Folic acid attenuates cobalt chloride-induced PGE2 production in HUVECs via the NO/HIF-1alpha/COX-2 pathway

Yuming Lianga, Xiaozhou Zhena, Kaiwen Wanga, Jing Maa,*


Prostaglandin E2 (PGE2), an important lipid inflammatory mediator involved in the progression of vascular diseases, can be induced by hypoxia in many cell types. While folic acid has been shown to protect against inflammation in THP-1 cells duringpo hypoxia and hypoxia-induced endothelial cell injury, whether it might do so by attenuating PGE2 production remains unclear. To investigate this we constructed a hypoxia-induced injury model by treating human umbilical vein endothelial cells (HUVECs) with cobalt chloride (CoCl2), which mimics the effects of hypoxia. In CoCl2-treated HUVECs, folic acid significantly attenuated PGE2 production and increased vasoprotective nitric oxide (NO) content. Folic acid also decreased cyclooxygenase-2 (COX-2) and hypoxia-inducible factor 1-alpha (HIF-1α) expression and altered endothelial nitric oxide synthase (eNOS) signaling by increasing p-eNOS(Ser1177) and decreasing p-eNOS(Thr495) in a dose-dependent manner. Further investigation of the pathway demonstrated that treatment with 2-Methoxyestradiol (2-MeOE2) and celecoxib both decreased CoCl2-induced COX-2 expression but only 2-MeOE2 decreased HIF-1α expression. The ability of folic acid to down-regulate HIF-1α and COX-2 protein levels was dramatically abrogated by L-NAME treatment, which also decreased eNOS mRNA and NO production. The NO donor sodium nitroprusside also dose-dependently down-regulated HIF-1α and COX-2 protein levels. Overall, these findings suggest a novel application for folic acid in attenuating CoCl2-induced PGE2 production in HUVECs via regulation of the NO/HIF-1α/COX-2 pathway.

Folic acid; Human umbilical vein endothelial cells; Hypoxia; Cobalt chloride; Prostaglandin E2

1. Introduction

Prostaglandin E2 (PGE2) is one of the most significant lipid mediators involved in multiple aspects of chronic inflammation and plays important roles in the realm of vascular diseases. Atherosclerosis, arterial aneurism, and angiogenesis involve production of PGE2 from arachidonic acid through an enzyme cascade that includes cyclooxygenase (COX) and PGE2 synthase[1,2]. Considerable research has demonstrated that induction of the COX-2 isoform with inflammatory mediators such as tumor necrosis factor α (TNFα), interleukin-1 (IL-1), and lipopolysaccharide (LPS) can lead to increased production of PGE2[3,4]. Interestingly, growing evidence suggests that hypoxia can also serve as an inflammatory signal to induce COX-2 and activate PGE2 production[5,6,7].
Hypoxia is medically defined as a reduction in the normal level of tissue oxygen tension resulting from an inadequate supply of oxygen and it is often associated with the pathogenesis of cancer, obesity, atherosclerosis, and other chronic inflammatory diseases[8,9,10]. The most important transcription factor activated by hypoxia is hypoxia-inducible factor 1 (HIF-1), composed of HIF-1α and HIF-1β subunits. By binding to hypoxia response elements (HREs) of target genes, HIF-1 has been shown to mediate angiogenesis as well as cell proliferation, glycolysis, apoptosis, and migration[9]. Hypoxia may also activate COX-2 expression in a HIF-1-dependent manner, as functional HREs have been identified in the COX-2 promoter sequence[11,12,13].
In contrast to PGE2, endothelial-derived nitric oxide (NO), generated by endothelial nitric oxide synthase (eNOS), serves as a vasoprotective molecule by contributing to vasodilation and inhibiting platelet aggregation and leukocyte adhesion to vessel walls[14]. Hypoxia can suppress the expression or activity of eNOS and eNOS-derived NO production via epigenetic, transcriptional, post-transcriptional, and post-translational mechanisms, leading to endothelial dysfunction[15]. At the same time, NO can blunt HIF stabilization and activity during hypoxia by inhibiting mitochondrial cytochrome c oxidase (CcO), resulting in oxygen redistribution and eventual reductions in HIF-1α stability[16,17].
Treatment with folic acid (vitamin B9) has been shown to help prevent and treat elevated blood levels of homocysteine[18,19], an independent risk factor of cardiovascular and cerebrovascular diseases including atherosclerosis[20], stroke[21], and Alzheimer’s disease[22]. The active form of folic acid, 5-methyl tetrahydrofolate (5-MTHF), can also reduce inflammation by promoting eNOS recoupling and activity through dihydrofolate reductase (DHFR) recycling of dihydrobiopterin (BH2) to tetrahydrobiopterin (BH4)[23,24]. Moreover, administration of folic acid has been shown to restore DHFR levels, NO bioavailability, and BH4 levels under hypoxic conditions in human pulmonary artery endothelial cells[25]. Mechanistically, folic acid enhances eNOS activity by promoting eNOS dephosphorylation at a negative regulatory site (Thr495) and increasing phosphorylation at a positive regulatory site (Ser1177)[26].
Based on these reports, we hypothesized that folic acid treatment would restore endothelial NO production and reduce HIF-1α stability to repress COX-2 expression and PGE2 production in human umbilical vein endothelial cells (HUVECs) during hypoxia. Here, we aimed to determine the effect and potentially the underlying mechanism of folic acid on endothelial PGE2 production induced by the classic hypoxia mimic cobalt chloride (CoCl2)

2. Materials and methods

2.1. Reagents

2-Methoxyestradiol (2-MeOE2), Celecoxib and L-NAME HCL were purchased from Selleck Chemicals (Houston, TX, USA) and sodium nitroprusside (SNP) was purchased from Beyotime Biotechnology (China).

2.2. Cell culture

HUVECs were obtained from ScienCell Research Labs and cultured in Endothelial Cell Medium (ECM) containing 5% fetal bovine serum (FBS), 1% endothelial cell growth supplement (ECGS) and 1% penicillin/streptomycin (P/S) (ScienCell, San Diego, CA) under normal oxidative condition (5% CO2, 95% air). Cells between passages 3 and 6 were used for all experiments.

2.3. Viability assay

To study the toxic effects of folic acid and CoCl2 (Sigma-Aldrich, St. Louis, MO) on HUVECs, we conducted a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. 5×104 cells/well were seeded into 96-well plates, cultured in serum-free ECM for 24 h, and then treated with increasing concentrations of folic acid (0-400 µg/ml) and CoCl2 (0-400 µM) for 24 h. After incubation, 20 µl MTT (5 mg/ml) was added to each well and cells were incubated for a further 4 h at 37OC before the supernatants were removed and 150 µl DMSO (Sigma-Aldrich) was added to each well. Crystals were dissolved by agitating the plate for 10 min and optical density was measured at 570 nm using a microplate reader (Bio-Rad, San Diego, CA).

2.4. Western blot analysis

Total cellular protein was extracted from cultured cells following standard procedures, subjected to 8% SDS-PAGE electrophoresis, and transferred onto PVDF membranes (Millipore, Bedford, MA). Membranes were blocked with 5% non-fat milk at room temperature for 1 h, followed by incubation with primary antibodies diluted 1:1000 for HIF-1α (BD Bioscience NJ), p-eNOS(Ser1177), p-eNOS(Thr495), eNOS, COX-2 (Cell Signaling Technology, Danvers, MA) or 1:10000 for β-actin (Proteintech Group Inc, Chicago, IL) overnight at 4OC. After washing blots were incubated with HRP-conjugated secondary antibodies diluted 1:2000 at room temperature for 1 h. Protein bands were visualized with ECL reagent (Thermo Fisher, Rockford, USA).

2.5. RNA preparation and RT-qPCR

RNA extraction was performed using Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. cDNA was synthesized from 2 µg RNA using a PrimeScriptTMRT Master Mix (TaKaRa Bio Co. Inc, Dalian, China) as described in the manufacturer’s protocol. RT-qPCR was performed using a SYBR Fast qPCR Mix (TaKaRa Bio Co. Inc, Dalian, China) on a Vii7 system (ABI, Carlsbad, CA, USA) using the indicated primer sequences (Table 1). For all primers a two-step real-time PCR protocol consisting of denaturation at 95OC for 30 s and 40 cycles of 95OC for 5 s followed by 60OC for 30 s. Relative gene expression was calculated using the comparative Ct method (2-∆∆Ct) and all experiments were performed in triplicate.

2.6. Quantification of PGE2 and NO levels

The PGE2 concentration in culture supernatants was determined by ELISA (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions. Total NO content was determined as the nitrite concentration in culture supernatants using a total nitrite oxide assay kit (Beyotime Biotechnology, China) based on the Griess reaction.

2.7. Statistical analysis

All experiments were performed at least three times and data are expressed as means ± SEM. Differences among groups were assessed by performing one-way ANOVA combined with the least significant difference (LSD) test using SPSS 19.0 software (SPSS Inc, Chicago, IL, USA). P<0.05 was considered statistically significant 3. Results 3.1. Determination of folic acid and CoCl2 toxicity In order to assess dose-dependent toxicity of folic acid (0-400 µg/ml) and CoCl2 (0-400 µM) in HUVECs, cell viability was determined via MTT assay. HUVEC viability was not significantly affected by folic acid doses at 40 µg/ml or below, but HUVECs treated with 200-400 µM CoCl2 showed significant decreases in viability (Supplementary Figure 1). Based on these results, 0.4, 4, and 40 µg/ml folic acid and 100 µM CoCl2 were used in subsequent experiments. 3.2. Effects of folic acid on PGE2 and NO production in CoCl2-treated HUVECs HUVECs pretreated with increasing doses of folic acid (0-40 µg/ml) for 30 min were treated with CoCl2 for 24 h to induce chemical hypoxia. In response to CoCl2, PGE2 levels increased significantly, while NO levels decreased significantly (Fig. 1A and B). Treatment with folic acid reversed these changes in a dose-dependent manner (P< 0.05). 3.3. Effect of folic acid on eNOS, HIF-1α and COX-2 expression in CoCl2-treateded HUVECs To investigate the mechanism underlying folic acid’s ability to decrease PGE2 and increase NO concentration under hypoxic condition, eNOS, HIF-1α, and COX-2 expression in folic acid-treateded HUVECs was determined by western blot analysis and RT-qPCR. Compared with controls, 24 h treatment with CoCl2 significantly increased HIF-1α and COX-2, but not total eNOS protein expression (Fig. 1C and D). Pretreatment with folic acid for 30 min caused a robust, dose-dependent decrease in HIF-1α and COX-2 expression but had no effect on total eNOS expression at any concentration. Similarly, folic acid significantly reduced CoCl2-induced COX-2 mRNA in a dose-dependent manner. In contrast, neither HIF-1α nor eNOS mRNA expression were altered under any conditions (Fig. 1F). Because total eNOS expression was unaltered by folic acid treatment despite its effect on NO production, we next investigated eNOS phosphorylation status by western blot. CoCl2 decreased eNOS phosphorylation at Ser1177 while increasing phosphorylation at Thr495, modifications that down-regulate eNOS activity (Fig. 1E). Treatment with all concentrations of folic acid reversed this effect, increasing p-eNOS(Ser1177) and decreasing p-eNOS(Thr495) to restore eNOS activity. 3.4. COX-2 expression and PGE2 production under chemical hypoxia is facilitated by HIF-1α Next, we analyzed the pathways involved in CoCl2-induced increases in COX-2 expression. HIF has been reported to enhance COX-2 expression by interacting with functional HREs in the COX-2 promoter sequence[13]. As expected, pretreating HUVECs with 10 µM 2-MeOE2, a selective HIF-1α inhibitor, for 30 min prior to 24 h incubation with CoCl2 prevented increases in HIF-1α protein as well as COX-2 protein and mRNA expression (Fig. 2C, D and F). CoCl2-induced PGE2 production also decreased in response to 2-MeOE2, while NO levels were not significantly altered (Fig. 2A and B). Pretreatments with 20 µM celecoxib, a selective COX-2 inhibitor, for 30 min prior to 24 h CoCl2 treatment similarly decreased COX-2 protein and mRNA expression and abrogated chemical hypoxia-induced PGE2 production but had little effect on HIF-1α protein or NO production (Fig. 2A-D and F). However, neither 2-MeOE2 nor celecoxib pretreatment significantly altered phosphorylation of eNOS or changed eNOS and HIF-1α mRNA in CoCl2-treated HUVECs (Fig. 2E and F). These data indicate that CoCl2 increases HIF-1α accumulation to promote COX-2 expression and induce secretion of PGE2. 3.5. A folic acid-driven decrease in HIF-1α protein expression during chemical hypoxia is related to eNOS-derived NO Several publications have linked NO production with its effect on HIF-1α stability and activity[16,17], prompting us to investigate the involvement of NO in folic acid-mediated HIF-1α and COX-2 protein reduction using the selective eNOS inhibitor L-NAME. Pretreatment with 100 µM L-NAME for 30 min prior to 24 h CoCl2 treatment decreased eNOS protein levels and abolished the ability of folic acid to down-regulate HIF-1α and COX-2 protein levels (Fig. 3C and D). As a result, L-NAME prevented folic acid treatment from decreasing PGE2 and increasing NO concentrations during chemical hypoxia (Fig. 3A and B). Interestingly, at the transcriptional level neither HIF-1α nor eNOS mRNA were significantly affected by L-NAME (Fig. 3E). To further probe the inhibition of HIF-1α by NO we investigated the effect of adding SNP, a NO donor, to HUVECs undergoing chemical hypoxia. While total eNOS, p-eNOS(Ser1177), and p-eNOS(Thr495) levels were not altered significantly by pretreatment with SNP for 30 min, HIF-1α and COX-2 expression declined gradually in a dose-dependent manner (Fig. 4B-D). Similar to our folic acid treatment data, only COX-2 mRNA was down-regulated by SNP (Fig. 4E), and SNP inhibited PGE2 production in a dose-dependent manner (Fig. 4A). Together, these results suggest that functional eNOS and eNOS-derived NO are required for the regulation of HIF-1α and COX-2 expression by folic acid in CoCl2-treated HUVECs. 4. Discussion Hypoxia is thought to be critically important as a pathophysiological condition involved in the development of chronic inflammation, cancer, obesity, atherosclerosis and other diseases[8,9,10]. Both in vivo and in vitro hypoxia has been shown to induce production of inflammatory cytokines including IL-1, IL-1 receptor antagonist (IL-1RA), IL-6, and C-reactive protein (CRP), and increased levels of these likely play a role in development of pathology[27,28,29]. At the same time, hypoxia has long been known to induce COX-2 expression through activation of the NF-κB p65 transcription factor in HUVECs[5], and reports have increasingly linked this with enhanced production of PGE2, a bioactive lipid associated with inflammation[6,7]. While we have previously shown that folic acid exerts anti-inflammatory effects during hypoxia by inhibiting the PI3K/Akt/HIF-1α pathway and reducing IL-1β, IL-8 and TNF-α production[30], whether it might also repress PGE2 production remained to be determined. Thus, we decided to further investigate the protective properties of folic acid during chemical hypoxia in HUVECs. Following treatment with CoCl2 for 24 h to induce chemical hypoxia, HUVECs significantly increased their production of PGE2 while decreasing NO production. Here, we demonstrate for the first time that folic acid reverses this response in a dose-dependent manner, simultaneously decreasing PGE2 and increasing NO levels. Because NO is indispensable for maintaining normal vascular physiology, decreases in its concentration can contribute critically to endothelial dysfunction and its levels may serve as a marker for the risk of future cardiovascular events[31]. Recent studies have suggested folic acid may protect cells from hypoxia-related injury by reducing reactive oxygen species (ROS) levels and increasing NO content[32]. Therefore, increase of NO production may be one of the mechanisms by which folic acid attenuates PGE2 production induced by CoCl2 in HUVECs. While our results clearly demonstrated a dramatic, dose-dependent decrease in CoCl2-induced COX-2 and HIF-1α protein levels in response to folic acid, at the transcriptional level only COX-2, and not HIF-1α, mRNA expression increased in response to CoCl2 and demonstrated a corresponding decrease with folic acid treatment. However, this is consistent with previous reports in which neither CoCl2 nor folic acid treatment affected HIF-1α mRNA levels[30,33,34]. One explanation is that folic acid may decrease HIF-1α protein by promoting its degradation, more rapidly than it can be replaced. In our experiments using the selective COX-2 inhibitor celecoxib and the HIF-1α specific inhibitor 2-MeOE2 we found that both decreased COX-2 expression but only 2-MeOE2 decreased HIF-1α. These results suggest degradation of HIF-1α might be involved in the down-regulation of COX-2 expression by folic acid. As outlined in a number of recent reviews[15,17], the ability of NO to regulate HIF activation remains controversial, and hypoxia has been shown to both up- and down-regulate eNOS expression and activity and thus levels of eNOS-derived NO in various reports. In our study both CoCl2 and folic acid had little effect on eNOS protein or gene expression in HUVECs, but folic acid essentially reversed the decrease in p-eNOS(Ser1177) and increase in p-eNOS(Thr495) induced by CoCl2, suggesting folic acid may contribute to elevated NO production by altering the phosphorylation state and activity of eNOS. 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