Nitric Oxide Production In Human Umbilical Vein Endothelial Cells Research Articles

Pregnancy-Specific Glycoprotein 9 Enhances Store-Operated Calcium Entry and Nitric Oxide Release in Human Umbilical Vein Endothelial Cells

We explored changes in pregnancy-specific glycoprotein 9 (PSG9) levels in the serum of patients with preeclampsia and the effects and underlying mechanisms of PSG9 effects on calcium (Ca2+) homeostasis and nitric oxide (NO) release in human umbilical vein endothelial cells (HUVECs). Western blotting was used to detect protein expression levels, and an NO fluorescence probe was used to examine NO production. Intracellular Ca2+ concentrations were measured using a Ca2+-sensitive fluorescent dye under a fluorescence microscope. Compared with those in healthy pregnant women, serum PSG9 levels were significantly decreased in patients with preeclampsia. PSG9 (0.1 μg/mL) treatment of HUVECs significantly enhanced the expression levels of store-operated calcium entry (SOCE) channel proteins Orai1 and Orai2, but not Orai3, and of endothelial nitric oxide synthase (eNOS) and NO production. Pretreatment with an inhibitor of SOCE (BTP2) abolished PSG9-enhanced Orai1, Orai2, and eNOS expression levels and NO production in HUVECs. The mechanisms underlying SOCE that were PSG9 enhanced in HUVECs appear to involve the Ca2+/eNOS/NO signaling pathway. These findings suggest that serum PSG9 levels may be a potential biomarker for monitoring the occurrence or development of preeclampsia in pregnancy and that PSG9 may be a potential therapeutic target for the treatment of preeclampsia.

Enhancement of the In Vitro Antitumor Effects of Berberine Chloride When Encapsulated within Small Extracellular Vesicles.

Berberine hydrochloride (BRB) is an isoquinoline alkaloid with promising anticancer efficacies. However, application of BRB had been hampered by its poor aqueous solubility, low gastrointestinal absorption, and rapid metabolism. The present study takes advantage of small extracellular vesicles (sEVs) to increase both stability and efficacy of BRB. sEVs from immature dendritic cells were produced and loaded with BRB. Proliferation, migration and Matrigel assay were performed, cycle arrest and nitric oxide (NO) production were evaluated in human breast cancer cell line (MDA-MB-231) and human umbilical vein endothelial cells (HUVECs). sEVs loaded with BRB formed a stable and homogenous population with a drug entrapment efficiency near to 42%. BRB loaded into sEVs was more potent than free BRB for MDA-MB-231 and endothelial proliferation, migration, and capillary-like formation in HUVECs. The mechanisms involved a blockade of cell cycle in G0/G1 phase, increased S phase and decreased of G2/M in MDA-MB-231 and HUVECs, and inhibition of NO production in HUVECs. Altogether, sEV-loaded BRB displayed higher effects than free BRB on different steps leading to its antitumor activity and anti-angiogenic properties in vitro. Thus, sEV formulation may be considered as an innovative approach and promising delivery of BRB to prevent tumorigenesis and angiogenesis.

Decreased NO production in endothelial cells exposed to plasma from ME/CFS patients

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a debilitating disease characterized by severe and persistent fatigue. Along with clinical studies showing endothelial dysfunction (ED) in a subset of ME/CFS patients, we have recently reported altered ED-related microRNAs in plasma from affected individuals. Inadequate nitric oxide (NO), mainly produced by the endothelial isoform of nitric oxide synthase (eNOS) in endothelial cells (ECs), is a major cause of ED. In this study, we hypothesized that plasma from that cohort of ME/CFS patients induces eNOS-related ED in vitro. To test this, we cultured human umbilical vein endothelial cells (HUVECs) in the presence of plasma from either ME/CFS patients (ME/CFS-plasma, n = 11) or healthy controls (HC-plasma, n = 12). Then, we measured the NO production in the absence and presence of tyrosine kinase and G protein-coupled receptors agonists (TKRs and GPCRs, respectively), well-known to activate eNOS in ECs. Our data showed that HUVECs incubated with ME/CFS-plasma produced less NO either in the absence or presence of eNOS activators compared to ones in presence of HC-plasma. Also, the NO production elicited by bradykinin, histamine, and acetylcholine (GPCRs agonists) was more affected than the one triggered by insulin (TKR agonist). Finally, inhibitory eNOS phosphorylation at Thr495 was higher in HUVECs treated with ME/CFS-plasma compared to the same treatment with HC-plasma. In conclusion, this study in vitro shows a decreased NO production in HUVECs exposed to plasma from ME/CFS patients, suggesting an unreported role of eNOS in the pathophysiology of this disease.

Glossogyne tenuifolia Extract Increases Nitric Oxide Production in Human Umbilical Vein Endothelial Cells.

The vascular nitric oxide (NO) system has a protective effect in atherosclerosis. NO is generated from the conversion of L-arginine to L-citrulline by the enzymatic action of endothelial NO synthase (eNOS). Compounds with the effect of enhancing eNOS expression are considered to be candidates for the prevention of atherosclerosis. In this study, extracts from the aerial, root, and whole plant of Glossogyne tenuifolia (GT) were obtained with ethanol, n-hexane, ethyl acetate (EA), and methanol extraction, respectively. The effects of these GT extracts on the synthesis of NO and the expression of eNOS in human umbilical vein endothelial cells (HUVECs) were investigated. NO production was determined as nitrite by colorimetry, following the Griess reaction. The treatment of HUVECs with EA extract from the root of GT and n-hexane, methanol, and ethanol extract from the aerial, root, and whole plant of GT increased NO production in a dose-dependent manner. When at a dose of 160 μg/mL, NO production increased from 0.9 to 18.4-fold. Among these extracts, the methanol extract from the root of GT (R/M GTE) exhibited the most potent effect on NO production (increased by 18.4-fold). Furthermore, using Western blot and RT–PCR analysis, treatment of HUVECs with the R/M GTE increased both eNOS protein and mRNA expression. In addition, Western blot analysis revealed that the R/M GTE increased eNOS phosphorylation at serine1177 as early as 15 min after treatment. The chemical composition for the main ingredients was also performed by HPLC analysis. In conclusion, the present study demonstrated that GT extracts increased NO production in HUVECs and that the R/M GTE increased NO production via increasing eNOS expression and activation by phosphorylation of eNOS at serine1177.

N‑acetyl cysteine inhibits lipopolysaccharide‑induced apoptosis of human umbilical vein endothelial cells via the p38MAPK signaling pathway.

Lipopolysaccharide (LPS) can regulate the expression of apoptotic factors, including caspase‑3, Bcl‑2 and Bcl‑2‑associated X protein (Bax). Nitric oxide (NO) plays an important role in apoptosis. N‑acetyl cysteine (NAC) has been shown to exhibit antioxidant effects invitro. However, the effects of NAC on LPS‑induced apoptosis of human umbilical vein endothelial cells (HUVECs) and the associated mechanisms are not well characterized. The present study explored the effect of NAC on LPS‑induced apoptosis of HUVECs and determined the participation of the p38 mitogen‑activated protein kinase (MAPK) pathway in the process of apoptosis. Cell viability was assessed using the Cell Counting Kit‑8 (CCK‑8) assay. The expression of caspase‑3, Bax, Bcl‑2, phosphorylated (p)‑p38MAPK/total (t‑)p38MAPK and p‑endothelial e nitric oxide synthase (eNOS)/t‑eNOS proteins were determined by western blotting. The expression levels of caspase‑3, Bax and Bcl‑2 mRNA were determined using reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR). The rate of apoptosis was determined using flow cytometry. An NO detection kit (nitric reductase method) was used to determine NO concentration. The results of CCK‑8 and flow cytometric analyses showed that pretreatment of HUVECs with NAC or p38MAPK inhibitor (SB203580) attenuated LPS‑induced decrease in cell viability and increase in cell apoptosis. RT‑qPCR and western blotting showed that LPS promoted caspase‑3 and Bax expression, but inhibited that of Bcl‑2 in HUVECs; however, these effects were attenuated by pretreatment with NAC or SB203580. LPS stimulation significantly enhanced the expression of p‑p38MAPK protein and reduced the expression of p‑eNOS protein; however, these effects were attenuated by pretreatment with NAC or SB203580. NAC pretreatment attenuated LPS‑induced inhibition of NO synthesis, which was consistent with the effects of SB203580. The results demonstrated that NAC pretreatment alleviated LPS‑induced apoptosis and inhibition of NO production in HUVECs. Furthermore, these effects were proposed to be mediated via the p38MAPK signaling pathway.

Native low density lipoprotein increases the production of both nitric oxide and reactive oxygen species in the human umbilical vein endothelial cells.

Nitric oxide synthases (NOSs) are a unique family of enzymes that catalyze the production of nitric oxide (NO) from L-arginine. Atherogenic action of oxidized low-density lipoproteins (oxLDL) may be mediated partly by the formation of NO in endothelial cells. The objective of this study was to identify sources of reactive oxygen species (ROS) causing native LDL (nLDL)-induced senescence of cultured human umbilical vein endothelial cells (HUVECs). HUVECs were treated with nLDL and NO production was assessed using Griess reagent as substrate and spectrophotometry in the absence or presence of specific inhibitors of endothelial NOS (eNOS) and inducible NOS (iNOS). In addition, expression levels of eNOS and iNOS were measured with ELISA and western blotting, and ROS was evaluated using 2',7'-dichlorofluorescin diacetate (DCF-DA) and a fluorescence microplate reader. NO formation in nLDL-treated HUVECs was significantly increased. Long-term treatment with nLDL up-regulated both eNOS and iNOS proteins. Such increase of NO production in HUVECs induced by nLDL was significantly suppressed by treatment with iNOS-selective inhibitor 1400W, but not by the eNOS-selective inhibitor L-NIO. Native LDL treatment uncoupled Hsp90, the regulatory binding protein of eNOS, from the enzyme in HUVECs. Native LDL also significantly increased ROS production in HUVECs. These findings suggest that oxidative stress originated from induction of iNOS and eNOS could be a causative factor for nLDL-induced senescence of HUVECs.

TRPC1 stimulates calcium‑sensing receptor‑induced store‑operated Ca2+ entry and nitric oxide production in endothelial cells.

Store-operated Ca2+ entry (SOCE) via store-operated Ca2+ channels (SOCC), encoded by transient receptor potential canonical (TRPC) channel proteins, is an important underlying mechanism regulating intracellular Ca2+ concentration ([Ca2+]i) and various intracellular functions in endothelial cells (ECs). TRPC1, the probable candidate for SOCC, is expressed in ECs. Ca2+-sensing receptor (CaSR) is functionally expressed in vascular endothelium and is important in Ca2+ mobilization and cardiovascular functions. To date, there have been no reports demonstrating an association between CaSR and TRPC1 in ECs. The present study investigated the effects of TRPC1 on CaSR-induced Ca2+ influx and nitric oxide (NO) production in human umbilical vein ECs (HUVECs). TRPC1 and CaSR proteins in HUVECs were measured by immunostaining and western blot analysis. [Ca2+]i levels were measured using the Fura-2-acetoxymethyl ester method. The indicator 3-amino, 4-aminomethyl-2, 7-difluorescein diacetate was used to measure NO production in HUVECs. The expression of TRPC1 protein in HUVECs was silenced by transfecting HUVECs with small interfering RNA (siRNA) against TRPC1. Although changes in extracellular Ca2+ failed to alter [Ca2+]i in HUVECs, the CaSR agonist spermine increased [Ca2+]i and NO production in HUVECs. NO production in HUVECs was diminished in Ca2+-free medium or following treatment with a CaSR negative allosteric modulator (Calhex231), SOCC inhibitor (MRS1845) or TRPC inhibitor (SKF96365). The spermine-induced increases in [Ca2+]i and NO production were reduced in HUVECs transfected with TRPC1 siRNA. These results suggested that TRPC1 is a primary candidate in forming SOCC that stimulates CaSR-induced SOCE and NO production in HUVECs and is a potential therapeutic target for vascular diseases.

Sodium tanshinone IIA sulfonate suppresses heat stress-induced endothelial cell apoptosis by promoting NO production through upregulating the PI3K/AKT/eNOS pathway.

Heat shock is a life-threatening disease involving systematic inflammation that is closely related to endothelial injury and can lead to multiple organ dysfunction syndrome. Sodium tanshinone IIA sulfonate (STS) has various functions in the vascular endothelium. In the present study, STS is presented to suppress heat stress‑induced apoptosis of human umbilical vein endothelial cells (HUVECs) and high ambient temperature‑induced systematic inflammation in Sprague Dawley rats. In addition, the STS apoptosis‑suppression mechanism was explored. The results presented in the present study demonstrated that the PI3K/AKT pathway was stimulated by STS treatment and that eNOS phosphorylation at Ser‑1177 was also upregulated, resulting in increased nitric oxide production in HUVECs under heat stress. Using specific inhibitors, the authors confirmed that STS‑induced endothelial nitric oxide synthase (eNOS) phosphorylation at Ser‑1177 was activated by protein kinase B phosphorylation at Ser‑473, involving activation of phosphatidylinositol‑3 kinase (PI3K). The results suggested that STS suppresses heat stress‑induced apoptosis of HUVECs via the PI3K/AKT/eNOS pathway and may be used in heat shock treatment as a vascular endothelial protection mechanism.

Rutin protects endothelial dysfunction by disturbing Nox4 and ROS-sensitive NLRP3 inflammasome.

High glucose induced endothelial dysfunction is blamed for initiation of vascular complication in Type 2 diabetes mellitus. Rutin has been described in possessing comprehensive pharmacological activities, but the mechanism involved in endothelial protection through regulating oxidative stress and damage is still ambiguous. In the present study, rutin was evaluated in high glucose stimulated human umbilical vein endothelial cells (HUVECs) and high glucose diet-treated SD rats were applied to explored the molecular mechanism in rutin counteracting oxidative stress and damage. Firstly, 30 and 100μM rutin effectively increased HUVECs viability in high glucose challenge. Then we found that rutin could dose-dependently reduced high glucose mediated mRNA and protein expressions of Nox4. With Nox4 and Nox2 inhibitors, we further confirm that Nox4, but not Nox2, was responsible for reactive oxygen species (ROS) production in high glucose environment. Moreover, rutin and Nox4 inhibitor significantly ameliorated ROS generation and TXNIP, NLRP3, caspase-1 and IL-1β proteins expression in vivo. Furthermore, rutin substantially recovered nitric oxide production in HUVECs under high glucose condition. However, rutin could not inhibited inflammasome protein expressions and improved nitric oxide production in Nox4-overexpressed HUVECs under high glucose challenges. Finally, we found that rutin restored phenylephrine-mediated contractions and acetylcholine induced relaxations in aortic tissue of high glucose diet treated rats. In vitro, expressions of TXNIP, NLRP3, and caspase-1 in aortic tissue of high glucose diet treated rats were decreased under rutin administration. In summary, rutin may protect endothelial dysfunction through inhibiting Nox4 responsive oxidative stress and ROS-sensitive NLRP3 signaling pathway under high glucose stress both in vivo and vitro.

Arsenic and fluoride induce apoptosis, inflammation and oxidative stress in cultured human umbilical vein endothelial cells

Excessive amount of inorganic arsenic (iAs) and fluoride (F) coexist in drinking water in many regions, which is associated with high risk of vascular diseases. However, the underlying mechanisms are not well studied. The present study was to evaluate the effects of iAs and F individual or combined exposure on endothelial activation and apoptosis in vitro. Primary human umbilical vein endothelial cells (HUVECs) were exposed to 5 μM As2O3 and/or 1 mM NaF. Changes in endothelial cell apoptosis, inflammation, oxidative stress and nitric oxide (NO) production were analyzed. The results showed that iAs and/or F induced significant increase in endothelial cell apoptosis and inflammation as indicated by the increase of mRNA and protein expression of vascular cell adhesion molecule-1, intercellular adhesion molecule-1, and pentraxin 3. Furthermore, iAs and/or F exposure induced intracellular reactive oxygen species and malondialdehyde generation. Results showed iAs and/or F exposure increased the activity of NADPH oxidase (NOX) and up-regulated the mRNA expression of NOX subunits p22phox. The results indicated that activation of NOX was related to oxidative stress induced by iAs and/or F. Also, iAs and/or F reduced NO production in HUVECs. The up-regulation of inflammation genes expression and oxidative stress in iAs and F co-exposed ECs were less pronounced as compared to single F-exposed cells, which showed an antagonistic effect between iAs and F. In conclusion, endothelial activation and apoptosis induced by iAs and/or F are potential mechanisms in their vascular toxicity. Oxidative stress and impaired NO production are involved in their pro-inflammatory and pro-apoptotic effects.

Additive effects of low concentrations of estradiol-17β and progesterone on nitric oxide production by human vascular endothelial cells through shared signaling pathways

Potential cardiovascular benefits of low-dose formulations of estrogens and progesterone (P4) for treating climacteric symptoms in postmenopausal women remain unclear because information is lacking on their combined vascular effects. Protective effects of low concentrations (5nM) of P4 and estradiol-17β (E2), alone and in combination (P4+E2), were investigated in a nongenomic model of vascular protection which measured acute increases in nitric oxide (NO) production by cultured human umbilical vein endothelial cells (HUVECs). Treatment with 5nM P4+E2 for twenty minutes significantly increased NO production and endothelial NO synthase (eNOS) phosphorylation, whereas 5nM treatments with either steroid alone were ineffective. The 5nM P4+E2 treatment also increased phosphorylation of ERK and Akt, mimicking the effects of higher concentrations of P4 and E2 alone. Pre-treatment with inhibitors of PI3K (wortmannin), Akt (ML-9), and MAP kinase (AZD6244 and U0126) completely blocked the NO response to 5nM P4+E2. Combined 5nM treatments with specific estrogen and progesterone receptor agonists showed an involvement of membrane progesterone receptor alpha (mPRα, also known as PAQR7), G protein-coupled estrogen receptor 1 (GPER), and estrogen receptor alpha (ERα), but not ERβ, in P4+E2 stimulation of NO production. P4+E2 also exerted genomic actions, increasing mPRα, GPER, cyclooxygenase-1, and prostacyclin-synthase mRNA levels. Taken together, the results show that a low concentration of P4+E2 rapidly increases NO production in HUVECs through mPRα, ERα, and GPER and involves common signaling pathways, PI3K/Akt and MAP kinase. These in vitro findings suggest that low doses of E2 and P4 may also have some beneficial cardiovascular effects in vivo when administered as hormone replacement therapy (HRT) for post-menopausal women.

In vitro vasorelaxation mechanisms of Isoapiole extracted from Lemonfragrant Angelica Root on rat thoracic aorta

Ethnopharmcological relevanceLemonfragrant Angelica (Ostericum citriodorum (Hance) C. Q. Yuan & Shan) is a traditional Chinese herb for treatment of angina pectoris, stomach pain and abdominal pain. However, its active components and mechanisms of action were not well understood. Aims of the studyIn this study, we investigated whether the isoapiole extracted from Lemonfragrant Angelica Root (LAR) could directly stimulate the production of nitric oxide (NO) in vascular endothelial cells (VECs) and lead to the vascular relaxation Materials and methodsVascular activity experiments were performed in aortic rings isolated from Wistar rats using standard muscle bath procedures. Isoapiole was added with different concentrations (0.75, 2.5, 5μg/mL), and vessel relaxation of rat aortic rings pre-contracted with norepinephrine (NE) or potassium chloride was recorded. NO release from aortic rings exposed to isoapiole (5μg/mL) was measured by Griess method. The endothelial nitric oxide synthase (eNOS) expression in primary human umbilical vein endothelial cells (HUVECs) incubated with isoapiole was determined using Western blot and microplate reader assay. Classical receptor antagonists, channel and enzymatic inhibitors were used to check the mechanisms involved. ResultsIsoapiole (0.75, 2.5, 5μg/mL) inhibited norepinephrine-induced contraction in endothelium-intact rat aortic rings. However, a very weak relaxation of aortic rings was obtained in endothelium-denuded preparations. Isoapiole (0.75, 2.5, 5μg/mL) did not have vascular relaxative effect on neither endothelium-intact nor endothelium-denuded aortas pre-contracted with KCl (60mmol/L). The vasorelaxation effect of isoapiole on rat aortic rings was attenuated by the eNOS inhibitor, NG-nitro-L-arginine methyl ester (L-NAME). This result suggested that suggested that the isoapiole action was at least partially mediated by promoting eNOS expression. It was further found that isoapiole (5μg/mL) increased NO production in isolated rat thoracic aorta rings. Isoapiole increased eNOS expression leading to NO production in HUVECs. ConclusionIsoapiole stimulates NO production in the endothelium, leading to vascular dilatation.

Activating transcription factor 3 SUMOylation is involved in angiotensin II-induced endothelial cell inflammation and dysfunction

Activating transcription factor 3 (ATF3) is an adaptive-response protein induced by various environmental stresses and is implicated in the pathogenesis of many disease states. However, the role of ATF3 SUMOylation in hypertension-induced vascular injury remains poorly understood. Here we investigated the function of ATF3 SUMOylation in vascular endothelial cells (ECs). The expression of ATF3 and small ubiquitin-like modifier 1 (SUMO1) was increased in angiotensin II (Ang II)-induced human umbilical vein endothelial cells (HUVECs). Microscopic analyses further revealed that the expression of ATF3 and SUMO1 is upregulated and colocalized in the endothelium of thoracic aortas from Ang II-induced hypertensive mice. However, Ang II-induced upregulation of ATF3 and SUMO1 in vitro and in vivo was blocked by Ang II type I receptor antagonist olmesartan. Moreover, Ang II induced ATF3 SUMOylation at lysine 42, which is SUMO1 dependent. ATF3 SUMOylation attenuated ATF3 ubiquitination and in turn promoted ATF3 protein stability. ATF3 or SUMO1 knockdown inhibited Ang II-induced expression of inflammatory molecules such as tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-8. Wild type ATF3 but not ATF3-K42R (SUMOylation defective mutant) reduced the production of nitric oxide (NO), a key indicator of EC function. Consistently, ginkgolic acid, an inhibitor of SUMOylation, increased NO production in HUVECs and significantly improved vasodilatation of aorta from Ang II-induced hypertensive mice. Our findings demonstrated that ATF3 SUMOylation is involved in Ang II-induced EC inflammation and dysfunction in vitro and in vivo through inhibiting ATF3 ubiquitination and increasing ATF3 protein stability.

Progesterone increases nitric oxide synthesis in human vascular endothelial cells through activation of membrane progesterone receptor-α.

Progesterone exerts beneficial effects on the human cardiovascular system by inducing rapid increases in nitric oxide (NO) production in vascular endothelial cells, but the receptors mediating these nongenomic progesterone actions remain unclear. Using human umbilical vein endothelial cells (HUVECs) as a model, we show that progesterone binds to plasma membranes of HUVECs with the characteristics of membrane progesterone receptors (mPRs). The selective mPR agonist Org OD 02-0 had high binding affinity for the progesterone receptor on HUVEC membranes, whereas nuclear PR (nPR) agonists R5020 and medroxyprogesterone acetate displayed low binding affinities. Immunocytochemical and Western blot analyses confirmed that mPRs are expressed in HUVECs and are localized on their plasma membranes. NO levels increased rapidly after treatment with 20 nM progesterone, Org OD 02-0, and a progesterone-BSA conjugate but not with R5020, suggesting that this progesterone action is at the cell surface and initiated through mPRs. Progesterone and Org OD 02-0 (20 nM) also significantly increased endothelial nitric oxide synthase (eNOS) activity and eNOS phosphorylation. Knockdown of mPRα expression by treatment with small-interfering RNA (siRNA) blocked the stimulatory effects of 20 nM progesterone on NO production and eNOS phosphorylation, whereas knockdown of nPR was ineffective. Treatment with PI3K/Akt and MAP kinase inhibitors blocked the stimulatory effects of progesterone, Org OD 02-0, and progesterone-BSA on NO production and eNOS phosphorylation and also prevented progesterone- and Org OD 02-0-induced increases in Akt and ERK phosphorylation. The results suggest that progesterone stimulation of NO production in HUVECs is mediated by mPRα and involves signaling through PI3K/Akt and MAP kinase pathways.

Role of Sigma-1 Receptor in Cocaine-Induced Endothelial Cell Damage

Background :Cocaine abuse is associated with an increased risk of cardiac and cerebrovascular events, such as myocardial infarction, sudden cardiac death, and ischemic stroke. The underlying mechanisms leading to these complications are not fully understood although intravascular thrombus formation and accelerated atherosclerosis are prominent findings. We showed that chronic cocaine use is associated with markers of endothelial dysfunction (ED) (Sáez et al. Thromb Res 2011; 128:18). The hallmark of ED is a change to a prothrombotic and proadhesive phenotype associated with a decreased bioavailability of nitric oxide (NO). Several experimental studies have shown that cocaine induces ED in vitro and in vivo; however, the receptor and the signaling pathways involved in the interaction of cocaine with endothelial cells (EC) are unknown. Sigma-1 receptor (sigma-1R) belongs to the family of nonopioid sigma receptors that regulate a variety of cellular functions in neuronal, glial and peripheral cells and are involved in pathological conditions such as neurodegenerative diseases, drug addiction, smooth muscle cell contraction and ischemia. Cocaine binding to sigma-1R has been demonstrated in microglial cell lines, where is located in the lipid raft microdomains of the endoplasmic reticulum being translocated into the plasma membrane upon activation. The effect of cocaine on sigma-1R expression and function in systemic endothelial cells EC are largely unknown. We hypothesize that activation of sigma-1R in EC and its downstream signaling pathway may be involved in the pathogenesis of cocaine-associated endothelial dysfunction, a key phenomenon in the onset and progression of atherosclerosis.Objectives:The main aim of this study was to demonstrate the presence of sigma-1R in EC and its role in cocaine-induced EC damage/activation.Methods:Human umbilical vein endothelial cells (HUVECs) were cultured under standard conditions and exposed to cocaine (10μM) or vehicle in the presence or absence of BD1047 (sigma-1 receptor antagonist). Sigma-1R was demonstrated by western blotting using rabbit-anti-OPRS1 polyclonal antibody. Sub-cellular distribution of sigma-1R and von Willebrand factor (VWF) bound to the membrane were studied by indirect immunofluorescence (IF) microscopy. The production of NO was determined by fluorometric assay using DAF-2DA (5μM) and results expressed as arbitrary fluorescence units (AFU). Platelet adhesion under static conditions was assessed by co-incubation of resting platelets with cultured HUVEC; platelets and EC were identified and quantified by IF microscopy using anti-glycoprotein Ib and anti-VE-cadherin antibodies, respectively.Results:HUVEC expressed sigma-1R protein as demonstrated by Western blot analysis. By IF the receptor showed a perinuclear pattern in untreated HUVEC. Treatment with cocaine resulted in translocation to the cytoplasm and suggest co-localization with actin. These changes were abrogated completely by pre-incubation of the cells with BD1047. HUVEC treated with cocaine exhibited an increase in VWF deposited on the cell membrane with the formation of strings, phenomena that were inhibited by incubation with BD1047. With respect to platelet adhesion, cocaine induced a significant increase in the number of platelets adhered to HUVEC as compared with the controls (46±24 versus 13.3±7 platelets per cell, respectively), which was drastically reduced by BD1047 (10.5± 6.3 platelets per cell). NO production in HUVEC exposed to cocaine was significantly reduced with respect to cells treated with vehicle (10.7±0.67 vs 19.5± 6.4 nM/ml, P=0.002), the use of BD1047 only partially restored NO production (12.1±2.5 nM/ml).Conclusions: Our results show that in cultured HUVEC expressed sigma 1-R with a sub-cellular distribution and changes upon cell activation similar to those observed in other cell systems. The blockade of sigma-1R inhibited significantly the pro-adhesive and pro-thrombotic changes observed in HUVEC exposed to cocaine, which support the notion that the receptor may play an important role in cocaine-induce endothelial cell dysfunction. These findings have implications for the management of drug addiction, since sigma-1R antagonists may provide a novel therapeutic intervention to protect the endothelium from the deleterious effects associated with cocaine abuse. DisclosuresNo relevant conflicts of interest to declare.

Inhibition of calpain reduces oxidative stress and attenuates endothelial dysfunction in diabetes.

AimsThe present study was to investigate the role of calpain in reactive oxygen species (ROS) production in endothelial cells and endothelium-dependent vascular dysfunction under experimental conditions of diabetes.Methods and resultsExposure to high glucose activated calpain, induced apoptosis and reduced nitric oxide (NO) production without changing eNOS protein expression, its phosphorylation and dimers formation in primary human umbilical vein endothelial cells (HUVECs). These effects of high glucose correlated with intracellular ROS production and mitochondrial superoxide generation. Selectively scavenging mitochondrial superoxide increased NO production in high glucose-stimulated HUVECs. Inhibition of calpain using over-expression of calpastatin or pharmacological calpain inhibitor prevented high glucose-induced ROS production, mitochondrial superoxide generation and apoptosis, which were concurrent with an elevation of NO production in HUVECs. In mouse models of streptozotocin-induced type-1 diabetes and OVE26 type-1 diabetic mice, calpain activation correlated with an increase in ROS production and peroxynitrite formation in aortas. Transgenic over-expression of calpastatin reduced ROS production and peroxynitrite formation in diabetic mice. In parallel, diabetes-induced reduction of endothelium-dependent relaxation in aortic ring was reversed by over-expression of calpastatin in mouse models of diabetes. However, the protective effect of calpastatin on endothelium-dependent relaxation was abrogated by eNOS deletion in diabetic mice.ConclusionsThis study suggests that calpain may play a role in vascular endothelial cell ROS production and endothelium-dependent dysfunction in diabetes. Thus, calpain may be an important therapeutic target to overcome diabetes-induced vascular dysfunction.

Role of rutin on nitric oxide synthesis in human umbilical vein endothelial cells.

Nitric oxide (NO), produced by endothelial nitric oxide synthase (eNOS), is a major antiatherogenic factor in the blood vessel. Oxidative stress plays an important role in the pathogenesis of various cardiovascular diseases, including atherosclerosis. Decreased availability of endothelial NO promotes the progression of endothelial dysfunction and atherosclerosis. Rutin is a flavonoid with multiple cardiovascular protective effects. This study aimed to investigate the effects of rutin on eNOS and NO production in cultured human umbilical vein endothelial cells (HUVEC). HUVEC were divided into four groups: control; oxidative stress induction with 180 μM H2O2; treatment with 300 μM rutin; and concomitant induction with rutin and H2O2 for 24 hours. HUVEC treated with rutin produced higher amount of NO compared to control (P < 0.01). In the oxidative stress-induced HUVEC, rutin successfully induced cells' NO production (P < 0.01). Rutin promoted NO production in HUVEC by inducing eNOS gene expression (P < 0.05), eNOS protein synthesis (P < 0.01), and eNOS activity (P < 0.05). Treatment with rutin also led to increased gene and protein expression of basic fibroblast growth factor (bFGF) in HUVEC. Therefore, upregulation of eNOS expression by rutin may be mediated by bFGF. The results showed that rutin may improve endothelial function by augmenting NO production in human endothelial cells.

Fo Shou San, an Ancient Chinese Herbal Decoction, Protects Endothelial Function through Increasing Endothelial Nitric Oxide Synthase Activity

Fo Shou San (FSS) is an ancient herbal decoction comprised of Chuanxiong Rhizoma (CR; Chuanxiong) and Angelicae Sinensis Radix (ASR; Danggui) in a ratio of 2∶3. Previous studies indicate that FSS promotes blood circulation and dissipates blood stasis, thus which is being used widely to treat vascular diseases. Here, we aim to determine the cellular mechanism for the vascular benefit of FSS. The treatment of FSS reversed homocysteine-induced impairment of acetylcholine (ACh)-evoked endothelium-dependent relaxation in aortic rings, isolated from rats. Like radical oxygen species (ROS) scavenger tempol, FSS attenuated homocysteine-stimulated ROS generation in cultured human umbilical vein endothelial cells (HUVECs), and it also stimulated the production of nitric oxide (NO) as measured by fluorescence dye and biochemical assay. In addition, the phosphorylation levels of both Akt kinase and endothelial NO synthases (eNOS) were markedly increased by FSS treatment, which was abolished by an Akt inhibitor triciribine. Likewise, triciribine reversed FSS-induced NO production in HUVECs. Finally, FSS elevated intracellular Ca2+ levels in HUVECs, and the Ca2+ chelator BAPTA-AM inhibited the FSS-stimulated eNOS phosphorylation. The present results show that this ancient herbal decoction benefits endothelial function through increased activity of Akt kinase and eNOS; this effect is causally via a rise of intracellular Ca2+ and a reduction of ROS.

Abstract 237: VEGF-Mediated Phosphorylation of eNOS Regulates Angioblast and Endothelial Cell Proliferation

To evaluate when nitric oxide (NO) is first expressed in the EC lineage, the expression pattern of eNOS, phosphorylated eNOS (P-eNOS), and key proteins that define the endothelial lineage (i.e., Flk-1, TAL-1, CD31) were assessed in 7.0-8.5dpc mouse embryos. Immunohistochemical analyses revealed that embryonic endothelial cells (Flk-1 + /TAL-1 + / CD31 + ) expressed eNOS prior to their investment by smooth muscle cells while isolated angioblasts (Flk-1 + /TAL-1 + / CD31 - ) did not express eNOS. Based on eNOS expression we identified a cell type, transitional angioblasts ( eNOS + / FLK-1 + /TAL-1 + / CD31 - ), intermediate between embryonic endothelial cells (EECs) and angioblasts. Transitional angioblasts are further distinguished from angioblasts by their initiation of cell-cell contacts with other eNOS + Flk-1 + /TAL-1 + /CD31 - cells or with EECs. Analysis of P-eNOS and phospho-histone H3 expression in transitional angioblasts and EECs showed a tight correlation between P-eNOS expression and cell proliferation. This correlation was also observed in cultured human umbilical vein endothelial cells (HUVECs) treated with nocodazole to induce a G2/M cell cycle block. Given that VEGF mediates both EC division and NO production, we evaluated whether VEGF-mediated NO production is critical for neovascular processes (vasculogenesis/angiogenesis). We show using allantoic cultures that VEGF-mediated mitosis in Flk-1 + cells is decreased by the eNOS inhibitors L-NIO and 17-AAG, suggesting that VEGF-mediated eNOS phosphorylation is regulating cell proliferation. Supporting such a role, we found that the alterations in VEGF-mediated vascular patterning we observed in response to eNOS inhibitors were consistent with an overall reduction in the EEC numbers and that VEGF-induced NO production in HUVECs increased the S-nitrosylation of proteins important for the regulation of the G2/M transition, such as cyclin B. Taken together, our findings suggest that VEGF-mediated eNOS phosphorylation regulates transitional angioblast, EEC, and EC proliferation.

Angiotensin II type 1 receptor, but no type 2 receptor, interferes with the insulin-induced nitric oxide production in HUVECs

ObjectiveTwo subtypes of angiotensin II (ATII) receptor have been defined on the basis of their differential pharmacological and biochemical properties: ATII-type1 receptors (AT1-R) and ATII-type2 receptors (AT2-R). It has been hypothesized that part of the protective effects on the cardiovascular system of AT1-R blockers is mediated by an ATII-mediated overstimulation of AT2-R.We hypothesized that the inhibition of AT1-R has a stronger impact on insulin-induced nitric oxide (NO) production than ATII-mediated overstimulation of AT2-R. Therefore we studied the effect of the inhibition of AT1-R and AT2-R on ATII-mediated actions in Human Umbilical Vein Endothelial Cells (HUVECs). MethodsWe analyzed the phosphorylation state of IRS1 at Ser616 and Ser312 and on tyrosines after preincubation with PD123319, an inhibitor of AT2-R, alone and in combination whit losartan, an inhibitor of AT1-R. In addition we measured eNOS and Akt activation through the evaluation of their phosphorylation at Ser1177 and Ser473 sites respectively. ResultsATII induces IRS-1 phosphorylation at Ser312 and Ser616 through the activation of JNK and ERK 1/2, resulting in the inhibition of the insulin-induced phosphorylation of IRS1 tyrosines, Akt and eNOS. Treatment of HUVECs with AT1-R inhibitor restored the insulin signaling leading to NO production, whereas AT2-R inhibitor did not have effects on NO production in presence of ATII. ConclusionOur results demonstrate that in presence of AT1-R antagonist, the AT2-R blockage does not modify the effect obtained with the AT1-R inhibition alone. Therefore, a possible positive role of an AT2-R overstimulation in condition of AT1-R antagonism seems to be irrelevant.