Fig 2: Exposure to hyperglycemic conditions, cigarette smoke extract and both combined promotes the pro-inflammatory and anti-oxidant defense activation in HCMEC/D3 BBB endothelial cells. a1 PECAM-1 expression in membrane fraction was significantly increased only in endothelial cultures following 12 h exposure to HG and HG + CSE. The magnitude of the effect significantly increased at 24 h exposure showing a consistent additive pattern between tested conditions (b1). a2 12 h HG, CSE and HG + CSE triggered upregulation of ICAM-1 membrane fraction. By contrast, although statistically significant, the upregulation of ICAM-1 did not show a longitudinal response pattern to the tested conditions (b2). c1, c2 Release of IL-6 modestly increased in HG-CSE treated cultures while an additive and more robust increased in IL-8 release was observed under the same conditions at 24 h. d Representative Western blot showing the translocation of Nrf-2 from cytosol to nucleus of HCMEC/D3 cells in response to the above mentioned tested conditions. The graph bars clearly show the nuclear vs. cytosol ratios of Nrf-2 under the tested conditions. n = 3–4 biological replicates. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. control; $ p < 0.05, $$ p < 0.01, $$$$ p < 0.0001 vs. HG and # p < 0.05, #### p < 0.0001 vs. CSE.

Fig 3: Working model: TNFa stimulation increases the association of MLKL and RBM6.Consequently, MLKL-RBM6 complex stabilizes the mRNA of ICAM1 VCAM1 and E-selectin and promotes the adhesion molecules protein expression.

Fig 4: Exosome-transmitted miR-25 increases the levels of inflammatory factors in endothelial cells. (A) Bars show relative luciferase activity of NFκB in HEK293 cells treated with exosomes isolated from H. pylori-infected or uninfected GES-1 cells. (B) Bars show relative mRNA expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), interleukin 6 (IL-6), and monocyte chemoattractant protein-1 (MCP-1) in HUVECs with or without H. pylori infection. (C,D) Bars show protein levels of IL-6 and MCP-1 released by HUVECs treated with exosomes isolated from H. pylori-infected or uninfected GES-1 cells. (E) Western blotting shows protein expression of VCAM-1 and ICAM-1 in HUVECs treated with exosomes isolated from H. pylori-infected or uninfected GES-1 cells. GAPDH served as the internal reference. (F) Bars show relative luciferase activity of NFκB in HEK293 cells transfected with the miR-25 negative control (NC) or miR-25 mimics. (G) Bars show mRNA expression of VCAM-1, ICAM-1, IL-6, and MCP-1 in HUVECs transfected with the miR-25 NC or miR-25 mimics. (H) Bars show relative luciferase activity of NFκB in HEK293 cells treated with exosomes isolated from GES-1 cells transfected with the miR-25 NC, mimics, and miR-25 inhibitors. (I) Bars show mRNA expression of VCAM-1, ICAM-1, IL-6, and MCP-1 in HUVECs treated with exosomes isolated from GES-1 cells transfected with the miR-25 NC, mimics, and miR-25 inhibitors. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig 5: The expression of ICAM1 was significantly different between naturally developed big and small aggregates of the H9 cells. A Representative images of big and small aggregates of the H9 cells on day 5 in 3D suspension culture condition without any extra treatment; these two groups were separated by natural settlement. Scale bar = 200 µm. B Comparison of average diameter of big and small aggregates of the H9 cells on day 5. C Diameter distribution of big and small aggregates of the H9 cells on day 5. D–F Gene expression analysis by qRT-PCR for pluripotent genes (D), CAMs-related genes (E), and ICAM1 (F), relative gene expression represents data normalized to GADPH and expressed relative to untreated H9 aggregates. G The protein levels of E-cad and ICAM1 were determined by western blotting in big and small aggregates of the H9 cells. H, I The densitometry for the protein levels of E-cad (H) and ICAM1 (I) were quantitated in G; GAPDH was used as a loading control. Data represent the mean ± SD. *P < 0.05 and **P < 0.01 and ***P < 0.001