Nicotine induces endothelial dysfunction and promotes atherosclerosis via GTPCH1

2.1 Materials

Nicotine, sepiapterin, BH4, actinomycin D (ActD), acetylcholine (ACh), and phenylephrine (PE) were from Sigma-Aldrich (St. Louis, MO). CMLD-2 was from Calbiochemical (San Diego, CA). The Magna RIP kit was from Millipore (Billerica, MA). Nucleoprotein Extraction Kit was from Sangon Biotech (Shanghai, China). Commercial kits for determining NO and malondialdehyde (MDA) were from Jiancheng Bioengineering Institute (Nanjing, China). Lentivius expressing GFP or GTPCH1 (LV.GFP and LV.GTPCH1) was generated by Vigenebio Co. (Jinan, China).

2.2 Cell culture

Human umbilical vein endothelial cells (HUVECs) were obtained from Clonetics (Walkersville, MD). Cells were grown in endothelial basal medium supplemented with 2% foetal bovine serum, 100 U/mL penicillin and 10 mg/mL streptomycin. Cultured cells between passages three and eight were used for experiments. All cells were incubated in a humidified atmosphere of 5% CO₂ and 95% air at 37°C. When 70%-80% confluent, cells were treated with nicotine at the indicated dose.

2.3 Western blot analysis

Protein extracts were prepared by lysing cells in RIPA lysis buffer (Santa Cruz Biotechnology). Cleared lysates were separated on 10% Tris-glycine gels, then transferred to nitrocellulose membranes, which were blocked in 5% skim milk, then incubated with primary antibodies for GAPDH (1:1000, Cell Signaling Technology, USA), GTPCH1 (1:1000, Sigma); HuR (1:1000) and p-HuR S221 (1:1000, Millipore); and Lamin B1 (1:1000, Cell Signaling Technology). After a wash and incubation with horseradish peroxidase-conjugated secondary antibodies, protein bands were visualized using chemiluminescent HRP substrate (Millipore, USA). The intensity of individual bands was measured by densitometry (Model GS-700, Imaging Densitometer; Bio-Rad, Hercules, CA). All experiments were repeated four times and mean values were derived.

2.4 Quantitative real-time PCR (RT-PCR)

Total RNA was extracted from cells (kept at −80°C) using RNA Extraction Reagent (Vazyme Biotech Co., Nanjing), and 1 μg of RNA was reverse-transcribed into cDNA using Hiscript Reverse Transcriptase (Vazyme Biotech Co.). PCR amplification involved the SYBR PCR mix (Bio-Rad). The oligonucleotide primer sequences were for GTPCH1, 5′-CCAGGTGCAGCAATGGGTTC-3′ and 5′-TTCAACCACTACCCCGACTC-3′; and GAPDH, 5′-AGCTAAGAGAAGGGCGGAAC-3′ and 5′-CATCTGCAGGCTGACATTGA-3′.

2.5 Detection of reactive oxygen species (ROS) and intracellular BH4

Intracellular ROS levels were measured by dihydroethidium (DHE) fluorescence. Briefly, cells were incubated with 10 μmol/L DHE for 30 minutes, then washed with serum-free DMEM for three times. Dihydroethidium fluorescence intensity was recorded using a fluorescent reader at 480 nm excitation and 525 nm emission. Concentrations of BH4 were measured using an ELISA kit.

2.6 RNA immunoprecipitation (IP) assays

The Magna RIP kit was used for RNA IP assay. Briefly, whole-cell lysates were incubated at 4°C overnight with magnetic protein A/G beads precoated with 5 μg rabbit IgG or HuR antibody (Millipore). Beads were washed and incubated with proteinase K buffer (30 minutes at 55°C), followed by RNA isolation from the immunoprecipitates, then cDNA synthesis. PCR involved use of the primers for quantitative RT-PCR.

2.7 Animals and experimental protocols

Male ApoE^−/− mice (C57BL/6J genetic background, 8-12 weeks old) were from Vital River Laboratories (Distributor of Jackson Laboratory, Beijing) and housed under specific pathogen-free conditions on a 12-hour light/12-hour dark cycle with food and water freely available. The procedures were in accordance with institutional guidelines and were approved by the Animal Care and Use Committee of Shandong University.

In the first part of the animal study, ApoE^−/− mice fed a high-fat diet (HFD; 47% calories from fat and 0.2% from cholesterol) were divided randomly into four groups (n = 10 in each group): (a) the sham group fed an HFD, (b) the nicotine group fed an HFD+nicotine (100 mg/L in drinking water), (c) the BH4 group fed an HFD and received BH4 (10 mg/kg per day, intraperitoneal injection) and (d) the nicotine+BH4 group fed an HFD+nicotine and received BH4.

In the second part of the animal study, ApoE^−/− mice fed an HFD were divided randomly into four groups (n = 10 in each group) and received the following treatments for 12 weeks: (a) the GFP group injected intravenously with LV.GFP (1 × 10⁹ plaque-forming units [PFU] in 200 μL phosphate buffered saline) and fed an HFD, (b) the GFP+nicotine group injected intravenously with LV.GFP and fed an HFD+nicotine, (c) the GTPCH1 group injected intravenously with LV.GTPCH1 (1 × 10⁹ PFU) and fed an HFD and (d) the GTPCH1+nicotine group injected intravenously with LV.GTPCH1 and fed an HFD+nicotine.

2.8 Measurement of endothelium-dependent vasorelaxation

The endothelium-dependent vasorelaxation was measured as follows. Mice were anaesthetized with isoflurane (2%) and isolated aorta rings were immersed in Krebs bicarbonate buffer (118 mmol/L NaCl, 4.7 mmol/L KCl, 25 mmol/L NaHCO₃, 1.2 mmol/L KH₂PO₄, 1.2 mmol/L MgSO₄, 2.5 mmol/L CaCl₂, and 5 mmol/L glucose) and then suspended by two tungsten wires mounted in a vessel myograph system (Danish Myotechnologies, Aarhus, Denmark). After undergoing an equilibration period, rings were treated with 1 μmol/L phenylephrine for induction of contraction. At the plateau of contraction, acetylcholine (ACh) (10⁻⁸-10⁻⁴ mol/L) was added to elicit endothelium-dependent vasorelaxation. The relaxation was calculated as a ratio of ACh-induced vasodilation to phenylephrine-elicited vasoconstriction, and the ratio at one was set to 100% of relaxation.

2.9 Oil-red O staining

Atherosclerotic lesions were quantified in aortic-root cross sections from frozen OCT-embedded hearts. Briefly, serial 6-μm-thick cryostat sections were prepared from the origin of the aortic valve cusps, and cross-sectional analysis for atherosclerotic lesion was performed every 60 μm over 360 μm by staining with Oil-red O (Sigma-Aldrich). The extent of atherosclerosis was expressed as the percentage of aortic root covered by lesions. Images were captured digitally with a video camera and analysed using Image-Pro Plus (Media Cybernetics). Samples were coded, and acquisition of images and analysis of lesions were performed in a blinded fashion.

2.10 Histological analysis of aortic lesions

The thoracic aorta was fixed in 4% paraformaldehyde overnight, embedded in paraffin and sectioned at 4 μm. The deparaffinized, rehydrated sections from thoracic aortas and cryosections from aortic roots (5 μm) were microwaved in citrate buffer for antigen retrieval, incubated in endogenous peroxidase (DAKO) and protein block buffer and then primary antibodies overnight at 4°C. Slides were rinsed with washing buffer and incubated with labelled polymer-horseradish peroxidase-conjugated anti-mouse/anti-rabbit antibodies followed by DAB+ chromogen detection (DAKO). Data were analysed using Image-Pro Plus.

2.11 Measurements of serum NO and MDA

Serum NO and MDA levels were assayed using commercial kits.

2.12 Statistical analysis

All analyses involved use of SPSS v23 (SPSS Inc., Chicago, IL). Data are represented as mean ± SD. Comparison of two groups of continuous data involved Student t test and multiple groups one-way ANOVA, followed by the Scheffe post-hoc test. Statistical significance was determined at P < 0.05.

3.1 Nicotine inhibits GTPCH1 expression in endothelial cells

GTPCH1 is important for BH4 synthesis, which contributes to NO production and endothelial function. To investigate the effect of nicotine on GTPCH1 expression, HUVECs were treated with different doses of nicotine for 48 hour. GTPCH1 protein level was decreased by nicotine in dose-dependent manner (Figure 1A and B). Besides, HUVECs were exposed to 1 μmol/L nicotine for different times. GTPCH1 protein level was significantly decreased by nicotine during 48 hour of exposure (Figure 1C), with maximal 2-fold reduction at 48 hour (Figure 1D). Nicotine also decreased GTPCH1 mRNA level (Figure 1E). As expected, the levels of BH4 in endothelial cells and NO in supernatant were reduced after nicotine exposure (Figure 1F and G).

Considering about blood vessels in the body exposure 5%-7% O₂ based on vessel type, HUVECs were cultured in the 5% O₂ incubator and treated with 1 μmol/L nicotine. The levels of GTPCH1, BH4 and NO were also decreased by nicotine in the environment with low oxygen (Figure S1).

Under conditions of limited BH4, eNOS functions in an “uncoupled” state in which reduced nicotinamide-adenine dinucleotide phosphate-derived electrons, rather than L-arginine, are added to molecular oxygen, which leads to ROS production.5 Therefore, we evaluated the effect of nicotine on ROS production. As expected, nicotine time-dependently increased the levels of ROS in HUVECs (Figure 1H). Taken together, nicotine inhibited GTPCH1 expression and increased ROS production in endothelial cells.

3.2 GTPCH1 overexpression rescues nicotine-reduced BH4 and -increased ROS levels

To determine the role of GTPCH1 in the nicotine-reduced BH4 level, HUVECs were infected with a lentivirus-expressing GFP (LV.GFP) or LV.GTPCH1 for overexpression, then treated with nicotine. LV.GTPCH1 increased the GTPCH1 level in HUVECs with or without nicotine treatment (Figure 2A and B). Meanwhile, GTPCH1 overexpression rescued the nicotine-reduced BH4 level in HUVECs (Figure 2C). Also, nicotine-increased ROS levels were attenuated by GTPCH1 overexpression (Figure 2D and E). These results confirm the role of GTPCH1 in nicotine-reduced BH4 and -increased ROS levels. Thus, nicotine exposure inhibited GTPCH1 levels, which reduced BH4 and increased ROS levels.

3.3 Sepiapterin attenuates nicotine-reduced NO and -increased ROS levels

Because BH4 is a cofactor for eNOS and contributes to NO production, we detected whether BH4 supplement could rescue the nicotine-reduced NO level. Human umbilical vein endothelial cells were pretreated with sepiapterin, a stable precursor of BH4, then exposed to nicotine. Nicotine decreased NO production, which was rescued by sepiapterin pretreatment (Figure 3A). Similarly, nicotine-increased ROS levels were attenuated by sepiapterin (Figure 3B and C). These results further support the roles of BH4 in nicotine-reduced NO and -increased ROS levels.

3.4 Nicotine regulates GTPCH1 by suppressing HuR translocation

To better understand the regulation of GTPCH1 expression by nicotine, we examined its 5′ untranslated region (UTR) and 3′ UTR to assess whether GTPCH1 may be a target of posttranscriptional modification. We identified seven conserved adenylate-uridylate-rich elements (AREs) in the 3′ UTR of human GTPCH1 mRNA and nine AREs in the 3′ UTR of mouse GTPCH1 mRNA. Thus, GTPCH1 mRNA may be a target of HuR. We first evaluated the effect of nicotine on HuR translocation. Nicotine decreased cytoplasmic HuR level (Figure 4A) but did not alter total HuR level (Figure 4B), so nicotine suppressed HuR translocation from nuclei to cytosol. HuR translocation is regulated by its phosphorylation at S221, which elevates its cytoplasmic abundance.14 We detected HuR phosphorylation after nicotine treatment and found that nicotine decreased the level of phospho-HuR at S221 (Figure 4B), which explained the reduced cytoplasmic HuR level after nicotine treatment.

We assessed the ability of HuR to bind with GTPCH1 mRNA. RNA IP with an anti-HuR antibody or control IgG showed that HuR could bind to GTPCH1 mRNA and nicotine reduced this binding (Figure 4C). Next, we examined the effect of HuR on the stability of GTPCH1 mRNA. Human umbilical vein endothelial cells were pre-treated with CMLD-2, an HuR-specific inhibitor,15 then actinomycin D, a transcriptional inhibitor. The half-life of GTPCH1 mRNA decreased from 9.5 to 7 hour after HuR inhibition (Figure 4D), so HuR enhanced GTPCH1 mRNA stability. As expected, CMLD-2 also dose-dependently decreased GTPCH1 protein level (Figure 4E). These results indicate that nicotine suppressed HuR translation to decrease GTPCH1 level.

3.5 BH4 supplement reduces nicotine-accelerated atherosclerosis

To determine the effect of nicotine and BH4 on the ability to induce atherosclerosis, ApoE^−/− mice were fed an HFD supplemented with or without BH4 or nicotine for 12 weeks later, then sacrificed, and the size of atherosclerotic lesions was analysed. The lesion size in aortic roots was greater with nicotine than vehicle treatment (Figure 5A and B), so nicotine accelerated the development of atherosclerosis and was reduced with BH4 treatment (Figure 5A and B). We further determined the effect of BH4 or nicotine on the endothelial function in ApoE^−/− mice. ACh-induced relaxation in the aortic artery was greatly reduced with nicotine treatment and improved with BH4 supplement (Figure 5C). As expected, nicotine also reduced serum NO level and increased serum MDA level in mice (Figure 5D and E). All of these negative nicotine-induced effects were reversed with BH4 supplement (Figure 5D and E), which further suggests that BH4 is required for nicotine-mediated uncoupling of eNOS in vivo. Moreover, nicotine increased 4-hydroxynonenal (4-HNE) staining (a marker for ROS) in aortic plaques from ApoE^−/− mice, which was reduced with BH4 supplement (Figure 5F). These data suggest that nicotine induced endothelial dysfunction and accelerated the development of atherosclerosis and that BH4 supplement could reverse the endothelial dysfunction and attenuate atherosclerosis.

3.6 GTPCH1 overexpression reduces nicotine-accelerated atherosclerosis

To evaluate the role of GTPCH1 in the development of atherosclerosis, we used lentivirus overexpression of GPF or GTPCH1 with LV.GFP or LV.GTPCH1 in ApoE^−/− mice fed an HFD with or without nicotine. The lesion size in aortic roots was greater in ApoE^−/− mice with than without nicotine and was reduced with GTPCH1 overexpression (Figure 6A and B). On immunohistochemistry, the expression of GTPCH1 in the aortic endothelium was reduced with nicotine treatment, and GTPCH1 overexpression in ApoE^−/− mice increased endothelial GTPCH1 level (Figure 6C). The protein expression of GTPCH1 in whole aorta was increased with LV.GTPCH1 injection compared with LV.GFP injection (Figure 6D), which further confirmed the efficiency of lentiviral transduction of GTPCH1 in vivo. Similarly, nicotine reduced serum NO level in mice, which was reversed with GTPCH1 overexpression (Figure 6E).

Nicotine increased 4-HNE staining, indicating ROS levels, in aortic plaques from ApoE^−/− mice, which was reduced with GTPCH1 overexpression (Figure 7A). Also, serum MDA level was increased with nicotine treatment and suppressed with GTPCH1 overexpression (Figure 7B). In the development of atherosclerosis, adhesion proteins induce the recruitment of monocytes,16 so we evaluated the effect of nicotine and GTPCH1 on the expression of adhesion proteins such as intracellular adhesion molecule 1 (ICAM1) and vascular cell adhesion molecule 1 (VCAM1). The levels of ICAM1 and VCAM1 were reduced with nicotine treatment and increased with GTPCH1 overexpression (Figure 7C and D). Taken together, nicotine induced eNOS uncoupling and accelerated the development of atherosclerosis, which was reduced by GTPCH1 overexpression.