Aerobic Exercise Training Prevents the Onset of Endothelial Dysfunction via Increased Nitric Oxide Bioavailability and Reduced Reactive Oxygen Species in an Experimental Model of Menopause

Objective

Previous studies have shown that estrogen deficiency, arising in postmenopause, promotes endothelial dysfunction. This study evaluated the effects of aerobic exercise training on endothelial dependent vasodilation of aorta in ovariectomized rats, specifically investigating the role of nitric oxide (NO) and reactive oxygen species (ROS).

Methods

Female Wistar rats ovariectomized (OVX – n=20) or with intact ovary (SHAM – n=20) remained sedentary (OVX and SHAM) or performed aerobic exercise training on a treadmill 5 times a week for a period of 8 weeks (OVX-TRA and SHAM-TRA). In the thoracic aorta the endothelium-dependent and –independent vasodilation was assessed by acetylcholine (ACh) and sodium nitroprusside (SNP), respectively. Certain aortic rings were incubated with L-NAME to assess the NO modulation on the ACh-induced vasodilation. The fluorescence to dihydroethidium in aortic slices and plasma nitrite/nitrate concentrations were measured to evaluate ROS and NO bioavailability, respectively.

Results

ACh-induced vasodilation was reduced in OVX rats as compared SHAM (Rmax: SHAM: 86±3.3 vs. OVX: 57±3.0%, p<0.01). Training prevented this response in OVX-TRA (Rmax: OVX-TRA: 88±2.0%, p<0.01), while did not change it in SHAM-TRA (Rmax: SHAM-TRA: 80±2.2%, p<0.01). The L-NAME incubation abolished the differences in ACh-induced relaxation among groups. SNP-induced vasodilation was not different among groups. OVX reduced nitrite/nitrate plasma concentrations and increased ROS in aortic slices, training as effective to restore these parameters to the SHAM levels.

Conclusions

Exercise training, even in estrogen deficiency conditions, is able to improve endothelial dependent vasodilation in rat aorta via enhanced NO bioavailability and reduced ROS levels.     

An NO Donor Approach to Neuroprotective and Procognitive Estrogen Therapy Overcomes Loss of NO Synthase Function and Potentially Thrombotic Risk

Selective estrogen receptor modulators (SERMs) are effective therapeutics that preserve favorable actions of estrogens on bone and act as antiestrogens in breast tissue, decreasing the risk of vertebral fractures and breast cancer, but their potential in neuroprotective and procognitive therapy is limited by: 1) an increased lifetime risk of thrombotic events; and 2) an attenuated response to estrogens with age, sometimes linked to endothelial nitric oxide synthase (eNOS) dysfunction. Herein, three 3^rd generation SERMs with similar high affinity for estrogen receptors (ERα, ERβ) were studied: desmethylarzoxifene (DMA), FDMA, and a novel NO-donating SERM (NO-DMA). Neuroprotection was studied in primary rat neurons exposed to oxygen glucose deprivation; reversal of cholinergic cognitive deficit was studied in mice in a behavioral model of memory; long term potentiation (LTP), underlying cognition, was measured in hippocampal slices from older 3×Tg Alzheimer''s transgenic mice; vasodilation was measured in rat aortic strips; and anticoagulant activity was compared. Pharmacologic blockade of GPR30 and NOS; denudation of endothelium; measurement of NO; and genetic knockout of eNOS were used to probe mechanism. Comparison of the three chemical probes indicates key roles for GPR30 and eNOS in mediating therapeutic activity. Procognitive, vasodilator and anticoagulant activities of DMA were found to be eNOS dependent, while neuroprotection and restoration of LTP were both shown to be dependent upon GPR30, a G-protein coupled receptor mediating estrogenic function. Finally, the observation that an NO-SERM shows enhanced vasodilation and anticoagulant activity, while retaining the positive attributes of SERMs even in the presence of NOS dysfunction, indicates a potential therapeutic approach without the increased risk of thrombotic events.     

Estrogen regulates hepcidin expression via GPR30-BMP6-dependent signaling in hepatocytes

Hepcidin, a liver-derived iron regulatory protein, plays a crucial role in iron metabolism. It is known that gender differences exist with respect to iron storage in the body; however, the effects of sex steroid hormones on iron metabolism are not completely understood. We focused on the effects of the female sex hormone estrogen on hepcidin expression. First, ovariectomized (OVX) and sham-operated mice were employed to investigate the effects of estrogen on hepcidin expression in an in vivo study. Hepcidin expression was decreased in the livers of OVX mice compared to the sham-operated mice. In OVX mice, bone morphologic protein-6 (BMP6), a regulator of hepcidin, was also found to be downregulated in the liver, whereas ferroportin (FPN), an iron export protein, was upregulated in the duodenum. Both serum and liver iron concentrations were elevated in OVX mice relative to their concentrations in sham-operated mice. In in vitro studies, 17β-estradiol (E(2)) increased the mRNA expression of hepcidin in HepG2 cells in a concentration-dependent manner. E(2)-induced hepatic hepcidin upregulation was not inhibited by ICI 182720, an inhibitor of the estrogen receptor; instead, hepcidin expression was increased by ICI 182720. E(2) and ICI 182720 exhibit agonist actions with G-protein coupled receptor 30 (GPR30), the 7-transmembrane estrogen receptor. G1, a GPR30 agonist, upregulated hepcidin expression, and GPR30 siRNA treatment abolished E(2)-induced hepcidin expression. BMP6 expression induced by E(2) was abolished by GPR30 silencing. Finally, both E(2) and G1 supplementation restored reduced hepatic hepcidin and BMP6 expression and reversed the augmentation of duodenal FPN expression in the OVX mice. In contrast, serum hepcidin was elevated in OVX mice, which was reversed in these mice with E(2) and G1. Thus, estrogen is involved in hepcidin expression via a GPR30-BMP6-dependent mechanism, providing new insight into the role of estrogen in iron metabolism.     

G-Protein Coupled Receptor 30 (GPR30): A Novel Regulator of Endothelial Inflammation

Estrogen, the female sex hormone, is known to exert anti-inflammatory and anti-atherogenic effects. Traditionally, estrogen effects were believed to be largely mediated through the classical estrogen receptors (ERs). However, there is increasing evidence that G-protein coupled receptor 30 (GPR30), a novel estrogen receptor, can mediate many estrogenic effects on the vasculature. Despite this, the localization and functional significance of GPR30 in the human vascular endothelium remains poorly understood. Given this background, we examined the subcellular location and potential anti-inflammatory roles of GPR30 using human umbilical vein endothelial cells as a model system. Inflammatory changes were induced by treatment with tumor necrosis factor (TNF), a pro-inflammatory cytokine involved in atherogenesis and many other inflammatory conditions. We found that GPR30 was located predominantly in the endothelial cell nuclei. Treatment with the selective GPR30 agonist G-1 partially attenuated the TNF induced upregulation of pro-inflammatory proteins such as intercellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). This effect was completely abolished by the selective GPR30 antagonist G-15, suggesting that it was indeed mediated in a GPR30 dependent manner. Interestingly, estrogen alone had no effects on TNF-treated endothelium. Concomitant activation of the classical ERs blocked the anti-inflammatory effects of G-1, indicating opposing effects of GPR30 and the classical ERs. Our findings demonstrate that endothelial GPR30 is a novel regulator of the inflammatory response which could be a potential therapeutic target against atherosclerosis and other inflammatory diseases.     

Estrogen dependence of the renal vasodilatory effect of nicotine in rats: role of α7 nicotinic cholinergic receptor/eNOS signaling

AimsWe recently reported that acute exposure to nicotine vasodilates the renal vasculature of male rats via facilitation of endothelial nitric oxide synthase (eNOS). In this study, we investigated whether this effect of nicotine is sexually dimorphic and the role of estrogen in modulating the nicotine effect.Main methodsNicotine-evoked vasodilation was evaluated in phenylephrine-preconstricted perfused kidneys obtained from male, proestrus female, ovariectomized (OVX) and estrogen-replaced OVX (OVXE₂) rats.Key findingsNicotine infusion (5 × 10^? 5, 1 × 10^? 4, and 5 × 10^? 4 M) produced greater concentration-dependent reductions in the renal perfusion pressure (RPP) in an isolated kidney from proestrus females than from males. Inhibition of NOS by N^G-nitro-l-arginine abolished the nicotine-evoked reduction in RPP and abolished the gender difference in the nicotine effect. Nicotine vasodilation was also attenuated in kidneys isolated from OVX and diestrus rats, models characterized by reduced estrogen levels. Further, estrogen or l-arginine supplementation in OVX rats largely restored the renal vasodilatory response to nicotine. Estrogen receptor blockade by tamoxifen abrogated the enhanced nicotine-evoked vasodilation elicited by E₂ in OVX rats. The nitrite/nitrate levels and protein expressions of eNOS and α₇ nicotinic cholinergic receptor (α₇ nAChRs) were significantly higher in renal tissues of OVXE₂ compared with OVX rats, suggesting a facilitatory effect for E₂ on α₇ nAChRs/eNOS signaling.SignificanceEstrogen-dependent facilitation of NOS signaling mediates the enhanced vasodilator capacity of nicotine in the renal vasculature of female rats. Preliminary evidence also suggests a potential role for α₇ nAChRs in this estrogen-dependent phenomenon.     

Chronic Activation of the G Protein-Coupled Receptor 30 with Agonist G-1 Attenuates Heart Failure

G protein-coupled receptor (GPR) 30 is a novel estrogen receptor. Recent studies suggest that activation of the GPR30 confers rapid cardioprotection in isolated rat heart. It is unknown whether chronic activation of GPR30 is beneficial or not for heart failure. In this study we investigated the cardiac effect of sustained activation or inhibition of GPR30. Female Sprague–Dawley rats were divided into 7 groups #2Q1: sham surgery (Sham), bilateral ovariectomy (OVX), OVX+estrogen (E₂), OVX+isoproterenol (ISO), OVX+ISO+G-1, OVX+ISO+E₂+G15, OVX+ISO+E₂. ISO (85 mg/kg×17 day, sc) was given to make the heart failure models. G-1(120 µg/kg·d×14 day) was used to activate GPR30 and G15 (190 µg/kg·d×14 day) was used to inhibit GPR30. Concentration of brain natriuretic peptide in serum, masson staining in isolated heart, contractile function and the expression of β₁ and β₂- adrenergic receptor (AR) of ventricular myocytes were also determined. Our data showed that ISO treatment led to heart failure in OVX rats. G-1 or E₂ treatment decreased concentration of brain natriuretic peptide, reduced cardiac fibrosis, and enhanced contraction of the heart. Combined treatment with β₁ (CGP20712A) and β₂-AR (ICI118551) antagonist abolished the improvement of myocardial function induced by G-1. We also found that chronic treatment with G-1 normalized the expression of β₁-AR and increased the expression of β₂-AR. Our results indicate that chronic activation of the GPR30 with its agonist G-1 attenuates heart failure by normalizing the expression of β₁-AR and increasing the expression of β₂-AR.     

2-Methoxyestradiol Induces Vasodilation by Stimulating NO Release via PPARγ/PI3K/Akt Pathway

The endogenous estradiol metabolite 2-methoxyestradiol (2-ME) reduces atherosclerotic lesion formation, while the underlying mechanisms remain obscure. In this work, we investigated the vasodilatory effect of 2-ME and the role of nitric oxide (NO) involved. In vivo studies using noninvasive tail-cuff methods showed that 2-ME decreased blood pressure in Sprague Dawley rats. Furthermore, in vitro studies showed that cumulative addition of 2-ME to the aorta caused a dose- and endothelium-dependent vasodilation. This effect was unaffected by the pretreatment with the pure estrogen receptor antagonist ICI 182,780, but was largely impaired by endothelial nitric oxide synthase (eNOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME) or by phosphoinositide 3-kinase (PI3K) inhibitor wortmannin (WM). Moreover, 2-ME（10^{−7 ∼}10⁻⁵ M）enhanced phosphorylation of Akt and eNOS and promoted NO release from cultured human umbilical endothelial cells (HUVECs). These effects were blocked by PI3K inhibitor WM, or by the transfection with Akt specific siRNA, indicating that endothelial Akt/eNOS/NO cascade plays a crucial role in 2-ME-induced vasodilation. The peroxisome proliferator-activated receptor γ (PPARγ) mRNA and protein expression were detected in HUVECs and the antagonist GW9662 or the transfection with specific PPARγ siRNA inhibited 2-ME-induced eNOS and Akt phosphorylation, leading to the impairment of NO production and vasodilation. In conclusion, 2-ME induces vasodilation by stimulating NO release. These actions may be mediated by PPARγ and the subsequent activation of Akt/eNOS cascade in vascular endothelial cells.     

Buthionine sulfoximine causes endothelium dependent hyper-relaxation and hypoadiponectinemia

A close relationship between oxidative stress, endothelial dysfunction, and hypoadiponectinemia has been observed. The present study was performed to investigate how glutathione depletion via buthionine sulfoximine (BSO) administration affects endothelial function and adiponectin levels in rats. Acetylcholine (Ach)-induced vasodilation was significantly enhanced in BSO-treated rats, compared with control rats. This was completely abolished by L-NAME, and Ach-induced vasodilation was not observed in the aorta without endothelium. These results suggest that Ach-induced hyper-relaxation of the aorta in BSO-treated rats is completely dependent on the presence of endothelium and mediated by changes in eNOS activity. Catalase significantly inhibited this relaxation to Ach and no effect of catalase on sodium nitroprusside-induced relaxation of the aorta without endothelium was observed in BSO-treated rats. Thus, hyper-relaxation of the aorta in BSO-treated rats is likely caused by H₂O₂ in addition to NO produced by the endothelium via an eNOS-dependent mechanism. Hypoadiponectinemia and decreased levels of adiponectin mRNA in adipose tissue were observed in BSO-treated rats. Protein expression of eNOS and SODs (SOD-1 and SOD-2) in the aorta was increased and plasma NOx levels were decreased in BSO-treated rats. Our results suggest that oxidative stress induced by BSO causes eNOS uncoupling and hyper-relaxation by producing H₂O₂, and that BSO-induced oxidative stress causes hypoadiponectinemia, probably by increasing H₂O₂ production in adipose tissue.     

Age impairs Flk-1 signaling and NO-mediated vasodilation in coronary arterioles

Impairment of flow-induced vasodilation in coronary resistance arterioles may contribute to the decline in coronary vasodilatory reserve that occurs with advancing age. This study investigated the effects of age on flow-induced signaling and activation of nitric oxide (NO)-mediated vasodilation in coronary resistance arterioles. Coronary arterioles were isolated from young (approximately 6 mo) and old (approximately 24 mo) male Fischer-344 rats to assess vasodilation to flow, vascular endothelial growth factor (VEGF), and ACh. Flow- and VEGF-induced vasodilation of coronary arterioles was impaired with age (P相似文献   

17β-雌二醇对去卵巢胰岛素抵抗大鼠主动脉舒缩功能损伤的保护作用

为观察17β-雌二醇（17beta-estradiol,17β-E2）对去卵巢胰岛素抵抗（insulin resistance,IR）大鼠主动脉结构和舒缩功能的影响及其可能机制,成年雌性Sprague-Dawley大鼠卵巢切除后,高果糖喂养8周诱导IR,同时给予生理剂量的17β-E2（30μg／kg）,每天皮下注射一次,并检测IR相关指标。大鼠胸主动脉石蜡切片,HE染色,图像分析系统测定其结构。采用血管环灌流法,观察各组大鼠胸主动脉环对新福林（L-phenylephrine,PE）的收缩反应和对ACh、硝普钠（sodium nitroprusside,SNP）的舒张反应以及一氧化氮合酶（nitric oxide synthase,NOS）抑制剂N-硝基-L-精氨酸甲脂（N-nitrl-L-arginine methylester,L-NAME）对卵巢切除＋果糖喂养＋17β-E2组大鼠胸主动脉ACh的舒张反应的影响;检测各组大鼠一氧化氮（nitric oxide,NO）含量。结果显示：（1）17β-E2能防止高果糖诱导的去卵巢IR人鼠收缩压升高、高胰岛素血症和胰岛素敏感性下降;（2）各组火鼠胸主动脉的结构无显著性差异;（3）卵巢切除＋果糖喂养组大鼠与卵巢切除组或果糖喂养组相比,血清NO显著降低,胸主动脉对PE的收缩反应显著增强,对ACh的舒张反应显著降低,17β-E2能逆转上述改变,L-NAME可部分阻断17β-E2的这种作用;（4）各组大鼠胸主动脉对SNP的舒张反应和去内皮后对PE的收缩反应均无显著差异。以上结果表明,17β-E2能抑制高果糖诱导的去卵巢IR大鼠血管舒缩功能的紊乱,其机制一方面可能是部分通过血管内皮细胞NOS途径促进NO的释放,保护内皮细胞;另一方面可能是通过降低血压,血清胰岛素水平,改善IR所致。     

Diabetes abolishes the vascular protective effects of estrogen in female rats

Estrogen is known to exert a protective effect against cardiovascular disease. However, women with diabetes have three times the risk as compared with age-matched non-diabetic women. Our previous study on aortic rings of ovariectomized (OVX) female rats treated with 17-beta-estradiol (E2) demonstrated that the beneficial effect of estrogen is related to the basal release of NO from endothelial cells. In the present study, in order to understand why estrogen protection is abolished in diabetes, we tested vascular responses in OVX, streptozotocin-diabetic female rats and their non-diabetic controls receiving or not E2 replacement. Concentration-response curves to norepinephrine (NE) showed attenuation of the contractile response in E2-treated diabetic, with respect to non-diabetic preparations. This response was further impaired in diabetic, E2-deprived rats. The basal release of NO, as evaluated by concentration-related responses to N(G)-methyl-L-arginine acetate in NE-precontracted aortic rings, was found to be impaired in E2-treated diabetic rats, no further effect being induced by E2 deprivation. The endothelium-dependent relaxation produced by carbachol did not change between groups, whereas the relaxation produced by histamine was enhanced by both diabetes and E2 deprivation. However, E2 treatment counteracted the response to histamine only in preparations from non-diabetic animals. Finally, the relaxation induced by sodium nitroprusside, an endothelium-independent relaxant agent, was comparable between groups. These findings suggest that the lack of protective effects of estrogen in diabetes may be mainly ascribed to the failure of estrogen to reverse the impaired basal release of NO and the abnormal relaxation to histamine, which are observed in the aorta of diabetic rats.     

Nitric-oxide-dependent pial arteriolar dilation in the female rat: effects of chronic estrogen depletion and repletion

In this study, we compared endothelial nitric oxide synthase (eNOS)-mediated cerebral vasodilating responses in intact female rats, chronically ovariectomized (OVX) rats, and OVX rats treated for 2 weeks with 17beta-estradiol (E(2)). Under anesthesia, using intravital microscopy and a closed cranial window system, pial arteriolar diameter changes were monitored during sequential cortical suffusions of an eNOS-dependent dilator [acetylcholine (ACh)] and a direct NO donor [S-nitrosoacetylpenicillamine (SNAP)]. In separate rats from the same groups, we compared eNOS and caveolin-1 (CAV-1) protein abundance in pial arterioles (via immunofluorescence analyses). In untreated and low-dose E(2)-treated (1.0 microg x kg(-1) x day(-1)) OVX rats, ACh-induced vasodilations were virtually absent. High-dose E(2) treatment (100 microg x kg(-1) x day(-1)) restored ACh-induced pial arteriolar dilations to levels seen in intact females. The vasodilations elicited by SNAP and ADO were unaffected by chronic estrogen changes, indicating no direct estrogen influence on vascular smooth muscle (VSM) reactivity. Pial arteriolar eNOS protein abundance was diminished by ovariectomy and restored by high-dose E(2) treatment. Pial arteriolar CAV-1 expression was higher in OVX versus intact and E(2)-treated OVX females. These results suggest that long-term changes in estrogen directly influence brain eNOS functional activity. The estrogen-related changes in eNOS-dependent vasodilating function appear to be related, in part, to a capacity for E(2) to increase eNOS protein expression and, in part, to an E(2)-associated diminution in endothelial CAV-1 expression.     

17Beta-estradiol inhibits ovariectomy-induced expression of inducible nitric oxide synthase in rat aorta in vivo

The objective of this study was to elucidate a role of ovarian steroid hormones in the production of immunologic nitric oxide (NO) synthases in the female rat aorta in vivo. Aortic homogenates were analyzed by using western blot with isoform-specific antibodies against endothelial NOS (eNOS) and inducible NOS (iNOS). Two weeks after ovariectomy (OVX), rats (10-week-old) were treated with 17beta-estradiol (E2) and/or progesterone (P4) for 5 days, and aortae were obtained from these rats on the following day. OVX markedly increased the levels of iNOS protein in abdominal aorta, whereas treatment with E2 or a combination of E2 and P4 inhibited the induction of iNOS in the aorta. The present findings indicate that endogeneous estrogen negatively regulates the expression of iNOS in abdominal aorta, and suggest that changes in the levels of circulating estrogen may affect vascular function.     

Assessment of cell line competence for studies of pharmacological GPR30 modulation

Context/objective: Cell lines used to study the role of the G protein-coupled receptor 30 (GPR30) or G protein-coupled estrogen receptor (GPER) as a mediator of estrogen responses have yielded conflicting results. This work identified a simple assay to predict cell line competence for pharmacological studies of GPR30.

Materials and methods: The phosphorylation or expression levels of ERK1/2, Akt, c-Fos and eNOS were evaluated to assess GPR30 activation in response to known agonists (17β-estradiol and G-1) in MCF-7 and T-47D breast cancer cell lines and in bovine aortic endothelial cells. GPR30 expression was analyzed by qRT-PCR and Western blot with two distinct antibodies directed at its carboxy and amino terminals.

Results: None of the agonists, at any of the concentrations tested, activated any of those target proteins. Additional experiments excluded the disruption of the signaling pathway, interference of phenol red in the culture medium and constitutive proteasome degradation of GPR30 as possible causes for the lack of response of the three cell lines. Analysis of receptor expression showed the absence of clearly detectable GPR30 species of 44 and 50–55?kDa previously identified in cell lines that respond to 17β-estradiol and G-1.

Discussion and conclusion: Cells that do not express the 44 and 50–55?kDa species do not respond to GPR30 agonists. Thus, the presence or absence of these GPR30 species is a simple and rapid manner to determine whether a given cell line is suitable for pharmacological or molecular studies of GPR30 modulation.     

Aging and Estrogen Status: A Possible Endothelium-Dependent Vascular Coupling Mechanism in Bone Remodeling

Bone loss with aging and menopause may be linked to vascular endothelial dysfunction. The purpose of the study was to determine whether putative modifications in endothelium-dependent vasodilation of the principal nutrient artery (PNA) of the femur are associated with changes in trabecular bone volume (BV/TV) with altered estrogen status in young (6 mon) and old (24 mon) female Fischer-344 rats. Animals were divided into 6 groups: 1) young intact, 2) old intact, 3) young ovariectomized (OVX), 4) old OVX, 5) young OVX plus estrogen replacement (OVX+E2), and 6) old OVX+E2. PNA endothelium-dependent vasodilation was assessed in vitro using acetylcholine. Trabecular bone volume of the distal femoral metaphysis was determined by microCT. In young rats, vasodilation was diminished by OVX and restored with estrogen replacement (intact, 82±7; OVX, 61±9; OVX+E2, 90±4%), which corresponded with similar modifications in BV/TV (intact, 28.7±1.6; OVX, 16.3±0.9; OVX+E2, 25.7±1.4%). In old animals, vasodilation was unaffected by OVX but enhanced with estrogen replacement (intact, 55±8; OVX, 59±7; OVX+E2, 92±4%). Likewise, modifications in BV/TV followed the same pattern (intact, 33.1±1.6; OVX, 34.4±3.7; OVX+E2, 42.4±2.1%). Furthermore, in old animals with low endogenous estrogen (i.e., intact and old OVX), vasodilation was correlated with BV/TV (R² = 0.630; P<0.001). These data demonstrate parallel effects of estrogen on vascular endothelial function and BV/TV, and provide for a possible coupling mechanism linking endothelium-dependent vasodilation to bone remodeling.     

Oxidant stress from uncoupled nitric oxide synthase impairs vasodilation in fetal lambs with persistent pulmonary hypertension

Persistent pulmonary hypertension of newborn (PPHN) is associated with decreased NO release and impaired pulmonary vasodilation. We investigated the hypothesis that increased superoxide (O(2)(*-)) release by an uncoupled endothelial nitric oxide synthase (eNOS) contributes to impaired pulmonary vasodilation in PPHN. We investigated the response of isolated pulmonary arteries to the NOS agonist ATP and the NO donor S-nitroso-N-acetylpenicillamine (SNAP) in fetal lambs with PPHN induced by prenatal ligation of ductus arteriosus and in sham-ligated controls in the presence or absence of the NOS antagonist nitro-L-arginine methyl ester (L-NAME) or the O(2)(*-) scavenger 4,5-dihydroxy-1,3-benzenedisulfonate (Tiron). ATP caused dose-dependent relaxation of pulmonary artery rings in control lambs but induced constriction of the rings in PPHN lambs. L-NAME, the NO precursor L-arginine, and Tiron restored the relaxation response of pulmonary artery rings to ATP in PPHN. Relaxation to NO was attenuated in arteries from PPHN lambs, and the response was improved by L-NAME and by Tiron. We also investigated the alteration in heat shock protein (HSP)90-eNOS interactions and release of NO and O(2)(*-) in response to ATP in the pulmonary artery endothelial cells (PAEC) from these lambs. Cultured PAEC and endothelium of freshly isolated pulmonary arteries from PPHN lambs released O(2)(*-) in response to ATP, and this was attenuated by the NOS antagonist L-NAME and superoxide dismutase (SOD). ATP stimulated HSP90-eNOS interactions in PAEC from control but not PPHN lambs. HSP90 immunoprecipitated from PPHN pulmonary arteries had increased nitrotyrosine signal. Oxidant stress from uncoupled eNOS contributes to impaired pulmonary vasodilation in PPHN induced by ductal ligation in fetal lambs.     

Acacetin improves endothelial dysfunction and aortic fibrosis in insulin-resistant SHR rats by estrogen receptors

The aim of the work was to investigate the effects of acacetin on endothelial dysfunction and aortic fibrosis in insulin-resistant SHR rats and explore its mechanism. Seven-week-old male spontaneously hypertensive rats (SHR) were selected to establish a rat model of hypertension with insulin resistance induced by 10% fructose. The nuclear factor kappa B p65 (NF-κB p65) and Collagen I were observed by Immunohistochemistry. Immunofluorescence was used to observe estrogen receptor-alpha (ERα), estrogen receptor-beta (ERβ), and G protein-coupled receptor 30 (GPR30). Western blotting was used to detect interleukin (IL-1β), Arginase 2 (ARG2), Nostrin, endothelial nitric oxide synthase (eNOS), TGF-β, Smad3, ERK pathway proteins such as p-c-Raf, p-MEK1/2, p-ERK, ERK, p-P90RSK and p-MSK1. We found that acacetin did have an improvement on endothelial dysfunction and fibrosis. Meanwhile, it was also found to have a significant effect on the level of estrogen in this model by accident. Then, the experiment of uterine weight gain in mice confirmed that acacetin had a certain estrogen-like effect in vivo and played its role through the estrogen receptors pathway. In vitro experience HUVEC cells were stimulated with 30 mM/L glucose and 100 mM/L NaCl for 24 h to establish the endothelial cell injury model. HUVEC cells were treated with 1 μM/L estrogen receptors antagonist (ICI 182780) for 30 min before administration. Cell experiments showed that acacetin could reduce the apoptosis of HUVEC cells, the levels of inflammatory cytokines and the expression of TGF-β, Collagen I and Smad3 in endothelial cell injury model. After treatment with ICI 182780, the improvement of acacetin was significantly reversed. The results showed that acacetin relieved endothelial dysfunction and reduced the aortic fibrosis in insulin-resistant SHR rats by reducing the release of inflammatory factors and improving vasodilatory function through estrogen signaling pathway.

     

C-peptide inhibits leukocyte-endothelium interaction in the microcirculation during acute endothelial dysfunction.

C-peptide is a cleavage product that comes from processing proinsulin to insulin that induces nitric oxide (NO) -mediated vasodilation. NO modulates leukocyte-endothelium interaction. We hypothesized that C-peptide might inhibit leukocyte-endothelium interaction via increased release of endothelial NO. Using intravital microscopy of the rat mesentery, we measured leukocyte-endothelium interactions after administration of C-peptide to the rat. Superfusion of the rat mesentery with either thrombin or L-NAME consistently and significantly increased the number of rolling, adhering, and transmigrated leukocytes. C-peptide significantly attenuated either thrombin- or L-NAME-induced leukocyte-endothelium interactions in rat mesenteric venules. A control scrambled sequence of C-peptide characterized by the same amino acid composition in a randomized sequence failed to inhibit leukocyte-endothelium interactions. These effects of C-peptide were associated with decreased surface expression of the cell adhesion molecules P-selectin and ICAM-1 on the microvascular endothelium. Endothelial nitric oxide synthase (eNOS) mRNA levels were increased in rats injected with C-peptide. This enhanced eNOS expression was associated with a marked increase in basal NO release from the aorta of C-peptide-treated rats. We conclude that C-peptide is a potent inhibitor of leukocyte-endothelium interaction and that this effect is specifically related to inhibition of endothelial cell adhesion molecules via maintenance of NO release from the vascular endothelium.     

Resveratrol Ameliorates High Glucose and High-Fat/Sucrose Diet-Induced Vascular Hyperpermeability Involving Cav-1/eNOS Regulation

Vascular endothelial hyperpermeability is one of the manifestations of endothelial dysfunction. Resveratrol (Res) is considered to be beneficial in protecting endothelial function. However, currently, the exact protective effect and involved mechanisms of Res on endothelial dysfunction-hyperpermeability have not been completely clarified. The aim of present study is to investigate the effects of Res on amelioration of endothelial hyperpermeability and the role of caveolin-1 (Cav-1)/endothelial nitric oxide synthase (eNOS) pathway. Adult male Wistar rats were treated with a normal or high-fat/sucrose diet (HFS) with or without Res for 13 weeks. HFS and in vitro treatment with high glucose increased hyperpermeability in rat aorta, heart, liver and kidney and cultured bovine aortic endothelial cells (BAECs), respectively, which was attenuated by Res treatment. Application of Res reversed the changes in eNOS and Cav-1 expressions in aorta and heart of rats fed HFS and in BAECs incubated with high glucose. Res stimulated the formation of NO inhibited by high glucose in BAECs. Beta-Cyclodextrin (β-CD), caveolae inhibitor, showed the better beneficial effect than Res alone to up-regulate eNOS phosphorylative levels, while NG-Nitro-77 L-arginine methyl ester (L-NAME), eNOS inhibitor, had no effect on Cav-1 expression. Our studies suggested that HFS and in vitro treatment with high glucose caused endothelial hyperpermeability, which were ameliorated by Res at least involving Cav-1/eNOS regulation.