The anti-angiogenic activity of rPAI-1(23) inhibits fibroblast growth factor-2 functions.
Many angiogenesis inhibitors are breakdown products of endogenous extracellular matrix proteins. Plasmin and matrix metalloproteinase-3 generate breakdown products of matrix-bound plasminogen activator inhibitor-1 (PAI-1). We produced a truncated form of PAI-1, rPAI-1(23), that possesses significant anti-angiogenic activity and stimulates high levels of apoptosis in quiescent arterial endothelial cells. Quiescent endothelial cells are less susceptible to apoptosis than angiogenic endothelial cells. The present study was designed to determine the mechanism of the rPAI-1(23) effects in bovine aortic endothelial cells. Apoptosis was measured in annexin V and caspase 3 assays. Expression of death and survival signaling molecules were examined by Western blot and kinase activity. Fibroblast growth factor 2 (FGF2) functions were analyzed in angiogenesis assays. The early response to rPAI-1(23) was an increase in annexin V-positive cells and phosphorylated (p) JNK isoform expression followed by an increase in p-Akt and p-c-Jun expression. Caspase 3 was activated at 4 h, whereas p-Akt was reduced to control levels. By 6 h of rPAI-1(23) treatment cell number was reduced by 35%, and p-c-Jun and p-JNK were degraded by proteasomes. Confocal microscopic images showed increased amounts of FGF2 in the extracellular matrix. However, rPAI-1(23) blocked FGF2 signaling through FGF receptor 1 and syndecan-4, inhibiting cell migration, tubulogenesis, and proliferation. Exogenous FGF2 stimulation could not reverse these effects. We conclude that rPAI-1(23) stimulation of apoptosis in BAEC triggers a cascade of death versus survival events that includes release of FGF2. The rPAI-1(23) anti-angiogenic activity inhibits FGF2 pro-angiogenic functions by blocking FGF2 signaling through FGF receptor 1 and syndecan-4 and downstream effectors p-Akt, p-JNK, and p-c-Jun.
Drinane
Mary
M
Department of Surgery, Vascular Section, Dartmouth Medical School, Lebanon, NH 03756, USA.
Walsh
Jannine
J
Mollmark
Jessica
J
Simons
Michael
M
Mulligan-Kehoe
Mary Jo
MJ
D020533:Angiogenesis Inhibitors / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002417:Cattle; D002478:Cells, Cultured; D002645:Chickens; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism*; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D010446:Peptide Fragments / Q000494:pharmacology*; D010766:Phosphorylation; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology; D015398:Signal Transduction
D020533:Angiogenesis Inhibitors; D010446:Peptide Fragments; D017395:Plasminogen Activator Inhibitor 1; D011994:Recombinant Proteins; D016222:Fibroblast Growth Factor 2; D051057:Proto-Oncogene Proteins c-akt; D048031:JNK Mitogen-Activated Protein Kinases; D046988:Proteasome Endopeptidase Complex