Hyperuricemia induces endothelial dysfunction and accelerates atherosclerosis by disturbing the asymmetric dimethylarginine/dimethylarginine dimethylaminotransferase 2 pathway

Figures

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  Fig. 1. Excess uric acid increases intracellular levels of ADMA by reducing DDAH-2 expression in ECs. HAECs were treated with the indicated concentrations of uric acid (UA) for 24 h. (A) Intracellular ADMA was evaluated using a commercial assay kit. (B) Western blot analysis of DDAH-1, DDAH-2, and α-tubulin. Data are expressed as means ± SEMs from five independent experiments. *Statistically significant difference (P < 0.05) relative to vehicle group.

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  Fig. 2. Excess uric acid induces endothelial dysfunction. HAECs were treated with indicated concentrations of uric acid (UA) for 24 h. (A) NO bioavailability was assessed using Griess's assay, and (B) intracellular level of cGMP was evaluated using an ELISA kit. (C and D) HAECs were treated with UA (12 mg/dL) for indicated time period (0, 3, 6, 9 12, and 18 h). Protein expression of ICAM-1, VCAM-1, and α-tubulin was examined by conducting Western blot analysis. (E and F) Cells were treated with indicated concentrations of UA for 24 h and then coincubated with BCECF-AM-labeled human monocyte THP-1 cells (1 × 105) for an additional 1 h. (E) Cellular lysates were subjected to fluorometry, and (F) cell images were photomicrographed. Data are expressed as means ± SEMs from five independent experiments. *Statistically significant difference (P < 0.05) relative to vehicle group.

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  Fig. 3. NOX/ROS pathway is crucial in the excess UA-induced deregulation of ADMA−DDAH-2 system and EC dysfunction. HAECs were treated with 12 mg/dL uric acid (UA) for indicated time period (min). (A) The changes in NADP+/NADPH ratio was examined using the NADP+/NADPH assay kit. The fold induction is defined as the ratio of NADP+/NADPH at the indicated times relative to that the time zero group set as 1. (B and C) Intracellular levels of superoxide and hydrogen peroxide were evaluated. (D–F) HAECs were pretreated with the ROS scavenger NAC (10 mM) or the NADPH oxidase inhibitor apocynin (150 μM) for 2 h and subsequently treated with 12 mg/dL UA for 24 h. (D) Intracellular ADMA was assessed using a commercial assay kit. (E) NO production was assessed using Griess's assay. (F) Monocyte–EC adhesion assay was conducted by performing fluorometry and photomicrography. Data are expressed as means ± SEMs from five independent experiments. *Statistically significant difference (P < 0.05) relative to vehicle group; #Statistically significant difference (P < 0.05) relative to UA-treated group.

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  Fig. 4. DDAH-2 overexpression prevents excess UA-induced increases in ADMA production and EC dysfunction. (A) HAECs were infected with adenovirus expressing human ddah‐2 (Ad-ddah-2, 0–20 MOI) for 24 h. Western blot analysis of protein levels of DDAH‐2 and α‐tubulin. (B–D) HAECs were infected with Ad‐vector (Ad‐null) or Ad‐ddah-2 (20 MOI) for 24 h, then treated with UA (12 mg/dL) for 24 h. (B) Intracellular ADMA was assessed using a commercial assay kit. (C) NO production was assessed using Griess's assay. (D) Monocyte–EC adhesion assay was conducted by performing fluorometry and photomicrography. Data are expressed as means ± SEMs from five independent experiments. *Statistically significant difference (P < 0.05) relative to vehicle group; #Statistically significant difference (P < 0.05) relative to UA-treated group.

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  Fig. 5. Treatment with oxonic acid deregulates ADMA/DDAH-2 pathway and induces atherosclerosis progression in apoe−/− mice. Four-month-old apoe−/− mice received daily treatment with oxonic acid (10 mg/kg body weight) or saline (vehicle control) through gastric gavage for 4 weeks. (A) Protein levels of DDAH-1, DDAH-2, and α-tubulin in aortas. (B) Body weight. (C) Plasma levels of uric acid. (D) Plasma levels of ADMA. (E) Atherosclerotic lesion at the aortic root. (F–I) Serum levels of total cholesterol, non-HDL cholesterol, HDL cholesterol, and triglycerides. Data are expressed as means ± SEMs of 10 mice. *Statistically significant difference (P < 0.05) relative to vehicle-treated apoe−/− mice.

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  Fig. 6. Treatment with oxonic acid increases level of oxidative stress and inflammatory mediators in aortas of apoe−/− mice. Levels of (A) MDA, (B) TNF-α, IL-1β, IL-6, MCP-1, and MIP-2. (C) Western blots of ICAM-1, VCAM-1, and α-tubulin in aortas. Data are expressed as means ± SEMs of 10 mice. *Statistically significant difference (P < 0.05) relative to vehicle-treated apoe−/− mice.

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  Fig. 7. DDAH‐2 overexpression in ECs prevents detrimental effect of UA on ADMA/DDAH-2 pathway and atherosclerosis in apoe−/− mice. Four-month-old male apoe−/− mice and apoe−/−/EC-ddah-2 Tg mice were treated daily with oxonic acid (10 mg/kg body weight) through gastric gavage for 4 weeks. (A) Body weight. (B) Plasma levels of uric acid. (C) Plasma levels of ADMA. (D) Protein levels of DDAH-1, DDAH-2, and α-tubulin in aortas. (E) Atherosclerotic lesion at the aortic root. (F–I) Serum levels of total cholesterol, non-HDL cholesterol, HDL cholesterol, and triglycerides. Data are expressed means ± SEMs of 10 mice. *Statistically significant difference (P < 0.05) relative to oxonic acid–treated apoe−/− mice.

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  Fig. 8. DDAH‐2 overexpression in ECs inhibits deleterious effect of UA on oxidative stress and proinflammatory response in aortas of apoe−/− mice. Levels of (A) MDA, (B) TNF-α, IL-1β, IL-6, MCP-1, and MIP-2. (C) Western blots of ICAM-1, VCAM-1, and α-tubulin in aortas. Data are expressed as means ± SEMs of 10 mice. *Statistically significant difference (P < 0.05) relative to oxonic acid-treated apoe−/− mice.