PPAR Delta Agonist GW501516 Inhibits MMP-2 Expression And Cell Apoptosis In HUVEC

BACKGROUND:Matrix Metalloproteinases (MMPs) belong to the incision enzyme family that depending on Zn2+ and Ca2+, which can combine with the extracellar matric (ECM) gredients specifically to degrade them and thus play a role in vascular remodeling and pathological impair of diseases. Recently, many research have demonstrated that MMPs affect the progression of atherosclerosis by promoting vascular neogenesis and smooth muscle cells immigration and phenotypic alternation, vascular remodeling, sustaining plaque stability and so on. MMP-2 is a typeⅣcollagenase, which is one of the MMPs family and can degrade ECM includingⅣcollagen and partially denaturatedⅠ,Ⅱ,Ⅲcollagen, whose content and activity have close correlation with the formation and stability of atherosclerotic plaque.PPARδis one of the three isoforms of Peroxisome Proliferator-Activated Receptor(PPARs) which is expressed ubiquitously. PPARδcan enhance fatty acid catabolism and energy uncoupling in adipose tissue and muscles, suppress macrophage-induced inflammation decrease atherosclerosis and is involved in the progression of many diseases. PPARδligands are divided into two kinds, natural ligands and synthetic ligands. GW501516 is a synthetic ligand which belongs to the phenoxy acetic acid derivative. When it binds to PPARδ, PPARδwill be activated and heterodimers wil be formed together with retinoid X receptors (RXRs) and bind to peroxisome proliferator response element (PPRE) to modulate transcription and translation of the target genes downstream and regulate biological functions such as control weight gain, enhance physical endurance, and improve insulin sensitivity.Our aim is to investigate the effect of PPARδon MMP-2 expression induced by oxLDL or high glucose in HUVEC and its influence on endothelial cell function. Part I. Effect of PPARδagonist GW501516 on MMP-2 expression induced by oxLDL and Glucose in HUVECObjective: To identify the effect of GW501516 on MMP-2 expression induced by oxLDL and high glucose in HUVEC and its influence on cells apoptosis and proliferation viability.Methods: 1. HUVEC were treated with oxLDL at different concentrations (0 mg/L, 25 mg/L, 50 mg/L, 100 mg/L) for 24h, HUVEC were treated with high glucose at different concentrations (0mM, 15mM, 30mM, 45mM)for 24h, MMP-2 mRNA level were determined by Rea1-time PCR.2. HUVEC were pre-treated with GW501516 at different concentrations(0nM, 1nM, 10nM, 100nM, 1μM)for 1h and then were co-incubated with 50mg/L oxLDL and 30mM glucose for 24h, respectively. Unmixed HUVEC were used as control. MMP-2 mRNA and protein level were determinded by Rea1-time PCR and Western blotting respectively. HUVEC were pre-treated with GW501516 at different concentrations(0nM, 1nM, 10nM, 100nM, 1μM)for 1h and then were co-incubated with 100mg/L oxLDL or 30mM glucose for 24h, respectively, flow cytometer (FCM) was used to examine apoptosis of HUVEC, cell proliferation ability was analyzed using MTT.Results: OxLDL significantly enhanced expression of MMP-2 mRNA in a dose-dependent manner and peaked at the concentration of 50mg/L. expression of MMP-2 mRNA were increased by Glu in a dose-dependent manner within 0-45mM for 24h, and peaked at 30mM. MMP-2 expression in both protein and mRNA levels induced by oxLDL or high glucose was obviously down-regulated by GW501516 at different concentrations(0nM, 1nM, 10nM, 100nM, 1μM)for 24h(P<0.05). GW501516 could decrease apoptosis induced by high glucose or oxLDL. MTT method demonstrated that GW501516 could weaken the effect influcence of high glucose or oxLDL on cell viability.Part II. Effect of siRNA on PPARδand MMP-2 expression in HUVECObjective: To investigate the effect of siRNA on PPARδexpression and whether the inhibition of GW501516 on oxLDL and high glucose-induced MMP-2 expression will be influenced with the alteration of PPARδexpression, in order to show the role of PPARδpathway in the regulation of GW501516 on MMP-2 expression in HUVEC.Methods: Four pairs of siRNA( NC 591 621 972)were designed and HUVEC were intervened with siRNA Expression Cassette segments in vitro. 24h after transfection, fluorescence microscope was used to observe the transfection efficiency. Transfected HUVEC were pretreated by GW501516 for 1h and co-incubated with 50mg/L oxLDL and 30mM glucose, respectively. Irrelevant siRNA transfected cells were used as control. PPARδand MMP-2 mRNA and protein expression were detected by quantitative Real-time PCR and Western blotting respectively.Results : Three pairs of the designed siRNA Expression Cassette (591 621 972) could inactivate PPARδexpression efficiently, among which 621 is the most effective one. MMP-2 mRNA and protein expression were both increased in the transfected HUVEC, which suggested that PPARδsiRNA depressed the inhibiton of GW501516 on oxLDL and high glucose-induced MMP-2 expression.Conclusions :1. PPARδspecific agonist GW501516 down-regulated MMP-2 expression which was induced by oxLDL or high glucose in HUVEC.2. PPARδspecific agonist GW501516 suppressed HUVEC apoptosis and proliferation ability induced by oxLDL or high glucose.3. PPARδsiRNA suppressed down-regulation of GW501516 on MMP-2 expression.