| Aim: Pigment epithelium-derived factor (PEDF) is a multifunctional protein withanti-inflammatory, anti-oxidant, antithrombotic, anti-angiogenic, anti-tumorigenic,nertrophic and neuroprotective properties. The present study concentrated on evaluatingthe relationship between plasma levels of PEDF and acute coronary syndrome (ACS).In vivo, we observed PEDF expression on oxidative low density lipoprotein(ox-LDL)-induced injury of human umbilical vein endothelial cells (HUVECs) andinvestigated possible mechanism of D-4F, an apolipoprotein A-Ⅰmimetic peptide, onthe regulation of PEDF expression.Methods:1. Clinical research: Plasma PEDF levels of two hundred consecutivehospitalized ACS patients, who were admitted to the Chinese PLA General Hospital,and one hundred and sixty healthy control subjects were measured using a specific andquantitative commercially available enzyme-linked immunosorbent assay (ELISA) kitand the differences were compared in two groups. Logistic regression analysis wasperformed to determine whether PEDF was an independently protective factor againstACS. All ACS patients were followed up and the short-term major adversecardiovascular events (MACE) were obtained. Kaplan-Meier survival curve was used toanalyze the relationship between PEDF levels and short-term MACE.2. Research onmechanism:①The cultured HUVECs were treated with ox-LDL at differentconcentrations (6.25,12.5,25,50,100,150mg/L) for24h. Then apoptotic rate andintracellular reactive oxygen species (ROS) were measured by flow cytometry, and cellviability was analyzed with MTT assay. PEDF levels were assessed by Western blot andquantitative real-time PCR, respectively. Pearson regression was used to analyze therelationship between PEDF expression and ROS content.②The cultured HUVECswere pretreated with D-4F at different concentrations (12.5,25,50mg/L) or25mg/L sD-4F for2h, followed by exposure to100mg/L ox-LDL for24h except for the controlgroup. Apoptotic rate and intracellular ROS were measured by flow cytometry, and cellviability was analyzed with MTT assay. The levels of LDH, NO, intracellular MDA andSOD were measured using an assay kit according to the manufacturer’s protocol. Inaddition, PEDF levels were assessed by Western blot and quantitative real-time PCR,respectively.③The cultured HUVECs were pretreated with PEDF-siRNA, which wasused to inhibit the expression of PEDF gene, followed by pretreated with100mg/Lox-LDL in the absence or presence of50mg/L D-4F for24h. Western blot and RT-PCRwere used to determinate whether PEDF expression had been successfully silenced.Flow cytometry analyzed apoptotic rate and intracellular ROS. The levels of LDH, NO,intracellular MDA and SOD were measured using an assay kit according to themanufacturer’s protocol, respectively.Results:1.The plasma levels of PEDF in ACS group and control group were (7.36±2.12)μg/ml and (8.44±2.13) μg//ml, respectively, and the differences were significant(P=0.001). Logistic regression analysis showed that PEDF was an independentprotective factor among a set of influencing variables for ACS (OR=0.76,95%CI0.623to0.935, P=0.001). Average follow-up time was6months (mean5.6±1.4months)during which a total of22patients suffered short-term MACE. The mean PEDFconcentration of the patients with occurrence of short-term MACE was6.05±2.18μg/ml,which was lower than in patients without cardiovascular events (7.52±2.07μg/ml, P=0.031). Furthermore, all ACS patients were divided into two groups according to theirPEDF levels: the high (H) group and the low (L) group. Short-term MACE occurred in12patients in the L group (n=86), including4patients who died due to ACS recurrence.In the H group (n=114),10patients were rehospitalized due to recurrent chest pain.However, the Kaplan-Meier survival curves suggested that the difference was notsignificant (P=0.477).2. The results showed that25mg/L ox-LDL significantly inducedapoptosis and increased intracellular ROS levels, but cell viability started to reducewhen ox-LDL concentration was12.5mg/L. In addition, the Western blot and RT-PCRshowed that PEDF expression in ox-LDL-induced HUVECs was decreased in a concentration-dependent manner (P＜0.05), and the relationship between PEDFprotein and mRNA expression and ROS content was negative linear correlation(r=-0.121,95%CI-0.156～-0.087; r=-0.319,95%CI-0.398～-0.239, respectively).3.Pre-incubation with25mg/L and50mg/L D-4F markedly decreased the apoptotic ratio(P＜0.01), restored cell viability (P＜0.01), inhibited LDH release (P＜0.01), reducedintracellular ROS levels and MDA content (P＜0.05), increased the activity ofintracellular SOD and NO release (P＜0.05), and attenuated the ox-LDL-induceddownregulation of PEDF expression (P＜0.01) compared with the100mg/Lox-LDL-alone group.4. Western blot and RT-PCR were undertaken to verify whetherthe transfection with PEDF siRNA could abolish PEDF expression, and the resultsshowed that the protein and mRNA levels of PEDF were largely eliminated by PEDFsilencing (P＜0.05). We found that treatment of cells with PEDF siRNA clearlyincreased the percentage of apoptotic cells, LDH release, intracellular ROS levels andMDA content, but decreased SOD activity and NO release (both P＜0.01) comparedwith50mg/L D-4F-alone group, which was not pre-treated with PEDF siRNA.Conclusions: The plasma PEDF levels are independent protective factor against ACS,and cardiovascular events of ACS patients increase with the decrease of PEDF levels.PEDF protein and mRNA expression in HUVECs is down-regulated through anincreased ox-LDL-induced intracellular production of ROS. Preventing thedown-regulation of PEDF expression is possible mechanism that apolipoprotein A-Ⅰmimetic peptide D-4F effectively protects vascular endothelial cells againstox-LDL-induced injury. |
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