Dulbecco’s Modified Eagle Medium (DMEM) and RPMI-1640 medium were purchased from Fisher Scientific (Pittsburg, PA). Penicillin-Streptomycin and L-Glutamine were purchased from Mediatech (Herdon, VA). RNase inhibitor, MultiScribe Reverse Transcriptase and SyBr green master mix were purchased from Applied Biosystems (Foster City, CA). Oligonucleotides were purchased from Operon Technologies, Inc. (Alameda, CA). Fetal Bovine Serum (FBS) was obtained from Hyclone (Logan, UT). TNFα was purchased from R&D systems (Minneapolis, MN). AA (n-6 FA) and EPA (n-3 FA) were obtained from Cayman Chemical (Ann Arbor, MI). Vybrant cell adhesion assay kit and Hoescht stain for nuclear staining were obtained from Invitrogen (Eugene, OR). Albumin was purchased from Sigma (St. Louis, MO). Phosphate saline buffer solution (PBS) was from the University of California San Francisco Cell Culture Facility (San Francisco, CA).
The cellular model used in this experiment included EA.hy.926 (for ECs) and U937 monocytes. EA.hy.926 cells are a fusion of human umbilical vein endothelial cells (HUVEC) and the A549 epidermal carcinoma line, that retains many of the characteristics of primary endothelial cells12
. The EA.hy.926 cells were a kind gift from Dr. Cora-Jean S. Edgell from the University of North Carolina, Chapel Hill12
. U937 cells are a human monocyte cell line and were obtained from the American Type Cell Collection13
. The ECs were maintained in a 37°C/5% CO2
incubator in DMEM with 10% FBS, 2mM L-glutamine, 1% (v/v) penicillin-streptomycin-neomycin antibiotic mix, 4.5 g/L D-Glucose and 1% (v/v) Amphotericin. The monocytes were maintained in a 37°C/5% CO2
incubator in RPMI-1640 medium containing 10% FBS, 2 mM L-glutamine, 1% (v/v) penicillin-streptomycin-neomycin antibiotic mix. Medium was changed every 2 days. Cell concentration was adjusted to 5×105
cells/ml at each medium change. Cell counts were performed with a hemacytometer (Improved Neubauer, Reichert, NY).
Treatment of endothelial cells for adhesion assays
Confluent ECs were trypsinized and plated at 5000 cells per well in a 96-well plate, then grown to confluency prior to experimental treatments. FA and cytokine treatments were performed as follows: ECs were incubated with either 5μg/ml EPA, 5μg/ml AA, 100ng/ml TNFα, vehicle alone, or a combination of these for 4 or 24 hours prior to the adhesion assay. The dose of TNF-α was selected based on dose-response curves leading to the greatest activation of the ECs based on adhesion molecule expression. The doses of FAs were selected based on the human physiological dose range as well as dose-response curves performed in our laboratory. Vehicle contained DMEM with 2% FBS, 2mM L-glutamine, 1% (v/v) penicillin-streptomycin-neomycin antibiotic mix, 4.5 g/L D-Glucose, 1% (v/v) Amphotericin, and 1.25mg/ml FA-free albumin. Monocytes were labeled with Calcein-AM14
according to the Molecular Probes Vybrant cell adhesion assay kit manufacturer’s instructions and washed twice with Phosphate saline buffer solution. Labeled monocytes were resuspended in vehicle with 0.1% FBS and incubated for 20 minutes with ECs. The 96-well plate was gently washed 1–2 times with Phosphate Saline Buffer solution. Fluorescence per well (Ex 488 nm, Em 515 nm) was measured using a Fluoroscan II plate reader.
In order to investigate a possible pathway for the actions of PUFAs (i.e. the cyclooxygenase pathway), experiments with the n-6 FA AA were performed in the presence of a COX-2 inhibitor. For pre-treatment with the COX-2 inhibitor, adhesion assays were repeated using the same conditions mentioned above, with pre-treatment for 1 hour with indomethacin (50 uM) prior to treatment with the n-6 FA.
RNA was isolated using RNeasy™ Mini kit (QIAGEN, Valencia, CA) according to the manufacturer’s protocol. For RNeasy™ Mini kit RNA isolation, cells were seeded in 6-well plates with DMEM media supplemented with 10%, 2mM L-glutamine, 1% (v/v) penicillin-streptomycin-neomycin antibiotic mix, 4.5 g/L D-Glucose and 1% (v/v) Amphotericin until they reached confluence. Once confluent, media was changed to DMEM supplemented with 2% FBS while all other supplements remained the same. The cells were then treated with the different FAs and TNFα [5μg/ml EPA, 5μg/ml AA, 100ng/ml TNFα, a combination of AA and TNF-α or vehicle alone] for a period of 4 hours. Upon removal of media, the ECs were washed two times with PBS and then 350ul of buffer RLT (supplied in kit) were added to each well. Cells were scrapped off the plate with a cell lifter. The lysate was then placed into a QIA shredder homogenizer (QIAGEN, Valencia, CA) and the flow through was isolated using the Qiagen RNeasy™ Mini kit and processed as per manufacturer’s instruction. The samples were then stored at −80°C until further analysis.
Reverse Transcription (RT)
RNA (0.3ug) was added to 30ul reverse transcriptase (RT) reaction buffer containing 5 mM MgCl2, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 4 mM dNTPs, 2.5 μM oligo d(T) primer, 2.5 U/μl of MultiScribe, and 20 U/μl of RNase inhibitor. The RT reaction was incubated at room temperature for 10 minutes, 42°C for 30 minutes, inactivated at 99°C for 5 minutes, and cooled at 5°C for 5 minutes.
Real-time Quantitative RT-PCR (qRTPCR)
cDNA (2 μl) from the RT reaction was added to 20 μl real-time quantitative polymerase chain reaction (qPCR) mixture containing 10 μl of 2x SYBR®
Green PCR Master Mix (Applied Biosystems, Foster City, CA) and 12 pmol oligonucleotide primers. PCRs were carried out in a Bio-Rad MyiQ Single-Color Real-Time PCR Detection System (Bio-Rad, Hercules, CA). The thermal profile was 50°C for 2 minutes and 95°C for 10 minutes to activate the Taq polymerase, followed by 50 amplification cycles, consisting of denaturation at 95°C for 1 minute 40 seconds, annealing at 63°C for 1 minute 10 seconds and elongation at 72°C for 1 minute 40 seconds. Fluorescence was measured and used for quantitative purposes. At the end of the amplification period, melting curve analysis was performed to confirm the specificity of the amplicon. RNA samples were normalized to cyclophilin (CPHI)
internal standard. Relative quantification of gene expression was calculated by using the 2−(Ct gene T − Ct CPHI T)−(Ct gene 0hr −Ct CPHI 0hr)
equation, where “Ct
gene T” represents the calculated threshold cycle (Ct
) of a time point of each sample other than 0 hour, or each treatment other than control. Relative gene abundance was calculated using 2(Ct gene T − Ct CPHI T)
. Some primer sequences have been previously used15–17
-and are presented in . All data derived using qRTPCR were from independent biological samples (n= 4).
Primers used for qRTPCR in Endothelial Cells
Confluent ECs were tryptinized and plated on a coverslips in 96-well plates, then grown to confluency. FAs and cytokine treatments were performed as described above for the 96-well plate assay. Calcein-AM labeled monocytes were incubated with ECs for 30 minutes, before the coverslips were gently dipped in PBS 4 times. ECs and monocytes were imaged using Zeiss Axioscop Fluorescent Microscope (Carl Zeiss, Germany) and an Orca-ER CCD camera (Hamamatsu Corporation, Bridgewater, NJ).
Data were assessed for normality and outliers. Means and standard deviations are presented. ANOVAs were used to compare the means of the different treatment groups. The p-values presented were corrected for multiple-hypothesis testing using the Bonferroni method. Statistical analyses were performed using Stata/SE 12 (StataCorp, College Station, TX).