Fetal bovine serum was provided by Hyclone Laboratories, Inc. (Logan, UT). HAM's F-12K and HEPES buffer were from Corning Inc. (Corning, NY). Sodium selenite, insulin, heparin, and transferrin were from Sigma-Aldrich (St. Louis, MO). Antibiotics/antimycotics, glutamine, trypsin-EDTA, and bovine collagen were purchased from Life Technologies (Carlsbad, CA). Indomethacin, 15-F_2t-IsoP (formerly 8-isoPGF_2α), and 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH) were purchased from Cayman Chemical (Ann Arbor, MI). Lipopolysaccharide (LPS) purified from E. coli 0111:B4 was purchased from Invivogen (San Diego, CA).

Primary BAEC were used to model systemic vascular acute inflammation. The methods used to isolate BAEC have been previously described by Mavangira and coworkers (8). Isolated EC were distinguished from other cell types with von Willebrand factor staining and morphological analysis. Cells were stored in liquid nitrogen at pass 4 until being thawed out for experiments. Upon thawing cells, BAEC were grown to 75-80% confluency in flasks (Corning Inc., Corning, NY) incubated at 5% CO2 and 37°C, and were used from passages 6-9. Cells were cultured in BAEC media containing HAM's F-12K, 20 mM HEPES, 10% fetal bovine serum, 100 U/mL antibiotic/antimycotic consisting of penicillin, streptomycin, and amphotericin B, 300 mg/mL L-glutamine, 10 ng/mL sodium selenite, 10 μg/mL insulin, 100 μg/mL heparin, and 5 μg/mL transferrin.

Conditions of increased ROS generation and acute inflammation were generated using AAPH or LPS. Treatments were as follows: untreated media control, vehicle control with 0.05% ethanol by volume, positive control of either 5 mM AAPH or LPS (15 or 25 ng/mL) as an agonist, physiologically relevant doses of 15-F_2t-IsoP (10–500 nM), and co-culture of either AAPH or LPS with 15-F_2t-IsoP. Incubation times varied by assay, ranging from 1 to 24 h. All BAEC treatments were delivered in F-12K media with either 10% or 0% FBS. A graphical summary of the experimental design is in Figure 1.

Cellular viability was assessed with Promega CellTiter-Glo Luminescent Cell Viability Assay (Madison, WI). This assay produces a luminescent signal proportional to the amount of ATP generated from metabolically active cells present in a well. Cells were plated in white flat-bottom 96-well plates at a density of 4.0 × 10⁴ cells/well. The assay was then performed according to manufacturer's instructions. Luminescence was read on a Tecan Infinite 200 Pro (Männedorf, Switzerland).

To determine if IsoP were influencing apoptosis in BAEC, a Promega Caspase-Glo 3/7 kit was employed. Briefly, caspase activation of the substrate releases aminoluciferin. Subsequent interaction of the free aminoluciferin with luciferase results in a luminescent signal proportional to caspase 3/7 activity. Similar to the viability assay, cells were plated in a white flat-bottom 96-well plate at a density of 4.0 × 10⁴ cells/well. Manufacturer's instructions were followed to carry out the assay. Luminescence was read on a Tecan Infinite 200 Pro.

Total intracellular RONS production was assessed with Cell Biolabs OxiSelect Intracellular RONS kit (San Diego, CA). All reactive species of the sample were measured utilizing a dichlorodihydrofluorescin DiOxyQ (DCFH-DiOxyQ) probe. In brief, RONS will interact with the highly reactive DCFH, which rapidly oxidizes to 2,7'-dichlorodihydrofluorescein, producing a fluorescent signal. Therefore, fluorescence is directly proportional to the amount of reactive species in a sample. Cells were plated at 4.0 × 10⁴ cells/well in black clear-bottom 96-well plates. After 18 hr, 10% FBS media was removed and replaced with 0% treatment media. The assay was then performed according to manufacturer's instructions. A Tecan Infinite 200 Pro was employed to read fluorescence at 480 nm excitation and 530 nm emission.

Endothelial cell barrier integrity was assessed utilizing an electric cell-substrate impedance sensing (ECIS) ZTheta system (Applied Biophysics, Troy, NY). Cells were seeded (density = 1.0 × 10⁵) on a 96-well array (96W10idf, Applied Biophysics) in 5% FBS F-12K media and allowed to grow for ~18 h until resistances stabilized, suggesting a confluent monolayer of endothelial cells. Once confluency was reached, media was changed to 0% FBS and the resistances were allowed to equilibrate for ~4 h. Treatments as listed above in the experimental design section were added. The ECIS system took measurements every 180 s at multiple frequencies for 24 h. The resistance measurements taken at 4,000 Hz after treatment were utilized for analysis.

Cells were cultured in 6-well plates for RNA extraction. Wells were seeded at 1.0 × 10⁶ cells and grown to 75–80% confluency. Each well was washed twice with HBSS and then 300 μL Buffer RLT (Qiagen, Hilden, Germany) was added to lyse the cells. The buffer was collected from each tube and then added to a 1.5 mL microcentrifuge tube and cell lysate was stored at −20°C pending RNA extraction within 1 mo of collection.

Extraction of RNA occurred utilizing a Promega Maxwell RSC Instrument following the manufacturer's protocol. Quantification and the quality of RNA was assessed with a Nanodrop ND-1000 spectrophotometer (ThermoFisher, Waltham, MA) and then stored at −20°C until cDNA was generated. The A_260/280 ratios for all samples were between 1.9 and 2.1. Prior to cDNA generation, all samples were standardized with nuclease-free water to 100 ng/μL. An equal volume of master mix containing 10 × reverse-transcription buffer, 25 × dNTP, 10 × random primers, Multiscribe reverse transcriptase, RNase inhibitor, and RNase nuclease-free water from a high-capacity cDNA reverse-transcription kit with RNase inhibitor (Applied Biosystems High Capacity cDNA Archive Kit, Waltham, MA) was added to each standardized RNA sample. Samples were placed in a MiniAmp Plus Thermal Cycler (Applied Biosystems, Waltham, MA) programmed with the following settings: 25°C for 10 min, followed by 37°C for 2 h, then 85°C for 5 min, finishing with 4°C hold until samples are removed. Samples were then stored at −20°C until qRT-PCR was completed.

Real-time PCR was carried out with predesigned TaqMan primers and FAM-MGB probes (Applied Biosystems). Genes that have demonstrated alterations in the face of IsoP challenge and are also relevant to mastitis pathophysiology were selected for analysis (20–22). Samples were assessed for nitric oxide synthase 2 (NOS2, Bt03249586_m1), peroxisome proliferator activated receptor alpha (PPARA, Bt03220821_m1), peroxisome proliferator activated receptor gamma (PPARG) (Bt03217547_m1), nuclear factor kappa B subunit 1 (NFKB1, Bt03243457_m1), thromboxane A2 receptor (TBXA2R, Bt04301659_m1), and beta-2-microglobulin (B2M, endogenous control, Bt03251630_g1). Genes were evaluated in triplicate with 2 × TaqMan Gene Expression Master Mix (Applied Biosystems), 20 × TaqMan Gene Expression Assay Mix (Applied Biosystems), sample cDNA (50 ng/well), and nuclease-free water for a total of 10 μL per reaction well. Thermal cycling conditions for the Fast 2-step PCR system were as follows: stage 1, 95°C for 20 s; stage 2, 95°C for 3 s; stage 3, 60°C for 30 s, with 40 cycles of stages 2 and 3. Data were recorded and compiled using ThermoFisher ExpressionSuite Software version 1.3.

Results for all analyses except for PCR are presented as the ratio of least squares means in treated cells to the untreated control + standard deviation. The results for PCR were analyzed with a modified ΔΔCt method and are graphically represented as 2^−ΔΔCt (23). Sample size was calculated a priori based on unpublished preliminary apoptosis data. The PROC POWER statement was used in SAS 9.4 (Cary, North Carolina) with the following syntax: procpower; onewayanova; groupmeans = 1 (untreated media control), 1.11 (ethanol vehicle control), 1 (10 nM 15-F_2t-IsoP), 18.6 (25 ng/mL LPS), 15.2 (25 ng/mL LPS + 10 nM 15-F_2t-IsoP); stddev = 1.15; alpha = 0.05; npergroup =.; power = 0.9. The calculation suggested a sample size of at least 3 was sufficient to detect a difference of 1-fold change in apoptotic response. Data were analyzed for normality by visualizing Q-Q plots and confirmed with Shapiro-Wilk normality test. For commercial assays and PCR, a one-way ANOVA with Tukey's HSD posthoc test was performed. For analysis of endothelial cell barrier resistance, a two-way ANOVA was performed with the factors being time and treatment. Two-way ANOVA was followed by Tukey's HSD posthoc test. All statistical analyses were carried out with SAS 9.4.

Viability results at 4, 8, and 12 h were all similar and therefore, only 12 hr data are presented here. The viability of cells treated only with 10-500 nM 15-F_2t-IsoP was not different from untreated cells (P = 0.64), suggesting that IsoP do not have a detrimental effect on BAEC viability (Figure 2A). In BAEC treated with LPS, viability was 85% of that seen in untreated cells (P < 0.0001). Furthermore, up to 500 nM 15-F_2t-IsoP doses did not increase cell viability relative to LPS (P = 0.85; Figure 2B). Therefore, while 15-F_2t-IsoP does not affect viability on its own, it also does not appear to reduce cell death during LPS challenge.

As above, 12 hr data were chosen to represent the effect of IsoP on apoptosis at all timepoints. Complimentary to our viability data, apoptosis in BAEC treated with only 15-F_2t-IsoP was not altered from untreated controls (P = 0.64; Figure 2C). When cells were treated with LPS, apoptosis increased 389% after 12 hr compared to untreated cells (P < 0.0001). However, treating BAEC with LPS and 10-500 nM 15-F_2t-IsoP did not decrease apoptosis relative to LPS only (P = 0.65; Figure 2D). Thus, 15-F_2t-IsoP does not have an effect on BAEC apoptosis alone nor in the presence of LPS.

As with viability and apoptosis, only the 12 hr data are presented given 4, 8, and 12 hr results were similar. Compared to untreated controls, 15-F_2t-IsoP did not alter RONS production on its own in BAEC (P = 0.87; Figure 2E). When challenged with the free radical generator AAPH, a 10-fold increase in BAEC RONS production was seen compared to media controls (P = 0.0015). When treated with 15-F_2t-IsoP in addition to AAPH, RONS production was not different from AAPH alone (P = 0.99). However, RONS production was only 9-fold (P=0.007) and 8.5-fold (P = 0.02) higher than untreated cells at 50 and 100 nM doses of IsoP, respectively. These data therefore demonstrate a relative decrease of RONS production at intermediate doses (Figure 2F).

As maintaining endothelial cell barrier integrity is a critical component of appropriate inflammatory responses, we were interested in assessing if IsoP could improve the resistance across a BAEC monolayer under LPS challenge. A significant time-treatment interaction was detected (P < 0.0001; Figure 3). Between 8 and 24 hr, barrier integrity was significantly decreased in LPS-treated cells compared to the untreated media control (P < 0.0001). A relative increase of barrier integrity was noted in LPS + 15-F_2t-IsoP-treated BAEC compared to LPS only between 8 and 24 hr (P = 0.06).

One possible mechanism involved with the relative increase in barrier integrity described above could be due to alterations in inflammatory gene regulation. In relevant inflammatory genes, IsoP did not have an appreciable effect on the gene expression of BAEC challenged with LPS. As the data were similar across genes and timepoints, TBXA2R, NFKB1, and NOS2 at 4 hr were selected as representatives to be reported herein (Figures 4A–C, respectively). Table 1 shows the P values for all genes and timepoints assessed. There was a higher expression of NFKB1 and NOS2 in LPS-treated cells compared to those without the agonist (P = 0.001 and 0.002, respectively). However, no differences were noted between LPS and LPS + 15-F_2t-IsoP groups (P > 0.05). Therefore, IsoP did not influence inflammatory gene expression in the face of LPS challenge.

To ensure the lack of change in inflammatory gene expression was not due to prolonged time in cell culture or low dosing of IsoP, we also evaluated TBXA2R, NFKB1, and NOS2 expression at 1 hr with 10 and 100 nM IsoP concentrations. Similar to the 4, 8, and 12 hr timepoints, no differences were noted between any treatment group for any of the genes (P > 0.14). However, numerical decreases in NFKB1 (Figure 5A) and NOS2 (Figure 5B) expression were demonstrated in LPS and 15-F_2t-IsoP compared to LPS treatments (P = 0.64 and 0.52, respectively). Moreover, although a relative increase of NOS2 expression was seen in ethanol-treated cells compared to untreated controls at 1 hr, the IsoP treatments that were delivered with the vehicle did not appear to be affected to a similar degree (P = 0.63).