Hydrogen sulfide reduces the activity of human endothelial cells

Abstract

INTRODUCTION:

The volatile endogenous mediator hydrogen sulfide (H₂S) is known to impair thrombus formation by affecting the activity of human platelets. Beside platelets and coagulation factors the endothelium is crucial during thrombogenesis.

OBJECTIVE:

This study evaluates the effect of the H₂S donor GYY4137 (GYY) on human umbilical vein endothelial cells (HUVECs) in vitro.

METHODS:

Flow cytometry of resting, stimulated or GYY-treated and subsequently stimulated HUVECs was performed to analyse the expression of E-selectin, ICAM-1 and VCAM-1. To study a potential reversibility of the GYY action, E-selectin expression was further assessed on HUVECs that were stimulated 24 h after GYY exposure. A WST-1 assay was performed to study toxic effects of the H₂S donor. By using the biotin switch assay, protein S-sulfhydration of GYY-exposed HUVECs was assessed. Further on, the effects of GYY on HUVEC migration and von Willebrand factor (vWF) secretion were assessed.

RESULTS:

GYY treatment significantly reduced the expression of E-selectin and ICAM-1 but not of VCAM-1. When HUVECs were stimulated 24 h after GYY treatment, E-selectin expression was no longer affected. The WST-1 assay revealed no effects of GYY on endothelial cell viability. Furthermore, GYY impaired endothelial migration, reduced vWF secretion and increased protein S-sulfhydration.

CONCLUSIONS:

Summarizing, GYY dose dependently and reversibly reduces the activity of endothelial cells.

Keywords

Thrombosis E-selectin S-sulfhydration migration gaseous transmitter

1 Introduction

Hydrogen sulfide (H₂S) is known as the third endogenous gaseous transmitter along with nitric oxide and carbon monoxide [1]. It is enzymatically synthetized by cystathione-ß-synthase, cystathionine-γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase [2].

The effects of H₂S on the cardiovascular system are pleiotropic, including anti-hypertensive, anti-thrombotic, anti-oxidative and anti-atherosclerotic actions [3 –5]. Vice versa, a lack of H₂S is associated with accelerated atherosclerosis [6]. As a lipophilic mediator H₂S easily permeates membranes without the need for specific transporters. In platelets multiple anti-aggregatory effects are mediated by reduced expression of the pro-thrombotic adhesion molecules P-selectin, glycoprotein receptors GP_IIb/IIIa and GP_Ib, reduced exocytosis of intracellular granules and impaired morphological response to pro-thrombotic stimuli [7 –9].

In the vasculature H₂S further reacts as an endothelium derived relaxing factor [10, 11]. Via protein S-sulfhydration of adenosintriphosphate-depending potassium channels H₂S mediates the tone of vascular smooth muscle cells, thus vasodilatation and subsequently the systemic blood pressure. Endothelial cells express CSE and therefore synthesize H₂S locally [12]. The endothelium significantly affects haemostasis as it regulates the vascular tone, smooth muscle cells proliferation, thrombus formation and platelet activity [13]. Damage or dysfunction of the endothelium contribute to the pathogenesis of cardiovascular diseases, such as hypertension, coronary artery disease or atherosclerosis [14]. Previous studies addressing the effects of H₂S on the endothelium used NaHS as a donor [7 , 15–18]. This donor is characterized by an unphysiological instant liberation of H₂S. Therefore, this study aimed to study the effects of the slow releasing H₂S donor GYY4137 (GYY) on endothelial activation, including expression of adhesion molecules, von Willebrand factor (vWF) secretion, migration and protein S-sulfhydration. GYY is characterized by a continuous liberation of H₂S for at least three hours that mimics the liberation by the respective enzymes more physiologically [19].

2 Material and methods

2.1 Cell culture

Human umbilical vein endothelial cells (HUVECs) were used and obtained from PromoCell (PromoCell, Heidelberg, Germany) and grown in endothelial cell medium (C-22110, PromoCell, Heidelberg, Germany) from passage 2–7. The low-serum (2%) fetal calf serum (FCS) medium was supplemented with the respective supplement mix (PromoCell), penicillin, streptomycin and FCS to a final concentration of 10% FCS. Cells were cultured in different uncoated tissue culture plates with sizes respective to the performed assay.

2.2 WST-1 assay

To assess the cellular activity of HUVECs, a colorimetric in vitro assay (WST-1 assay; Roche, Mannheim, Germany) was used. The WST-1 assay was carried out according to the manufacturer’s instructions. In short, 4×10³ HUVECs were seeded in 96-wells. After 24 hours cells were treated with either TNF-α at 10 ng/ml for 3 hours, the hydrogen sulphide donor GYY(Enzo Life Sciences GmbH, Lörrach, Germany) at 1, 5 or 10 mM for 1 hour or the vehicle for 1 hour. Untreated HUVECs served as reference. Afterwards, WST-1 reagent was added to each well and the absorption was measured after 1 hour at 37°C, at 450 nm with 620 nm as reference using a microplate reader and corrected to blank values (wells without cells). n = 4 independent experiments.

2.3 HUVEC exposure to TNF-a and flowcytometric analysis of E-selectin, ICAM-1 and VCAM-1

For flowcytometric analysis of pro-thrombotic adhesion molecules on endothelial cells 1×10⁵ HUVECs were seeded on 6-wells for 24 hours. Cells were exposed to GYY at 1, 5 or 10 mM for 1 hour at 37°C. Vehicle-treated endothelial cells served as control. Afterwards medium was changed and HUVECs were exposed to TNF-α at 10 ng/ml, for 3 hours to study E-selectin expression or for 24 hours to study ICAM-1 and VCAM-1 expression, at 37°C. This was followed by washing with PBS and resuspension of the cells in fresh medium to study the respective receptor expression. Cells were trypsined with 1 ml Tryp/EDTA (Promocell, Heidelberg, Germany) for 3 minutes, followed by washing with 2 ml trypsin neutralisation solution and centrifugation for 4 minutes at 220 g. After decantation the pellet was resuspended in 1 ml FACS-buffer (PBS containing 1% BSA) and centrifuged again like before. After decantation antibody incubation (20μl) of the pellet was performed for 40 minutes in the dark. Incubation was stopped with 1 ml FACS-buffer, followed by centrifugation as described above and subsequent resuspension in 500μl FACS buffer before flowcytometric analysis was performed. n = 6-9 independent experiments.

2.4 Antibody treatment

Expression of E-selectin was investigated by direct immunofluorescence using a monoclonal APC-coupled anti-human anti-CD62E antibody (no. 551144, BD Pharmingen™, Franklin Lakes, New Jersey, USA). In addition, a monoclonal APC-coupled anti-human anti-CD54 antibody (no. 559771, BD Pharmingen™, Franklin Lakes, New Jersey, USA) directed against ICAM-1 was used. VCAM-1 expression on HUVECs was assessed using a monoclonal FITC-coupled anti-human CD106 antibody (no. 551146, BD Pharmingen™, Franklin Lakes, New Jersey, USA). Respectively APC- or FITC-coupled IgG1 isotype-matched control antibodies (no. 555748 and 555751 respectively, both BD Pharmingen™, Franklin Lakes, New Jersey, USA) were used to exclude nonspecific binding. Vehicle-treated HUVECs served as control.

FACScan flowcytometer (Becton Dickinson) was calibrated with fluorescent standard microbeads (CaliBRITE Beads; Becton Dickinson) for accurate instrument setting. HUVECs were identified by their characteristic forward and sideward scatter light and selectively analysed for their fluorescence properties with the CellQuest program (Becton Dickinson), assessing 20.000 events per sample. The relative fluorescence intensity of a given sample was calculated by subtracting the signal obtained when cells were incubated with the isotype-specific control antibody from the signal generated by cells incubated with the test antibody.

2.5 Migration analysis

To evaluate the effect of H₂S on endothelial cell migration, 0.5×10⁵ HUVECs were seeded onto 35 mm dishes containing a cell culture insert (Ibidi^®, Munich, Germany). Since the scratch and therefore injury of the seeded cells, like performed in classical scratch assays for migration analysis [20], could lead to liberation of intracellular mediators that might affect migration, the Ibidi^® assay was used. It is different as no scratch is performed. The cells are seeded in dishes with 35 mm in diameter, containing an incorporated silicon insert. When cells were grown to confluence after 24 h, the silicon insert was removed which created a standardized 500μm gap between the cell patches for assessment of migration. After removal of the cell culture insert, treatment with pure cell medium or medium and either 10 ng/ml VEGF, 1 mM GYY, an equimolar volume of the vehicle or 10 ng/ml VEGF and 1 mM GYY was performed. Herein VEGF was used as positive stimulus. Sham-treated cells served as control. The dishes were photographed immediately and at 12 and 24 hours after removal of the insert. The digitized images were taken in grayscale format and analysed by the CapImage software (Dr. Zeintl Software, Heidelberg, Germany). n = 3–5 independent experiments.

2.6 ELISA of vWF

To assess vWF secretion from activated HUVECs an ELISA (ab189571, abcam, Cambridge, UK) was performed analysing the vWF concentration in the endothelial cell medium. For this purpose, 2×10⁵ HUVECs were seeded in 96 well plates and grown to confluence for 24 hours, followed by exposure to 100μM histamin for 60 minutes or to GYY at 5 or 10 mM for 30 minutes and subsequent treatment with 100μM histamin for another 60 minutes. For the ELISA 50μl of the cell medium were used. After incubation for 1 hour at 37°C, absorption was measured at 450 nm with 620 nm as reference using a microplate reader (VICTOR™ X3, PerkinElmer, Waltham, Massachusetts, US) and corrected to blank values (wells without cells). n = 7–8 independent experiments.

2.7 Biotin switch assay

The assay for assessment of S-sulfhydration of endothelial proteins by GYY was performed as described [21]. In brief, vehicle-treated and with 1, 5 or 10 mM GYY-exposed HUVECs were homogenized in HEN buffer (250 mM Hepes-NaOH; ph 7.7, 1 mM EDTA, and 0.1 mM neocuproine) supplemented with 100μM deferoxamine and centrifuged at 13.000 g for 10 minutes at 4°C. Cell lysate was added to blocking buffer (HEN buffer adjusted to 2.5% SDS and 20 mM methyl methanethiosulfonate (MMTS)) at 50°C for 20 minutes and frequently vortexed. The MMTS was then removed by 2 vol of acetone and the proteins were precipitated at –20°C for 20 minutes. After acetone removal, the proteins were resuspended in HENS buffer (HEN buffer adjusted to 1% SDS), followed by adding 4 vol of 4 mM biotin-HPDP in DMSO. After incubation for 2 hours at RT, biotinylated proteins were precipitated by 2 vol of acetone. Acetone was removed and proteins were resuspended in HENS buffer before immunoblotting. Biotinylated proteins were diluted with an equal volume of 2x SDS/PAGE loading buffer and heated to 95°C for 5 minutes. Total proteins (15μg) were separated by SDS/PAGE (12% gels) and transferred onto polyvinyldifluoride membranes. The immobilized proteins were then blocked in 2% BSA (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and the membranes were incubated over night at 4°C with a monoclonal anti-biotin antibody (1 : 1.000; Sigma-Aldrich) followed by a secondary peroxidase-linked anti-mouse antibody (1 : 60.000; Sigma-Aldrich). Protein expression was visualized by means of luminol-enhanced chemiluminescence (ECL plus; Amersham Pharmacia Biotech, Freiburg, Germany) and digitalized with ChemiDoc™ XRS System (Bio-Rad Laboratories, Munich, Germany). Signals were densitometrically assessed (Quantity One; Bio-Rad Laboratories) and normalized to the β-actin signals (mouse monoclonal anti-β-actin antibody; 1 : 20.000; Sigma-Aldrich). n = 5 independent experiments.

2.8 Statistical analysis

Differences between groups were assessed using one-way ANOVA, followed by the appropriate post hoc comparison tests. For post hoc comparison of vWF concentration and E-selectin expression the Dunn’s Method was used while the expression of ICAM-1 and VCAM-1 as well as the endothelial cell migration and protein S-sulfhydration were compared by the Holm-Sidak method. All data were expressed as mean and standard error of mean (SEM, in case of normality) or median and interquartile range (i.e. 25% and 75% percentile, in case of failing normality) and overall statistical significance was set at p < 0.05.

3 Results

3.1 Endothelial viability

The effect of GYY treatment on cell viability of HUVECs was assessed at concentrations of 1, 5 and 10 mM GYY. Photospectrometric analysis of formazan formation using the WST-1 assay revealed that neither increasing concentrations of GYY nor the vehicle at a dosage corresponding the dosage applied with 10 mM GYY, or TNF-α affected endothelial cell viability (data not shown). Mean absorbance in all studied groups ranged from 100% in untreated HUVECs to 87±7% in vehicle-treated endothelial cells (p = 0.547).

3.2 Analysis of adhesion molecule expression on HUVECs

In contrast to vehicle-treated resting HUVECs, TNF-α activation caused a marked upregulation of E-selectin and ICAM-1 as well as a slight increase in VCAM-1 expression (Fig. 1 A, C and D). Concomitant exposure of HUVECs to the lowest GYY concentration of 1 mM did not affect endothelial adhesion molecule expression (Figs 1A, C and D). However, higher concentrations, i.e. 5 and 10 mM GYY reduced the fraction of TNF-α-induced E-selectin expression to 39±3% and 24±5%, respectively (p < 0.05 and p < 0.001 vs. TNF-α: 57±3%, respectively; Fig. 1A). Moreover, ICAM-1 expression decreased as well and showed a mean fluorescence shift from 875±139 (TNF-α) to 658±151 and 311±29 (p = 0.58 and p < 0.05 vs. TNF-α, respectively) in the presence of 5 and 10 mM GYY, respectively (Fig. 1C). In contrast, increased GYY application had no significant effect on VCAM-1 expression (Fig. 1D).

Fig. 1

HUVEC adhesion molecule expression. Flowcytometric analysis of HUVECs for expression of E-selectin (A and B), ICAM-1 (C) and VCAM-1 (D). HUVECs were exposed to TNF-α alone or after preincubation with 1 mM, 5 mM or 10 mM GYY. To assess the reversibility of the GYY effect, TNF-α stimulation of HUVECs was performed immediately (A) and 24 hours after GYY treatment (B). Untreated platelets without TNF-α exposure served as resting controls. ANOVA followed by appropriate post hoc comparison test. Values are given as means±SEM. ^∗p < 0.05 vs. TNF-α / Ø GYY, ^∗∗∗p < 0.001 vs. TNF-α/Ø GYY. n = 6–9 independent experiments.

To assess a possible reversibility of the GYY effect on HUVECs, E-selectin expression upon TNF-α was further assessed 24 hours after GYY treatment in a separate set of experiments. FACS analysis revealed a comparable E-selectin expression when TNF-α activation was performed 24 hours after GYY exposure compared to only TNF-α-treated endothelial cells (p = 0.758; Fig. 1B).

3.3 GYY reduces endothelial migration

In order to assess the effect of GYY on another form of cellular activation, the migration of HUVECs was studied and given as percentage of covered area (Fig. 2). After 12 hours sham- and vehicle-treated endothelial cells showed comparable migration of 11±1% and 10±1%. The positive control VEGF significantly accelerated endothelial migration to 14.9±1% (p < 0.05 vs. sham and vehicle, respectively). Exposure of HUVECs to 1 mM GYY significantly reduced cell migration to 4.1±1% (p < 0.05 vs sham, vehicle and VEGF, respectively). GYY treatment further impaired endothelial cell migration when HUVECs were simultaneously VEGF-exposed to 11±1% (p < 0.05 vs VEGF), while the simultaneous VEGF treatment still accelerated cellular migration compared to GYY treatment alone (p < 0.05 vs GYY).

Fig. 2

Assessment of cell migration in vitro. Planimetric analysis of sham-, VEGF- or GYY- and VEGF-treated HUVEC migration at 0 h, 12 h and 24 h after treatment (A). Quantitative assessment of migration after 12 h in endothelial cells that were either sham-treated or exposed to GYY, to its vehicle, to VEGF alone or to GYY and VEGF (B). ANOVA followed by appropriate post hoc comparison test. Values are given as means±SEM. ^∗p < 0.05 vs sham, ^#p < 0.05 vs vehicle, ^§p < 0.05 vs VEGF, ^$p < 0.05 vs GYY. n = 3–5 independent experiments.

3.4 Assessment of vWF secretion upon GYY treatment

Analysis of vWF concentration in endothelial cell medium revealed a concentration of 1.2 IU/ml (25% percentile: 1.0 IU/ml; 75% percentile: 3.6 IU/ml) when resting HUVECS were vehicle-treated (Fig. 3). Histamin markedly increased vWF concentration to 6.83 IU/ml (25% percentile: 4.73 IU/ml; 75% percentile: 9.95 IU/ml). Pretreatment with 5 or 10 mM GYY followed by histamin stimulation reduced vWF concentration to 1.65 IU/ml (25% percentile: 1.0 IU/ml; 75% percentile: 4.23 IU/ml) and 1.45 IU/ml (25% percentile: 1.05 IU/ml; 75% percentile: 2.8 IU/ml, both p < 0.05, respectively).

Fig. 3

Measurement of vWF secretion from HUVECs. Quantification of vWF concentration in the medium of vehicle-, histamine-, 5 mM or 10 mM GYY-treated endothelial cells. Data are given as box plots indicating the median with the 25th and 75th percentiles. ANOVA followed by appropriate post hoc comparison test. ^∗p < 0.05 vs histamine / Ø GYY. n = 7–8 independent experiments.

3.5 GYY treatment increases protein S-sulfhydration of endothelial cells

Densitometric Western-blot analysis showed that 1 mM GYY significantly increased the relative intensity of the extent of S-sulfhydrated protein binding to biotin from 0.54 (25% percentile: 0.34; 75% percentile: 0.73) upon vehicle-treatment to 2.72 (25% percentile: 1.49; 75% percentile: 3.07, p < 0.05). In contrast, 5 and 10 mM GYY did not further affect protein S-sulfhydration (Fig. 4).

Fig. 4

S-sulfhydration of HUVEC proteins. Quantitative densitometric analysis of sulfhydrated proteins of vehicle or GYY-exposed (1 mM, 5 mM, 10 mM) HUVECs. Protein S-sulfhydration is visualized by biotin binding in accordance to the biotin switch method. Values are presented as relative intensity of S-sulfhydrated proteins binding biotin/b-actin. Data are given as means±SEM. ANOVA followed by appropriate post hoc comparison test. ^∗p < 0.05 vs. Ø GYY. n = 5 independent experiments.

4 Discussion

This study demonstrates that endothelial cell treatment with the H₂S donor GYY (i) reversibly reduces the expression of extracellular adhesion molecules and (ii) impairs cellular migration. It further shows a reduced secretion of vWF (iii) as a marker for impaired granular exocytosis upon H₂S exposure. All in all, these findings account for a decreased activity of endothelial cells which might be due to H₂S-mediated protein S-sulfhydration (iv).

As shown by our and other groups, H₂S is a potent anti-thrombotic mediator as it impairs platelet activity and therefore platelet aggregation [8 , 23]. H₂S further modulates hemostatic plasma parameters [24] and impairs both venous and arterial thrombosis [25, 26]. This study focused on the endothelium. Referring to the Virchow triad, endothelial damage is one of the main triggers for thrombus formation [27]. Therefore, it was reasonable to study the effects on pro-thrombotic endothelial adhesion molecules. H₂S dose-dependently reduced the expression of E-selectin in vitro. This would affect thrombus formation as E-selectin modulates neutrophil and monocyte activity directly and further affects fibrin content of a thrombus [28, 29]. Reduced E-selectin expression, especially in combination with reduced P-selectin expression, was shown to reduce thrombus formation and inflammation in the vessel wall [29]. In parallel to P-selectin, E-selectin is stored intracellular in granules, i.e. the Weibel-Palade bodies, that also contain vWF [30]. The reduced E-selectin expression and vWF secretion might be due to an impaired exocytosis of these intracellular granules, which would account for a reduced endothelial cell activity. This effect was also found in platelets, where granular exocytosis upon pro-thrombotic stimuli was impaired as well by H₂S [9]. Regardless the exact mechanism behind this, the effect of H₂S on E-selectin expression was found to be reversible as H₂S-treated HUVECs expressed E-selectin comparable to non-H₂S-treated cells when the activation with TNF-α was performed 24 hours after H₂S exposure. This finding is quite important as it shows that the H₂S effect is not due to an irreversible cellular damage, which further accounts for a non-toxic concentration of H₂S released from its donor GYY. This is in line with the results of WST-1 assay that revealed no statistically significant effect of H₂S exposure to endothelial cell viability. To our knowledge this is the first demonstration of the reversibility of a H₂S-mediated effect with respect to the endothelium. Although it was only shown for E-selectin, reversible effects are an important aspect for a potential clinical application of H₂S donors.

Serum levels of ICAM-1 are a marker for local endothelial and leukocyte activity and are related to thrombosis [31, 32]. Therefore, the dose-dependently reduced ICAM-1 expression upon H₂S exposure further accounts for a reduced endothelial activity. In addition, ICAM-1 mediates leukocyte-endothelial binding [33]. This is important as leukocytes promote platelet aggregation and -secretion [34], enhance thrombin and tissue factor generation [32, 35], and therefore increase thrombus stability. Another way how leukocytes support thrombus stability is the formation of neutrophil extracellular traps (NETs) during neutrophil activation [36]. NETs were shown to promote thrombus formation, causing platelet adhesion, activation, and aggregation [37]. In turn, it might be reasonable to conclude that a reduced ICAM-1-mediated leukocyte binding to activated endothelial cells could be another mechanism of H₂S to impair thrombus formation and increase thrombolysis like previously assessed in vivo [8, 9].

Reduced E-selectin and ICAM-1 expression upon H₂S treatment further supports the findings of previous studies showing that NaHS reduces ICAM-1 expression in Apo-E-knockout mice via inhibition of NFkB signalling [38] as well as ICAM-1 and E-selectin expression by reduction of TNF-a induced intracellular production of reactive oxygen species, inhibition of p38-signalling and induction of hemeoxygenase-1 expression [39].

Although the reduced endothelial cell migration upon H₂S exposure further accounts for a reduced cellular activity, this result is contrary to recently published data showing an increased migration of HUVECs upon H₂S exposure [15 –18]. All these studies used the H₂S donor NaHS at 100μM [17, 18], 25–100μM [16] or 400μM [15]. As shown by Li et al. 1 mM GYY leads to an almost 40×lower H₂S release in vitro compared to 100μM NaHS [19]. This might account for a dose-dependent effect of H₂S on endothelial migration. Although GYY liberates H₂S continuously over several hours, the release is lower compared to NaHS, which releases H₂S instantly within seconds [19]. The presented data further show that the reduced migration is not due to the vehicle, that does not affect migration compared to sham treatment, and that HUVECs still react on stimulation as VEGF increased endothelial migration in presence and absence of H₂S.

A potential mechanism behind all these effects on HUVECs might be the S-sulfhydration of endothelial proteins induced by H₂S. This posttranscriptional modulation of protein function was shown to affect multiple enzymes, receptors, transcription factors and ion channels in the cardiovascular system [3]. By combining densitometric analysis and the biotin switch assay, an increased degree of protein S-sulfhydration upon HUVEC exposure to H₂S was demonstrated. As the protein S-sulfhydration is limited by the number of cystein-SH groups of the protein, a dose response of protein S-sulfhydration by GYY could not be observed. This finding is in line with previous published data on platelet protein S-sulfhydration [9].

Although S-sulfhydration of the different endothelial proteins was not studied in detail, it is reasonable to conclude that the descripted effects of H₂S could be due to S-sulfhydration of specific protein-1 (SP-1). SP-1 is an important transcription factor that modulates endothelial phenotypes and can be S-sulfhydrated on cysteines Cys⁶⁸ and Cys⁷⁵⁵ [40].

In summary, this study demonstrates a reduced endothelial activation upon H₂S exposure. It is reasonable be conclude that this effect contributes to the anti-thrombotic actions of H₂S in vivo. The demonstrated reversibility is an important aspect for a potential therapeutic use of H₂S in human and should be studied in more detail in further studies.

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