Fig 2: sCAMs are increased in the plasma samples from ZIKV+ patients. (a) ZIKV-infected patients from the CARBO cohorts. (b) CXCL10 levels in healthy blood donors and ZIKV+ patients were measured by ELISAs. Results are expressed as means ± SEMs (24 healthy, 24 ZIKV+ plasma samples [12 neuro and 12 non-neuro]) and analyzed using a Wilcoxon-Mann-Whitney test. ****, P < 0.0001 compared to healthy plasma. (c and d) Soluble ICAM-1 and VCAM-1 levels in healthy blood donors and ZIKV+ patients were measured by ELISAs. Results are expressed means ± SEMs (24 healthy, 24 ZIKV+ plasma samples [12 neuro and 12 non-neuro]) and analyzed using a Wilcoxon-Mann-Whitney test. *, P < 0.05; **, P < 0.01; ****, P < 0.0001 compared to healthy plasma samples.

Fig 3: Schematic showing a role for Tpl2 in endothelial function the cellular response to VEGF-A. Step 1: VEGF-A stimulation promotes increased Tpl2 expression and activation via an unknown transcription factor (TF-X). Step 2: Tpl2 maintains phosphorylation of residue T71 on ATF-2 stabilizes ATF-2 levels. Step 3: VEGF-A-stimulated hyperphosphorylation of residue T71 on ATF-2 stimulates VCAM-1 gene transcription leading to increased VCAM-1 levels and increased endothelial-leukocyte interaction. Step 4: basal Tpl2 activity promotes phosphorylation of residue S473 on Akt, causing Akt activation leading to eNOS-S1177 phosphorylation. Step 5: eNOS phosphorylation and activation leads to NO production and increased endothelial cell permeability. Grey arrows depict undefined mechanism.

Fig 4: Greek honey extracts inhibit TNF-α-induced adhesion molecule expression.Greek honey extracts (H1–H12) inhibit TNF-α-induced ICAM-1 (A) and VCAM-1 (B) protein expression in HAEC. HAEC were incubated in the absence of TNF-α or compounds (control), with αT3 (25 µΜ), or with different concentrations (20–500 µg/ml) of honey extracts for 18 h, followed by stimulation with TNF-α (1 ng/mL) for up to 24 h. Adhesion molecules were measured by cell ELISA. Results are expressed as percent of control. A *p<0.05 value was considered statistically significant when compared to TNF-α-treated cells (**p<0.01, ***p<0.001). Values represent mean ± SD based on three independent experiments performed in triplicate.

Fig 5: Depletion of H19 facilitates inflammatory activation of ECs. (A) The expression of ICAM-1 and VCAM-1 upon H19 depletion in unstimulated HUVECs was analysed by qRT-PCR 48 h after transfection. Results are represented as mean ± SEM from n = 7 and n = 12 biological replicates per group. Unpaired t-test (ICAM-1) and the Mann–Whitney test (VCAM-1) were used to determine statistical significance. (B) The cell surface abundance of ICAM-1 and VCAM-1 on HUVECs was analysed by flow cytometry 48 h after siRNA-mediated depletion of H19 and 16 h after stimulation with 100 ng/mL IL-6 and sIL6-Rα. Results are represented as mean ± SEM from n = 6 biological replicates per group. Unpaired t-test was used to determine statistical significance. (C) Adhesion of monocytes to HUVECs was measured upon siRNA-mediated depletion of H19 and stimulation with 100 ng/mL IL-6 and sIL6-Rα for 16 h in HUVECs, 48 h after transfection. Results are represented as mean ± SEM from n = 9 biological replicates per group. Unpaired t-test was used to determine statistical significance. (D) The number of CD45+ cells in the soleus muscle of endothelial-specific inducible H19iEC-KO mice and Ctrl littermates was analysed 21 days after hindlimb ischaemia surgery. CD45+ cells are displayed in red, cell membranes are displayed in white. The scale bar denotes 100 µm. Results are represented as mean ± SEM from five mice per group. Unpaired t-test was used to determine statistical significance. (E) H19 expression in wild-type HUVECs was analysed by qRT-PCR at 1 h, 2 h, 4 h, 8 h, and 24 h after stimulation with tumor necrosis factor α. Results are represented as mean ± SEM from n = 5 to n = 7 biological replicates per group. A post-test for linear trend was used to determine statistical significance.; *P < 0.05, **P < 0.01, and ***P < 0.001.