Role of eNOS and NOX containing microparticles in the endothelial
dysfunction in patients with obesity

MP preparation and flow cytometry analysis

MP preparation Citrated blood samples were collected from 46 subjects (14 controls and 32 subjects with obesity) and processed within 2h. Platelet-rich plasma (PRP) was collected after centrifugation at 1,500g for 15min at room temperature (RT). Plateletfree plasma (PFP) was obtained by further centrifugation at 13,000g at RT for 2min, and the MP pellet after ultracentrifugation of the PFP samples at 20,000g, 4°C for 90 min. MP and PFP aliquots were stored at -80°C until analysis (13)

MP quantification by flow cytometry

MP samples were analyzed using an Accuri C6 flow cytometer and software (Accuri Cytometers, Ann Arbor, MI) to define the MP gate and to quantify the absolute numbers of MPs per µl of plasma. Regions corresponding to MPs were identified in forward and side-angle light scatter intensity dot plot representation set at logarithmic gain, based on the diameter using standard microbeads (0.1 and 1.1µm) (14). 

Vascular reactivity measurement

Rat aorta rings from 3-month-old Wistar rats were incubated for 5h in Dulbecco’s modified eagle medium/Krebs-HEPES solution (117mM NaCl, 74.5mM KCl, 84mM NaHCO3, 1.5mM KH2PO4, 1.7mM MgCl2, 21mM HEPES, 11.1mM glucose) in the absence (n=8) or presence of MPs from controls (n=6) or subjects 114 with obesity (n=7). The final MP concentration corresponded to the MP circulating plasma level. Aorta rings were then mounted in organ chambers filled with Krebs solution warmed at 37°C and continuously aerated with a 95%O2-5%CO2 gas mixture. Aorta rings were connected to an isometric force transducer (EMKA Technologies, EMKA Paris, France), linked to an amplifier (EMKA Technologies, EMKA Paris, France) and changes in isometric force were recorded on a computer equipped with a data acquisition system. Resting tension was adjusted to the maximal length tension of 2g and aorta rings were allowed to equilibrate for 1h. Contractions were produced by addition of 10-4 M phenylephrine and the endothelium-dependent response was studied by addition of ACh at different concentrations (5.10-9 to 10-4 M). Finally, 10-5 M sodium nitroprusside (SNP) was used to reach the maximum response and to test the endothelium-independent response (16). MP effect on the ACh-sensitivity of aorta rings was evaluated by calculating the ACh concentration inducing the half-maximal response (EC50). Care and use of laboratory animals were according to the European Community standards. 

Western blot analysis

MP pellets from controls (n=6) and subjects with obesity (n=6) were suspended in lysis buffer (25mM Tris pH=7.6, 150mM NaCl, 1% Triton X100) containing phosphatase inhibitor (Fisher Scientific) and protease inhibitor cocktails (Sigma Aldrich). Proteins were transferred to PVDF membranes and probed with mouse monoclonal anti-eNOS (1:300; BD Biosciences) and anti-eNOS-PSer1177 (1:200; BD Biosciences) and goat polyclonal anti-gp91-phox (1:200; Santa Cruz Biotechnology), anti-p47-phox (1:200; Santa Cruz Biotechnology), anti-NOX4 (1:200; Santa Cruz Biotechnology) and rabbit polyclonal anti-αȕ tubulin (1:γ000; BD Biosciences) antibodies. Immunodetection was carried out using the ECL System 115 (Super Signal West Pico Chemiluminescence Substrate, Thermo Scientific) followed by exposure to X-ray films for revelation. Statistical analysis Data analysis was performed using the SPSS 17.0 software (SPSS Inc, Chicago, IL, USA). Results are expressed as the mean ± SEM. The data normal distribution was tested using the Kolmogorov-Smirnov test. The independentsamples t-test was used after investigation of variances equality by using the Levene’s test. Vasoreactivity data were analyzed using a two-way ANOVA and Dunnett’s multiple comparisons test. Correlations were obtained using the Pearson correlation test. Statistical significance was inferred when the two-tailed p-value was lower than 0.05.