Cells and culture
Pooled, neonatal dermal human microvascular endothelial cells (HMVECs) were purchased from Lonza (Basel, Switzerland). HMVECs were grown in Endothelial Growth Media-2 (Lonza) supplemented with 5% fetal bovine serum (FBS). Cells were plated at a density of approximately 30,000 cells/cm2 and grown to confluence. For a positive control for iNOS expression, murine vascular smooth muscle cells from strain C57BL/6 were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% FBS. Experiments were conducted between the 2nd and 5th passages. Media was exchanged every 3 days.
Agonist and inhibitor reagents
The following reagents and concentrations were used in experiments: 10 μg/mL Poly I:C (Sigma Aldrich, St. Louis, MO, USA), 100 μmol/L L-NG-Nitroarginine methyl ester (L-NAME, Sigma Aldrich, St. Louis, MO, USA), 10 μmol/L SB203580 (Cell Signaling Technology, Danvers, MA, USA). For a positive control of iNOS induction, 10 ng/mL tumor necrosis factor alpha (TNFα, Sigma Aldrich, St. Louis, MO, USA) was used. Agonists, inhibitors and vehicle controls were provided to cells in the presence of culture media.
Western blot and electrophoresis
For siRNA experiments, cell lysates were collected at 90 min after agonist exposure. This time point was based on previously published data of protein phosphorylation kinetics [11]. In a separate set of experiments examining for NOS proteins, cell lysates were collected at 90 min, 6 h and 16 h after agonist exposure. In addition, for a positive control for iNOS expression, murine vascular smooth muscle cells were exposed to TNFα or vehicle control for 24 h prior to lysate collection. Protein extracts (50 μg/sample) were separated by SDS electrophoresis on a polyacrylamide gel (10%) and transferred to nitrocellulose membranes. Membranes were blocked with Odyssey Blocking Buffer (LI-COR Biosciences, Lincoln, NE, USA) for 1 h at room-temperature. Membranes were incubated with primary antibodies overnight at 4 °C on a rocker. Antibodies were as follows: IKKαβ, phospho-IKKβ, p-p38, p38, p-ERK, ERK, p-IRF7, IRF7, p-eNOS (S1177), eNOS (Cell Signaling Technology, Danvers, MA, USA), iNOS (R&D Systems, Minneapolis, MN, USA), IRF3 (Santa Cruz Biotechnology, Inc., Dallas, TX, USA), p-IRF3 (EMD Milipore, Billerica, MA, USA), and αTubulin (Vanderbilt Antibody Core, Nashville, TN, USA). Afterwards, membranes were incubated with fluorescent secondary antibodies and analyzed using the Odyssey Imaging System (LI-COR Biosciences, Lincoln, NE, USA). Protein quantification was performed via densitometry and normalized as a ratio of expressed protein to αTubulin or phosphorylated protein to respective total protein.
siRNA transfection
HMVECs were treated with siRNA (scrambled siControl, sieNOS) according to the manufacturer’s recommendations. In brief, siRNA was procured from Dharmacon (Lafayette, CO, USA). siRNA (25 nmol/L) was incubated with Dharmafect (Dharmacon) in serum-free medium for 20 min. The resultant complex of siRNA-Dharmafect was added to the cells in 5% FBS media without antibiotics for 6 h. Afterwards, the transfection media was replaced with complete media including antibiotics for another 66 h for a total of 72 h siRNA incubation time prior to agonist exposure. For conditions using TEER, cells were exposed to siRNA for 6 h with removal of siRNA containing media afterwards and replacement of fresh media. Cells were permitted to remain undisturbed for another 42 h. Then cells were plated on the CellZScope and allowed to settle for 24 h to achieve confluence and complete 72 h siRNA incubation time prior to agonist exposure. Percent protein knockdown was determined by western blot analysis as described above.
Cytokine and chemokine production
Supernatants from cells cultures were collected at the completion of the agonist exposure (6 or 16 h) based on the timing of cytokine secretion from previously published data [12]. Collected supernatants were stored at − 80 °C until analyzed. Supernatant IL-6 (eBioScience, San Diego, CA, USA), IL-8, IP-10 and soluble VCAM (sVCAM) (R&D Systems, Minneapolis, MN, USA) concentrations were assessed using a commercially available enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer’s specifications.
Transendothelial electrical resistance (TEER) assay
Determination of TEER was performed utilizing the CellZScope2 (nanoAnalytics GmbH, Münster, Germany). For assays involving siRNA, treatment of siRNA was done prior to plating cells to reduce the impact of washing on monolayer formation and resistance. HMVECs were plated on ThinWell Cell Culture inserts (0.4 μm pore diameter, Greiner Bio-One, Monroe, NC) coated with poly-L-lysine (2x) for 20 min then washed with Dulbecco’s phosphate-buffered saline (D-PBS), glutaraldehyde 50% (1000x) for 15 min then washed with D-PBS, and lastly gelatin (40x) then washed again with D-BPS. Afterwards 70% ethanol was applied for 30 min and washed followed by an aldehyde scavenger for another 30 min in medium and then removed. Cells were then plated at 40,000 cells/well and allowed to grow to confluence over 24 h. Afterwards, cells were treated with Poly I:C for 16 to 24 h and examined for TEER in 1 h increments. In some experiments, 30 min prior to Poly I:C exposure, cells were treated with L-NAME, SB203580 or vehicle control (DMSO < 1%) and remained for the duration of the Poly I:C exposure.
Proximity ligation assay (PLA)
Proximity Ligation Assays were performed using a commercially available kit according to the manufacturer’s instructions (Duolink PLA, Sigma Aldrich, St. Louis, MO, USA). In brief, 96-well glass bottom plates were utilized for the experiment and pretreated with poly-L-lysine, glutaraldehyde 50%, gelatin and an aldehyde scavenger in the same concentrations and times and described above. HMVECs were then plated and exposed to siRNA for 72 h total incubation time as previously described. Cells were then exposed to Poly I:C for 90 min. Afterwards, the media containing Poly I:C was removed, cells were washed with PBS and exposed to a formaldehyde containing fixing solution (8 parts water, 1 part 40% formaldehyde, 1 part 10x PBS) for 15 min and permeabilized with 0.1% Triton X-100 in PBS for 15 min. The solution was then removed and 40 μL of Duolink Blocking Solution (Sigma Aldrich, St. Louis, MO, USA) was placed in each well and incubated at 37 °C for 1 h. The blocking solution was then replaced with Duolink Antibody Diluent containing the following primary antibodies at a 1:100 dilution: eNOS rabbit mAb (clone #D9A5L, Cell Signaling Technology, Danvers, MA, USA), p38α mouse mAb (clone #9F12, Novus Biologicals, Littleton, CO, USA). Plates were then sealed and incubated overnight at 4 °C.
The next day, the primary antibodies were removed, and the cells were washed with Duolink Wash Buffer. Duolink PLA probes (anti-rabbit secondary antibody with plus oligonucleotides and anti-mouse secondary antibody with minus oligonucleotides) were diluted in a 1:5 ratio in Duolink Antibody Diluent and 40 μL of probe containing solution was added to each well for 1 h at 37 °C. Kit ligase was added at a 1:40 dilution to Duolink Ligation Buffer, which contained pre-mixed concentrations of bridging oligonucleotides, and added to each well for 30 min at 37 °C. Next, cells were washed and kit provided polymerase was added in a 1:80 dilution in Duolink Amplification Buffer and applied to each well for 100 min at 37 °C. The amplification buffer included pre-mixed concentrations of Duolink Orange fluorescent probes (ex 554/em 576). Cells were then washed and Duolink Wash Buffer containing 1 drop NucBlue (ex 360/em 460, Thermo Fischer Scientific, Waltham, MA, USA) per mL of buffer was added to each well at room temperature for 10 min. The wash buffer was then removed and replaced with 120 μL of Live Cell Imaging Solution (Thermo Fischer Scientific, Waltham, MA, USA) and imaged at the appropriate wavelength using an inverted confocal microscope (Zeiss LSM 880, Carl Zeiss AG, Oberkochen, Germany). Fluorescent reactions and cell number were counted by averaging two random fields per well using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
Intracellular nitric oxide quantification
NO was determined using the fluorescent probe 4,5-Diaminofluorescein Diacetate (DAF-2 DA, Sigma Aldrich, St. Louis, MO, USA). Cells were grown on 96-well glass bottom plates and treatments applied as described above. After reaching confluence, cells were exposed to Poly I:C for 90 min or 6 h. Cells treated with no agonist or L-NAME for 90 min served as controls. Afterwards, supernatants were removed, cells were washed with media and exposed to 5 μmol/L DAF-2 DA for 40 min in the dark at 37 °C. The media containing DAF-2 DA was then removed and replaced with fresh phenol-red free media and the cells remained in the dark for an additional 30 min at 37 °C. Media was then replaced with 120 μL of Live Cell Imaging Solution (Thermo Fischer Scientific, Waltham, MA, USA) supplemented with 5% FBS and 5 mmol of glucose and imaged for retained intracellular probe (ex 485/em 520) using an inverted confocal microscope (Zeiss LSM 880, Carl Zeiss AG, Oberkochen, Germany). Treated wells (L-NAME or agonist) were normalized against fluorescent intensity in the control, untreated wells.
Gene profiles of human biopsies
Gene array profiles of omental adipose tissue biopsies were obtained from the publicly available National Centers for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) database. Values were obtained from GEO Dataset (GSE20950) initially collected by Hardy OT, et al. “Morbidly obese insulin-resistant patients: omental and subcutaneous adipose tissue” [13]. The database was queried for NOS3 (eNOS, ID: NM_000603), MAPK14 (p38, ID: L35253) and TLR3 (TLR3, ID: NM_003265) from samples run on Affymetrix Human Genome U133 Plus 2.0 Array.
Statistical analysis
For endothelial cell culture experiments, data are expressed as means ± SE of multiple, individual experiments. Comparisons of treatment groups and conditions were done via unpaired t-test for single comparisons and one-way ANOVA, with Bonferroni correction, for multiple-group comparisons. For gene profile expression of biopsy samples, data are expressed as medians (5–95%) ± SE of individual cohorts. Comparisons of groups were done via a Mann-Whitney test and linear regression for direct expression comparison with 95% CI. All analysis was done using GraphPad Prism 5.03 statistical software (GraphPad Software Inc., La Jolla, CA). A p value of < 0.05 was considered statistically significant.