Immunoelectron Microscopic Localization of 'LLpe 1 Plasmhogen
Activator Inhibitor in the Extracellular Matrix of Wansforming Growth Factor-P-Activated Endothelial Cells THOMAS J. PODOR' Departwent of P a t h o b
MciUiasm Univenity Hamiltwr, Onturio, Canada L8N 325
DAVID J. LOSKUTOFF Commitac on V i c u h BW@ The &n"s Research InstitUte La / o h , Cid@rnia 92037 INTRODUCTION Transforming growth ictor-beta (TGF-8) is a polypeptide growth fictor which regulates numerous cell-matrix interactions including both fibrosis and angiogenesis in pipg.1J Purified or platelet-derived E F - P stimulates the biosynthesis of type 1 plasminogen activator inhibitor (PAI-1) in various cultured cells, including bovine aortic endothelial cells (BAEs) We have recently demonstrated that activation of quiescent human umbilical vein endothelial cells (HUVECs) or BAEs with inflammatory mediators increases the accumulation of active PAI-1 on the culture substratumattached extracellular matrix (ECM) and the luminal cell This interaction with the culture substratum-attached ECM is influenced by serumderived vitronecth6 However, the culture substratum-attached ECM is, in part, artifactual as it is largely composed of serumderived proteins ( i t . ,vitronectin, fibronectin) adsorbed to the culture plastic intedce and thus does not accurately represent the more complex, physiologically relevant subendothelid cell E M in vivo.7 In this study we analyzed the effects of E F - P on the distribution of BAE PAI-1 in cellular-ocganized, fibrous ECM networks utilizing immunoassays, and immunogold electron microscopy employing a monoclonal antibody to bovine PAI-1.8 .394
a Address correspondence to Thomas Podor, Ph.D. at Hamilton Civic Hospitals Research Centre, Henderson General Division, 711 Concession St., Hamilton, Ontario, L8V lC3; Tel.: (416) 575-2630; FAX: (416) 575-2639.
46
PODOR & LOSKUTOFF: PAI-1 IN THE EXTRACELLULAR MATRIX
47
RESUL.. AND DISCUSSION Confluent, serum-deprivedBAES were treated for various times with purified TGF/3 in the presence or absence of 10% fetal bovine serum and the distribution of PAI-1 antigen quantitated in the conditioned media, Triton-soluble cell lysates and Tritoninsoluble ECM as previously des~ribed.5,~ In the absence of serum, EF-/3 stimulates accumulation of PAI-1 associated with the BAE Triton X-100 insoluble ECM prior to its detection (
Activator Inhibitor in the Extracellular Matrix of Wansforming Growth Factor-P-Activated Endothelial Cells THOMAS J. PODOR' Departwent of P a t h o b
MciUiasm Univenity Hamiltwr, Onturio, Canada L8N 325
DAVID J. LOSKUTOFF Commitac on V i c u h BW@ The &n"s Research InstitUte La / o h , Cid@rnia 92037 INTRODUCTION Transforming growth ictor-beta (TGF-8) is a polypeptide growth fictor which regulates numerous cell-matrix interactions including both fibrosis and angiogenesis in pipg.1J Purified or platelet-derived E F - P stimulates the biosynthesis of type 1 plasminogen activator inhibitor (PAI-1) in various cultured cells, including bovine aortic endothelial cells (BAEs) We have recently demonstrated that activation of quiescent human umbilical vein endothelial cells (HUVECs) or BAEs with inflammatory mediators increases the accumulation of active PAI-1 on the culture substratumattached extracellular matrix (ECM) and the luminal cell This interaction with the culture substratum-attached ECM is influenced by serumderived vitronecth6 However, the culture substratum-attached ECM is, in part, artifactual as it is largely composed of serumderived proteins ( i t . ,vitronectin, fibronectin) adsorbed to the culture plastic intedce and thus does not accurately represent the more complex, physiologically relevant subendothelid cell E M in vivo.7 In this study we analyzed the effects of E F - P on the distribution of BAE PAI-1 in cellular-ocganized, fibrous ECM networks utilizing immunoassays, and immunogold electron microscopy employing a monoclonal antibody to bovine PAI-1.8 .394
a Address correspondence to Thomas Podor, Ph.D. at Hamilton Civic Hospitals Research Centre, Henderson General Division, 711 Concession St., Hamilton, Ontario, L8V lC3; Tel.: (416) 575-2630; FAX: (416) 575-2639.
46
PODOR & LOSKUTOFF: PAI-1 IN THE EXTRACELLULAR MATRIX
47
RESUL.. AND DISCUSSION Confluent, serum-deprivedBAES were treated for various times with purified TGF/3 in the presence or absence of 10% fetal bovine serum and the distribution of PAI-1 antigen quantitated in the conditioned media, Triton-soluble cell lysates and Tritoninsoluble ECM as previously des~ribed.5,~ In the absence of serum, EF-/3 stimulates accumulation of PAI-1 associated with the BAE Triton X-100 insoluble ECM prior to its detection (
