Fig 1: Neutrophil elastase (NE) promotes foam cell formation by inhibiting macrophage cholesterol efflux capacity. A, NE deficiency results in decreased foam cell formation in vivo. Peritoneal-naive macrophages were isolated from wild-type (WT) and NE-/-/Apolipoprotein E-/- double knockout (NE_KO) mice fed a high-fat diet for 12 weeks and subjected to Oil Red “O” staining. Representative foam cell image and quantitative data of the integrated optical density (IOD) per cell for Oil Red “O” staining are presented (n=5 mice per group). B, NE promotes foam cell formation from bone marrow–derived macrophages (BMMs). Bone marrow monocytes isolated from WT or NE_KO mice were induced to differentiate to BMMs by macrophage colony-stimulating factor, followed by incubation with 5 µg/mL DiI-Ac-LDL in the absence or presence of 1 µg/mL active human neutrophil elastase (HNE; Abcam, ab91099) for 5 hours at 37°C. Cells were fixed and counterstained with 4',6-diamidino-2-phenylindole (DAPI). Representative foam cell image and quantitative data of the ratio of mean fluorescent intensity (MFI) of DiI-Ac-LDL (red) and DAPI (blue) from 10 to 12 images are presented. C, NE protein detection in BMMs conditioned culture medium. Serum-starved WT and NE_KO BMMs were cultured in serum-free fresh RPMI1640 medium for 24 hours. Cell supernatant was harvested and used for NE protein concentration measurement. D, NE secreted from BMMs promotes foam cell formation. NE_KO BMMs were incubated with WT BMMs conditioned culture medium in the presence of 1 µg/mL IgG or anti-NE antibody for 6 hours, then subjected to foam cell formation assay as described in (B). E, NE plays no significant role in DiI-Ac-LDL binding to peritoneal-naive macrophages. F, NE inhibits peritoneal naïve macrophage cholesterol efflux capacity. Peritoneal-naive macrophages were isolated from WT and NE_KO mice and subjected to cholesterol efflux assays using 10 µg/mL Apolipoprotein A-I as cholesterol acceptor. Data presented are an average of 5 independent experiments (n=5 in C, E, and F). *P<0.05 (WT vs NE_KO or HNE treatment vs vehicle control).
Fig 2: The effect of NE on apoptosis, inflammation and activation of pVICs. A TUNEL assay was used to value apoptosis of pVICs treated with HNE for 24 h. Scar bar = 100 µm. B MTT assay detection of proliferation in pVICs treated with NE for 24 h. C–E Western blot analysis of Bcl-2 and Bax protein expression. F, G RT-qPCR detection of RUNX2, a-SMA, Collagen I, TNF-a, IL-6 and IL-1ß mRNA expression in pVICs treated with NE for 12, 24 and 48 h. *P < 0.05, **P < 0.01 vs Blank group. NS meant no significant difference. The measurement data are expressed as the mean ± SD. All experiments were independently repeated three times
Fig 3: Neutrophil elastase (NE) promotes ABCA1 protein degradation in macrophages. A and B, ABCA1 protein but not the mRNA expression level in bone marrow–derived macrophages (BMMs) was inhibited by NE. Bone marrow monocytes isolated from wild-type (WT) or NE-/-/Apolipoprotein E-/- double knockout (NE_KO) mice were induced to differentiate to BMMs by macrophage colony–stimulating factor, followed by serum starvation overnight. Serum-starved BMMs were treated with vehicle control or 1 µg/mL human neutrophil elastase (HNE) for 4 hours at 37°C. Protein and RNAs were harvested and subjected to Western blot (A) and real-time quantitative polymerase chain reaction (B) analyses, respectively. Representative blots (A) and quantitative data of the relative ABCA1 protein (A) or RNA (B) expression levels from 5 independent experiments (n=5) are presented. *P<0.05 (NE_KO vs WT); # P<0.05 (HNE vs vehicle control). C, NE promotes ABCA1 protein degradation. NE_KO BMMs were incubated with vehicle control or 1 µg/mL HNE for 0, 1, 2, 4, or 8 hours in the presence of 10 µg/mL cycloheximide (CHX) as indicated. Representative blots and quantitative data of the relative ABCA1 protein expression levels from 5 independent experiments (n=5) are presented. *P<0.05 (HNE vs vehicle control at the indicated time points), # P<0.05 (vs 0 hour) (2-way ANOVA test with Bonferroni post hoc test). D and E, NE promotes ABCA1 protein degradation, partially through lysosomal system. NE_KO BMMs were incubated with vehicle control or 1 µg/mL HNE for 1 hour in the presence of 10 µg/mL CHX, and in the absence or presence of various inhibitors as indicated. 40 µg/mL Calpeptin, a cell-permeable calpain inhibitor; 100 nmol/L Bortezomib, a reversible inhibitor of the 26S proteasome; 10 µmol/L GM6001, a pan-matrix metallopeptidases inhibitor; 2.5 mmol/L NH 4Cl, a typical lysosomal inhibitor; and 100 µmol/L Chloroquine, a specific inhibitor of the lysosomal system. Data in (D and E) were representative blots and quantitative data of the relative ABCA1 protein expression levels from 5 independent experiments (n=5) presented as median with interquartile range. *P<0.05 (vs vehicle control); # P<0.05 (inhibitors vs vehicle control in the presence of HNE) (Kruskal–Wallis 1-way ANOVA with a Dunn post hoc test).
Supplier Page from Abcam for Native human Neutrophil Elastase protein (Active)