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. 2012 Nov;16(11):2777-88.
doi: 10.1111/j.1582-4934.2012.01595.x.

Monocytic microparticles promote atherogenesis by modulating inflammatory cells in mice

Friedrich Felix Hoyer  1 , Meike Kristin Giesen, Carolina Nunes França, Dieter Lütjohann, Georg Nickenig, Nikos Werner
Affiliations

Monocytic microparticles promote atherogenesis by modulating inflammatory cells in mice

Friedrich Felix Hoyer et al. J Cell Mol Med. 2012 Nov.

Abstract

Microparticles (MP) are generated during a vast number of biological processes such as inflammation, cell activation and apoptosis. Increasing evidence points towards an important role of MP as intercellular messengers of biological information. During atherogenesis, monocytes infiltrate the vascular wall and foster inflammation, accompanied by the release of monocytic MP (mono-MP). To date, only little is known about the biological function of mono-MP in the vascular wall. Here, we investigated the role of mono-MP during atherogenesis. Mono-MP were generated by starvation of THP-1 monocytes and isolated by ultracentrifugation. To investigate whether mono-MP influence atherogenesis, ApoE(-/-) mice were fed a high-fat, cholesterol-rich diet for 8 weeks and simultaneously treated with mono-MP or vehicle twice a week. Mice treated with mono-MP showed significantly increased monocyte and T-cell infiltration into the vessel wall, as assessed by Moma-2 and CD3 staining, and enhanced plaque formation, as assessed by oil-red-O staining. However, atherosclerotic plaque composition was not influenced by mono-MP application. In vitro, incubation of mono-MP with murine macrophages and endothelial cells resulted in the uptake of calcein-labelled mono-MP. Mono-MP uptake initiated the generation of intracellular reactive oxygen species. Murine macrophages pre-treated with mono-MP showed significantly enhanced expression of CCR2, migration to MCP-1 and increased release of pro-inflammatory interleukin-6. Co-incubation of mono-MP with endothelial cells resulted in significantly increased expression of ICAM-1, as assessed by RT-PCR and ELISA. Mono-MP act as paracrine messengers that intensify inflammation during atherogenesis by stimulating vascular-bound and inflammatory cells in their vicinity.

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Figures

Fig. 1
Fig. 1
Blood pressure, heart rate, total cholesterol. Blood pressure, heart rate and cholesterol levels were determined after 8 weeks of a high-fat, cholesterol-rich diet. No differences were observed between both groups. (A) Cholesterol [mono-MP: 882.8 ± 92.42 mg/dl (n = 7) versus vehicle: 1032 ± 50.97 mg/dl (n = 5), P > 0.05] (B) Heart rate [mono-MP: 673.8 ± 13.54 bpm (n = 7) versus vehicle: 662.4 ± 7.3 bpm (n = 5), P > 0.05] (C) Blood pressure [mono-MP: 134.2 ± 4.1 mmHg (n = 7) versus vehicle: 140.7 ± 2.4 mmHg (n = 5), P > 0.05].
Fig. 2
Fig. 2
Atherosclerotic lesion formation and leucocyte infiltration. After 8 weeks of a high-fat, cholesterol-rich diet atherosclerotic burden was assessed in the aortic sinus by means of oil-red-O staining. Atherosclerotic lesion formation was increased in ApoE−/− mice treated with 1 × 106 mono-MP twice a week (n = 7) compared with ApoE−/− mice treated with vehicle (n = 5) (0.29 ± 0.03 mm2 versus 0.19 ± 0.03 mm2, * indicates P < 0.05) (A). Monocyte/macrophage accumulation in the vascular wall was assessed by means of Moma-2 staining. ApoE−/− mice treated with mono-MP showed significantly increased vascular accumulation of macrophages compared with control (0.35 ± 0.03 versus 0.25 ± 0.03, ratio Moma-2 positive area/vessel wall, * denotes P < 0.05) (B). The accumulation of T cells was determined using anti-CD3 staining. Mono-MP treatment significantly increased CD3+ T-cell accumulation (193 ± 25 versus 103 ± 14 CD3+ cells/vessel wall, *P < 0.05). T cells are depicted as little red spots (C). Mono-MP application did not influence plaque composition such as collagen content, as assessed by picrosirius red staining (0.49 ± 0.05 versus 0.48 ± 0.05, ratio collagen/vessel wall, ns indicates P > 0.05) (D).
Fig. 3
Fig. 3
Mono-MP Uptake. (A) To investigate whether mono-MP were incorporated into target cells, cell culture experiments were performed. Calcein-labelled mono-MP were co-cultured with murine macrophages and engulfed in a time-dependent manner with a peak at 4 hrs of co-cultivation. Results were analysed using an ELISA reader at 485 nm (excitation) and 530 nm (emission). Heat denaturation of mono-MP was associated with a significant reduction in mono-MP uptake (4 hrs mono-MP: 277 ± 26% versus heat-inactivated mono-MP: 157 ± 6%, n = 4, *P < 0.05). (B) Calcein-labelled mono-MP uptake into macrophages. Calcein-labelled mono-MP are represented in green, CD68 and Cy3 staining of macrophages in red, the cell nucleus is coloured in blue by the use of Dapi. (C) PKH-26-labelled mono-MP uptake into macrophages. PKH-26 fluoresces in red, CD68 and Cy2-stained macrophages appear in green, cell nucleus in blue (Dapi). (D) Calcein-labelled mono-MP uptake into HUVEC. Calcein-labelled mono-MP appear in green, HUVEC counterstained with CD31 and Cy3 are demonstrated in red, the cell nucleus is represented in blue (Dapi).
Fig. 4
Fig. 4
Migration of macrophages and CCR2. Chemotactic and migratory effects of mono-MP on murine macrophages were measured via modified Boyden chamber experiments. Murine macrophages pre-treated with mono-MP or control were allowed to migrate to mono-MP or control (A). No differences were detected between both groups [mono-MP to mono-MP: 111.2 ± 3.3%; mono-MP to heat-inactivated mono-MP: 108.8 ± 5.8%; mono-MP to vehicle (control): 100.0 ± 3.2%, n = 5, P > 0.05]. Next, migration to MCP-1 of murine macrophages pre-treated with mono-MP or control was determined (B). Migration of murine macrophages to MCP-1 was significantly enhanced after 24 hrs co-incubation with mono-MP compared with control (mono-MP: 156 ± 7% versus hi mono-MP: 115 ± 5%, n = 5, * denotes P < 0.05). CCR2 levels were analysed using quantitative RT-PCR. Macrophages, stimulated with mono-MP for 24 hrs, showed significantly increased CCR2 levels, compared with control (macrophages + mono-MP: 293.5 ± 50.21% versus macrophages+hi mono-MP: 106.5 ± 49.35%, n = 14, *P < 0.05) (C).
Fig. 5
Fig. 5
Apoptosis and proliferation of murine macrophages. Apoptotic effects of mono-MP on murine macrophages were assessed and analysed using a caspase-3 assay. Murine bone marrow-derived macrophages were cultured and stimulated with mono-MP or vehicle for 24 hrs on day 6 (1:1-relation mono-MP: cells). Mono-MP application did not influence apoptosis of target cells. Apoptosis of macrophages induced by either 1 μmol or 5 μmol camptothecin was not further influenced by mono-MP pre-stimulation as well (mono-MP: 1484 ± 183 versus heat inactivated mono-MP: 1346 ± 127, DEVD-Cleavage in AU, n = 4, P > 0.05) (A). The influence of mono-MP on the proliferation of murine macrophages was analysed using BrdU-FACS experiments. Macrophages were incubated with BrdU on day 6 of differentiation and concomitant stimulated with mono-MP or heat-inactivated mono-MP (1:1-relation mono-MP: cells). Mono-MP stimulation did not influence proliferation of murine macrophages: BrdU (control): 100.0 ± 4.2%; BrdU + mono-MP: 97.63 ± 21.76%; BrdU + heat-inactivated mono-MP: 74.65 ± 7.29%; BrdU + vehicle: 103.5 ± 10.42%, n = 3, P > 0.05 (B).
Fig. 6
Fig. 6
IL-6 release of murine macrophages. Mono-MP treatment of murine macrophages for 24 hrs induced a significantly increased liberation of IL-6 compared with control, as assessed by ELISA [mono-MP: 238.2 ± 17.64% versus vehicle (control): 100.0 ± 3.7%, n = 3, *P < 0.05].
Fig. 7
Fig. 7
Generation of reactive oxygen species in murine macrophages.To assess the influence of mono-MP on the generation of intracellular oxidative stress in macrophages, C2′,7′-dichlorodihydrofluorescin-diacetate experiments were performed. Murine bone marrow-derived macrophages were stimulated with mono-MP or vehicle for 4 hrs on day 7 of differentiation. Cells pre-treated with mono-MP showed significantly increased intracellular ROS generation compared with control (mono-MP: 120.2 ± 4.9% versus heat inactivated mono-MP: 104.6 ± 3.9%, n = 4, * denotes P < 0.05).
Fig. 8
Fig. 8
Mono-MP up-regulate ICAM-1. HUVEC pre-treated with mono-MP for 24 hrs showed significantly enhanced ICAM-1 level compared with control in quantitative RT-PCR (HUVEC+mono-MP: 313.5 ± 60.14% versus HUVEC+ heat-inactivated mono-MP: 130.8 ± 23.60%, n = 4, P < 0.05) (A). ELISA experiments confirmed the upregulation of ICAM-1, measuring soluble ICAM-1 in HUVEC supernatant after stimulation with mono-MP or control. ICAM-1 generation was significantly increased after mono-MP stimulation compared with control (mono-MP: 0.33 ± 0.024 ng/ml versus hi mono-MP: 0.24 ± 0.018 ng/ml versus vehicle: 0.32 ± 0.10 ng/ml, n = 4, * denotes P < 0.05) (B).
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