title: Thrombomodulin Induces a Quiescent Phenotype and Inhibits Migration in Vascular Smooth Muscle Cells In Vitro.
abstract: BACKGROUND
  Loss of critical endothelial cell function and subsequent vascular smooth muscle cell (VSMC) migration is central to the pathology of injury-induced neointimal hyperplasia and recurrent stenosis. Thrombomodulin (TM), well known for its function as an endothelial surface anticoagulant, may have an unknown direct effect on VSMC physiology that would be lost after injury. Here, we examined a novel effect of TM on VSMC by testing the hypothesis that direct application of TM induces favorable changes to the morphology of VSMC and inhibits their migration.
  
  
  METHODS
  Primary human VSMC were harvested using the explant technique and used in early passage (1-4) for all experiments. Laser-scanning confocal fluorescent imaging was performed to assess the effect of soluble TM on VSMC morphology. In vitro, migration of VSMC was measured using: (1) a 4-hr modified Boyden chemotaxis assay and (2) a 24-hr electric cell-substrate impedance sensing injury migration assay. Migration experiments were conducted with VSMC exposed to increasing doses of soluble recombinant TM. Recombinant thrombin served as a positive control and serum-free media as a negative control for all experimentation. Data were analyzed using a Student's t-test or repeated measures analysis of variance where appropriate (α < 0.05).
  
  
  RESULTS
  VSMC exposed to TM clearly demonstrated a quiescent morphology with organized stress fibers consistent with a quiescent, differentiated, contractile phenotype; whereas, thrombin stimulation led to an activated, dedifferentiated, synthetic phenotype. VSMC demonstrated a low, baseline level of migration in unstimulated serum-free conditions. Thrombin significantly stimulated VSMC migration as expected. TM, independent of thrombin, significantly inhibited baseline VSMC migration in a dose-response fashion. The maximal inhibition was observed at (5 μg/mL) with 70% reduction (56 ± 1.7 vs. 18 ± 3.5 cells/5 high-power fields, P = 0.0005).
  
  
  CONCLUSIONS
  TM has a direct effect on VSMC resulting in a quiescent, differentiated and contractile phenotype, and inhibition of migration. This effect is independent of the presence of thrombin. These findings provide new knowledge in understanding the pathophysiology of vascular injury and support a strategy focused on restoring key endothelial function to prevent intimal hyperplasia.
authors.affiliation: Department of Molecular Pharmacology and Physiology, University of South Florida Morsani College of Medicine, Tampa, FL.
authors.forename: Heather M
authors.initials: HM
authors.lastname: Bass
authors.affiliation: Department of Molecular Pharmacology and Physiology, University of South Florida Morsani College of Medicine, Tampa, FL.
authors.forename: Richard S
authors.initials: RS
authors.lastname: Beard
authors.affiliation: Department of Molecular Pharmacology and Physiology, University of South Florida Morsani College of Medicine, Tampa, FL.
authors.forename: Byeong J
authors.initials: BJ
authors.lastname: Cha
authors.affiliation: Department of Molecular Pharmacology and Physiology, University of South Florida Morsani College of Medicine, Tampa, FL.
authors.forename: Sarah Y
authors.initials: SY
authors.lastname: Yuan
authors.affiliation: Department of Molecular Pharmacology and Physiology, University of South Florida Morsani College of Medicine, Tampa, FL; Division of Vascular and Cardiothoracic Surgery, Department of Surgery, University of South Florida Morsani College of Medicine, Tampa, FL. Electronic address: pnelson1@health.usf.edu.
authors.forename: Peter R
authors.initials: PR
authors.lastname: Nelson
mesh_terms: D018929:Cell Culture Techniques; D002465:Cell Movement / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D006801:Humans; D009131:Muscle, Smooth, Vascular / Q000187:drug effects* / Q000473:pathology / Q000503:physiopathology; D032389:Myocytes, Smooth Muscle / Q000187:drug effects* / Q000473:pathology / Q000502:physiology; D010641:Phenotype; D013917:Thrombin; D018180:Thrombomodulin
chemical_list: D018180:Thrombomodulin; D013917:Thrombin