Ct values for gene transcripts.

Endothelial cells synthesize biochemical signals to coordinate a response to insults, resolve inflammation and restore barrier integrity. Vascular cells release a variety of vasoactive bioactive lipid metabolites during the inflammatory response and produce pro-resolving mediators (e.g., Lipoxin A4, LXA4) in cooperation with leukocytes and platelets to bring a halt to inflammation. Aspirin, used in a variety of cardiovascular and pro-thrombotic disorders (e.g., atherosclerosis, angina, preeclampsia), potently inhibits proinflammatory eicosanoid formation. Moreover, aspirin stimulates the synthesis of pro-resolving lipid mediators (SPM), so-called Aspirin-Triggered Lipoxins (ATL). We demonstrate that cytokines stimulated a time- and dose-dependent increase in PGI2 (6-ketoPGF1α) and PGE2 formation that is blocked by aspirin. Eicosanoid production was caused by cytokine-induced expression of cyclooxygenase-2 (COX-2). We also detected increased production of pro-resolving LXA4 in cytokine-stimulated endothelial cells. The R-enantiomer of LXA4, 15-epi-LXA4, was enhanced by aspirin, but only in the presence of cytokine challenge, indicating dependence on COX-2 expression. In contrast to previous reports, we detected arachidonate 5-lipoxygenase (ALOX5) mRNA expression and its cognate protein (5-lipoxygenase, 5-LOX), suggesting that endothelial cells possess the enzymatic machinery necessary to synthesize both pro-inflammatory and pro-resolving lipid mediators independent of added leukocytes or platelets. Finally, we observed that, endothelial cells produced LTB4 in the absence of leukocytes. These results indicate that endothelial cells produce both pro-inflammatory and pro-resolving lipid mediators in the absence of other cell types and aspirin exerts pleiotropic actions influencing both COX and LOX pathways.</div

Antibodies.

Polyclonal or monoclonal antibodies were used at the indicated dilutions. Where possible, positive controls (THP-1, HL-60 cell lysates for 5-LOX, 15-LOX, LTA4H and FLAP proteins) or recombinant proteins for COX-1 and COX-2. In addition, negative controls also consisted of pre-absorption of primary antibodies with blocking peptides where available. (DOCX)</p

M199 media supplemented with 0.5% charcoal-stripped serum (0.5% FCS) and 5% Endothelial Cell Growth Supplement (5% ECGS) not exposed to cells was analyzed for lipid analytes listed in the table.

All analytes were below the lower limit of detection. Duplicate samples were analyzed in two separate experiments. (DOCX)</p

Aspirin inhibits synthesis of pro-inflammatory, vasoactive eicosanoids in endothelial cells.

HUVEC were stimulated with IL−1β (A, 0.2 ng/ml or B, 2 ng/ml) M199+0.5% charcoal-stripped FCS for 24 hr in the presence of ASA (10–4–10–10 M) or vehicle (0.1% EtOH) and media were analyzed for PGI2 (6-ketoPGF1α and PGE2) by ELISA. The data are expressed as mean±SEM (ng/mg protein, n = 6–12 samples/concentration). Experiment was replicated three times.</p

IL−1β promotes LTB₄ biosynthesis in endothelial cells that is lipoxygenase-dependent and augmented by aspirin.

HUVEC were incubated in medium supplemented with 20 μM arachidonic acid (AA) and stimulated for 24 hr with IL−1β (2 ng/ml) or vehicle (PBS/0.1% BSA) in the presence or absence of ASA (0.1 mM). Media were analyzed for LTB4 by ELISA. The data are the mean±SEM (n = 6–12), analyzed by two-tailed t-tests and the experiment was conducted twice. (A) Blue bars, no ASA treatment (p4H. After a 24-hr incubation with IL−1β (2 ng/ml) in the presence or absence of ASA, total RNA was extracted and RT-PCR carried out using Taqman™ chemistry. Transcript expression was computed by the 2-ΔΔCt method using GAPDH as a housekeeping reference gene and fold change relative to untreated controls was calculated. The data are the mean±SEM (n = 4–6 biological replicates) and the experiment was conducted twice.</p

PCR primer sets.

All qRT-PCR experiments were conducted using Taqman™ chemistry and data were analyzed using the 2-ΔΔCt method. (DOCX)</p

Cytokines stimulate eicosanoid production in endothelial cells.

(A) Kinetics: HUVEC were stimulated with human recombinant IL−1β (2 ng/ml, blue circles) or TNFα (20 ng/ml, red triangles) for 0.25–24 hr in DMEM+0.5% FBS and assayed for 6-ketoPGF1α (upper panel) or PGE2 (lower panel). Control cells were incubated with vehicle (PBS/0.1% BSA, open circles). (B) Dose-response: HUVEC were stimulated with IL−1β (0.01–10 ng/ml) or TNFα (0.1–50 ng/ml) for 24 hr in DMEM+0.5% FBS and assayed for 6-ketoPGF1α (stable metabolite of PGI2) and PGE2. Control cells were incubated with vehicle (PBS/0.1% BSA, 6-ketoPGF1α, blue bar; PGE2, red bar). Data are expressed as mean±SEM (n = 6). Experiment was replicated three times. (A)*p<0.01; **p<0.0001. (B, upper panel) *p<0.0128; **p<0.0001; (B, lower panel) *p<0.0004; **p<0.0001. (C) Western blot: HUVEC extracts from vehicle or IL−1β (2 ng/ml)-stimulated cells were probed with COX-1, COX-2, PGIS or mPGES-1 antibodies. Blots were re-probed with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a loading control. Immunoreactive proteins were visualized by chemiluminescence and the ratio of target:β−actin was computed by densitometry (D). The data are presented as the mean±SEM and represent of 3 biological replicates and experiment was conducted twice. The data were analyzed by one-way analysis of variance followed by Dunnett’s test for multiple differences (p values are shown and a minimum p value of <0.05 was considered significant).</p

5-LOX/FLAP pathway inhibitors attenuate pro-inflammatory (A, B, top panel) and pro-resolving (C, D, lower panel) lipids.

HUVEC were stimulated for 24 hr with IL−1β (2 ng/ml, blue bars) or vehicle (PBS/0.1% BSA, red bars). Cells were then preincubated for 60 min with 20 μM arachidonic acid in the presence or absence of inhibitors (aspirin, 0.2 mM, Zileuton, 0.2 μM or MK-886, 0.2 μM) followed by a 30-min challenge with calcium ionophore (A23187, 50 μM). Lipid production was measured by ELISA. The data are mean±SEM (n = 6–12 samples/treatment) and the data were analyzed by one-way ANOVA followed by Tukey’s test for differences. The data are representative of two experiments. * Denotes that PGE2 level was below Lower Limit of Quantification (LLOQ).</p

Sodium salicylate does not inhibit pro-inflammatory eicosanoids in endothelial cells.

HUVEC were stimulated with IL−1β (2 ng/ml) M199+0.5% charcoal-stripped FCS for 24 hr in the presence of 10−4–10−10 M ASA (circles), SA (triangles) or vehicle (0.1% EtOH) and media were analyzed for PGI2 (6-ketoPGF1α and PGE2) by ELISA. The data are expressed as mean±SEM (ng/mg protein, n = 6–12 samples/concentration). Experiment was replicated two times.</p

Proposed model of single cell synthesis of LTB₄ and LXA₄ in endothelial cells.

The conventional model suggests that endothelial cells and leukocytes (infiltrating neutrophils or monocyte/macrophages or platelets) cooperate in transcellular synthesis of chemoattractant LTB4 and pro-resolving LXA4 during acute inflammation (A). Our updated model suggests that endothelial cells express modest levels of the enzyme machinery necessary for leukocyte/platelet-independent lipid synthesis (B).</p