title: The effect of endothelial cell overexpression of plasminogen activator inhibitor-1 on smooth muscle cell migration.
abstract: BACKGROUND
  Plasminogen activator inhibitor-1 (PAI-1), a known inhibitor of plasminogen activators, may regulate smooth muscle cell migration (SMC) through alteration in matrix metalloproteinase (MMP) activity.
  
  
  METHODS
  To study the effect of endothelial cell (EC) PAI-1 overexpression on SMC migration, RT-PCR was used to clone the full length PAI-1 gene, which was ligated into the pCMV/myc/ER expression vector. With electroporation, bovine aortic ECs were transfected with either the PAI-1 construct or the empty vector as control. EC PAI-1 overexpression was shown with a specific PAI-1 activity assay and enzyme-linked immunosorbent assay. The effect of EC PAI-1 overexpression on SMC migration was measured with a modified Boyden-chamber assay. SMC MMP expression was measured with zymography.
  
  
  RESULTS
  Selected clones (EC9, EC21) had a three-fold to five-fold increase in PAI-1 activity compared with untransfected EC and empty vector EC (ECC). Similarly, enzyme-linked immunosorbent assay results showed a 3.5-fold to 5.5-fold increase in PAI-1 levels in EC9 and EC21 versus ECC. Untransfected EC and ECC had similar effects on SMC migratory patterns. Migration of SMC exposed to PAI-1 overexpressing EC was inhibited by 35% to 57% compared with ECC. This inhibitory effect was reversed with addition of exogenous urokinase-type plasminogen activator (uPA). Zymography showed downregulation of MMP-2 and MMP-9 in SMCs exposed to PAI-1 overexpressing EC.
  
  
  CONCLUSIONS
  PAI-1 overexpression with transfected EC inhibits SMC migration. This effect may be mediated through decreased SMC MMP activity.
authors.affiliation: Department of Surgery, Dartmouth-Hitchcock Medical Center, Dartmouth Medical School, Lebanon, NH 03756, USA.
authors.forename: Richard R
authors.initials: RR
authors.lastname: Proia
authors.forename: Peter R
authors.initials: PR
authors.lastname: Nelson
authors.forename: Mary Jo
authors.initials: MJ
authors.lastname: Mulligan-Kehoe
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Wagner
authors.forename: Arthur J
authors.initials: AJ
authors.lastname: Kehas
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Powell
mesh_terms: D000818:Animals; D001013:Aorta, Thoracic / Q000166:cytology / Q000378:metabolism; D002417:Cattle; D002465:Cell Movement / Q000187:drug effects*; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology* / Q000187:drug effects*; D004797:Enzyme-Linked Immunosorbent Assay; D020778:Matrix Metalloproteinase 2 / Q000096:biosynthesis / Q000187:drug effects; D020780:Matrix Metalloproteinase 9 / Q000096:biosynthesis / Q000187:drug effects; D023421:Models, Animal; D009131:Muscle, Smooth, Vascular / Q000166:cytology* / Q000187:drug effects*; D017395:Plasminogen Activator Inhibitor 1 / Q000096:biosynthesis* / Q000494:pharmacology*; D015842:Serine Proteinase Inhibitors / Q000096:biosynthesis* / Q000494:pharmacology*; D013997:Time Factors
chemical_list: D017395:Plasminogen Activator Inhibitor 1; D015842:Serine Proteinase Inhibitors; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9