UA (>99% pure, without endotoxin), polyethylene glycol-superoxide dismutase (PEG-SOD), 4-phenylbutyric acid (4-PBA), and polymyxin B were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fluo-3, acetoxymethyl (AM) was purchased from Molecular Probes, Inc. (Eugene, OR, USA). Antibodies against p-eNOS-Thr⁴⁹⁵ (ab138430), p-eNOS-Ser¹¹⁷⁷ (ab195944), eNOS (ab66127), caspase-12 (ab62484), CHOP (ab11419), and ATF-6 (ab11909) were obtained from Abcam (Cambridge, UK). Primary monoclonal antibody against human CaM (05-173) was from Millipore Corp. (Billerica, MA, USA). Primary polyclonal antibodies against PKC (#2056) and p-PKC (#9375) and HRP-linked secondary antibodies against rabbit IgG (#7074), mouse IgG (#7076) were purchased from Cell Signaling Technology, Inc. (Boston, MA, USA). The Annexin V-FITC Apoptosis Detection kit was from BD Pharmingen (San Diego, CA, USA), and the Nitric Oxide Synthase Assay kit was from Beyotime Biotech (Jiangsu, China). All chemicals were of analytical grade.

Primary HUVECs were purchased from PromoCell (Heidelberg, Germany) and cultured at 37°C in 5% CO₂ in Endothelial Cell Growth Medium (Cat. no. C-22010; PromoCell), which contains 2% fetal bovine serum, 5 ng/ml epidermal growth factor (EGF), 10 ng/ml basic fibroblast growth factor (bFGF), 20 ng/ml Long R3 IGF-1, and 0.5 ng/ml vascular endothelial growth factor (VEGF). Cells at passage 3–7 were used for experiments.

The UA solution was prepared as follows: 0.4 g of NaOH was dissolved in 10 ml ultrapure water to obtain 1 mM NaOH solution. Subsequently, 150 mg UA was added, fully dissolved in a warm water bath under agitation. The final UA concentration was 15 mg/ml. The solution was filtered using a 0.22-µm filter under a sterile hood for eventual removal of microorganisms. The solution was stored at room temperature. Prior to the experiment, the stock solution was used to obtained culture media at 6, 9 and 12 mg/dl.

UA was added to HUVEC cultures 24 h after passage, at final concentrations of 6, 9 or 12 mg/dl. Where indicated, 30 min prior to the addition of UA, the following inhibitors were added at the indicated final concentrations: 100 U/ml of the PEG-SOD antioxidant, 10 mM of the ER stress inhibitor 4-PBA, or 20 µg/ml of the PKC inhibitor polymyxin B. Untreated HUVECs were used as the control.

Trypsin-dispersed HUVECs (5×10⁴/sample) were resuspended in 200 µl Annexin V-FITC and 10 µl propidium iodide and subsequently incubated at room temperature (20–25°C) in the dark for 20 min. Staining was then analyzed using a Becton-Dickinson flow cytometer (Becton-Dickinson, San Diego, CA, USA).

Cell culture supernatant NO content was measured using a Total Nitric Oxide Assay kit (nitrate/nitrite assay kit; Beyotime Biotech) according to the manufacturer's instructions. Total NO production was estimated according to the accumulation of nitrite and nitrate in the cell culture supernatant using Griess reagent (Beyotime Biotech). Nitrate was converted enzymatically into nitrite by nitrate reductase. Nitrite is a stable metabolite of NO, and thus is an indicator of released NO in the media, as previously described (30).

eNOS activity was measured using a Nitric Oxide Synthase Assay kit (Beyotime Biotech) according to the manufacturer's instructions. As previously described (31), HUVECs were seeded in a 96-well plate. Cells were resuspended in 100 µl NOS detection buffer and 100 µl reaction buffer containing NOS substrates (5 µl 0.1 mM NADPH and L-arginine, respectively) and a cell-permeable fluorescent substance (5 µl 3-amino 14-aminomethyl-2′,7′-difluorescein diacetate) and incubated at 37°C in the dark for 40 min. Fluorescence intensity (FI) was measured at Ex 495 nm/Em 515 nm on a microplate reader (Molecular Devices, LLC, Sunnyvale, CA, USA). The relative activity (RA) of eNOS was calculated as: RA = FI ( sample ) − FI ( negetive ) FI ( control ) − FI ( negetive )

The intracellular ROS levels were measured using CM-H2DCFDA (Sigma, Carlsbad, CA, USA). HUVECs were seeded on 35-mm confocal culture dishes at 4×10⁴ cells/ml in 2 ml media per dish 24 h before UA stimulation. After stimulation, culture media was replaced with dichloro-dihydro-fluorescein diacetate (DCFH-DA), diluted in serum-free media to a final concentration of 10 µM, and cells were incubated at 37°C in the dark. After 30 min, the cells were washed with serum-free media three times and FI was measured using a Nikon A1 confocal microscope (purchased from Nikon Instruments, Inc., Tokyo, Japan) at Ex 488 nm/Em 515 nm.

The intracellular level of Ca²⁺ was measured using the Ca²⁺-specific fluorescent probe Fluo-3, AM, as previously described (26). HUVECs were cultured in the presence or absence of 12 mg/dl of UA for 6, 12 or 24 h, then incubated with 50 µM Fluo-3, AM at 37°C in the dark. After 1 h, cells were washed three times in Hanks' solution to remove unloaded probes. The FI of Fluo-3, AM was measured at Ex 488 nm/Em 525 nm using a Nikon A1 confocal microscope (Nikon Instruments, Inc.).

HUVECs were harvested with cell lysis buffer containing protease inhibitors (#5871; Cell Signaling Technology, Inc., Danvers, MA, USA) on ice for 30 min. The supernatant protein lysates were collected after 30 min of centrifugation at 1,200 × g. Lysate (500 µg) was incubated overnight with 3 µg eNOS-directed antibody or preimmune IgG (Cell Sciences, Canton, MA, USA) at 4°C. Labeled lysates were incubated with 25 µl of protein A agarose beads (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) at 4°C. After 3 h, protein A-agarose beads were washed three times with lysis buffer and resuspended in 15 µl of 2X SDS loading buffer, boiled for 5 min, and analyzed by SDS-PAGE and western blot analysis.

HUVECs were lysed in RIPA buffer with phosphatase inhibitor cocktail (100X) (#5870; Cell Signaling Technology, Inc., Danvers, MA, USA) and stored at −70°C after determining the protein concentration using the BCA reagent (Beyotime Biotech). As described previously (32), proteins were separated on SDS-PAGE with a 5% stacking gel and a 12% resolving gel. After separation, proteins were electrophoretically transferred onto PVDF membranes (Millipore Corp.), and incubated in blocking buffer (Beyotime Biotech) for 2 h at room temperature. Blots were probed with primary antibodies (against p-eNOS-Thr495, p-eNOS-Ser1177, eNOS, caspase-12, CHOP, ATF-6, CaM, PKC, p-PKC and β-actin) overnight at 4°C, then corresponding HRP-labeled secondary antibodies (against rabbit IgG or mouse IgG), and developed using enhanced chemiluminescence reagents (Millipore Corp.). Band intensity was analyzed using the Touch Gel Dock system (UVP LLC, Upland, CA, USA). β-actin was used as the internal reference; the relative expression of protein was represented as the ratio of the target protein to β-actin (target protein/β-actin).

All data in the present study are presented as the means ± standard deviation (SD). Statistical analysis was performed using SPSS. Group characteristics were compared by one-way ANOVA, followed by the Student-Newman-Keuls (SNK-q) post hoc test. A P-value <0.05 was considered to indicate a statistically significant difference.

As shown in Fig. 1A, in the present study we examined the effect of UA on vascular endothelial cellular function and NO production by treating in vitro cultured HUVECs with various concentrations of UA for different durations of time. We found that the rate of apoptosis was increased in HUVECs incubated with UA in a time- and concentration-dependent manner. Significantly increased HUVEC apoptosis was detected within 48 h in HUVECs incubated with 6 mg/dl of UA, within 24 h in HUVECs incubated with 9 mg/dl of UA, and within 12 h in HUVECs incubated with 12 mg/dl of UA (all P<0.05) (Fig. 1A).

Incubation of HUVECs with UA reduced NO production in a time- and concentration-dependent manner (Fig. 1B) and reduced eNOS activity (Fig. 1E and F), whereas even 12 mg/dl UA barely affected the protein expression of eNOS (Fig. 1C and D).

Oxidative stress is known to play an important role in UA-induced endothelial dysfunction (12,26). We measured intracellular ROS levels in order to estimate oxidative stress using a specific probe, CM-H2DCFDA, in primary HUVEC cultures. Intracellular ROS levels were significantly increased 3 h after incubation with 12 mg/dl of UA, and remained significantly elevated for 24 h (P<0.05) (Fig. 2A and C). Pretreatment of HUVECs with the cell-permeable ROS scavenger PEG-SOD (26) for 30 min significantly ameliorated the UA-induced increase in intracellular ROS accumulation (Fig. 2B and D).

UA has previously been reported to regulate cell function through the ER stress-signaling network in glomerular mesangial cells and hepatocytes, and oxidative stress is one of the key drivers of ER stress (20,21). To investigate the role of ER stress in UA-induced endothelial dysfunction, we measured the cellular content of the ER stress biomarkers ATF-6 and CHOP, and the ER stress-induced apoptotic marker caspase-12, by western blot analysis. UA (12 mg/dl) increased the levels of ER stress markers in HUVECs in a time-dependent manner (Fig. 3A). The cellular content of ATF-6 and CHOP was significantly increased after 6 h of incubation with UA, rose again at 12 h, and remained elevated for 24 h; for CHOP, expression peaked at 12 h and for ATF-6 at 24 h. Cellular levels of caspase-12 were significantly increased at 12 h and peaked at 24 h. HUVECs were pre-treated with the antioxidant PEG-SOD or ER stress inhibitor 4-PBA in order to explore the association between UA-induced oxidative stress and ER stress. Both PEG-SOD and 4-PBA effectively inhibited UA-induced ATF-6, CHOP, and caspase-12 upregulation (Fig. 3B). UA-induced oxidative stress was observed after 3 h, and ER stress was observed after 6 h. These results suggest that UA stimulation induces oxidative stress, which triggers ER stress in HUVECs.

To further confirm the roles of oxidative stress and ER stress in UA-induced apoptosis and endothelial dysfunction, we assessed the rate of apoptosis and NO production in HUVECs pre-treated with PEG-SOD and 4-PBA. While HUVECs incubated with 12 mg/dl UA for 48 h exhibited increased apoptosis and reduced NO production (P<0.05), in cells pre-incubated with PEG-SOD or 4-PBA this effect was not observed (Fig. 4), suggesting that UA induces endothelial dysfunction by triggering oxidative stress and ER stress.

Ca²⁺, CaM, eNOS, and eNOS phosphorylation state are known to act as key regulators of eNOS activity (22,33) and were thus examined in HUVECs in the present study. HUVECs incubated with 12 mg/dl UA for 6, 12 or 24 h contained higher levels of eNOS and CaM. Although the intracellular Ca²⁺ concentration, the rate of eNOS phosphorylation at Ser¹¹⁷⁷, and CaM expression were almost unaltered (Fig. 5A–C), eNOS phosphorylation at Thr⁴⁹⁵ was increased in a time-dependent manner between 6 and 24 h (P<0.05) (Fig. 5A). Binding of eNOS and CaM was assessed by co-immunoprecipitation, and we noted that UA also reduced eNOS and CaM binding in a time-dependent manner (P<0.05) (Fig. 5D), which was negatively correlated with increased phosphorylation of eNOS at Thr⁴⁹⁵. These findings indicated that UA inhibits eNOS activity through phosphorylation of eNOS at Thr⁴⁹⁵ and prohibits its interaction with CaM.

PKC activates eNOS enzymatic activity by phosphorylating eNOS at Thr⁴⁹⁵ (28). To further investigate the mechanism by which UA modulates eNOS activity, PKC expression and phosphorylation, HUVECs were incubated with the antioxidant PEG-SOD, the ER stress inhibitor 4-PBA, and the PKC inhibitor polymyxin B prior to incubation with UA. The PKC expression level was almost unaltered by incubation with UA, whereas the level of p-PKC was elevated after 6 h and peaked at 12 h (P<0.05) (Fig. 6A). Moreover, PEG-SOD, 4-PBA, or polymyxin B pretreatment inhibited UA-induced PKC phosphorylation (Fig. 6B–D). These results suggest that UA triggers ER stress and PKC phosphorylation by inducing ROS generation. Active PKC increases phosphorylation of eNOS at Thr⁴⁹⁵, and reduces the interaction between eNOS and CaM, thus inhibiting eNOS activity and NO production.