title: Transforming growth factor-beta1 modulates extracellular matrix production, proliferation, and apoptosis of endothelial progenitor cells in tissue-engineering scaffolds.
abstract: BACKGROUND
  Valvular endothelial cells and circulating endothelial progenitor cells (EPCs) can undergo apparent phenotypic change from endothelial to mesenchymal cell type. Here we investigated whether EPCs can promote extracellular matrix formation in tissue engineering scaffolds in response to transforming growth factor (TGF)-beta1. Method and Results- Characterized ovine peripheral blood EPCs were seeded onto poly (glycolic acid)/poly (4-hydroxybutyrate) scaffolds for 5 days. After seeding at 2 x 10(6) cells/cm2, scaffolds were incubated for 5 days in a roller bottle, with or without the addition of TGF-beta1. After seeding at 15 x 10(6) cells/cm2, scaffolds were incubated for 10 days in a roller bottle with or without the addition of TGF-beta1 for the first 5 days. Using immunofluorescence and Western blotting, we demonstrated that EPCs initially exhibit an endothelial phenotype (ie, CD31+, von Willebrand factor+, and alpha-smooth muscle actin (SMA)-) and can undergo a phenotypic change toward mesenchymal transformation (ie, CD31+ and alpha-SMA+) in response to TGF-beta1. Scanning electron microscopy and histology revealed enhanced tissue formation in EPC-TGF-beta1 scaffolds. In both the 10- and 15-day experiments, EPC-TGF-beta1 scaffolds exhibited a trend of increased DNA content compared with unstimulated EPC scaffolds. TGF-beta1-mediated endothelial to mesenchymal transformation correlated with enhanced expression of laminin and fibronectin within scaffolds evidenced by Western blotting. Strong expression of tropoelastin was observed in response to TGF-beta1 equal to that in the unstimulated EPC. In the 15-day experiments, TGF-beta1-stimulated scaffolds revealed dramatically enhanced collagen production (types I and III) and incorporated more 5-bromodeoxyuridine and TUNEL staining compared with unstimulated controls.
  
  
  CONCLUSIONS
  Stimulation of EPC-seeded tissue engineering scaffolds with TGF-beta1 in vitro resulted in a more organized cellular architecture with glycoprotein, collagen, and elastin synthesis, and thus noninvasively isolated EPCs coupled with the pleiotropic actions of TGF-beta1 could offer new strategies to guide tissue formation in engineered cardiac valves.
authors.affiliation: Department of Cardiac Surgery, Children's Hospital Boston, 300 Longwood Ave, Boston, Massachusetts 02115, USA.
authors.forename: Virna L
authors.initials: VL
authors.lastname: Sales
authors.forename: George C
authors.initials: GC
authors.lastname: Engelmayr
authors.forename: Bret A
authors.initials: BA
authors.lastname: Mettler
authors.forename: John A
authors.initials: JA
authors.lastname: Johnson
authors.forename: Michael S
authors.initials: MS
authors.lastname: Sacks
authors.forename: John E
authors.initials: JE
authors.lastname: Mayer
mesh_terms: D000199:Actins / Q000032:analysis; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured / Q000187:drug effects / Q000378:metabolism; D003094:Collagen / Q000096:biosynthesis; D004261:DNA Replication / Q000187:drug effects; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D005109:Extracellular Matrix / Q000187:drug effects* / Q000378:metabolism; D006025:Glycosaminoglycans / Q000096:biosynthesis; D059630:Mesenchymal Stem Cells / Q000187:drug effects* / Q000378:metabolism; D008855:Microscopy, Electron, Scanning; D010641:Phenotype; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000032:analysis; D011091:Polyesters; D012756:Sheep; D023822:Tissue Engineering / Q000295:instrumentation / Q000379:methods*; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D053773:Transforming Growth Factor beta1
chemical_list: D000199:Actins; D006025:Glycosaminoglycans; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D011091:Polyesters; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; C512419:poly(glycolic acid)-co-poly(4-hydroxybutyrate); C013786:A73025; D003094:Collagen