SULODEXIDE PROTECTS ENDOTHELIAL CELLS AGAINST 4-HYDROXYNONENAL-INDUCED OXIDATIVE STRESS AND GLUTATHIONE-DEPENDENT REDOX IMBALANCE BY MODULATION OF SESTRIN2/NUCLEAR FACTOR
ERYTHROID 2-RELATED FACTOR 2 PATHWAY

Sulodexide (SDX) is a mixture of glycosaminoglycans containing 80% heparan sulfate and 20% dermatan sulfate (1). Besides anticoagulant effect, SDX also has antithrombotic, profibrinolytic, antiaggregant, angioprotective and antihyperlipidemic properties (2). SDX is known for its effectiveness in treating chronic venous disease and prophylaxis of deep vein thrombosis recurrence (3). In addition, same clinical and experimental studies proved renoprotective (4), neuroprotective (5) and cardioprotective (6) effects of SDX. It was also found SDX has antioxidant (7), anti-senescent (8), anti-inflammatory (9) and anti-proteolytic (10) features.

A significant number of studies point to the vascular endothelium as the main site of SDX’s pharmacological action. It was reported that SDX protects the endothelial glycocalyx (11), improves the ability of endothelial cells to control vascular tone (12), and prevents the increase of reactive oxygen species (ROS) caused by hyperglycemia (7). However, there are only a few studies that have been conducted at the molecular level to elucidate the SDX antioxidant mechanism on endothelial cells (7, 10, 13, 14).

4-hydroxynonenal (HNE) is one of the lipid peroxidation products that may be involved in the induction of vascular endothelial cell damage in hypercholesterolemia and atherosclerosis. HNE is the most abundant and reactive carbonyl species, formed from hydroperoxides of n-6 polyunsaturated fatty acids (n-6 PUFAs), such as linoleic acid (18:2, n-6) and arachidonic acid (20:4, n-6) (15). An increased level of HNE (>20 μM) promotes oxidative stress, endothelial dysfunction and apoptosis by affecting the expression of various pro-apoptotic proteins and the activation of stress signaling pathways (16, 17). HNE forms adducts with proteins, lipids and DNA, leading to endothelial cell damage, e.g. mitochondrial disturbances, proteasome inactivation, an increase of pro-inflammatory mediators, dysfunction of antioxidant systems and an increase of ROS formation (18-20). The rate of HNE metabolism is closely related to the intracellular level of reduced glutathione (GSH).

GSH (γ-L-glutamyl-L-cysteinyl-glycine) is important for preventing ROS increase and maintaining redox balance. It is also the main target of HNE (21). Within cells, glutathione exists in reduced (GSH) and oxidized (GSSG) states. The GSH:GSSG ratio is a significant indicator of the cellular redox status and contributes to the determination of the molecular mechanisms underlying apoptosis (22). De novo synthesis of GSH is a two-step process mediated by glutamate-cysteine ligase (GCL) and glutathione synthase (GSS). GCL catalyzes the formation of γ-glutamylcysteine (GGC) from glutamate and cysteine, while GSS couples GGC with glycine to form GSH. GCL is composed of catalytic (GCLc) and modifier (GCLm) subunits. GCLc exhibits the catalytic ability to form GGC, while GCLm modulates the affinity of GCLc for its substrates and inhibitors (23).

HNE conjugates with GSH, generating HNE-GSH adducts in a spontaneous or glutathione S-transferase-mediated reaction. The HNE-GSH adduct formation always leads to a decrease in intracellular GSH level (19). Therefore, some of the cellular effects of HNE could be secondary to GSH depletion, which reduces antioxidant defenses, enhances both oxidative stress and cellular lipid peroxidation, and triggers apoptosis (18, 24, 25). HNE-induced oxidative stress and decreasing GSH availability lead to increased permeability of the endothelium, glycocalyx damage, apoptosis, and rapid atherosclerosis progression (26, 27). High levels of HNE and redox imbalance were reported in various diseases related to oxidative stress (28). For this reason, finding drugs with antioxidant and carbonyl scavenging properties against HNE may be an effective way to prevent endothelial dysfunction in atherosclerosis and vascular diseases.

4-HNE is a strong inducer of nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response elements (ARE) signaling pathway, according to several studies. Upon exposure to HNE along with GSH depletion, Nrf2 dissociates from the Kelch-like ECH-associated protein-1 (Keap-1) rapidly and translocates to the nucleus (17). Nrf2 binding to ARE leads to the transcription of genes that encode key antioxidant proteins, including enzymes involved in GSH synthesis (13). There is also evidence that cell stimulation with 4-HNE can increase Nrf2 mRNA levels (29). Moreover, it was found that Sestrin2 protein (SESN2) is one of the key regulators of Nrf2 (30). SESN2 promotes autophagy-dependent degradation of Keap-1 and thus activates the Nrf2 signaling pathway (31). There is evidence that SESN2 expression is positively correlated with the severity of oxidative stress, suggesting that SESN2 levels are increased when free radical production increases (32). Recent experimental and clinical studies have shown SESN2 involvement in various cardiovascular disease progression (33, 34). However, the exact role of SESN2 in regulation of HNE-induced oxidative stress in endothelial cells is still not clearly understood.

In this work, the protective functions of SDX regarding apoptosis, redox status and oxidative damage of HNE-treated human umbilical vein endothelial cells (HUVECs) were investigated to further explain its antioxidant effects. SDX restored GSH levels and prevented the downregulation of GCLc and GSS proteins by HNE. Our results also revealed SDX’s modulation of the SESN2/Nrf2 pathway is important in preventing HNE-induced oxidative damage to endothelial cells. In addition, we identified the role of intracellular GSH as a negative regulator of the SESN2/Nrf2 pathway.

MATERIALS AND METHODS

Cell culture

HUVECs and the Clonetics™ EGM™-2 BulletKit™ were purchased from Lonza (Verviers, Belgium). The cells were cultured as recommended by the supplier in a humidified incubator (5% CO2, 37ºC). HUVECs were detached and subcultured when approx. 90% confluence was reached, and passages 3 to 5 were used for all experiments.

Cell treatments

For induction of the cytotoxic effect, we applied HNE (Calbiochem, San Diego, CA, USA) to the culture medium at the final concentrations of 5, 10, 25, and 50 μM (dissolved in ethanol; final medium concentration: 0.01% ethanol) for 4 hours. Ethanol at a concentration up to 0.1% (v/v) did not affect the viability of the cells (data not shown). Again, to investigate the time-dependent effect of HNE on HUVECs, cells were treated with HNE for different time intervals: 2, 4, 6, and 8 h, with a final concentration of 25 μM. Incubations with HNE were performed in a serum-free environment to allow HNE to bind to intracellular proteins. Due to its chemical structure, HNE has a strong affinity for free amino acids (e.g. lysine, cysteine, histidine) present in the complete medium. The optimal HNE concentration and incubation time were confirmed when the cell viability was close to 50%.

HUVECs were treated with 25 μM HNE or HNE combined with 0.5 LRU/mL SDX (Vessel Due F, Alfasigma S.p.A., Bologna, Italy) for 4 h, based on preliminary data (Fig. 1) and available literature (14, 35). The untreated cells in the standard culture medium served as a control. As needed, experiments were also performed with antioxidant N-acetylcysteine (100 μM, NAC) and precursor of GSH γ-glutamylcysteine (100 μM, GGC). Both compounds were obtained from Sigma-Aldrich (St. Louis, MO, USA). NAC and GGC concentrations were selected based on the results of our previous study and published data demonstrating their effectiveness in cellular models of oxidative stress (36, 37).

MTT assay

Cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-terazolium bromide (MTT) test. Briefly, HUVECs in 96-well plates (5×103) were treated with 4-HNE as indicated, and after incubation, 100 μL of MTT (0.25 mg/mL) was added for an additional 3 hours. After that, the formazan crystals formed by living cells were dissolved in 100 μL of DMSO. The absorbance was measured at 570 nm using a Multiskan Ascent microplate reader (Labsystems, Helsinki, Finland).

Fluorescence microscopy

HUVECs (5×104) were seeded in 35-mm plates and treated with HNE alone or HNE combined with SDX. Prior to the fluorescence imaging, the cells were labeled by two fluorescence dyes simultaneously. Hoechst 33342 (Sigma-Aldrich, St. Louis, MO, USA) was used to image the cell nuclei (excitation wavelength, 405 nm; emission wavelength, 420 nm). The production of intracellular ROS was determined using the fluorogenic CellROX® Green Reagent (excitation wavelength, 485 nm; emission wavelength, 520 nm) from Life technologies (Molecular Probes, Eugene, OR, USA). After treatment, 5 μM CellROX® and 5 μg/mL Hoechst 33342 were added to the cells and incubated for 30 min at 37ºC. After removing CellROX® and Hoechst 33342 and washing three times with phosphate-buffered saline (PBS), the cells were fixed in 4% formaldehyde for 20 min in the dark. Fluorescence images were captured using Nikon TS-100 F fluorescence microscope equipped with Nikon DS Ri1-U2 camera and NIS-BR imaging software (Nikon, Tokyo, Japan). The mean fluorescence intensity (MFI) was analyzed using ImageJ software (1.48v, NIH, USA; http://imagej.nih.gov.ij/) and the mean fluorescence in arbitrary units (A.U.) was calculated.

Western blot

Cells destined for Western blot analysis were sieved at the density of 1×106/dish onto 100-mm dishes. After treatment, HUVECs were rinsed twice with ice-cold PBS and lysed by incubation in RIPA lysis buffer (Sigma-Aldrich, St. Louis, MO, USA) supplemented with Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific, Waltham, MA, USA). Proteins (20 μg) were resolved by 10% SDS-polyacrylamide gel electrophoresis and transferred to a PVDF membranes (GE Healthcare, Amersham, UK). Non-specific binding sites were blocked at room temperature (RT) for 1 h in 5% fat-free milk in Tris-buffer saline containing 0.05% (v/v) Tween-20 (TBS-T, Sigma-Aldrich, St. Louis, MO, USA). Membranes were incubated overnight at 4ºC with primary antibodies (1:1000) against Bax, cleaved caspase-3, Keap-1, Nrf2, Lamin B and GAPDH (Cell Signaling Technology, Danvers, MA, USA). After several washings with TBS-T, membranes were incubated with secondary antibody (1:10,000) conjugated with horseradish peroxidase (Santa Cruz Biotech, Santa Cruz, CA, USA) for 1 h in RT. The immunoreactive bands were visualized with the PierceTM ECL Western Blotting Substrate (Pierce, Waltman, MA, USA). Chemiluminescent signals of immunoblots were detected with a ChemiDoc-IT 410 system (Ultra-Violet Products Ltd, Upland, CA, USA). ImageJ software (1.48v, NIH, USA; http://imagej.nih.gov.ij/) was used to quantify protein expression.

Reduced glutathione and oxidized glutathione measurements

Total (tGSH) and oxidized GSH (GSSG) levels were determined using a Glutathione Colorimetric Detection Kit (InvitrogenTM, Life Technologies Co, Frederick, MD, USA), as previously described (14). Briefly, the cell culture monolayer (an initial density of 5×104 cells/35-mm dish) with or without treatment was washed with ice-cold PBS and precipitated in ice-cold 5% (w/v) 5-sulfo-salicylic acid (SSA, Sigma-Aldrich, St. Louis, MO, USA). Cells were scraped then with a rubber policeman and centrifuged (10 min, 14,000, 4ºC). The pellets were dissolved in RIPA lysis buffer (Sigma-Aldrich, St. Louis, MO, USA) plus 0.1 M NaOH for the determination of total protein content. The clear deproteinized supernatants were analyzed for tGSH (GSH and GSSG). For GSSG measurements, 2-vinylpirydine (2VP, Sigma-Aldrich, St. Louis, MO, USA) was added to the supernatants to block free GSH and other thiols. Samples were incubated with 2-VP for 1 h at RT. The absorbance was read at 405 nm in a microplate reader (Multiskan Ascent, Labsystems, Helsinki, Finland). tGSH and GSSG concentrations were interpolated on calibration curves specific to each run and normalized to total protein levels. The amount of GSH was obtained by subtracting GSSG from tGSH. Values are expressed in nmol/mg protein. The GSH:GSSG ratio was used as an indicator of the redox state (38).

Enzyme-linked immunosorbent assay (ELISA)

Determinations of SESN2, GCLc and GSS proteins were performed in cell culture lysates using the Human SESN2 (Sestrin2), Human GCLC (glutamate-cysteine ligase catalytic subunit) ELISA Kit and Human GSS (glutathione synthetase) ELISA Kit (Wuhan Fine Biological Technology Co., Ltd., Wuhan, China). Briefly, cells were seeded at 1×104/well on 24-well plates. After treatment exposures, SESN2, GCLc and GSS in cell lysates were determined according to the manufacturer’s instructions. The microplate reader (Multiskan Ascent, Labsystems, Helsinki, Finland) was employed to measure the absorbance at 450 nm. Standard curves were applied to calculate analyte concentrations. The SESN2, GCLc and GSS levels were normalized to total protein concentrations in cell lysates and expressed as ng/mg protein.

Quantitative real-time PCR (qRT-PCR)

The HUVECs (an initial density of 1×106/100 mm-dish) were collected and total RNA extracted with TRI Reagent (Sigma-Aldrich, St. Louis, MO, USA). cDNA was generated from 1 μg of total RNA using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA, USA) according to the manufacturer’s instructions. Primers used for qRT-PCR (Table 1) were retrieved from the database RTPrimerDB (http://www.rtprimerdb.org) and synthesized by Genomed S.A. Company (Warsaw, Poland). All PCR assays were performed on the Roche LightCycler 480 using SYBR Select Master Mix (Applied Biosystems, Waltham, MA, USA) and according to the manufacturer’s instructions, using the following cycling conditions: i) UDP activation for 1 cycle at 50ºC for 2 min, ii) preheat for 1 cycle at 95ºC for 2 min, iii) amplification for 40 cycles: 95ºC for 15 s, followed by the optimal annealing temperature of each primer (Table 1) for 1 min, iv) melting curve for 1 cycle: 95ºC for 5 s, 65 to 97ºC for 1 min, v) cooling to 37ºC for 30 s. A negative control without the cDNA template was included in each assay. GAPDH expression from the same sample was used for data normalization. The relative mRNA expression levels were normalized to GAPDH using the 2–ΔΔCt method (39). The product of each primer pair was verified by electrophoresis analysis on a 2% agarose gel to check for amplicon size. Information on PCR primer sets and PCR products are summarized in Table 1.

Table 1. Primers used in the qRT-PCR analysis.

*Annealing temperature in qRT-PCR.

Nrf2 silencing

Silencer Select Pre-designed siRNA against Nrf2 and Silencer Select Negative Control siRNA were obtained from Ambion (Austin, TX, USA). HUVECs were seeded at the density of 1×105/well on 6-well plates. Cell transfection was performed in Opti-MEM Medium with lipofectamine RNAiMAX Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. After 24 h incubation, the transient transfected HUVECs were treated with HNE alone or HNE with SDX for 4 hours. Relative expression of Nrf2, GCLc and GSS was assessed by qRT-PCR.

Preparation of nuclear and cytosolic fractions

Nuclear and cytosolic proteins were isolated from HUVECs (an initial density of 1×106 cells/100-mm dish) using Nuclear Extraction Kit (Abcam, Cambridge, UK) following the manufacturer’s instructions.

Determination of protein concentration

Bradford Reagent (Sigma-Aldrich, St. Louis, MO, USA) was used to determine total protein concentration. Bovine serum albumin (head shock fraction, ≥98%) (Sigma-Aldrich, St. Louis, MO, USA) serves as a protein standard. Absorbance was measured at 595 nm using a microplate reader (Multiskan Ascent, Labsystems, Helsinki, Finland).

Statistical analysis

All statistical analyses were performed using R software. Normality and homogeneity of variance were tested with Shapiro-Wilk and Levene tests, respectively. Statistically significant differences (p<0.05) were determined by analysis of variance (one-way ANOVA) followed by subsequent Tukey multiple range test as post-hoc analysis. Correlations between the assayed parameters were assessed using the Pearson correlation coefficient.

RESULTS

4-hydroxynonenal induces cell death of HUVECs

In HUVECs, we determined the HNE concentration that caused a 50% decrease in cell viability (Fig. 1). Treatment of cells with 5 and 10 μM HNE for 4 h did not affect cell viability, whereas higher concentrations (25 and 50 μM) triggered cell death (Fig. 1A). In addition, a time-dependent increase in cytotoxicity was observed when the cells were treated with 25 μM HNE for 2, 4, 6, and 8 hours (Fig. 1B). MTT assay showed that incubation with 25 μM HNE for 4 h could reduce viability of HUVECs to 52.48±6.00% compared to the control (p<0.001). Therefore, HNE concentration of 25 μM and incubation time of 4 h were chosen for subsequent experiments.

Fig. 1. Effect of 4-hydroxynonenal on viability of human endothelial cells. HUVECs were treated with the indicated concentrations of HNE for 4 h (A) or with 25 μM HNE for indicated time periods (B). Cell viability was analyzed by MTT assay. Data are presented as mean ±SD (n=12). One-way ANOVA and Tukey’s post-hoc test were used; **p<0.01; ***p<0.001 vs. control. CTRL, control; HNE, 4-hydroxynonenal.

Effect of sulodexide on 4-hydroxynonenal-induced apoptosis and intracellular reactive oxygen spaecies production

Nuclear condensation, activation of pro-apoptotic proteins (Bax family) and subsequent activation of caspases are apoptosis’s key events (40). Thus, in the present study, cells were analyzed for apoptosis after visualization of nuclei morphology with DNA-binding dye Hoechst 33342 and immunoblot analysis of two apoptotic markers (Bax and cleaved caspase-3). To assess intracellular scavenging potency of SDX, the ROS biosensor CellROX® Green Reagent was used. CellROX® exhibits green fluorescence upon oxidation and subsequent binding to DNA, with a signal localized primarily in the nucleus and mitochondria.

Nontreated cells, which served as control, acquired the typical blue Hoechst 33342 fluorescence in their nuclei. However, HNE treatment led to a significant increase in nuclear condensation with increasing fluorescence intensity of Hoechst 33342 (Fig. 2A, upper panel). The same population of cells is also stained intensely with CellROX® green (Fig. 2A, middle panel). The green signal emitted by the CellROX® green probe was superposed on Hoechst 33342 nucleus staining, confirming their cellular specific localization (Fig. 2A, bottom panel).

Fig. 2. Effects of sulodexide (SDX) on 4-hydroxynonenal-induced apoptosis and oxidative stress in human endothelial cells. HUVECs were incubated with 25 μM HNE for 4 h in the absence or presence of SDX (0.5 LRU/mL). (A): Representative fluorescence images of nuclei stained with Hoechst 33342 (blue), ROS accumulation detected with CellROX Green Reagent (green) and merged images (×200). In the images, arrows indicate the nuclei of apoptotic cells (top panel), and arrowheads indicate the nuclei of cells with increased oxidative stress (middle panel). (B): Quantification of mean fluorescence intensity from Hoechst 33342 (Nuclear MFI) and CellROX Green Reagent (CellROX MFI). Data are presented as the mean ±SD (n=9 images per group). (C): Correlation between the intensity of Hoechst 33342 and CellROX Green fluorescence. Pearson’s correlation coefficient R2=0.79 was calculated from the linear regression analysis between Nuclear MFI and CellROX MFI. One-way ANOVA and Tukey’s post-hoc test in (B) were used; *p<0.05; **p<0.01; ***p<0.001. CTRL, control; HNE, 4-hydroxynonenal; HNE+SDX, 4-hydroxynonenal + sulodexide; ROS, reactive oxygen species; Nuclear MFI, nuclear mean fluorescence intensity; CellROX MFI, CellROX mean fluorescence intensity; A.U., arbitrary units.

As presented in Fig. 2A, SDX significantly protected HNE-treated HUVECs from apoptosis. These cells also had weaker CellROX® nuclear fluorescence compared to the HNE group, suggesting that SDX prevented HNE-induced oxidative stress (Fig. 2A).

We then measured the mean fluorescence intensities (MFI) of Hoechst 33342 (nuclear MFI) and CellROX Green Reagent (CellROX MFI) in each experimental group (Fig. 2B). Compared to the control group, HNE-treated cells showed a marked increase in the nuclear MFI (82.55±12.27 A.U. vs. 175±16.58 A.U., p<0.001). However, the HNE-induced increase in nuclear MFI was significantly blocked by SDX (107.93±7.99 A.U., p<0.001). Moreover, exposure to 4-HNE led to an increase in CellROX MFI in HUVECs (113.97±20.58 A.U.) compared to control (48.71±7.87 A.U., p<0.001). Additional treatment with SDX significantly ameliorated the 4-HNE-induced oxidation of the CellROX Green Reagent (58.33±7.11 A.U., p<0.001). Significant linear correlations were seen between attenuation of Nuclear MFI and decreased ROS levels (Fig. 2C, R2=0.79 and p<0.001).

Next, the protein expression levels of apoptotic markers, including Bax and cleaved caspase-3, were evaluated by Western blot (Fig. 3A). Compared with the control cells, immunoblot analysis revealed that HNE increased the expressions of Bax and cleaved caspase-3 (3.27±0.67, p<0.01 and 5.68±0.8-fold, p<0.001, respectively), and these effects were attenuated by SDX treatment (1.97±0.36, p<0.05 and 1.96<0.92-fold, p<0.01, respectively) (Fig. 3B and 3C).

Fig. 3. Effect of sulodexide (SDX) on Bax and cleaved caspase-3 protein expression in human endothelial cells treated with 4-hydroxynonenal. HUVECs were incubated with 25 μM HNE for 4 h in the absence or presence of SDX (0.5 LRU/mL). (A): Western blot analysis of Bax and cleaved caspase-3 expression. The relative expression of Bax (B) and cleaved caspase-3 (C) was quantified by densitometry and normalized to GADPH. Data are shown as the mean ±SD (n=3). One-way ANOVA and Tukey’s post-hoc test in (B) and (C) were used; *p<0.05; **p<0.01; ***p<0.001. CTRL, control; HNE, 4-hydroxynonenal; HNE+SDX, 4-hydroxynonenal + sulodexide.

Furthermore, SDX was found to protect endothelial cells against HNE-induced damage, leading to an improvement in cell viability of up to 84.91±7.47% (Fig. 4A, p<0.001). The study also established a negative linear correlation between the cell viability and apoptosis (Fig. 4B, R2=0.85 and p<0.001) or ROS production (Fig. 4C, R2=0.78 and p<0.001).

Fig. 4. Effects of sulodexide (SDX) on viability of human endothelial cells. HUVECs were incubated with 25 μM HNE for 4 h in the absence or presence of SDX (0.5 LRU/mL). (A): Cell viability analysis by MTT assay. Data are presented as mean ±SD (n=12). (B): Correlation between formazan absorbance and Hoechst 33342 fluorescence intensity. Pearson’s correlation coefficient R2=0.85 was calculated from the linear regression analysis between optical density at 570 nm (OD570) and Nuclear MFI. (C): Correlation between formazan absorbance and CellROX Green Reagent fluorescence intensity. Pearson’s correlation coefficient R2=0.78 was calculated from the linear regression analysis between optical density at 570 nm (OD570) and CellROX MFI. One-way ANOVA and Tukey’s post-hoc test in (A) were used; ***p<0.001. CTRL, control; HNE, 4-hydroxynonenal; HNE+SDX, 4-hydroxynonenal + sulodexide; Nuclear MFI, nuclear mean fluorescence intensity; CellROX MFI, CellROX mean fluorescence intensity; A.U., arbitrary units.

Effect of sulodexide on 4-hydroxynonenal-induced glutathione depletion and redox imbalance

To further confirm the SDX protective effects in HNE-injured HUVECs, we determined the concentrations of GSH and GSSG, as well as the GSH:GSSG ratio. Moreover, we compared the antioxidant capacity of SDX with N-acetylcysteine (NAC) and γ-glutamylcysteine (GGC). NAC exhibits highly protective scavenging properties against HNE (41). NAC replenishes GSH by providing cysteine, reduces disulfide bonds in proteins and restores small thiol pools that regulate the redox state (42). GGC is a direct precursor of GSH, which, thanks to the content of cysteine residue, has antioxidant properties. It was reported that GGC protects human endothelial cells from oxidative stress and activates the antioxidant defense function of GSH (43).

The results presented in Fig. 5A show a significant decrease in GSH level from 22.79±0.36 nmol/mg protein in control cells to 15.99±0.53 nmol/mg protein after administration of HNE (p<0.001). Co-treatment of HUVECs with HNE and SDX significantly elevated intracellular GSH (20.8±0.65 nmol/mg protein) in comparison with the HNE-only group (p<0.001). Furthermore, NAC and GGC caused a significant increase in GSH (27.18±0.89 and 24.23±0.36 nmol/mg protein, respectively) in comparison both to the untreated cells and HNE group (Fig. 5A).

Fig. 5. Effect of sulodexide (SDX), N-acetylcysteine (NAC) and γ-glutamylcysteine (GGC) on intracellular concentrations of glutathione, redox state as well as GCLc and GGS protein levels in human endothelial cells treated with 4-hydroxynonenal. HUVECs were incubated with 25 μM HNE for 4 h in the absence or presence of SDX (0.5 LRU/mL), NAC (100 μM) and GGC (100 μM). The intracellular concentrations of reduced glutathione (GSH) (A) and oxidized glutathione (GSSG) (B) were measured by colorimetric assay (n=5). The GSH and GSSG levels were normalized to total protein concentrations and expressed as nmol/mg protein. The GSH:GSSH ratio (C) for each experimental group was calculated. (D-E): Quantitative analysis of GCLc (D) and GSS (E) in cell lysates performed by ELISA (n=6). The GCLc and GSS protein concentrations in cell lysates were normalized to total protein concentration and expressed as ng/mg protein. Data are shown as the mean ±SD. One-way ANOVA and Tukey’s post-hoc test were used; *p<0.05; **p<0.01; ***p<0.001. CTRL, control; HNE, 4-hydroxynonenal; HNE+SDX, 4-hydroxynonenal + sulodexide; HNE+NAC, 4-hydroxynonenal + N-acetylcysteine; HNE+GGC, 4-hydroxynonenal + γ-glutamylcysteine; GCLc, glutamate-cysteine ligase catalytic subunit; GSS, glutathione synthase.

There was no increase in GSSG concentrations in cells treated with HNE alone or HNE with SDX (1.06±0.01 and 1.05±0.02 nmol/mg protein, respectively) compared to control (1.13±0.02 nmol/mg protein). However, treatment of HNE-injured HUVECs with NAC or GGC was associated with a significant increase in GSSG levels (1.74±0.07, p<0.001 and 1.3±0.06 nmol/mg protein, p<0.05, respectively) (Fig. 5B).

Glutathione-dependent redox balance was expressed as GSH:GSSG ratio (Fig. 5C). The GSH:GSSG proportion is an indicator of the overall redox environment in the cell. A higher GSH:GSSG redox status could make the endothelial cells more resistant to oxidative stress and apoptosis (44). The intracellular GSH:GSSG ratio after exposure to HNE was found to be significantly lower (15.16±0.43) than in control cells (20.27±0.99, p<0.001). The redox balance shifts toward more reducing state in HNE-injured HUVECs treated with SDX or GGC (19.77±0.68, p<0.001 and 18.66±0.78, p<0.001, respectively). Meanwhile, additional incubation with NAC had no noticeable effect on GSH:GSSH ratio (15.64±0.4) compared with 4-HNE treatment alone due to an increase in intracellular GSSG levels (Fig. 5B and 5C).

GSH synthesis is catalyzed by GCL and GSS, we thus measured the protein levels of GCLc and GSS by ELISA as a surrogate for their enzymatic activity (Fig. 5D and 5E). Some studies have shown a correlation between the protein content and the enzymatic activity of GCL and GSS (45). Results showed that HNE significantly reduced GCLc protein levels (6.32±0.38 ng/mg protein) compared to the control group (11.68±1.82 ng/mg protein, p<0.001). The treatment of HNE-injured cells with SDX, NAC or GGC raised GCLc protein levels (13.07±1.34, p<0.001; 14.45±0.78, p<0.001 and 16.78±0.81 ng/mg protein, p<0.001, respectively) (Fig. 5D).

As shown in Fig. 5E, the GSS concentration was decreased in cells treated with HNE alone (44.98±6.72 ng/mg protein) compared to the control (57.47±3.09 ng/mg protein, p<0.001). Co-treatment of HUVECs with HNE and SDX, NAC or GGC significantly increased GSS protein levels (57.81±3.64, p<0.001; 73.14±2.11, p<0.001 and 62.26±2.52 ng/mg protein, p<0.001, respectively).

Effect of sulodexide on 4-hydroxynonenal-induced increase in SESN2 protein level

We next measured the intracellular level of the stress-inducible protein SESN2. The SESN2 levels are usually elevated in the oxidatively damaged cells and tissues, compared to control conditions. Also, there is correlation between SESN2 protein expression and the severity of the damage (33). Some studies have identified a protective effect of SESN2 against HNE-induced cytotoxicity (46, 47). As expected, SESN2 protein concentration was significantly increased in the cells exposed to HNE alone (median 24.78 ng/mg protein, range 23.08–28.45) compared to the control (median 17.1 ng/mg protein, range 15.79–17.56, p<0.001). The co-administration of HNE and SDX also resulted in an increase in SESN2 protein (median 21.21 ng/mg protein, range 19.34–22.63, p<0.001), whereas co-treatments of HNE with NAC or GGC markedly reduced SESN2 concentrations in HUVECs (median 12.49 ng/mg protein, range 11.76–13.01, p<0.001; median 14.74 ng/mg protein, range 14.4–17.56, p<0.001, respectively) (Fig. 6).

Fig. 6. Effect of sulodexide (SDX), N-acetylcysteine (NAC) and γ-glutamylcysteine (GGC) on SESN2 protein level in human endothelial cells treated with 4-hydroxynonenal. HUVECs were incubated with 25 μM HNE for 4 h in the absence or presence of SDX (0.5 LRU/mL), NAC (100 μM) and GGC (100 μM). Quantitative analysis of SESN2 level in cell lysates was performed by ELISA (n=6). The SESN2 protein concentrations in cell lysates were normalized to total protein concentration and expressed as ng/mg protein. Data are box-plots representing the median and quartiles with the upper and lower limits. One-way ANOVA and Tukey’s post-hoc test were used; ***p<0.001; [•]: the outlier. CTRL, control; HNE, 4-hydroxynonenal; HNE+SDX, 4-hydroxynonenal + sulodexide; HNE+NAC, 4-hydroxynonenal + N-acetylcysteine; HNE+GGC, 4-hydroxynonenal + γ-glutamylcysteine.

In addition, after administration of HNE or HNE+SDX, SESN2 levels were positively correlated with ROS production (Fig. 7A, R2=0.66 and p<0.001) and negatively correlated with intracellular GSH levels (Fig. 7B, R2=0.81 and p<0.001). A significant inverse correlation between SESN2 and GSH was also obtained for cells treated with a combination of HNE and NAC (Fig. 7C, R2=0.95 and p<0.001) or GGC (Fig. 7D, R2=0.93 and p<0.001).

Fig. 7. Linear regression analysis of the relationship between SESN2 protein level and ROS production after sulodexide (SDX) treatment (A) as well as between SESN2 and GSH concentrations after SDX (B), N-acetylcysteine (NAC) (C) or γ-glutamylcysteine (GGC) (D) treatment. Each panel represents a scatterplot and the regression line. Pearson’s correlation coefficient (R2) and p values are plotted in every graphic. CTRL, control; HNE, 4-hydroxynonenal; HNE+SDX, 4-hydroxynonenal + sulodexide; HNE+NAC, 4-hydroxynonenal + N-acetylcysteine; HNE+GGC, 4-hydroxynonenal + γ-glutamylcysteine.

Effect of sulodexide on 4-hydroxynonenal-induced increase in Nrf2, GCLc and GGS mRNA levels

Previous studies have demonstrated an important role for Nrf2 in HNE-induced endogenous adaptive antioxidant pathways (17). With this in mind, we determined whether the combined administration of HNE and SDX also modulates Nrf2 mRNA level as well as two other genes that are regulated by Nrf2 signaling and involved in GSH synthesis (GCLc and GSS).

As shown in Fig. 8A-8C, both HNE alone and HNE+SDX treatment increased mRNA levels of Nrf2 (median 1.74-fold, range 1.56–1.89, p<0.001 and median 1.31-fold, range 1.23–1.54, p<0.01 respectively), GCLc (median 1.8-fold, range 1.67–2.62, p<0.001 and median 1.59-fold, range 1.55–1.62, p<0.001, respectively) and GSS (median 2.01-fold, range 1.77–2.47, p<0.001 and median 1.68-fold, range 1.55–1.92, p<0.001, respectively) compared to the control group. However, it should be noted that SDX significantly reduced HNE-stimulated Nrf2, GCLc and GSS mRNA levels (p<0.01, p<0.05 and p<0.01, respectively). Further analysis showed that the protein levels of SESN2 were positively correlated with mRNA levels of Nrf2 (Fig. 8D, R2=0.73 and p<0.001), GCLc (Fig. 8E, R2=0.69 and p<0.001) and GSS (Fig. 8F, R2=0.73 and p<0.001).

Fig. 8. Effect of sulodexide (SDX) on Nrf2, GCLc and GGS mRNA levels in human endothelial cells treated with 4-hydroxynonenal. HUVECs were incubated with 25 μM HNE for 4 h in the absence or presence of SDX (0.5 LRU/mL). The relative expression of Nrf2 (A), GCLc (B) and GSS (C) were determined by quantitative real-time PCR and normalized to GAPDH. The control (untreated cells) is given as normalized to a value of 1 (continuous line section). Data are box-plots representing the median and quartiles with the upper and lower limits. One-way ANOVA and Tukey’s post-hoc test were used; *p<0.05, **p<0.01, ***p<0.001; [•]: the outlier. (D-F): Linear regression analysis of the relationship between the fold change of SESN2 protein levels and mRNA expression for Nrf2 (D), GCLc (E) and GSS (F). Each panel represents a scatterplot and the regression line. Pearson’s correlation coefficient (R2) and p values are plotted in every graphic. CTRL, control; HNE, 4-hydroxynonenal; HNE+SDX, 4-hydroxynonenal + sulodexide.

Silencing Nrf2 decreases GCLc and GGS mRNA levels

To assess whether Nrf2 silencing downregulated Nrf2, GCLc and GSS gene expressions induced by HNE administration with or without SDX, we measured their mRNA levels. As shown in Fig. 9A, the qRT-PCR results confirmed that Nrf2 expression was successfully silenced (p<0.001) in HUVECs (0.37±0.1-fold compared to negative control siRNA).

After incubation with Nrf2 siRNA, the cells treated with HNE alone or HNE+SDX showed a 0.75±0.11, p<0.001 and 0.58±0.14-fold, p<0.001 decrease in Nrf2 mRNA, respectively. Moreover, as shown in Fig. 9B and C, Nrf2 siRNA significantly attenuated the induction of both GCLc and GSS caused by exposure to HNE (p<0.001) as well as the combined administration of HNE and SDX (p<0.01 and p<0.001, respectively).

Fig. 9. Inhibition of Nrf2 by siRNA attenuates sulodexide (SDX)-mediated induction of Nrf2, GCLc and GGS genes in human endothelial cells treated with 4-hydroxynonenal. HUVECs were transfected with negative control siRNA or Nrf2 siRNA for 24 h and then incubated with 25 μM HNE for 4 h in the absence or presence of SDX (0.5 LRU/mL). The relative expressions of Nrf2 (A), GCLc (B) and GSS (C) were determined by quantitative real-time PCR and normalized to GAPDH (n=3). The negative control siRNA (Silencer Select Negative Control siRNA) is given as normalized to a value of 1. Data are shown as the mean ±SD. One-way ANOVA and Tukey’s post-hoc test were used; *p<0.05; **p<0.01; ***p<0.001. siNC, negative control siRNA; siNrf2, Nrf2 siRNA; HNE, 4-hydroxynonenal; HNE+SDX, 4-hydroxynonenal + sulodexide.

Effect of sulodexide on 4-hydroxynonenal-induced Keap-1 degradation and Nrf2 nuclear translocation

Finally, the protein expression levels of Keap-1 and Nrf2 were measured by immunoblotting. As shown in Fig. 10, the cytoplasmic expression levels of Keap-1 and Nrf2 were significantly reduced in the HNE-injured HUVECs (0.35±0.11, p<0.01 and 0.3±0.15–fold, p<0.001, respectively), and these effects were attenuated by SDX treatment (0.8±0.17, p<0.01 and 0.65±0.07-fold, p<0.05, respectively). In parallel, HNE significantly increased the expression of nuclear Nrf2 (1.84±0.26-fold, p<0.01). Additional treatment with SDX significantly ameliorated the increase of nuclear Nrf2 in response to HNE (1.4±0.06-fold, p<0.05).

Fig. 10. Effect of sulodexide (SDX) on Keap-1 and Nrf2 protein expression in human endothelial cells treated with 4-hydroxynonenal. HUVECs were incubated with 25 μM HNE for 4 h in the absence or presence of SDX (0.5 LRU/mL). (A): Western blot analysis of Keap-1 and Nrf2 expression in cytosolic and nuclear fractions. (B): The relative expression of Keap-1 and Nrf2 in cytosolic fraction was quantified by densitometry and normalized to GADPH. Data are shown as the mean ±SD (n=3). (C): The relative expression of Nrf2 in nuclear fraction was quantified by densitometry and normalized to Lamin B. Data are shown as the mean ±SD (n=3). One-way ANOVA and Tukey’s post-hoc test in (B) and (C) were used; *p<0.05; **p<0.01; ***p<0.001. CTRL, control; HNE, 4-hydroxynonenal; HNE+SDX, 4-hydroxynonenal + sulodexide.

Fig. 11. Schematic diagram illustrating the protective effects of sulodexide (SDX) against 4-hydroxynonenal-induced apoptosis and oxidative stress in endothelial cells. HNE, 4-hydroxynonenal; ROS, reactive oxygen species; CYS, cysteine; GLU, glutamate; GCLc, catalytic subunit of glutamate-cysteine ligase; GGC, γ-glutamylcysteine; GLY, glycine; GSS, glutathione synthase; GSH, reduced form of glutathione; GSSG, oxidized form of glutathione; SESN2, Sestrin2; →, induction; ┤, inhibition.

DISCUSSION

In the present study, we investigated whether SDX has potential antioxidant activity in endothelial cells exposed to HNE. Our results showed that SDX at a clinically relevant concentration (0.5 LRU/mL) (35) significantly attenuated the cytotoxic effect of HNE in HUVEC cell culture. The protective mechanisms of SDX were accompanied by a decrease in the induction of the apoptotic markers (Bax and cleaved caspase-3), the reduction of ROS production, and the increase in GSH level. More importantly, SDX can maintain the proper redox balance and regulate the SESN2/Nrf2 pathway in HNE-treated endothelial cells.

HNE can directly affect the cellular redox status by depleting GSH, which can then induce ROS production and subsequent activation of caspases (48, 49). On the other hand, an increase in intracellular GSH and redox balance are essential to maintaining cell viability (50). This suggests that antioxidants that increase GSH and regulate glutathione redox status may be effective in preventing HNE-induced endothelial cell apoptosis. In our study, HNE was shown to reduce intracellular GSH levels and this effect was clearly associated with an increase in Bax and cleaved caspase-3 expression (Fig. 3). Additionally, treatment of HUVECs with HNE decreased GCLc and GSS protein level. The decline in GCLc protein in response to HNE may be due to increased degradation by caspase-3 during apoptosis. Previous studies have shown that GSH deficiency in apoptotic cells was largely due to reduced expression of the GCLc protein, and caspase-3 is responsible for its degradation (51). It has also been shown that changes in GCLc mRNA do not immediately translate into protein levels and that post-translational modification of GCLc by caspases plays an important role in regulating GSH synthesis during apoptosis (51, 52). This is also confirmed by our results (Fig. 5D and 8B) and suggests that GCLc protein is a target for caspase-3-mediated cleavage during HNE-induced apoptotic cell death. At the same time, HNE reduced the level of GSS protein (Fig. 5E). The role of GSS protein in decreased GSH concentration is less obvious. There are no confirmed reports of post-translational modifications of the GSS. Nevertheless, a correlation was noted between a decrease in GSH levels in the liver and a reduction in GSS activity. It is known that GCL and GSS may be regulated coordinately and possibly interdependently through a protein-protein interaction (53). As observed in our study, the decrease in GCLc protein concentration must sufficiently explain the decrease in GSH in HNE-damaged endothelial cells. The molecular mechanism underlying the HNE-induced decline in GSS protein levels in HUVECs remains unclear.

Our results suggest that SDX protects HNE-injured endothelial cells by increasing the intracellular GSH concentration and redox state, which inhibit the signaling pathways leading to caspase-3-dependent apoptosis (48). The impact of SDX on apoptosis has been reported in some papers (14, 54-57). Similar to our study, the inhibition of caspase-3 activation and ROS production in HUVECs under metabolic stress has been observed for SDX (58). However, in this paper, we showed for the first time that SDX, unlike NAC, has the ability to enhance the redox state in HUVECs exposed to HNE and is comparable to GGC in this respect (Fig. 5C). These results are consistent with observations suggesting that the GSH:GSSG ratio does not change following NAC treatment due to a corresponding increase in GSSG (59). Furthermore, we showed that SDX treatment restored GCLc and GSS protein levels, although its stimulatory effect was less significant than that of NAC and GGC (Fig. 5D and 5E). This also supports our hypothesis that SDX may prevent post-translational modifications of GCLc that occur during apoptosis.

We found that the protective effect of SDX in HNE-injured HUVECs is based on its anti-oxidant capacity through modulation of SESN2/Nrf2 signaling. SESN2 may regulate ROS production and be involved in endothelial cell response to oxidative stress conditions (60). It was shown that SESN2 levels are usually elevated in patients with cardiovascular or metabolic diseases and positively correlated with the severity of the disease (33). Moreover, a study by Yi et al. (46) on HUVECs reported the activation of SESN2 after treatment with angiotensin II. However, the question remains how endothelioprotective drugs, such as SDX, used in treating various cardiovascular diseases can modulate the functions of SESN2 and whether it is related to their antioxidant activity. Our study showed that the intracellular level of SESN2 was significantly increased in HNE-injured HUVECs. Importantly, we also found that administration of SDX decreased SESN2 levels in HNE-treated HUVECs, correlating with ROS production (Fig. 6 and 7A). This study revealed a negative correlation between SESN2 levels and GSH, which significantly decreased with increasing GSH concentrations (Fig. 7). HNE-induced oxidative stress and GSH depletion, which may cause SESN2 up-regulation, appear to have resolved after administration of SDX as well as NAC and GGC.

When oxidative stress occurs, antioxidant defense systems, such as Nrf2/GSH pathway, can be activated to block oxidative stress-induced endothelial cell injury (61). HNE has been found to have direct properties to modify cysteine residues on Keap-1, leading to the dissociation and activation of Nrf2, followed by the induction of antioxidant genes (62). In addition to the dissociation of Nrf2 from Keap-1, regulation of Nrf2 signaling also occurs at the transcriptional level. Furthermore, it was found that in endothelial cells, Nrf2 could be transcriptionally activated by 4-HNE (63). There is evidence that Nrf2 transcription is activated by an autoregulation mechanism through the ARE-like element located in the proximal region of its promoter (64). It has been proven that HNE rapidly stimulates Nrf2 nuclear translocation in HUVECs. This causes an increase in the level of Nrf2 in the cell nucleus, which can act on the ARE of the Nrf2 promoter, thereby increasing Nrf2 mRNA expression (63). However, higher concentrations of HNE cause apoptosis in endothelial cells due to dysregulation of oxidative and antioxidant balance and loss of compensation (17). In this study, HNE significantly upregulated SESN2 protein, increasing expression of Nrf2, GCLc and GSS genes. We also found that SDX reduced SESN2 content and downregulated Nrf2 mRNA induced by HNE, which may be related to the inhibition of oxidative stress by SDX. The administration of SXD also significantly decreased GCLc and GSS mRNA levels in comparison with HNE group, suggesting that the damaging effect of HNE-induced oxidative stress on HUVECs could be suppressed (Fig. 8). Furthermore, Nrf2 silencing reduced the mRNA levels of these two Nrf2-regulated genes involved in GSH synthesis and abolished their induction, indicating that they are regulated by the Nrf2 signaling pathway both in response to HNE alone, and in co-treatment with HNE and SDX (Fig. 9).

As mentioned above, HNE activates Nrf2 primarily by inhibiting Keap1-dependent Nrf2 degradation, through alkylation of specific cysteine residues on Keap-1 (62). It was demonstrated that Cys273/Cys288 play a fundamental role in HNE sensing and are probably the critical redox-sensitive cysteine residues for the Keap1-Nrf2 interaction. Keap-1 is recognized as a molecular ROS sensor, which detects the redox state inside the cell and transmits it to Nrf2 (65). The simultaneous increase in ROS production, GSH depletion and redox imbalance caused by HNE lead to the oxidation of Keap-1, stabilization of Nrf2 and enhanced transcription of target genes (66). Therefore, we hypothesize that SDX, by restoring the normal redox state and attenuating oxidative stress, reduces the ability of HNE to oxidize Keap-1, thereby leading to attenuation of HNE-induced Nrf2-dependent gene expression. Our results also seem important in the context of findings indicating that prolonged over-activation of Nrf2 can cause endothelial dysfunction and have a proatherogenic effect (67). Recently, several studies have linked the endothelial protective effects of both natural and synthetic antioxidants to modulation of the Nrf2/ARE pathway via interaction with cysteine residues in Keap-1. For example, rutin has been shown to react with reactive cysteine residue in Keap-1, activating Nrf2 and protecting HUVECs from oxidative stress (68). Similarly, treatment of HUVECs with other compounds such as sulforaphane, withaferin A, and curcumin showed that they interact with cysteine residues on Keap-1, thus promoting Nrf2 nuclear accumulation and ameliorating endothelial dysfunction (69-71).

However, the fact that HNE binds to the Keap-1 protein does not exclude the possibility that it may also influence p62-dependent autophagic degradation of Keap-1. This mechanism has been shown to be mediated by SESN2 (31). HNE dysregulates autophagy, resulting in increased expression of the autophagic substrate p62. Accumulated p62 together with SESN2 leads to the sequestration of Keap-1 protein in autophagosomes, thus promoting its degradation and activation of Nrf2 (72). This is especially prominent under conditions when p62 is phosphorylated (73). Interestingly, SESN2 facilitates the phosphorylation of p62, which translates into a prolonged accumulation of Nrf2 and transcriptional upregulation of its target genes. In turn, Nrf2 increases the expression of SESN2 and p62 in a positive feedback manner (72). We have reported that there are positive correlations between SESN2 protein and expression of Nrf2, GCLc and GSS genes (Fig. 8). Since it has been shown that SDX in endothelial cells exposed to various stressors significantly down-regulates p62 protein expression and increases autophagic flux (58), these effects may translate into the weakening of the SESN2/Nrf2 pathway observed in our study (Fig. 10). Testing this hypothesis will require further experimental approaches.

In summary, our study shows that SDX administration restores GSH:GSSG balance and levels of GCLc and GSS proteins in HNE-damaged endothelial cells. In addition, SDX may mitigate the effects of oxidative stress on SESN2 and regulate the SESN2/Nrf2/GSH pathway (summarized mechanistic pathways are shown in Fig. 11). These results indicate that modulation of the SESN2/Nrf2 pathway may be the basis for the development of new pharmacological strategies in the treatment of atherosclerosis and vascular diseases.

Funding: This research was funded by National Science Centre, Poland, grant number 2019/35/N/NZ7/03071 (Preludium-18, K.B.) and Medical University of Silesia in Katowice, Poland, grant number PCN-2-062/N/0/O (K.B.)

Conflict of interests: None declared.

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