title: Endothelin Receptor Down-Regulation Mediated Ligand Regulation Mechanisms Protect Against Cellular Hypoxia Injury in Rat Vascular Endothelial Cells.
abstract: OBJECTIVE
  Investigation of the effect of endothelin receptor A (ETaR)-targeting small interfering RNA (siRNA) on rat vascular endothelial cellular hypoxia injury, as well as its underlying mechanism.
  
  
  METHODS
  An in vitro rat vascular smooth muscle cells - endothelial cells co-culture model was established and transfected with ETaR siRNA before hypoxia treatment. Cell culture supernatant, cellular protein and RNA were collected and examined at 0.5hrs, 1hrs, 2hrs, 4hrs, 8hrs, 16hrs, 24hrs and 48hrs of hypoxia with 1% oxygen. The time point at which the best silencing effect was achieved was chosen, eNOS inhibitor L-NAME was added, and post hypoxia cell culture supernatant, cellular protein and RNA was collected for further examination.
  
  
  RESULTS
  After hypoxic treatment, endothelial-1 (ET-1) and ETaR expression levels gradually increased as oxygen deprivation extended. ET-1 and ETaR expression levels were significantly lower in the ETaR siRNA group compared with the Hypoxia group (P<0.001). Such difference peaked at 4hrs of hypoxia. ELISA examination of cell culture supernatant revealed that the amount of ET-1 and TGF-βin the ETaR siRNA group were significantly lower compared to the Hypoxia group at all times, while the amount of NO and eNOS was higher. After 4 hrs of hypoxia, Smad2, Smad3, HIF-1, TNF-α, IFN-γ, IL-6, MCP-1, NF-κb, ET-1 and ANG II mRNA expression in endothelial cells and ETaR mRNA expression in A-10 cells of the ETaR siRNA group were lower than those of the Hypoxia siRNA group, while such results were much higher in the L-NAME group. Western Blot results showed lower expression of ETaR in the ETaR siRNA group compared with the hypoxia and negative siRNA groups, as well as significantly higher ETaR expression in the L-NAME group compared with the ETaR siRNA group. PI3K and p-AKT expression levels were mildly elevated after mild oxygen deprivation, and ETaR siRNA was able to enhance such elevation induced by hypoxia. In the L-NAME group, PI3K and p-AKT expression was much higher than the ETaR siRNA group. PKG and sGC expression levels significantly descended after mild oxygen deprivation. While such levels were higher in the ETaR siRNA group, compared with the hypoxia and negative siRNA groups, the L-NAME group had lower levels of PKG and sGC compared with the ETaR siRNA group.
  
  
  CONCLUSIONS
  ETaR siRNA is capable of down-regulating the expression of inflammatory and transcription factors among endothelial cells treated with hypoxia. Down-regulation of ET-1 is triggered by altered nucleus transcription factor activity through the sGC/PKG signal pathway, and results in enhanced eNOS activity through the PI3K/Akt signal pathway. We suspect this to be the mechanism of the protective effect of ETaR siRNA.
authors.affiliation: Department of Urology, Zhongshan Hospital, Fudan University, Shanghai, China.
authors.forename: Long
authors.initials: L
authors.lastname: Li
authors.forename: Mushuang
authors.initials: M
authors.lastname: Hu
authors.forename: Long
authors.initials: L
authors.lastname: Zheng
authors.forename: Chao
authors.initials: C
authors.lastname: Zhang
authors.forename: Jiawei
authors.initials: J
authors.lastname: Li
authors.forename: Ruiming
authors.initials: R
authors.lastname: Rong
authors.forename: Tongyu
authors.initials: T
authors.lastname: Zhu
authors.forename: Yichen
authors.initials: Y
authors.lastname: Jia
mesh_terms: D000804:Angiotensin II / Q000378:metabolism; D000818:Animals; D015687:Cell Hypoxia / Q000187:drug effects; D018920:Coculture Techniques; D015536:Down-Regulation / Q000187:drug effects; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology*; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism; D007249:Inflammation / Q000473:pathology; D007371:Interferon-gamma / Q000378:metabolism; D015850:Interleukin-6 / Q000378:metabolism; D007668:Kidney / Q000187:drug effects / Q000473:pathology; D008024:Ligands; D008297:Male; D008954:Models, Biological; D016328:NF-kappa B / Q000378:metabolism; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D020011:Protective Agents / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D017207:Rats, Sprague-Dawley; D044022:Receptor, Endothelin A / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D051900:Smad3 Protein / Q000378:metabolism; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D015850:Interleukin-6; D008024:Ligands; D016328:NF-kappa B; D020011:Protective Agents; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D044022:Receptor, Endothelin A; D051900:Smad3 Protein; D014409:Tumor Necrosis Factor-alpha; D000804:Angiotensin II; D007371:Interferon-gamma; D019331:NG-Nitroarginine Methyl Ester