cytoplasm of endothelial cells lining the cavernous arteries. The cGMP-dependent relaxation of cavernous arteries is not only dependent on the normal function of the peripheral autonomic nervous system but also on the functional integrity of the vascular endothelium. The expression of the cGMP-specific PDE5 and the ability of the PDE5 inhibitor sildenafil to reverse the adrenergic tension of isolated segments of HCA underline the important role of the NO/cGMP pathway in the control of smooth muscle tone of human trabecular smooth musculature and penile cavernous arteries. Our results also suggest a significance of the cAMP-dependent signaling mechanisms in the regulation of tension of central HCAs. The present findings are also in support of the hypothesis of interactions between the cGMP- and cAMP-mediated signaling pathways in HCAs. Further investigations are indicated in order to outline potential differences between central HCAs and helicine resistance arteries. This may help to understand better the relations between structural and functional changes in the penile erectile tissue in patients with cardiovascular diseases and endothelial dysfunction.

Oxidative stress is closely associated with the onset of diabetes mellitus (DM). Diabetic urethropathy is one of the most common complications of DM, but few studies have been conducted to investigate the role of oxidative stress in diabetic urethropathy. Grape seed proanthocyanidin extract (GSPE) has been previously reported to reduce oxidative injury. The present study aimed to investigate the role of oxidative stress and the protective effects of GSPE on urethral dysfunction using a streptozotocin-induced DM rat model. Female Wistar rats were divided into a control group (n=36), a DM group (n=36) and a DM + GSPE group (n=36). Urodynamic testing was performed using a PowerLab data acquisition device. The expression of neuronal nitric oxide synthase (nNOS), 3-nitrotyrosine and nuclear factor erythroid 2-related factor 2 (Nrf2) was determined using western blot analysis. The expression of 3-nitrotyrosine was also determined using immunohistochemistry. Nitric oxide (NO), cyclic guanosine monophosphate (cGMP), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and malondialdehyde (MDA) were measured using commercial ELISA kits. A significant increase was observed in the intravesical pressure thresholds for inducing urethral relaxation and the urethral perfusion pressure nadir in DM rats compared with the control group. GSPE was observed to reverse the increase of these parameters compared with the DM group. In addition, GSPE could reverse the downregulation of nNOS, NO and cGMP expression, and the decreased activities of antioxidant enzymes (SOD and GSH-Px). GSPE reversed the upregulation of 3-nitrotyrosine and MDA in DM rats. GSPE also activated Nrf2, which is a key antioxidative transcription factor. The findings of the present study demonstrated that GSPE protects urethra function in DM rats through modulating the NO-cGMP signaling pathway. The protective roles of GSPE may be associated with activation of the Nrf2 defense pathway.

This study with recombinant reconstituted system mimicking the cellular conditions of the native cones documents that photoreceptor ROS-GC1 is modulated by gaseous CO.sub.2. Mechanistically, CO.sub.2 is sensed by carbonic anhydrase (CAII), generates bicarbonate that, in turn, directly targets the core catalytic domain of ROS-GC1, and activates it to increased synthesis of cyclic GMP. This, then, functions as a second messenger for the cone phototransduction. The study demonstrates that, in contrast to the Ca.sup.2+-modulated phototransduction, the CO.sub.2 pathway is Ca.sup.2+-independent, yet is linked with it and synergizes it. It, through R.sup.787C mutation in the third heptad of the signal helix domain of ROS-GC1, affects cone-rod dystrophy, CORD6. CORD6 is caused firstly by lowered basal and GCAP1-dependent ROS-GC1 activity and secondly, by a shift in Ca.sup.2+ sensitivity of the ROS-GC1/GCAP1 complex that remains active in darkness. Remarkably, the first but not the second defect disappears with bicarbonate thus explaining the basis for CORD6 pathological severity. Because cones, but not rods, express CAII, the excessive synthesis of cyclic GMP would be most acute in cones.

Background Dialysis membranes that release nitric oxide (NO) from their surface, mimicking one of the functions of endothelial cells, may suppress platelet reactivity during hemodialysis treatment. The aim of the present study was to examine whether the addition of a NO donor to the dialysis fluid can suppress platelet reactivity during dialysis. Methods Porcine whole blood was circulated for 4 h through a polysulfone (PS) dialyzer or polymethylmethacrylate (PMMA) dialyzer. After the blood was circulated through the blood circuit and dialyzer, sodium nitroprusside was added to the dialysis fluid as a NO donor. The changes in the platelet reactivity, measured by the platelet aggregation activity by the addition of adenosine diphosphate or collagen in the blood sample, were evaluated during ex vivo dialysis experiments in the presence of a dialysis fluid containing or not containing a NO donor. Results The platelet aggregation activity was significantly decreased at 30 min after the start of the experiment in the case where nitroprusside was added to the dialysis fluid (the NO (+) condition) as compared to the case where no nitroprusside was added to the dialysis fluid (the NO (−) condition), for both the PS and PMMA membranes. The suppression of the platelet reactivity in the NO (+) condition was sustained until the end of the experimental period (240 min). The platelet cyclic guanosine monophosphate level was also significantly increased in the NO (+) condition as compared to the NO (−) condition. Conclusions NO in the dialysis fluid appears to be capable of suppressing the increase of the platelet reactivity observed during dialysis.

In this Review, Chen and colleagues discuss recent advances in understanding of the cGAS–STING pathway, focusing on the regulatory mechanisms and roles of this pathway in heath and disease. The recognition of microbial nucleic acids is a major mechanism by which the immune system detects pathogens. Cyclic GMP-AMP (cGAMP) synthase (cGAS) is a cytosolic DNA sensor that activates innate immune responses through production of the second messenger cGAMP, which activates the adaptor STING. The cGAS–STING pathway not only mediates protective immune defense against infection by a large variety of DNA-containing pathogens but also detects tumor-derived DNA and generates intrinsic antitumor immunity. However, aberrant activation of the cGAS pathway by self DNA can also lead to autoimmune and inflammatory disease. Thus, the cGAS pathway must be properly regulated. Here we review the recent advances in understanding of the cGAS–STING pathway, focusing on the regulatory mechanisms and roles of this pathway in heath and disease.

The invasion of mammalian cytoplasm by microbial DNA from infectious pathogens or by self DNA from the nucleus or mitochondria represents a danger signal that alerts the host immune system 1 . Cyclic GMP–AMP synthase (cGAS) is a sensor of cytoplasmic DNA that activates the type-I interferon pathway 2 . On binding to DNA, cGAS is activated to catalyse the synthesis of cyclic GMP–AMP (cGAMP) from GTP and ATP 3 . cGAMP functions as a second messenger that binds to and activates stimulator of interferon genes (STING) 3 – 9 . STING then recruits and activates tank-binding kinase 1 (TBK1), which phosphorylates STING and the transcription factor IRF3 to induce type-I interferons and other cytokines 10 , 11 . However, how cGAMP-bound STING activates TBK1 and IRF3 is not understood. Here we present the cryo-electron microscopy structure of human TBK1 in complex with cGAMP-bound, full-length chicken STING. The structure reveals that the C-terminal tail of STING adopts a β-strand-like conformation and inserts into a groove between the kinase domain of one TBK1 subunit and the scaffold and dimerization domain of the second subunit in the TBK1 dimer. In this binding mode, the phosphorylation site Ser366 in the STING tail cannot reach the kinase-domain active site of bound TBK1, which suggests that STING phosphorylation by TBK1 requires the oligomerization of both proteins. Mutational analyses validate the interaction mode between TBK1 and STING and support a model in which high-order oligomerization of STING and TBK1, induced by cGAMP, leads to STING phosphorylation by TBK1. The cryo-electron microscopy structure of human TBK1 in complex with cyclic GMP–AMP-bound chicken STING reveals a binding mode that suggests that STING phosphorylation by TBK1 requires oligomerization of both proteins.