title: Interleukin-1β mediates high glucose induced phenotypic transition in human aortic endothelial cells.
abstract: BACKGROUND
  Previous studies have shown that high glucose (HG) induced endothelial cell (EC) damage via a phenotypic transition of EC. There is increasing evidence suggesting the role of inflammatory cytokines in mediated HG-induced EC damage. However, little is known about the potential role of interleukin-1β (IL-1β) in the process. The aim of present study was to investigate whether IL-1β mediated HG-induced phenotypic transition in human aortic endothelial cells (HAECs) and to determine the possible underlying mechanism.
  
  
  METHODS
  Primary HAECs were exposed to normal glucose (NG, 5.5 nM), high glucose (HG,30 nM), IL-1β (10 ng/ml), HG + IL-1β (10 ng/ml) and HG + anti-IL-1β antibodies (1000 ng/ml) or HG + IL-1β small interfering RNA (siRNA). Pathological changes were investigated using confocal microscopy and electron microscopy. Confocal microscopy was performed to detect the co-expression of CD31 and fibroblast specific protein 1 (FSP1). To study the effect of protein kinase C-β (PKCβ) activation on IL-1β in HAECs, HAECs were stimulated with 30 nM PMA (PKCβ activator) and 0.3 μM PKCβ inhibition (LY317615) for 48 h in the NG or HG group. The expressions of PKCβ and IL-1β were detected by RT-PCR and Western blot. And the concentration of IL-1β in the supernatant of HAECs was measured by ELISA. The expressions of FSP1, a-SMA and CD31 were detected by Western blot.
  
  
  RESULTS
  It was shown that the HG resulted in significant increase in the expressions of PKCβ and IL-1β in dose-and time-dependent manners. The HG or exogenous IL-1β alone inhibited the expression of CD31 and markly increased the expressions of FSP1 and α-SMA. Furthermore, we observed that the HG and IL-1β synergistically increased FSP1 and a-SMA expressions compared with the HG or IL-1β alone group (P < 0.05). Confocal microscopy revealed a colocalization of CD31 and FSP1 and that some cells acquired spindle-shaped morphologies and a loss of CD31 staining. Electron microscopy showed that the HG resulted in the increased microfilamentation and a roughened endoplasmic reticulum structure in the cytoplasm. However, the changes above were attenuated by the intervention of anti-IL-1β antibodies or IL-1β siRNA (P < 0.05). In addition, the PMA induced the expressions of PKCβ and IL-1β in HAECs. The PKCβ activation may mediate the effect of the HG on IL-1β production, which could be attenuated by the PKCβ selective inhibitor (LY317615) (P < 0.05).
  
  
  CONCLUSIONS
  Our findings suggested that HG-induced phenotypic transition of HAECs might require IL-β activation via the PKCβ pathway.
authors.affiliation: Institute of Nephrology, Zhongda Hospital, Southeast University School of Medicine, Nanjing, 210009, China. zdd_1023@163.com.
authors.forename: Dong-Dong
authors.initials: DD
authors.lastname: Zhu
authors.affiliation: Institute of Nephrology, Zhongda Hospital, Southeast University School of Medicine, Nanjing, 210009, China. trnvictory77@163.com.
authors.forename: Ri-Ning
authors.initials: RN
authors.lastname: Tang
authors.affiliation: Institute of Nephrology, Zhongda Hospital, Southeast University School of Medicine, Nanjing, 210009, China. lvlinli2000@hotmail.com.
authors.forename: Lin-Li
authors.initials: LL
authors.lastname: Lv
authors.affiliation: Institute of Nephrology, Zhongda Hospital, Southeast University School of Medicine, Nanjing, 210009, China. wy3511362@126.com.
authors.forename: Yi
authors.initials: Y
authors.lastname: Wen
authors.affiliation: Institute of Nephrology, Zhongda Hospital, Southeast University School of Medicine, Nanjing, 210009, China. jstzliu@sina.com.
authors.forename: Hong
authors.initials: H
authors.lastname: Liu
authors.affiliation: Institute of Nephrology, Zhongda Hospital, Southeast University School of Medicine, Nanjing, 210009, China. tonyxlz@163.com.
authors.forename: Xiao-Liang
authors.initials: XL
authors.lastname: Zhang
authors.affiliation: Institute of Nephrology, Zhongda Hospital, Southeast University School of Medicine, Nanjing, 210009, China. klma05@163.com.
authors.forename: Kun-Ling
authors.initials: KL
authors.lastname: Ma
authors.affiliation: Institute of Nephrology, Zhongda Hospital, Southeast University School of Medicine, Nanjing, 210009, China. liubc64@163.com.
authors.forename: Bi-Cheng
authors.initials: BC
authors.lastname: Liu
mesh_terms: D000199:Actins / Q000378:metabolism; D001011:Aorta / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D004789:Enzyme Activation; D005786:Gene Expression Regulation; D005947:Glucose / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D053583:Interleukin-1beta / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D010641:Phenotype; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000378:metabolism; D064546:Protein Kinase C beta / Q000037:antagonists & inhibitors / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D034622:RNA Interference; D000071999:S100 Calcium-Binding Protein A4; D015398:Signal Transduction / Q000187:drug effects; D014162:Transfection
chemical_list: C541116:ACTA2 protein, human; D000199:Actins; D002135:Calcium-Binding Proteins; C508585:IL1B protein, human; D053583:Interleukin-1beta; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D047428:Protein Kinase Inhibitors; D000071999:S100 Calcium-Binding Protein A4; C077591:S100A4 protein, human; C579054:PRKCB protein, human; D064546:Protein Kinase C beta; D005947:Glucose