Monocyte Derived Microvesicles Deliver a Cell Death Message via Encapsulated Caspase-1

Figure 4

Microvesicle encapsulated exogenous caspase-1 directly induces VSMC apoptosis.

A) Monocytes were either pretreated with YVAD-cmk or DMSO for 30 min. Cells were then rinsed with PBS and then stimulated with LPS (1 µg/ml) for 15 min. Microvesicles were isolated from the conditioned medium. Additionally, VSMCs were also pretreated with either saline, or with IL-1RA, IL-18 bp or sFAS for 30 min (* indicates VSMC pretreatments). VSMCs were then subjected to the isolated microvesicles from LPS stimulated monocytes. (▪) VSMC+ MV from control monocytes; () VSMC+ MV from LPS/YVAD monocytes; () VSMC treated with IL-1RA+ MV from LPS treated monocytes; (□) VSMC treated with IL-18 bp + MV from LPS treated monocytes; () VSMC treated with sFAS+ MV from LPS treated monocytes. Apoptosis of VSMC was measured using Annexin V/PI assay. Graph represents average of n = 2 experiments. B) HEK-293 cells were transfected with either wild type or mutant caspase-1 (0.2 µg) along with ASC (0.2 µg) using Lipofectamine 2000. Transfection was normalized using vector controls. Supernatants were collected from the transfected cells and subjected to VSMC. Apoptosis was measured using Annexin V/PI assays. THP-1 cells were lysed and cell lysates were either kept at 4°C or incubated at 30°C for 1 h. Cell lysates were then centrifuged at 15,000 X g for 20 min. Both lipid and non-lipid fractions were measured for caspase-1 activity using C) immunoblot and D) WEHD-enzymatic assay. E) Both fractions were also subjected to VSMC and cell death was analyzed by Annexin V/PI assays.

doi: https://doi.org/10.1371/journal.pone.0007140.g004