XML Template (2015) 22.4.20152:53pm 112 [601891]

XML Template (2015) [22.4.2015–2:53pm] [1–12]
//blrnas3.glyph.com/cenpro/ApplicationFiles/Journals/SAGE/3B2/ACBJ/Vol00000/150023/APPFile/SG-ACBJ150023.3d (ACB) [PREPRINTER stage]
Original Article
Sucrose non-fermenting AMPK related kinase/Pentraxin 3
and DNA damage axis: a gateway to cardiovascular
disease in systemic lupus erythematosus among
Egyptian patients
Doaa Hussein Zineldeen1, Walaa Arafa Keshk1, Ahmed Hamza Ghazy2and
Amal Mohamed El-Barbary3
Abstract
Background: Systemic lupus erythematosus is a chronic multisystemic autoimmune disease characterized by chronic
inflammatory processes and failure of immune-regulatory mechanisms. Systemic lupus erythematosus is associated with
increased risk for cardiovascular disease. In view of immunometabolic derangements of systemic lupus erythematosus,
we investigated the roles of sucrose non-fermenting AMPK related kinase, Pentraxin 3, and DNA damage in thepathogenesis of systemic lupus erythematosus complicated with cardiovascular disease.Methods: Forty systemic lupus erythematosus women with cardiovascular disease (systemic lupus erythematosus
cases), 40 systemic lupus erythematosus women without cardiovascular disease, and 40 healthy controls were enrolledin this study. Demographic and clinical data were recorded. Plasma concentrations of sucrose non-fermenting AMPKrelated kinase and Pentraxin 3 were immunoassayed. Carotid intima media thickness, atherogenic, and DNA damageindices were also assessed.
Results: Plasma sucrose non-fermenting AMPK related kinase and Pentraxin 3 concentrations were increased in sys-
temic lupus erythematosus cases with cardiovascular disease compared to systemic lupus erythematosus controls and
healthy controls ( P<0.0001). In systemic lupus erythematosus cases, there was a positive correlation between sucrose
non-fermenting AMPK related kinase and Pentraxin 3 (r ¼0.57, P<0.002).
Conclusions: These data highlight a novel role of sucrose non-fermenting AMPK related kinase/Pentraxin 3 axis in
systemic lupus erythematosus pathogenesis. Sucrose non-fermenting AMPK related kinase/Pentraxin 3 combined rolein immunometabolic signaling and DNA damage response is proposed to accelerate cardiovascular complications insystemic lupus erythematosus patients.
Keywords
Systemic lupus erythematosus, Sucrose non-fermenting AMPK related kinase, Pentraxin 3, DNA damage, oxidativestress, cardiovascular disease, intima media thickness
Accepted: 26th February 2015
Introduction
Systemic lupus erythematosus (SLE) is an autoimmune
inflammatory disease, characterized by B/T1Department of Medical Biochemistry and Molecular Biology, Faculty of
Medicine, Tanta University, Tanta, Egypt
2National Heart Institute, Cairo, Egypt
3Department of Rheumatology & Rehabilitation, Faculty of Medicine,
Tanta University, Tanta, Egypt
Corresponding author:
Doaa Hussein Zineldeen, Department of Medical Biochemistry and
Molecular Biology, Faculty of Medicine, Tanta University, El-GeishStreet, Tanta, El-Gharbia, Egypt.Email: [anonimizat], [anonimizat] of Clinical Biochemistry
0(0) 1–12
!The Author(s) 2015
Reprints and permissions:
sagepub.co.uk/journalsPermissions.navDOI: 10.1177/0004563215578190acb.sagepub.com Ann Clin Biochem OnlineFirst, published on April 27, 2015 as doi:10.1177/0004563215578190
by guest on April 28, 2015 acb.sagepub.com Downloaded from

XML Template (2015) [22.4.2015–2:53pm] [1–12]
//blrnas3.glyph.com/cenpro/ApplicationFiles/Journals/SAGE/3B2/ACBJ/Vol00000/150023/APPFile/SG-ACBJ150023.3d (ACB) [PREPRINTER stage]
lymphocytes, macrophages, and dendritic cells dys-
function in addition to production of autoantibo-dies.
1SLE patients have elevated concentrations of
pro-inflammatory cytokines such as IL-6, TNF- a
and IL-1 which contribute to the inflammatorypro-cess.
2Cardiovascular disease (CVD) risk is at least
doubled among SLE patients compared with thegeneral population.
3Atherosclerosis, a major cause
of CVD, is accelerated in SLE patients due to trad-itional risk factors such as hypertension and dyslipi-demia.
4SLE patients possess various mechanisms to
develop accelerated atherosclerosis independent ofother traditional CVD risk factors, such as endothe-lial dysfunction,
5immune complex generation, oxi-
dant/anti-oxidant imbalance, and cytokineactivation.
6SLE metabolomic studies reveal evidence
of heightened oxidative stress, reduced energy gener-ation, and altered lipid profile. Persistent cellularmetabolic signals promote chronic inflammationand autoimmunity.
7The molecular basis underlying
immunometabolic signaling in SLE pathogenesisremains largely elusive.
7The Sucrose Non-
Fermenting AMPK Related Kinase (SNARK) is amember of the AMP-dependent kinase family ofserine/threonine kinases, which are involved in regu-lation of smooth muscle contractility in addition toits role as a sensor of cellular energy, and metabol-ism, yet its role in autoimmunity has not been clar-ified.
8,9SNARK RNA transcripts are induced by
ultraviolet (UV) radiation in rat keratinocytes.10
Exposure to UV radiation is among the environmen-tal factors that have been implicated in pathogenesisof SLE.
11Microarray data suggest that SNARK is
the only kinase substantially induced in endothelialcells by TNF- a, under NF-k B transcriptional con-
trol.
12The long pentraxin 3 (PTX3) is a member
of pentraxin superfamily that plays an importantrole in immune system
13and more recently is con-
sidered as a potential marker of atherosclerotic andcardiovascular disorders.
14The proximal promoter of
PTX3 contains a NF- kB binding site that is essential
for its induction by TNF- a.15PTX3 is produced by
smooth muscle cells as well as many innate immunecells such as neutrophils, fibroblasts, epithelial cells,and vascular endothelial cells. Increased concentra-tions of PTX3 have been detected in some auto-
immune and degenerative disorders.
14,16The
molecular basis of SLE pathogenesis which lead to
accelerated CVD remains to be clarified.4This study
was performed to elucidate the possible role ofSNARK and PTX3 as surrogate biomarkers of ath-erosclerosis and CVD in Egyptian SLE patients inthe context of heightened oxidative and environmen-tal stress, altered lipid profiles, and DNA damage
response.Materials and methods
Study population
Informed written consent was obtained from all sub-
jects enrolled in this study. This study was approved bythe Research Ethical Committee of Tanta University.
Eighty female SLE patients were recruited from the
Department of Rheumatology & Rehabilitation,Tanta University Hospitals in the period from 2011to 2013. Patients with SLE fulfilled the AmericanCollege of Rheumatology criteria.
17Patients were sub-
divided into two groups: Group I (n ¼40) SLE cases
who survived one or more manifestations of CVDdefined as a history of myocardial infarction (non-ST-
segment elevation myocardial infarction; n ¼18, ST-
segment elevation myocardial infarction; n ¼12; five
of whom underwent stenting), stable angina (n ¼7),
and vasculitis (n ¼3). Group II (n ¼40) age- and sex-
matched SLE controls with no clinical manifestations
of CVD. In addition, age- and sex-matched healthyvolunteers within the same geographical distributionserved as a healthy control group III (n ¼40). All indi-
viduals were premenopausal and subjected to the samesunlight exposure and clothing conditions. Exclusioncriteria included: other autoimmune diseases, tobaccosmoking, oral prednisolone greater than 10 mg/day,abnormal liver or kidney function, previous transientischemic attacks or cerebrovascular accident, and pastor concurrent history of malignancy. For each subject,the SLE Disease Activity Index (SLEDAI) was
assessed.
18Assessment of SLE-related disease damage
was according to the Systemic Lupus International
Collaborating Clinics (SLICC) damage index.19None
of SLE patients without CVD or apparent healthy con-trols were taking antihypertensive or lipid loweringdrugs during the study period.
Methods
Venous blood samples were collected after an overnightfast into EDTA tubes, centrifuged at 3000 r/min for15 min. The recovered plasma aliquots were stored at–80
/C14C for further analysis. All chemicals were pur-
chased from Sigma-Aldrich, St. Louis, MO, USA,unless otherwise stated.
Routine laboratory measurements
Measurements of the following analytes were underta-ken using the methods indicated: C-reactive protein(EMIT; Merck Diagnostica, Zurich, Switzerland);anti-dsDNA by ELISA kits (Immulisa, IMMCODiagnostic, Inc., Buffalo, NY, USA); antinuclear anti-body assay (Kallestad HEP-2 Kit, Biorad, USA);
plasma glucose by a glucose oxidase method2 Annals of Clinical Biochemistry 0(0)
by guest on April 28, 2015 acb.sagepub.com Downloaded from

XML Template (2015) [22.4.2015–2:53pm] [1–12]
//blrnas3.glyph.com/cenpro/ApplicationFiles/Journals/SAGE/3B2/ACBJ/Vol00000/150023/APPFile/SG-ACBJ150023.3d (ACB) [PREPRINTER stage]
(Biodiagnostic, Cairo, Egypt), total lipid profile
(cholesterol [TC], triglycerides [TG], and HDL-cholesterol [HDL-C] by enzymatic-colorimetric meth-ods (Biodiagnostic, Cairo, Egypt). LDL cholesterol(LDL-C) concentration was calculated according tothe Friedewald equation.
20Atherogenic Index was cal-
culated using the formula LDL-C/HDL-C ratio21and
Coronary Risk Index was calculated using the formulaTC/HDL-C ratio.
22
Doppler ultrasound examination
Carotid ultrasound was performed using B-mode ultra-sound (Siemens G60S, Munich, Germany) equipped
with a linear transducer and carotid intima media
thickness (CIMT) was determined in millimeters as asurrogate measure of atherosclerosis as described.
23
Enzyme linked immunosorbent assays
Plasma PTX3 was determined using a commercialsolid-phase sandwich enzyme-linked immunosorbent
assay (ELISA) (Quantikine DPTX 30; R&D Systems
Inc., Minneapolis, MN, USA) according to the manu-facturer’s instructions. For SNARK detection, a home-made sandwich ELISA assay was developed. Briefly,flat-bottomed ELISA plates (96 wells; Corning GlassWorks, Corning, NY) were coated with 10 mg/L ofanti-SNARK monoclonal antibody (sc-374348, SantaCruz Biotechnology, Inc., Santa Cruz, CA, USA)
diluted in coating buffer (70 mmol/L Na
2CO 3,
30 mmol/L NaHCO 3, pH 9) overnight at 4/C14C.
Subsequently, plates were washed three times with
0.05% Tween-phosphate buffered saline (PBST), andblocked by incubation with 3% bovine serum albumin(BSA-PBST) for 1 h at room temperature. A total of100mL of calibrators and undiluted plasma samples
were added and incubated for 2 h at room temperature
or overnight at 4
/C14C. SNARK recombinant protein
(kindly provided by M. Nakanishi) was diluted at 0,
0.0156, 0.0312, 0.0625, 0.125, 0.25, 0.5, 1 ug/L inPBST dilution buffer and served as calibrators for gen-eration of a standard curve. After washing with PBST,100mL of the 1:500 v/v diluted anti-SNARK rabbit
polyclonal antibody (11592-1-AP, Proteintech Group,Inc., Chicago, IL, USA) in blocking buffer was added
and incubated for 2 h at room temperature. The wells
were then washed and incubated with horseradish-per-oxidase conjugated anti-rabbit IgG (MBL) (100 mLo f
1:1000 v/v in blocking buffer for 1 h at room tempera-ture. After washing 50 mL of HRP substrate TMB
(3,3
0,5,50-tetramethylbenzidine, R&D) was added to
each well and incubated for 15–30 min. Color wasmonitored by absorbance at 450 nm with a microplate
reader (Stat Fax 2100, NY, USA).Cell culture and immunoblotting
HeLa cells (American Type Culture Collection,
Manassas, VA, USA) were cultured and maintainedin Dulbecco’s modified Eagle’s medium (Gibco,Invitrogen, USA) supplemented with glutamine(2 mmol/L), 10% fetal bovine serum (Equitech-Bio,
Inc., USA), penicillin (100,000 units/L; Gibco), and
streptomycin (0.1 g/L; Gibco) at 37
/C14C in a 5% CO 2
incubator. HeLa cells were either stimulated withrecombinant TNF- a(20mg/L) for 2 h, or UV (50 J/
m
2) (UVTEC, Cambridge), then harvested at the indi-
cated time points. Whole cell extracts and western blot-ting were performed as described previously.
24Blots
were incubated with anti-SNARK antibody (11592-1-
AP, ProteinTech Group, Inc., Chicago, IL, USA) at
1:10 dilution. /C12-actin (8226; Abcam, Cambridge, UK)
was used as a loading control. Blots were washed andthen incubated with respective horseradish peroxidase-conjugated secondary antibodies (1:3000) (MBL).After washings, protein expression concentrationswere detected using ECL detection kit (AmershamBiosciences, Little Chalfont, UK) and chemilumines-
cence was detected by gel documentation system
(Biometra, Goettingen, Germany).
In situ detergent extraction and immunofluorescence
analysis
HeLa cells grown onto glass coverslips were treated
with 400 mmol/L H 2O2at 37/C14C for 3 h. Double
immunofluorescence staining on paraformaldehyde-fixed cells was performed as previously described
25
using anti-phosphohistone (S139) H2AX (05-636,Millipore, Upstate, Temecula, USA) and anti-SNARK (11592-1-AP, ProteinTech Group, Inc.,Chicago, IL, USA) antibodies at 1:10,000 and 1:300dilutions, respectively. Secondary antibodies weregoat anti-rabbit IgG, Alexa Fluor 488 conjugated and
goat anti-rabbit IgG, Alexa Flour 594 conjugated
(Molecular probes, Carlsbad, CA, USA). Nuclei werevisualized with DAPI (4
0,6-Diamidino-2-Phenylindole,
Dilactate, Invitrogen, Carlsbad, USA). Slides wereexamined under fluorescent microscope (OlympusBX51, Tokyo, Japan).
DNA damage indices
Lipid peroxidation product malondialdehyde (MDA)was assayed as previously described.
26The activity of
superoxide dismutase (SOD) in plasma was assayed bycommercial kit (Biodiagnostic, Cairo, Egypt).
27For
comet assay, peripheral blood mononuclear cells wereisolated by Ficoll-Hypaque gradient from SLE patientsor their allied controls. Alkaline comet assay was per-
formed using a commercial kit (Trevigen’s CometZineldeen et al. 3
by guest on April 28, 2015 acb.sagepub.com Downloaded from

XML Template (2015) [22.4.2015–2:53pm] [1–12]
//blrnas3.glyph.com/cenpro/ApplicationFiles/Journals/SAGE/3B2/ACBJ/Vol00000/150023/APPFile/SG-ACBJ150023.3d (ACB) [PREPRINTER stage]
Assay kit, 4250-050-k, Gaithersburg, MD, USA)
according to the manufacturer’s instructions. DNAwas stained with PI and slides were digitally photo-graphed (Olympus BX51, Tokyo, Japan).Tail momentsand tail DNA contents of captured comet images wereanalyzed as in Park et al.
28using TriTek (Comet Score
Freeware program, Sumerduck, VA, USA).
Statistical analysis
Results are reported as mean /C6SD. Multiple compari-
sons were performed by one-way analysis of variancefollowed by Tukey’s multiple comparison test.Comparison between any two groups was analyzed by
the unpaired Student’s t-test or Mann–Whitney test
using GraphPad Prism 5.00 software (GraphPad
Software, San Diego, USA). Correlations were ana-lyzed using Spearman rank or Pearson’s correlationcoefficients. Statistical significance was consideredwhen the Pvalue was <0.05.
Results
Clinical and metabolic characteristics
The demographic variables and clinical characteristics
of the studied groups are shown (Table 1). There wasno significant difference in age or body mass index,between patients and controls. Disease duration,SLEDAI, and SLICC were statistically significantlyhigher in SLE cases than controls. Systolic/diastolicblood pressure and CIMT were higher in SLE groups(Table 2). There was a statistically significant increasein TG, C reactive protein (CRP), and fasting plasma
glucose concentrations and atherogenic ratios in
SLE cases when compared to SLE controls and healthysubjects (Table 2).
SNARK assessment
Plasma SNARK concentrations were increased in allSLE patients when compared to healthy control sub-
jects ( P<0.0001). Furthermore, SNARK concentra-
tions in SLE cases showed a statistically significant
increase when compared to SLE controls,(P<0.0001) (Table 2, Figure 1(a)). HeLa cells treated
with TNF- aunder serum starved or asynchronous con-
ditions revealed increased SNARK expression withpeak after 4 h of TNF- atreatment (Figure 4(a)).
Markers of CVD
The mean CIMT was statistically higher in SLE casesas compared to SLE controls and in the latter com-pared to healthy subjects (Table 1). In SLE cases withCVD, plasma PTX3 concentrations were significantly
Table 1. Basic and demographic characteristics of the studied groups.
Healthy controls
(n¼40)SLE-cases(CVD)(n¼40)SLE-controls(n¼40)P value
–NSTEMI ( n¼18)
STEMI ( n¼12)
Stable angina ( n¼7)
Vasculitis ( n¼3) ––
Age (years) 45.15 /C65.35
a46.2/C65.28a44.7/C65.8a0.6621 NS
Disease duration (years) – 10.5 /C61.36b5.025 /C61.61c<0.0001 S
Body mass index (kg/m2) 24.9 /C62.2a25.87 /C61.8a24.89 /C62.1a0.2394 NS
SBP(mm Hg) 125.7 /C610.5a138.2 /C66.7b125.2 /C610.2a<0.0001 S
DBP(mm Hg) 79.25 /C66.35a88.45 /C64.85b79.95 /C64.9a<0.0001 S
ANA(%positive) – 46%b33%b0.0601 NS
Anti-dsDNA (IU/mL) – 89.5 /C610.4b85.4/C69.2b0.0645 NS
SLEDAI – 12.9 /C62.3b12.5/C64.8b0.7397 NS
SLICC damage score – 2.63 /C60.44b1.28/C60.32c<0.0001 S
Current prednisone (%) – 77%b67%b0.7585 NS
CIMT (mm) 0.5 /C60.09a1.1/C60.43b0.8/C60.30c<0.0001 S
Data presented as mean /C6SD. (S) statistically significant difference. NS: non-significant, Pwas calculated by chi square test or one-way ANOVA test
followed by Turkey’s post hoc test. Identical superscript letters indicate non-significant differences while different superscript ones show statistically
significant results. Pwas considered significant at <0.05.
ANA: antinuclear antibody; CIMC: carotid intima media thickness; DBP: diastolic blood pressure; NSTEMI: non-ST -segment elevation myocardial
infarction; SBP: systolic blood pressure; SLEDAI: SLE Disease Activity Index; SLICC: Systemic Lupus International Collaborating Clinics; STMI: S T-
segment elevation myocardial infarction.4 Annals of Clinical Biochemistry 0(0)
by guest on April 28, 2015 acb.sagepub.com Downloaded from

XML Template (2015) [22.4.2015–2:53pm] [1–12]
//blrnas3.glyph.com/cenpro/ApplicationFiles/Journals/SAGE/3B2/ACBJ/Vol00000/150023/APPFile/SG-ACBJ150023.3d (ACB) [PREPRINTER stage]
higher than those of SLE controls and healthy subjects
(P<0.0001) (Figure 1(b), Table 2).
DNA damage and redox status
DNA damage in peripheral blood mononuclear cells was
determined by computerized assessment of comet assay
parameters and expressed as percent of tail DNA andtail moment (Figure 2(A), (a) to (c)). DNA damage wasincreased in cells from SLE patients (SLE cases and SLEcontrols) compared to healthy control subjects (Figure2(B) and (C)). SLE cases and SLE controls had pro-oxidant/anti-oxidant imbalance represented by increasedplasma concentrations of the oxidative stress biomarker
MDA and a decrease in SOD activity compared to
healthy control subjects. SLE cases with CVD hadlower SOD activity than SLE controls (Table 2).
SNARK is involved in DNA damage response
One of the first cellular responses to double strandbreaks (DSBs) is the rapid phosphorylation of the his-
tone H2AX at serine-139 at the sites of DSBs leading to
the formation of distinct gamma-H2AX foci.
29Since
SLE patients exhibited higher concentrations ofSNARK as well as oxidative DNA damage, weexplored the role of SNARK in DNA damageresponse. HeLa cells grown onto glass cover slipswere treated with H
2O2or left untreated. By means of
double immunostaining with SNARK and gH2AXantibodies (DSB marker), most of the human endogen-ous SNARK was localized mainly at the cytoplasm andinterphase nuclei in untreated cells (Figure 3(a)).However, when cells were treated with H
2O2,
SNARK translocated into the nucleus in a foci ofimmunoreactivity, that co-localized with gamma
H2AX foci (Figure 3(a)), and the number of cells show-
ing positive nuclear SNARK foci co-localized withgH2AX was significantly higher than cells having cyto-
plasmic SNARK when cells were treated with H
2O2,
P¼0.0022 (Figure 3(b)), suggesting a role of
SNARK in oxidative DNA damage response. In add-ition, we checked SNARK protein expression afterUV-mediated DNA damage by means of western blot-
ting. Increased SNARK protein expression was
observed in HeLa cells treated with UV and reachesits maximum concentration 24 h post-treatment com-pared to untreated ones (Figure 4(b)).
Correlations of SNARK and PTX3 with some
cardiovascular risk factors
All correlations are summarized in Tables 3 and 4. In
SLE cases, SNARK concentrations correlated posi-tively with PTX3 concentrations and SLEDAI/SLICCindices. Among both SLE groups SNARK correlatedpositively with systolic blood pressure, plasma concen-trations of CRP, DNA damage, MDA, atherogenicindices, TC, LDL-C, and CIMT and negatively corre-lated with HDL-C and SOD activity (Table 3). In SLE
Table 2. Biochemical findings of the studied groups.
Healthy controls
(n¼4)SLE cases (CVD)(n¼40)SLE controls(n¼40)OverallPvalue
TC (mmol/L) 4.3 /C60.54
a6.1/C60.57b4.97/C60.39c<0.0001 S
TG (mmol/L) 1.25 /C60.13a1.69/C60.18b1.5/C60.24c<0.0001 S
HDL-C (mmol/L) 1.58 /C60.061a1.15/C60.14b1.48/C60.12c<0.0001 S
LDL-C (mmol/L) 2.14 /C60.54a4.15/C60.63b2.79/C60.43c<0.0001 S
Atherogenic indices
TC/HDL-C (AI)2.72/C60.39a5.29/C60.98b3.37/C60.31c<0.0001 S
LDL-C/HDL-C (CRI) 1.36 /C60.37a3.61/C60.88b1.9/C60.33c<0.0001 S
FPG (mmol/L) 4.6 /C60.43a5.3/C61.09b5.2/C60.71a0.0106 S
SOD activity(U/mL) 316.49 /C656.72a191.68 /C633.57b262.47 /C669.65c0.0071 S
MDA ( mmol/L) 1.58 /C60.40a7.37/C62.92b6.08/C63.22b<0.0001 S
CRP (mg/L) 3.4 /C60.27a25.1/C65.82b14.8/C67.49c<0.0001 S
PTX3 ( mg/L) 1.05 /C60.9a11.19 /C64.82b2.21/C61.19a<0.0001 S
SNARK (ng/L) 5.18 /C61.32a18.40 /C63.2b11.02 /C62.43c<0.0001 S
Data presented as means /C6SD. (S) significant difference. NS: non-significant, Pwas calculated by one-way ANOVA test followed by T urkey’s post hoc
test. Identical superscript letters indicate non-significant differences while different superscript ones show statistically significant results. Pwas con-
sidered significant at <0.05.
AI: atherogenic Index; CRI: Coronary Risk Index; CRP: C reactive protein; FPG: fasting plasma glucose; HDL-C: high density lipoprotein cholesterol;
LDL-C: low density lipoprotein cholesterol; MDA: malondialdehyde; SOD: superoxide dismutase; TC: total cholesterol; TG: triglycerides.Zineldeen et al. 5
by guest on April 28, 2015 acb.sagepub.com Downloaded from

XML Template (2015) [22.4.2015–2:53pm] [1–12]
//blrnas3.glyph.com/cenpro/ApplicationFiles/Journals/SAGE/3B2/ACBJ/Vol00000/150023/APPFile/SG-ACBJ150023.3d (ACB) [PREPRINTER stage]
cases, PTX3 concentrations positively correlated with
systolic blood pressure, CRP, SLEDAI/SLICC, CIMT,
SNARK, and negatively with SOD activity (Table 4).In SLE cases PTX3 showed positive correlations withTC, atherogenic indices, and MDA (Table 4).
Discussion
SLE is a chronic inflammatory autoimmune disease,
where persistent cellular metabolic signals promote
chronic inflammation and loss of immune toler-ance.
30,31To the best of our knowledge, this study is
the first to report a role for the stress/UV up-regulatedkinase
10SNARK in SLE pathogenesis. The present
study clearly shows a significant increase in plasmaSNARK concentrations in SLE patients relative tocontrols with higher values among SLE patients with
CVD. SNARK expression with antiapoptotic featureshas been reported to be induced by CD95 via NF- kB
signaling.
32Interestingly, previous studies described
increased expression of antiapoptotic genes in SLE
patients in response to CD95 stimulation,33suggesting
a role of SNARK in pathogenesis of SLE. SNARK isactivated by cellular ATP depletion caused by disrup-
tion of mitochondrial or glycolytic ATP synthesis.
34
Therefore, our observed increase of SNARK concen-
trations in SLE patients may be due to persistent cel-lular metabolic signals that promote ongoing chronicinflammation and loss of immune tolerance. This is inagreement with a recent report that describes disturb-ances in metabolic and organelle homeostasis in SLEpatients that lead to abnormal T-cell signaling and acti-
vation.
35In our study, we observed positive correl-
ations between SNARK and CRP, systolic blood
pressure, atherogenic and DNA damage indices, aswell as CIMT in SLE patients, raising a possible rolefor SNARK in promoting CVD complications in SLEpatients. The role of SNARK in the pathogenesis ofatherosclerosis remains to be clarified; however, it hasbeen reported that SNARK mRNA expression is
increased in skeletal muscle biopsies from obese
humans and in cultured myotubes in response toTNF-a stimulation.
36This is consistent with our
immunoblotting results and others who detectedincreased SNARK expression in response to TNF- a
stimulation.
37Increased TNF-a concentrations in
SLE patients has been implicated in promoting athero-genesis.
38Moreover, increased TNF-a has been linked
to coronary artery atherosclerosis with poor progno-sis.
39Intriguingly, in our cohort of SLE patients with
CVD, PTX3 exhibited the highest concentrations andcorrelated well with SNARK concentrations, CIMT,atherogenic and DNA damage indices, as well as dis-ease activity.
PTX3 has a role in modulation of the immunoin-
flammatory response associated with atherosclerosis
and cardiovascular injury. Hollan et al.
40reported
that circulating PTX3 could likely be used as a bio-
marker for severity of CVD in inflammatory rheumaticdisease. Tombetti et al.
41reported increased PTX3 con-
centration in SLE and Takayasu arteritis and mightrepresent a biomarker of actual arteritis. Consistentwith our results Shimada et al.
42reported increased
PTX3 concentrations in SLE patients that was signifi-
cantly associated with disease activity, however a sig-
nificant correlation between PTX3 concentrations andcarotid atherosclerosis was not found. This discrepancymight be attributed to different study populations andgeographical distribution. In view of the ability ofTNF-a to induce expression of SNARK as well as
PTX3 via the transcription factor NF- kB,
15,32
SNARK signaling could represent a good axis for
accelerated vascular damage in SLE patients.
Figure 1. SNARK/PTX3 values in SLE patients. Graph repre-
sents mean /C6SD (error bars); *** P<0.0001 using ANOVA fol-
lowed by Tukey’s post hoc test, NS: non-significant. (a)
Comparison of SNARK concentrations between the indicatedgroups. (b) Comparison of PTX3 concentrations between the
groups.6 Annals of Clinical Biochemistry 0(0)
by guest on April 28, 2015 acb.sagepub.com Downloaded from

XML Template (2015) [22.4.2015–2:53pm] [1–12]
//blrnas3.glyph.com/cenpro/ApplicationFiles/Journals/SAGE/3B2/ACBJ/Vol00000/150023/APPFile/SG-ACBJ150023.3d (ACB) [PREPRINTER stage]
Excessive generation of reactive oxygen species (ROS)
in SLE has the potential to initiate modification and
damage to lipids, proteins, and DNA with generation
of auto-antigens which can elicit autoimmune responsesleading to chronic inflammation and CVD.
43Lipid per-
oxidation generates a variety of relatively stable endproducts such as MDA that induce DNA damage.
44
In this study, increased MDA concentrations werenoted in cases with CVD, a finding that is in accordancewith that obtained previously.
45Furthermore, the pre-
sent study shows evidence of reduction in SOD activityin SLE cases with CVD compared to other groups. Thiscould be due to the presence of autoantibodies againstSOD which lead to the formation of immune com-plexes.
46Additionally, we detect higher concentrations
of DNA damage among SLE cases. In support of ourfindings, prior studies have revealed an abnormal DNAdamage response in autoimmune diseases including
SLE.
47Our observed positive correlation between
SNARK and DNA damage indices drives us to char-
acterize a possible role for SNARK in DNA damageresponse. We demonstrated that SNARK is translo-cated to the nucleus in response to oxidative DNAdamage and is recruited to chromatin at sites of DSB,predicting a role of SNARK in oxidative DNA damageresponse and strengthening the notion that SNARK
may act as a modulator of gene expression throughits nuclear localization and chromatin binding.
48
Environmental stresses, such as UV have been impli-cated in SLE pathogenesis,
49by inhibiting DNA
methylation that convert normal antigen-specific
CD4țT lymphocytes into autoreactive, cytotoxic, pro-
inflammatory cells and consequently accelerates lupusdevelopment and activity.
49This is consistent with our
observation, where SNARK protein expression isincreased in response to UV treatment. This could bemediated via NF-kB activation that in turn increased
SNARK transcription
32and mechanistically unravel a
new role for SNARK in SLE pathogenesis.
Importantly, our study demonstrates altered lipid pro-files and increased atherogenic ratios among all SLEpatients studied. These changes are associated withincreased incidence of CVD in the general popula-tion.
50Previous work stated that endothelial dysfunc-
tion in SLE patients was associated with abnormal lipid
profiles.23
In SLE patients with CVD, SNARK concentra-
tions correlated positively with CIMT and LDL con-
centrations that explain its possible role in promotingearly atherogenesis and cardiovascular complicationsin SLE patients. Surprisingly, our SLE cases hadhigher CIMT values than SLE controls, who hadbeen on long-term steroid therapy indicating that
steroid therapy alone may not be associated with
Figure 2. (A) Photomicrographs of alkaline single cell electrophoresis (comet assay) of peripheral blood mononuclear cells from
healthy controls (a), SLE cases with CVD (b), and SLE controls (c); scale bar 10 mm. (B) Tail DNA percentages in healthy control
subjects, SLE cases and SLE control subjects. (C) Tail moment percentages in healthy control subjects, SLE cases and SLE control
subjects. Mean /C6SD, *statistically significant, P<0.0001 by one-way ANOVA test followed by Turkey’s post hoc test. n ¼50 cells
scored cells from five independent experiments.Zineldeen et al. 7
by guest on April 28, 2015 acb.sagepub.com Downloaded from

XML Template (2015) [22.4.2015–2:53pm] [1–12]
//blrnas3.glyph.com/cenpro/ApplicationFiles/Journals/SAGE/3B2/ACBJ/Vol00000/150023/APPFile/SG-ACBJ150023.3d (ACB) [PREPRINTER stage]
premature CVD in SLE, a finding that has been pre-
viously reported51and allows the possibility that cer-
tain signaling pathway(s) might initiate atherogenesis
and CVD risk in certain subsets of lupus patients.
Rho-associated protein kinase (ROCK) has beenimplicated in autoimmunity and SLE pathogenesis.
52
ROCK mediates its actions partially through phos-phorylation of the smooth muscle regulatory subunitmyosin phosphatase target protein-1 (MYPT1) thatpromotes endothelial cell contraction, permeability,and atherosclerosis.
53Interestingly, SNARK also
phosphorylates MYPT1 on other sites,37and regu-
lates actin stress fibers formation,9highlighting an
atherogenic potential of SNARK mediated by modu-lation of endothelial function and smooth musclecontractility. A schematic model, depicting our pro-posed SNARK/PTX3 signaling is reported inSupplementary Figure 1, where we suggest that vary-ing signals such as ATP depletion, TNF- a, UV light,
and ROS might result in increased SNARK
Figure 3. SNARK-mediated DNA damage response. (a) Immunofluorescence pictures of HeLa cells treated with 400 mmol/L H 2O2
for 3 h (upper panel) or left untreated (lower panel), fixed and double immunostained with endogenous SNARK antibody, gH2AX
antibody (DSB marker). DAPI was used to stain nuclei. Inset shows co-localization of SNARK with gH2AX at the sites of DSB. Scale
bar 5 mm. (b) Graph showing percentage of cells from (a) with nuclear SNARK co-localized with gH2AX at DSBs. Data expressed as
mean /C6SD, (n) ¼100 and results are means of three independent experiments. Pvalue¼0.0022 by Mann–Whitney test. * Statistically
significant at P<0.005.
Figure 4. SNARK is expressed in response to TNF- astimula-
tion or cellular stress. (a) HeLa cells stimulated with 20 mg/L
TNF-a and cells harvested at 0, 2, 4 h post-treatment, then
immunoblotted with anti-SNARK antibody. (b) HeLa cells treated
with UV (50 J/m2) and harvested at 30 min, 120 min, and 24 h
postirradiation, then immunoblotted with anti-SNARK antibody.
(–); non-UV, /C12-actin was used as a loading control.8 Annals of Clinical Biochemistry 0(0)
by guest on April 28, 2015 acb.sagepub.com Downloaded from

XML Template (2015) [22.4.2015–2:53pm] [1–12]
//blrnas3.glyph.com/cenpro/ApplicationFiles/Journals/SAGE/3B2/ACBJ/Vol00000/150023/APPFile/SG-ACBJ150023.3d (ACB) [PREPRINTER stage]
Table 3. Relationships of plasma SNARK concentration ( mg/L) to inflammatory and cardiovascular risk factors.
Healthy Controls SLE cases (CVD) SLE controls
Rp r p Rp
Age (years) –0.16 0.5005 0.5472 0.0125 0.1884 0.4262
SLEDAI – – 0.6357 0.0012 0.3557 0.1238
SLICC damage score – – 0.53271 0.0161 0.2912 0.2129
Disease Duration – – 0.1957 0.4083 0.3604 0.1186
BMI (kg/m2) 0.01432 0.9522 0.268449 0.252458 0.206337 0.382773
SBP –0.2085 0.3777 0.5313 0.0159 0.4964 0.026
DBP –0.3256 0.1612 0.2316 0.3258 0.2739 0.2425
FPG (mmol/L) 0.003646 0.9878 0.3043 0.192 0.2344 0.3198
CRP (mg/L) 0.3682 0.1102 0.6562 0.0017 0.4929 0.0272
CIMT (mm) 0.1936 0.4133 0.6023 0.005 0.7238 0.0003
PTX3 (ng/L) 0.2771 0.2369 0.7117 0.0004 0.1797 0.4483
TC (mmol/L) 0.6857 0.0008 0.8375 <0.0001 0.825 <0.0001
TG (mmol/L) 0.3013 0.1968 0.1824 0.4415 –0.3351 0.1487
HDL (mmol/L) –0.4256 0.0613 –0.5831 0.007 –0.02374 0.9209
LDL (mmol/L) 0.706 0.0005 0.8848 <0.0001 0.8399 <0.0001
LDL/HDL 0.7402 0.0002 0.8575 <0.0001 0.7219 0.0003
TC/HDL 0.7471 0.0002 0.8411 <0.0001 0.6982 0.0006
SOD activity (U/mL) 0.1824 0.4415 –0.8452 <0.0001 –0.8244 <0.0001
MDA ( mmol/L) 0.2238 0.3428 0.7328 0.0002 0.5412 0.0137
Tail DNA (%) 0.8752 <0.0001 0.9883 <0.0001 0.8614 <0.0001
Tail moment (%) 0.9207 <0.0001 0.9883 <0.0001 0.8614 <0.0001
Values are Pearson or spearman rank correlation coefficients. Bold font marks significance at P<0.05.
BMI: body mass index; DBP: diastolic blood pressure; FPG: fasting plasma glucose; SBP: systolic blood pressure.
Table 4. Relationships of plasma PTX3 concentration ( mg/L) to inflammatory and cardiovascular risk factors.
Healthy Controls SLE cases (CVD) SLE controlsRp r p r p
Age (years) –0.1568 0.5092 0.5378 0.0145 0.4038 0.0775
SLEDAI – – 0.4507 0.0461 0.3535 0.1263
SLICC damage score – – 0.5344 0.0342 0.1242 0.6018
Disease duration – – 0.2609 0.2665 0.09687 0.6845
BMI (kg/m
2) 0.01432 0.9522 0.117846 0.620727 0.069732 0.770189
SBP –0.3304 0.1548 0.6638 0.0014 0.2643 0.2601
DBP –0.3106 0.1826 0.2348 0.3189 0.1971 0.4048
FPG (mmol/L) 0.2424 0.3031 0.3506 0.1296 0.1665 0.483
CIMT (mm) 0.1854 0.4338 0.7285 0.0003 0.3686 0.1098
CRP (mg/L) 0.05207 0.8274 0.4658 0.0385 0.4164 0.0678
SNARK (ng/L) 0.05161 0.8289 0.7117 0.0004 0.1797 0.4483
TC (mmol/L) 0.244 0.2998 0.8182 <0.0001 0.7106 0.0004
TG (mmol/L) 0.3057 0.1899 0.2716 0.2466 –0.1433 0.5468
HDL (mmol/L) 0.06906 0.7724 –0.3328 0.1517 0.0175 0.9416
LDL (mmol/L) 0.2037 0.3891 0.788 <0.0001 0.6759 0.0011
(continued)Zineldeen et al. 9
by guest on April 28, 2015 acb.sagepub.com Downloaded from

XML Template (2015) [22.4.2015–2:53pm] [1–12]
//blrnas3.glyph.com/cenpro/ApplicationFiles/Journals/SAGE/3B2/ACBJ/Vol00000/150023/APPFile/SG-ACBJ150023.3d (ACB) [PREPRINTER stage]
expression. SNARK involvement in SLE pathogenesis
may affect B-cell homeostasis, control smooth musclecontractility, or control gene expression of some tar-get(s). The synergetic increase of SNARK/PTX3 viaTNF-a/NF- kB signaling and DNA damage response
may mediate SLE pathogenesis, atherogenesis, and
CVD development. The novel proposed role of
SNARK/PTX3 opens the door for further diagnosticand therapeutic approaches that ensure modi Ecation
of metabolic and immune-modulatory status to pre-vent premature cardiovascular complications in SLEpatients.
Acknowledgements
We would like to thank Dr Safwat M and Prof Nakanishi M
(Nagoya City University, Japan) for providing fluorescent
antibodies and human recombinant Nuak2 protein, Dr
Miura Y, (Nagoya City University, Japan) for help in provid-ing chemicals and reagents, Dr Zaytoun H, (Radiology
Department, Tanta University) for Doppler ultrasound
examination.
Conflict of interests
None declared.
Funding
This research received no specific grant from any
funding agency in the public, commercial, or not-for-profit
sectors.
Ethical approval
The Ethical Committee of Tanta Faculty of Medicineapproved this study (2868/11/14).
Guarantor
DHZ.Contributorship
DHZ researched literature and conceived the study, per-
formed experimental work, and wrote the first draft paper.
AME-B was involved in protocol development, gaining eth-ical approval, patient recruitment, and data analysis. WAK
contributed to experimental work, statistical analysis, and to
the writing of the manuscript. AHG helped in clinical cardio-vascular assessment of data. All authors reviewed and edited
the manuscript and approved the final version of the
manuscript.
References
1. Oaks Z and Perl A. Metabolic control of the epigenome in
systemic Lupus erythematosus. Autoimmunity 2014; 47:
256–264.
2. Jiang W and Gilkeson G. Sex differences in monocytes and
TLR4 associated immune responses; implications for sys-
temic lupus erythematosus (SLE). J Immunother Appl
2014; 1: 1.
3. Schoenfeld SR, Kasturi S and Costenbader KH. The epi-
demiology of atherosclerotic cardiovascular disease amongpatients with SLE: a systematic review. Semin Arthritis
Rheum 2013; 43: 77–95.
4. Lo ´pez-Pedrera C, Aguirre M, Barbarroja N, et al.
Accelerated atherosclerosis in systemic lupus erythema-tosus: role of proinflammatory cytokines and thera-
peutic approaches. J Biomed Biotechnol 2010; 2010: ii.
DOI: 10.1155/2010/607084.
5. Zardi EM and Afeltra A. Endothelial dysfunction and vas-
cular stiffness in systemic lupus erythematosus: are theyearly markers of subclinical atherosclerosis? Autoimmun
Rev2010; 9: 684–686.
6. Avalos I, Rho YH, Chung CP, et al. Atherosclerosis in
rheumatoid arthritis and systemic lupus erythematosus.
Clin Exp Rheumatol 2008; 26: S5–13.
7. Wu T, Xie C, Han J, et al. Metabolic disturbances asso-
ciated with systemic lupus erythematosus. PLoS One 2012;
7: e37210.
8. Towler MC and Hardie DG. AMP-activated protein
kinase in metabolic control and insulin signaling. Circ
Res2007; 100: 328–341.Table 4. Continued.
Healthy Controls SLE cases (CVD) SLE controls
Rp r p r p
LDL-C/HDL-C 0.1859 0.4327 0.6673 0.0013 0.5565 0.0108
TC/HDL-C 0.2047 0.3866 0.6521 0.0018 0.5606 0.0101
SOD (U/mL) 0.4852 0.0301 –0.6981 0.0006 –0.2212 0.3486
MDA (m mol/L) 0.04193 0.8607 0.6663 0.0013 0.5062 0.0228
Tail DNA (%) 0.5183 0.0192 0.9460 <0.0001 0.2820 0.2284
Tail moment (%) 0.5715 0.0085 0.9026 <0.0001 0.0854 0.7202
Values are Pearson or spearman rank correlation coefficients. Bold font marks significance at P<0.05.
BMI: body mass index; DBP: diastolic blood pressure; SBP: systolic blood pressure.10 Annals of Clinical Biochemistry 0(0)
by guest on April 28, 2015 acb.sagepub.com Downloaded from

XML Template (2015) [22.4.2015–2:53pm] [1–12]
//blrnas3.glyph.com/cenpro/ApplicationFiles/Journals/SAGE/3B2/ACBJ/Vol00000/150023/APPFile/SG-ACBJ150023.3d (ACB) [PREPRINTER stage]
9. Vallenius T, Vaahtomeri K, Kovac B, et al. An associ-
ation between NUAK2 and MRIP reveals a novel mech-
anism for regulation of actin stress fibers. J Cell Sci 2011;
124: 384–393.
10. Rosen CF, Poon R and Drucker DJ. UVB radiation-
activated genes induced by transcriptional and posttran-scriptional mechanisms in rat keratinocytes. Am J Physiol
1995; 268: C846–855.
11. Barbhaiya M and Costenbader KH. Ultraviolet radiation
and systemic lupus erythematosus. Lupus 2014; 23:
588–595.
12. Goto K, Lin W, Zhang L, et al. The AMPK-related
kinase SNARK regulates hepatitis C virus replication
and pathogenesis through enhancement of TGF- /C12signal-
ing.J Hepatol 2013; 59: 942–948.
13. Goodman AR, Cardozo T, Abagyan R, et al. Long pen-
traxins: an emerging group of proteins with diverse func-
tions. Cytokine Growth Factor Rev 1996; 7: 191–202.
14. Knoflach M, Kiechl S, Mantovani A, et al. Pentraxin-3 as
a marker of advanced atherosclerosis results from the
Bruneck, ARMY and ARFY Studies. PLoS One 2012;
7: e31474.
15. Basile A, Sica A, d’Aniello E, et al. Characterization of
the promoter for the human long pentraxin PTX3. Role
of NF-kappaB in tumor necrosis factor-alpha and inter-leukin-1beta regulation. J Biol Chem 1997; 272:
8172–8178.
16. Wang H, Wang K, Wang C, et al. Increased plasma levels
of pentraxin 3 in patients with multiple sclerosis and neu-romyelitis optica. Mult Scler 2012; 19(7): 926–931.
17. Tan EM, Cohen AS, Fries JF, et al. The 1982 revised
criteria for the classification of systemic lupus erythema-tosus. Arthritis Rheum 1982; 25: 1271–1277.
18. Bombardier C, Gladman DD, Urowitz MB, et al.
Derivation of the SLEDAI. A disease activity index forlupus patients. The Committee on Prognosis Studies in
SLE. Arthritis Rheum 1992; 35: 630–640.
19. Gladman DD, Urowitz MB, Ong A, et al. Lack of cor-
relation among the 3 outcomes describing SLE: disease
activity, damage and quality of life. Clin Exp Rheumatol
1996; 14: 305–308.
20. Friedewald WT, Levy RI and Fredrickson DS.
Estimation of the concentration of low-density lipopro-tein cholesterol in plasma, without use of the preparative
ultracentrifuge. Clin Chem 1972; 18: 499–502.
21. Abbott RD, Wilson PW, Kannel WB, et al. High density
lipoprotein cholesterol, total cholesterol screening, andmyocardial infarction. The Framingham Study.Arteriosclerosis 1988; 8: 207–211.
22. Shanmugasundaram KR, Visvanathan A, Dhandapani
K, et al. Effect of high-fat diet on cholesterol distributionin plasma lipoproteins, cholesterol esterifying activity inleucocytes, and erythrocyte membrane components stu-
died: importance of body weight. Am J Clin Nutr 1986;
44: 805–815.
23. Svenungsson E, Cederholm A, Jensen-Urstad K, et al.
Endothelial function and markers of endothelial activa-
tion in relation to cardiovascular disease in systemiclupus erythematosus. Scand J Rheumatol 2008; 37:
352–359.24. Shimada M, Niida H, Zineldeen DH, et al. Chk1 is a
histone H3 threonine 11 kinase that regulates DNAdamage-induced transcriptional repression. Cell 2008;
132: 221–232.
25. Green CM and Almouzni G. Local action of the chro-
matin assembly factor CAF-1 at sites of nucleotide exci-sion repair in vivo. EMBO J 2003; 22: 5163–5174.
26. Draper HH and Hadley M. Malondialdehyde determin-
ation as index of lipid peroxidation. Methods Enzymol
1990; 186: 421–431.
27. Kakkar P, Das B and Viswanathan PN. A modified spec-
trophotometric assay of superoxide dismutase. Indian J
Biochem Biophys 1984; 21: 130–132.
28. Park JH, Park EJ, Lee HS, et al. Mammalian SWI/SNF
complexes facilitate DNA double-strand break repair bypromoting gamma-H2AX induction. EMBO J 2006; 25:
3986–3997.
29. Pilch
DR, Sedelnikova OA, Redon C, et al.
Characteristics of gamma-H2AX foci at DNA double-strand breaks sites. Biochem Cell Biol 2003; 81: 123–129.
30. Barbosa Ve S, Re ˆ go J and Anto ˆ nio da Silva N. Possible
role of adipokines in systemic lupus erythematosus andrheumatoid arthritis. Rev Bras Reumatol 2012; 52:
278–287.
31. de Leeuw K, Freire B, Smit AJ, et al. Traditional and
non-traditional risk factors contribute to the develop-
ment of accelerated atherosclerosis in patients with sys-
temic lupus erythematosus. Lupus 2006; 15: 675–682.
32. Legembre P, Schickel R, Barnhart BC, et al.
Identification of SNF1/AMP kinase-related kinase as
an NF-kappaB-regulated anti-apoptotic kinase involved
in CD95-induced motility and invasiveness. J Biol Chem
2004; 279: 46742–46747.
33. Andre J, Cimaz R, Ranchin B, et al. Overexpression of
the antiapoptotic gene Bfl-1 in B cells from patients withfamilial systemic lupus erythematosus. Lupus 2007; 16:
95–100.
34. Lefebvre DL and Rosen CF. Regulation of SNARK
activity in response to cellular stresses. Biochim Biophys
Acta 2005; 1724: 71–85.
35. Spies CM, Straub RH and Buttgereit F. Energy metab-
olism and rheumatic diseases: from cell to organism.Arthritis Res Ther 2012; 14: 216.
36. Rune A, Osler ME, Fritz T, et al. Regulation of skeletal
muscle sucrose, non-fermenting 1/AMP-activated proteinkinase-related kinase (SNARK) by metabolic stress and
diabetes. Diabetologia 2009; 52: 2182–2189.
37. Yamamoto H, Takashima S, Shintani Y, et al.
Identification of a novel substrate for TNFalpha-induced
kinase NUAK2. Biochem Biophys Res Commun 2008;
365: 541–547.
38. Svenungsson E, Fei GZ, Jensen-Urstad K, et al. TNF-
alpha: a link between hypertriglyceridaemia and inflam-mation in SLE patients with cardiovascular disease.
Lupus 2003; 12: 454–461.
39. Dopheide JF, Knopf P, Zeller GC, et al. Low IL-10/
TNF aratio in patients with coronary artery disease and
reduced left ventricular ejection fraction with a poorprognosis after 10 years. Inflammation 2014; 38(2):
911–922.Zineldeen et al. 11
by guest on April 28, 2015 acb.sagepub.com Downloaded from

XML Template (2015) [22.4.2015–2:53pm] [1–12]
//blrnas3.glyph.com/cenpro/ApplicationFiles/Journals/SAGE/3B2/ACBJ/Vol00000/150023/APPFile/SG-ACBJ150023.3d (ACB) [PREPRINTER stage]
40. Hollan I, Bottazzi B, Cuccovillo I, et al. Increased levels
of serum pentraxin 3, a novel cardiovascular biomarker,
in patients with inflammatory rheumatic disease. Arthritis
Care Res (Hoboken) 2010; 62: 378–385.
41. Tombetti E, Di Chio M, Sartorelli S, et al. Systemic pen-
traxin-3 levels reflect vascular enhancement and progres-sion in Takayasu arteritis. Arthritis Res Ther 2014; 16:
479.
42. Shimada Y, Asanuma YF, Yokota K, et al. Pentraxin 3 is
associated with disease activity but not atherosclerosis in
patients with systemic lupus erythematosus. Mod
Rheumatol 2014; 24: 78–85.
43. Wang G, Pierangeli SS, Papalardo E, et al. Markers of
oxidative and nitrosative stress in systemic lupus erythe-
matosus: correlation with disease activity. Arthritis
Rheum 2010; 62: 2064–2072.
44. Cline SD, Riggins JN, Tornaletti S, et al.
Malondialdehyde adducts in DNA arrest transcriptionby T7 RNA polymerase and mammalian RNA polymer-ase II. Proc Natl Acad Sci USA 2004; 101: 7275–7280.
45. Frostega ˚rd J, Svenungsson E, Wu R, et al. Lipid perox-
idation is enhanced in patients with systemic lupus ery-thematosus and is associated with arterial and renal
disease manifestations. Arthritis Rheum 2005; 52:
192–200.
46. Mansour RB, Lassoued S, Gargouri B, et al. Increased
levels of autoantibodies against catalase and superoxide
dismutase associated with oxidative stress in patients withrheumatoid arthritis and systemic lupus erythematosus.Scand J Rheumatol 2008; 37: 103–108.
47. Evans MD, Cooke MS, Akil M, et al. Aberrant process-
ing of oxidative DNA damage in systemic lupus erythe-matosus. Biochem Biophys Res Commun 2000; 273:
894–898.
48. Kuga W, Tsuchihara K, Ogura T, et al. Nuclear localiza-
tion of SNARK; its impact on gene expression. Biochem
Biophys Res Commun 2008; 377: 1062–1066.
49. Somers EC and Richardson BC. Environmental expos-
ures, epigenetic changes and the risk of lupus. Lupus
2014; 23: 568–576.
50. Lupattelli G, Lombardini R, Schillaci G, et al. Flow-
mediated vasoactivity and circulating adhesion moleculesin hypertriglyceridemia: association with small, denseLDL cholesterol particles. Am Heart J 2000; 140:
521–526.
51. Falaschi F, Ravelli A, Martignoni A, et al. Nephrotic-
range proteinuria, the major risk factor for early athero-sclerosis in juvenile-onset systemic lupus erythematosus.
Arthritis Rheum 2000; 43: 1405–1409.
52. Stirzaker RA, Biswas PS, Gupta S, et al.
Administration of fasudil, a ROCK inhibitor, attenuatesdisease in lupus-prone NZB/W F1 female mice. Lupus
2012; 21: 656–661.
53. Barandier C, Ming XF and Yang Z. Small G proteins as
novel therapeutic targets in cardiovascular medicine.
News Physiol Sci 2003; 18: 18–22.12 Annals of Clinical Biochemistry 0(0)
by guest on April 28, 2015 acb.sagepub.com Downloaded from