Coenzyme Q10 Prevents Senescence and Dysfunction Caused by Oxidative Stress in Vascular Endothelial Cells

Abstract

Oxidative damage in endothelial cells is proposed to play an important role in endothelial dysfunction and atherogenesis. We previously reported that the reduced form of coenzyme Q10 (CoQ₁₀H₂) effectively inhibits oxidative stress and decelerates senescence in senescence-accelerated mice. Here, we treated human umbilical vein endothelial cells (HUVECs) with H₂O₂ and investigated the protective effect of CoQ₁₀H₂ against senescence, oxidative damage, and reduction in cellular functions. We found that CoQ₁₀H₂ markedly reduced the number of senescence-associated β-galactosidase-positive cells and suppressed the expression of senescence-associated secretory phenotype-associated genes in H₂O₂-treated HUVECs. Furthermore, CoQ₁₀H₂ suppressed the generation of intracellular reactive oxygen species (ROS) but promoted NO production that was accompanied by increased eNOS expression. CoQ₁₀H₂ prevented apoptosis and reductions in mitochondrial function and reduced migration and tube formation activity of H₂O₂-treated cells. The present study indicated that CoQ₁₀H₂ protects endothelial cells against senescence by promoting mitochondrial function and thus could delay vascular aging.

1. Introduction

Cardiovascular diseases (CVD) continue to be a leading cause of death and disability worldwide [1]. This trend is particularly pronounced among aged populations in many countries. Since CVD mortality rates increase with patient age, the aging process is recognized to be the largest risk factor for the development of CVD [2], especially atherosclerosis [3].

The term senescence, a process encompassing age-related and irreversible reductions in physiological functions and increasing mortality rate, is often used to distinguish these processes from chronological aging. Senescence is complex and involves metabolic changes and destruction of molecular and cellular homeostasis that can eventually lead to organ failure and death [4]. Recent studies in humans and animal models suggest that vascular aging/senescence leads to impaired endothelial functions associated with elevated oxidative stress and a proinflammatory phenotype [5]. Senescent cells can activate various types of proinflammatory cytokines, chemokines, growth factors, and proteases, which together form the senescence-associated secretory phenotype (SASP) [6].

Hayflick and Moorhead first described cellular senescence in 1961 when they observed a limited ability of cells to replicate in vitro, which they referred to as replicative senescence [7]. At the cellular level, senescence is a complex pathophysiological process that includes various factors, such as accumulation of DNA damage [8], oxidative stress [9], and activation of signaling pathways involved in the general aging process. Several treatments were subsequently identified that promote cellular senescence and induce the phenotype known as stress-induced premature senescence (SIPS). Reactive oxygen species (ROS) are formed as natural products of metabolism and have important roles in several cellular signaling and metabolic pathways, including mitochondrial oxidative phosphorylation, cell proliferation, and cell cycle arrest [10]. However, massive production of ROS during environmental stresses can damage cellular proteins and nucleic acids, as well as peroxidize lipids, which together can ultimately lead to cell death [11]. Previous research on the relationship between oxidative stress and aging indicated that oxidative stress is the origin of cellular senescence [12]. For example, the oxidative stressor hydrogen peroxide (H₂O₂) can induce an oxidative environment that rapidly leads to premature senescence [13].

The vascular endothelium comprises a thin layer of endothelium that lines the inner surface of blood vessels. Endothelial cells produce the vasorelaxant nitric oxide (NO) generated by NO synthase in endothelial cells (eNOS) that plays an important role in vasodilation. Aging of vascular endothelium in the elderly induces multifactorial dysfunctions in addition to reduced vasodilation [14] during the development of atherosclerosis, which is more pronounced with age [15, 16]. Thus, aging and endothelial cell dysfunction are closely related and insights into the mechanisms that are responsible for this dysfunction are important for delaying aging and enhancing the overall health of the elderly population.

Human umbilical vein endothelial cells (HUVECs) originate from the endothelium of human umbilical cord veins. HUVECs are used as a classic model system to study many aspects of endothelial function and disease, such as oxidative stress- and inflammation-related pathways in endothelia under normal and pathological conditions [17].

Coenzyme Q10 (CoQ₁₀) is a ubiquitous lipid-soluble molecule found in many eukaryotic cells [18] that is essential for mitochondrial oxidative phosphorylation and electron transport chain activity [19]. With increasing age, CoQ10 concentrations in organisms decrease gradually and this decrease can be accompanied by the onset of physical dysfunction and the emergence of disease [20]. The critical role of CoQ₁₀ in mitochondrial function [21] and its status as a lipid-soluble antioxidant have led to its use in therapeutic applications and clinical trials for CVD treatments [22]. Most CoQ₁₀ in circulation and tissues exists in its reduced form (CoQ₁₀H₂), which acts as an antioxidant through its oxidation to an oxidized form (oxCoQ₁₀) [23]. In addition, recent studies showed that the nonmitochondrial CoQ₁₀-forming enzyme in Golgi membranes has specific cardiovascular protective functions that modulate eNOS activity through redox reactions involving CoQ₁₀ [24]. NO synthetized by eNOS is known to be an essential factor for cardiovascular function in vertebrates [25] and prevents the progression of age-related dysfunction in endothelial cells [26]. Our previous studies showed that CoQ₁₀H₂ increases levels of cyclic adenosine monophosphate (cAMP) in tissues and cells and also enhances mitochondrial antioxidant function by activating SIRT1 and PGC-1α that in turn delays senescence and incidence of related diseases [27]. SIRT1 is recognized as an important deacetylase that is related to increasing eNOS-derived nitric oxide (NO) for prevention of endothelial senescence [28, 29]. In addition, we found that KKAy mice fed diets supplemented with CoQ₁₀H₂ reduced white adipose tissue and enhanced the function of brown adipose tissue by promoting the expression of SERCA2 and decreasing cytoplasmic Ca²⁺ levels in liver cells. These mice also had an enhanced fat metabolic rate through inhibition of the CaMKII-MEK1/2-ERK1/2 signaling pathway and increased cAMP levels [30]. In HUVECs, CoQ₁₀H₂ has been reported to have an anti-inflammatory function and to delay SASP acquisition in senescence status by attenuating miR-146a expression [31].

Here, our results showed that preincubation of HUVECs with CoQ₁₀H₂ prevented H₂O₂-induced premature senescence and ameliorated declines in physiological functions of endothelial cells. This action could be mediated by enhancing mitochondrial and endothelial function via the SIRT1-eNOS pathway and upregulating expression of antioxidant enzymes and decreasing intracellular ROS production.

2. Materials and Methods

2.1. Cell Culture

Human umbilical vein endothelial cells (HUVECs) were purchased from the Japanese Cancer Research Resources Bank (http://cellbank.nibiohn.go.jp/english/). The cells were grown in endothelial growth medium (EGM-2; Lonza Walkersville, MD, USA) at 37°C under a humidified atmosphere of 5% CO₂, and the medium was changed every 2 days. HUVECs were passaged when they reached 80% confluence, and cells from passages 2–8 were used for all experiments. When HUVECs reached 90% confluence, the cells were divided into 4 experimental groups: (1) control group: untreated HUVECs; (2) CoQ₁₀H₂ group: cultured HUVECs incubated for 24 hours in medium with 10 μM CoQ₁₀H₂ and then cultured for an additional 12 hours in medium lacking CoQ₁₀H₂; (3) H₂O₂ group: HUVECs cultured for 24 hours in medium without CoQ₁₀H₂ and cultured for another 12 hours in medium containing 100 μM H₂O₂ and lacking CoQ₁₀H₂; and (4) CoQ₁₀H₂ + H₂O₂ group: HUVECs cultured for 24 hours in medium with 10 μM CoQ₁₀H₂ and then cultured for 12 hours in medium with 100 μM H₂O₂ and lacking CoQ₁₀H₂. We also incubated HUVECs in medium containing high glucose (30 mM; HG medium) to induce senescence. We pretreated HUVECs with 10 μM CoQ₁₀H₂ and then cultured for an additional 72 hours in HG medium.

2.2. Real-Time RT-PCR

HUVECs were collected by scraping and the total RNA was extracted from cells using TRIzol Reagent (Invitrogen, CA) followed by treatment with DNA-Free (Applied Biosystems, CA) to remove contaminating DNA. Total RNA was subjected to reverse transcription using an Omniscript RT kit (Applied Biosystems, CA) with random primers (Applied Biosystems, CA). Quantitative real-time RT-PCR analysis was carried out using an ABI PRISM 7500 Sequence Detection System (Applied Biosystems, CA) with SYBR Green (Takara Bio, Tokyo, Japan). Primer sequences are listed in Table S1.

2.3. Senescence-Associated Galactosidase (SA-β-Gal) Staining

After 12 hours incubation in medium with or without H₂O₂, or 72 hours incubation in medium with or without HG, HUVECs were washed twice with PBS, fixed for 10 minutes with 10% formalin at room temperature, and washed twice in PBS. The cells were then incubated at 37°C for 12 hours with a staining solution (40 mM citric acid/sodium phosphate buffer pH 6.0, 0.5 mg/mL X-Gal, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM NaCl, and 2 mM MgCl₂). SA-β-Gal-positive cells were observed by microscopy, and over 300 cells were counted in three independent fields to calculate the proportion of positive cells.

2.4. Analysis of Apoptosis

Apoptosis was assessed using an Annexin V-FITC Apoptosis Detection kit (Enzo Life Sciences, NW). HUVECs in each experimental group were washed with PBS after 12 hours incubation in medium with or without H₂O₂ and then collected by trypsinization. The collected cells were washed in PBS with gentle shaking and resuspended with 195 μL of a specific binding buffer containing 5 μL annexin V-FITC. After incubation for 10 minutes in the dark at room temperature, the cells were washed in PBS, resuspended in 190 μL binding buffer, and then stained with 10 μL propidium iodide (PI) (20 μg/mL). Cellular fluorescence was analyzed with a FACSCanto II flow cytometer system (BD Biosciences, NJ), and the data were analyzed using BD FACSDiva™ software. HUVECs were classified as follows: normal healthy cells (Annexin V⁻/PI⁻), early apoptotic cells (Annexin V⁺/PI⁻), late apoptotic cells (Annexin V⁺/PI⁺), and necrotic cells (Annexin V⁻/PI⁺).

2.5. Total Reactive Oxygen Species (ROS) and Superoxide Production

Total ROS and superoxide production in HUVECs was determined using a total ROS/superoxide detection kit (Enzo Life Sciences, NW). HUVECs in each experimental group were washed with PBS after 12 hours incubation in medium with or without H₂O₂ and then stained according to the kit manufacturer's instructions. Then, cells were assessed by a FACSCalibur flow cytometer system (BD Biosciences, NJ) using the FL1 and FL2 channels to detect signals from ROS- and superoxide-sensitive reagents, respectively. We set the gate for 4 quadrants such that >99% of unstained untreated control HUVECs were grouped in the negative area. Over 89% of positive control cells (pyocyanin-treated) stained only with Oxidative Stress Detection Reagent (green) were gated into the ROS-positive area, and >89% of positive control cells (pyocyanin-treated) stained only with Superoxide Detection Reagent (orange) were gated into the superoxide-positive area. Each analysis was continued until 5000 cells were recorded. The obtained data were analyzed using BD CellQuest™ Pro software.

2.6. Measurement of Free Nitric Oxide (NO)

HUVECs were seeded directly into 12-well plates and after reaching 90% confluency, they were treated with CoQ₁₀H₂ and H₂O₂ as described above. NO production was measured by a ROS-ID NO Detection kit (Enzo Life Science, NW) on a LSM 5 EXCITER laser scanning microscope (Carl Zeiss Microscopy, Jena Deutschland) according to the manufacturer's instructions and analyzed with LSM Software ZEN 2009.

2.7. Analysis of Mitochondrial Membrane Potential

The probe 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) was used to measure mitochondrial membrane potential and depolarization in HUVECs. HUVECs were placed in 12-well plates, cultured to 90% confluence, and treated or not with CoQ₁₀H₂ and H₂O₂. Then the HUVECs were stained with a JC-1 Mitochondrial Membrane Potential Detection kit according to the manufacturer's instructions (Biotium Inc., CA). Green fluorescence (JC-1 as a monomer at low membrane potentials) and red fluorescence (JC-1 as “J-aggregates” at higher membrane potentials) were monitored with a LSM 5 laser scanning microscope (Carl Zeiss Microscopy, Jena Deutschland). Mitochondrial depolarization was indicated by a decrease in the red/green fluorescence intensity ratio.

2.8. Migration Assay

A scratch (wound healing) assay was performed to evaluate HUVEC migration activity. Treated HUVECs in 12-well plates were scratched with 200 μL pipette tips. At 0 hour and 12 hours after scratching, images were taken under an inverted microscope to assess the ability of the cells to migrate into the wound area. The ratio of wound closure was calculated by analyzing images taken 0 and 12 hours after scratching with an image processing program (NIH ImageJ software, version 1.61) [32].

2.9. Cell Tube Formation Assay

Matrigel Matrix 356237 (Corning Inc., Life Sciences, MA) was coated on 15-well μ-angiogenesis slides at 10 μL/well (ibidi GmbH, Planegg, Germany). The coated slides were incubated for 15 minutes at 37°C. Treated HUVECs (10,000 cells/well) were harvested and seeded into the Matrigel-containing wells and incubated for 6 hours at 37°C to allow tube formation. The wells were then imaged for capillary-like structures using an inverted microscope (Life Technologies). Quantification of the tubes was performed by taking 4 images of each chamber, which were then analyzed by ImageJ for in vitro angiogenesis [33].

2.10. Determination of Cellular Ca²⁺

Free cytosolic Ca²⁺ levels were determined with the fluorescent probe Fluo-3 (Dōjindo Laboratories), and the effect of CoQ₁₀H₂ on H₂O₂-induced changes in Ca²⁺ levels was monitored using real-time laser scanning confocal microscopy. Cells were cultivated in 35 mm plates and treated for 24 hours with vehicle (control) or 10 μM CoQ₁₀H₂. The cells were then loaded for 30 minutes with 3 μM Fluo-3, and medium was replaced with loading buffer to assess whether the CoQ₁₀H₂ effect was due to Ca²⁺ entry or release from intracellular stores. Cells were imaged with a LSM 7 laser scanning confocal microscope (Carl Zeiss Microscopy, Jena Deutschland), and the results were analyzed with LSM Software ZEN 2010. The baseline was established within the first minute of recording after which 100 μL H₂O₂ was added to the plate. Epifluorescence images were then recorded. Results were analyzed with software accompanying the laser scanning confocal microscope. Results show the ratio of averaged profiles versus baseline for each treatment [34].

2.11. Analysis of the Concentration of CoQ₁₀H₂ and oxCoQ₁₀

After incubation, cells were washed twice in PBS, pelleted, resuspended at 3 × 10⁶ in 700 μL 2-propanol, frozen in acetone in dry ice, and stored on dry ice. For analysis, 150 μL of 2-propanol containing an internal standard of ubiquinone-9 at 100 ng/mL was added to 200 μL of the cell suspension, stirred with a vortex mixer for 30 seconds, and centrifuged for 5 minutes at 12,000 rpm. The supernatant was diluted 5-fold in 2-propanol : methanol (4 : 5, v/v), and 10 μL of the diluted solution was injected into a LC/MS/MS system.

CoQ₁₀H₂ and oxCoQ₁₀ levels in the cells were determined using a LC/MS/MS method described by Ruiz-Jimenez et al. [35] with a minor modification. Briefly, detection and quantification were performed using a QTRAP 5500 LC-MS/MS System (AB SCIEX, Framingham, MA, USA) equipped with a Turbo Ion Spray electrospray ionization (ESI) source and a Prominence UFLC system (Shimadzu, Kyoto, Japan). Chromatographic separation was performed on a YMC-UltraHT Pro C18 column, 50 mm × 2.0 mm I.D., 2.0 μm particle size (YMC, Kyoto, Japan) maintained at 30°C. The mobile phase was methanol containing 5 mM ammonium formate : 2 -propanol : ultrapure water (50 : 47 : 3, v/v/v) pumped at a rate of 0.5 mL/minute. The run time was 5 minutes per injection. Calibration curves were derived from the peak area ratios (analyte/internal standard) using weighted linear least-squares regression of the peak area ratio versus the concentration of the standards. The limits of quantification of ubiquinol-10 and ubiquinone-10 were both 1.5 ng/10⁶ cells.

2.12. Statistical Analysis

All data are presented as means ± SD. Data were analyzed using one-way ANOVA followed by Tukey's test or Student's t-test using SPSS for Windows software (version 13.0; SPSS Inc., IL). P < 0.05 was considered to be statistically significant.

3. Results

3.1. Effect of CoQ₁₀H₂ on H₂O₂-Induced SA-β-Gal Activity and Senescence-Associated Gene Expression

HUVECs were incubated in medium containing different concentrations (0–30 μM) of CoQ₁₀H₂ or oxCoQ₁₀ for 24 hours, and the expression of SIRT1 mRNA [27] was assessed by RT-PCR (Figure S1A–D). SIRT1 mRNA levels were the highest with 10 μM CoQ₁₀H₂. Next, HUVECs were incubated in medium containing 10 μM CoQ₁₀H₂ or oxCoQ₁₀ for different time periods (0–48 h). Under these conditions, expression levels of SIRT1 mRNA were the highest after 24 hours incubation. Meanwhile, mRNA expression levels of eNOS and plasminogen activator inhibitor-1 (PAI-1) in HUVECs first treated with different concentrations of H₂O₂ (0–100 μM) for 1 hour decreased and increased, respectively, in a dose-dependent manner (Figure S1E). Cell viability markedly decreased during incubation in medium containing 100 μM H₂O₂ (0–48 hours), but preincubation with 10 μM CoQ₁₀H₂ for 24 hours significantly promoted cell viability at 12 hours (Figure S1F). We used a 24-hour preincubation with 10 μM CoQ₁₀H₂ followed by a 12-hour incubation with 100 μM H₂O₂ for subsequent experiments. Notably, 10 μM is almost the same as the plasma concentration in individuals who take oral CoQ10 supplements [36]. We observed that H₂O₂ treatment of HUVECs strongly decreased cell proliferation as evidenced by a reduction in the number of H₂O₂-treated cells relative to control group cells per field of vision. This effect could be prevented by preincubation with CoQ₁₀H₂ (Figure S2).

The SA-β-Gal activity and PAI-1 mRNA expression rate were detected to examine the senescent phenotype of HUVECs (Figures 1(a) to 1(c)). After treatment with H₂O₂ for 12 hours, about 72% of the cells were SA-β-Gal-stain positive in the H₂O₂ group but only 33% of the cells were positive in the CoQ₁₀H₂ + H₂O₂ group (Figures 1(a) and 1(b)). Increases in the mRNA expression levels of PAI-1 in the H₂O₂ group were prevented in the CoQ₁₀H₂ + H₂O₂ group (Figure 1(c)). RT-PCR analysis of SASP-related gene expression (P14, p16^INK4a/CDKN2A, P53, P21, IL-1α, IL-1β, IL-6, TNF-α, MMP-1, MMP-3, and MMP-13) showed that most genes, including p14, p16 (CDKN2A), P53, P21, IL-6, TNF-α, MMP-1, and MMP-3, had increased expression after treatment with 100 μM H₂O₂, but these adverse effects of H₂O₂ could be rescued upon preincubation with CoQ₁₀H₂ (Figure 1(d)).

Figure 1.

Preincubation with CoQ₁₀H₂ prevented H₂O₂-induced senescence of HUVECs. (a) Representative images of SA-β-Gal-stained cells from each group (control, CoQ₁₀H₂, H₂O₂, and CoQ₁₀H₂ + H₂O₂). (b) Percentage of SA-β-Gal-positive cells (n = 4). (c) Expression levels of PAI-1 mRNA (n = 10). (d) Expression of genes involved in the senescence-associated secretory phenotype. Histograms show fold change in mRNA level relative to control cells (n = 6–9). ^∗ P < 0.05, ^∗∗ P < 0.01, and ^∗∗∗ P < 0.001; one-way ANOVA followed by Tukey's test.

3.2. Effect of CoQ₁₀H₂ on HG-Induced SA-β-Gal Activity and Senescence-Associated Gene Expression

SA-β-Gal activity was detected to examine the HG-induced senescent phenotype of HUVECs [37, 38] (Figures 2(a) and 2(b)). After treatment with HG for 72 hours, ~26% of the cells were SA-β-Gal-stain positive in the HG group but only 10% of the cells were positive in the CoQ₁₀H₂ + HG group. Then, senescence-associated gene expression was detected and the results showed that several genes, including PAI-1and P53, had increased expression after treatment with 30 mM glucose (HG), but the adverse effects of HG could be rescued by preincubation with CoQ₁₀H₂ (Figure 2(c)).

Figure 2.

Preincubation with CoQ₁₀H₂ prevented HG-induced senescence of HUVECs. (a) Representative images of SA-β-Gal-stained cells from each group (control, CoQ₁₀H₂, HG, and CoQ₁₀H₂ + HG). (b) Percentage of SA-β-Gal-positive cells (n = 3). (c) Expression levels of senescence-associated mRNA and histograms show fold change in mRNA level relative to control cells (n = 3). ^∗ P < 0.05, ^∗∗ P < 0.01; one-way ANOVA followed by Tukey's test.

3.3. CoQ₁₀H₂ Prevented H₂O₂-Induced Apoptosis and Necrosis

H₂O₂ was previously reported to promote endothelial tissue injury by inducing cell apoptosis and necrosis [39]. Here, we explored the effect of H₂O₂ on apoptosis and necrosis in HUVECs. Incubation of HUVECs with H₂O₂ for 12 hours increased the percentage of apoptosis-positive cells from 1.65% to 6.73%, indicating a modest but significant increase in apoptosis. Meanwhile, the percentage of apoptotic HUVECs preincubated with CoQ₁₀H₂ followed by incubation with H₂O₂ was below that for control cells (Figures 3(a) and 3(b)). Treatment with H₂O₂ increased the necrosis rate of HUVECs to 9%, but preincubation with CoQ₁₀H₂ could in part rescue H₂O₂-induced cell necrosis (Figure 3(c)). Relative to control cells, levels of mRNA for proapoptotic BAX were increased in the H₂O₂ group and decreased in the CoQ₁₀H₂ + H₂O₂ group. mRNA expression of antiapoptotic BCL-2 decreased in the H₂O₂ group, whereas the levels in the CoQ₁₀H₂ + H₂O₂ group were similar to control cells and cells incubated with CoQ₁₀H₂ alone (Figure 3(d)). The BAX/BCL-2 ratio dramatically increased in the H₂O₂ group but not in the CoQ₁₀H₂ + H₂O₂ group (Figure 3(e)). Preincubation with CoQ₁₀H₂ also inhibited an increase in levels of free cytosolic Ca²⁺ induced by H₂O₂ treatment of HUVECs (Figure S3).

Figure 3.

Preincubation with CoQ₁₀H₂ protects HUVECs from H₂O₂-induced apoptosis and necrosis. (a) Apoptotic cells were stained with annexin V-FITC and PI and evaluated by flow cytometry. Representative graphs of flow cytometric outputs for each group are shown. (b and c) Bar diagram representing apoptotic and necrotic cell populations (n = 6). (d) Analysis of BAX and BCL-2 gene expression. Histograms show fold change in mRNA level relative to control cells (n = 9). (e) Histograms show BAX/BCL-2 ratio (n = 9). ^∗ P < 0.05, ^∗∗ P < 0.01, and ^∗∗∗ P < 0.001; one-way ANOVA followed by Tukey's test.

3.4. CoQ₁₀H₂ Inhibited H₂O₂-Induced ROS Production

Next, we used flow cytometry to examine the effect of CoQ₁₀H₂ and H₂O₂ on intracellular ROS production in HUVECs (Figure 4(a)). Treatment with H₂O₂ decreased the percentage of ROS/superoxide-negative cells compared with the control group from 64.44% to 37.48%, whereas 24 hours preincubation with CoQ₁₀H₂ prevented this decrease such that the values were similar to that of control cells. Treatment with H₂O₂ induced the production of ROS, which was prevented by preincubation with CoQ₁₀H₂. However, the percentage of superoxide-positive cells and total ROS (tROS) was not affected by H₂O₂ treatment (Figures 4(b) and 4(e)), although preincubation with CoQ₁₀H₂ did increase mRNA expression of the ROS scavenger enzyme SOD2 compared with the control group (Figure 4(f)). Meanwhile, treatment with H₂O₂ resulted in significant downregulation of SOD2 mRNA expression, which was significantly relieved by preincubation with CoQ₁₀H₂.

Figure 4.

Preincubation with CoQ₁₀H₂ decreased H₂O₂-induced ROS production in HUVECS. (a) Cells were stained with two color oxidative stress detection reagents for determining ROS production and superoxide levels. Representative pictures of flow cytometry output show the cell population in the four fractions separated by ROS and superoxide (n = 6). (b, c, d, and e) Histograms show percentage of cells in negative fractionation, ROS-positive fraction, double-positive fraction, and superoxide-positive fraction. (f) Real-time RT-PCR analysis of SOD2 mRNA expression in cells. Histograms show fold change in mRNA level relative to control cells (n = 6). ^∗ P < 0.05, ^∗∗ P < 0.01, and ^∗∗∗ P < 0.001; one-way ANOVA followed by Tukey's test.

3.5. CoQ₁₀H₂ Prevented a Decrease in NO Production Induced by H₂O₂

NO production is an important feature of endothelial cell function. Although laser scanning confocal microscopy showed weaker (~44%) NO-specific red fluorescent signals in the cytosol of H₂O₂-treated HUVECs compared with the control group, preincubation with CoQ₁₀H₂ increased red fluorescent signals to similar levels as that for the control group (Figures 5(a) and 5(b)). H₂O₂ treatment also reduced levels of eNOS mRNA compared with the control group, whereas preincubation with CoQ₁₀H₂ resulted in a significant upregulation of eNOS mRNA compared with the H₂O₂-treated group (Figure 5(c)), but treatment with either CoQ₁₀H₂ or H₂O₂ did not affect the level of iNOS mRNA compared to the control cells or cells treated with CoQ₁₀H₂ alone (Figure 5(d)).

Figure 5.

Preincubation with CoQ₁₀H₂ prevents H₂O₂-induced suppression of NO production in HUVECs. (a) Representative images of staining with DAPI (blue) and a NO-specific fluorometric probe (red) acquired using a laser scanning microscope. The two images in each row were captured within the same field and then merged. (b) The intracellular NO level was calculated from the fluorescence intensity in each cell. Histograms show fold change in NO levels relative to control cells (n = 6). (c and d) Analysis of eNOS and iNOS gene expression. Histograms show fold change in mRNA level relative to control cells (n = 10). ^∗ P < 0.05 and ^∗∗∗ P < 0.001; one-way ANOVA followed by Tukey's test.

3.6. Effect of CoQ₁₀H₂ on H₂O₂-Dependent Reductions in Mitochondrial Membrane Potential

We next analyzed HUVECs in each group using the mitochondrial membrane potential-sensitive dye JC-1. In control cells and cells that were preincubated with CoQ₁₀H₂, JC-1 aggregates (red fluorescence) in the cytosol were dispersed, whereas in cells exposed to H₂O₂, green fluorescence, indicative of the monomeric form of JC-1, was more prominent (Figure 6(a)). Moreover, HUVECs pretreated with CoQ₁₀H₂ displayed a significant upregulation in the ratio of red/green fluorescence relative to the control group, whereas reductions in the ratio of red/green fluorescence seen for H₂O₂-treated cells indicated a deterioration in mitochondrial membrane polarization. Preincubation with CoQ₁₀H₂ reduced these H₂O₂-dependent decreases in mitochondrial membrane potential (Figure 6(b)). To test whether the CoQ₁₀H₂-dependent preservation of mitochondrial membrane potential was associated with upregulated expression of genes involved in mitochondrial function, we analyzed SIRT1, SIRT3, and PGC-1α (PPARGC1A) mRNA levels in each group. H₂O₂ treatment reduced SIRT1, SIRT3, and PGC-1α mRNA levels compared with the control group, whereas cells preincubated with CoQ₁₀H₂ prior to H₂O₂ treatment showed significant upregulation of SIRT1 and SIRT3 mRNA relative to cells treated with H₂O₂ alone (Figure 6(c)).

Figure 6.

Preincubation with CoQ₁₀H₂ prevents H₂O₂-induced deterioration of mitochondrial membrane potential in HUVECs. (a) Representative laser scanning microscopy images of aggregated (red) and monomeric (green) JC-1. The two images in each row were captured within the same field and then merged. (b) Mitochondrial depolarization was demonstrated by a change in JC-1 fluorescence from red to green (aggregate/monomer) (n = 6). (c) Real-time RT-PCR analysis of SIRT1, SIRT3, and PGC-1α (PPARGC1A) mRNA expression. Histograms show fold change in mRNA level relative to the control cells (n = 6–9). ^∗ P < 0.05, ^∗∗ P < 0.01, ^∗∗∗ P < 0.001; one-way ANOVA followed by Tukey's test.

3.7. CoQ₁₀H₂ Restored Migration Activity and Tube Formation Inhibited by H₂O₂ or HG Treatment

Cell migration and tube formation are involved in angiogenesis, so we next assessed the effect of CoQ₁₀H₂ treatment on impairments of these functions in HUVECs treated with 100 μM H₂O₂. H₂O₂ treatment of HUVECs strongly decreased the migration activity of these cells as evaluated by a standard cell migration assay (Figures 7(a) and 7(b)). Similarly, treatment with H₂O₂ resulted in a 30% reduction in capillary-like tube formation by HUVECs; both of these effects could be prevented by preincubation with CoQ₁₀H₂ (Figures 7(c) and 7(d)). We also observed a similar effect of CoQ₁₀H₂ treatment in HUVECs treated with 60 μM H₂O₂ (Figure S4).

Figure 7.

Preincubation with CoQ₁₀H₂ prevented H₂O₂ or HG-induced reduction in migration and tube formation by HUVECs. (a) Representative images of cell migration analysis evaluated using a wound-healing assay conducted over 12 hours in H₂O₂-induced reduction of migration. (b) Histograms show fold change in migration activity relative to control cells (n = 9). (c) Representative images from a H₂O₂-induced tube formation assay after a 6-hour incubation. (d) Histograms show fold change in total cell tube length relative to control cells (n = 6). (e) Representative images of cell migration over 12 hours evaluated using a wound-healing assay in HG-induced reduction of migration. (f) Histogram shows fold change in migration activity relative to control cells (n = 3). ^∗ P < 0.05, ^∗∗ P < 0.01, and ^∗∗∗ P < 0.001; mean ± SD, one-way ANOVA followed by Tukey's test.

We next assessed the effect of CoQ₁₀H₂ treatment on HG-induced impairment of cell migration function in HUVECs. HG treatment of HUVECs evidently decreased the migration activity of these cells as evaluated by a standard cell migration assay, and this reduction could be rescued by preincubation with CoQ₁₀H₂ (Figures 7(e) and 7(f)).

There was no protective effect of incubating cells with 10 μM CoQ₁₀H₂ for 24 hours after treatment with 100 μM H₂O₂ for 12 hours (Figures S5A and B). However, when the incubation time for 100 μM H₂O₂ was shortened to 3 hours, postincubation with CoQ₁₀H₂ did promote a significant protective effect (Figures S5C and D).

3.8. CoQ₁₀H₂ and oxCoQ₁₀ Concentrations in CoQ₁₀H₂- or H₂O₂-Treated HUVECs

Incubation of HUVECs in medium containing 10 μM CoQ₁₀H₂ led to small and dramatic increases in the intracellular levels of CoQ₁₀H₂ and oxCoQ₁₀, respectively (Figure 8(a)), and the percentage of CoQ₁₀H₂ relative to total CoQ₁₀ was increased compared with the control group (Figure 8(b)). Interestingly, the concentration of oxCoQ₁₀ was markedly increased in H₂O₂-treated cells compared with the control group. However, CoQ₁₀H₂ preincubation prevented the increase in oxCoQ₁₀ and increased the ratio of CoQ₁₀H₂ to oxCoQ₁₀ (CoQ₁₀H₂/oxCoQ₁₀) in the CoQ₁₀H₂ + H₂O₂-treated group.

Figure 8.

Intracellular CoQ₁₀H₂ and oxCoQ₁₀ levels in HUVECs changed dramatically after treatment with H₂O₂ and CoQ₁₀H₂. (a) The amount of reduced (CoQ₁₀H₂) and oxidized (oxCoQ₁₀) forms of coenzyme Q10 in whole cell extracts was determined. (b) The percentage of CoQ₁₀H₂ in total coenzyme Q10 (Total CoQ₁₀) was calculated (n = 4). ^∗∗ P < 0.01, ^∗∗∗ P < 0.001; one-way ANOVA followed by Tukey's test.

We also analyzed H₂O₂-induced changes in CoQ₁₀H₂ and oxCoQ₁₀ concentrations. HUVECs were treated with 100 μM H₂O₂ for various times (0–24 hours), and cellular CoQ₁₀H₂ and oxCoQ₁₀ levels were determined for each time point. The oxCoQ₁₀ levels in H₂O₂-treated HUVECs increased in a time-dependent manner, while the percentage of CoQ₁₀H₂ in the total coenzyme Q10 (Total CoQ₁₀) decreased (Figures S6A and B). The mRNA expressions of the CoQ₁₀ biosynthesis genes PDSS2 and COQ₂ were both markedly increased in H₂O₂-treated HUVECs after 12 hours compared with the 0-hour group (Figure S6C). The cell number and protein concentration of HUVECs per culture flask were decreased for the 12-hour group relative to the 0-hour group, but the changes were not significant (Figure S6D).

4. Discussion

The main findings of our study are that CoQ₁₀H₂ prevented functional damage of endothelial cells that accompany premature senescence caused by oxidative stress. Specifically, we examined the effect of preincubation with CoQ₁₀H₂ prior to H₂O₂ exposure on cell senescence, cell proliferation, apoptosis, and oxidative damage as well as how these compounds affected various endothelial cell functions, including eNOS production, mitochondrial activity and migration, and tube formation activities. In addition, we assessed the effect of preincubation with CoQ₁₀H₂ prior to HG exposure on cell senescence.

In addition to its bioenergetic role as a proton carrier in the mitochondrial respiratory chain, CoQ₁₀H₂ has a protective role by inhibiting lipid peroxidation in membranes and lipoproteins [19, 40]. Oxidative stress and a proinflammatory state are major characteristics of aging and many age-related disorders, including CVD [41]. During aging of humans and animals, levels of oxidative stress increase, whereas the capacity of antioxidant defense systems and levels of CoQ₁₀ decline [20, 42]. CoQ₁₀ supplementation has beneficial effects for both aging subjects and CVD patients by enhancing endothelial function [43]. At a cellular level, the antioxidant effect of CoQ₁₀ on endothelial functions is thought to be mediated through protection against mitochondrial dysfunction [44], although the details of these mitochondria-related mechanisms are unclear, and mitochondria may not be the only cellular site that is affected by CoQ10. Indeed, nonmitochondrial effects of CoQ₁₀ in endothelial cells include regulating NO production and signaling in the Golgi compartment mediated through altered eNOS activity and membrane redox status [24]. eNOS is known to be a critical regulator of cardiovascular functions through its generation of NO, which can slow the progression of endothelial cell senescence and dysfunction [26, 45]. During aging, NO production by human endothelial cells does decrease [46]. Meanwhile, mRNA expression levels of PAI-1, which is associated with atherosclerosis, and SA-β-Gal activity, a marker of senescence, were increased in senescent endothelial cells and aortas from aged mice, respectively [47]. H₂O₂ can induce premature senescence and apoptosis in endothelial cells [48, 49], and H₂O₂ acts as an oxidative stressor to modulate levels of endogenous oxidants and ROS products [50, 51].

Our previous studies revealed that dietary supplementation with the reduced form of CoQ₁₀H₂ could effectively improve mitochondrial functions, inhibit oxidative stress in the liver, and decelerate senescence in senescence-accelerated mice (SAMP1 strain) [27]. Subsequently, we also found that dietary supplementation with CoQ₁₀H₂ of KKAy mice reduced the amount of white adipose tissue and enhanced the function of brown adipose tissue by promoting lipid metabolism [30]. In these studies, we showed that CoQ₁₀H₂ decreases cytoplasmic Ca²⁺ in liver cells by enhancing activity of Ca²⁺ pumps in the endoplasmic reticulum, which in turn led to increased cAMP levels and enhanced activity of mitochondrial antioxidant defense systems via upregulated gene expression of AMPK, SIRT1, and PGC1-α signaling proteins [30]. Here, we determined the optimal concentration and times for preincubation with CoQ₁₀H₂ and incubation with H₂O₂ in HUVECs using SIRT1 expression and cell viability, respectively, as indices.

The SASP includes expression of proinflammatory cytokines, tumor necrosis factors, and matrix metalloproteinases in body tissues and is a hallmark of biological and premature aging [52]. Cellular senescence progression is thought to be regulated by the tumor suppressor pathways p14ARF/p53/p21 and p16INK4a/retinoblastoma (Rb) [53, 54]. Treatment with H₂O₂ negatively impacts cellular functions as well induces senescence and apoptosis in various cells, including endothelial cells [48, 55]. CoQ10 has been reported to decrease expression of proinflammatory cytokines in replicative senescent HUVECs and p53, p21, and p16^INK4a expression in induced premature mesenchymal stem cells [31, 56]. Our results showed that preincubation of HUVECs with CoQ₁₀H₂ protected against senescence, apoptosis, and necrosis induced by H₂O₂ and also promoted rescue from ROS overproduction, mitochondria hypofunction, and diminished NO production. Furthermore, CoQ₁₀H₂-mediated inhibition of increased intracellular Ca²⁺ levels after H₂O₂ treatment (Figure S3) is consistent with our previous results obtained with hepatocarcinoma HepG2 cells [30].

Using the fluorescent dye JC-1 [57] to analyze functional damage of mitochondria by assessing changes in mitochondrial membrane potential (MMP), we found that pretreatment with CoQ₁₀H₂ could mitigate H₂O₂-induced reductions in MMP, which must be preserved to avoid apoptosis [58]. SIRT1 is proposed to be a critical gene in aging and age-related diseases [59] that affects mitochondrial biogenesis through regulation of PGC-1α expression [60], whereas mitochondrial SIRT3 activates mitochondrial antioxidant defense systems in the presence of caloric restriction (CR) [61]. Consistent with these earlier findings, here, we showed that CoQ₁₀H₂ enhanced activity of the mitochondrial antioxidative system in HUVECs.

Angiogenesis is the physiological process wherein new capillaries grow from existing vessels. Endothelial cell functions such as migration and tube formation play important roles in the formation of these vessel sprouts [62, 63]. Our results suggested that CoQ₁₀H₂ can enhance activity of microvascular endothelial cells to promote wound repair while also impairing formation of tube-like structure induced by oxidative stress.

CoQ₁₀ is endogenously produced in all cells, and its reduced form (CoQ₁₀H₂) has important antioxidative effects [19, 40]. In this study, we detected dramatic increases in cellular CoQ₁₀H₂ concentrations following supplementation with CoQ₁₀H₂ in media for HUVEC culture, which is similar to previous findings [64]. Exposure of HUVECs to H₂O₂ resulted in a rapid increase in cellular oxCoQ₁₀ (Figure 8, Figure S6A), and this effect was prevented in part by pretreatment with CoQ₁₀H₂ (Figure 8). We analyzed the mRNA expression of PDSS2 and COQ2 and the important enzymes for CoQ₁₀ biosynthesis and found that the expression levels for both increased significantly (Figure S6C). Additional studies will be needed to fully characterize the effect of H₂O₂ on CoQ₁₀ biosynthesis, which is known to involve at least 13 genes (e.g., PDSS2, COQ1, COQ2, and COQ3) [65]. Increased amounts of total CoQ₁₀ induced by H₂O₂ treatment could indicate that oxidative damage promotes increased biosynthesis of oxCoQ₁₀ and that CoQ₁₀H₂ supplementation curbs the effect of H₂O₂. The limited cellular incorporation of CoQ₁₀ in the surviving cell population in the presence of oxidative stress induced by H₂O₂ treatment suggests that surviving cells may invoke an adaptive response mediated through upregulation of CoQ₁₀ synthesis. However, increased amounts of oxCoQ₁₀ might not translate to enhanced viability and functionality in light of the very high percentage of oxCoQ₁₀ that could disrupt the optimal reduced state of CoQ₁₀ in H₂O₂-exposed cells. Taken together, our results indicate that CoQ₁₀H₂ plays a critical role in cellular processes and CoQ₁₀ synthesis, but further studies are needed to elucidate the mechanism and biological significance of the observed CoQ₁₀ upregulation.

Finally, we selected the concentration of H₂O₂ (100 μM) based on previous reports [48, 49]. We showed that 100 μM H₂O₂ affected eNOS and PAI-1 mRNA expression in HUVECs and reduced cell viability to 20% (Figures S1E and F). To determine cell viability, we used the MTT method, which can provide information not only for cytotoxicity and cell proliferation but also for mitochondrial activation [66–68]. Thus, it is clear that the mechanism by which CoQ₁₀H₂ preincubation protects against the negative effects of H₂O₂ treatment is complex, and for a selected population of HUVECs, cellular senescence and mitochondrial function may also be involved. Additional investigations will be needed to characterize the detailed effects of CoQ₁₀H₂ on dysfunction induced by oxidative stress in endothelial cells.

In conclusion, H₂O₂ can induce HUVEC senescence through oxidative stress and increased ROS production. CoQ₁₀H₂ can markedly increase resistance to oxidative damage by enhancing mitochondrial function as well as prevent senescence and diminished function of endothelial cells treated with H₂O₂. Our results would facilitate another perspective for the investigation of the potential of CoQ₁₀H₂ to protect against age-associated exacerbation of CVD. In vivo studies will be needed to study how CoQ₁₀H₂ affects the development of endothelial dysfunction and the mechanisms by which CoQ₁₀H₂ regulates endothelial cell aging in both older individuals and CVD patients.

Conflicts of Interest

The authors declare that they have no competing interests.

Authors' Contributions

Jia Huo, Masayuki Mori, Jinko Sawashita, and Keiichi Higuchi conceived and designed the experiments. Jia Huo, Zhe Xu, Hiroki Miyahara, and Jian Dai performed the experiments. Jia Huo, Zhe Xu, Hiroki Miyahara, Kazunori Hosoe, and Jinko Sawashita analyzed the data. Kazunori Hosoe and Hiroshi Kubo contributed reagents and materials. Jia Huo and Keiichi Higuchi wrote the paper.

Supplementary Materials

Supplementary Materials

Table S1: primer sequences for real-time RT-PCR. Figure S1: H₂O₂, CoQ₁₀H₂, and oxCoQ₁₀ affect HUVEC-related gene expression and CoQ₁₀H₂ prevents H₂O₂-mediated decreases in cell viability. Figure S2: preincubation with CoQ₁₀H₂ prevented H₂O₂-induced reduction of cell proliferation in HUVECs. Figure S3: CoQ₁₀H₂ blocks H₂O₂-induced increases in free cytosolic Ca²⁺ levels in endothelial cells. Figure S4: CoQ₁₀H₂ treatment prevented H₂O₂-induced reduction in migration and tube formation. Figure S5: CoQ₁₀H₂ affects H₂O₂-dependent reduction in endothelial cell migration. Figure S6: CoQ₁₀H₂ and oxCoQ₁₀ levels in HUVECs increased after H₂O₂ treatment.