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. 2014 Sep;66(9):2532-2544.
doi: 10.1002/art.38703.

V体育官网 - Neutrophil extracellular trap-derived enzymes oxidize high-density lipoprotein: an additional proatherogenic mechanism in systemic lupus erythematosus

Carolyne K Smith #  1 , Anuradha Vivekanandan-Giri #  2 , Chongren Tang  3 , Jason S Knight  2 , Anna Mathew  2 , Robin L Padilla  4 , Brenda W Gillespie  4 , Carmelo Carmona-Rivera  1 , Xiaodan Liu  2 , Venkataraman Subramanian  5 , Sarfaraz Hasni  1 , Paul R Thompson  5 , Jay W Heinecke  3 , Rajiv Saran  2   4 , Subramaniam Pennathur  2 , Mariana J Kaplan  1
Affiliations

Neutrophil extracellular trap-derived enzymes oxidize high-density lipoprotein: an additional proatherogenic mechanism in systemic lupus erythematosus

Carolyne K Smith et al. Arthritis Rheumatol. 2014 Sep.

"V体育ios版" Abstract

Objective: Oxidative stress and oxidized high-density lipoprotein (HDL) are implicated as risk factors for cardiovascular disease (CVD) in systemic lupus erythematosus (SLE). Yet, how HDL is oxidized and rendered dysfunctional in SLE remains unclear VSports手机版. Neutrophil extracellular traps (NETs), the levels of which are elevated in lupus, possess oxidant-generating enzymes, including myeloperoxidase (MPO), NADPH oxidase (NOX), and nitric oxide synthase (NOS). We hypothesized that NETs mediate HDL oxidation, impairing cholesterol efflux capacity (CEC). .

Methods: Plasma MPO levels and CEC activity were examined in controls and lupus patients, and 3-chlorotyrosine (MPO specific) and 3-nitrotyrosine (derived from reactive nitrogen species) were quantified in human HDL. Multivariable linear models were used to estimate and test differences between groups V体育安卓版. HDL was exposed to NETs from control and lupus neutrophils in the presence or absence of MPO, NOX, NOS inhibitors, and chloroquine (CQ). Murine HDL oxidation was quantified after NET inhibition in vivo. .

Results: SLE patients displayed higher MPO levels and diminished CEC compared to controls. SLE HDL had higher 3-nitrotyrosine and 3-chlorotyrosine content than control HDL, with site-specific oxidation signatures on apolipoprotein A-I. Experiments with human and murine NETs confirmed that chlorination was mediated by MPO and NOX, and nitration by NOS and NOX. Mice with lupus treated with the NET inhibitor Cl-amidine displayed significantly decreased HDL oxidation V体育ios版. CQ inhibited NET formation in vitro. .

Conclusion: Active NOS, NOX, and MPO within NETs significantly modify HDL, rendering the lipoprotein proatherogenic. Since NET formation is enhanced in SLE, these findings support a novel role for NET-derived lipoprotein oxidation in SLE-associated CVD and identify additional proatherogenic roles of neutrophils and putative protective roles of antimalarials in autoimmunity. VSports最新版本.

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"VSports" Figures

Figure 1
Figure 1. HDL isolated from SLE patients is significantly oxidized and dysfunctional
Plasma and HDL were purified from healthy controls (N=20), and SLE patients (N=40). (A) ABCA1-mediated cholesterol efflux capacity (determined in J774 cells loaded with radiolabeled [3H]-cholesterol and then incubated with SLE or control apoB depleted plasma). (B) MPO-specific 3-chlorotyrosine (Cl-Tyr) levels and RNS- and MPO-mediated 3-nitrotyrosine (N-Tyr) levels in lupus and control HDL (in log base 10). The length of the box defines the interquartile range (IQR). Medians (IQR) are on raw scale. (C) Box blots display distributions of isotopically labeled chlorinated or nitrated tyrosines. Native apoA-1 peptides were spiked into HDL samples from control (N=20, white) and SLE patients (N=40, shaded). Extracted ion chromatograms from specific fragment ions were used for quantitative analysis. Values are expressed in log base 10 and significant differences between controls and SLE are denoted by an asterisk (*p<0.05). The length of the box defines the IQR. (D) For all subjects, correlations between levels of N-Tyr and Cl-Tyr HDL oxidation, MPO and Cl-Tyr HDL oxidation, MPO and N-Tyr HDL oxidation, and MPO and chlorination levels at tyrosine 192 within apoA1 from patients. Figure D displays the scatter plot and least square regression line.
Figure 2
Figure 2. NET-derived MPO, NOS and NOX modify human HDL
Immunofluorescent staining and immunoblotting was used to identify possible enzymatic sources of HDL nitration. (A) NOX subunits, p22 and p47, and two NOS isoforms, eNOS and iNOS, were detected in human NETs. Control neutrophil and lupus LDG NETs were added to control, unoxidized HDL. Resulting levels of (B) MPO-specific Cl-Tyr, and (C) RNS-mediated N-Tyr oxidation were quantified by mass spectrometry. 3-AT, NMMA and DPI were added to block MPO, NOS and NOX activity, respectively. The PAD inhibitor Cl-Am was added to LDGs to block spontaneous NETosis. For control neutrophils, absence of PMA stimulation was used as a negative control for NETosis. (N= 6/group; ** p<0.007, *** p=0.0002, **** p< 0.0001. Data are displayed as mean ± SEM).
Figure 3
Figure 3. Chloroquine abrogates NET formation
(A) Control neutrophils or lupus LDGs purified from whole blood and treated with or without chloroquine (CQ) were assessed for DNA externalization via Sytox assay. Immunofluorescent staining of these treated cells was used to quantify (B) NET production and (C) MPO, iNOS and p22 externalization on the NETs. (N=5/group; * p=0.01, *** p=0.0001, **** p<0.0001. Data are displayed as mean ± SEM.)
Figure 4
Figure 4. NET-derived NOS and NOX induce HDL oxidation in mice, while NET inhibition decreases HDL oxidation in vivo
Immunofluorescent staining and immunoblotting was used to identify possible enzymatic sources of HDL nitration. (A) NOX subunits, p22 and p47, and two NOS isoforms, eNOS and iNOS, were detected in murine NETs. Control Balb/c and lupus NZM NETs were added to control, unoxidized murine HDL. (B) Resulting levels of MPO-specific Cl-Tyr oxidation in the absence or presence of isolated NETs, as quantified by mass spectrometry (N=4/group). (C) Resulting levels of NOS- and NOX-mediated N-Tyr oxidation as quantified by mass spectrometry (N=8/group). 3-AT, NMMA and DPI were added to block MPO, NOS and NOX activity, respectively. Absence of PMA stimulation was used as a negative control for NET formation. (D) Levels of plasma and HDL N-Tyr content in 26-week old NZM mice that received daily injections of PBS (N= 10) or Cl-Am (N=10) for 14 weeks. (For C, comparisons were made within the same mouse strain; ** p<0.01, **** p<0.0001. Data are displayed as mean ± SEM).
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