Kaposi Sarcoma Herpesvirus Induces HO-1 during De Novo Infection of Endothelial Cells via Viral miRNA-Dependent and -Independent Mechanisms

ABSTRACTKaposi sarcoma (KS) herpesvirus (KSHV) infection of endothelial cells (EC) is associated with strong induction ofheme oxygenase-1(HO-1), a stress-inducible host gene that encodes the rate-limiting enzyme responsible for heme catabolism. KS is an angioproliferative tumor characterized by the proliferation of KSHV-infected spindle cells, and HO-1 is highly expressed in such cells. HO-1 converts the pro-oxidant, proinflammatory heme molecule into metabolites with antioxidant, anti-inflammatory, and proliferative activities. Previously published work has shown that KSHV-infected ECin vitroproliferate in response to free heme in a HO-1-dependent manner, thus implicating virus-enhanced HO-1 activity in KS tumorigenesis. The present study investigated the molecular mechanisms underlying KSHV induction of HO-1 in lymphatic EC (LEC), which are the likely spindle cell precursors. In a time course analysis of KSHV-infected cells, HO-1 expression displays biphasic kinetics characterized by an early transient induction that is followed by a more sustained upregulation coincident with the establishment of viral latency. A viral microRNA miR-K12-11 deletion mutant of KSHV was found to be defective for induction of HO-1 during latency. A potential mechanism for this phenotype was provided by BACH1, a cellularHO-1transcriptional repressor targeted by miR-K12-11. In fact, in KSHV-infected LEC, theBACH1message level is reduced, BACH1 subcellular localization is altered, and miR-K12-11 mediates the inverse regulation ofHO-1andBACH1during viral latency. Interestingly, the data indicate that neither miR-K12-11 norde novoKSHV gene expression is required for the burst of HO-1 expression observed at early times postinfection, which suggests that additional virion components promote this phenotype.IMPORTANCEWhile the mechanisms underlying KSHV induction of HO-1 remain unknown, the cellular mechanisms that regulate HO-1 expression have been extensively investigated in the context of basal and pathophysiological states. The detoxifying action of HO-1 is critical for the protection of cells exposed to high heme levels. KS spindle cells are erythrophagocytic and contain erythrocyte ghosts. Erythrocyte degeneration leads to the localized release of heme, creating oxidative stress that may be further exacerbated by environmental or other cofactors. Our previous work showed that KSHV-infected cells proliferate in response to heme and that this occurs in a HO-1-dependent manner. We therefore hypothesize that KSHV induction of HO-1 contributes to KS tumor development via heme metabolism and propose that HO-1 be evaluated as a therapeutic target for KS. Our present work, which aimed to understand the mechanisms whereby KSHV induces HO-1, will be important for the design and implementation of such a strategy.

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Kaposi sarcoma-associated herpesvirus (KSHV) induces heme oxygenase-1 expression and activity in KSHV-infected endothelial cells

Blood ◽

10.1182/blood-2003-08-2781 ◽

2004 ◽

Vol 103 (9) ◽

pp. 3465-3473 ◽

Cited By ~ 63

Author(s):

Shane C. McAllister ◽

Scott G. Hansen ◽

Rebecca A. Ruhl ◽

Camilo M. Raggo ◽

Victor R. DeFilippis ◽

...

Keyword(s):

Endothelial Cells ◽

Heme Oxygenase ◽

Cell Biology ◽

Molecular Mechanisms ◽

Expression Profiles ◽

Kaposi Sarcoma ◽

Heme Oxygenase 1 ◽

Spindle Cell Proliferation

Abstract Kaposi sarcoma (KS) is the most common AIDS-associated malignancy and is characterized by angiogenesis and the presence of spindle cells. Kaposi sarcoma-associated herpesvirus (KSHV) is consistently associated with all clinical forms of KS, and in vitro infection of dermal microvascular endothelial cells (DMVECs) with KSHV recapitulates many of the features of KS, including transformation, spindle cell proliferation, and angiogenesis. To study the molecular mechanisms of KSHV pathogenesis, we compared the protein expression profiles of KSHV-infected and uninfected DMVECs. This comparison revealed that heme oxygenase-1 (HO-1), the inducible enzyme responsible for the rate-limiting step in heme catabolism, was up-regulated in infected endothelial cells. Recent evidence suggests that the products of heme catabolism have important roles in endothelial cell biology, including apoptosis and angiogenesis. Here we show that HO-1 mRNA and protein are up-regulated in KSHV-infected cultures. Comparison of oral and cutaneous AIDS-KS tissues with normal tissues revealed that HO-1 mRNA and protein were also up-regulated in vivo. Increased HO-1 enzymatic activity in vitro enhanced proliferation of KSHV-infected DMVECs in the presence of free heme. Treatment with the HO-1 inhibitor chromium mesoporphyrin IX abolished heme-induced proliferation. These data suggest that HO-1 is a potential therapeutic target for KS that warrants further study. (Blood. 2004;103: 3465-3473)

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Hericium erinaceusInhibits TNF-α-Induced Angiogenesis and ROS Generation through Suppression of MMP-9/NF-κB Signaling and Activation of Nrf2-Mediated Antioxidant Genes in Human EA.hy926 Endothelial Cells

Oxidative Medicine and Cellular Longevity ◽

10.1155/2016/8257238 ◽

2016 ◽

Vol 2016 ◽

pp. 1-15 ◽

Cited By ~ 9

Author(s):

Hebron C. Chang ◽

Hsin-Ling Yang ◽

Jih-Hao Pan ◽

Mallikarjuna Korivi ◽

Jian-You Pan ◽

...

Keyword(s):

Endothelial Cells ◽

Transcriptional Activation ◽

Molecular Mechanisms ◽

Nuclear Translocation ◽

Intercellular Adhesion Molecule ◽

Heme Oxygenase 1 ◽

Related Factor ◽

Dependent Manner ◽

Antioxidant Genes ◽

Anti Inflammatory

Hericium erinaceus(HE) is an edible mushroom that has been shown to exhibit anticancer and anti-inflammatory activities. We investigated the antiangiogenic and antioxidant potentials of ethanol extracts of HE in human endothelial (EA.hy926) cells upon tumor necrosis factor-α- (TNF-α-) stimulation (10 ng/mL). The underlying molecular mechanisms behind the pharmacological efficacies were elucidated. We found that noncytotoxic concentrations of HE (50–200 μg/mL) significantly inhibited TNF-α-induced migration/invasion and capillary-like tube formation of endothelial cells. HE treatment suppressed TNF-α-induced activity and/or overexpression of matrix metalloproteinase-9 (MMP-9) and intercellular adhesion molecule-1 (ICAM-1). Furthermore, HE downregulated TNF-α-induced nuclear translocation and transcriptional activation of nuclear factor-κB (NF-κB) followed by suppression of I-κB (inhibitor-κB) degradation. Data from fluorescence microscopy illustrated that increased intracellular ROS production upon TNF-α-stimulation was remarkably inhibited by HE pretreatment in a dose-dependent manner. Notably, HE triggered antioxidant gene expressions of heme oxygenase-1 (HO-1),γ-glutamylcysteine synthetase (γ-GCLC), and glutathione levels, which may contribute to inhibition of ROS. Increased antioxidant status was associated with upregulated nuclear translocation and transcriptional activation of NF-E2related factor-2 (Nrf2) in HE treated cells. Our findings conclude that antiangiogenic and anti-inflammatory activities ofH. erinaceusmay contribute to its anticancer property through modulation of MMP-9/NF-κB and Nrf2-antioxidant signaling pathways.

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Linoleyl hydroperoxide transcriptionally upregulates heme oxygenase-1 gene expression in human renal epithelial and aortic endothelial cells.

Journal of the American Society of Nephrology ◽

10.1681/asn.v9111990 ◽

1998 ◽

Vol 9 (11) ◽

pp. 1990-1997

Author(s):

A Agarwal ◽

F Shiraishi ◽

G A Visner ◽

H S Nick

Keyword(s):

Endothelial Cells ◽

Renal Disease ◽

Heme Oxygenase ◽

Molecular Mechanisms ◽

De Novo ◽

Oxidized Ldl ◽

Heme Oxygenase 1 ◽

Aortic Endothelial Cells ◽

Mrna Induction ◽

Aortic Endothelial

Atherogenic lipoproteins such as oxidized LDL are implicated in the pathogenesis of atherosclerosis and renal disease. Fatty acid hydroperoxides and phospholipids such as linoleyl hydroperoxide (LAox or 13-HPODE) and lysophosphatidylcholine (lyso-PC), abundant components of oxidized LDL, mediate the effects of atherogenic lipids. Oxidized LDL has been shown to induce heme oxygenase-1 (HO-1), a microsomal enzyme that is involved in heme detoxification and is a major endogenous source of carbon monoxide. HO-1 is also induced by many other stimuli that shift cellular redox. To identify the constituents and molecular mechanisms of oxidized LDL-mediated HO-1 induction, human renal epithelial cells and aortic endothelial cells were exposed to LAox and lyso-PC. Exposure to LAox (25 microM) showed an approximately 16-fold induction of HO-1 mRNA, whereas exposure to lyso-PC (25 microM) showed only an approximate 2.6-fold increase. Treatment with actinomycin-D (4 microM), a transcriptional inhibitor, as well as nuclear run-on assays, demonstrated that LAox-mediated HO-1 gene induction is dependent on de novo transcription. Cycloheximide did not affect LAox-mediated HO-1 mRNA induction, suggesting that new protein synthesis is not required for transcriptional induction. Transfection of a human HO-1 promoter-reporter gene construct showed that LAox upregulation of HO-1 occurs via mechanisms different from those of known inducers, heme and cadmium. These studies are the first demonstration that LAox induces HO-1 by transcriptional mechanisms and may have implications in the pathogenesis of cell injury in atherosclerosis and progressive renal disease.

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Thrombin stimulates albumin transcytosis in lung microvascular endothelial cells via activation of acid sphingomyelinase

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00157.2015 ◽

2016 ◽

Vol 310 (8) ◽

pp. L720-L732 ◽

Cited By ~ 18

Author(s):

Wolfgang M. Kuebler ◽

Claudia Wittenberg ◽

Warren L. Lee ◽

Eike Reppien ◽

Neil M. Goldenberg ◽

...

Keyword(s):

Endothelial Cells ◽

Lipid Rafts ◽

Molecular Mechanisms ◽

De Novo ◽

Acid Sphingomyelinase ◽

Microvascular Endothelial Cells ◽

Microvascular Endothelial Cell ◽

Dependent Manner ◽

Proinflammatory Mediators ◽

Microvascular Endothelial

Transcellular albumin transport occurs via caveolae that are abundant in lung microvascular endothelial cells. Stimulation of albumin transcytosis by proinflammatory mediators may contribute to alveolar protein leak in lung injury, yet the regulation of albumin transport and its underlying molecular mechanisms are so far incompletely understood. Here we tested the hypothesis that thrombin may stimulate transcellular albumin transport across lung microvascular endothelial cells in an acid-sphingomyelinase dependent manner. Thrombin increased the transport of fluorescently labeled albumin across confluent human lung microvascular endothelial cell (HMVEC-L) monolayers to an extent that markedly exceeds the rate of passive diffusion. Thrombin activated acid sphingomyelinase (ASM) and increased ceramide production in HMVEC-L, but not in bovine pulmonary artery cells, which showed little albumin transport in response to thrombin. Thrombin increased total caveolin-1 (cav-1) content in both whole cell lysates and lipid rafts from HMVEC-L, and this effect was blocked by inhibition of ASM or de novo protein biosynthesis. Thrombin-induced uptake of albumin into lung microvascular endothelial cells was confirmed in isolated-perfused lungs by real-time fluorescence imaging and electron microscopy of gold-labeled albumin. Inhibition of ASM attenuated thrombin-induced albumin transport both in confluent HMVEC-L and in intact lungs, whereas HMVEC-L treatment with exogenous ASM increased albumin transport and enriched lipid rafts in cav-1. Our findings indicate that thrombin stimulates transcellular albumin transport in an acid sphingomyelinase-dependent manner by inducing de novo synthesis of cav-1 and its recruitment to membrane lipid rafts.

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Decoding the temporal nature of brain GR activity in the NFκB signal transition leading to depressive-like behavior

Molecular Psychiatry ◽

10.1038/s41380-021-01016-1 ◽

2021 ◽

Author(s):

Young-Min Han ◽

Min Sun Kim ◽

Juyeong Jo ◽

Daiha Shin ◽

Seung-Hae Kwon ◽

...

Keyword(s):

Inhibitory Action ◽

Time Course ◽

Molecular Mechanisms ◽

Late Phase ◽

Fine Tuning ◽

Dependent Manner ◽

Middle Phase ◽

Temporal Shift

AbstractThe fine-tuning of neuroinflammation is crucial for brain homeostasis as well as its immune response. The transcription factor, nuclear factor-κ-B (NFκB) is a key inflammatory player that is antagonized via anti-inflammatory actions exerted by the glucocorticoid receptor (GR). However, technical limitations have restricted our understanding of how GR is involved in the dynamics of NFκB in vivo. In this study, we used an improved lentiviral-based reporter to elucidate the time course of NFκB and GR activities during behavioral changes from sickness to depression induced by a systemic lipopolysaccharide challenge. The trajectory of NFκB activity established a behavioral basis for the NFκB signal transition involved in three phases, sickness-early-phase, normal-middle-phase, and depressive-like-late-phase. The temporal shift in brain GR activity was differentially involved in the transition of NFκB signals during the normal and depressive-like phases. The middle-phase GR effectively inhibited NFκB in a glucocorticoid-dependent manner, but the late-phase GR had no inhibitory action. Furthermore, we revealed the cryptic role of basal GR activity in the early NFκB signal transition, as evidenced by the fact that blocking GR activity with RU486 led to early depressive-like episodes through the emergence of the brain NFκB activity. These results highlight the inhibitory action of GR on NFκB by the basal and activated hypothalamic-pituitary-adrenal (HPA)-axis during body-to-brain inflammatory spread, providing clues about molecular mechanisms underlying systemic inflammation caused by such as COVID-19 infection, leading to depression.

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Cyclooxygenase-2 Glycosylation Is Affected by Peroxynitrite in Endothelial Cells: Impact on Enzyme Activity and Degradation

Antioxidants ◽

10.3390/antiox10030496 ◽

2021 ◽

Vol 10 (3) ◽

pp. 496

Author(s):

Sonia Eligini ◽

Susanna Colli ◽

Aida Habib ◽

Giancarlo Aldini ◽

Alessandra Altomare ◽

...

Keyword(s):

Endothelial Cells ◽

De Novo ◽

Cyclooxygenase 2 ◽

Nitric Oxide Donors ◽

Metabolic Labeling ◽

Dependent Manner ◽

De Novo Synthesis ◽

Human Endothelial Cells ◽

Prolonged Incubation ◽

Cox 2

The exposure of human endothelial cells to 3-morpholinosydnonimine (SIN-1) induced the expression of cyclooxygenase-2 (COX-2) in a dose- and time-dependent manner. Interestingly, after a prolonged incubation (>8 h) several proteoforms were visualized by Western blot, corresponding to different states of glycosylation of the protein. This effect was specific for SIN-1 that generates peroxynitrite and it was not detected with other nitric oxide-donors. Metabolic labeling experiments using 35S or cycloheximide suggested that the formation of hypoglycosylated COX-2 was dependent on de novo synthesis of the protein rather than the deglycosylation of the native protein. Moreover, SIN-1 reduced the activity of the hexokinase, the enzyme responsible for the first step of glycolysis. The hypoglycosylated COX-2 induced by SIN-1 showed a reduced capacity to generate prostaglandins and the activity was only partially recovered after immunoprecipitation. Finally, hypoglycosylated COX-2 showed a more rapid rate of degradation compared to COX-2 induced by IL-1α and an alteration in the localization with an accumulation mainly detected in the nuclear membrane. Our results have important implication to understand the effect of peroxynitrite on COX-2 expression and activity, and they may help to identify new pharmacological tools direct to increase COX-2 degradation or to inhibit its activity.

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Abstract 3380: Acetylation-dependent Regulation Of Notch1 Signaling In Endothelial Cells

Circulation ◽

10.1161/circ.118.suppl_18.s_415-c ◽

2008 ◽

Vol 118 (suppl_18) ◽

Author(s):

Virginia Guarani ◽

Franck Dequiedt ◽

Andreas M Zeiher ◽

Stefanie Dimmeler ◽

Michael Potente

Keyword(s):

Endothelial Cells ◽

Notch Signaling ◽

Cell Fate ◽

Molecular Mechanisms ◽

Trichostatin A ◽

Notch Pathway ◽

Dependent Manner ◽

Class Iii ◽

Cell Fate Decisions ◽

Knock Down

The Notch signaling pathway is a versatile regulator of cell fate decisions and plays an essential role for embryonic and postnatal vascular development. As only modest differences in Notch pathway activity suffice to determine dramatic differences in blood vessel development, this pathway is tightly regulated by a variety of molecular mechanisms. Reversible acetylation has emerged as an important post-translational modification of several non-histone proteins, which are targeted by histone deacetylases (HDACs). Here, we report that specifically the Notch1 intracellular domain (NICD) is itself an acetylated protein and that its acetylation level is tightly regulated by the SIRT1 deacetylase, which we have previously identified as a key regulator of endothelial angiogenic functions during vascular growth. Coexpression of NICD with histone acetyltransferases such as p300 or PCAF induced a dose- and time-dependent acetylation of NICD. Blocking HDAC activity using the class III HDAC inhibitor nicotinamid (NAM), but not the class I/II HDAC inhibior trichostatin A, resulted in a significant increase of NICD acetylation suggesting that NICD is targetd by class III HDACs for deacetylation. Among the class III HDACs with deacetylase activity (SIRT1, 2, 3, 5), knock down of specifically SIRT1 resulted in enhanced acetylation of NICD. Moreover, wild type SIRT1, but not a catalytically inactive mutant catalyzed the deacetylation of NICD in a nicotinamid-dependent manner. SIRT1, but SIRT2, SIRT3 or SIRT5, associated with NICD through its catalytic domain demonstrating that SIRT1 is a direct NICD deacetylase. Enhancing NICD acetylation by either overexpression of p300 or inhibition of SIRT1 activity using NAM or RNAi-mediated knock down resulted in enhanced NICD protein stability by blocking its ubiquitin-mediated degradation. Consistent with these results, loss of SIRT1 amplified Notch target gene expression in endothelial cells in response to NICD overexpression or treatment with the Notch ligand Dll4. In summary, our results identify reversible acetylation of NICD as a novel molecular mechanism to control Notch signaling and suggest that deacetylation of NICD by SIRT1 plays a key role in the dynamic regulation of Notch signaling in endothelial cells.

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Abstract 660: Local Hemodynamic Forces and Aqueous Cigarette Smoke Extract Affect Development of Endothelial Dysfunction

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.34.suppl_1.660 ◽

2014 ◽

Vol 34 (suppl_1) ◽

Author(s):

Sindy Giebe ◽

Coy Brunssen ◽

Melanie Brux ◽

Natalia Cockcroft ◽

Katherine Hewitt ◽

...

Keyword(s):

Endothelial Cells ◽

Endothelial Dysfunction ◽

Laminar Flow ◽

Tobacco Smoking ◽

Molecular Mechanisms ◽

Low Flow ◽

Dependent Manner ◽

Flow Conditions ◽

Hemodynamic Forces ◽

Plaque Phenotype

Endothelial dysfunction is one of the first steps in the development of atherosclerosis. This proinflammatory phenotype is associated with decreased bioavailability of nitric oxide and a corresponding expression profile in the endothelial cells. Tobacco smoking promotes development of atherosclerotic plaques and local hemodynamic forces are key stimuli in this process. Low laminar flow is involved in the development of an unstable plaque phenotype, while high laminar flow has atheroprotective role. The molecular mechanisms controlling plaque stability in response to tobacco smoking remain largely unknown so far. Therefore, we exposed human endothelial cells to cigarette smoke extract (CSEaq) under disturbed flow conditions. Primary human endothelial cells were stimulated with increasing dosages of CSEaq for 24h. Cell viability was reduced by CSEaq in a dose-dependent manner. The impact of specific flow conditions and different doses of CSEaq on the expression of atherosclerosis-related genes was investigated using a cone-and-plate viscometer. High laminar flow induced elongation of endothelial cells in the direction of flow, increased eNOS expression and NO release in a time-dependent manner. This increase was inhibited by CSEaq. Low laminar flow showed no effect on eNOS expression and NO release. The NRF2 antioxidative defense system was also induced by high laminar flow. NRF2 and NRF2 target genes HMOX1 and NQO1 were strongly activated by CSEaq. Furthermore, we monitored the expression of proinflammatory genes. CSEaq strongly induced adhesion molecule ICAM-1. Interestingly, VCAM-1 was unaffected by CSEaq. Induction of endothelial NADPH oxidase isoform 4 by CSEaq was prevented by high laminar flow. Catalase expression was not affected by flow and CSEaq, whereas CSEaq transiently increased SOD1 expression. Endothelial wound healing was improved by atheroprotective high laminar flow. Low flow did not affect wound healing. Furthermore, high laminar flow decreased adhesion of monocytes to endothelial cells, compared to low flow. We suggest novel molecular mechanisms how tobacco smoking promotes the development of endothelial dysfunction. This can contribute to the formation of an unstable atherosclerotic plaque phenotype.

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Comparative transcriptomic analysis of flowering induction in pitaya induced by supplementary lighting

10.21203/rs.2.9246/v1 ◽

2019 ◽

Author(s):

Rui Xiong ◽

Liu Chengli ◽

Min Xu ◽

Shuang-Shuang Wei ◽

Hua Tang

Keyword(s):

Time Course ◽

Molecular Mechanisms ◽

De Novo ◽

Floral Induction ◽

Transcriptomic Analysis ◽

Pcr Analysis ◽

Reverse Transcription Pcr ◽

Transcription Factor Genes ◽

Supplementary Lighting ◽

Transcriptomic Level

Abstract Background Pitayas are currently attracting considerable interest as a fruit with many health benefits. However, the lack of natural light after November in Hainan, China, severely restricts the production of pitaya in winter. To further explore the molecular mechanisms regulating flowering in pitaya, we used de novo RNA sequencing-based transcriptomic analysis for four stages of pitaya subjected to light induction. Results We assembled 68113 unigenes in total, comprising 29782 unigenes with functional annotations in the NR database, 20716 annotations in SwissProt, 18088 annotations in KOG, and 11059 annotations in KEGG. Comparison between different samples revealed different numbers of significantly differentially expressed genes (DEGs). A number of DEGs involved in energy metabolism-related processes and plant hormones were detected. Moreover, we discovered many CONSTANS-LIKE, FLOWERING LOCUS T and other DEGs involved in direct regulation of flowering, along with CDF and TCP, which function as typical transcription factor genes in the flowering process. At the transcriptomic level, we confirmed 13 DEGs with different functions in the time-course response to light-induced flowering by quantitative reverse-transcription PCR analysis. Conclusions These DEGs may include some key genes that control the floral-induction network, increasing our understanding of the molecular mechanism of floral regulation in pitaya. These findings will also aid the development of biotechnologies aimed at creating a variant of pitaya that is less sensitive to light conditions and blooms throughout the year.

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Vascular Protective Effects of Xanthotoxin and Its Action Mechanism in Rat Aorta and Human Vascular Endothelial Cells

Journal of Vascular Research ◽

10.1159/000509112 ◽

2020 ◽

Vol 57 (6) ◽

pp. 313-324

Author(s):

Li-Hua Cao ◽

Ho Sub Lee ◽

Zhe-Shan Quan ◽

Yun Jung Lee ◽

Yu Jin

Keyword(s):

Endothelial Cells ◽

Cell Adhesion ◽

Adhesion Molecule ◽

Molecular Mechanisms ◽

Umbilical Vein ◽

Neuroprotective Effect ◽

Intercellular Adhesion Molecule ◽

Dependent Manner ◽

Protective Effects ◽

Concentration Dependent Manner

Objective: Xanthotoxin (XAT) is a linear furanocoumarin mainly extracted from the plants Ammi majus L. XAT has been reported the apoptosis of tumor cells, anti-convulsant, neuroprotective effect, antioxidative activity, and vasorelaxant effects. This study aimed to investigate the vascular protective effects and underlying molecular mechanisms of XAT. Methods: XAT’s activity was studied in rat thoracic aortas, isolated with aortic rings, and human umbilical vein endothelial cells (HUVECs). Results: XAT induced endothelium-dependent vasodilation in a concentration-dependent manner in the isolated rat thoracic aortas. Removal of endothelium or pretreatment of aortic rings with L-NAME, 1H-[1,2,4]-oxadiazolo-[4,3-a]-quinoxalin-1-one, and wortmannin significantly inhibited XAT-induced relaxation. In addition, treatment with thapsigargin, 2-aminoethyl diphenylborinate, Gd3+, and 4-aminopyridine markedly attenuated the XAT-induced vasorelaxation. XAT increased nitric oxide production and Akt- endothelial NOS (eNOS) phosphorylation in HUVECs. Moreover, XAT attenuated the expression of TNF-α-induced cell adhesion molecules such as intercellular adhesion molecule, vascular cell adhesion molecule-1, and E-selectin. However, this effect was attenuated by the eNOS inhibitors L-NAME and asymmetric dimethylarginine. Conclusions: This study suggests that XAT induces vasorelaxation through the Akt-eNOS-cGMP pathway by activating the KV channel and inhibiting the L-type Ca2+ channel. Furthermore, XAT exerts an inhibitory effect on vascular inflammation, which is correlated with the observed vascular protective effects.

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