Uric acid enhances PKC-dependent eNOS phosphorylation and mediates cellular ER stress: A mechanism for uric acid-induced endothelial dysfunction

Abstract

The mechanism by which hyperuricemia induced-endothelial dysfunction contributes to cardiovascular diseases (CVDs) is not yet fully understood. In the present study, we used uric acid (UA) to trigger endothelial dysfunction in cultured endothelial cells, and investigated the effects of induced reactive oxygen species (ROS) generation, endoplasmic reticulum (ER) stress induction, and the protein kinase C (PKC)-dependent endothelial nitric oxide synthase (eNOS) signaling pathway. Human umbilical vein endothelial cells (HUVECs) were incubated with 6, 9 or 12 mg/dl UA, ROS scavenger polyethylene glycol-superoxide dismutase (PEG‑SOD), ER stress inhibitor 4-phenylbutyric acid (4-PBA), and PKC inhibitor polymyxin B for 6-48 h. Nitric oxide (NO) production, eNOS activity, intracellular ROS, ER stress levels, and the interaction between eNOS and calmodulin (CaM) and cytosolic calcium levels were assessed using fluorescence microscopy and western blot analysis. Apoptosis was assessed by annexin V staining. UA increased HUVEC apoptosis and reduced eNOS activity and NO production in a dose- and time-dependent manner. Intracellular ROS was elevated after 3 h, while ER stress level increased after 6 h. UA did not alter intracellular Ca2+, CaM, or eNOS concentration, or eNOS Ser1177 phosphorylation. However, PKC-dependent eNOS phosphorylation at Thr495 was greatly enhanced, and consequently interaction between eNOS and CaM was reduced. Cellular ROS depletion, ER stress inhibition and PKC activity reduction inhibited the effect of UA on eNOS activity, NO release and apoptosis in HUVECs. Thus, we concluded that UA induced HUVEC apoptosis and endothelial dysfunction by triggering oxidative and ER stress through PKC/eNOS-mediated eNOS activity and NO production.

Figure 1

Uric acid (UA) induces apoptosis and decreases nitric oxide (NO) release and endothelial nitric oxide synthase (eNOS) activity in human umbilical vein endothelial cells (HUVECs). Primary cultured HUVECs were incubated with 6, 9 and 12 mg/dl of UA for the indicated periods of time. (A) Apoptosis was measured by Annexin V/PI staining, and detected by flow cytometry. (B) NO release was measured using Griess reagent. (C and D) eNOS protein levels were measured by western blot analysis in HUVECs incubated with 12 mg/dl UA. (E and F) eNOS activity was measured using a NOS activity assay kit. *P<0.05 vs. control; ^#P<0.05 vs. 12 h and 6 mg/dl; ^&P<0.05 vs. 24 h and 9 mg/dl; n=3–5.

Figure 2

Effect of uric acid (UA) on intracellular reactive oxygen species (ROS) generation in human umbilical vein endothelial cells (HUVECs). (A and C) Intracellular ROS levels were detected using the specific probe CM-H2DCFDA. (B and D) HUVECs were pre-treated with or without 100 U/ml polyethylene glycol-superoxide dismutase (PEG-SOD) for 30 min before incubation with UA. ^*P<0.05 vs. control; ^#P<0.05 vs. 3 h and UA; ^&P<0.05 vs. 6 h; n=3.

Figure 3

Effect of uric acid (UA) on endoplasmic reticulum (ER) stress in human umbilical vein endothelial cells (HUVECs). (A) HUVECs were incubated with 12 mg/dl of UA for 3, 6, 12 and 24 h. Activating transcription factor 6 (ATF-6), CCAAT-enhancer-binding protein homologous protein (CHOP), and caspase-12 protein expression was assessed by western blot analysis. (B) HUVECs were incubated with 100 U/ml of polyethylene glycol-superoxide dismutase (PEG-SOD) or 10 mM of 4-phenylbutyric acid (4-PBA) for 30 min, prior to incubation with or without UA for 24 h. ^*P<0.05 vs. control; ^#P<0.05 vs. 6 h and UA; ^&P<0.05 vs. 12 h; n=3. NC, HUVECs not stimulated with UA.

Figure 4

Effect of polyethylene glycol-superoxide dismutase (PEG-SOD) and 4-phenylbutyric acid (4-PBA) on cell apoptosis and nitric oxide (NO) release in human umbilical vein endothelial cells (HUVECs). NC indicates HUVECs which were incubated without UA stimulation, just with different pharmacological pretreatment. (A and B) HUVECs were incubated with 12 mg/dl of uric acid (UA) with or without a 30-min pre-incubation with PEG-SOD or 4-PBA. HUVEC apoptosis was measured with an Annexin V-FITC kit. (C and D), and endothelial nitric oxide (NO) synthase (eNOS) activity and NO release were measured using a NOS activity assay kit and Griess reagent, respectively. ^*P<0.05 vs. control; ^#P<0.05 vs. UA; n=3. NC, HUVECs not stimulated with UA.

Figure 5

Uric acid (UA) decreases endothelial nitric oxide synthase (eNOS) activity by phosphorylation at Thr⁴⁹⁵. Human umbilical vein endothelial cells (HUVECs) were incubated with 12 mg/dl of UA for 6, 12 and 24 h. (A) Cellular content of p-eNOS-Ser¹¹⁷⁷, p-eNOS-Thr⁴⁹⁵ and eNOS was assessed by western blot analysis. (B) Intracellular (Ca²⁺)_cyto was assessed using the specific probe Fluo-3, AM by laser confocal microscope. (C) Cellular content of calmodulin (CaM) was assessed by western blot analysis. (D) Co-immunoprecipitation of eNOS and CaM from lysates of UA-treated HUVECs using an eNOS-directed antibody, and detected using a CaM-directed antibody. ^*P<0.05 vs. control; ^#P<0.05 vs. 6 h; ^&P<0.05 vs. 12 h; n=3.

Figure 6

Effect of pharmacological inhibition on the protein kinase C (PKC)/endothelial nitric oxide synthase (eNOS) pathway in uric acid (UA)-stimulated human umbilical vein endothelial cells (HUVECs). NC indicates HUVECs which were incubated without UA stimulation, just with different pharmacological pretreatment. (A) HUVECs were incubated with 12 mg/dl of UA for the indicated time periods. Cellular content of PKC and p-PKC was assessed by western blot analysis. (B) HUVECs were incubated with 12 mg/dl of UA with or without a 30-min pre-incubation with polyethylene glycol-superoxide dis-mutase (PEG-SOD) or 4-phenylbutyric acid (4-PBA). Cellular content of PKC and p-PKC was assessed by western blot analysis. (C) HUVECs were incubated with 12 mg/dl of UA with or without a 30-min pre-incubation with PEG-SOD, 4-PBA, or polymyxin B. Cellular content of p-eNOS-Ser¹¹⁷⁷, p-eNOS-Thr⁴⁹⁵, and eNOS was assessed by western blot analysis. (D) eNOS activity was detected in HUVECs incubated with 12 mg/dl UA with or without a 30-min pre-incubation with PEG-SOD, 4-PBA, or polymyxin B. ^*P<0.05 vs. control; ^#P<0.05 vs. 6 h and UA; n=3. NC, HUVECs not stimulated with UA.