Increased eNOS mRNA Expression Research Articles

Isoliquiritigenin: a potential drug candidate for the management of erectile dysfunction.

This study aimed to assess the erectogenic properties of isoliquiritigenin taking sildenafil (SDF) as the standard. The binding affinity of isoliquiritigenin (ISL) with the erectile marker proteins (endothelial nitric oxide synthase [eNOS] and enzyme phosphodiesterase type 5 [PDE5]) was investigated using Autodock Vina, which was validated using molecular dynamics simulation. Furthermore, the effect of ISL on the eNOS and PDE5 messenger ribonucleic acid (mRNA) expression and the sexual behavior of mice was investigated, along with the assessment of the pharmacokinetics of ISL. The results revealed that the binding affinity of ISL-eNOS/PDE5 and SDF-eNOS/PDE5 was in the range of -7.5 to -8.6 kcal/mol. The ISL-eNOS/PDE5 complexes remained stable throughout the 100 ns simulation period. Root mean square deviation, Rg, SASA, hydrogen, and hydrophobic interactions were similar between ISL-eNOS/PDE5 and SDF-eNOS/PDE5. Analysis of mRNA expressions in paroxetine (PRX)-induced ED mice showed that the co-administration of PRX with ISL reduced PDE5 and increased eNOS mRNA expression, similar to the co-administered group (PRX+SDF). The sexual behavior study revealed that the results of PRX+ISL were better than those of the PRX+SDF group. Pharmacokinetic evaluation further demonstrated that ISL possesses drug-like properties. The results showed that ISL is equally potent as SDF in terms of binding affinity, specific pharmacological properties, and modulating sexual behavior.

Endothelial nitric oxide synthase in rat brain is downregulated by sub-chronic antidepressant treatment.

Nitric oxide (NO) is a neurotransmitter that may be related to major depressive disorder (MDD) because the selective neuronal NO synthase (NOS) inhibitor, 7-nitroindazole, induces a dose-dependent antidepressant-like effect. NO modulates major neurotransmitters involved in the neurobiology of MDD, such as norepinephrine, serotonin, dopamine, and glutamate. In this study, we investigated the effects of antidepressants as NO modulators in acute and sub-chronic treatments. Rats were injected with the SSRI paroxetine (PAR, 10mg/kg), the SNRI milnacipran (MIL, 30mg/kg), or the NaSSA mirtazapine (MIR, 10mg/kg) for acute (1h) or sub-chronic (3weeks) treatment prior to analysis of nine brain regions (frontal cortex, temporal cortex, striatum, thalamus, hippocampus, midbrain, pons, cerebellum, and olfactory bulb). The mRNA expression levels of three NOS subtypes (neuronal: nNOS, inducible: iNOS, and endothelial: eNOS) were analyzed using real-time PCR with Taqman probes. Acute MIR treatment significantly increased nNOS mRNA expression in the hippocampus, midbrain, cerebellum and olfactory bulb, and iNOS mRNA expression in the frontal cortex and midbrain. Acute PAR and MIR treatments significantly increased eNOS mRNA expression in most brain regions. Conversely, sub-chronic treatment with all types of antidepressants resulted in significant decreases of eNOS mRNA expression in most brain regions. Sub-chronic treatment with the three types of antidepressants consistently decreased eNOS mRNA expression levels in the rat brain. These effects may be associated with the involvement of the NO system in the mechanism of action of antidepressants.

Erythropoietin Reverses Sepsis-Induced Vasoplegia to Norepinephrine Through Preservation of α1D-Adrenoceptor mRNA Expression and Inhibition of GRK2-Mediated Desensitization in Mouse Aorta.

We investigated the effect of erythropoietin (EPO) posttreatment on survival time and vascular functions in a mouse model of sepsis. Sepsis was induced by cecal ligation and puncture. After 20 ± 2 hours of sepsis, thoracic aorta was isolated for assessing its reactivity to norepinephrine (NE) and acetylcholine (ACh). We also measured the tissue nitric oxide (NO) level, inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS), G protein-coupled receptor kinase 2 (GRK2), and α1D adrenoceptor messenger RNA (mRNA)/protein expression. In septic mice, EPO moderately improved the survival time from 19.68 ± 0.75 to 34.7 ± 3.2 hours. Sepsis significantly decreased the aortic contractile response to NE along with reduced α1D mRNA and protein expression. Erythropoietin significantly preserved the α1D receptor expression and restored NE-induced contractions to control levels in septic mice. Further, it attenuated the aortic α1D receptor desensitization in sepsis which was evident from reduced GRK2 mRNA expression. Accordingly, a selective GRK2 inhibitor markedly restored the contractile responses to NE in sepsis. Erythropoietin treatment attenuated iNOS mRNA expression and iNOS-induced overproduction of NO, but improved endothelium-dependent relaxation to ACh associated with increased eNOS mRNA expression. In conclusion, EPO seems to reverse sepsis-induced vasoplegia to NE through the preservation of α1D adrenoceptor mRNA/protein expression, inhibition of GRK2-mediated desensitization, and attenuation of NO overproduction in the mouse aorta.

Nitric oxide (NO) reversed TNF-α inhibition of trophoblast interaction with endothelial cellular networks

IntroductionThe interaction between trophoblast cells and maternal uterine endothelium is important for placental vascular modelling. Nitric oxide (NO) is a potent vasorelaxant that regulates systemic blood pressure and is reduced in preeclampsia. NO may affect placental cell interaction. ObjectivesThis study was to examine whether NO plays a role in regulating TNF-α induced inhibition of trophoblast cell integration into endothelial cellular networks in-vitro. MethodsRed fluorescent-labelled human uterine myometrial microvascular endothelial cells (UtMVECs) were seeded on Matrigel. After endothelial cellular networks formed, green fluorescent-labelled HTR-8/SVneo trophoblast cells were co-cultured with endothelial cells, together with/without TNF-α (0.5 ng/ml) and/or NO donor, sodium nitroprusside dihydrate (SNP) (100 μM). Images were captured after 24 h. The effects on HTR-8/SVneo cell integration were quantified by Image Analysis software. The cells were then recovered from Matrigel to extract mRNA. Quantitative PCR was performed to evaluate the expression of eNOS, VCAM-1 and E-selectin. The concentrations of sVCAM-1 and sE-selectin in the conditioned medium were measured by ELISA. ResultsTNF-α inhibited HTR-8/SVneo trophoblast cell integration into endothelial cellular networks, as well as decreased eNOS mRNA expression. Increases in VCAM-1 and E-selectin in cellular mRNA and protein concentrations in the conditioned medium were also seen. The NO donor reversed the inhibitory effect of TNF-α on trophoblast integration and increased eNOS mRNA expression. SNP also reduced sE-selectin and sVCAM-1 expressions which were increased by TNF-α in the conditioned medium. ConclusionOur data suggest the inhibitory effect of TNF-α on trophoblast integration may be mediated by NO, via reducing endothelial cell activation.

REMEDIAL EFFECTS OF VITAMIN E AND L-ARGININE ON PERIPHERAL NEUROPATHY IN STREPTOZOTOCIN-INDUCED DIABETIC RATS

It was shown that hyperglycemia in diabetic patient s is the main factor of diabetic peripheral neuropathies. Various pathways related to oxidative stress, vascular defect and defective endothelium dependent relaxation have been implicated in the de velopment of diabetic peripheral neuropathy. A substantial number of studies have shown that antio xidant treatment are promising therapeutics that ca n prevent or correct reduced motor nerve conduction i n diabetic rats by acting on these mechanisms. This study was designed to investigate the possible role of insulin treatment along with or without vitamin E or L-arginine on diabetic neuropathy. This goal was accessed by examining nerve conduction, parameters of oxidative stress and lipid peroxidation as well as the expression level of endothelial nitric oxide synthase in the sciatic nerve of control and strept ozotocine-induced diabetic rats. Data showed that diabetic rats showed increased levels of serum gluc ose (382.5%) and sciatic nerve lipid peroxidation Marker (MDA, 261.6%) with a concomitant decrease in the expression of sciatic nerve eNOS mRNA as compared to control rats. The nerve conduction stud ies of the sciatic nerves of these rats showed decr ease conduction as evident by delayed NCV (63.6%) and low Amplitude of Muscle Contraction (AMC, 36.4%). Solitary insulin treatment (but not vitamin -E or L-arginine) corrected serum glucose to contro l values and corrected nerve conduction parameters in the sciatic nerve. However, treating diabetic rats with different doses of vitamin E (300 mg kg -1 and 600 mg kg -1 ) significantly reduced oxidative stress by decreasing MDA and increasing GPx activity, corrected NCV by reducing the latency and improving AMC and increased eNOS mRNA expression in sciatic nerve with a more profound effect to seen with the high dose (600 mg kg -1 ). However, the maximum potent ameliorating effect of the vitamin E on these parameters was seen when administered in combination with insulin. On the other hand, L-arginine treatment alone or in combination with insulin had no effect on the oxidative stress markers nor eNOS expression but significantly and maximally improved NCV through reducing the conduction latency and increasing AMC. This study supported the notion tha t diabetic peripheral neuropathy is a multifactoria l complication, caused by hyperglycemia, oxidative st ress and vascular impairment. It is concluded that conjugate treatment with vitamin-E, especially in h igher doses, with insulin could be of great value. Moreover correction of impaired nerve blood flow by drugs that induce nitric oxide has proved to be efficient in the protection against and correction of experimental diabetic peripheral neuropathy.

Effects of heat exposure during fixed time on thoracic aorta contractility in rats

Objective To investigate the effect of heat exposure during fixed time on the thoracic aorta contractile properties of rats and the related mechanism.Methods Male Wistar rats were randomly divided into control group,random-time heat exposure group and fixed-time heat exposure group(ambient temperature to 24℃;the two heat exposure groups were exposed to heat \ 6 h daily). After 7 days of heat exposure,the body core temperature of rats was measured by telemetry technology.The response of isolated thoracic aorta to noradrenaline(NA) was determined by organ bath system.Expression of aorta eNOS mRNA and plasma NOx(NO2-and NO3-) content were also examined.Results Heat exposure showed no significant effects on body core temperature of rats(P0.05).Compared with control group,the fixed-time heat exposure group had significantly lowered thoracic aortic vascular reactivity to NA(P0.01).Compared with control group and random-time heat exposed group,the fixed-time heat exposure group had significantly increased eNOS mRNA expression and plasma NOx content(P0.05). ConclusionFixed-time exposure to heat can change the reactivity of the thoracic aorta in rats,and vascular endothelial cell-derived NOS pathway may be involved in the vasodilatory effects.

Epigenetic Regulation of Endothelial Lineage Committed Genes in Pro-Angiogenic Hematopoietic and Endothelial Progenitor Cells

Proangiogenic hematopoietic and endothelial progenitor cells (EPCs) contribute to postnatal neovascularization, but the mechanisms regulating differentiation to the endothelial lineage are unclear. To elucidate the epigenetic control of endothelial gene expression in proangiogenic cells and EPCs. Here we demonstrate that the endothelial nitric oxide synthase (eNOS) promoter is epigenetically silenced in proangiogenic cells (early EPCs), CD34(+) cells, and mesoangioblasts by DNA methylation and prominent repressive histone H3K27me3 marks. In order to reverse epigenetic silencing to facilitate endothelial commitment, we used 3-deazaneplanocin A, which inhibits the histone methyltransferase enhancer of zest homolog 2 and, thereby, reduces H3K27me3. 3-Deazaneplanocin A was not sufficient to increase eNOS expression, but the combination of 3-deazaneplanocin A and the histone deacetylase inhibitor Trichostatin A augmented eNOS expression, indicating that the concomitant inhibition of silencing histone modification and enhancement of activating histone modification facilitates eNOS expression. In ischemic tissue, hypoxia plays a role in recruiting progenitor cells. Therefore, we examined the effect of hypoxia on epigenetic modifications. Hypoxia modulated the balance of repressive to active histone marks and increased eNOS mRNA expression. The reduction of repressive H3K27me3 was associated with an increase of the histone demethylase Jmjd3. Silencing of Jmjd3 induced apoptosis and senescence in proangiogenic cells and inhibited hypoxia-mediated up-regulation of eNOS expression in mesoangioblasts. These findings provide evidence that histone modifications epigenetically control the eNOS promoter in proangiogenic cells.

Repeated and long-term treatment with physiological concentrations of resveratrol promotes NO production in vascular endothelial cells

In the present study, we examined the effect of repeated and long-term treatment with resveratrol on NO production in endothelial cells as a model of routine wine consumption. Repeated treatment with resveratrol for 5 d resulted in an increase in endothelial NO synthase (eNOS) protein content and NO production in human umbilical vein endothelial cell (HUVEC) in a concentration-dependent manner. A significant increase in functional eNOS protein content was observed with resveratrol, even at 50 nm. In contrast, eNOS phosphorylation was not stimulated and inducible NO synthase (iNOS) was not detected after resveratrol treatment. Both eNOS protein and mRNA expression were promoted by 50 nm-resveratrol in a time-dependent manner. Increased eNOS mRNA expression in response to resveratrol was not decreased by an oestrogen receptor (ER) antagonist ICI182780, a PPARα inhibitor MK886 or a sirtuin inhibitor Salermide. However, a combination of ICI182780 and MK886 significantly inhibited resveratrol-induced eNOS mRNA expression. Salermide had no effect even in the presence of ICI182780 or MK886. These results demonstrate that resveratrol within the physiological range increases eNOS mRNA and protein expression through ER and PPARα activation, thereby promoting NO production in endothelial cells. eNOS induction might result from the accumulative effect of nanomolar concentrations of resveratrol. The present study results can account in part for the observation that cardiovascular benefits of red wine are experienced with routine consumption, but not with acute consumption.

Effect of high soy diet on the cerebrovasculature and endothelial nitric oxide synthase in the ovariectomized rat

High soy (HS) diets are neuroprotective and promote vascular dilatation in the periphery. We hypothesized that an HS diet would promote vascular dilatation in the cerebrovasculature by mimicking estradiol's actions on the endothelial nitric oxide synthase (eNOS) system including increasing eNOS expression and decreasing caveolin-1 expression to increase nitric oxide (NO) production. Ovariectomized rats were fed HS or a soy-free diet (SF) ± low physiological estradiol (E2) for 4 weeks. Neither E2 nor HS altered middle cerebral artery (MCA) structure or vascular responses to acetylcholine, serotonin, or phenylephrine. Estradiol enhanced bradykinin-induced relaxation in an eNOS-dependent manner. Although E2 and HS increased eNOS mRNA expression in the brain and cerebrovasculature, they had no effect on eNOS protein expression or phosphorylation in the MCA. However, E2 decreased caveolin-1 protein in the MCA. In MCAs neither E2 nor HS altered estrogen receptor (ER) alpha expression, but E2 did reduce ER beta levels. These data suggest that HS diets have no effect on vascular NO production, and that E2 may modulate basal NO production by reducing the expression of caveolin-1, an allosteric inhibitor of NOS activity. However, the effects of E2 and HS on the cerebrovasculature are small and may not underlie their protective actions in pathological states.

Piper Sarmentosum Increases Nitric Oxide Production in Oxidative Stress: A Study on Human Umbilical Vein Endothelial Cells

OBJECTIVE:Nitric oxide produced by endothelial nitric oxide synthase (eNOS) possesses multiple anti-atherosclerotic properties. Hence, enhanced expression of eNOS and increased Nitric oxide levels may protect against the development of atherosclerosis. Piper sarmentosum is a tropical plant with antioxidant and anti-inflammatory activities. This study aimed to investigate the effects of Piper sarmentosum on the eNOS and Nitric oxide pathway in cultured human umbilical vein endothelial cells (HUVECs).METHODS:HUVECs were divided into four groups: control, treatment with 180 μM hydrogen peroxide (H2O2), treatment with 150 μg/mL aqueous extract of Piper sarmentosum, and concomitant treatment with aqueous extract of PS and H2O2 for 24 hours. Subsequently, HUVECs were harvested and eNOS mRNA expression was determined using qPCR. The eNOS protein level was measured using ELISA, and the eNOS activity and Nitric oxide level were determined by the Griess reaction.RESULTS:Human umbilical vein endothelial cells treated with aqueous extract of Piper sarmentosum showed a marked induction of Nitric oxide. Treatment with PS also resulted in increased eNOS mRNA expression, eNOS protein level and eNOS activity in HUVECs.CONCLUSION:Aqueous extract of Piper sarmentosum may improve endothelial function by promoting NO production in HUVECs.

Role of oxidative stress in elevated blood pressure induced by high free fatty acids

The aim of the study is to investigate the possible mechanism of oxidative stress in the high free fatty acids (FFAs)-induced hypertension. Male Sprague-Dawley rat models were established and classified into three groups, namely the control group (NC group), the FFA group, and the N-acetylcysteine (NAC) group. Blood pressure (BP) was recorded. An organ chamber experiment was performed to determine endothelium-dependent/-independent vasodilation (EDV/EIV). Reactive oxygen species (ROS), nitrotyrosine, reduced glutathione hormone (GSH) and NO(2)(-)/NO(3)(-) levels were measured in plasma. Endothelial nitric oxide synthase (eNOS) mRNA expression in endothelial cells was evaluated by real-time PCR. The following results were observed: (1) In the FFA group, BP increased after 4 h infusion of Intralipid+heparin. In the NAC group, systolic and diastolic BP remained the same. (2) In the FFA group, the aortic rings tended to show impaired EDV in response to acetylcholine (ACh). There was no difference of EDV response in the NAC and NC groups. (3) In the FFA group, NO(2)(-)/NO(3)(-) levels were significantly reduced, and eNOS mRNA expression and activity were significantly decreased compared with the NC group. NAC administration increased eNOS mRNA expression and activity. (4) ROS and nitrotyrosine concentrations in the FFA group were higher than in the NC group, and GSH concentrations in the FFA group were lower than in the NC group. Elevated FFAs can induce elevated BP, potentially through FFA-induced impairment of EDV resulting from decreased eNOS mRNA expression and activity. Oxidative stress may also play an important role in potential mechanisms of this high FFA-induced elevated BP.

Effects of Metformin on Penile Expression of Nitric Oxide Synthase in OLETF Rats

Purpose: It is well known that metformin, an oral biguanide insulinsensitizing agent, targets AMP-activated protein kinase (AMPK), which activates endothelial nitric oxide synthase (eNOS), which may be associated with erectile dysfunction. The aim of this study was to evaluate whether metformin activates eNOS in the penile tissue of a genetically obese rat model. Materials and Methods: We measured phospho-eNOS levels and eNOS expression in the penile tissue of Otsuka Long Evans Tokushima Fatty (OLETF) rats after 3 days of treatment with metformin (150 or 300 mg) to evaluate whether metformin activates eNOS in the penile tissue of this genetically obese rat model. Seven-month-old OLETF rats were used, and eNOS expression was analyzed by real-time polymerase chain reaction (PCR). The experimental groups were compared with use of the KruskalWallis or Mann-Whitney test. Results: Seven-month-old OLETF rats had severe visceral fat deposition and elevated serum leptin concentrations. eNOS expression analyzed by real-time PCR was lower in the penile tissue of OLETF rats than in Long Evans Tokushima Otsuka (LETO) rats. Short-term treatment with metformin did not change visceral fat mass or serum leptin levels. However, metformin treatment increased eNOS mRNA expression in the penile tissue as determined by real-time PCR. The levels of phospho-eNOS and phospho-AMPK (pAMPK) in penile tissue revealed a dose-dependent tendency to increase with metformin treatment; however, there was no statistical difference by Kruskal-Wallis test among the experimental groups. The pAMPK level was dose-dependently elevated in the soleus by metformin treatment. There was no significant change in pSTAT3 by metformin treatment in the soleus. Conclusions: Metformin activates eNOS mRNA expression in the penile tissue of OLETF rats. Further study on the relation between erectile function and eNOS levels is needed for clinical application. (Korean J Urol 2009;50:179-185) 󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏

NO‐dependent Molecular Mechanism in Relaxing Effect of Cilnidipine (CIL) in Human Internal Mammary Arteries (IMA)

CIL, the novel long‐lasting L/N‐type dihydropyridine calcium channel blocker, induces both endothelium‐dependent and independent relaxation in human IMA. We investigated the underlying molecular mechanism on nitric oxide (NO)‐cyclic guanosine monophosphate (cGMP) pathway. The expression level of eNOS mRNA in IMA incubated with/without (n=5/6) 3 μ CIL for 30 min followed by 10 μM norepinephrine (NE) stimulation for 4 hrs was assayed by Real‐Time quantitative PCR. The phosphorylation of eNOS at Ser1177 (p‐eNOS) was determined by Western blotting in IMA (n=4) simulated by 10 μM NE for 15 min followed by incubation for 5 min with 3 μM CIL or 50 nM bradykinin. The CIL‐induced relaxation was examined in endothelium‐denuded (n=7) and ‐intact IMA (n=7) pretreated with/without LNNA (300 μM, n=8) and ODQ (3 μM, n=8). CIL 3 μM increased eNOS mRNA expression by 42.4% and p‐eNOS by 55.0% in the NE‐stimulated IMA. Endotheliumdenudation (47.7 ± 7.0%, p < 0.05), L‐NNA (48.6 ± 6.1%, p < 0.05), and ODQ (41.6 ± 3.8%, p< 0.01) remarkably reduced the CIL‐induced relaxation to 40 mM K+‐induced contraction, comparing to endothelium‐intact control (73.9 ± 6.4 %). Thus, CIL‐induced endothelium‐dependent relaxation in human IMA is mediated by NOcGMP signal pathway with strong molecular evidence.Supported by St. Vincent Foundation, Hong Kong RGC (CUHK4383/03M) & CUHK (4450171, 4450231, 2041305).

Effect of dietary sodium on vasoconstriction and eNOS-mediated vascular relaxation in caveolin-1-deficient mice

Changes in dietary sodium intake are associated with changes in vascular volume and reactivity that may be mediated, in part, by alterations in endothelial nitric oxide synthase (eNOS) activity. Caveolin-1 (Cav-1), a transmembrane anchoring protein in the plasma membrane caveolae, binds eNOS and limits its translocation and activation. To test the hypothesis that endothelial Cav-1 participates in the dietary sodium-mediated effects on vascular function, we assessed vascular responses and nitric oxide (NO)-mediated mechanisms of vascular relaxation in Cav-1 knockout mice (Cav-1-/-) and wild-type control mice (WT; Cav-1+/+) placed on a high-salt (HS; 4% NaCl) or low-salt (LS; 0.08% NaCl) diet for 16 days. After the systolic blood pressure was measured, the thoracic aorta was isolated for measurement of vascular reactivity and NO production, and the heart was used for measurement of eNOS expression and/or activity. The blood pressure was elevated in HS mice treated with NG-nitro-l-arginine methyl ester and more so in Cav-1-/- than WT mice and was significantly reduced during the LS diet. Phenylephrine caused vascular contraction that was significantly reduced in Cav-1-/- (maximum 0.25 +/- 0.06 g/mg) compared with WT (0.75 +/- 0.22 g/mg) on the HS diet, and the differences were eliminated with the LS diet. Also, vascular contraction in response to membrane depolarization by high KCl (96 mM) was reduced in Cav-1-/- (0.27 +/- 0.05 g/mg) compared with WT mice (0.53 +/- 0.12 g/mg) on the HS diet, suggesting that the reduced vascular contraction is not limited to a particular receptor. Acetylcholine (10(-5) M) caused aortic relaxation in WT mice on HS (23.6 +/- 3.5%) and LS (23.7 +/- 5.5%) that was enhanced in Cav-1-/- HS (72.6 +/- 6.1%) and more so in Cav-1-/- LS mice (93.6 +/- 3.5%). RT-PCR analysis indicated increased eNOS mRNA expression in the aorta and heart, and Western blots indicated increased total eNOS and phosphorylated eNOS in the heart of Cav-1-/- compared with WT mice on the HS diet, and the genotypic differences were less apparent during the LS diet. Thus Cav-1 deficiency during the HS diet is associated with decreased vasoconstriction, increased vascular relaxation, and increased eNOS expression and activity, and these effects are altered during the LS diet. The data support the hypothesis that endothelial Cav-1, likely through an effect on eNOS activity, plays a prominent role in the regulation of vascular function during substantial changes in dietary sodium intake.

Ex vivo pretreatment of bone marrow mononuclear cells with endothelial NO synthase enhancer AVE9488 enhances their functional activity for cell therapy

Bone marrow mononuclear cells (BMC) from patients with ischemic cardiomyopathy (ICMP) show a reduced neovascularization capacity in vivo. NO plays an important role in neovascularization, and NO bioavailability is typically reduced in patients with ICMP. We investigated whether the impaired neovascularization capacity of ICMP patient-derived progenitor cells can be restored by pretreatment with the novel endothelial NO synthase (eNOS) transcription enhancer AVE9488 (AVE). Ex vivo pretreatment of BMC from patients with ICMP with AVE significantly increased eNOS mRNA expression by 2.1-fold (P < 0.05) and eNOS activity as assessed by ESR by >3-fold (P < 0.05). The increased eNOS expression was associated with an enhanced migratory capacity in vitro (P < 0.01) and improved neovascularization capacity of the infused BMC in an ischemic hind limb model in vivo (P < 0.001). The improvement in ischemic limb perfusion after infusion of AVE-pretreated BMC resulted in an increase in swimming time (P < 0.05). The enhancement of limb perfusion by AVE-treated BMC was abrogated by ex vivo pretreatment with the eNOS inhibitor N(G)-nitro-l-arginine methyl ester. Consistently, AVE showed no effect on the impaired migratory capacity of BMC derived from eNOS-deficient mice, documenting the specific involvement of NO. The reduced neovascularization capacity of BMC from patients with ICMP may limit their therapeutic potential in cell therapy studies. Here, we show that pharmacological enhancement of eNOS expression with AVE at least partially reverses the impaired functional activity of BMC from ICMP patients, highlighting the critical role of NO for progenitor cell function.

Protective effects of propofol on lipopolysaccharide-activated endothelial cell barrier dysfunction

Propofol has been widely used in intravenous anesthesia. It possesses antioxidant and immunomodulating effects. This study aimed to investigate whether propofol may attenuate lipopolysaccharide (LPS)-induced endothelial cell barrier dysfunction and the possible mechanisms of such modulation. Cultured human umbilical vein endothelial cells (HUVECs) were used to assess the following treatments: (i) no additives (negative control), (ii) LPS alone (1 and 10 microg/ml), (iii) propofol alone (20 microg/ml), (iv) intralipid (a solvent of propofol) alone (20 microg/ml), (v) LPS (10 microg/ml) combination with propofol (4 and 20 microg/ml) and (vi) LPS (10 microg/ml) combination with intralipid (20 microg/ml). Changes of cell permeability and filamentous actin (F-actin) were determined. Expression of nitrotyrosine proteins and activity of nuclear factor kappaB (NF-kappaB) were analyzed by Western blot and immunocytochemistry. Expression of endothelial nitric-oxide synthase (eNOS) and inducible nitric-oxide synthase (iNOS) were analyzed by reverse transcriptase-polymerase chain reaction. LPS markedly increased the permeability of endothelial cells, the formation of peroxynitrite and depolymerization of F-actin in HUVECs. LPS also significantly increased mRNA of iNOS, protein level of NF-kappaB and decreased mRNA of eNOS (P < 0.05). Propofol at both concentrations (4 and 20 microg/ml) significantly inhibited the LPS-induced increase in cell permeability and alteration in F-actin organization. Propofol also reduced the LPS-enhanced iNOS mRNA and NF-kappaB protein levels whilst it increased eNOS mRNA expression (P < 0.05). This study demonstrated that propofol, both at therapeutic concentrations and 5 times therapeutic concentrations, inhibited NF-kappaB activation in LPS-stimulated endothelial cells and was found to protect endothelial cells against LPS-induced barrier dysfunction.

Repeated Sauna Therapy Increases Arterial Endothelial Nitric Oxide Synthase Expression and Nitric Oxide Production in Cardiomyopathic Hamsters

Vascular endothelial dysfunction is involved in the pathophysiology of chronic heart failure (CHF). It has been reported that sauna therapy, which allows thermal vasodilation, improves vascular endothelial dysfunction in patients with CHF. The present study investigates the mechanisms through which sauna therapy improves endothelial dysfunction induced by CHF. Normal control and male TO-2 cardiomyopathic hamsters were used. Thirty-week-old TO-2 hamsters were treated daily with an experimental far infrared-ray dry sauna system for 15 min at 39 degrees C followed by 20 min at 30 degrees C. This procedure raised the rectal temperatures by about 1 degrees C. Arterial endothelial nitric oxide (NO) synthase (eNOS) mRNA and protein expressions were examined, and serum concentrations of nitrate were measured. The expression of eNOS mRNA in the aortas of normal controls did not change, whereas those of the TO-2 hamsters decreased with age. Four weeks of sauna therapy significantly increased eNOS mRNA expression in the aortas of TO-2 hamsters compared with those that did not undergo sauna therapy. Sauna therapy also upregulated aortic eNOS protein expression. Serum nitrate concentrations of the TO-2 hamsters were increased by 4 weeks of sauna therapy compared with those that did not undergo sauna. Repeated sauna therapy increases eNOS expression and NO production in cardiomyopathic hamsters with heart failure.

Oscillatory shear stress upregulation of endothelial nitric oxide synthase requires intracellular hydrogen peroxide and CaMKII

We have previously shown that hydrogen peroxide (H 2O 2) upregulates endothelial nitric oxide synthase (eNOS) expression via a calcium/calmodulin-dependent protein kinase II (CaMKII)-mediated mechanism whereas it also acutely activates eNOS enzyme. We hypothesized that oscillatory shear stress (OSS), which stimulates endogenous H 2O 2, would have effects on eNOS expression and function similar to that of exogenous H 2O 2. Exposure of bovine aortic endothelial cells to OSS (±15 dynes/cm 2) increased eNOS mRNA expression by 3-fold. Pretreatment with either polyethylene glycol-catalase (PEG-CAT, a scavenger of H 2O 2) or KN93, an inhibitor of CaMKII, abolished this response. OSS activated CaMKII in an H 2O 2-dependent fashion whereas unidirectional laminar shear stress (LSS) inhibited CaMKII phosphorylation. Inhibition of c-Src (essential for LSS upregulation of eNOS) had no effect on OSS upregulation of eNOS. Additionally, OSS stimulated NO • production acutely. Scavenging of H 2O 2 by PEG-CAT attenuated OSS stimulation of NO • by 50% whereas it had no effect on LSS regulation of NO • production. These data suggest that intracellular H 2O 2 and CaMKII mediate OSS upregulation of eNOS. The acute activation of eNOS by OSS also partially requires H 2O 2. As OSS has been shown previously to stimulate sustained production of superoxide (O 2 •–) which would inactivate NO •, these responses may represent attempted compensation to restore NO • bioavailability in areas exposed to OSS. Simultaneous stimulation of O 2 •– and NO • by this mechanism, however, could facilitate peroxynitrite formation and protein nitration, which may enhance atherosclerotic lesion formation. Both OSS and LSS upregulate eNOS expression but via different signaling mechanisms.

Dexamethasone blocks hypoxia-induced endothelial dysfunction in organ-cultured pulmonary arteries.

We assessed the effects of dexamethasone (DEX) on hypoxia-induced dysfunction of the pulmonary endothelium using organ-cultured rabbit intrapulmonary arteries; 3-microM DEX inhibited the 7-day hypoxia (5% oxygen)-induced impairments of endothelial-dependent relaxation, cGMP accumulation, and increase in intracellular Ca(2+) level under substance P-stimulated conditions. Treatment with DEX over the final 3 days of the 7-day hypoxic exposure period also restored the decreased endothelium-dependent relaxation. Although chronic hypoxia did not change the mRNA expression of endothelial nitric oxide synthase (eNOS), 3 microM of DEX increased eNOS mRNA expression in both the hypoxic and normoxic (20% oxygen) pulmonary endothelium. On the other hand, eNOS protein expression was not changed in any of the arteries. We next assessed the effects of DEX on the eNOS activation pathway. Chronic hypoxia impaired eNOS phosphorylation and Akt phosphorylation under both the nonstimulated and substance P-stimulated conditions, and 3-microM DEX restored these phosphorylations. Morphologic study revealed that 3-microM DEX inhibited chronic hypoxia-induced atrophy of endothelial cells and eNOS protein condensation into plasma membranes. These results suggest that DEX exerts beneficial effects on chronic hypoxia-induced impairments of nitric oxide-mediated arterial relaxation by increasing eNOS mRNA expression and inhibiting hypoxia-induced impairments in eNOS activation pathway with atrophy of endothelial cells.

Flavonoids from Artichoke (Cynara scolymus L.) Up-Regulate Endothelial-Type Nitric-Oxide Synthase Gene Expression in Human Endothelial Cells

Nitric oxide (NO) produced by endothelial nitric-oxide synthase (eNOS) represents an antithrombotic and anti-atherosclerotic principle in the vasculature. Hence, an enhanced expression of eNOS in response to pharmacological interventions could provide protection against cardiovascular diseases. In EA.hy 926 cells, a cell line derived from human umbilical vein endothelial cells (HUVECs), an artichoke leaf extract (ALE) increased the activity of the human eNOS promoter (determined by luciferase reporter gene assay). An organic subfraction from ALE was more potent in this respect than the crude extract, whereas an aqueous subfraction of ALE was without effect. ALE and the organic subfraction thereof also increased eNOS mRNA expression (measured by an RNase protection assay) and eNOS protein expression (determined by Western blot) both in EA.hy 926 cells and in native HUVECs. NO production (measured by NO-ozone chemiluminescence) was increased by both extracts. In organ chamber experiments, ex vivo incubation (18 h) of rat aortic rings with the organic subfraction of ALE enhanced the NO-mediated vasodilator response to acetylcholine, indicating that the up-regulated eNOS remained functional. Caffeoylquinic acids and flavonoids are two major groups of constituents of ALE. Interestingly, the flavonoids luteolin and cynaroside increased eNOS promoter activity and eNOS mRNA expression, whereas the caffeoylquinic acids cynarin and chlorogenic acid were without effect. Thus, in addition to the lipid-lowering and antioxidant properties of artichoke, an increase in eNOS gene transcription may also contribute to its beneficial cardiovascular profile. Artichoke flavonoids are likely to represent the active ingredients mediating eNOS up-regulation.