Nitric oxide inhibition assay and the respective target identification of an aptamer designed to control atherosclerosis

Introduction: Aptamers are emerging newer therapeutics and diagnostics can be designed to bind any kind of target proteins. Vascular endothelial damage by the excess amount of nitric oxide production in systemic circulation leads to the secretion of inflammatory chemoattractant and cell adhesion, which is the prime pro-atherogenic events in the formation of plagues at atrial intimal layers due to oxidation – sensitive mechanisms. Nitric oxide inhibition assay is one of the valuable qualitative anti-atherosclerosis matrices. Methods: In this research, Nitric oxide inhibition efficiency of a ssDNA aptamer on cell lines was studied and the respective targets of that aptamer were identified by network analysis. The aptamer used here was originally designed for Selectin P Ligand Protein to control atherogenic process. 20 nM of aptamer solution in LipofectamineTM 2000 shows the highest level of 70.5 % inhibition of nitric oxide liberation on 24 hours cultured medium of Lipopolysaccharide stimulated murine macrophage RAW 264.7 cell lines. Results: Protein interaction network analysis on the nitric oxide synthesis pathway interactors and the molecular docking analysis with network resulted proteins such as AKT Serine/Threonine Kinase 1, Calmodulin, Estrogen Receptor 1, and Nitric Oxide Synthase-3 confirms that the G – quadruplex Model of 18-mer sequence effectively binds on the active sites of Estrogen Receptor 1, and Nitric Oxide Synthase-3. Conclusion : The aptamer designed for atherosclerotic target have also exert significant nitric oxide inhibition to control the atherogenic events through the proteins, AKT1, NOS3 and ESR1.

NOS3 is a differentially expressed gene in brain metastatic human breast cancer.

Metastasis to the brain is a clinical problem in patients with breast cancer (1-3). We mined published microarray data (4, 5) to compare primary and metastatic tumor transcriptomes for the discovery of genes associated with brain metastasis in humans with metastatic breast cancer. We found that nitric oxide synthase 3, encoded by NOS3, was among the genes whose expression was most different in the brain metastases of patients with metastatic breast cancer as compared to primary tumors of the breast. NOS3 mRNA was present at increased quantities in brain metastatic tissues as compared to primary tumors of the breast. Expression of NOS3 in primary tumors was significantly correlated with patient overall survival. Modulation of NOS3 expression may be relevant to the biology by which tumor cells metastasize from the breast to the brain in humans with metastatic breast cancer.

Loss of ITGB3 in ovine conceptuses decreases conceptus expression of NOS3 and SPP1: implications for the developing placental vasculature†

During the peri-implantation period of pregnancy in sheep, there is an initial period of loose apposition of the elongating conceptuses (embryos and associated placental membranes) to the endometrial luminal epithelium (LE) that is followed by adhesion of the conceptus trophectoderm to the endometrial LE for implantation. Integrins and maternal extracellular matrix (ECM) molecules are major contributors to stable adhesion at implantation, and the β3 integrin subunit (ITGB3) is implicated in the adhesion cascade for implantation in several species including the sheep. We blocked mRNA translation for trophectoderm-expressed ITGB3 by infusing morpholino antisense oligonucleotides into the uterine lumen of pregnant ewes on Day 9 to assess effects on conceptus elongation, and on Day 16 to assess effects on early placental development in sheep. Results indicate that sheep conceptuses elongate and implant to the uterine wall in the absence of ITGB3 expression by the conceptuses; however, loss of ITGB3 in conceptuses decreased the growth of embryos to Day 24 of gestation, and decreased expression of secreted phosphoprotein 1 (SPP1) and nitric oxide synthase 3 (NOS3). Abundant SPP1 was localized around the blood vessels in the placental allantoic membrane in normal sheep pregnancies. We hypothesize that NOS3 and SPP1 positively influence the development of the vasculature within the allantois, and that decreased expression of NOS3 and SPP1, in response to knockdown of ITGB3 in conceptuses, alters development of the vasculature in the allantois required to transport nutrients from the endometrium to support growth and development of the embryo.

Influence of NOS3 rs2070744 genotypes on hepatocellular carcinoma patients treated with lenvatinib

We investigated whether or not nitric oxide synthase 3 (NOS3) rs2070744 genotypes can affect the response for lenvatinib treatment in patients with hepatocellular carcinoma (HCC). We evaluated the relation of the NOS3 rs2070744 genotypes to the tumor response, progression-free survival (PFS), and overall survival (OS) as the response for lenvatinib. We also examined the association between fibroblast growth factor receptor (FGFR) gene polymorphisms, a potential feature of lenvatinib, and the response. There were no significant differences between the studies for either PFS or OS, even though patients with the TT genotype had a longer mean PFS (hazard ratio [HR] 0.60; p = 0.069) and mean OS (HR 0.46; p = 0.075) than those with the TC/CC genotypes. However, patients with a single-nucleotide polymorphism (SNP) combination pattern of the NOS3 rs2070744 TC/CC and FGFR4 rs351855 CT/TT genotypes had a significantly shorter mean PFS (HR 2.56; p = 0.006) and mean OS (HR 3.36; p = 0.013) than those with the other genotypes. The NOS3 rs2070744 genotypes did not influence the clinical response. However, the SNP combination pattern of the NOS3 rs2070744 and FGFR4 rs351855 genotypes may be helpful as treatment effect predictors and prognostic factors for HCC patients treated with lenvatinib.

Carvedilol induces biased β1 adrenergic receptor-nitric oxide synthase 3-cyclic guanylyl monophosphate signalling to promote cardiac contractility

Aims β-blockers are widely used in therapy for heart failure and hypertension. β-blockers are also known to evoke additional diversified pharmacological and physiological effects in patients. We aim to characterize the underlying molecular signalling and effects on cardiac inotropy induced by β-blockers in animal hearts. Methods and results Wild-type mice fed high-fat diet (HFD) were treated with carvedilol, metoprolol, or vehicle and echocardiogram analysis was performed. Heart tissues were used for biochemical and histological analyses. Cardiomyocytes were isolated from normal and HFD mice and rats for analysis of adrenergic signalling, calcium handling, contraction, and western blot. Biosensors were used to measure β-blocker-induced cyclic guanosine monophosphate (cGMP) signal and protein kinase A activity in myocytes. Acute stimulation of myocytes with carvedilol promotes β1 adrenergic receptor (β1AR)- and protein kinase G (PKG)-dependent inotropic cardiac contractility with minimal increases in calcium amplitude. Carvedilol acts as a biased ligand to promote β1AR coupling to a Gi-PI3K-Akt-nitric oxide synthase 3 (NOS3) cascade and induces robust β1AR-cGMP-PKG signal. Deletion of NOS3 selectively blocks carvedilol, but not isoproterenol-induced β1AR-dependent cGMP signal and inotropic contractility. Moreover, therapy with carvedilol restores inotropic contractility and sensitizes cardiac adrenergic reserves in diabetic mice with minimal impact in calcium signal, as well as reduced cell apoptosis and hypertrophy in diabetic hearts. Conclusion These observations present a novel β1AR-NOS3 signalling pathway to promote cardiac inotropy in the heart, indicating that this signalling paradigm may be targeted in therapy of heart diseases with reduced ejection fraction.

NOS3 is differentially expressed in hepatosplenic T-cell lymphoma.

Hepatosplenic T-cell lymphoma (HSTCL) is a rare peripheral T-cell lymphoma affecting the spleen and liver (1). By mining published microarray datasets (2-3) we found that the nitric oxide synthase 3 (NOS3) was among the genes most differentially expressed in HSTCL when compared to a different peripheral T-cell lymphoma, NKT Lymphoma. Analysis of a separate dataset revealed that when comparing HSTCL tissues and cell lines based on cytogenetic profile, NOS3 expression was associated with isochromosome 7q-positivity. NOS3 may be relevant to the biology of HSCTL.

Sinapic Acid Attenuates Cardiovascular Disorders in Rats by Modulating Reactive Oxygen Species and Angiotensin Receptor Expression

The main avoidable risk factor for cardiovascular conditions is high blood pressure (hypertension). At global level, hypertension is believed to be responsible for a 54% stroke-related mortality rate and a 47% mortality rate associated with coronary heart disease. It is postulated that sinapic acid (SA) could help in hypertension management because it displays robust antioxidant, antihyperglycemic, and peroxynitrite scavenging effects. To explore this hypothesis, this work examined the effect of SA on oxidative stress and cardiovascular disease in rats with hypertension by comparison against captopril. For this purpose, 50 male rats were used and equally allocated to five groups, namely, normal control, positive control (L-NAME), L-NAME with concomitant captopril administration, L-NAME with concomitant SA administration, and L-NAME with concomitant administration of both SA and captopril. Results showed that, by contrast to control, L-NAME exhibited marked elevation in serum CK-MB, total cholesterol, triglycerides, VLDL-C, LDL-C, Ang II, AT2R, ET-1, and angiopoietin-2; on the other hand, L-NAME exhibited marked reduction in serum HDL-C, superoxide dismutase (SOD) activity, nitric oxide synthase 3 (NOS3), and glutathione (GSH). Furthermore, joint administration of SA and captopril ameliorated hypertension, enhanced cardiovascular function, hindered hyperlipidemia, and decreased oxidative stress and myocardial hypertrophy displayed by rats with hypertension. Based on such findings, better chemopreventive or therapeutic approaches can be devised to manage hypertension and cardiovascular conditions.

Association of nitric oxide synthase 3 gene rs1799983 G/T polymorphism with idiopathic asthenozoospermia in Iranian Azeri males: a case-control study

ObjectivesRecently, oxidative stress (OS) has been described extensively as an important cause of men infertility. The nitric oxide synthase 3 (NOS3) gene expression involved in normal spermatogenesis regulation in testis. Several single nucleotide polymorphisms (SNPs) on NOS3 gene are reported in association with sperm function and spermatogenesis impairment in infertile men. In present study, we investigated association of NOS3 gene rs1799983 G/T polymorphism in Iranian Azeri male with idiopathic asthenozoospermia (AZS).MethodsIn this case-control study, we collected 50 males with idiopathic AZS as a case group and 50 age and ethnically matched male as healthy controls from East Azerbaijan area, Iran. The case and control groups genotyping was performed using tetra-primer amplification refractory mutation system-polymerase chain reaction (Tetra-ARMS PCR) method.ResultsGenotype frequency in AZS patients was 40% GG, 60% GT, and 0% TT, whereas in healthy controls were 60% GG, 30% GT, and 10% TT. Statistical analysis showed that the GT heterozygous genotype frequency of NOS3 gene rs1799983 G/T polymorphism in AZS patients was significantly more than healthy controls (p>0.05).ConclusionsWe demonstrated that NOS3 gene rs1799983 G/T polymorphism was associated with AZS in Iranian Azeri men. However, more studies on different geographic areas, races and ethnicities are required to determine exact role of NOS3 gene rs1799983 G/T polymorphism in idiopathic AZS.