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Phlebology OnlineFirst, published on May 2, 2014 as doi:10.1177/0268355514531951

Original Article

Treatment of endothelial cell with flavonoids modulates transendothelial leukocyte migration

Phlebology 0(0) 1–7 ! The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0268355514531951 phl.sagepub.com

Isabella Werner1, Fengwei Guo1, Arndh H Kiessling1, Eva Juengel2, Borna Relja3, Peter Lamm4, Ulrich A Stock1, Anton Moritz1 and Andres Beiras-Fernandez1

Abstract Objective: This study aimed to investigate the influence of the flavonoid oxerutin (VenorutonÕ , Novartis, Basel, Switzerland) on endothelial cell apoptosis and transendothelial migration of peripheral blood mononuclear cells and to elucidate the potential mechanisms affecting these processes. Methods: Human endothelial cells were treated with Venoruton to assess the potential effect on apoptosis and on the transendothelial migration process. Endothelial nitric oxide synthase and inducible nitric oxide synthase expression in endothelial cell after Venoruton treatment as well as reactive oxygen species levels were analyzed. Results: Low-dose Venoruton shows a protective effect on endothelial cells and inhibits transendothelial migration of peripheral blood mononuclear cells through an endothelial monolayer, but high-dose Venoruton inversely elevated transendothelial migration of peripheral blood mononuclear cells. Meanwhile, a dose-dependent action of Venoruton on endothelial cell apoptosis could be observed. Endothelial nitric oxide synthase and inducible nitric oxide synthase expression were gradually increased in endothelial cells with increasing Venoruton dosage. In addition, reactive oxygen species were significantly reduced by 0.1 mM and 0.5 mM Venoruton and elevated after high dose treatment. Conclusion: These data suggest that the increased transendothelial migration of peripheral blood mononuclear cells is related to the excessive activation of the nitric oxide-axis and subsequent relaxation of the endothelial cells.

Keywords Flavonoids, VenorutonÕ , transendothelial migration, nitric oxide-axis, apoptosis

Introduction Chronic venous disease (CVD) is very common and includes a range of chronic disorders of the venous system. The ineffectiveness of venous valves is central to the venous hypertension that seems to be the reason for most or all signs of CVD.1 Stepwise improvements have been made in understanding the inflammatory mechanisms of CVD manifestations, such as edema, venous eczema, or hyperpigmentation of skin of the ankle.1 The analysis of vein specimens from patients with CVD and appropriate controls from Ono et al.2 suggest that venous valve damage in refluxing saphenous veins is associated with leukocyte infiltrate. Furthermore, it has been proposed that the magnitude of leukocyte infiltration into the vein wall and in the base of the valve leaflet may be important in the genesis of primary venous dysfunction.2

Transendothelial migration (TEM) of leukocytes plays a critical role in the normal immune response 1 Department of Thoracic and Cardiovascular Surgery, University Hospital Frankfurt, Frankfurt, Germany 2 Department of Urology, University Hospital Frankfurt, Frankfurt, Germany 3 Department of Trauma Surgery, University Hospital Frankfurt, Frankfurt, Germany 4 Department of Cardiac Surgery, Chirurgische Klinik Dr. Rinecker, Munich, Germany

The first two authors contributed equally to this work, therefore sharing first authorship. Corresponding author: Isabella Werner, Department of Thoracic and Cardiovascular Surgery, University Hospital Frankfurt, Theodor Stern Kai 7, 60590 Frankfurt am Main, Germany. Email: [email protected]

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and the development of inflammation and under certain conditions even in the development of cardiovascular diseases.3 Leukocytes can use sophisticated mechanisms to cross vascular endothelium through a well-characterized sequence of rolling, activation, and firm adhesion events.4 This process is initiated and modulated by a variety of molecules and signaling cascades triggered by the cross-talk between leukocytes and endothelium. TEM can not only occur through endothelial borders (paracellular migration) but also occurs through the endothelial cell body (transcellular migration).5 But, the critical factors that influence the route preference for TEM are still largely unknown. The endothelium and its junctions are important regulators of endothelial permeability and leukocyte transmigration is facilitated by increased endothelial permeability.6 Nitric oxide (NO) produced by eNOS appears to be a homeostatic regulator of numerous essential cardiovascular functions.7 However, the role of NO in microvascular permeability is still controversial. Flavonoids are a large family of heterogeneous polyphenolic compounds, secondary metabolites of plants, and can be found in fruits, vegetables, roots, stems, flowers, wine, and tea.8 At present, several thousand different flavonoids have been identified.9 Some previous studies indicated that flavonoids exert numerous medicinal properties such as acting anti-inflammatory, because of their ability to scavenge reactive oxygen and nitrogen species and capacity to modulate TNF-a and IL-6 plasma levels in humans,10,11 exhibiting antioxidant characteristics12 as well as acting against tumors.13,14 Currently, flavonoids are used to treat different entities like cancer, atherosclerosis,15 as well as to reduce inflammation. Recent investigations revealed that flavonoids may be vascular protective agents by directly inducing NO production and arterial relaxation.16 Furthermore, flavonoids from artichokes can up-regulate eNOS gene expression in human endothelial cells.17 VenorutonÕ , 0-(beta-hydroxyethyl)-rutosides, is a standardized mixture, which belongs to the class of flavonoids, extracted from Sophora Japonica, a plant used in traditional medicine in China.18 Presently, it is used to treat patients with CVD as it relieves signs and symptoms of chronic venous insufficiency (CVI), varicose veins, and deep venous disease.19,20 Venoruton is also used as prophylaxis of flight edema,21,22 which is mainly characterized by edema of the lower limbs. Edema is a phenomenon occurring during long-haul flights which may affect both patients with venous disease and normal subjects. The LONFLIT4-Venoruton study aimed to evaluate the protective effects on developing flight edema using Venoruton in patients with venous insufficiency traveling in the economy

section on long-haul flights (8–9 h). The data of this study support the hypothesis that Venoruton appears to control flight edema in most subjects (>95%) with mild-moderate venous insufficiency without causing any problem (side effect or tolerability).21,22 However, many potential mechanisms are involved such as inhibiting adhesion of neutrophils and platelets to the venous endothelium,23 antioxidant action on the endothelial membrane,20 and the inhibition of the lipooxygenase.24 However, the influences of Venoruton upon TEM of peripheral blood mononuclear cells (PBMCs) and the mechanism affecting TEM of PBMCs are far from being understood. Thus, this study aimed to investigate the influence of Venoruton upon TEM of PBMCs and the effect of Venoruton on nitric oxide synthase (eNOS and iNOS), as well as the effect of Venoruton on endothelial cell apoptosis.

Materials and methods Ethics statement This study was approved by the ethics committee on human research of the University of Frankfurt, Germany (GN: 227/13). All blood samples (n ¼ 3) were obtained under informed consent and according to the declaration of Helsinki. All participants provided their written informed consent to participate in this study.

Cell culture and reagents Human microvascular endothelial cells-1 (HuMEC-1) (kindly provided by Dr. V. Mirakaj, University Tu¨bingen, Department of Anesthesiology and Intensive Care Medicine) were cultured in MCDB-131 (Life Technologies, Darmstadt, Germany) supplemented with 10% fetal calf serum (Gibco, Karlsruhe, Germany), 1% glutamine (Gibco, Karlsruhe, Germany), 1% Pen/Strep solution (Sigma Chemical Co. St. Louis, USA), 10 ng/ml Epidermal Growth Factor (Sigma Chemical Co. St. Louis, USA), and 1 mg/ml hydrocortisone (Sigma Chemical Co. St. Louis, USA) at 37 C and 5% CO2 atmosphere.

Isolation and preparation of PBMCs Peripheral blood was drawn from healthy volunteers (n ¼ 3) and collected in heparinized polypropylene tubes. Mononuclear cells were isolated by density gradient centrifugation using PolymorphprepTM according to the manufacturer’s protocol. Briefly, the fresh blood was underlain with 5 ml PolymorphprepÕ (Axis-Shield, Oslo, Norway), for density gradient centrifugation, and centrifuged at 450g for 35 min continuously.

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The PBMC layer was transferred to a new tube and washed twice with phosphate-buffered saline (PBS) (Gibco, Karlsruhe, Germany). PBMCs were stained with calcein-AM (ABD-Bioquest, Biomol, Hamburg, Germany) for 40 min. The calcein-labeled PBMCs were then used for transmigration assays.

Endothelial cell apoptosis assay HuMEC-1 apoptosis was analyzed by positive Annexin-V and negative propidium iodide staining. Confluent HuMEC-1 monolayers were treated with three different doses (0.1 mM, 0.5 mM, and 1 mM) Venoruton for 1 h or remained untreated to serve as control. Then the cells were gently detached with alfazyme (PAA, Pasching, Austria) and rapidly stained with an allophycocyanin (APC)-labeled Annexin-V Detection Kit (eBioscience, San Diego, USA) according to the manufacturer’s instructions. Sample tubes were acquired and analyzed on a fluorescence-activated cell sorter (FACS) CantoII flow cytometer with FACSDiva 6.1.2 software (BD, Heidelberg, Germany).

TEM assay HuMEC-1 (passages 2–5, 1 105 cells/filter) were suspended in the upper compartment of fibronectin – coated 3 mm – pore transwell filters (Fluoroblok inserts, BD, Heidelberg, Germany) and cultured for 24 h to form an endothelial monolayer. HuMEC-1 were treated with Venoruton (Novartis, Basel, Switzerland) after monolayers were cultured on transwell filters but previously to the transmigration assay. HuMEC-1 monolayers were either treated with 0.1 mM, 0.5 mM, or 1 mM Venoruton for 1 h. Untreated cells served as control. Then, calcein-labeled PBMCs (3  105/cell) were suspended in RPMI 1640 serum-free medium and added into the upper compartment of the transwell filter. The lower compartment contained RPMI 1640 supplemented with 0.5% BSA. The transmigration setup was incubated at 37 C in 5% CO2 for 2 h. The fluorescence signal of cells migrated to the bottom surface of the filter was measured with a microplate reader and expressed as percent of control (transmigration index).

RNA isolation, cDNA synthesis, and reverse transcription polymerase chain reaction HuMEC-1 were harvested after no treatment (control) or 1 h treatment with three different doses of Venoruton (0.1 mM, 0.5 mM, and 1 mM). HuMEC-1 were washed twice with cold PBS. RNA was extracted using the ZR RNA MiniPrepTM Kit (Zymo Research, Irvine, USA) according to the manufacturer’s instructions.

Appropriate quantity of total RNA was transcribed into cDNA using the High Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Life Technologies, Applied Biosystems, Darmstadt, Germany) according to the manufacturer’s protocol. Reverse transcription polymerase chain reaction (RTPCR) analysis of specific gene transcripts was carried out using the specific oligonucleotide primers for iNOS and eNOS (both SABioscience, Hilden, Germany) using MX3005P PCR cycler (Stratagene, Santa Clara, USA). GAPDH served as housekeeping gene in comparisons of gene expression data of iNOS and eNOS (SABioscience, Hilden, Germany).

Cellular reactive oxygen species detection assay Cellular reactive oxygen species (ROS) detection assay kit (Abcam, Cambridge, UK) was used according to the manufacturer’s protocol to detect ROS after Venoruton. Briefly, HuMEC-1 were harvested and seeded into dark, clear bottom 96-well microplates with 25,000 cells per well. The cells were allowed to adhere overnight. HuMEC-1 were stained by adding 100 ml/well of the DCFDA (20 ,70 -dichlorofluorescein diacetate) solution and incubated for 45 min at 37 C in the dark. After the staining, the cells were treated with Venoruton (0.1 mM, 0.5 mM, and 1 mM) for 1 h. Finally, the fluorescence signal was measured on a plate reader with excitation wavelength at 485 nm and emission wavelength at 535 nm. Positive control samples were treated with 50 mM TBHP (Tert-Butyl Hydrogen Peroxide) for 3 h to assure appropriate assay performance.

Statistical analysis All experiments were performed at least in three separate replicates. The results are described as mean  SEM. The statistical differences were determined by unpaired Student’s t-test and one-way ANOVA and corrected with the help of Bonferroni’s multiple comparisons test, as appropriate, using GraphPad Prism 6 software. p Values less than 0.05 were considered statistically significant.

Results HuMEC-1 apoptosis is positively influenced by Venoruton The analysis of endothelial cell apoptosis after Venoruton treatment with Venoruton (0.1 mM, 0.5 mM, and 1 mM Venoruton for 1 h) revealed no increase in Annexin-V-positive and propidium iodidenegative cells compared to non-treated control cells

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(control 3.44  0.22% vs.0.1 mM Venoruton 2.71  0.05%, 0.5 mM Venoruton 2.14  0.13%, 1 mM Venoruton 2.74  0.54%). The 0.5 mM Venoruton group even showed a significant decrease in HuMEC1 apoptosis compared to control group (control 3.44  0.22% vs. 0.5 mM Venoruton 2.14  0.13%) (Figure 1, Table 1).

Transmigration of PBMCs is dose dependently modulated by Venoruton TEM of PBMCs was significantly decreased after 0.1 mM and 0.5 mM Venoruton treatment compared to control group. In contrast, transmigration of PBMCs was significantly increased after 1 mM Venoruton treatment compared to control group. Further significant differences can be observed between the 0.1 mM Venoruton group and 1 mM Venoruton group as well as between the 0.5 mM Venoruton group and 1 mM Venoruton group (Figure 2).

Venoruton causes an up-regulation of eNOS and iNOS expressions in endothelial cells The eNOS expression was significantly increased after 0.5 mM and 1 mM Venoruton treatment compared to untreated controls. Additionally, a significant difference was found between 0.1 mM Venoruton group in comparison with 1 mM Venoruton group and 0.5 mM Venoruton group compared with 1 mM Venoruton group (Figure 3(a)). The iNOS expression was significantly increased in 0.5 mM and 1 mM Venoruton group compared with the control group. A significant elevation of iNOS expression between 0.1 mM Venoruton treatment and 1 mM Venoruton treatment as well as between 0.5 mM and 1 mM Venoruton treatment was detected by RT-PCR analysis (Figure 3(b)).

ROS production is suppressed in endothelial cell after low-dose Venoruton treatment Venoruton treatment of endothelial cells with 0.1 mM and 0.5 mM caused a significant drop of ROS levels compared to the control group (Figure 4). Even though 1 mM Venoruton dosage caused an increase in ROS compared to control by trend, no significant difference was detectable. However, by comparing lowand mid-dose Venoruton treatment with high 1 mM Venoruton treatment, a strong up-regulation in ROS levels was found (Figure 4). Positive control samples treated with 50 mM TBHP for 3 h evoked a drastic increase of ROS compared to the control group (ctr 1.00  0.0 ROS vs. positive ctr 4.40  1.48 ROS, p ¼ 0.03; data not shown in Figure 4), proving an appropriate assay performance.

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Figure 1. Endothelial cells apoptosis after Venoruton treatment. The number of APC-positive/PI-negative HuMEC-1 after Venoruton stays constantly low with increasing Venoruton dosages. (a) Unstained and untreated controls served as initial point for the gating strategy. (b) Untreated control. (c) HuMEC-1 treated with 0.1 mM Venoruton for 1 h. (d) HuMEC-1 treated with 0.5 mM Venoruton for 1 h. (e) HuMEC-1 treated with 1 mM Venoruton for 1 h (n ¼ 3).

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Table 1. Endothelial cell apoptosis after Venoruton treatment.

Treatment

APC-positive/ PI-negative Concentration Time (h) cells (%)

Non-treated control Venoruton 0.1 mM 0.5 mM 1 mM

1 1 1

3.44  0.22* 2.71  0.05 2.14  0.13* 2.74  0.54

The number of apoptotic human microvascular endothelial cells significantly decreases after 0.5 mM Venoruton treatment compared to control but remains stable regarding the 0.1 mM and 1 mM treatment (* p

Treatment of endothelial cell with flavonoids modulates transendothelial leukocyte migration.

This study aimed to investigate the influence of the flavonoid oxerutin (Venoruton®, Novartis, Basel, Switzerland) on endothelial cell apoptosis and t...
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