The Roles And Mechanism Of ER Stress In High Uric Acid Induced Endothelial Injury

BackgroundUric acid (UA) is the end product of purine metabolism in humans and higher primates. During primate evolution, mutations in the hepatic enzyme uricase prevent efficient metabolism of uric acid into allantoin, and as a result, humans tend to have higher blood uric acid levels than other mammals. Previous studies suggest that uric acid is an antioxidant and has protective effects on the heart, blood vessels and nerve cells. However, in recent years, further in-depth study found that high concentrations of uric acid not only lost the protective effect as an antioxidant, but also lead to vascular endothelial cell injury.Furthermore, hyperuricemia has recently been reported to be an independent risk factor for cardiovascular disease(CVD), such as acute myocardial infarction(AMI), heart failure, hypertension and so on.Allopurinol lowers uric acid and improves endothelial function, insulin resistance and systematic inflammation in hyperuricemia. However, mechanisms by which hyperuricemia contributes to cardiovascular dysfunction are not fully understood.Endothelial dysfunction, is characterized by decreased NO levels and cellular activity, and contributes to the pathogenesis of various CVDs.Studies have shown that increased uric acid can induce apoptosis of many kinds of cells, such as renal tubular epithelial cells, islet cells, liver cells and endothelial cells, but in different cells, the uric acid concentration causing cell apoptosis are not the same.American Rheumatism Association recommends:target serum level of UA is 6mg/dl; in patients who combined with other risk factors or severe gout, target serum UA level should be less than 6mg/dl. However, in our clinical work, there is a serious shortage of attention to the early intervention of asymptomatic hyperuricemia. One reason of this phenomenon is that, hyperuricemia is often asymptomatic, mostly be found in health examination, or in patients with gout arthritis or gout nephropathy. The other reason is that, uric acid with low concentration has no cell injury effect, and it may has a protective effect; uric acid with high concentration can lead to endothelial cell injury, however, the critical level of uric acid concentration which began to induce endothelial damage is inconclusive. Therefore, in this study we used primary cultured human umbilical vein endothelial cells to establish uric acid injury model in vitro, observed the effect of uric acid with different concentrations on endothelial cell proliferation, apoptosis, and NO generating capacity, provide data support for early clinical intervention of hyperuricemia.Purpose1.To establish the uric acid induced cell injury model in primary cultured human umbilical vein endothelial cells in vitro.2.To verify the influence of uric acid at different concentrations on cell activity of vascular endothelial cells, and to screen out the lowest concentration of uric acid which can lead to endothelial cell injury.Methods1.The primary culture of HUVECsPrimary HUVECs were purchased from PromoCell (Heidelberg, Germany) and cultured at 37 ? in 5% CO2 in Endothelial Cell Growth Medium. Cells at passage 3-5 were used for experiments.2. Identification of HUVECsThe expression of surface markers Von Willebrand factor (vWF), CD31 and Smooth muscle alpha-actin (alpha SMA) were detected by immunofluorescence staining and flow cytometry to identify the primary cultured cells.3.Proliferations were detected with MTT methodHUVECs were incubated with 4,6,9,12 mg/dl uric acid for 12h,24h or 48h, then cell proliferations were detected by MTT method.4.Apoptosis analysisHUVECs were incubated with 4,6,9,12 mg/dl uric acid for 12h,24h or 48h, then cell apoptosis were detected using Annexin V-FITC detecting kit.5. Measurement of NO releaseHUVECs were incubated with 4,6,9,12 mg/dl uric acid for 12h,24h or 48h, then cell culture supernatant NO content was measured using nitrate reductase assay.6.Statistical analysis.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 difference, A P-value <0.01 was considered to indicate a statistically significant difference.Results1. The primary culture and identify of HUVECsPrimary cultured HUVECs is irregular, flat, oval or polygon, cell monolayer when fusion is typical "aura" appearance, immunofluorescence staining and flow cytometry testing results show that the expression of vWF and CD31 are positive, the expression of alpha SMA is negative, conform to the endothelial cell characteristics.2.The effect of uric acid on cell proliferationMTT testing results show that, compared with control group, no obvious cell proliferation changes were discovered in cells stimulated with 4mg/dl and 6mg/dl uric acid, cell proliferation were decreased in cells stimulated with 9mg/dl uric acid at 48h,12 mg/dl uric acid at 24h and 48h.3. The effect of uric acid on cell apoptosisApoptosis analysis results show that, compared with control group, there were no significant difference in cell apoptosis in cells stimulated with 4mg/dl uric acid, cell apoptosis were increased in cells stimulated with 6mg/dl,9mg/dl or 12mg/dl uric acid in a dose-and time-dependent manner.4. The effect of uric acid on cell NO productionNO detecting results show that, compared with control group, there were no significant difference in NO production in cells stimulated with 4mg/dl uric acid, NO productions were decreased in cells stimulated with 6mg/dl,9mg/dl or 12mg/dl uric acid in a dose-and time-dependent manner.Conclusion1. In primary cultured HUVECs,4mg/dl uric acid had no obvious effect on cell apoptosis and proliferation.2. When the concentration is higher than 6mg/d1, uric acid increased cell apoptosis, reduced cell proliferation in a dose- and time-dependent manner.3. When the concentration is higher than 6mg/dl, uric acid decreased NO production in a dose- and time-dependent manner.BackgroundEndothelial dysfunction, is characterized by decreased NO levels and cellular activity, and contributes to the pathogenesis of various CVDs. Hyperuricemia has been reported to induce endothelial dysfunction in several studies. In endothelial cells, high concentration of uric acid have been reported to increase ROS level, and decrease eNOS activity and NO production in a time- and dose-dependent manner. However, the molecular mechanism by which uric acid influences eNOS activity reduction remains elusive. In the hyperuricemia rat model, vascular endothelial cells edema and shed off from the blood vessel wall, the cell gap is enlarged, the inflammatory cell aggregated and located in the vessel wall.Oxidative stress and other stress factors trigger ER stress, via a highly evolutionally conserved cellular response. ER stress activates a complex signaling network referred to as the Unfolded Protein Response (UPR)to reduce ER stress and restore homeostasis. However, sustained and unresolvable ER stress promotes apoptosis, elevates CCAAT-enhancer-binding protein homologous protein (CHOP) and Activating transcription factor 6 (ATF-6) expressions, inducing endothelial dysfunction. ER stress also activates PKC and AKT signaling pathways to mediate downstream effects. Uric acid was reported to induce ER stress in glomerular mesangial cells and hepatocytes. Meanwhile in endothelial cells, physiological and pathological stimulation such as homocysteine thiolactone, urea, and Ox-LDL cause endothelial dysfunction by triggering ER stress. Thus, we hypothesize that uric acid may activate the ER stress signaling network to induce endothelial dysfunction and inflammation. In in vitro primary cultured endothelial cells, we investigated the potential roles of reactive oxygen species (ROS) generation; endoplasmic reticulum (ER) stress and the PKC-dependent endothelial nitric oxide synthase (eNOS) signaling pathway.Purpose1.This study was to observe the affection of uric acid stimulation on cell activity and function in primary cultured endothelial cells in vitro.2.To explore the role and mechanism of oxidative stress, endoplasmic reticulum stress and related signaling pathways in endothelial cell injury induced by uric acid.Methods1. UA stimulation and pharmacological inhibitionUA was added to HUVEC cultures 24h 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 nal concentrations:100 U/ml of the PEG-SOD antioxidant,10 mM of the ER stress inhibitor 4-PBA, or 20 ?g/ml of the PKC inhibitor polymyxin B. Untreated HUVECs were used as the control.2. Measurement of ROSHUVECs were cultured with or with out 12mg/dl UA for 3h,6h,12h and 24h respectively, intracellular ROS levels were detected using specific fluorescent probe CM-H2DCFDA.3. Western blot analysisHUVECs were cultured with or with out 12mg/dl UA for 3h,6h,12h and 24h respectively, cells were harvested with cell lysis buffer to extract cell total protein. The the protein expressions of CHOP, ATF-6, Caspase-12, eNOS, PKC, CaM, and the phosphorylation level of eNOS and PKC were detected using western blotting method.4. Co-immunoprecipitation assayHUVECs were cultured with or with out 12mg/dl UA for 3h,6h,12h and 24h respectively, cells were harvested with cell lysis buffer to extract cell total protein. The interactions of eNOS and CaM were detected using immune precipitation test.5. Measurement of Ca2+ concentrationHUVECs were cultured with or with out 12mg/dl UA for 3h,6h,12h and 24h respectively, the cytoplasmic calcium levels were detected using specific fluorescent probe Fluo-3 AM.6. Measurement of eNOS activity and NO productionHUVECs were pretreated with oxygen scavenger PEG-SOD, endoplasmic reticulum stress reliever 4PBA, or PKC inhibitors polymyxin B respectively, then cultured with or without UA for 48h, eNOS activity was measured using a Nitric Oxide Synthase Assay kit, NO production was measured using nitrate reductase assay.7. Statistical analysis.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 signi cant difference. A P-value <0.01 was considered to indicate a statistically significant differenceResultsl.The effect of uric acid on intracellular ROS productionIntracellular ROS level were significantly increased 3h after incubation with 12 mg/dl uric acid, and remained significantly elevated for 24 h. Pretreatment of HUVECs with ROS scavenger cell permeable PEG-SOD for 30 min significantly ameliorated the uric acid induced increase in intracellular ROS accumulation.2. The effect of uric acid on ER stressUric acid (12 mg/dl) increased the level of ER stress markers in HUVECs in a time-dependent manner. Cellular content of ATF6 and CHOP was significantly increased after 6h incubation with uric acid, peaked at 12h and remained elevated for 24h. Cellular levels of caspase-12 were significantly increased at 12h and peaked at 24h. Both PEG-SOD and 4-PBA effectively inhibited uric acid induced ATF6, CHOP and caspase-12 up-regulation.3. ROS scavenging and ER stress inhibition attenuate UA-induced apoptosis and decrease of NO production.We assessed the rate of apoptosis and NO production in HUVECs pretreated with PEG-SOD and 4-PBA. While HUVECs incubated with 12 mg/d1 UA for 48h exhibited increased apoptosis and reduced NO production, in cells preincubated with PEG-SOD or 4-PBA this effect was not observed.4. UA decreases eNOS activity by increasing Thr495 phosphorylationResults shown that, compared with control, there were no significant changes in the expression of total protein levels of eNOS and CaM afer been incubated with 12 mg/dl UA for 6,12 or 24 h in HUVECs. Although the intracellular Ca2+ concentration, the rate of eNOS phosphorylation at Ser1177 were almost unaltered, eNOS phosphorylation at Thr495 was increased in a time-dependent manner between 6 and 24 h. Binding of eNOS and CaM was assessed by coimmunoprecipitation, and we noted that UA also reduced eNOS and CaM binding in a time-dependent manner, which was negatively correlated with increased phosphorylation of eNOS at Thr495.5. UA decreases eNOS activity through the ER stress/PKC pathway in HUVECsResults shown that, compared with control, PKC expression level was not altered by incubation with uric acid, while the level of p-PKC was elevated after 6 h and peaked at 12 h. Meanwhile, PEG-SOD,4-PBA or polymyxin B pretreatment ameliorated uric acid induced PKC phosphorylation, restored uric acid induced decrease of eNOS activity.Conclusion1.High levels of UA induce cell oxidative stress, trigger ER stress, and induce cell apoptosis through ER stress way.2. High levels of UA enhance eNOS phosphorylation at Thr495, decrease the eNOS/CaM interaction, which reduce eNOS activity and NO production in HUVECs.3. High levels of UA induced ER stress activates the PKC pathway to regulate eNOS activity.4. ER stress inhibitor 4PBA effectively inhibite high uric acid induced endothelial dysfunction in HUVECs.