p-eNOS Expression Research Articles

SAT-175 (-)-Epicatechin Modulates Cellular Signaling and Mitochondrial Redox in Endothelium

Diabetes mellitus affects 451 million people worldwide, and people with diabetes are 3-5 times more likely to develop cardiovascular disease. In vascular tissue, mitochondrial function is important for vasodilation and arterial stiffness, both of which are abnormal in diabetes. Diabetes-mediated generation of excess reactive oxygen species (ROS) may be contributing to this problem. We have identified (-)-epicatechin (EPICAT), a plant compound and known vasodilator, as a potential therapy. We hypothesized that mitochondrial ROS accumulation in cells treated with high glucose (HG) or antimycin A (AA, a compound targeting complex III) could be diminished by EPICAT. We further hypothesized that reduction of ROS would correlate with improved mitochondrial dynamics and cellular signaling. To test this hypothesis, we employed human umbilical vein endothelial cells (HUVEC) treated with HG, AA, and/or 0.1 or 1.0 µM of EPICAT, and measured mitochondrial and cellular superoxide, mitochondrial dynamics, and cellular signaling upstream of mitochondrial function. AA significantly increased mitochondrial width (p=0.012) and decreased mitochondrial length (p=0.0001), but we observed no impact of EPICAT. We measured superoxide production by electron paramagnetic resonance spectroscopy using two spin probes specific to mitochondrial (Mito-TEMPO-H) and total cell (CMH) superoxide. No impact of HG treatment was detected in the total cell superoxide; however, an increase in mitochondrial superoxide was measured in cells treated with HG as compared to control (0.42 µM superoxide vs. 0.31 µM superoxide, respectively). We found that both concentrations of EPICAT significantly attenuated mitochondrial superoxide production when treated with HG (HG alone=0.44 µM superoxide, HG + 0.1 µM EPICAT=0.34 µM superoxide, HG + 1.0 µM EPICAT= 0.34 µM superoxide, p=0.02 EPICAT effect), but not with AA treatment. We found that in combination with HG, 0.1 µM EPICAT significantly increased eNOS expression (p<0.05). At 1.0 µM, EPICAT also increased peNOS expression (p<0.05) and attenuated a decrease in pAMPK (p<0.05) in response to HG. EPICAT at both concentrations significantly increased mitochondrial complex II expression (p<0.05 for both). In summary, HG-induced mitochondrial ROS is responsive to EPICAT, whereas AA-induced ROS is not, suggesting that EPICAT is useful in mitigating moderate ROS concentrations and may be modulating mitochondrial activity upstream of complex III. Our data further illustrate that EPICAT may be a tool to modulate both cellular and mitochondrial glucose toxicity through the regulation of AMPK and eNOS, upstream of mitochondrial activity, as well as bolstering endogenous antioxidant defenses.

Piperazine ferulate exerts antihypertensive effect and improves endothelial function in vitro and in vivo via the activation of endothelial nitric oxide synthase

To investigate the effect of piperazine ferulate (PF) on hypertension and endothelial function, and to assess the possible underlying mechanism. Human umbilical vein endothelial cells (HUVEC), adult male Wistar Kyoto (WKY) rats aged 12 to 14 weeks, and spontaneously hypertensive (SH) and Sprague Dawley (SD) rats were used for this study. Cell viability, activities of angiotensin-converting enzyme (ACE) and heme oxygenase-1 (HO-1), in vivo NO synthesis, arterial systolic blood pressure, vascular function, expressions of endothelial NO synthase (eNOS) and phosphorylated-eNOS (p-eNOS) were determined or assessed as appropriate. The results of MTT assay showed the number of viable cells were significantly increased with increase in PF concentration (p &lt; 0.05). The level of expression of ACE was significantly reduced with increase in PF concentration (p &lt; 0.05), while the level of HO-1 expression significantly increased (p &lt; 0.05). Results of DAF-FM fluorescent staining showed that the amounts of NO synthesized in vivo was significantly higher in aortic rings of SH and SD rats treated with PF than in the corresponding control groups (p &lt; 0.05). Treatment with PF in vivo significantly improved impaired acetylcholine-induced aortic relaxation in SH rats. Total eNOS expression was significantly increased after treatment with PF (p &lt; 0.05). The expressions of total eNOS and p-eNOS in both groups were not affected by PF when compared to the control group. These results indicate that PF exerts antihypertensive effect and improves endothelial function in vitro and in vivo via the activation of eNOS.

Chronic administration of sildenafil improves endothelial function in spontaneously hypertensive rats by decreasing COX-2 expression and oxidative stress

AimsSpontaneously hypertensive rats (SHR) exhibit impaired endothelial vasodilation and enhanced vasoconstriction. The phosphodiesterase 5 (PDE5) inhibitor sildenafil (Sild) potentiates the nitric oxide (NO)-mediated effects exerting antioxidative and anti-inflammatory actions. In the present study, we hypothesized that Sild could improve endothelial function in SHR. Materials and methodsMale rats were treated daily for 60 days by oral gavage with Sild (45 mg/kg) before the onset of the hypertensive state (pre-hypertensive protocol). The aortic relaxation to acetylcholine (ACh), sodium nitroprusside (SNP) and the phenylephrine (Phe)-induced contraction was evaluated in SHR. Protein expression of eNOS, p-eNOS, caveolin, COX-1, COX-2, ERK and p-ERK was measured by Western blot. Key findingsResting blood pressure was not modified by Sild administration. Treatment with Sild did not alter the relaxation response to SNP but improved the ACh-induced relaxation and reduced Phe-induced contraction in aortic rings from SHR. This protective effect of Sild could be attributed to reduced superoxide anions (O2−) generation, cyclooxygenase type 2 (COX-2) protein downregulation and increased NO bioavailability. SignificanceSild improves endothelial function in SHR aorta without affecting resting blood pressure values. These results indicate that PDE5 inhibition has a potential role in the improvement of vascular function and could be an adjuvant in the treatment of essential hypertension.

CTRP3 Alleviates Ox-LDL-Induced Inflammatory Response and Endothelial Dysfunction in Mouse Aortic Endothelial Cells by Activating the PI3K/Akt/eNOS Pathway.

C1q/tumor necrosis factor-related protein-3 (CTRP3) is a novel, certified, adipokine that beneficially regulates metabolism and inflammation in the cardiovascular system. Atherosclerotic plaque rupturing and secondary thrombosis cause vascular disorders, such as myocardial infarction and unstable angina. However, the underlying role of CTRP3 in atherosclerosis remains unclear. In this study, we aimed to elucidate whether and how CTRP3 ameliorates inflammation and endothelial dysfunction caused by oxidized low-density lipoprotein (ox-LDL). We first confirmed that CTRP3 expression was inhibited in ApoE-/- mice, compared to normal mice. Then, pcDNA-CTRP3 and siCTRP3 were transfected into mouse aortic endothelial cells after ox-LDL stimulation, and we observed that enhanced CTRP3 remarkably downregulated CRP, TNF-α, IL-6, CD40, and CD40L. We also observed that overexpression of CTRP3 elevated cell activity and decreased lactated hydrogenase release, accompanied by a marked reduction in cell apoptosis induced by ox-LDL. Meanwhile, overexpressed CTRP3 caused a decrease in Ang II, ICAM-1, and VCAM-1 expression, and it restored the balance between ET-1 and NO. Mechanism analysis confirmed that incremental CTRP3 upregulated p-PI3K, p-Akt, and p-eNOS expression, indicating that CTRP3 facilitated activation of the PI3K/Akt/eNOS pathway. On the contrary, siCTRP3 exerted the opposite effect to this activation. Blocking these pathways using LY294002 or L-NAME attenuated the protective role of CTRP3. Overall, these results suggest that CTRP3 can efficiently inhibit the inflammatory response and endothelial dysfunction induced by ox-LDL in mouse aortic endothelial cells, perhaps by activating the PI3K/Akt/eNOS pathway, indicating a promising strategy against atherosclerosis.

AMPK agonist AICAR ameliorates portal hypertension and liver cirrhosis via NO pathway in the\xa0BDL rat model

Recent studies have indicated that the Adenosine 5‘-monophosphate (AMP)-activated protein kinase (AMPK) pathway is closely involved in liver fibrosis and other fibrotic diseases. However, whether targeting the AMPK pathway can rescue liver fibrosis and its complications, such as portal hypertension, is unknown. This study aimed to explore the therapeutic value of AICAR (5-aminoimidazole-4-carboxyamide ribonucleoside), an agonist of the AMPK pathway, on liver fibrosis and portal hypertension in bile duct ligation (BDL) rats. In vitro experiments showed that the gene expression levels of TGF-b, a-SMA, and collagen 1 in primary rat hepatic stellate cells (HSCs) were significantly decreased after AICAR treatment. The p-eNOS expression and nitric oxide (NO) production were increased by AICAR administration in sinusoidal endothelial cells (SECs). For in vivo animal studies, AICAR acutely decreased portal pressure in the BDL and CCL4 fibrotic rats, but not in the partial portal vein ligation (PVL) rats, without changes in systemic hemodynamics. It was also observed by using intravital fluorescence microscopy that AICAR led to sinusoidal vasodilation in situ experiment. We propose that the relevant mechanisms may be related to the activation of the AMPK/NO pathway in SECs and that this activation promoted NO production in the liver, thereby promoting hepatic sinusoid microcirculation and decreased intrahepatic resistance. The results were verified using the NO inhibitor L-NAME. Chronic AICAR treatment also showed profound beneficial effects on the BDL model rats. The hemodynamic condition was greatly improved, but the positive effect could be partially blocked by L-NAME. Moreover, AICAR also decreased hepatic fibrogenesis in the BDL rats.Key messagesAcute and chronic use of AICAR could alleviate portal pressure without changing systemic hemodynamics.AICAR induced sinusoidal vasodilation by improving NO bioavailability and ameliorating endothelial dysfunction in vivo and in vitro.AICAR could alleviate liver cirrhosis in the BDL model rats.

Abstract TP320: Endothelial Nitric Oxide Synthase (eNOS) Inhibition Abolishes the Exercise-Induced Improvement in Neurovascular and Neurobehavioral Stroke Outcomes

Background: Clinically, it was shown that increased physical activity reduces the risk of stroke and severity of outcomes. However, the underlying mechanism is not clear. Endothelial nitric oxide synthase (eNOS) activation was shown to improve vascular function and reduce the risk of cardiovascular diseases. However, the role of eNOS in mediating short term exercise-induced improvement in stroke outcome was not previously studied. Accordingly, we tested the hypothesis that acute short term pre-stroke exercise improves stroke outcomes through increased eNOS activity, and that eNOS inhibition abolishes these beneficial effects. Methods: Male Wistar rats (300 g) were subjected to high intensity interval training (HIIT) through treadmill running sessions for four days (25 minutes/day), break for 2 days and then one acute bout for 30 minutes. Exercised animals were subjected to thromboembolic stroke at 1h, 6h, 24h or 72h after the last exercise session. At 24h after stroke, sedentary and exercised rats were tested for neurological outcomes, infarct size and cerebral edema. Western Blotting was used to measure the expression of active eNOS (p-S1177-eNOS) in brain and cerebral vessels. Additional control and 1h exercise groups were treated with eNOS inhibitor (L-NIO (L-N 5 -1-Iminoethyl ornithine)) right after stroke (I.P. injection, dose: 20 mg/Kg). Results: Acute exercise significantly reduced infarct size, edema and improved functional outcomes, and increased the expression of peNOS in the brain and cerebral vessels. eNOS inhibition significantly increased the infarct size, edema and worsened the neurobehavioral outcomes in exercised animals compared to non-treated group (table). Conclusion: Short term acute pre-stroke exercise significantly reduced neurovascular injury and improved neurobehavioral and functional outcomes after stroke, and eNOS inhibition abolished these beneficial effects.

ANXA4 promotes trophoblast invasion via the PI3K/Akt/eNOS pathway in preeclampsia.

The inadequate trophoblast invasion is associated with the development of preeclampsia (PE). Considering that annexin A4 (ANXA4) enhances tumor invasion, we aimed to explore the functional role of ANXA4 in trophoblast cells and to examine the underlying mechanism. ANXA4 expression in PE placentas was analyzed using immunohistochemistry and Western blotting. Cell proliferation, invasion, and apoptosis were determined using a MTT assay, Transwell assay, and flow cytometry, respectively. The expression levels of matrix metalloproteinase (MMP)-2, MMP-9, phosphoinositide 3-kinase (PI3K), Akt, phosphorylated (p)-Akt, and phosphorylated endothelial nitric oxide synthase (p-eNOS) were detected by Western blotting. Placentas were prepared for pathological examination using hematoxylin and eosin staining and apoptosis determination using the TUNEL method. Expression of ANXA4, PI3K, p-Akt and p-eNOS was downregulated in human PE placentas and PE placenta-derived extravillous cytotrophoblasts (EVCTs). Furthermore, ANXA4 overexpression promoted cell proliferation and invasion, inhibited cell apoptosis, and upregulated protein expression of PI3K, p-Akt, and p-eNOS in human trophoblast cells HTR-8/SVneo and JEG-3. By contrast, ANXA4 knockdown exerted the opposite effects. Furthermore, inhibition of the PI3K/Akt pathway by LY294002 abrogated the ANXA4 overexpression-mediated effects on trophoblast behavior. Furthermore, eNOS knockdown abrogated the ANXA4 overexpression-induced promotion of cell invasion and MMP2/9 expression. Additionally, in N-nitro-l-arginine methyl ester (l-NAME)-induced PE rats, ANXA4 overexpression alleviated PE progression, accompanied by an increase in expression of PI3K, p-Akt, and p-eNOS in rat placentas. Our findings demonstrate that ANXA4 expression is downregulated in PE. ANXA4 may promote trophoblast invasion via the PI3K/Akt/eNOS pathway.

Vasodilation effect and mechanism of extraction of Tongmai Yangxin Pills (TMYX) on isolated rat mesenteric artery

The aim of the present study is to evaluate the vasodilation effects of Tongmai Yangxin Pills (TMYX) on rat mesenteric artery as well as its mechanism of action. The relaxation effects of TMYX extracts with different concentrations were determined on isolated rat mesenteric artery in normal condition as well as pretreating by phenylephrine and KCl. Vascular relaxation effects of TMTX were also determined in mesenteric artery preincubated with L-ANME and indomethacin or in endothelium denuded mesenteric artery. Moreover, effects of TMYX by 50 mg·L⁻¹ on NO secretion and the phosphorylation of eNOS in a cellular model of human umbilical vein endothelial cell (HUVEC) pretreated with or without L-NAME were also observed. The experimental results showed that TMYX has no obvious effect on vasodilation of arteries in normal or KCl pretreated condition, while it can dose-dependently relax the rat mesenteric artery with intact endothelium stimulated with phenylephrine at a maximal diastolic rate of (64.71±10.03)%. After preincubating with L-NAME for 15 min or removal of mesenteric artery endothelium, the maximal diastolic rate was decreased to (35.77±8.93)% and (25.85±10.84)% respectively. However, preincubating with indomethacin had no inhibitory effect on TMYX induced vascular relaxation. Meanwhile, TMYX at 50 mg·L⁻¹ could increase the expression of P-eNOS and the secretion of NO in HUVEC. L-NAME significantly inhibited NO release and phosphorylation of eNOS induced by TMYX. The results suggested TMYX exerted endothelium-dependent relaxation effects against PE-induced contractions of isolated rat mesenteric artery through NO-cGMP signaling pathway.

Salvianolic acid inhibits the effects of high glucose on vascular endothelial dysfunction by modulating the Sirt1-eNOS pathway.

Salvianolic acid (SA) is known for improving blood circulation, scavenging hydroxyl radicals, and preventing platelet aggregation. The research explored whether SA can protect against cardiovascular disease induced by high glucose conditions. Our results indicate that SA significantly increases cells viability and nitric oxide levels while decreasing reactive oxygen species generation. SA upregulated the expression levels of Bcl-2 and decreased the levels of Bax, cleaved caspase-3, and cleaved caspase-9. Furthermore, the expression levels of Sirtuin 1 (Sirt1) and p-endothelial nitric oxide synthase (eNOS) were markedly increased in response to SA treatment. Moreover, exposure of human umbilical vein endothelial cells to Ex527 resulted in reducing expression of p-eNOS. However, the beneficial effects of SA were abolished partially when Ex527 was added. These findings suggest that SA can be used as a potential therapeutic to protect against high glucose-induced endothelial injury by modulating Sirt1-eNOS pathway.

PO-144 Intermittent Exercise Activates NRG1-SERCA2a Pathway to Improve Cardiac Function in Myocardial Infarction Rats

Objective Intermittent exercise can improve cardiac function in rats with myocardial infarction. The Neuregulin-1(NRG1)/SERCA2a palys a critical role in maintain cardiac function. We want to investigate the effect of Neuregulin-1 (NRG1) on NRG1-SERCA2a signaling pathway activated by intermittent exercise and on improves cardiac function in rats with MI.&#x0D; Methods 32 male sprague-dawley rats were randomly divided into four groups (n=8): Sham-operated group (S), sedentary MI group (MI), MI with interval training group (ME), ME with inhibitor AG1478 group (MA). ME and MA model after the MI model was established by ligation of the left anterior descending coronary artery, and began training 1 week after MI surgery. The S model only by threading without ligation. Rats in ME and MA model taken one week adaptive training, then began 8-week interval training. MA model were injected with inhibitor AG1478, once every two days. The 24h after training, rats were anesthetized, the LVSP, LVEDP, ±dp/dt max were tested by carotid artery intubation which in order to evaluate cardiac function. The protein expression of NRG1, PI3K, Akt, eNOS, PKG, PLN, SERCA2a in myocardium were measured by Westernblotting, themRNA expression of serca2a were tested by RT-qPCR.&#x0D; Results Compared with S, the protein expression of NRG1, PKG, peNOS, pAkt, pPLN, pPI3K and SERCA2a decreased, serca2a mRNA expression decreased, LVSP and ±dp/dt max significantly decreased, LVEDP significantly increased; Compared with MI, the protein expression of NRG1, PKG, peNOS, pAkt, pPLN, pPI3K and SERCA2a increased, serca2a mRNA expression increased, LVSP and ±dp/dt max significantly increased, LVEDP decreased, and the effect of exercise were weaken by inhibitor AG1478. Correlation analysis showed that the myocardial pPLN and SERCA2a protein expression both were positively correlated with LVSP, ±dp/dtmax, and negatively correlated with LVEDP.&#x0D; Conclusions Intermittent exercise can increased myocardial NRG1 protein expression and activates NRG1-SERCA2a signaling pathway, improve myocardial infarction cardiac function.

A0625 Cinnameldehyde Attenuated Endothelial Dysfunction through Nrf2 Activation as a TRPA1 Agonist

Objectives: Advancing age is characterized with the development of vascular endothelial dysfunction which is the major risk factor for the development of cardiovascular diseases. TRPA1 is involved in lifespan and its agonist cinnameldehyde(CA)mediates endothelial vasorelaxation. Therefore, we hypothesized that TRPA1 is involved in aging-related endothelial dysfunction and CA administration would improve endothelial function. Methods: Human umbilical vein endothelial cells (HUVECs) were cultured until 14th passage and the 6th passage were used as control. Twenty-four months-old male Sprague Dawley (SD) rats were given dietary CA (0.2%) and six months-old rodents were used as young control. Rodent carotid arteries vasorelaxation was detected by wire myograph. TRPA1, Nrf2, UCP2 and its target genes as well as eNOS, p-eNOS were analysed by immunoblotting. Results: Immunoblotting and immunofluorescence confirmed the TRPA1 expression in HUVECs and vascular endothelium. CA(10 μM) promoted Nrf2 nuclear translocation and eNOS phosphorylation, led to the up-regulation of HO-1, GPx-1, NQO-1 and a reduction in ROS production in HUVECs. However, these effects of CA could be reversed by TRPA1 antagonist, HC030031 (10 μM), Nrf2 inhibitor brusatol (40 nM) or UCP2 inhibitor. NO level was found decreased, and enthdothelium dependent relaxation was impaired in carotid arteries of aged rats, but both improved after 12 weeks of CA administration. Immunoblotting showed the expression of TRAP1, Nrf2, UCP2 and p-eNOS significantly decreased in vascular tissue of aged rats but partly restored after CA treatment. Conclusion: TRPA1 may be involved in aging related endothelial dysfunction and CA improved endothelial dependent vasorelaxation through Nrf2 activation as a TRPA1 agonist.

Protective effects of Danzhi jiangtang capsule on vascular endothelial damages induced by high-fat diet and palmitic acid

Danzhi jiangtang capsule (DJC) is mainly used to treat type 2 diabetes mellitus and vascular complication. Our aim was to investigate whether the protective effects of DJC by oral administration on high-fat diet (HFD) and palmitic acid-induced damages of endothelial cells are related to oxidative stress and endoplasmic reticulum (ER) stress. Male Sprague-Dawley rats were randomly divided into standard chow diet (SCD), HFD, HFD plus DJC-low dose (HFD + DJCL) and HFD plus DJC-high dose treatment groups (HFD + DJCH). The latter three groups were given HFD feeding for three months, then the HFD + DJCL and HFD + DJCH rats were treated with DJC (500 and 1000 mg/kg/day) via gavage for another two months respectively. Endothelium-dependent relaxations induced by acetylcholine were observed in isolated rat thoracic aortic ring preparation. Malondialdehyde (MDA), total-antioxidant capacity (T-AOC), superoxide dismutase (SOD), interleukin 1β (IL-1β), tumour necrosis factor α (TNFα), free fatty acids (FFA), endothelin-1 (ET-1) and nitric oxide (NO) levels in serum were assayed. The pathological changes, protein expression of endothelial NO synthase (eNOS), phosphorylated eNOS (p-eNOS) and ER stress-related genes in the thoracic aorta were measured. Human umbilical vein endothelial cells (HUVEC) were treated with serum-medicated DJC and then with palmitic acid to detect the reactive oxygen species (ROS) levels and C/EBP homologous protein (CHOP) distribution, expression of p-eNOS and ER stress-related genes. DJC therapy exhibited a potent antioxidant activity and effectively prevented the endothelial dysfunction (ED) manifested by promoting p-eNOS expression and enhancing NO release, decreasing lipid deposition (Oil-red O staining, CPT1b and ACC) and inflammation (IL-1β, TNFα, CD68 and p-JNK), alleviating oxidative and ER stress, and decreasing the apoptosis of endothelial cells (TUNEL, BCL-2 and BAX) induced by HFD and palmitic acid respectively. These findings suggest that the administration of DJC had endothelial protective effects against HFD-induced ED, inflammation and apoptosis by alleviating lipid deposition, oxidative and ER stress.

Effect of low androgen levels on IKca and SKca3 channels in rat penile corpus cavernosum.

We investigated whether low androgen levels affected erectile function by regulating the expressions of intermediate-conductance Ca2+ -activated K+ channel (IKca) and small-conductance Ca2+ -activated K+ channel 3 (SKca3) in corpus cavernous of rats. Thirty-six healthy male SD rats were randomly divided into the 4-week control group, 4-week castration group, 4-week androgen replacement after castration group, 8-week control group, 8-week castration group and 8-week androgen replacement after castration group, respectively. The rats in the androgen replacement groups were subcutaneously injected with testosterone (3mg/kg) every other day after castration. After 4 and 8weeks, maximum intracavernous pressure/mean arterial pressure (ICPmax /MAP) was measured. Expressions of IKca, SKca3, endothelial nitric oxide synthase (eNOS) and P-eNOS in penile corpus cavernosum were detected. ICPmax /MAP decreased significantly in the castration groups as compared to the control groups and the androgen replacement groups (p<0.01). mRNA expressions of IKca and SKca3 decreased significantly in the castration groups as compared to the control groups and androgen replacement groups (p<0.01). Protein expressions of eNOS, P-eNOS, IKca and SKca3 in the castration groups were significantly reduced as compared to the control groups and androgen replacement groups (p<0.01). Under low androgen levels, ICPmax /MAP can be reduced by down-regulating the expressions of SKca3 and IKca, inhibiting P-eNOS/eNOS and reducing eNOS bioactivity.

Protective effect of resveratrol on methylglyoxal-induced endothelial dysfunction in aged rats.

The cardiovascular benefits of resveratrol (RSV) have been well established by previous experimental and clinical studies. The aim of this study was to investigate the effectiveness of RSV administration on the impaired endothelial function induced by methylglyoxal (MGO), and to elucidate the role of endothelial nitric oxide synthase (eNOS) on its protective effect. Aged Wistar rats (80weeks old, n = 15) were used in this study. The thoracic aorta was isolated and cut into rings for organ culture. Aortic segments of rats were incubated with MGO (420µM) in the presence or absence of RSV (30µM) for 4h (short-term) or 24h (long-term). Isometric tension studies were performed by an isolated organ bath in response to acetylcholine (ACh, an endothelium-dependent vasodilator) and sodium nitroprusside (SNP, an endothelium-independent vasodilator). Beside, expressions of eNOS and phospho-eNOS (p-eNOS) (Ser 1177) in thoracic aorta rings were evaluated by immunohistochemistry. Both short-term and long-term MGO incubation significantly inhibited the relaxation response induced by ACh, while the relaxation to SNP was not significantly altered. In addition, eNOS and p-eNOS expressions decreased significantly in arteries incubated with MGO. The impaired endothelial reactivity as well as decreased expressions of eNOS and p-eNOS in MGO-incubated vessels were significantly improved by RSV treatment. Endothelium-dependent vasodilatation of the thoracic aorta was significantly inhibited by MGO administration, and RSV may improve vascular endothelial function. The protective effect of RSV against MGO-induced endothelial dysfunction seems to be via increased eNOS expression and activity.

Effects of ginsenoside Rb1 on oxidative stress injury in rat spinal cords by regulating the eNOS/Nrf2/HO‑1 signaling pathway

The present study aimed to investigate whether ginsenoside Rb1 (G-Rb1) attenuates spinal cord injury-associated oxidative stress in rats by regulating the endothelial nitric oxide synthase eNOS/nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase (HO)-1 signaling pathway. Sprague Dawley rats were randomly divided into the sham operation group (S group), spinal cord injury group (SCI group), G-Rb1 treatment group (G-Rb1 group) and SCI+G-Rb1+Inhibitor L-name group (L-name group). The posterior limb function was evaluated via the Basso, Beattie and Bresnahan scoring method. The levels of superoxide dismutase (SOD), malondialdehyde (MDA), catalase (CAT) and glutathione (GSH) in serum were measured by ELISA. The pathological changes in the spinal cord were observed by H&E staining. Reverse transcription-quantitative polymerase chain reaction and western blot analyses were used to detect eNOS, phosphorylated (p)-eNOS, heat shock protein (HSP)90, Nrf2 and NAD(P)H quinone dehydrogenase 1 (Nqo1) at the mRNA and protein level. Immunohistochemistry was used to detect the expression of Nrf2 and p-eNOS. Compared with the S group, the scores of spinal cord function in the SCI group were significantly lower, and the levels of MDA were significantly increased, while the levels of SOD, CAT and GSH protein in spinal cord were significantly decreased (P<0.05). The spinal cord tissue exhibited hemorrhage, neuronal degeneration/necrosis, as well as mononuclear cell and lymphocyte infiltration. The eNOS, HSP90, Nrf2, Nqo1 and HO-1 mRNA levels were decreased (P<0.05). Compared with those in the SCI group, the spinal cord function score in the G-Rb1 group were significantly higher and the serum MDA content was significantly decreased, while the activity of SOD, CAT and GSH was significantly increased (P<0.05). The degeneration/necrosis of spinal cord neurons was attenuated, inflammatory cell infiltration was significantly reduced and the levels of eNOS, HSP90, Nrf2, Nqo1 and HO-1 were significantly upregulated (P<0.05). In the group that was administered the eNOS inhibitor L-name, the levels of eNOS, HSP90, Nrf2, Nqo1 and HO-1 were significantly decreased. In conclusion, G-Rb1 attenuates oxidative stress in injured spinal cords. The mechanism may at least in part involve the eNOS/Nrf2/HO-1 pathway.

Downregulated PI3K-Akt-eNOS expression is related to increased atrial fibrillation inducibility in diabetic rats

Objective: To explore the impact of PI3K-Akt-eNOS signaling on atrial fibrillation inducibility in diabetic rats. Method: Eight-week-old male diabetic rats were randomized assigned into GK group, IGF group and L-NAME group (n=8 each) which respectively received normal saline (NS), insulin like growth factor (IGF-1) or L-NAME+IGF-1 through tail vein daily for 4 weeks. Eight 8-week-old male homologous Wister-Kyoto(WKY) rats treated with intravenous NS served as control group (WKY group). Blood glucose was measured once per week. The left atrial diameter (LAD) was measured by echocardiography, the atrial electrical parameters, including the P-wave duration, the atrial effective refractory period (AERP) and its dispersion (AERP-d), the incidence and the duration of atrial fibrillation induced by atrial burst pacing, were evaluated by electro-physiological instrument at 4 weeks post various treatments. Rats were then sacrificed, left atrial (LA) cell morphology was determined on HE stained sections, LA interstitial collagen was determined on Masson stained sections. The protein expression of phosphatidylinositol 3-kinase (PI3K) and phosphate endothelial nitric oxide synthase (p-eNOS) were detected by Western blot. Results: (1) At the beginning of the study, the random blood glucose (GLU) level was significantly higher and LAD was large in GK, IGF and L-NAME groups than in WKY group;after 4 weeks, GLU level and LAD dimension of IGF group were lower than GK and L-NAME groups (P<0.01 or 0.05). (2) One rat in L-NAME group died during operation. Four weeks later, the incidence of atrial fibrillation in GK group, IGF group, L-NAME group and WKY group was 7/8, 2/8, 6/7 and 3/8. The median duration of atrial fibrillation in GK group, IGF group, L-NAME group and WKY group was 11.9(9.3, 13.1), 0(0, 1.8), 11.5(4.4, 12.0), and 0(0, 3.0) s. Compare with WKY group, the P-wave duration and PR interval were significantly longer, AERP-d, incidence, and duration of atrial fibrillation were significantly higher in GK group (P<0.01), these changed were significantly reversed in IGF group compared to GK and L-NAME groups (all P<0.01). Heart rate and AERP were similar among the 4 groups on (P>0.05). (3) Four weeks later, the CSA and CVF of LA were significantly larger in GK group than in WKY group (P<0.01), which were significantly reversed in IGF group (P<0.01 vs. GK group), and the beneficial effects of IGF disappeared by co-treatment with L-NAME (P<0.01 vs. IGF group). (4) Four weeks later, compare with WKY group, the protein expression of PI3K (P<0.01) and p-eNOS (P<0.05) of LA were significantly downregulated in GK group, which could be significantly upregulated by IGF (P<0.01 and 0.05 vs. GK group), these effects diminished by co-treatment with L-NAME (P<0.01 or 0.05 vs. IGF group). Conclusion: Increased atrial fibrillation susceptibility in diabetic rat is linked with structural and electrical remodeling in LA, possibly mediated through downregulated PI3K-Akt-eNOS signaling.

Protective effect of exendin-4 treatment on erectile dysfunction induced by chronic methylglyoxal administration in rats

ObjectiveThe aim of this study was to investigate the effect of chronic exendin-4 (Ex-4) treatment on corpus cavernosum (CC) dysfunction in methylglyoxal (MGO) administered rats. MethodsMale rats were divided into four groups as control, MGO (75 mg/kg/day in drinking water for 12 weeks), MGO + low-dose Ex-4 (0.1 μg/kg twice daily subcutaneously for 12 weeks concomitant with MGO), and MGO + high-dose Ex-4 (1 μg/kg twice daily subcutaneously for 12 weeks concomitant with MGO). Nitric oxide (NO)-mediated endothelium-dependent and neurogenic CC relaxations were evaluated by acetylcholine (ACh) and electrical field stimulation (EFS), respectively. Apoptosis was determined by TUNEL. Endothelial nitric oxide synthase (eNOS), phosphorylated eNOS (p-eNOS), NADPH oxidase subunit gp91phox (NOX2), and Rho kinase (ROCK2) expressions in CC were investigated by immunohistochemistry. Levels of the malondialdehyde (MDA) and advanced oxidation protein products (AOPP) were also measured. ResultsIn MGO administered rats, both endothelium-dependent and neurogenic CC relaxations were significantly impaired as compared to controls. Apoptotic cell death and levels of MDA and AOPP increased significantly in MGO administered rats. eNOS and p-eNOS expressions decreased significantly in MGO group, while gp91phox expressions increased significantly. The diminished relaxation in response to ACh or EFS as well as the changes in expression of proteins in MGO groups were significantly improved by exendin-4 treatment. TUNEL-positive cells, and levels of MDA and AOPP in MGO group rats were also significantly reduced by exendin-4. ConclusionExendin-4 treatment improves NO-mediated CC relaxations in MGO administered rats probably by inhibiting NADPH oxidase.

Chronic intermittent hypobaric hypoxia protects vascular endothelium by ameliorating autophagy in metabolic syndrome rats

AimsThe study aimed to investigate the protective effect of chronic intermittent hypobaric hypoxia (CIHH) on endothelium function and relaxation of mesenteric artery in metabolism syndrome (MS) rats. Main methodsMale adult Sprague-Dawley rats were randomly divided into control (CON), CIHH (treated with 28-days hypobaric hypoxia simulating an altitude of 5000 m, 6 h daily), MS (induced by high fat diet and 10% fructose water feeding), and MS + CIHH groups. Body weight, systolic arterial pressure, blood biochemical and the endothelium dependent relaxation (EDR) of mesenteric arteries were measured. The expression of phosphor-endothelial nitric oxide synthase (p-eNOS), endoplasmic reticulum (ER) stress-related proteins and autophagy-related proteins in mesenteric arteries was assayed. Key findingsThe MS rats displayed hypertension, obesity, metabolic abnormity and insulin resistance, EDR was attenuated, p-eNOS expression was down-regulated, the expressions of ER stress-related proteins were up-regulated, and autophagy dysfunction occurred. All aforementioned abnormalities in MS rats were ameliorated in MS + CIHH rats. Furthermore, the improvement of CIHH on EDR and p-eNOS was cancelled by the ER stress inducer, and the autophagy inhibitor. SignificanceIn conclusion CIHH protects endothelium function and enhances relaxation in mesenteric arteries of MS rats through improving autophagy function, reducing ER stress and up-regulating p-eNOS.

The effect of exercise on purinergic receptor‐mediated cerebrovascular dysfunction in Alzheimer's disease

IntroductionCerebrovascular dysfunction is a critical initial step of Alzheimer's disease (AD). Adenosine triphosphate (ATP) regulates cerebral blood flow through purinergic receptors. The aim of this study is to investigate the protective effect of exercise training on ATP‐induced purinergic receptor‐mediated cerebrovascular dysfunction in the AD and its underlying mechanisms.MethodsWe used the control mice (CON; C57BL/6), CON with exercise training (CON‐EX), AD mice (AD; APP/PS1) and AD with EX (AD‐EX). At 7 to 9 months of age, CON‐EX and AD‐EX mice underwent 10–12 weeks of aerobic exercise training, which consisted of 1 hour of treadmill running at 15m/min at a 5% grade. To test the purinergic receptor‐dependent vasoreactivity, a posterior cerebral artery (PCA) was isolated and ATP (2‐MeS‐ATP, 1 nmol/L to 10 μmol/L)‐induced dose‐dependent vascular dilation/constriction response was determined at 10–12 months of age. Western blotting was utilized to analyze the expression of P2Y2 receptors (P2Y2R), total eNOS (t‐eNOS) and phosphorylated eNOS (p‐eNOS) in the brain.ResultsATP induced vasodilation of PCA in CON and CON+EX mice, but it induced vasoconstriction in AD mice. Notably, exercise training reverses ATP‐induced vasoconstriction in the AD to vasodilation (AD+EX) comparable to CON mice. Western blotting data demonstrated that P2Y2R and p‐eNOS expression were significantly diminished in AD mice compared to CON mice, but they were elevated in AD‐EX mice brain. There was no significant difference in t‐eNOS expression among mice groups.ConclusionOur findings suggest that ATP‐induced purinergic receptor‐dependent cerebrovascular function is impaired in the AD. However, exercise training reverses ATP‐induced purinergic receptor‐dependent vasoconstriction to vasodilation mainly through the alteration of P2Y2R and p‐eNOS expression in the AD‐EX.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Preliminary Study of Mechanisms of Fluoride-Induced Suppression of Nitric Oxide Synthesis in Human Umbilical Vein Endothelial Cells.

This study was aimed to determine if excessive exposure to fluoride could suppress the synthesis of nitric oxide (NO) and to detail the mechanisms involved. With the exception of the control group, human umbilical vein endothelial cells (HUVECs) were treated with sodium fluoride (NaF) (1.2μg/mL) for 24h, with or without a 2-h pretreatment with 100nM insulin-like growth factor 1 (IGF-1, a PI3K/AKT agonist), or 10μM histamine (HIS, a eNOS agonist). The levels of NO in culture fluids, as well as the expressions of eNOS, p-eNOS, PI3K, AKT, and p-AKT, were compared. The levels of NO significantly decreased in all experimental groups; however, the levels of NO were obviously higher in the NaF + HIS and NaF + IGF-1 groups, compared to the NaF group. The p-eNOS/eNOS ratios dropped clearly in NaF and NaF + HIS groups, while that in the NaF + HIS group was distinctly higher than that in the NaF group. The p-AKT/AKT ratios went down apparently in NaF and NaF + IGF-1 groups, while that in the NaF + IGF-1 group was overtly higher than that of the NaF group. Excessive exposure to fluoride inhibited the synthesis of NO. The PI3K/AKT/eNOS pathway played a crucial role in the reduced expression of NO caused by excessive fluoride exposure.