Endothelial nitric oxide synthase deficiency influences normal cell cycle progression and apoptosis in trabecular meshwork cells

AIM： To clarify how the endothelial nitric oxide synthase （eNOS, NOS3） make effect on outflow facility through the trabecular meshwork （TM）. METHODS： Inhibition of NOS3 gene expression in human TM cells were conducted by three siRNAs. Then the mRNA and protein levels of NOS3 in siRNA-treated and negative control （NC） cells were determined, still were the collagen, type IV, alpha 1 （COL4A1） and fibronectin 1 by real-time PCR and Western blot analysis. In addition, NOS3 concentrations in culture supernatant fluids of TM cells were measured. Cell cycle and cell apoptosis analysis were performed using flow cytometry. RESULTS： The mRNA level of NOS3 was decreased by three different siRNA interference, similar results were obtained not only of the relative levels of NOS3 protein, but also the expression levels of COL4A1 and fibronectin 1. The number of cells in S phase was decreased, while contrary result was obtained in G2 phase. The number of apoptotic cells in siRNA-treated groups were significant increased compared to the NC samples. CONCLUSION： Abnormal NOS3 expression can make effects on the proteins levels of extracellular matrix component （e.g. fibronectin 1 and COL4A1）. Reduced NOS3 restrains the TM cell cycle progression at the G2/ M-phase transition and induced cell apoptosis.

Effects of rotational culture on morphology,nitric oxide production and cell cycle of endothelial cells

Devices for the rotational culture of cells and the study of biological reactions have been widely applied in tissue engineering.However,there are few reports exploring the effects of rotational culture on cell morphology,nitric oxide(NO)production,and cell cycle of the endothelial cells from human umbili-cal vein on the stent surface.This study focuses on these parameters after the cells are seeded on the stents.Results showed that covering of stents by endothelial cells was improved by rotational culture.NO produc-tion decreased within 24 h in both rotational and static culture groups.In addition,rotational culture signifi-cantly increased NO production by 37.9%at 36 h and 28.9%at 48 h compared with static culture.Flow cytometry showed that the cell cycle was not obviously influenced by rotational culture.Results indicate that rotational culture may be helpful for preparation of cell-seeded vascular grafts and intravascular stents,which are expected to be the most frequently implanted materials in the future.

INSULIN INDUCES NITRIC OXIDE PRODUCTION IN BOVINE AORTIC ENDOTHELIAL CELLS

Objective To examine the effects of insulin on cell proliferation, nitric oxide (NO) release and nitric oxide synthase (NOS) gene expression in bovine aortic endothelial cells ( BAEC ) . Methods The mi togenesis was assessed by MTT method; the products of NO in the culture media, by Griess reaction; and the levels of NOS mRNA in BAEC , by RT/PCR tech nique. Results BAEC were not responsive to the growth-promoting effects of insulin. Stimulation with insulin resulted a dose-dependent rise of NO in the culture supernatants 2h later, with a maximum at 12～24h and a decline at 24h. This rise was inhibited by an inhibitor of NOS (L-NAME). NOS mRNA increased slightly in BAEC without statistical significance. Conelu sion The study suggested that the insulin-induced NO release might be caused directly by NOS activation.

Martentoxin, a large-conductance Ca^(2+)-activated K^+ channel inhibitor, attenuated TNF-α-induced nitric oxide release by human umbilical vein endothelial cells

Martentoxin, a 4,046 Da polypeptide toxin purified from the venom of the scorpion Buthus martensii Karsch, has been demonstrated to block large-conductance Ca2＋-activated K＋ （BKca） channels; however, its biological roles are still largely unknown. In the present study, we investigated the pharmacological effects of martentoxin on regulating the production of nitric oxide induced by TNF-a in human umbilical vein endothelial cells （HU- VECs）. We found that, 1, 10 and 100 ~tmol/L martentoxin decreased nitric oxide production by HUVECs ex- posed to 10 ng/mL TNF for 6, 12 and 24 hours. We further demonstrated that martentoxin inhibited the activity of iNOS and retarded the down-regulation of eNOS mRNA induced by TNF-a. Therefore, martentoxin could be a potential therapeutic agent for vascular diseases.

Effect of IBD sera on expression of inducible and endothelial nitric oxide synthase in human umbilical vein endothelial cells

AIM： To study the expression of endothelial and inducible nitric oxide synthases （eNOS and iNOS） and their role in inflammatory bowel disease （IBD）. METHODS： We examined the effect of sera obtained from patients with active Crohn＇s disease （CD） and ulcerative colitis （UC） on the function and viability of human umbilical vein endothelial cells （HUVEC）. HUVECs were cultured for 0-48 h in the presence of a medium containing pooled serum of healthy controls, or serum from patients with active CD or UC. Expression of eNOS and iNOS was visualized by immunofluorescence, and quantified by the densitometry of Western blots. Proliferation activity was assessed by computerized image analyses of Ki-67 immunoreactive cells, and also tested in the presence of the NOS inhibitor, 10^-4 mol/L L-NAME. Apoptosis and necrosis was examined by the annexin-V-biotin method and by propidium iodide staining, respectively. RESULTS： In HUVEC immediately after exposure to UC, serum eNOS was markedly induced, reaching a peak at 12 h. In contrast, a decrease in eNOS was observed after incubation with CD sera and the eNOS level was minimal at 20 h compared to control （18%±16% vs 23%± 15% P〈0.01）. UC or CD serum caused a significant increase in iNOS compared to control （UC： 300%±21%; CD： 275% ± 27% vs 108% ± 14%, P〈0.01）. Apoptosis/necrosis characteristics did not differ significantly in either experiment. Increased proliferation activity was detected in the presence of CD serum or after treatment with L-NAME. Cultures showed tube-like formations after 24 h treatment with CD serum. CONCLUSION： IBD sera evoked changes in the ratio of eNOS/iNOS, whereas did not influence the viability of HUVEC. These involved down-regulation of eNOS and up-regulation of iNOS simultaneously, leading to increased proliferation activity and possibly a reduced antiinflammatory protection of endothelial cells.

Changes in Human Umbilical Vein Endothelial Cells Induced by Endothelial Nitric Oxide Synthase Traffic Inducer

This study investigated the changes in human umbilical vein endothelial cells （HUVECs） induced by overexpression of endothelial nitric oxide synthase traffic inducer （NOSTRIN） and its role in cellular injury. Recombinant NOSTRIN-expressing and empty vectors were transfected into cultured HUVECs, and factor Ⅷ-related antigen was examined by using immunohistochemical analysis. Growth curves were generated for both transfected and untransfected cells and these indicated that the prolifera- tive ability of cells overexpressing NOSTRIN was significantly decreased. The expression of NOSTRIN and eNOS proteins was detected by using Western blot analysis, endothelial NOS （eNOS） activity was assayed by using spectrophotometry, and NO2-/NO3- levels were measured usin~ nitrate reductase. Immunohistochemical analysis demonstrated that all groups expressed NOSTRIN in the plasma mem- brane and cytoplasm, and Western blot analysis confirmed that NOSTR1N levels were significantly higher in cells transfected with the NOSTR1N plasmid （P〈0.01）. The activity of eNOS and the levels of NO2-/NO3 were significantly decreased in NOSTRIN overexpressing cells as compared with empty vector and untransfected cells （P〈0.01 and P〈0.01, respectively）. Morphological and ultrastructural changes were observed under light and electron microscopy, and it was found that NOS- TRIN-overexpressing cells were elongated with deformities of the karyotheca, injury to the plasma membrane, increased lipids in the cytoplasm, and shortened microvilli. This study showed that overex- pression of NOSTRIN had a significant effect on eNOS activity in HUVECs and resulted in significant cellular damage.

Shear stress effect on endothelial nitric oxide synthase in cultured human umbilical vein endothelial cells

Background: Low shear stress caused by disturbed or turbulent flow at arterial branch points is known to associate with atherosclerosis. However, shear stress at the venous valve location and its association with deep vein thrombosis are less understood due to the complex and poorly understood bi-directional flow in the valve pocket region. We investigated how venous endothelial cells respond to flow shear stress around the venous valve region using a novel in vitro system that mimics venous flow. Results: Human umbilical vein EAhy. 926 cells were cultured on a flexible silastic membrane that mimicked venous tissue. Confluent cells were exposed to sinusoidal uni-and bi-directional pulsatile shear stress (0.1 to 1 dyne/cm2) for up to 6 h. Western-blot analyses indicated that endothelial nitric oxide (eNOS) expression levels decreased regardless of all tested flow patterns, stress magnitude, and shearing time. In contrast, the expression levels of inhibitor of κB (kappa B) and α (alpha)-tubulin were unaffected by the shear stress. Conclusions: Our results indicate that shear stress causes a decrease specifically in eNOS expression, suggesting that it may play a significant role in regulating inflammation related protein expression in endothelial cells.

Adhesion of different cell cycle human hepatoma cells to endothelial cells and roles of integrin β_1

AIM: To investigate the adhesive mechanical properties of different cell cycle human hepatoma cells (SMMC-7721) to human umbilical vein endothelial cells (ECV-304), expression of adhesive molecule integrinβ1 in SMMC-7721 cells and its contribution to this adhesive course. METHODS: Adhesive force of SMMC-7721 cells to endothelial cells was measured using micropipette aspiration technique. Synchronous G1 and S phase SMMC-7721 cells were achieved by thymine-2-deoxyriboside and colchicines sequential blockage method and double thymine-2-deoxyriboside blockage method, respectively. Synchronous rates of SMMC-7721 cells and expression of integrinβ1 in SMMC-7721 cells were detected by flow cytometer. RESULTS: The percentage of cell cycle phases of general SMMC-7721 cells was 11.01% in G2/M phases, 53.51% in G0/G1 phase, and 35.48% in S phase. The synchronous rates of G1 and S phase SMMC-7721 cells amounted to 74.09% and 98.29%, respectively. The adhesive force of SMMC-7721 cells to endothelial cells changed with the variations of adhesive time and presented behavior characteristics of adhesion and de-adhesion. S phase SMMC-7721 cells had higher adhesive forces than d phase cells [(307.65±92.10)×10-10N vs(195.42±60.72)×10-10N, P<0.01]. The expressive fluorescent intensity of integrinβ1 in G1 phase SMMC-7721 cells was depressed more significantly than the values of S phase and general SMMC-7721cells. The contribution of adhesive integrinβ1 was about 53% in this adhesive course. CONCLUSION: SMMC-7721 cells can be synchronized preferably in d and S phases with thymine-2-deoxyriboside and colchicines. The adhesive molecule integrinβ1 expresses a high level in SMMC-7721 cells and shows differences in various cell cycles, suggesting integrin β1 plays an important role in adhesion to endothelial cells. The change of adhesive forces in different cell cycle SMMC-7721 cells indicates that S phase cells play predominant roles possibly while they interact with endothelial cells.

Crosstalk among Oxidative Stress,Autophagy,and Apoptosis in the Protective Effects of Ginsenoside Rb1 on Brain Microvascular Endothelial Cells:A Mixed Computational and Experimental Study

Objective Brain microvascular endothelial cells (BMECs) were found to shift from their usually inactive state to an active state in ischemic stroke (IS) and cause neuronal damage. Ginsenoside Rb1 (GRb1),a component derived from medicinal plants,is known for its pharmacological benefits in IS,but its protective effects on BMECs have yet to be explored. This study aimed to investigate the potential protective effects of GRb1 on BMECs. Methods An in vitro oxygen-glucose deprivation/reperfusion (OGD/R) model was established to mimic ischemia-reperfusion (I/R) injury. Bulk RNA-sequencing data were analyzed by using the Human Autophagy Database and various bioinformatic tools,including gene set enrichment analysis (GSEA),Gene Ontology (GO) classification and enrichment analysis,Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis,protein-protein interaction network analysis,and molecular docking. Experimental validation was also performed to ensure the reliability of our findings. Results Rb1 had a protective effect on BMECs subjected to OGD/R injury. Specifically,GRb1 was found to modulate the interplay between oxidative stress,apoptosis,and autophagy in BMECs. Key targets such as sequestosome 1 (SQSTM1/p62),autophagy related 5 (ATG5),and hypoxia-inducible factor 1-alpha (HIF-1α) were identified,highlighting their potential roles in mediating the protective effects of GRb1 against IS-induced damage. Conclusion GRbl protects BMECs against OGD/R injury by influencing oxidative stress,apoptosis,and autophagy. The identification of SQSTM1/p62,ATG5,and HIF-1α as promising targets further supports the potential of GRb1 as a therapeutic agent for IS,providing a foundation for future research into its mechanisms and applications in IS treatment.

EFFECT OF TNF-a AND IFN-g ON THE EXPRESSION OF INDUCIBLE NITRIC OXIDE SYNTHASE GENE AND PROLIFERATION INHIBITION OF HUMAN COLON CANCER CELL LINE

Objective: To study the expression of the inducible nitric oxide synthase (iNOS) gene and the effects of tumor necrosis factor-α(TNF-a) and interferon-γ(IFN-g)on proliferation of the continuous cultured human colon cancer cell line CCL229. Methods: Using the molecular and biochemical techniques and electron microscopy to analyze the expression of iNOS, production of NO and growth characteristics of human colon cancer cells. Results: cytokine treatment can induce expression of the iNOS gene and production of nitric oxide was significantly higher after treatment of CCL229 cells with TNF-αor IFN-γ. Treatment with either cytokine or a combination of both significantly increased levels of Malondialdehyde (MDA) over control. Furthermore, cytokine treatment increased the proliferation inhibition rate as assessed in vitro and decreased the cell proliferation index on flow cytometry. Electron microscopy showed that cells treated with cytokines had fewer pseudopodia or cell processes than control cells and that cytokine treated cells had dilatation of the mitochondria and endoplasmic reticulum and dilated vesicular or tubular cisternae. Conclusion: Our findings indicate that TNF-α and IFN-γ induce the expression of iNOS gene in CCL229 cells, which increases the production of nitric oxide, inhibits proliferation, causes lipid peroxidation, and results in ultrastructural changes.

Inflammatory cytokines promote inducible nitric oxide synthase-mediated DNA damage in hamster gallbladder epithelial cells

AIM: To investigate the link between chronic biliary inflammation and carcinogenesis using hamster gallbladder epithelial cells. METHODS: Gallbladder epithelial cells were isolated from hamsters and cultured with a mixture of inflammatory cytokines including interleukin-1β,interferon-γ,and tumor necrosis factor-α. Inducible nitric oxide synthase (iNOS) expression,nitric oxide (NO) generation,and DNA damage were evaluated. RESULTS: NO generation was increased significantly following cytokine stimulation,and suppressed by an iNOS inhibitor. iNOS mRNA expression was demonstrated in the gallbladder epithelial cells during exposure to inflammatory cytokines. Furthermore,NO-dependent DNA damage,estimated by the comet assay,was significantly increased by cytokines,and decreased to control levels by an iNOS inhibitor. CONCLUSION: Cytokine stimulation induced iNOS expression and NO generation in normal hamster gallbladder epithelial cells,which was sufficient to cause DNA damage. These results indicate that NO-mediated genotoxicity induced by inflammatory cytokines through activation of iNOS may be involved in the process of biliary carcinogenesis in response to chronic inflammation of the biliary tree.

Production of reactive oxygen species and expression of inducible nitric oxide synthase in rat isolated Kupffer cells stimulated by Leptospira interrogans and Borrelia burgdorfen

AIM： To evaluate the production of reactive oxygen species （ROS） and the expression of inducible nitric oxide synthase （iNOS） in rat isolated Kupffer cells （KCs） stimulated by Leptospira interrogans and Borrelia burgdorferi. METHODS： Rat Kupffer cells were separated by perfusion of the liver with 0.05% collagenase, and purified by Percoll gradients. Pudfied Kupffer cells were tested in vitro with alive L.interogans and B. burgdorferi preparations. The production of ROS was determined by chemiluminescence, whereas iNOS protein expression was evaluated by Western blot assay using anti-iNOS antibodies. RESULTS： B. burgdorferi and to a less extent L. interrogans induced ROS production with a peak 35 min after infection. The chemiluminescence signal progressively diminished and was undetectable by 180 min of incubation. Leptospirae and borreliae induced an increased iNOS expression in Kupffer cells that peaked at 6 hours and was still evident 22 h after infection. CONCLUSION： Both genera of spirochetes induced ROS and iNOS production in rat Kupffer cells. Since the cause of liver damage both in leptospiral as well as in borrelial infections are still unknown, we suggest that leptospira and borrelia damage of the liver can be initially mediated by oxygen radicals, and is then maintained at least in part by nitric oxide.

Protection of Salvianolic Acid B for Human Endothelial Cells Against Hydrogen Peroxide-Induced Oxidative Damage

Salvianolic acid B(Sal B) is an active component of traditional Chinese medicine Salvia miltiorrhiza and is used to treat vascular diseases. To better understand its mechanism, the antioxidant capacities of Sal B was evaluated with human endothelial cells under oxidative stress. Human endothelial cells were pretreated with Sal B for 12 h followed by hydrogen peroxide for another 12 h. Production of reactive oxygen species (ROS), activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX), and concentration of glu-tathione were measured. Protective effect of Sal B on the endothelial cells from hydrogen peroxide-induced damage was observed, and ROS production in the cells was found significantly inhibited. Sal B remarkably enhanced the activities of antioxidant enzymes SOD, CAT and GPX. Furthermore, Sal B up-regulated the intracellular glu-tathione concentration. The results indicate that Sal B protected endothelial cells from oxidative stress by improving the redox status of the cells through enhancing the antioxidant enzyme activities and increasing the reductive glu-tathione concentration after the oxidative challenge.

INHIBITION OF NITRIC OXIDE SYNTHESIS INCREASES THE SECRETION OF ENDOTHELIN-1 IN VIVO AND IN CULTURED ENDOTHELIAL CELLS

The aim of this study is to investigate the effects of nitric oxide, formed from L-arginine, on the production of endothelin?1 in vivo and in cultured endothelial cells. In mechanically ventilated anesthetized dogs (n = 5), mean pulmonary arterial pressure (PAPm) and pulmonary vascular resistance (PVR) during hypoxic ventilation (FIO2 = 0.10) was 25 ?3.1 kPa and 68.7 ?10.2 kPa.s / L respectively. IG-nitro-L-arginine methylester (L-NAME), an inhibitor of nitric oxide synthase, increased the peak value of PAPm and PVR during hypoxic ventilation to 36.6 ?4.7 kPa and 158.4 ?25 kPa.s / L and its effect lasted for 2-3 hours. Meanwhile, plasma endothelin? level in the femoral artery increased by 20.9+ 7.1, 25.6?7.7, 28.6?7.9 pg / ml at the 60 th, 120th, 180th minute after the injection of L-NAME respectively (P<0.05 vs hypoxic control before the injection). In cultured endothelial cells from umbilical veins, endothelin-1 level of culture medium in control group was 35.1 ?.9 pg / 105 cells /ml (n=9). L-NAME increased endothelin-1 level to 42.8 ?4.9pg / 105 cells / ml (n = 9, P < 0.05) in case of 10-11 mol / L and to 43.0+ 4.7 pg / 105 cells / ml in case of 10 -7 mol/L (n=9, F<0.05). These findings indicate that endogenous nitric oxide is an inhibitory modulator of hypoxic pulmonary vasoconstriction and that nitric oxide inhibits the production of endothelin? in vivo and in cultured vascular endothelial cells.

Protective effects of icariin on human umbilical vein endothelial cell injured by angiotensin Ⅱ

To investigate the effects of icariin （ICA） on angiotensin Ⅱ（Ang Ⅱ）-induced injury in human umbilical vein endothelial cells line （ECV-304）. The ECV-304 cells were cultured in vitro. After 24 h incubating with icariin, the model of AngⅡ-induced injury in ECV-304 was established. The cell viability （MTT method）, Lactate dehydrogenase （LDH） release and Nitric oxide （NO） production in the medium, the capacity of scavenging superoxide anion radicals （O2^-） and hydroxyl radicals （.OH） were measured. The activities of superoxide dismutase （SOD）, total nitric oxide synthase （T-NOS）, inducible nitric oxide synthase （iNOS） and constitutive nitric oxide synthase （cNOS） in the cells were determined. Compared with the Ang Ⅱ-treated group, ICA can significantly raise the viability of EC, increase the activities of SOD, T-NOS and cNOS, increase the production of NO, enhance the capacity of scavenging superoxide anion radicals （ O2^- ） and hydroxyl radicals（.OH）, and lower LDH leakage and iNOS activity. The results suggest that ICA can protect endothelial cells （ECV-304） from Ang II-induced injury.

Implication of serum levels of interleukin-18 and nitric oxide in tumor growth and metastasis of non-small cell lung cancer

To study the effect of IL-18 and nitric oxide(NO) on the growth and metastasis of non-small cell lung cancer (NSCLC).Methods Serum IL-18 and nitrate and nitrite levels in 82 patients with NSCLC and 20 healthy control subjects were measured by using ELISA and Griess.Results The levels of serum IL-18 were (334.2±31.0)ng/L in NSCLC patients and (151.3±22.0)ng/L in control subjects,respectively.The levels of nitrate and nitrite were (237.1±21.0)μmol/L in NSCLC patients and (44.2±15.0)μmol/L in control subjects.The levels of serum IL-18 and nitrate and nitrite were not related with age,gender,histological types in patients with NSCLC.The levels of serum IL-18 was closely associated with TNM stage,lymph node metastasis and distal metastasis,but not with its degree and organ types of metastasis.There was a negative correlation between the levels of serum IL-18 and nitrate and nitrite.Conclusion Serum IL-18 and nitrate and nitrite levels may be useful to evaluate the prognosis of the patients with NSCLC.16 refs,2 tabs.

Effect of a nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester on invasion of human colorectal cancer cell line SL-174T

AIML To investigate the effect and mechanism of action of the nitric oxide synthase （NOS） inhibitor NG-nitro-L-arginine methyl ester （L-NAME） on invasion and metastasis of human colorectal cancer cell line SL-174T. METHODS： Human colorectal cancer cel4 line SL-174T was cultured and treated separately with four different dosages of L-NAME for 72 h, Nitric oxide （NO） production was measured with Griess reagent, The effect of L-NAME on invasion and migration of SL-174T cells were evaluated by using Transwell chambers attached with polycarbonate filters and reconstituted basement membrane （Matrigel）, RT-PCR was performed to determine the mRNA levels of matrix metalloproteinase-2 （MMP-2） and tissue inhibitor metalloproteinase-2 （TIMP-2）,RESULTS： L-NAME could significantly inhibit NO production of SL-174T in a dose-dependent manner. After being treated for 72 h with 0.2, 0.4, 0.8, and 1.0 mmol/L L- NAME, respectively, the ability of the L-NAME treated SL- 174T cells to invade the reconstituted basement membrane decreased significantly （t = 8.056, P〈0.05; t= 14.467, P〈0.01; t= 27.785, P〈0.01; and t= 29.405, P〈0.01, respectively） and the inhibition rates were 10.29%, 19.62%, 34.08%, and 42.23%, respectively. Moreover, L-NAME could inhibit migration of SL-174T cells, and the inhibition rates were 20.76%, 24.95%, 39.43%, and 46. 85% for L-NAME at 0.2, 0.4, 0.8, and 1.0 mmol/L, respectively （t = 15.116, P〈0.01）. In addition, after treatment with L-NAME, expression of MMP-2 mRNA was significantly decreased （t = 71.238, P〈0.01） and that of TIMP-2 mRNA was markedly increased （t = -13.020, P〈0.01）. CONCLUSION： L-NAME exerts anti-invasive and anti- metastatic effects on SL-174T cell line via downregulating MNP-2 mRNA expression and upregulating TIMP-2 mRNA expression.

Role of nitric oxide in the maintenance of pluripotency and regulation of the hypoxia response in stem cells

Stem cell pluripotency and differentiation are global processes regulated by several pathways that have been studied intensively over recent years. Nitric oxide(NO) is an important molecule that affects gene expression at the level of transcription and translation and regulates cell survival and proliferation in diverse cell types. In embryonic stem cells NO has a dual role, controlling differentiation and survival, but the molecular mechanisms by which it modulates these functions are not completely defined. NO is a physiological regulator of cell respiration through the inhibition of cytochrome c oxidase. Many researchers have been examining the role that NO plays in other aspects of metabolism such as the cellular bioenergetics state, the hypoxia response and the relationship of these areas to stem cell stemness.

Levels of biological markers of nitric oxide in serum of patients with squamous cell carcinoma of the oral cavity

The aim of the study was a determination of the levels of nitric oxide（NO）and its biological markers such as malonyldialdehyde（MDA）and nitrotyrosine in the serum of patients with squamous cell carcinoma（SCC）of the oral cavity and identification of the relationships between NO and those markers.These studies were performed on patients with SCC of the oral cavity before and after treatment.Griess reaction was used for the estimation of the total concentration of NO in serum.The nitrotyrosine level in serum was assessed with an enzyme-linked immunosorbent assay（ELISA）kit,and MDA level using a spectrophotometric assay.Higher concentrations of NO in blood serum were determined in patients with stage IV of the disease before treatment in comparison to the control group and patients with stages II and III of the disease.Moreover,higher concentrations of MDA and nitrotyrosine were determined in the serum of patients in all stages of the disease in comparison to healthy people.After treatment,lower concentrations of NO in the serum of patients with stage IV of the disease were observed in comparison to the amounts obtained prior to treatment.In addition,lower levels of nitrotyrosine in the serum of patients with all stages of the disease were recorded,whereas higher concentrations of MDA were determined in these patients in comparison to results obtained before treatment.The compounds formed with the contribution of NO,such as MDA and nitrotyrosine,may lead to cancer progression in patients with SCC of the oral cavity,and contribute to formation of resistance to therapy in these patients as well.Moreover,the lack of a relationship between concentrations of NO and MDA,and between NO and nitrotyrosine in serum suggests that the process of lipid peroxidation and nitration in patients with SCC does not just depend on NO.

Regulation of mitochondrial function and endoplasmic reticulum stress by nitric oxide in pluripotent stem cells

Mitochondrial dysfunction and endoplasmic reticulum stress(ERS) are global processes that are interrelated and regulated by several stress factors. Nitric oxide(NO) is a multifunctional biomolecule with many varieties of physiological and pathological functions, such as the regulation of cytochrome c inhibition and activation of the immune response, ERS and DNA damage; these actions are dose-dependent. It has been reported that in embryonic stem cells, NO has a dual role, controlling differentiation, survival and pluripotency, but the molecular mechanisms by which it modulates these functions are not yet known. Low levels of NO maintain pluripotency and induce mitochondrial biogenesis. It is well established that NO disrupts the mitochondrial respiratory chain and causes changes in mitochondrial Ca^(2+) flux that induce ERS. Thus, at high concentrations, NO becomes a potential differentiation agent due to the relationship between ERS and the unfolded protein response in many differentiated cell lines. Nevertheless, many studies have demonstratedthe need for physiological levels of NO for a proper ERS response. In this review, we stress the importance of the relationships between NO levels, ERS and mitochondrial dysfunction that control stem cell fate as a new approach to possible cell therapy strategies.