Fig 2: A Timeline of gene expression of SMPD1-3 in HCAECs after hyperglycemic injury, presented as 2-ddCT vs GAPDH, n = 6. B Acidic and neutral SMase enzyme activity in HCAECs after 72 h of hyperglycemic injury, n = 5–6. C + D Immunoblotting for nSMase2 and ß-Actin after 72 h of hyperglycemic injury, with quantification, n = 3. E MTT viability assay in HCAECs that were treated with lEVs from HCAECs after hyperglycemic injury, n = 6. F Flow-cytometric analysis of the induction of apoptosis in HCAECs that were treated with lEVs from HCAECs after hyperglycemic injury, (right) representative dot blots, n = 5. G MTT viability assay in HCAECs after treatment with mannitol, n = 6. H Flow-cytometric analysis of the induction of apoptosis in HCAECs that were treated with lEVs from HCAECs after osmotic injury with mannitol, n = 3. All data are presented as individual experiments with the mean ± SEM; n.s. not significant, *p < 0.05, ANOVA + Bonferoni’s multiple comparison test were used for A, E, G, and unpaired t-Test for B, D, F, H

Fig 3: Endothelial NLRP3 inflammasome activation in the carotid arteries of EC-specific Smpd1 transgenic mice during hypercholesterolemia. A: Representative fluorescent confocal microscope images displaying the yellow dots or patches showing the colocalization of FLICA (green) with vWF (red). B: The summarized data show the colocalization coefficient of FLICA with vWF. C: Representative microscopic images of tissue slide with immunohistochemical staining that shows IL-1β accumulation on the arterial wall. D: Summarized data showing the density of IL-1β stained with selective anti-IL-1β antibody. Data are expressed as means ± SEM, n = 5. ∗P < 0.05 is defined as significant. The scale bar represents 20 μm or 200 μm for A and 100 μm for C.

Fig 4: Formation of ASM-ceramide MR signaling platforms in the primary cultures of ECs from EC-specific Smpd1 transgenic mice. A: Representative fluorescent confocal microscopic images showing the colocalization of MR marker, flotillin (red) with ASM (green). B: Summarized data showing the colocalization coefficient of flotillin with ASM. C: Representative fluorescent confocal microscopic images showing the colocalization of MR marker, flotillin (red) with ceramide (green). D: Summarized data showing the colocalization coefficient of flotillin with ceramide. Data are expressed as means ± SEM, n = 5. ∗P < 0.05 is defined as significant. The scale bar represents 10 μm.

Fig 5: Effects of ASM blockade on NLRP3 inflammasome activation in the primary cultures of ECs from EC-specific Smpd1 transgenic mice. A: Representative Western blot gel documents showing the expression of cleaved caspase-1 in the primary cultures of carotid ECs from endothelium-specific Smpd1 transgenic mice. B: Summarized data showing the expression of cleaved caspase-1 in the primary cultures of carotid ECs from endothelium-specific Smpd1 transgenic mice. C: Summary data showing caspase-1 activity in the primary cultures of carotid ECs from endothelium-specific Smpd1 transgenic mice. D: Summarized data showing IL-1β production in the primary cultures of carotid ECs from endothelium-specific Smpd1 transgenic mice. Data are expressed as means ± SEM, n = 5. ∗P < 0.05 is defined as significant.