The role of hyperuricemia on vascular endothelium dysfunction

  • Authors:
    • Haitao Zhen
    • Fen Gui
  • View Affiliations

  • Published online on: August 9, 2017     https://doi.org/10.3892/br.2017.966
  • Pages:325-330
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Abstract

Hyperuricemia appears to be associated with an increased risk for cardiovascular disease and associated mortality. Population epidemiological data support a causal link between hyperuricemia and cardiovascular disease. Endothelium injury could be one of the potential mechanisms in hyperuricemia‑induced cardiovascular disease. However, the specific role of uric acid (UA) in the impairment of vascular relaxation and its signal transduction pathway has not been examined. The authors investigated the role of UA on vascular relaxation, nitric oxide (NO) production and expression of proinflammatory cytokines. Brachial flow‑mediated dilation and nitroglycerine‑mediated dilation were measured by B‑mode ultrasound with 10 megahertz linear‑array transducer from 21 patients with hyperuricemia and 16 control subjects. Human umbilical vein endothelial cells (ECs) were incubated with UA (5‑15 mg/dl) with or without nuclear factor (NF)‑κB inhibitor II. Hyperuricemia inhibited brachial flow‑mediated dilation. While UA significantly inhibited NO expression with time course‑ and dose‑ dependent manner in the cultured ECs, 10 mg/dl UA also increased expression of inflammation cytokine interleukin (IL)‑6, IL‑8 and tumor necrosis factor‑α in vitro. These abnormalities were associated with UA‑induced activation of transcription factor NF‑κB. Furthermore, NF‑κB inhibitor II prevented UA-induced reduction of NO and increased inflammation cytokines. These data suggested hyperuricemia‑induced endothelium injury and vascular dysfunction by a reduction of NO and expression of inflammatory cytokines through the NF-κB pathway.

Introduction

Hyperuricemia has been linked to cardiovascular disease (CVD) recent since it is regarded as an independent risk factors in the contribution of CVD including vascular disease, hypertension, metabolic syndrome and renal disease (1). Generally, uric acid (UA) levels >7 mg/dl in males and >6 mg/dl in females are considered as hyperuricemia in a clinical aspect (2). Risk factors for hyperuricemia include alcohol consumption, intake of high fat diet and refined carbohydrate, and the medicines of diuretics and angiotensin converting enzyme antagonists (1,2). Population epidemiological studies support the notion that there is a causal link between hyperuricemia and CVD. For example, Rotterdam (3) and a NHANES I study (4) found that there is an association between high levels of UA and myocardial infarctions and cardiovascular death even after adjustment for related factors such as age, dyslipidemia and obesity. Multiple center studies also suggested that hyperuricemia prompts hypertension and coronary heart disease, as well as vascular diseases such as cerebrovascular disease, preeclampsia, vascular dementia and kidney disease (5,6). Some of the potential pathophysiological mechanisms have been investigated to explain the association between UA and vascular injury. One study suggested that high dose UA significantly increased angiotensin II and oxidative stress in cultured endothelial cells (ECs) and this further prompted EC senescence and apoptosis (7). To this point, increase inflammation and reduction of nitric oxide (NO) are the characteristics of endothelium dysfunction (8). However, few studies have been conducted to investigate the role of UA on vascular relaxation, NO production, inflammation and its signaling pathway on ECs. In the current study, the authors hypothesized that UA-induced impairment of vascular relaxation is associated with a reduction of NO and expression of inflammatory cytokines through the nuclear factor (NF)-κB pathway.

Materials and methods

Study population

The study investigated 21 patients with hyperuricemia and 16 control subjects at The First Clinical Hospital of Hubei University of Science and Technology and Xianning Central Hospital in China, between February 2014 and March 2016. Blood samples were collected at 8:00 a.m. while subjects were regularly fasting. The study was approved by the institutional review board on human investigation at The First Clinical Hospital of Hubei University of Science and Technology and Xianning Central Hospital (Xianning, China). Informed consent was obtained from all individuals in the study.

Brachial flow-mediated dilation (FMD) and nitroglycerine-mediated dilation (NMD)

The diameter of the right brachial arteries in all subjects were measured on B-mode ultrasound with 10 megahertz linear-array transducer (Beijing ADSS Development Co., Ltd., Beijing, China) by one investigator at rest and sublingual spray nitroglycerin (350 µg), respectively. A pressure cuff was placed and automatically inflated on the forearm in order to initial forearm ischemia, the pressure cuff was then released after 5 min so that blood flow in the forearm could be increased. The maximum diameters were recorded after cuff release (9,10). The percentage increase in FMD and NMD were calculated as the increased diameters in brachial arteries compared with the corresponding baseline at rest (9,10).

EC culture

Primary human umbilical vein ECs were purchased from (cat. no. 211–500, Shanghai Biotechnology Co., Ltd, Shanghai, China) and cultured at cell incubator with 37°C in 5% CO2 atmosphere. The sub-cultured strains of ECs were used in experiments between passages 3–5. Prior to the experiments, ECs were serum-starved in Dulbecco's modified Eagle's medium for 24 h. UA and NF-κB inhibitor II were purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany).

Measurement of NO production

Nitrite, a stable metabolite of NO, is converted from nitrate by nitrate reductase and is an indicator of released NO in the media of cultured ECs (11). The NO productions in cultured ECs were measured using a nitrate/nitrite assay kit (cat. no. 23479; Sigma-Aldrich; Merck KGaA) following the protocol instructions (12).

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

The total RNA was isolated from the cultured human umbilical vein ECs using RNAzol (Sigma-Aldrich; Merck KGaA). After the first strand, cDNA was synthesized by using Improm II reverse transcription kit (Sigma-Aldrich; Merck KGaA), qPCR was done using 8 µl cDNA, 10 μl SYBR-Green PCR Master Mix and forward and reverse primers (10 pM/µl) using a qPCR system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The following primer sequences were used: interleukin (IL)-6 forward, 5′-ACAGTGTGGAATCATATATGA-3′ and reverse, 5′-GCGTCGTTATATGTTGAACGC-3′; IL-8 forward, 5′-CGTGGTTATGCTACAAATGT-3′ and reverse, 5′-ACGGGCAACCCGTGGAGAGC-3′; Tumor necrosis factor (TNF)-α forward, 5′-GCAGCCGTTAACGTAAACCT-3′ and reverse, 5′-TGTGTTAGGGCCTGTGGCAC-3′; 18S forward, 5′-GACGGTTGCCATCAAGTTCGA-3′ and reverse, 5′-TGCTGAGCATCGCTGGGAA-3′. The PCR cycling conditions were 2 min at 95°C for initial denaturation, 40 cycles of 45 sec at 95°C, 30 sec at 58°C and 45 sec at 72°C. The data were normalized to 18S ribosomal RNA that is a housekeeping gene.

Western blot analysis

Human umbilical vein ECs were collected and lysed with RIPA protein lysis buffer (cat. no. 20–188, Sigma-Aldrich; Merck KGaA) and the protein concentration was investigated by Bio Rad protein assay (Bio Rad Laboratories, Inc.). Proteins were resolved by 10% SDS-PAGE and transferred to nitrocellulose membranes (Sigma-Aldrich; Merck KGaA). Membranes were incubated with antibody targeting NF-κB (cat. no. SAB4502609; 1:1,000 dilution; Sigma-Aldrich; Merck KGaA) and horseradish peroxidase (HRP) conjugated secondary antibody (cat. no. A9542; 1:5000 dilution; Sigma-Aldrich; Merck KGaA) at room temperature for 1 hour. The HRP activity was examined by an enhanced chemiluminescence kit (Bio Rad Laboratories, Inc.). As a loading control, nuclear protein lamin B was probed in all the protein expression.

Activation of transcription factor NF-κB

Human umbilical vein ECs were transfected with 5 µg of pNF-κB-Luc (Clontech Laboratories, Inc., Mountainview, CA, USA). Transfection of human umbilical vein ECs was performed using the nucleofection device and solutions to deliver electrical stimuli to the ECs (Lonza Group, Basel, Switzerland) (13). Following 24 h transfection, 10 mg/dl UA was used to treat the cells for 3 h and luciferase activity was examined by using a Dual Luciferase Reporter Assay System and a luminometer (Promega Corporation, Madison, WI, USA) as per the manufacturer's protocol (14).

Statistical analysis

Results are calculated as the mean ± standard error of the mean. Data were analyzed by using SAS software (version, 9.4; SAS Institute Inc., Cary, NC, USA). Differences in data were determined using one-way analysis of variance or paired Student's t tests and P<0.05 was considered to indicate a statistically significant difference.

Results

Clinical characteristics of the subjects

There was no significant difference in age, sex, body mass index, blood pressure, plasma levels of creatinine, glucose, triglyceride and low/high-density lipoprotein between health control subjects and hyperuricemia patients (Table I). However, the plasma UA level is significantly higher in hyperuricemia patients than in control group (Table I).

Table I.

Clinical characteristic of the study population.

Table I.

Clinical characteristic of the study population.

VariableControl subjects (n=16)Hyperuricemia patients (n=21)
Age (years)65.2±7.2868.5±6.6
Males in population (%)50%47.6%
Body mass index (kg/m2)25.1±2.225.6.±1.7
Systolic blood pressure (mmHg)140±6142±10
Diastolic blood pressure (mmHg)85±486±4
Creatinine (mg/dl)0.78±0.110.77±0.14
Plasma uric acid (mg/dl)4.51±1.09 16.78±4.69
Plasma glucose (mg/dl)98.65±11.35100.47±9.69
Triglyceride (mg/dl)99.46±37.33128.44±51.22
Low-density lipoprotein121.25±27.98145.66±43.21
High-density lipoprotein (mg/dl)53.15±6.2362.22±8.09

{ label (or @symbol) needed for fn[@id='tfn1-br-0-0-966'] } Values are mean ± standard error of the mean.

† P<0.05 vs. control subjects.

Hyperuricemia induces arterial endothelium dysfunction

FMD and NMD are applied to access the endothelium-dependent and -independent arterial dilation, respectively and thus arterial endothelial function can be evaluated using FMD and NMD (9,10). In current study, the authors explored arterial endothelial function using FMD in hyperuricemia patients and control subjects to disclose whether an increase in serum UA is associated with arterial endothelial dysfunction. Hyperuricemia inhibited brachial FMD but not NMD (Figs. 1A and B). Furthermore, there was a positive correlation between hyperuricemia and impairment of FMD (R2=0.8907; Fig. 1C), suggesting that hyperuricemia impairs endothelium-dependent arterial dilation.

UA reduced NO production in cultured human umbilical vein ECs

The authors further investigated the effect of high dose of UA on NO production in cultured human umbilical vein ECs with various concentrations of UA treatments and different time points. As presented in Fig. 2, 5 mg/dl UA (normal dose) did not alter NO production. However, 10 and 15 mg/dl UA (high dose) significantly inhibited NO expression from 12–48 h. Compared to the 5 mg/dl group, both 10 and 15 mg/dl UA also significantly inhibited NO expression at 24 and 48 h (Fig. 2).

UA increased inflammation cytokine expression in cultured human umbilical vein ECs

To further confirm the roles of UA on the expression of inflammation cytokines, human umbilical vein ECs were incubated with 10 mg/dl UA for 24 h. UA significantly induced IL-6, IL-8, TNF-α expression (Fig. 3), suggesting that UA induces endothelium inflammation.

UA increased activation of transcription factor NF-κB in cultured human umbilical vein ECs

In order to investigate the role of UA on the activation of transcription factor NF-κB, human umbilical vein ECs were co-transfected with pNF-κB-Luc and 10 mg/dl UA. UA significantly induced activation of transcription factor NF-κB luciferase activity (Fig. 4A). Meanwhile, UA also induced NF-κB nuclear translocation at 5, 15 and 30 min in ECs (Fig. 4B), suggesting that UA increased transcription factor NF-κB activity.

NF-κB antagonist prevented UA-induced reduction of NO and increased inflammation cytokines

To determine the role of NF-κB in UA-induced EC dysfunction, human umbilical vein ECs were treated by 10 mg/dl UA with or without NF-κB inhibitor II (20 nmol/l) for 24 h. NF-κB inhibitor II prevented UA-induced reduction of NO (Fig. 5A). In addition, NF-κB inhibitor II prevented UA-induced increases in expression of IL6, IL8, and TNF-α in human umbilical vein ECs.

Discussion

Hyperuricemia is an independent risk factor in the pathogenesis of vascular disease, hypertension and metabolic syndrome (15). The current study further found that hyperuricemia inhibited endothelium-dependent arterial dilation and brachial FMD. While UA significantly inhibited NO expression with time course and dose dependent manner in the cultured HUVECs, high dose UA also increased expression of inflammation cytokine IL-6, IL-8 and TNF-α in vitro. These abnormalities were associated with UA-induced activation of transcription factor NF-κB. However, NF-κB inhibitor II, an antagonist of NF-κB, prevented UA-induced reduction of NO and increased inflammation cytokines. Thereby, these data implicated that activated NF-κB plays an important role in UA-induced endothelial inflammation and injury.

Hyperuricemia is a common clinical finding in patients with obesity, dyslipidemia, hypertension and other CVDs (16). Epidemiological studies have confirmed the association of hyperuricemia and CVD. Recent data from cross-sectional, interventional, and cohort studies have found that hyperuricemia is an independent risk factor for hypertension (17,18). To this point, endothelium injury and dysfunction is regarded as the first step to develop the hypertension, coronary heart disease and other CVDs (19). Thus, high dose UA has ability to initial the endothelium injury to development of CVD by increases in platelet aggregation and pro-inflammatory activity and inhibition of EC NO production (20). In the present study, FMD is used for assessing the vascular NO response and endothelial function in patients with hyperuricemia that have similar clinical characteristics with control individuals in age, body mass index, blood pressure, plasma levels of creatinine, glucose, triglyceride and low/high-density lipoprotein. Interestingly, hyperuricemia inhibited brachial FMD but not NMD and there is a positive correlation between hyperuricemia and impairment of FMD. Therefore, hyperuricemia induces the impairment of endothelium-dependent arterial dilation.

Inhibition of UA through targeting of xanthine oxidase has been found to decrease vascular dysfunction and improve cardiovascular function recent (21). The current study further found that high dose UA directly impaired NO generation in cultured human umbilical vein ECs, suggesting that hyperuricemia impairs vascular function by a mechanism of inhibition of NO production. In this regard, high dose may increase the activity of nicotinamide adenine dinucleotide phosphate oxidase that prompts oxidative stress and decreases the bioavailability of NO (22). Further, hyperuricemia impairs vascular function by increasing inflammation cytokine expression in IL-6, IL-8 and TNF-α. Therefore, UA-induced inflammatory immune response is as an important mechanism in UA's vascular effects.

The nuclear factor transcription factor NF-κB pathway is regarded as a proinflammatory signaling pathway since activated NF-κB mediates the expression of proinflammatory cytokines, chemokines, and adhesion molecules (23,24). In the current study, UA increased NF-κB activity and its nuclear protein translocation that further prompted the maladaptive immune response. Inhibition of NF-κB reduced the UA-induced inflammation cytokine expression of IL-6, IL-8 and TNF-α. Interestingly, inhibition of NF-κB also inhibited UA-induced reduction of NO. Indeed, NF-κB is a negative regulator of endothelial NO synthase expression via upregulation of microRNA155 under inflammatory conditions (25). Sustained activation of NF-κB decreases NO bioavailability in response to shear and endothelial inflammation (26). Therefore, activated NF-κB mediates UA-induced reduction of NO and increased inflammation response.

In conclusion, these data reveal that the NF-κB pathway mediates hyperuricemia-induced endothelium impairment and vascular dysfunction by a reduction of NO and expression of inflammatory cytokines. The link between activated NF-κB, reduction of NO, maladaptive immune responses and vascular dysfunction offers possibilities for identification of UA-induced vascular dysfunction and allows for development of novel therapeutic strategies for protection of cardiovascular disease.

Acknowledgements

The present research was supported by Hubei University of Science and Technology (grant. no. HK201608 to H.Z.).

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APA
Zhen, H., & Zhen, H. (2017). The role of hyperuricemia on vascular endothelium dysfunction. Biomedical Reports, 7, 325-330. https://doi.org/10.3892/br.2017.966
MLA
Zhen, H., Gui, F."The role of hyperuricemia on vascular endothelium dysfunction". Biomedical Reports 7.4 (2017): 325-330.
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Zhen, H., Gui, F."The role of hyperuricemia on vascular endothelium dysfunction". Biomedical Reports 7, no. 4 (2017): 325-330. https://doi.org/10.3892/br.2017.966