The Relationship Of Coronary Artery Lesions With Expression Of Peroxisome Proliferator-activated Receptor-γ And Serum Adiponectin In Kawasaki Disease

BackgroundKawasaki disease (Kawasaki disease, KD), also known as mucocutaneous lymphnode syndrome, is a non-specific systemic vasculitis, which occurs mainly in children.KD main clinical features are fever, bilateral conjunctival hyperemia, oral mucosalinflammation, cervical lymphadenopathy, rash, swelling of hands and feet, increasederythrocyte sedimentation rate and C-reactive protein, thrombocytosis,etc. Theinflammatory process of this disease preferentially involves the coronary arteries，potentially resulting in coronary arteritis. About20％-25％patients without curingwill get perpetuity coronary artery lesions(CAL)．About5％-10％of coronary arterylesions may develop ischemic heart disease. Currently, some more developed regions,Kawasaki disease is the most common cause of acquired coronary vesselabnormalities in children.Since the first report in1967, the etiology and pathogenesis of Kawasaki diseaseis still unclear. At present, most scholars believe KD is a super immune responsetriggered by the infection or superantigen, which could damage the endothelial cellsand other cells, thereby forming a systemic vasculitis. Recent studies have shown thatperoxisome proliferator-activated receptor-γ (PPAR-γ) and adiponectin (adiponectin,ADP) have anti-inflammatory and endothelial protective effects, which may beassociated with the formation and development of coronary artery lesions.Peroxisome proliferator-activated receptors (PPARs) were classified as orphanmembers of the nuclear receptor superfamily. To date, three PPAR subtypes have been discovered and characterized (PPARα, β/δ, γ). The expression of PPARs has beenshown in vascular endothelial cells, vascular smooth muscle cells, monocytes/macrophages and other cells. It has a variety of biological effects and plays animportant role in adipocyte differentiation, energy metabolism, glucose metabolism,lipid metabolism, atherosclerosis formation, inflammatory response and inhibition oftumor cell proliferation. PPAR-γ is closely related to inflammation and theproliferation, migration of vascular endothelial cells and smooth muscle fiber cell.PPARγ receptor can be activated by endogenous ligands (e.g.,prostaglandin, orhydroxyeicosatetraenoic acid), and synthetic ligands that include insulin sensitizingantidiabetic thiazolidinediones (TZD); nonsteroidal anti-inflammatory compoundsindomethacin, ibuprofen. Activation of PPAR-γ negatively influences the productionof inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), Interleukin(IL)-6, and IL-1by macrophages. In the early ischemic heart disease, PPARγ mayregulate and activate the release of proinflammatory cytokine. PPARγ agonistsdose-dependently inhibited the proliferation of endothelial cells, thereby inhibitingendothelial cell migration, blocking vascular endothelial growth factor expressintercellular adhesion molecule1, hindering the binding of monocytes and endothelialcells, finally limiting the blood vessel wall inflammation and protecting againstendothelial damage. Some studies demonstrated that decreased mRNA expression ofPPARγ was observd in coronary heart disease group than in normal ccontrol group.Negative correlation was found between mRNA expression of PPARγ and the Gensiniscore of coronary artery lesions. Recently, a study found that the mRNA expression ofPPARγ was significantly increased in acute and convalescent KD patients. Alsopointed out that The transcript abundance of PPARγ in KD patients may haveimportant clinical implications on the development of premature atherosclerosisADP, which is also referred to as adipocyte complement related protein,30kDa,AdipoQ and gelatin-binding protein28, is derived mainly from white adipose tissue.Human ADP is a244-amino acid protein that contains a collagen-like domain at itsN-terminus and a globular domain at its C-terminus. Research into this hormone hasrevealed it to have insulin-sensitizing, anti-inflammatory and cardio-protective roles.Some studies found that a significantly decreased tendency towards the levels of ADPwas observed in KD on acute phase. At the same time, researchers also found ADPhave been gathered at the damaged vascular endothelial cells. Miura et al reported expression of vascular cell adhesion molecules-1(VCAM-1), in the neovasculature ofKD coronary arteries. ADP may limit VCAM-1expression levels elicited by TNF-αand therefore protect the vascular wall from subsequent atherosclerotic lesions. KDpatients have been reported to have high levels of circulating TNF-α in the acutephase. ADP has a common amino acid sequence with complement Clq and TNF-α.Also ADP attenuates LPS-induced up-regulation of TNF-α and IL-6in culturedmacrophages, which is associated with decreased nuclear factor kappaB (NF-Κb)activation. APN potentiates nitric oxide (NO) through AdipoR1receptors favoringvasodilation. Adiponectin transcript levels are up-regulated in adipocytes upontreatment with thiazolindinediones (TZDs), which parallels increased secretion andelevated ADP levels in the circulation.ObjectivesTo test the expression of PPARγmRNA and serum ADP level, Lipids, C-reactiveprotein, erythrocyte sedimentation rate, platelet in children with KD, to explore therelationships between PPARγmRNA and serum ADP with coronary artery lesions inKD.Methods1. SubjectsForty-five simple KD children who accepted treatment and hospitalization inShenzhen Maternal and Child Health care Hospital during October2011to July2012were choosen as the KD group, including30cases of male,15cases of female; Aged0.17-6, the average age was (1.9±1.54); under the age of13cases (28.9%), l-year-oldto3years old in15cases (33.3%), over3years old in20cases (44.4%). All patientsmet Japan KD Research Committee revised2002diagnostic criteria for Kawasakidisease and rule out other diseases; clinical staging according to "PracticalPediatrics＂; All children taken echocardiography after admission, staging accordingto coronary artery involvement classification criteria of " Practical Pediatrics ";divided into coronary artery lesions (CAL) group18cases, which all were mildcoronary dilatation, normal coronary group (NCAL)27cases. These patients weretreated with oral aspirin30～50mg/kg d and1or2g/kg intravenous immuneglobulin after admission. After the heat back, oral aspirin reduction to3~5mg/kg d,for6to8weeks, if coronary artery disease, medication to coronary artery returned tonormal.Of which,32KD children were choosen for test of the expression of PPARγmRNA;21males,11females, aged0.25-4.6, mean age (1.75±1.02) years old.Twenty healthy children were randomly selected as control group during thesame period. Their body weights and heights were within the normal range, the liverfunction and renal function were normal, and without any chronic disease. Of which,there were14males and6females. The age arranged from0.25to6years old, meanage was (1.99±1.34). There were no statistical differences of age and gender betweenthe two groups.2. Sample collection and Biochemical measurementsAll tested children did not eat any foods in the midnight before test day. Three mlVenous blood were collected in fasting at8to9in the morning on the acute phase(within7days of fever), subacute phase (8-21days of fever) and convalescent phase(21to30days of fever):①Taken two ml blood in the drying tube, serum wascollected after centrifugation, the lipid measured by automatic biochemical analyzer(Beckman produced Unicel DXC8000PRO automatic biochemical analyzer producedby United States United States): including triglyceride (TG), total cholesterol (TC),high density lipoprotein (HDL-C), low density lipoprotein (LDL-C), apolipoprotein A(APOA), apolipoprotein B (APOB), lipoprotein (LPA), the remaining serum storgedat-80℃ultra-low temperature refrigerator for ADP testing.②Taken one ml bloodadding EDTA-Na2anticoagulant tube, examined platelets（PLT）, CRP by automaticblood analyzer (the U.S. company Beckman automatic blood analyzer); taken250ulanticoagulant blood added to750ul of TRIzol for extracting RNA.③taken two ml inanticoagulant heparin tubes, tested erythrocyte sedimentation ratio(ESR) byWestergren.3. Experimental method.3.1RT-qPCRTotal RNA was extracted using trizol. Reverse transcription was done with1μgRNA using RevertAid H Minus First strand cDNA synthesis Kit(Thermo).Quantitative PCR was done in ABI7300Real Time PCR System.(All of above wereconducted according to the manufacturer’s instructions).The data were automatically analysed by the SDS software of ABI7300according to automatic baseline and threshold values. Relative Quantification study need one sample as a control to calculate relative expression of other samples.Relative Quantification of genes of interest were2-ΔΔCt.3.2Enzyme Linked Immunosorbent Assay (ELISA)The concentration of sernm ADP were detected by Enzyme LinkedImmunosorbent Assay (ELISA) method,the kit was provided by American R&Dcompany.4. Statistical processingAll of the statistical analyses were performed with SPSS13.0. Mean and standarddeviation (SD) were given for data (X±S). Using T test or one-way ANOVA forMean comparison among the groups; Using Pearson correlation analysis forcorrelation analysis; There were statistically significant differences when P<0.05.Results1、The expression of PPAR-γ mRNA in32cases of KD on acute phase (3.29±2.67), subacute phase (2.60±2.26) and convalescent phase(2.12±1.90) weresignificantly higher than normal controls (1.05±0.56), all P <005. The expression ofPPAR-γ mRNA of KD on acute phase were significantly higher than subacute phaseand convalescent phase, all P <005. The expression of PPAR-γ mRNA in22cases ofKD without coronary artery lesion(NCAL) were significantly higher than10cases ofKD with coronary artery lesion(CAL) on acute phase (4.58±2.20vs.0.45±0.43),subacute phase (3.56±2.08vs.0.48±0.47) and convalescent phase(2.68±2.01vs.0.89±0.75), all P <005.2.45cases of KD on acute phase compared with control subjects, a significantlydecrease in ADP(5.21±2.30μg/ml vs.9.05±2.01μg/ml), TC(3.21±0.77mmol/L vs.4.16±0.25mmol/L)、 HDL_C(0.63±0.16mmol/L vs.1.04±0.14mmol/L)、LDL_C(2.02±0.73mmol/L vs.2.61±0.37mmol/L)、APOA(0.84±0.22g/L vs.1.22±0.10g/L) and APOB(0.84±0.22g/L vs.0.96±0.10g/L), all P<0.001. KD onsubacute phase compared with control subjects, a significantly decrease in ADP(5.79±2.29μg/ml vs.9.05±2.01μg/ml), HDL_C(0.81±0.17mmol/L vs.1.04±0.14mmol/L), a significantly increase in TG(1.48±0.78mmol/L vs.1.06±0.14mmol/L), all P<0.001. KD on convalescent phase compared with control subjects,a significantly decrease in ADP(7.73±2.36μg/ml vs.9.05±2.01μg/ml, P=0.03), APOB(0.81±0.19g/L vs.0.96±0.10g/L, P=0.00), a significantly increase inAPOA(1.41±0.26g/L vs.1.22±0.10g/L, P=0.00).3.27cases of NCAL compared with18cases of CAL, a significantly decrease inADP(4.53±1.83μg/ml vs.6.22±2.61μg/ml, P=0.01) on acute phase; a significantlyincrease in TG(1.69±0.92mmol/L vs.1.17±0.32mmol/L, P=0.01) on subacute phase;a significantly decrease in ADP(4.53±1.83μg/ml vs.6.22±2.61μg/ml, P=0.01) onconvalescent phase; a significantly increase in TG(1.05±0.52mmol/L vs.0.70±0.20mmol/L, P=0.00) on convalescent phase.4. The expression of PPAR-γ mRNA in32cases of KD were negative correctionwith PLT、ADP(r=-0.45，-0.34，P=0.00,0.05), were positive correction with coronaryartery lesion(r=0.80，P=0.00), were no correction with Age、gender、 CRP and ESR.5. ADP were positive correction with CRP (r=0.40,P=0.03), were negativecorrection with coronary artery lesion(r=-0.34,P=0.05), were no correction withAge、gender、PLTand ESR.Conclusion1、 The expression of PPAR-γ mRNA in KD were all higher than normalcontrols on acute phase, subacute phase and convalescent phase; inNCAL were also significantly higher than CAL.2、 The serum ADP of KD were significantly decrease than normalcontrols on acute phase, subacute phase and convalescent phase; in CALwere also significantly higher than NCAL.3、 There were the obvious blood lipid disorders exist in KD. KD comparedthan normal controls, a significantly decrease in HDL-C, Apo A, TC,Apo B on acute phase; still a significantly decrease in HDL-C, but asignificantly increase in TG on subacute phase; still a significantlydecrease in Apo B, but a significantly increase in Apo A onconvalescent phase. TG of CAL were significantly decrease than NCALon subacute phase and convalescent phase;4、 The expression of PPAR-γ mRNA in KD were negative correction withPLT、ADP, were positive correction with coronary artery lesion; Theserum ADP were positive correction with CRP, were negative correction with coronary artery lesion; This suggested that the expression level ofPPARγ mRNA and the serum ADP were related to coronary arteryinjury.