Human Endothelial Nitric Oxide Synthase Research Articles

Exploring the Role of Human Endothelial Nitric Oxide Synthase (eNOS) in Ischemic Heart Disease (IHD)

Background: Ischemic heart disease, also called coronary artery disease, is the term given to heart problems caused by narrowed heart (coronary) arteries that supply blood to the heart muscle. Aim of the Study: The aim of the study is to determine the levels of Nitric oxide synthase (eNOS) in patients with coronary artery disease. Patients and Methods: The current study is case-control study conducted in Tikrit city during the period between 10th of January to 10th of April 2023 on patients admitted to Coronary Care Unit of Salah Al-Din Teaching Hospital The study included 60 Iraqi patients with coronary heart disease with age range (37-66 years). The study also included 30 healthy individuals with same age range and from both sexes who apparently haven’t any acute or chronic diseases. Blood was collected from each patients for determination Nitric oxide synthase by enzyme-linked immunosorbent assay (ELISA) and lactate dehydrogenase (LDH) by chemiluminence technique. Results: The study showed that lowest mean of Human Endothelial Nitric Oxide Synthase (eNOS) was detected among IHD patients (2.41±0.74 ng/ml) and the maximum mean was within the control group (5.15±1.24 ng/ml). The differences were highly significant (P-value: 0.0001). The study confirmed that the peak mean of lactate dehydrogenase was detected among IHD patients (257.8±94.9 IU/L) and the deepest mean was in the control group (168.9±16.8 IU/L). The differences were highly significant (P-value: 0.0001). The study showed that the mean eNOS was 2.17 ng/ml in patients with hypertension, which was significantly lower than that in non-hypertensive IHD patients (2.76 ng/ml), at a P value of 0.024. The study showed that the mean LDH was 284.6 IU/L in patients with hypertension, which was significantly more than that in non-hypertensive IHD patients (217.5 IU/L), at a P value of 0.001. Conclusions: In conclusion, the findings of the study suggest that patients with ischemic heart disease (IHD) have reduced levels of serum eNOS compared to healthy individuals. More researches are needed to fully understand the relationship among eNOS, cardiovascular disease and to determine the clinical utility of their measuring in the diagnosis and management of these conditions.

In silico elucidation of potential drug targets against oxygenase domain of Human eNOS Dysfunction.

Nitric Oxide (NO) signaling pathway plays a vital role in various physiological and pathophysiological processes including vasodilation, neurogenesis, inflammation, translation and protein regulation. NO signaling pathway is associated with various diseases such as cardiovascular diseases, vision impairment, hypertension and Alzheimer's disease. Human Endothelial Nitric Oxide Synthase (eNOS) bound with calcium regulatory protein (calmodulin (CaM)) to produce NO which initiates cGMP pathway. The current study employs to screen the novel compounds against human eNOS independent of calcium regulatory protein (CaM). The current effort emphasized that the deficiency of CaM leads to dysfunction of cGMP signaling pathway. In this work, a hybrid approach of high-throughput virtual screening and comparative molecular docking studies followed by molecular dynamic simulation analyses were applied. The screening of top ranked two novel compounds against eNOS were reported that showed effective binding affinity, retrieved through the DrugBank and ZINC database libraries. Comparative molecular docking analyses revealed that Val-104, Phe-105, Gln-247, Arg-250, Ala-266, Trp-330, Tyr-331, Pro-334, Ala-335, Val-336, Tyr-357, Met-358, Thr-360, Glu-361, Ile-362, Arg-365, Asn-366, Asp-369, Arg-372, Trp-447 and Tyr-475 are potent residues for interactional studies. High-throughput virtual screening approach coupled with molecular dynamic simulation and drug likeness rules depicted that ZINC59677432 and DB00456 are potent compounds to target eNOS. In conclusion, the proposed compounds are potent against eNOS based on extensive in silico analyses. Overall, the findings of this study may be helpful to design therapeutic targets against eNOS.

Molecular dynamics study of in silico mutations in the auto-inhibitory loop of human endothelial nitric oxide synthase FMN sub-domain.

Structural flexibility of the peptide linker connecting two domains is essential for the functioning of multi-domain complex. Nitric oxide synthase (NOS) isoforms contain the oxygenase and the reductase domains connected by calmodulin binding linker (CBL) region. Additionally, the endothelial NOS (eNOS) isoform contain an auto-inhibitory loop (AI) in the FMN reductase sub-domain which represses the inter-domain electron transfer process. Binding of Ca2+-Calmodulin complex on the CBL region relieves the AI loop repression and facilitates electron transfer from FMN in the reductase domain to the heme in the oxygenase domain. Few experimental studies have reported that in vitro mutation of Serine-615 (S615D) and Serine-633 (S633D) in the FMN reductase sub-domain to aspartic acid increased NO production and increased Ca2+ sensitivity. To understand the role of AI loop in eNOS repression and activation in serine mutants (S615D and S633D), we modelled the FMN reductase sub-domain of human eNOS protein with and without the CBL region. Molecular dynamics simulations performed indicated that the mutant protein AI loop structure was stabilized by salt bridge formed between D615 and R602. It was also found that mutation increased the flexibility of C-terminal residues of eNOS CBL region. The hinge-like movement of the AI loop allowed rotation of the FMN sub-domain clockwise which may favour electron-transfer in the mutant protein. This study provides insight on mutation (S615D and S633D) induced changes in AI loop and increased flexibility of CBL region which may lead to the protein activation and may also facilitate Calmodulin binding at physiological Ca2+ concentration. Graphical Abstract Mutation of amino acid residues contribute to structural changes at molecular level leading to alteration in protein dynamics and its function. Serine-615 and Serine-633 in the auto-inhibitory loop of human eNOS reductase model was mutated to aspartic acid in silico and molecular dynamics simulations of the protein showed that steric hindrance due to mutation altered the auto-inhibitory loop rearrangement and the FMN sub-domain movement favouring electron transfer.

Inhibited Nitric Oxide Production of Human Endothelial Nitric Oxide Synthase by Nitrated and Oxygenated Polycyclic Aromatic Hydrocarbons.

Nitrated and oxygenated polycyclic aromatic hydrocarbons (NPAHs and OPAHs) from the direct atmospheric emission or the degradation of parent PAHs are increasingly recognized because of their potential health risks. Herein, we investigated the effects of four NPAHs/OPAHs (1-NNAP, 9-NANT, 9,10-AQ, and 9-FLU) and their parent PAHs (NAP, ANT, and FLU) on endothelium function with regard to endothelial nitric oxide synthase (eNOS) and endothelium-derived nitric oxide (NO) production in human umbilical vein endothelial cells. The eNOS enzymatic activity and NO production were promoted by NAP, ANT, and FLU; however, eNOS activity was dropped by 52.8, 52.1, 52.5, and 44.5%, and NO production was decreased by 31.1, 50.3, 65.0, and 35.0% after 24 h exposure to 0.01 μM 1-NNAP, 9-NANT, 9,10-AQ, and 9-FLU, respectively. The mRNA expression of eNOS and protein expression of phosphorylated eNOS (Ser1177) were increased by three PAHs but decreased by four NPAHs/OPAHs. The 100 ns molecular dynamics simulations reveal the conformational alteration in the key propionate of heme upon the binding of NPAHs/OPAHs. Our findings provide the first in silico and in vitro evidence for the potential endothelial dysfunction of nitrated and oxygenated PAHs. The health risk implications of NPAHs/OPAHs and corresponding parent PAHs warrant further research.

Structural and functional alterations of nitric oxide synthase 3 due to missense variants associate with high-altitude pulmonary edema through dynamic study

The human endothelial nitric oxide synthase (NOS3) is 28 Kbp at 7q36.1 and encodes protein comprising of 1280 amino acids. Being a major source of nitric oxide, the enzyme is crucial to the vascular homeostasis and thereby to be an important pharmaceutical target. We hence have been investigating this molecule in a high-altitude disorder namely, high-altitude pulmonary edema (HAPE). We performed a genome-wide association study (GWAS) in a case-control design of sojourners that included healthy controls and HAPE patients (n = 200) each. Four NOS3 missense SNPs i.e. rs1799983 (E298D), rs3918232 (V827M), rs3918201 (R885M) and rs3918234 (Q982L), were associated significantly with HAPE (P-value < 0.05). Furthermore, extensive in silico analyses were performed to predict the detrimental effect of the four variant types and their three most relevant co-factors namely, heme, flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) that are accountable for amendment of protein stability leading to structural de-construction. Subsequently, we validated the findings in a larger sample size of the two study groups. HAPE patients had a higher frequency of the four variants and significantly decreased levels of circulating nitric oxide (NO) (P-value < 0.001). The in silico and human subjects findings complement each other. This study explored the impact of HAPE-associated NOS3 variants with its protein structure stability and holds promise to be current and future drug targets.Communicated by Ramaswamy H. Sarma

Sensitive and quantitative method to evaluate DNA methylation of the positive regulatory domains (PRDI, PRDII) and cAMP response element (CRE) in human endothelial nitric oxide synthase promoter

Nitric oxide plays a prominent role in the cardiovascular system and much attention has been devoted in the last years on deciphering the regulation of human endothelial nitric oxide synthase (eNOS) expression. Epigenetic-based mechanisms have a key role in the eNOS expression and their pathologic perturbations may have profound effects on the steady state RNA levels in the endothelium. The human eNOS promoter lacks a canonical TATA box and it does not contain a proximal CpG island. A differentially DNA methylated region (DMR) in the native eNOS proximal promoter is involved in gene expression regulation. Here we describe a quantitative, sensitive and cost-effective method that, relying on a novel normalization strategy, allows the quantification of DNA methylation status of the positive regulatory domains (PRDI, PRDII) and cAMP response element (CRE) in human eNOS promoter. This technique will enable to explore the functional relevance of DNA methylation perturbations of eNOS promoter both under pathological and physiological conditions.

Docosahexaenoic acid reduces adenosine triphosphate-induced calcium influx via inhibition of store-operated calcium channels and enhances baseline endothelial nitric oxide synthase phosphorylation in human endothelial cells.

Docosahexaenoic acid (DHA), an omega-3-fatty acid, modulates multiple cellular functions. In this study, we addressed the effects of DHA on human umbilical vein endothelial cell calcium transient and endothelial nitric oxide synthase (eNOS) phosphorylation under control and adenosine triphosphate (ATP, 100 µM) stimulated conditions. Cells were treated for 48 h with DHA concentrations from 3 to 50 µM. Calcium transient was measured using the fluorescent dye Fura-2-AM and eNOS phosphorylation was addressed by western blot. DHA dose-dependently reduced the ATP stimulated Ca2+-transient. This effect was preserved in the presence of BAPTA (10 and 20 µM) which chelated the intracellular calcium, but eliminated after withdrawal of extracellular calcium, application of 2-aminoethoxy-diphenylborane (75 µM) to inhibit store-operated calcium channel or thapsigargin (2 µM) to delete calcium store. In addition, DHA (12 µM) increased ser1177/thr495 phosphorylation of eNOS under baseline conditions but had no significant effect on this ratio under conditions of ATP stimulation. In conclusion, DHA dose-dependently inhibited the ATP-induced calcium transient, probably via store-operated calcium channels. Furthermore, DHA changed eNOS phosphorylation suggesting activation of the enzyme. Hence, DHA may shift the regulation of eNOS away from a Ca2+ activated mode to a preferentially controlled phosphorylation mode.

Designing and Validation of One-Step T-ARMS-PCR for Genotyping the eNOS rs1799983 SNP.

Background: The transversion of G to T (G894T) in human endothelial nitric oxide synthase (eNOS) gene has profound effects such as male infertility, recurrent miscarriage, multiple sclerosis and cardiovascular diseases. Objectives: Development of a new Multiplex Tetra-Primer Amplification Refractory Mutation System - Polymerase Chain Reaction (T-ARMS-PCR) for detection of rs1799983 (G894T) in the human eNOS was sought. Materials and Methods: A T-ARMS-PCR for rs1799983 polymorphism in a single-step PCR was carried out, and the results were confirmed by PCR-RFLP technique in 82 infertile men with varicocele. Results: The results showed that GG (varicocele infertile men), GT and TT genotypes appear to be 53.65%, 34.14%, and 12.19%, respectively. Full accordance between PCR-RFLP and T-ARMS-PCR methods for genotyping of rs1799983 polymorphism was found. Conclusions: This is the first work that describes a rapid, relatively cheap, high throughput detection of G894T polymorphism in eNOS that can be used in large scale clinical studies.

Computational Prediction of the Effects of Single Nucleotide Polymorphisms of the Gene Encoding Human Endothelial Nitric Oxide Synthase

Computational Prediction of the Effects of Single Nucleotide Polymorphisms of the Gene Encoding Human Endothelial Nitric Oxide Synthase

Bioactivity of genistein: A review of in vitro and in vivo studies.

Genistein is a soy derived isoflavanoid compound with multitude of health benefits. This compound is found to be a potent agent in both prophylaxis and treatment of cancer and various other chronic diseases. Ranging from its antioxidant activity to its effect on various cancer types, genistein has been a compound of interest in a number of studies carried out so far. The great interest that has focused on genistein led to the identification of numerous intracellular targets of its action in the live cells. Retardation of atherogenic activity and increasing the antioxidant defense of a cell has been attributed to genistein while as it has also been reported that genistein possesses suppressive effects on both the cell-mediated and humoral components of the adaptive immune system. At the molecular level, genistein reduces the number of developing CD4(+) and CD8(+) thymocytes suggesting a possible mechanism for genistein effects on cell-mediated immunity. Genistein may inhibit cancer progression by inducing apoptosis or inhibiting proliferation. In addition, genistein has its prominent role in preventing the DNA damage. Apolipoprotein B secretion gets reduced when the subjects are administered with genistein. Genistein confers a better protection to ischemic conditions thereby giving a significant cardioprotection. At cellular level adipocyte differentiation is another property of genistein which makes it a better neutraceutical which can reduce the atherogenic condition and hypercholesterolemia. Expression of human endothelial nitric oxide synthase is associated with genistein supplementation. The advantage of using genistein is its multidirectional action and its lesser toxicity.

Role of nitric oxide in murine conventional outflow physiology.

Elevated intraocular pressure (IOP) is the main risk factor for glaucoma. Exogenous nitric oxide (NO) decreases IOP by increasing outflow facility, but whether endogenous NO production contributes to the physiological regulation of outflow facility is unclear. Outflow facility was measured by pressure-controlled perfusion in ex vivo eyes from C57BL/6 wild-type (WT) or transgenic mice expressing human endothelial NO synthase (eNOS) fused to green fluorescent protein (GFP) superimposed on the endogenously expressed murine eNOS (eNOS-GFPtg). In WT mice, exogenous NO delivered by 100 μM S-nitroso-N-acetylpenicillamine (SNAP) increased outflow facility by 62 ± 28% (SD) relative to control eyes perfused with the inactive SNAP analog N-acetyl-d-penicillamine (NAP; n = 5, P = 0.016). In contrast, in eyes from eNOS-GFPtg mice, SNAP had no effect on outflow facility relative to NAP (-9 ± 4%, P = 0.40). In WT mice, the nonselective NOS inhibitor N(G)-nitro-l-arginine methyl ester (l-NAME, 10 μM) decreased outflow facility by 36 ± 13% (n = 5 each, P = 0.012), but 100 μM l-NAME had no detectable effect on outflow facility (-16 ± 5%, P = 0.22). An eNOS-selective inhibitor (cavtratin, 50 μM) decreased outflow facility by 19 ± 12% in WT (P = 0.011) and 39 ± 25% in eNOS-GFPtg (P = 0.014) mice. In the conventional outflow pathway of eNOS-GFPtg mice, eNOS-GFP expression was localized to endothelial cells lining Schlemm's canal and the downstream vessels, with no apparent expression in the trabecular meshwork. These results suggest that endogenous NO production by eNOS within endothelial cells of Schlemm's canal or downstream vessels contributes to the physiological regulation of aqueous humor outflow facility in mice, representing a viable strategy to more successfully lower IOP in glaucoma.

Establishment of a rabbit model of coronary artery bypass graft and endothelial nitric oxide synthase gene transfection.

This study established an animal model of coronary artery bypass graft (CABG) surgery. The human endothelial nitric oxide synthase (eNOS) gene was transfected into grafted arterial walls to verify transfection efficiency. Forty rabbits were randomized into the following 4 equal groups: 1) eNOS gene transfection group (eNOS group); 2) empty eNOS gene transfection group (empty gene group); 3) control group; 4) normal femoral artery group. Grafted arteries, and normal carotid and femoral artery specimens were obtained 3 weeks later. Immunohistochemistry and analyses of tissue nitric oxide (NO) levels, eNOS activity, and eNOS protein western blotting were performed. The effectiveness and efficiency of transfection were observed and confirmed. All rabbits survived. The grafted arteries retained patency. Varying degrees of adaptability changes were observed in grafted arteries in each group. The eNOS group exhibited vascular wall thickening and significantly increased eNOS protein expression. The control and empty gene groups exhibited vessel wall degeneration, and eNOS protein was weakly or not expressed (P < 0.05). The arterial wall NO concentration and total eNOS activity in the eNOS group were significantly higher than those in the other groups (P < 0.05). Western blotting demonstrated that the vascular wall eNOS protein concentration was significantly greater than that in the other groups (P < 0.05). Furthermore, the eNOS gene transfection can increase eNOS expression and activity in vessel walls, increasing local NO concentration and expression.

Human blood platelets lack nitric oxide synthase activity

Reports on expression and functionality of nitric oxide synthase (NOS) activity in human blood platelets and erythrocytes are contradictory. We used a specific gas chromatography–mass spectrometry (GC–MS) method to detect NOS activity in human platelets. The method measures simultaneously [15N]nitrite and [15N]nitrate formed from oxidized 15N-labeled nitric oxide (15NO) upon its NOS-catalyzed formation from the substrate l-[guanidino-15N2]-arginine. Using this GC–MS assay, we did not detect functional NOS in non-stimulated platelets and in intact platelets activated by various agonists (adenosine diphosphate, collagen, thrombin, or von Willebrand factor) or lysed platelets. l-[guanidino-nitro]-Arginine-inhibitable NOS activity was measured after addition of recombinant human endothelial NOS to lysed platelets. Previous and recent studies from our group challenge expression and functionality of NOS in human platelets and erythrocytes.

The N-terminal portion of autoinhibitory element modulates human endothelial nitric-oxide synthase activity through coordinated controls of phosphorylation at Thr495 and Ser1177.

NO production catalysed by eNOS (endothelial nitric-oxide synthase) plays an important role in the cardiovascular system. A variety of agonists activate eNOS through the Ser1177 phosphorylation concomitant with Thr495 dephosphorylation, resulting in increased ·NO production with a basal level of calcium. To date, the underlying mechanism remains unclear. We have previously demonstrated that perturbation of the AIE (autoinhibitory element) in the FMN-binding subdomain can also lead to eNOS activation with a basal level of calcium, implying that the AIE might regulate eNOS activation through modulating phosphorylation at Thr495 and Ser1177. Here we generated stable clones in HEK-293 (human embryonic kidney 293) cells with a series of deletion mutants in both the AIE (Δ594–604, Δ605–612 and Δ626–634) and the C-terminal tail (Δ14; deletion of 1164–1177). The expression of Δ594–604 and Δ605–612 mutants in non-stimulated HEK-293 cells substantially increased nitrate/nitrite release into the culture medium; the other two mutants, Δ626–634 and Δ1164–1177, displayed no significant difference when compared with WTeNOS (wild-type eNOS). Intriguingly, mutant Δ594–604 showed close correlation between Ser1177 phosphorylation and Thr495 dephosphorylation, and NO production. Our results have indicated that N-terminal portion of AIE (residues 594–604) regulates eNOS activity through coordinated phosphorylation on Ser1177 and Thr495.

Analysis of human bradykinin receptor gene and endothelial nitric oxide synthase gene polymorphisms in end-stage renal disease among malaysians.

The aim of this study was to determine the association of the c.894G>T; p.Glu298Asp polymorphism and the variable number tandem repeat (VNTR) polymorphism of the endothelial nitric oxide synthase (eNOS) gene and c.181C>T polymorphism of the bradykinin type 2 receptor gene (B2R) in Malaysian end-stage renal disease (ESRD) subjects.A total of 150 ESRD patients were recruited from the National Kidney Foundation’s (NKF)dialysis centers in Malaysia and compared with 150 normal healthy individuals. Genomic DNA was extracted from buccal cells of all the subjects. The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was carried out to amplify the products and the restricted fragments were separated by agarose gel electrophoresis. Statistical analyses were carried out using software where a level of p <0.05 was considered to be statistically significant.The genotypic and allelic frequencies of the B2R gene (c.181C>T, 4b/a) and eNOS gene (c.894G>T) polymorphisms were not statistically significant (p >0.05) when compared to the control subjects. The B2R and eNOS gene polymorphisms may not be considered as genetic susceptibility markers for Malaysian ESRD subjects.

Functional role of NF-κB in expression of human endothelial nitric oxide synthase

The transcription factor NF-κB has an essential role in inflammation in endothelial cells. Endothelial nitric oxide synthase (eNOS)-derived nitric oxide (NO) prevents vascular inflammation. However, the molecular mechanism underlying NF-κB-mediated regulation of eNOS expression has not been clearly elucidated. We here found that NF-κB-activating stimuli, such as lipopolysaccharide, tumor necrosis factor-α (TNF-α), and interleukin-1β, suppressed eNOS mRNA and protein levels by decreasing mRNA stability, without affecting promoter activity. TNF-α-mediated suppression of eNOS expression, mRNA stability, and 3′-untranslated region (3′UTR) activity were inhibited by NF-κB inhibitors and Dicer knockdown, but not by p38 MAPK and MEK inhibitors, suggesting the involvement of NF-κB-responsive miRNAs in eNOS expression. Moreover, TNF-α increased MIR155HG expression and promoter activity as well as miR-155 biogenesis, and these increases were blocked by NF-κB inhibitors. Transfection with antagomiR-155 blocked TNF-α-mediated suppression of eNOS 3′UTR activity, eNOS mRNA and protein levels, and NO and cGMP production. These data provide evidence that NF-κB is a negative regulator of eNOS expression via upregulation of miR-155 under inflammatory conditions. These results suggest that NF-κB is a potential therapeutic target for preventing vascular inflammation and endothelial dysfunction induced by suppression of miR-155-mediated eNOS expression.

Sp1 modification of human endothelial nitric oxide synthase promoter increases the hypoxia-stimulated activity

Human endothelial nitric oxide synthase (eNOS) gene has a TATA-less weak promoter with a low activity. The aim of this study was to increase eNOS promoter activity by modification. Human eNOS promoter was modified by inserting a Sp1 element at a −74bp site and function of the modified promoter was investigated via a hypoxia model induced by cobalt chloride in human umbilical vein endothelial cells. The results demonstrated that the Sp1-modified promoter resulted in a significant increase of normalized luciferase activity in the presence of hypoxia. There was a correlation between the transcriptional activity of the Sp1-modified promoter and the level of eNOS expression with enhancement of nitric oxide production. Together, these data indicate that human eNOS promoter activity is increased by inserting Sp1 binding site into the GC-rich region of the promoter in response to hypoxia, suggesting that this provides an approach to ameliorate microcirculation barrier of some cardiovascular disease and to study its mechanistic process.

P38α subtype is a potential target to inhibit eNOS activity and NO production in human endothelial cells

Human endothelial nitric oxide synthase (eNOS) activity is important for maintaining blood pressure homeostasis and vascular integrity through nitric oxide (NO).The in vitro study aimed at investigating a role of p38α signaling in modulating NO production in human umbilical vein endothelial cell-12 (HUVEC-12). Consistent with the stimulation of lipopolysaccharide (LPS) or tumor necrosis factor (TNF)-α, the over-expression of p38α markedly down-regulated the eNOS promoter activity in HUVEC-12, which could be reversed by its negative mutant p38α (AF) or p38-specific inhibitor SB203580. Compared to the stimulation of LPS or TNF-α, p38α-targeting siRNA decreased the expressions of phosphorylated and non-phosphorylated p38α, and increased the promoter activity, an eNOS mRNA level and a phosphorylated eNOS protein expression with the enhancement of NO, which could be abrogated by the scrambled siRNA. The in situ eNOS protein expression in the cells treated by p38α-targeting siRNA was also higher than that of the control, following the corresponding attenuation of a p38 level, and mainly localized in the inner membrane and cytoplasm. These results indicate that the p38α subtype may be a potential target to down-regulate the eNOS activity and NO production in human endothelial cells.

An Intronic miRNA Regulates Expression of the Human Endothelial Nitric Oxide Synthase Gene and Proliferation of Endothelial Cells by a Mechanism Related to the Transcription Factor SP-1

ObjectiveThis study was to investigate the molecular mechanisms underlying the 27nt-miRNA-mediated regulation of expression of the endothelial nitric oxide synthase (eNOS) gene.MethodsCell lines overexpressing 27nt-miRNA or its mutant were established by transfecting the miRNA expression vector into the endothelial cells. eNOS mRNA and protein expression were examined by RT-PCR and Western Blotting, respectively. Luciferase activity reporter system was used to study the target of 27nt-miRNA.ResultsThe results showed that overexpression of 27nt-miRNA significantly inhibited eNOS mRNA level and protein expression, and reduced the eNOS transcriptional efficiency. Such inhibitory effects of 27nt-miRNA were attenuated by the sequence mutations in 27nt-miRNA. Interestingly, the transcription factor SP-1 expression was reduced by 27nt-miRNA. Meanwhile, overxpression of SP-1 protein partially restored eNOS expression, and rescued the 27nt-miRNA-mediated reduction of endothelial cell proliferation. Moreover, certain sites in the SP-1 mRNA were found to be the direct target of 27nt-miRNA by a luciferase reporter system.ConclusionsThese results demonstrate that the 27nt-miRNA suppresses eNOS gene expression and SP-1 expression in vascular endothelial cells. The 27nt-miRNA directly target to SP-1 mRNA, thereby contributing to proliferation of endothelial cells.

Active Stabilization of Human Endothelial Nitric Oxide Synthase mRNA by hnRNP E1 Protects against Antisense RNA and MicroRNAs

Human endothelial nitric oxide synthase (eNOS) mRNA is highly stable in endothelial cells (ECs). Posttranscriptional regulation of eNOS mRNA stability is an important component of eNOS regulation, especially under hypoxic conditions. Here, we show that the human eNOS 3' untranslated region (3' UTR) contains multiple, evolutionarily conserved pyrimidine (C and CU)-rich sequence elements that are both necessary and sufficient for mRNA stabilization. Importantly, RNA immunoprecipitations and RNA electrophoretic mobility shift assays (EMSAs) revealed the formation of heterogeneous nuclear ribonucleoprotein E1 (hnRNP E1)-containing RNP complexes at these 3'-UTR elements. Knockdown of hnRNP E1 decreased eNOS mRNA half-life, mRNA levels, and protein expression. Significantly, these stabilizing RNP complexes protect eNOS mRNA from the inhibitory effects of its antisense transcript sONE and 3'-UTR-targeting small interfering RNAs (siRNAs), as well as microRNAs, specifically, hsa-miR-765, which targets eNOS mRNA stability determinants. Hypoxia disrupts hnRNP E1/eNOS 3'-UTR interactions via increased Akt-mediated serine phosphorylation (including serine 43) and increased nuclear localization of hnRNP E1. These mechanisms account, at least in part, for the decrease in eNOS mRNA stability under hypoxic conditions. Thus, the stabilization of human eNOS mRNA by hnRNP E1-containing RNP complexes serves as a key protective mechanism against the posttranscriptional inhibitory effects of antisense RNA and microRNAs under basal conditions but is disrupted under hypoxic conditions.