Autologous apoptotic cell engulfment stimulates chemokine secretion by vascular smooth muscle cells

Abstract

Apoptosis of vascular smooth muscle cells (VSMCs) occurs in vivo under both physiological and pathological settings. The clearance of apoptotic cells may be accomplished in part by the surrounding normal VSMCs. However, the fate of internalized apoptotic cells, the rate of intracellular degradation, and the consequences of these processes to VSMC biology are unknown. Electron microscopy and confocal fluorescence imaging showed that rat VSMCs effectively bound and internalized autologous apoptotic VSMCs in vitro. Within 2 hours, the internalized apoptotic cells were delivered to lysosomes, and the majority of these internalized cells and their proteins were efficiently degraded by 24 hours. After degradation was completed, the phagocytic VSMCs remained viable with normal rates of proliferation. Clearance of apoptotic cells by VSMCs did not induce the release of vascular wall matrix proteases but was associated with a 1.6-fold increase in transforming growth factor-beta1 release. Interestingly, clearance of apoptotic cells stimulated VSMCs to secrete monocyte-chemoattractant protein-1 and cytokine-induced neutrophil chemoattractant. The coordinated release of transforming growth factor-beta1 and chemokines suggests that autologous apoptotic cell clearance stimulates VSMCs to release molecules that specifically recruit professional phagocytes while simultaneously dampening the inflammatory response and preventing vascular injury.

Figure 1

Phagocytosis of autologous apoptotic cells by VSMCs. a: Representative EM image of an apoptotic cell generated from VSMCs treated with LPS, TNF-α, IL-1β, and IFN-γ. Note the nuclear fragment (n) containing condensed chromatin (arrows). b to f: EM images of VSMCs were incubated with apoptotic cells for 2 hours (*, apoptotic cell; arrowheads, lysosomes containing apoptotic cell fragments). Bars = 2 μm in each frame. f: Magnified image of inset in e showing immunogold labeling with anti-p53 (arrows), confirming that these were apoptotic cell fragments.

Figure 2

Phagocytosis of apoptotic cells by VSMCs. a to h: Sequence of confocal optical sections showing apoptotic cells engulfed by a naïve VSMC in culture after 2 hours of co-incubation. The cytoskeleton of the VSMC was labeled with actin-conjugated with Cy3 (red), the nucleus was stained with DAPI (blue), and the apoptotic cell was labeled with FITC-conjugated annexin V (green) before internalization. Bar = 10 μm. i: xz projection of the image stack, showing phagocyted apoptotic cells within the cell (arrowhead).

Figure 3

Apoptotic cells contain iNOS. Apoptotic cells were generated by exposure of rat VSMCs to LPS, TNF-α, IL-1β, and IFN-γ and then fixed and stained with anti-iNOS antibody (a) and DAPI (b). c and d: Merged fluorescence and phase contrast images, respectively. Bar = 2 μm.

Figure 4

Degradation of iNOS and nitrated proteins after apoptotic cell phagocytosis. VSMCs were either untreated (a, f, k, and p) or incubated with apoptotic cells for 2 hours, followed by further incubation of 2 hours (b, g, i, and q), 4 hours (c, h, m, and r), 24 hours (d, i, n, and s), or 48 hours (e, j, o, and t) at 37°C. The cells were then fixed and stained with anti-iNOS (a to e), anti-nitrotyrosine (NTyr; f to j), and DAPI (k to o). Merged images are shown in p to t. At early incubation times, VSMC lysosomes contained both iNOS and nitrated proteins (arrowheads), however, by 24 to 48 hours, most of this material had been completely degraded. Bar = 10 μm.

Figure 5

Time course of lysosomal iNOS degradation. a: VSMCs were incubated for 2 hours at 37°C with apoptotic cells and then further incubated for varying amounts of time 37°C with or without pretreatment with chloroquine. The cells were then harvested, and iNOS expression was determined by immunoblot. GAPDH was used as a protein loading control. b: Densistometric analysis of three independent experiments (mean ± SD). Before incubation with apoptotic cells, VSMCs did not express detectable levels (nd) of iNOS. Chloroquine significantly inhibited iNOS degradation, indicating lysosomal degradation of iNOS. *P < 0.05, analysis of variance with Tukey post hoc analysis.

Figure 6

TGFβ1 and cytokine production by VSMCs. TGFβ1, MCP-1, and CINC-1 release into the culture medium by control and cells incubated for 48 hours with apoptotic cells. The levels of TGFβ1, MCP-1, and CINC-1 were assayed by ELISA and normalized to total cell protein (n = 6). The values represent the mean ± SD. The values for TGFβ1, MCP-1, and CINC-1 in the cells incubated with apoptotic cells were significantly different from controls (*P < 0.05, Student’s t-test).