title: Density-dependent accumulation of basic fibroblast growth factor in the subendothelial matrix.
abstract: Recent evidence indicates that basic fibroblast growth factor (bFGF), which lacks a conventional signal recognition sequence, is a component of the subendothelial matrix. However, the molecular mechanisms regulating its cellular release and subsequent matrix deposition remain equivocal. To examine the cellular and subcellular mechanisms regulating bFGF release and subendothelial sequestration, we generated polyclonal antibodies against a chemically cross-linked bFGF. We then used anti-bFGF IgG in conjunction with 3T3 cell [3H]thymidine incorporation assays, enzyme immunoassays and immunofluorescence to learn whether bFGF accumulation in the subendothelial matrix is dependent upon endothelial cell (EC)-cell contact, which coincides with growth arrest. In contrast to subconfluent cultures, which lacked any detectable extracellular matrix bFGF localization, bovine aortic and microvascular EC plated at confluent densities displayed a punctate extracellular staining pattern that was abolished when EC were pretreated with 10 micrograms/ml cycloheximide. Additionally, when EC were treated with either 1 mM beta-D xyloside, an inhibitor of proteoglycan assembly, or 100 micrograms/ml heparin, there was a 40% reduction in matrix-associated bFGF (quantified by image analysis of antibody stained cultures). 3T3 [3H]thymidine incorporation assays indicated that the beta-D xyloside-induced reduction of matrix-associated bFGF coincided with a significant increase in bFGF activity in the conditioned media. Neither sparsely-plated nor confluent EC cultures possessed specific bFGF localization of the nuclear compartment when cells were fixed using cold methanol; however, when EC were fixed in formaldehyde and lysed in isotonic buffers containing 0.1% Triton X-100 or absolute acetone, there was a marked decrease in anti-bFGF staining of the postconfluent extracellular matrix and a concomitant increase in nuclear fluorescence. Because bFGF-stimulated vascular cell growth has been implicated in controlling neointimal cell proliferation, we screened normal and atherosclerotic coronary blood vessels for bFGF, but we were unable to detect it either in lesioned or normal intima. In contrast, significant bFGF levels were observed in association with the EC and mesangial cells of the renal corpuscle, where heparan sulfate accumulates within the glomerular basement membrane. Our in vitro results suggest that bFGF accumulates within the proteoglycan-containing subendothelial matrix concomitant with the formation of cell-cell contacts. In situ, the composition of the microvascular matrix and the cellular phenotype may facilitate the selective accumulation of bFGF that we observed. This, in turn, may influence vascular morphogenesis and remodeling during angiogenesis.
authors: Program in Cell, Molecular and Developmental Biology, Tufts University Health Science Schools, Boston, MA 02111.
authors: A M
authors: AM
authors: Healy
authors: I M
authors: IM
authors: Herman
mesh_terms: D016475:3T3 Cells / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002452:Cell Count; D002478:Cells, Cultured / Q000187:drug effects; D004730:Endothelium, Vascular / Q000378:metabolism*; D005109:Extracellular Matrix / Q000737:chemistry*; D016222:Fibroblast Growth Factor 2 / Q000032:analysis / Q000378:metabolism*; D006027:Glycosides / Q000494:pharmacology; D006493:Heparin / Q000494:pharmacology; D007678:Kidney Glomerulus / Q000737:chemistry; D051379:Mice
chemical_list: D006027:Glycosides; C010807:xylosides; D016222:Fibroblast Growth Factor 2; D006493:Heparin