Nitric Oxide Synthase Type Research Articles

Novel Flavanone Naringenin Derivative with Anti-Inflammatory Activity

Naringenin is a flavonoid with several biological activities already reported but with few biological applications in the pharmaceutical field. In this work, a new flavanone, called carboxymidamide (DCHA), synthesized through the condensation reaction between naringenin and aminoguanidine is structurally confirmed through spectroscopic analysis of nuclear magnetic resonance, mass spectrometry and Fourier transform infrared. DCHA was not toxic to human fibroblasts and inhibited macrophage activation in vitro. In vivo, it suppressed leukocyte migration in lipopolysaccharide (LPS)-induced peritonitis and reduced zymosan-induced paw edema. Molecular docking simulations of DCHA in the active site of the enzymes inducible nitric oxide synthase, cyclooxygenase-2 and phosphodiesterase type 4 indicate that the anti-inflammatory activity of this new flavanone can be explained by the inhibition of these target enzymes. This is the first work to present the synthesis of a flavonoid derivative with aminoguanidine that showed a high anti-inflammatory potential.

Poly(ethylene Glycol) Methyl Ether Methacrylate-Based Injectable Hydrogels: Swelling, Rheological, and In Vitro Biocompatibility Properties with ATDC5 Chondrogenic Lineage.

Here, we present the synthesis of a series of chemical homopolymeric and copolymeric injectable hydrogels based on polyethylene glycol methyl ether methacrylate (PEGMEM) alone or with 2-dimethylamino ethyl methacrylate (DMAEM). The objective of this study was to investigate how the modification of hydrogel components influences the swelling, rheological attributes, and in vitro biocompatibility of the hydrogels. The hydrogels' networks were formed via free radical polymerization, as assured by 1H nuclear magnetic resonance spectroscopy (1H NMR). The swelling of the hydrogels directly correlated with the monomer and the catalyst amounts, in addition to the molecular weight of the monomer. Rheological analysis revealed that most of the synthesized hydrogels had viscoelastic and shear-thinning properties. The storage modulus and the viscosity increased by increasing the monomer and the crosslinker fraction but decreased by increasing the catalyst. MTT analysis showed no potential toxicity of the homopolymeric hydrogels, whereas the copolymeric hydrogels were toxic only at high DMEAM concentrations. The crosslinker polyethylene glycol dimethacrylate (PEGDMA) induced inflammation in ATDC5 cells, as detected by the significant increase in nitric oxide synthase type II activity. The results suggest a range of highly tunable homopolymeric and copolymeric hydrogels as candidates for cartilage regeneration.

Nitric oxide: an old drug but with new horizons in ophthalmology-a narrative review.

Based on basic knowledge and prior research on nitric oxide (NO), the potential of NO for treating eye diseases is reviewed, and the possibility of NO-based eye drops in clinical practice and the future potential of NO in ophthalmology are discussed. A PubMed search was performed for English-language original reports and reviews using the following key words: nitric oxide, eye, ocular, and drug. NO is synthesized in the human body by NO synthase (NOS) from L-arginine or through enzyme-dependent reduction of dietary nitrate. Three types of NOS (eNOS, nNOS, and iNOS) are abundantly expressed in the eye under normal physiologic or pathologic conditions. The biological effect of NO in the eye is dose dependent. Low intraocular NO concentrations, produced by eNOS or nNOS, have various cellular effects, including vasodilation, intraocular pressure (IOP) regulation, and neuroprotection. iNOS induced under pathologic ocular conditions produces high NO concentrations in the local environment and mediates tissue inflammation, ocular cell apoptosis, and neurodegeneration. In particular, increased iNOS has been reported in glaucoma and retinal ischemic or degenerative diseases. NO plays a vital role in ocular injury. NO can facilitate ocular surface wound healing while eradicating pathogens such as bacteria and Acanthamoeba in chemical burns or infectious keratitis. Furthermore, NO has antifibrotic activity via the cyclic guanosine monophosphate (cGMP) signaling pathway. NO causes smooth muscle relaxation, which can be used to inhibit myopia progression in children. NO can be a stem cell modulator and may help in treating ocular stem cell disorders. Because of its diverse biologic effects, NO can be a key player in regulating ocular inflammation in various ocular diseases, aiding ocular surface wound healing, controlling IOP in glaucoma, alleviating retinal disease, and suppressing myopia progression. Although there remain limitations to the effective use of highly unstable state, gaseous NO, the role of NO in the field of ophthalmology can be greatly expanded through the development of novel NO donors and effective delivery platforms.

Editorial introductions.

Editorial introductions.

Combined levothyroxine and testosterone treatment for restoring erectile dysfunction in propylthiouracil-induced hypothyroid rats.

Sexual dysfunction may indicate severe endocrine diseases. Recent research has suggested a link between hypothyroidism, low testosterone (T) levels, and erectile dysfunction (ED); however, the exact cause is unknown. We sought to investigate possible beneficial effects of levothyroxine and T alone or in combination on ED in propylthiouracil (PTU)-induced hypothyroid rats. Adult Wistar rats (n = 35) were divided into 5 groups: control, PTU-induced hypothyroidism, PTU + levothyroxine, PTU + Sustanon (a mixture of 4 types of T: propionate, phenylpropionate, isocaproate, and decanoate) and PTU + levothyroxine + Sustanon. PTU was given in drinking water for 6 weeks. Four weeks after PTU administration, levothyroxine (20μg microgram kg/day, oral) and Sustanon (10mg/kg/week, intramuscular) were given for 2weeks. Serum levels of total T, triiodothyronine (T3), and thyroxine (T4) were determined. In vivo erectile response and in vitro relaxant responses were measured. Localization of neuronal nitric oxide synthase (nNOS), endothelial NOS (eNOS), and phosphodiesterase type 5 (PDE5) were determined using immunohistochemical analysis. The relative area of smooth muscle to collagen was measured using Masson trichrome staining. Outcome variables included in vivo erectile function, in vitro relaxant and contractile responses of corpus cavernosum (CC) strips; protein localization of eNOS, nNOS, and PDE5; and smooth muscle content in penile tissue. The rat model of hypothyroidism showed a significant decline in serum levels of total T, T3, and T4. Levothyroxine increased T3 and T4 levels, whereas Sustanon normalized only total T levels. Combined treatment enhanced all hormone levels. Rats with hypothyroidism displayed the lowest erectile response (P < 0.001 vs controls). Combined treatment returned reduced responses, while partial amelioration was observed after levothyroxine and Sustanon treatment alone. Acetylcholine (P < 0.01 vs controls), electrical field stimulation (P < 0.001 vs controls), and sildenafil-induced relaxant responses (P < 0.05 vs controls) were decreased in the CC strips from hypothyroid rats. The combined treatment increased the reduction in relaxation responses. Levothyroxine and Sustanon restored decreases in eNOS and nNOS expression in the hypothyroid group. There was no significant difference in PDE5 expression among groups. Monotreatment partially enhanced reduced smooth muscle mass, while combined therapy completely recovered. The combination of thyroid hormones and T is likely to be a therapeutic approach for treatment of hypothyroidism-induced ED in men. Beneficial effects of levothyroxine and Sustanon treatment were shown in vitro and in vivo in PTU-induced hypothyroid rats. The main limitation of the study was the lack of measurement of androgen-sensitive organ weights and luteinizing hormone, follicle-stimulating hormone, and prolactin levels. These findings demonstrate that neurogenic and endothelium-dependent relaxation responses are reduced by hypothyroidism, which is detrimental to T levels and erectile responses. Levothyroxine and Sustanon combination medication was able to counteract this effect.

Priapism caused by partial deficiency of tetrahydrobiopterin through hypofunction of the sympathetic neurons in sepiapterin reductase gene‐disrupted mice

6R-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) is an essential cofactor for aromatic L-amino acid hydroxylases, including tyrosine hydroxylase (TH), alkylglycerol monooxygenase, and three types of nitric oxide (NO) synthases (NOS). Sepiapterin reductase (SPR) catalyzes the third step of BH4 biosynthesis. SPR gene-disrupted (Spr-/- ) mice exhibit a dystonic posture, low body weight, hyperphenylalaninemia, and unstable hypertension with endothelial dysfunction. In this study, we found that Spr-/- mice suffered from a high incidence of severe priapism. Their erections persisted for months. The biopterin, BH4, and norepinephrine contents, and TH protein levels in the penile tissue of Spr-/- mice without and with priapism were significantly reduced compared to those of Spr+/+ mice. In contrast, their neural NOS (nNOS) protein levels were increased, and the cyclic guanosine monophosphate (cGMP) levels were remarkably elevated in the penises of Spr-/- mice with priapism. The symptoms were relieved by repeated administration of BH4. The biopterin, BH4, and norepinephrine contents were increased in penile homogenates from BH4-supplemented Spr-/- mice, and the TH protein levels tended to increase, and their nitrite plus nitrate levels were significantly lower than those of vehicle-treated Spr-/- mice and were approximately the same as vehicle- and BH4-supplemented Spr+/+ mice. Thus, we deduced that the priapism of Spr-/- mice is primarily caused by hypofunction of the sympathetic neurons due to cofactor depletion and the loss of TH protein and, further, dysregulation of the NO/cGMP signaling pathway, which would be caused by disinhibition of nNOS-containing neurons and/or abnormal catabolism of cyclic nucleotides is suggested.

12 Sex-related differential susceptibility to ponatinib-induced cardiotoxicity and its relationship to modulation of notch signalling in a muine model

Abstract Aims Ponatinib (PON), a tyrosine kinase inhibitor approved in chronic myeloid leukaemia, has proven cardiovascular toxicity. Although sex is a risk factor for PON-induced cardiotoxicity in humans, little is known about its mechanisms, in general, and sex-related mechanisms in particular. To determine the mechanisms of sex-related PON-induced cardiotoxicity and identify potential rescue strategies in a murine model. Methods Twenty-four-month-old male and female C57B5 mice were treated with 3 mg/kg/day of PON or vehicle via oral gavage for 28 days, with/without siRNA-Notch1 or siRNA-scrambled via tail vein every 3 days. Results PON + scrambled siRNA-treated male mice had a higher number of TUNEL-positive cells, a higher percentage of senescence-associated β-galactosidase positive senescent cardiac areas, as well as a lower reactivity degree for the survival marker Bmi1 than female counterparts. Proteomics analysis of cardiac tissue showed upstream activation of nitric oxide synthase (NOS) type 2, downstream activation of cell death and production of reactive oxygen species in PON + scrambled siRNA- compared to vehicle or PON + Notch1 siRNA-treated male mice. Upstream analysis showed beta-estradiol activation, while downstream analysis showed activation of cell survival and inhibition of cell death in PON + scrambled siRNA compared to vehicle-treated female mice. PON + scrambled siRNA-treated mice also showed a down-regulation of cardiac actin, which was more marked in male; as well as vessel density, which was more marked in female mice. Female hearts showed a greater extent of cardiac fibrosis than male counterparts at baseline, with no significant changes after PON treatment. In contrast, PON + scrambled siRNA-treated mice had less fibrosis than vehicle or PON + Notch1-siRNA-treated mice. Left ventricular systolic dysfunction shown in PON + scrambled siRNA-treated male mice and—to a lesser extent—by female mice was similarly reversed in both PON + Notch1-siRNA-treated male and female mice (Table 1). Conclusions We found a sex-related differential susceptibility and Notch1 modulation in PON-induced cardiotoxicity. This can improve our understanding of sex-related differences and help identify biomarkers in PON cardiotoxicity.

Monomeric C reactive protein (mCRP) regulates inflammatory responses in human and mouse chondrocytes

AbstractC-reactive protein (CRP) is an acute-phase protein that is used as an established biomarker to follow disease severity and progression in a plethora of inflammatory diseases. However, its pathophysiologic mechanisms of action are still poorly defined and remain elusive. CRP, in its pentameric form, exhibits weak anti-inflammatory activity. On the contrary, the monomeric isoform (mCRP) exhibits potent pro-inflammatory properties in endothelial cells, leukocytes, and platelets. So far, no data exists regarding mCRP effects in human or mouse chondrocytes. This work aimed to verify the pathophysiological relevance of mCRP in the etiology and/or progression of osteoarthritis (OA). We investigated the effects of mCRP in cultured human primary chondrocytes and in the chondrogenic ATDC5 mouse cell line. We determined mRNA and protein levels of relevant factors involved in inflammatory responses and the modulation of nitric oxide synthase type II (NOS2), an early inflammatory molecular target. We demonstrate, for the first time, that monomeric C reactive protein increases NOS2, COX2, MMP13, VCAM1, IL-6, IL-8, and LCN2 expression in human and murine chondrocytes. We also demonstrated that NF-kB is a key factor in the intracellular signaling of mCRP-driven induction of pro-inflammatory and catabolic mediators in chondrocytes. We concluded that mCRP exerts a sustained catabolic effect on human and murine chondrocytes, increasing the expression of inflammatory mediators and proteolytic enzymes, which can promote extracellular matrix (ECM) breakdown in healthy and OA cartilage. In addition, our results implicate the NF-kB signaling pathway in catabolic effects mediated by mCRP.

PSD-95: An Effective Target for Stroke Therapy Using Neuroprotective Peptides

Therapies for stroke have remained elusive in the past despite the great relevance of this pathology. However, recent results have provided strong evidence that postsynaptic density protein-95 (PSD-95) can be exploited as an efficient target for stroke neuroprotection by strategies able to counteract excitotoxicity, a major mechanism of neuronal death after ischemic stroke. This scaffold protein is key to the maintenance of a complex framework of protein interactions established at the postsynaptic density (PSD) of excitatory neurons, relevant to neuronal function and survival. Using cell penetrating peptides (CPPs) as therapeutic tools, two different approaches have been devised and advanced to different levels of clinical development. First, nerinetide (Phase 3) and AVLX-144 (Phase 1) were designed to interfere with the coupling of the ternary complex formed by PSD-95 with GluN2B subunits of the N-methyl-D-aspartate type of glutamate receptors (NMDARs) and neuronal nitric oxide synthase (nNOS). These peptides reduced neurotoxicity derived from NMDAR overactivation, decreased infarct volume and improved neurobehavioral results in different models of ischemic stroke. However, an important caveat to this approach was PSD-95 processing by calpain, a pathological mechanism specifically induced by excitotoxicity that results in a profound alteration of survival signaling. Thus, a third peptide (TP95414) has been recently developed to interfere with PSD-95 cleavage and reduce neuronal death, which also improves neurological outcome in a preclinical mouse model of permanent ischemia. Here, we review recent advancements in the development and characterization of PSD-95-targeted CPPs and propose the combination of these two approaches to improve treatment of stroke and other excitotoxicity-associated disorders.

Nitric Oxide Synthase Type 1 Methylation Is Associated With White Matter Microstructure in the Corpus Callosum and Greater Panic Disorder Severity Among Panic Disorder Patients.

Objectives: Methylation of the neuronal nitric oxide synthase (NOS1/nNOS) gene has recently been identified as a promising biomarker of psychiatric disorders. NOS1 plays an essential role in neurite outgrowth and may thus affect the microstructure development of white matter (WM) in the corpus callosum (CC), which is known to be altered in panic disorder (PD). We examined the relationship between NOS1 methylation, WM tracts in the CC, and symptoms based on this finding.Methods: Thirty-two patients with PD and 22 healthy controls (HCs) were recruited after age, gender, and the education level were matched. The cell type used was whole-blood DNA, and DNA methylation of NOS1 was measured at 20 CpG sites in the promoter region. Although 25 patients with PD were assessed with the Panic Disorder Severity Scale (PDSS), diffusion tensor imaging (DTI) scans were only collected from 16 participants with PD.Results: We observed that the PD group showed lower methylation than did the HCs group and positive correlations between the symptom severity of PD and methylation at CpG4 and CpG9. In addition, CpG9 methylation was significantly correlated with the fractional anisotropy (FA) and mean diffusivity (MD) values of the CC and its major components (the genu and the splenium) in the PD group. Furthermore, path analyses showed that CpG9 methylation offers a mediating effect for the association between the MD values of the genu of the CC and PD symptom severity (95% CI = −1.731 to −0.034).Conclusions: The results suggest that CpG9 methylation leads to atypical development of the genu of the CC, resulting in higher PD symptom severity, adding support for the methylation of NOS1 as a future prognostic indicator of PD.

Sex-related differential susceptibility to ponatinib-induced cardiotoxicity and its relationship to modulation of Notch signaling in a murine model

Abstract Background Ponatinib (PON), a tyrosine kinase inhibitor approved in chronic myeloid leukemia, has proven cardiovascular toxicity. Although sex is a risk factor for PON-induced cardiotoxicity in humans, little is known about its mechanisms in general, and sex-related mechanisms in particular. Objectives To determine the mechanisms of sex-related PON-induced cardiotoxicity and identify potential rescue strategies in a murine model. Methods 24-months-old male and female C57B5 mice were treated with 3 mg/kg/day of PON or vehicle via oral gavage for 28 days, with/without siRNA-Notch1 or siRNA-scrambled via tail vein every 3 days. Results PON + scrambled siRNA-treated male mice had a higher number of TUNEL-positive cells, a higher percentage of senescence-associated β-galactosidase positive senescent cardiac areas, as well as a lower reactivity degree for the survival marker Bmi1 than female counterparts. Proteomics analysis of cardiac tissue showed upstream activation of nitric oxide synthase (NOS) type 2, downstream activation of cell death and production of reactive oxygen species in PON + scrambled siRNA- compared to vehicle or PON + Notch1 siRNA-treated male mice. Upstream analysis showed beta-estradiol activation, while downstream analysis showed activation of cell survival and inhibition of cell death in PON + scrambled siRNA compared to vehicle-treated female mice. PON + scrambled siRNA-treated mice also showed a downregulation of cardiac actin, which was more marked in male; as well as vessel density, which was more marked in female mice. Female hearts showed a greater extent of cardiac fibrosis than male counterparts at baseline, with no significant changes after PON treatment. In contrast, PON + scrambled siRNA-treated mice had less fibrosis than vehicle or PON + Notch1-siRNA-treated mice. Left ventricular systolic dysfunction shown in PON + scrambled siRNA-treated male mice and - to a lesser extent - by female mice was similarly reversed in both PON + Notch1-siRNA-treated male and female mice (Table 1). Conclusions We found a sex-related differential susceptibility and Notch1 modulation in PON-induced cardiotoxicity. This can improve our understanding of sex-related differences and help identify biomarkers in PON cardiotoxicity. Funding Acknowledgement Type of funding sources: None.

Effect of testosterone on endothelial function in men with type 2 diabetes mellitus

Objective: to study the effect of testosterone (T) levels on laboratory and instrumental markers of endothelial dysfunction (ED). Materials and methods: the study included 276 male patients with type 2 diabetes mellitus (DM). General clinical studies were carried out, analysis of parameters of carbohydrate metabolism, the content of hormones (total T, SHBG, free T, estradiol, LH, FSH, prolactin, TSH, DHEA) were performed. Endothelial secretory function was assessed using markers such as: nitric oxide (NO), endothelial NO synthase type 3, endothelin, ICAM-1, VCAM-1, p- and e-selectins, cadherin, PAI-1, VEGF-1. Additionally, the content of biologically active substances affecting endothelial function was studied: homocysteine B, C-reactive protein (CRP), osteoprotegerin, leptin, resistin, adiponectin. The vasomotor function of the endothelium was assessed by ultrasound examination of the endothelium-dependent vasodilation (EDVD) of the brachial artery (BA) during the reactive hyperemia test; in addition, the thickness of the intima-media complex (TIM) of the carotid arteries was measured. Correlation analysis was performed using Spearman’s method. Results: the level of total T is interrelated with the instrumental parameters of the endothelial function: the TIM of the carotid arteries (r = -0.26; p = 0.009), the time of maximum BA vasodilation development (r = -0.41; p &lt;0.001), EDVD (r = 0 , 28; p = 0.004), as well as laboratory markers of ED: ICAM-1 (r = -0.45; p &lt;0.001), VCAM-1 (r = -0.29; p &lt;0.001), cadherin (r = -0.36; p &lt;0.001), NO (r = 0.32; p = 0.002), VEGF (r = -0.23; p = 0.001), CRP (r = -0.29; p &lt;0.001) and adipohormones: leptin (r = -0.26; p = 0.01), resistin (r = -0.24; p &lt;0.001) and adiponectin (r = 0.28; p = 0.007). Conclusion: T deficiency is associated with a deterioration in the vasomotor function of the endothelium: a decrease in EDVD along with an increase in the time of maximum BA vasodilation development and impaired endothelial secretory function: an increase in the concentrations of VCAM-1, ICAM-1, cadherin, VEGF and, on the contrary, a decrease in NO levels. A decrease in T levels is accompanied by an increase in the content of CRP, resistin, leptin and a decrease in adiponectin, which aggravates the dysfunction of the endothelium.

POS0376 MONOMERIC C REACTIVE PROTEIN (mCRP) REGULATES INFLAMMATORY RESPONSES IN HUMAN AND MOUSE CHONDROCYTES

Background:C-reactive protein (CRP) is an acute-phase protein that is used as an established biomarker to follow disease severity and progression in a plethora of inflammatory diseases. However, its pathophysiologic mechanisms of action are still poorly defined and remain elusive. CRP, in its pentameric form, exhibits weak anti-inflammatory activity. On the contrary, the monomeric isoform (mCRP) exhibits potent pro-inflammatory properties in endothelial cells, leukocytes, and platelets. So far, no data exists regarding mCRP effects in human or mouse chondrocytesObjectives:This work aimed to verify the pathophysiological relevance of mCRP in the etiology and/or progression of osteoarthritis (OA)Methods:We investigated the effects of mCRP in cultured human primary chondrocytes and in the chondrogenic ATDC5 mouse cell line. We determined mRNA and protein levels of relevant factors involved in inflammatory responses and the modulation of nitric oxide synthase type II (NOS2), an early inflammatory molecular target.Results:We demonstrate, for the first time, that monomeric C reactive protein increases NOS2, COX2, MMP13, VCAM1, IL-6, IL-8, and LCN2 expression in human and murine chondrocytes. We also demonstrated that NF-kB is a key factor in the intracellular signaling of mCRP-driven induction of pro-inflammatory and catabolic mediators in chondrocytes.Conclusion:mCRP exerts a sustained catabolic effect on human and murine chondrocytes, increasing the expression of inflammatory mediators and proteolytic enzymes, which can promote extracellular matrix (ECM) breakdown in healthy and OA cartilage. In addition, our results implicate the NF-kB signaling pathway in catabolic effects mediated by mCRP.

Macrophages loaded with dendrimer-entrapped gold nanoparticles as a theranostic platform for CT imaging-guided combinational therapy of orthotopic osteosarcoma

Immune cell therapy has recently emerged as a promising treatment modality for malignant tumors. Specifically, nanoparticle-mediated cell-immunotherapy has attracted considerable attention due to the combined contributions of the biological regulation of nanoparticles and the intrinsic nature of immune cells. Herein, engineered macrophages with dendrimer-entrapped gold nanoparticles (Au [email protected]) were designed as a theranostic cell platform, for the first time, for CT imaging-guided cell immunotherapy and chemotherapy of osteosarcoma. In this study, Au DENPs were synthesized and used to activate macrophages into the anti-tumorigenic M1-like phenotype, characterized by the increased expression of CD86, the inducible type of nitric oxide synthase (iNOS), and tumor necrosis factor-α (TNF-α). After loading Au DENPs, the engineered macrophages showed cytostatic/pro-apoptotic effects against cancer cells and achieved CT imaging performance in vitro and in vivo. The combination of cell therapy and chemotherapy demonstrated a cooperative impact in enhancing cancer treatment. Our findings reveal that Au [email protected] can be used as a theranostic platform for osteosarcoma, which may present a brand‐new strategy for immune cell-combined chemotherapy of other cancer types.

Structural Determination, Biological Function, and Molecular Modelling Studies of Sulfoaildenafil Adulterated in Herbal Dietary Supplement.

Unapproved ingredients included in herbal medicines and dietary supplements have been detected as adulterated synthetic drugs used for erectile dysfunction. Extraction from a dietary supplement was performed to isolate the compounds by HPLC analysis. The structural characterization was confirmed using mass spectrometry (ESI-TOF/MS and LC-MS/MS), 1H NMR, and 13C NMR spectroscopy techniques. Results identified the thus-obtained compound to be sulfoaildenafil, a thioketone analogue of sildenafil. The biological activities of this active compound have been focused for the first time by the experimental point of view performance in vitro. The results revealed that sulfoaildenafil can affect the therapeutic level of nitric oxide through the upregulation of nitric oxide synthase and phosphodiesterase type 5 (PDE5) gene expressions. This bulk material, which displays structural similarity to sildenafil, was analyzed for the presence of a PDE5 inhibitor using a theoretical calculation. These unique features of the potential activity of PDE5 protein and its inhibitors, sildenafil and sulfoaildenafil, may play a key consideration for understanding the mode of actions and predicting the biological activities of PDE5 inhibitors.

Leptin ameliorates testicular injury by altering expression of nitric oxide synthases in diabetic rats.

We aimed to evaluate the effects of leptin and nitro-L-arginine methyl ester hydrochloride (L-NAME) on testicular damage and expression of nitric oxide synthase (NOS) types (neuronal, endothelial and inducible NOS) in streptozotocin (STZ) -induced diabetic and non-diabetic rats. Testicular damage was evaluated histologically and expression of NOSs was evaluated immunohistochemically in testis. Plasma leptin level and blood glucose level were assessed. Blood glucose levels increased in all diabetic groups. L-NAME reduced it, but leptin had no effect. Three types NOS expression were shown in germ cells immunohistochemically. Increased eNOS and iNOS expression and decreased nNOS expression was detected in diabetic group. Testicular damage was observed in diabetic groups. Leptin ameliorated the damage by reducing iNOS expression and L-NAME partially prevented injury by supressing excessive NO production in diabetic rats. It can be suggested that leptin and L-NAME partially prevents testicular damage by ameliorating histopathological changes by stimulating and /or supressing three types of NOS expression in diabetic rats. Leptin exhibited its effects by reducing iNOS expression in diabetic rats. This is the first report demonstrating the relationship between leptin and nitric oxide in testicular tissue in STZ-induced diabetic rats (Fig. 3, Ref. 33).

A Novel Endocrine Role for the BAT-Released Lipokine 12,13-diHOME to Mediate Cardiac Function.

Brown adipose tissue (BAT) is an important tissue for thermogenesis, making it a potential target to decrease the risks of obesity, type 2 diabetes, and cardiovascular disease, and recent studies have also identified BAT as an endocrine organ. Although BAT has been implicated to be protective in cardiovascular disease, to this point there are no studies that identify a direct role for BAT to mediate cardiac function. To determine the role of BAT on cardiac function, we utilized a model of BAT transplantation. We then performed lipidomics and identified an increase in the lipokine 12,13-dihydroxy-9Z-octadecenoic acid (12,13-diHOME). We utilized a mouse model with sustained overexpression of 12,13-diHOME and investigated the role of 12,13-diHOME in a nitric oxide synthase type 1 deficient (NOS1-/-) mouse and in isolated cardiomyocytes to determine effects on function and respiration. We also investigated 12,13-diHOME in a cohort of human patients with heart disease. Here, we determined that transplantation of BAT (+BAT) improves cardiac function via the release of the lipokine 12,13-diHOME. Sustained overexpression of 12,13-diHOME using tissue nanotransfection negated the deleterious effects of a high-fat diet on cardiac function and remodeling, and acute injection of 12,13-diHOME increased cardiac hemodynamics via direct effects on the cardiomyocyte. Furthermore, incubation of cardiomyocytes with 12,13-diHOME increased mitochondrial respiration. The effects of 12,13-diHOME were absent in NOS1-/- mice and cardiomyocytes. We also provide the first evidence that 12,13-diHOME is decreased in human patients with heart disease. Our results identify an endocrine role for BAT to enhance cardiac function that is mediated by regulation of calcium cycling via 12,13-diHOME and NOS1.

Testosterone positively regulates functional responses and nitric oxide expression in the isolated human corpus cavernosum.

Testosterone (T) deficiency is associated with erectile dysfunction (ED). The relaxant response of T on the corporal smooth muscle through a non-genomic pathwayhas been reported; however, the in vitro modulating effects ofTon human corpus cavernosum (HCC) have not been studied. To compare the effects of various concentrations of T on nitric oxide (NO)-dependent and nitric oxide-independent relaxation in organ bath studies and elucidate its mode of action, specifically targeting the cavernous NO/cyclic guanosine monophosphate (cGMP) pathway. Human corpus cavernosum (HCC) samples were obtained from men undergoing penile prosthesis implantation (n=9). After phenylephrine (Phe) precontraction, the effects of various relaxant drugs of HCC strips were performed using organ bath at low (150ng/dL), eugonadal (400ng/dL), and hypergonadal (600ng/dL) T concentrations. The penile tissue measurements of endothelial nitric oxide synthase (eNOS), neuronal (n)NOS, and phosphodiesterase type 5 (PDE5) were evaluated via immunostaining, Western blot, cGMP and nitrite/nitrate (NOx) assays. Relaxation responses to ACh and EFS in isolated HCC strips were significantly increased at all T levels compared with untreated tissues. The sildenafil-induced relaxant response was significantly increased at both eugonadal and hypergonadal T levels. Normal and high levels of T are accompanied by increased eNOS, nNOS, cGMP, and NOx levels, along with reduced PDE5 protein expression. This study reveals an important role of short-term and modulatory effects of different concentrations of T in HCC. T positively regulates functional activities, inhibition of PDE5 expression, and formation of cGMP and NOx in HCC. These results demonstrate that T indirectly contributes to HCC relaxation via downstream effects on nNOS, eNOS, and cGMP and by inhibiting PDE5. This action provides a rationale for normalizing T levels in hypogonadal men with ED, especially when PDE5 inhibitors are ineffective. T replacement therapy may improve erectile function by modulating endothelial function in hypogonadal men.

IL-22 Promotes IFN-γ-Mediated Immunity against Histoplasma capsulatum Infection.

Histoplasma capsulatum is the agent of histoplasmosis, one of the most frequent mycoses in the world. The infection initiates with fungal spore inhalation, transformation into yeasts in the lungs and establishment of a granulomatous disease, which is characterized by a Th1 response. The production of Th1 signature cytokines, such as IFN-γ, is crucial for yeast clearance from the lungs, and to prevent dissemination. Recently, it was demonstrated that IL-17, a Th17 signature cytokine, is also important for fungal control, particularly in the absence of Th1 response. IL-22 is another cytokine with multiple functions on host response and disease progression. However, little is known about the role of IL-22 during histoplasmosis. In this study, we demonstrated that absence of IL-22 affected the clearance of yeasts from the lungs and increased the spreading to the spleen. In addition, IL-22 deficient mice (Il22−/−) succumbed to infection, which correlated with reductions in the numbers of CD4+ IFN-γ+ T cells, reduced IFN-γ levels, and diminished nitric oxide synthase type 2 (NOS2) expression in the lungs. Importantly, treatment with rIFN-γ mitigated the susceptibility of Il22−/− mice to H. capsulatum infection. These data indicate that IL-22 is crucial for IFN-γ/NO production and resistance to experimental histoplasmosis.

Downregulation of thioredoxin-1-dependent CD95 S-nitrosation by Sorafenib reduces liver cancer

Hepatocellular carcinoma (HCC) represents 80% of the primary hepatic neoplasms. It is the sixth most frequent neoplasm, the fourth cause of cancer-related death, and 7% of registered malignancies. Sorafenib is the first line molecular targeted therapy for patients in advanced stage of HCC. The present study shows that Sorafenib exerts free radical scavenging properties associated with the downregulation of nuclear factor E2-related factor 2 (Nrf2)-regulated thioredoxin 1 (Trx1) expression in liver cancer cells. The experimental downregulation and/or overexpression strategies showed that Trx1 induced activation of nitric oxide synthase (NOS) type 3 (NOS3) and S-nitrosation (SNO) of CD95 receptor leading to an increase of caspase-8 activity and cell proliferation, as well as reduction of caspase-3 activity in liver cancer cells. In addition, Sorafenib transiently increased mRNA expression and activity of S-nitrosoglutathione reductase (GSNOR) in HepG2 cells. Different experimental models of hepatocarcinogenesis based on the subcutaneous implantation of HepG2 cells in nude mice, as well as the induction of HCC by diethylnitrosamine (DEN) confirmed the relevance of Trx1 downregulation during the proapoptotic and antiproliferative properties induced by Sorafenib. In conclusion, the induction of apoptosis and antiproliferative properties by Sorafenib were related to Trx1 downregulation that appeared to play a relevant role on SNO of NOS3 and CD95 in HepG2 cells. The transient increase of GSNOR might also participate in the deactivation of CD95-dependent proliferative signaling in liver cancer cells.