Fig 2: Airn maintained LSEC differentiation in vitro. A Primary LSECs were transfected with siAirn-1, siAirn-2, , 20μm. D Primary LSECs were infected with LV-Airn and LV-Control for 72 h. The RNA level of Airn, Vefgr2, eNos, Lyve-1, VE-cadherin, Angpt-2, and Laminin was detected by qRT-PCR. E The protein level of VEGFR2 and eNOS was determined by western blot and quantitatively compared with GAPDH as a reference control. F The expression of VE-Cadherin and LAMININ was determined by confocal microscopy and quantitatively compared. DAPI-stained nuclei blue; scale bar, 20 μm. The data are expressed as the mean ± SD for at least triplicate experiments, *p<0.05 stands for vs siRNA-Control or LV-Control

Fig 3: Effects of matrine on eNOS activity and PI3K/Akt/eNOS pathway‐related protein expression in ox‐LDL exposed HUVECs. A, Changes in eNOS activity in HUVECs exposed to ox‐LDL with or without matrine pretreatment along with matrine and/or LY‐294002/L‐NAME as measured using an eNOS activity assay kit. B‐E, Phosphorylation levels of Ser473Akt, Ser1177eNOS, Thr495eNOS, total Akt and the eNOS protein were measured by Western blotting. Representative images of three experiments; densitometric analysis of phosphorylated proteins was normalized to that of total proteins. Data are expressed as the mean ± SD of three independent experiments, *P < 0.05, **P < 0.01

Fig 4: ESM1 regulates CX40 expression. (A): Heat map of RNA level differences between induced pluripotent stem‐endothelial cells (iPS‐ECs) overexpressing ESM1 (EX‐ESM1) compared with control iPS‐ECs (EX‐mCherry). (B): Overexpression of ESM1 leading to a significant increase in CX40 48 hours after transfection with EX‐ESM1 compared with control (EX‐mCherry; data are means ± SEM [n = 3]; *, p < .05; **, p < .01). (C): Knockdown of ESM1 leading to a significant decrease in CX40 72 hours after lentiviral transduction with shESM1 compared with non‐targeting control (shNT; data are means ± SEM [n = 3]; **, p < .01; ***, p < .001). (D): Western blots showing increased protein levels in ESM1, eNOS, CX40, and NFKB 48 hours after ESM1 overexpression. (E): Immunofluorescent images of cells costained with ESM1 (red), CX40 (green), and DAPI (blue). Scale bars are 50 μm. The data presented are representative or means (±SEM) of three independent experiments.

Fig 5: ESM1 regulates endothelial cell (EC) marker expression in ECs from induced pluripotent stem (iPS) cells and ESM1 signaling. (A): Real‐time PCR data showing comparison of ESM1 mRNA expression levels between iPS cells, iPS‐ECs (at 3, 6, and 9 days of EC differentiation), and human umbilical vein endothelial cells (HUVECs; data are means ± SEM [n = 3]; *, p < .05; **, p < .01). (B): Immunofluorescent images showing costaining of ESM1 (red), EC markers KDR, eNOS, CD144 (green), and DAPI (blue). Scale bars are 50 μm. (C): Representative immunofluorescent images of iPS‐ECs overexpressing EX‐mCherry (red) and EX‐ESM1 (red) 48 hours after transfection with the corresponding plasmids. Scale bars are 100 μm. (D): Forty‐eight hours after ESM1 overexpression, a significant increase in mRNA expression of EC markers KDR, CD144, and eNOS was observed (data are means ± SEM [n = 3]; **, p < .01; ***, p < .001). (E): ESM1 protein concentration levels 48 hours after ESM1 overexpression were significantly increased in the cell culture media compared with control, as detected by Luminex assay (data are means ± SEM [n = 3]; *, p < .05). (F): ESM1 knockdown 72 hours after lentiviral transduction with shESM1, compared with nontargeting control (shNT), resulted in significantly decreased mRNA levels of ESM1 and EC markers KDR, CD144, and eNOS (data are means ± SEM [n = 3]; **, p < .01; ***, p < .001). (G): ESM1 protein concentration levels 72 hours after ESM1 knockdown were significantly decreased in the cell culture media compared with control, as detected by Luminex (data are means ± SEM [n = 3]; *, p < .05). (H): Western blot (left panel) and corresponding densitometry (right panel) showing decreased protein levels in ESM1 and eNOS in iPS‐ECs with ESM1 knockdown. The data presented are representative or means (±SEM) of three independent experiments (data are means ± SEM [n = 3]; *, p < .05; **, p < .01).