Endothelial dysfunction caused by circulating microparticles from patients with metabolic syndrome

Abstract

Microparticles are membrane vesicles that are released during cell activation and apoptosis. Elevated levels of microparticles occur in many cardiovascular diseases; therefore, we characterized circulating microparticles from both metabolic syndrome (MS) patients and healthy patients. We evaluated microparticle effects on endothelial function; however, links between circulating microparticles and endothelial dysfunction have not yet been demonstrated. Circulating microparticles and their cellular origins were examined by flow cytometry of blood samples from patients and healthy subjects. Microparticles were used either to treat human endothelial cells in vitro or to assess endothelium function in mice after intravenous injection. MS patients had increased circulating levels of microparticles compared with healthy patients, including microparticles from platelet, endothelial, erythrocyte, and procoagulant origins. In vitro treatment of endothelial cells with microparticles from MS patients reduced both nitric oxide (NO) and superoxide anion production, resulting in protein tyrosine nitration. These effects were associated with enhanced phosphorylation of endothelial NO synthase at the site of inhibition. The reduction of O2(-) was linked to both reduced expression of p47 phox of NADPH oxidase and overexpression of extracellular superoxide dismutase. The decrease in NO production was triggered by nonplatelet-derived microparticles. In vivo injection of MS microparticles into mice impaired endothelium-dependent relaxation and decreased endothelial NO synthase expression. These data provide evidence that circulating microparticles from MS patients influence endothelial dysfunction.

Figure 1

Circulating MP levels in patients with MS compared to healthy patients. Circulating MP levels (A) and different populations: procoagulant- (annexin V⁺, B), platelet- (CD41⁺, C), endothelium- (CD146⁺, D), and erythrocyte-derived (CD235a⁺, E) MPs, from healthy subjects (HS, n = 30) and MS patients (MS, n = 32). Results are expressed as events/μL of plasma (PFP) and given as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2

MPs from MS patients decrease NO production in endothelial cells via inhibition of NOS activity. Cells were incubated for 24 hours in the presence of vehicle (CTL), MPs from healthy subjects (HSMPs), or in the presence of MPs from MS patients (MSMPs). A: Quantification of the amplitude of the NO-Fe(DETC)₂ complex signal in human endothelial Eahy 926 cells. Values are expressed in units of amplitude/μg/μl of proteins of samples (n = 6). B–F: Western blotting using antibodies raised against eNOS (B), phospho-eNOS Ser 1177 (C), phospho-eNOS Thr 495 (D), or caveolin-1 (F). Immunoblots were quantified by densitometric analysis. Data are representative of four separate blots, and the densitometry values are expressed in arbitrary units (A.U.) as mean ± SEM. E: The ratio of phosphorylation of eNOS at the inhibitor site (Thr 495) and activation site (Ser 1177) is expressed in arbitrary units (A.U.) as mean ± SEM. *P < 0.05 versus CTL, **P < 0.01 versus CTL and HSMPs for (E) and ^††P < 0.01 versus HSMPs.

Figure 3

MPs from MS patients reduce O₂⁻ release in endothelial cells. Cells were incubated for 24 hours in the presence of either vehicle (CTL), MPs from healthy subjects (HSMPs), or in the presence of MPs from MS patients (MSMPs). A: Quantification of the amplitude of the O₂⁻-CMH complex signal in human endothelial Eahy 926 cells expressed in units of amplitude/μg/μl of proteins of samples as mean ± SEM (n = 6). B--I: Western blotting using antibodies raised against: NOX-1 (B), NOX-2 (C), NOX-4 (D), p47^phox (E), p67^phox (F), Mn SOD (G), Cu/Zn SOD (H), extracellular SOD (I). Immunoblots were quantified by densitometric analysis. Data are representative of five separate blots, and the densitometry values are expressed in arbitrary units (A.U.) as mean ± SEM. *P < 0.05 versus CTL and ^†P < 0.05 versus HSMPs.

Figure 4

MPs from MS patients increase nitration of proteins in endothelial cells. Cells were incubated for 24 hours in the presence of vehicle (CTL), MPs from healthy subjects (HSMPs), or in the presence of MPs from MS patients (MSMPs). A: The protein nitration was evaluated by Western blotting using antibody raised against nitrotyrosine-modified proteins. B: Immunoblots were quantified by densitometric analysis. Data are representative of four separate blots, and the densitometry values are expressed in arbitrary units (A.U.) as mean ± SEM. *P < 0.05 versus CTL.

Figure 5

MPs from MS patients impair endothelium-dependent relaxation in mouse aorta. A: Acetylcholine (Ach)-induced relaxation in vehicle- (CTL), healthy subjects MP- (HSMPs), and MS patients MP (MSMPs)-treated mouse aorta (n = 5 to 8). Results are expressed as a percentage of relaxation of U46619-induced precontraction. Western blotting of mouse aorta using antibodies raised against: eNOS (B), caveolin-1 (C). Immunoblots were quantified by densitometric analysis. Data are representative of four separate blots, and the densitometry values are expressed in arbitrary units (A.U.) as mean ± SEM. ***P < 0.001, **P < 0.01 versus CTL; ^†P < 0.05, ^††P < 0.01 versus HSMPs.

Figure 6

Nonplatelet-derived MS MPs decrease NO production in endothelial cells. Endothelial cells were incubated 24 hours with vehicle (anti-CD61 microbeads alone), platelet- or nonplatelet-derived MSMPs. Quantification of the amplitude of the NO-Fe(DETC)₂ complex signal in cells is expressed in units of amplitude/μg/μl of proteins of samples as mean ± SEM (n = 5). *P < 0.05.