15-oxoeicosatetraenoic acid mediates monocyte adhesion to endothelial cell

A great number of studies reported that 12/15-lipoxygenase (12/15-LO) played an important role in atherosclerosis. And its arachidonic acid(AA) metabolite, 15(S)-hydroperoxy-5,8,11,13-(Z,Z,Z,E)-eicosatetraenoic acid (15(S)-HETE), is demonstrated to mediate endothelial dysfunction. 15-oxo-5,8,11,13-(Z,Z,Z,E)-eicosatetraenoic acid (15-oxo-ETE) was formed from 15-hydroxyprostaglandin dehydrogenase (PGDH)-mediated oxidation of 15(S)-HETE. However, relatively little is known about the biological effects of 15-oxo-ETE in cardiovascular disease. Here, we explore the likely role of 15-lipoxygenase (LO)-1-mediated AA metabolism,15-oxo-ETE, in the early pathogenesis of atherosclerosis. The 15-oxo-ETE level in serum was detected by means of liquid chromatography and online tandem mass spectrometry (LC-MS/MS). And the underlying mechanisms were illuminated by molecular techniques, including immunoblotting, MTT assay, immunocytochemistry and Immunohistochemistry. Increased 15-oxo-ETE level is found in in patients with acute myocardial infarction (AMI). After 15-oxo-ETE treatment, Human umbilical vein endothelial cells (HUVECs) showed more attractive to monocytes, whereas monocyte adhesion is suppressed when treated with PKC inhibitor. In ex vivo study, exposure of arteries from C57 mice and ApoE−/−mice to 15-oxo-ETE led to significantly increased E-selectin expression and monocyte adhesion. This is the first report that 15-oxo-ETE promotes early pathological process of atherosclerosis by accelerating E-selectin expression and monocyte adhesion. 15-oxo-ETE -induced monocyte adhesion is partly attributable to activation of PKC.

There is substantial evidence for the pro-atherosclerotic effect of 15-LOX: (i) downregulation of 12/15-LOX reduced lipid oxidation and foam cell formation [8]; (ii) up-regulation of 15-LOX-2 in macrophages enhances inflammation and the recruitment of inflammatory cells in hypoxic atherosclerotic plaque [6]; (iii) 15-LOX is a key enzyme for the oxidization of low-density lipoprotein (LDL) [9][10][11]; (iv) disruption of 12/15-LOX gene decreased high-fat diet induced endothelial dysfunction and vascular inflammation [12]; (v) ALOX15B is highly expressed in carotid lesions in patients with cerebrovascular symptoms. 15-HETE, as the initial 15-LOX metabolite of AA, was first identified in human atherosclerotic plaque using high-performance liquid chromatography [13]. It is reported that 15S-HETE may be involved in endothelial dysfunction, monocyte/macrophage transmigration [12], vascular wall remodeling [14], and angiogenesis [15]. Moreover, a recent research showed that 15-HETE promoted oxidative stress and foam cell formation [16]. However, the evidence for the role of 15-oxoETE in atherosclerosis is unclear.
A large body of evidence suggests that endothelial dysfunction is a key variable in the pathogenesis of atherosclerosis [17][18][19]. Endothelial dysfunction, as a comprehensive index of the overall CVD risk factor burden includes three main consequences, exposure of adhesion molecules, the activation and aggregation of platelets, cholesterol accumulation [20]. Monocyte recruitment is the initial incident in atherosclerotic plaque formation, the present study was designed to explore the likely effect of 15-oxo-ETE on monocyte recruitment.

Study population
We enrolled patients with diagnosis of acute myocardial infarction (AMI) (n = 8) and patients without coronary artery lesion admitted for elective coronary diagnostic (n = 8). In addition, all the subjects didn't take aspirin or other NSAIDs until first medical contact in Beijing TianTan hospital.

Measurement of AA metabolites
Blood samples for 15-oxoETE assays were drawn into EDTA tubes from a radial artery approach before coronary angiography. After centrifugation, blood samples were stored at −80°C until AA derivative measurement. Plasma AA derivative levels were quantified by ultra-high performance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS). Briefly, Waters Oasis-HLB cartridges were washed with 1 ml methanol and 1 ml MilliQ water, and samples with internal standard mixture (each 5 ng) were loaded into the cartridges and washed with 5% methanol (1 ml). The aqueous plug was then pulled from the SPE cartridges under high vacuum for 20 min, and the analytes were eluted into tubes with 1 ml methanol. Samples were dried using a Thermo Speed Vac, dissolved in 120 μl 30% ACN, and filtered with a 0.22 μC for UPLC-MS/MS. A total of 31 AA mediators were detected.

Cell lines and cell culture
The human monocyte cell line THP-1 was purchased from Cell Resource Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Beijing, China. Cells were cultured in RPMI1640medium (Hyclone, USA) containing 10% fetal bovine serum. HUVECs were isolated from umbilical veins [21]. The cells were cultured in ECM (ScienCell, USA) containing 5% FBS, 1% endothelial cell growth supplement, and 1% penicillin/streptomycin solution. The cells were then cultured in a humidified incubator of 5% CO 2 at 37°C. HUVECs were used at passages 2-5.

In vitro adhesion assay
In brief, confluent HUVECs on 96-well plates were starved for 6 h and treated with 15-oxoETE (0, 0.5, 1, 2, 4, 8 μmol/L) for 6 h at 37°C. Thereafter, cells were exposed to human THP-1 monocytes (10 5 cells/well) for 40 min. Non-adherent THP-1 cells were removed by washing one time with PBS, the absorbance of adherent cells at 450 nm was measured. Six parallel wells were set up for each group. Adherent cells were counted in randomly selected optical fields taken from an inverted microscope (Leica). PKC inhibitor, staurosporine (2.5 nmol/ L), was used at the beginning of treatment [23].

PKC activity assay
The PKC activity in the HUVEC lysates was determined by PepTag® Non-Radioactive Protein Kinase Assays (Promega, V5330, USA). As described in the manufacturer's instructions, 5 × 10 6 cells were removed to prepare PKC extracts. The brightly colored, fluorescent peptide substrate was specially phosphorylated by PKC, and its net charge was altered from +1 to −1. This change in the net charge of the substrate allowed the phosphorylated and non-phosphorylated versions of the substrate to be rapidly separated on an agarose gel [22].

Immunoblotting
Bicinchoninic acidprotein assay kit was purchased from Thermo Fisher Scientific (USA). E-selectin (ab18981) and secondary antibodies was purchased from Cell Signaling Technology (USA). Details of immunoblotting have been described elsewhere [24].

Immunohistochemistry
Histology tissues were fixed in 4% paraformaldehyde and subsequently embedded in paraffin wax. 6-μm-thick sections were cut from each paraffin-embedded tissue and collected on microscope slides, and then dewaxed with xylene and rehydrated with ddH 2 O. Endogenous peroxidases were blocked by 0.3% H 2 O 2 solution (dilute with methanol) for 12 min. Antigen retrieval was conducted in EDTA working solution (10 mmol/L, pH 8.0) for 10 min at 92°C. The tissue sections were blocked by 5% goat serum for 1.5 h at 37°C, after which sections were incubated with primary antibodies (rabbit anti-ICAM-1 (PB0054, Boster), VCAM-1 (BA0406, Boster), or Eselectin (BA0615, Boster); dilution 1:200) overnight at 4°C. The tissue sections were then treated with appropriate HRP-conjugated secondary antibodies for 40 min at 37°C, and Antigen-antibody reactions were stained with 3, 3-diaminobenzidine. The adhesion molecules expression was examined with a Nikon Eclipse Ti microscope under high power (400×) fields. Adhesion molecules expression was quantified by Leica Q550CW image analysis system.

Ex vivo adhesion assay
For ex vivo adhesion experiments, 150 μl PBS and 150 μl 15-oxoETE (314 μM) were respectively given by tail vein injection. 24 h later, mice were anaesthetized with ketamine and 5% chloral hydrate. Under sterile conditions, the heart was exposed through a left thoracotomy, and perfused from the left ventricle with normal saline until the blood was washed out. Then Hoechstlabeled THP-1 monocytes (1 × 10 7 cells/ml) were perfused from the left ventricle to the aorta and incubated for 45 min at 37°C. Non-adherent THP-1 cells were removed by perfusion with normal saline. Aortas were harvested at the level of the aortic arch to the abdominal aorta beyond renal arteries and subsequently fixed in 4% paraformaldehyde solution. Adhesion was observed by confocal laser scanning microscope and quantified by calculating the areas of fluorescent monocytes attached to the vascular surface.

Statistical analysis
Statistical analysis was carried out using one way ANOVA with Dunnett's post-test and Student t test; p < 0.05 was considered significant. Error bars represent mean ± SD.  Table 1. The details of patients are provided in Additional file 1.

Cell viability of HUVECs undergoing 15-oxoETE treatment
To explore the influence of 15-oxoETE on cell viability, MTT assay were conducted on HUVECs treated with varying doses (0-40 μmol/L) of 15-oxoETE for a 24 h period. The cells exhibited a dose-dependent reduction in viability (Fig. 2a). Endogenous 15-oxoETE is so low that it can't kill cells in short period. Therefore, lower doses of 15-oxoETE (0-8 μmol/L) were added to cultured HUVECs under serum deprivation, and then the cell viability was assessed by MTT assay after 12 h. HUVECs showed no change when treated with concentrations of 0-8 μmol/L, which will be applied to the subsequent experiments (Fig. 2b).

15-oxo-ETE promotes monocyte adhesion and positively regulates E-selectin expression
Monocyte adhesion is a vital process in the pathogenesis of atherosclerosis. To uncover the effect of 15-oxo-ETE on monocyte adhesion to HUVECs, HUVECs were treated with various doses of 15-oxo-ETE for 6 h and co-cultured with THP-1 cells for an additional 40 min.  Adhesion of THP-1 cells to HUVECs was stimulated by 15-oxo-ETE in a dose-dependent manner as the amount of adherent THP-1 cells increased from 1.00 ± 0.15 to 1.28 ± 0.26 (Fig. 3a, b).
During the process of cell adhesion, cell adhesion molecules most likely play an important role in cell communication [25][26][27]. The cell ELISA results suggested that treatment of 15-oxo-ETE up-regulated E-selectin, but not ICAM-1 and VCAM-1 (Fig. 3c, d). When HUVECs were treated with 8 μmol/L of 15-oxo-ETE, expression of E-selectin reached 1.40 ± 0.32 fold increase compared with control (Fig. 3e). The protein level of E-selectin was also up-regulated as analyzed by Western blot (Fig. 3f ).

PKC activation is required for 15-oxoETE-induced monocyte adhesion
It has been known that the activity of PKC plays an important role in regulating endothelial dysfunction, including inflammation and adhesion [23]. PKC activity was examined in HUVECs treated with 15-oxoETE. In the present study, 15-oxoETE accelerated PKC phosphorylation compared with control in a dose-dependent manner (Fig. 5a). Interestingly, staurosporine treatment significantly diminished the increased monocytes adhesion in every concentration level of 15-oxoETE, especially in higher concentrations of 2-8 μmol/L (Fig. 5b). These data indicated that the activation of PKC was responsible for 15-oxoETE-induced monocytes recruitment.

Discussion
15-oxoETE is firstly found in rabbit lung, which is produced from 15-PGDH-mediated oxidation of 15-HETE [28]. Cong Wei et al. found that 15-oxoETE inhibited endothelial proliferation in a dose-dependent manner [29]. There was also a study suggested that 15-oxoETE promoted carotid artery construction during hypoxia [30]. These results indicated that 15-oxoETE might play an important role in atherosclerosis. Conversely, 15-oxoETE is found to protect pulmonary arterial smooth muscle cells against serum deprivation-induced apoptosis, indicating a potential therapeutical role of 15-oxoETE in pulmonary arterial hypertension [31]. What's more, 15-oxoETE was reported to mediate pro-inflammatory signaling by activating anti-inflammatory Nrf2 signaling and downregulating pro-inflammatory cytokine [32]. Taken together, this interesting metabolite of AA remains a biological curiosity in atherosclerotic pathophysiology. Thus, this study attempts to demonstrate that 15-oxoETE promote pathological process of atherosclerosis by accelerating monocytes recruitment.
More recently, 15-oxo-ETE was identified as a metabolite of AA in primary human monocytes with IL-4 treatment. They indicated that 15-oxoETE had a short half-life of only 11 min, and then crossed the cell membrane rapidly [29]. It may be the reason why people found 15-HETE but not 15-oxoETE is the main AA metabolite in atherosclerotic plaque. Interestingly, the present UPLC/MS/MS analysis showed that both 15-HETE and 15-oxoETE level is elevated in AMI patients ( Table 2, Fig. 1). Thus, we hypothesized 15-oxoETE was Vascular inflammation and monocyte adhesion are critical events in the initiation of atherosclerosis [25,33].
The increased expression of cellular adhesion molecules, such as VCAM-1 E-selectin and ICAM-1, plays an important role in endothelial inflammation and is essential to recruiting monocytes from the circulation [33,34]. The present study suggested that 15-oxoETE positively regulated E-selectin expression in HUVECs and increased monocyte adhesion to HUVECs (Fig. 3). Consistently, the result is further tested in both C57 and ApoE−/− mice. What's more, the adhesion change is more significant in ApoE−/− mice than C57 mice (Fig. 4). These data revealed that 15-oxoETE might be a risk factor of cardiovascular disease in both health groups and high-risk groups.
PKC activation strengthens the adhesion reaction of endothelial cells with monocytes [35,36]. Four PKC isoforms have been identified in human ECs, namely PKC-α, PKC-δ, PKC-ε, and PKC-ζ [27]. Although the present study did not explore the roles of different PKC isoforms in 15-OXO-ETE-treated ECs, we showed that total PKCs activity was increased in 15-oxoETE-treated ECs (Fig. 5a). Moreover, increased monocyte adhesion was significantly diminished by PKC inhibitor (Fig. 5b). Therefore, we speculate that PKC activation may play an important role in 15-oxoETE induced monocyte adhesion.

Conclusion
These studies provide a novel role of 15-oxoETE in atherosclerosis. 15-oxoETE promotes E-selectin expression and PKC-dependent monocyte adhesion, which indicating that 15-oxoETE is probably a potential risk factor of atherosclerosis. This question will be better clarified until abundant convictive clinical data will be given.  b Monocyte adhesion was measured in the presence or absence of staurosporine (2.5 nmol/L), the results are presented as the mean ± SD of three independent experiments (*P < 0.05, **P < 0.01, ***P < 0.001, compared with control)