receptors. Four distinct but structurally related re- ceptors, designated FGF receptor (FGFR)-1, -2, -3, or -4, have been identified.1 18 Multiple isoforms of.
American Journal ofPathology, Vol. 149, No. 6, December 1996 Copyright © American Societyfor Investigative Pathology
Postnatal Regulation of Fibroblast Growth Factor Ligand and Receptor Gene Expression in Rat Thoracic Aorta
Jeffrey A. Winkles,*t Gregory F. Alberts,* Kimberly A. Peifley,* Kenichi Nomoto,f Gene Liau,*§ and Mark W. Majeskyt From the Department of Molecular Biology,* Holland Laboratory, American Red Cross, Rockville, Maryland, the Departments of Biochemistry and Molecular Biologj and Anatomy and Cell Biology,§ George Washington University Medical Center, Washington, D.C., and the Departments of Pathology and Cell Biology,* Baylor College of Medicine,
Houston, Texas
Fibroblast growth factor (FGF)-1 and FGF-2 are potent angiogenic factors and vascular smooth muscle ceU (SMC) mitogens in vivo. Theyfunction via binding to afamily of structuraly related ceU surface receptors that possess intrinsic tyrosine kinase activity. Several studies have indicated that increased FGF and/or FGF receptor (FGFR) expression may correlate with adult SMCproliferation in viva In this study, we used Northern blot hybridization and reverse transcriptionpolymerase chain reaction assays to compare the FGF and FGFR mRNA levels in newborn rat aorta, where SMCs have a high replication index, to those in adult rat aorta, where SMCs are relatively quiescent. We found that FGF-2 and FGFR-2 mRNA expression was elevated 8.2- and 5.6-fold, respectively, in adult aorta. Increased FGF-2 protein expression in the adult aorta was confirmed by Western blot analysis. We also examined FGF and FGFR mRNA expression levels in SMC cultures derivedfrom newborn or adult rat aorta. FGF-1 transcripts were more abundant in newborn SMCs whereas FGF-2 and FGFR-1 mRNA expression was higher in adult SMCs. Furthermore, FGF-1 and FGF-2 mRNA expression levels were altered by ceU culture density and by serum treatment. We conclude that elevated FGF ligand and receptor expression does not always correlate with a high SMC proliferative index, that FGF-1 or FGF-2 may not be the primary mitogens
responsible for newborn SMC growth in vivo, and that FGF-1 and FGF-2 may serve nonmitogenic functions within the mature, adult vessel wall. (Am JPathol 1996, 149:2119-2131)
Fibroblast growth factor (FGF)-1 (acidic FGF) and FGF-2 (basic FGF) belong to a family of heparinbinding proteins that promote cellular proliferation, migration, and differentiation.1 2 They can stimulate vascular endothelial cell and smooth muscle cell (SMC) mitogenesis in vivo3-7 and are potent angiogenic factors.34'8-1 0 Furthermore, the FGF-1 and FGF-2 genes are expressed in human vascular cells as well as in monocyte/macrophages and T lymphocytes.11-15 Taken together, these findings indicate that FGF-1 and FGF-2 may play an important role in angiogenesis-dependent diseases, atherogenesis, hypertension, and restenosis after arterial injury. In support of this latter possibility, previous studies have demonstrated that FGF-2 is the major mitogen controlling medial SMC replication after balloon catheter injury of the rat carotid artery16 or rabbit abdominal aorta.17 The biological effects of FGF-1 and FGF-2 are mediated via binding to cell surface tyrosine kinase receptors. Four distinct but structurally related receptors, designated FGF receptor (FGFR)-1, -2, -3, or -4, have been identified.1 18 Multiple isoforms of the FGFR-1, -2, and -3 proteins can arise via alternative splicing of primary transcripts; in some cases, variants of a single receptor type can have distinct ligand-binding specificities. 19'20 FGF-1 can bind with high affinity to all of the FGFR family members and splice variants identified to date; in contrast, FGF-2 Supported in part by NIH grants HL-39727 (J. A. Winkles), HL37510 (G. Liau), and HL-47655 (M. W. Majesky). Accepted for publication July 29, 1996. Address reprint requests to Dr. Jeffrey A. Winkles, Department of Molecular Biology, Holland Laboratory, American Red Cross, 15601 Crabbs Branch Way, Rockville, MD 20855. Dr. Nomoto's present address is Eisai Co., Ltd., Ibaraki, Japan.
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binds preferentially to the FGFR-1 and FGFR-2 splice variants containing Ig domain IlIc and to FGFR4.19-23 The individual FGFR genes have different temporal and spatial expression patterns during development and are also expressed in a tissue-specific manner in adult animals. We reported previously that FGFR-1, -2, and -4 transcripts, but not FGFR-3 transcripts, are expressed in human arteries.14 The potential importance of the FGF ligand/receptor system in vascular SMC growth control has prompted several groups to examine FGF-1, FGF-2, and/or FGFR expression levels in animal models of atherogenesis,24 hypertension,25 or balloon catheter-induced vascular injury.26 32 Miano et a127 reported that both FGF-2 and FGFR-2 mRNA levels in SMCs were transiently induced after balloon catheterization of the rat aorta. Maximal transcript levels were apparent between 2 and 12 hours after injury, when previously quiescent medial SMCs begin to enter the cell cycle. Moreover, in situ hybridization26 and immunohistochemical29 studies have demonstrated elevated FGF-2 expression in replicating neointimal SMCs in injured rat carotid arteries. Finally, Casscells et a128 have shown that FGFR-1 levels increase after balloon injury to the rat carotid artery, and Daley and Gotlieb33 reported increased FGFR-1 expression during neointimal formation in porcine aortic organ cultures. These data indicate a possible positive correlation between FGF ligand and receptor expression levels and adult SMC proliferative activity. However, Olson et a134 and Tanaka et a132 have reported that FGF-2 expression is actually down-regulated in proliferating neointimal SMCs. In the present study, we have examined FGF Iigand and receptor gene expression during postnatal development of the rat aorta. It has been shown previously that rat aortic SMC hypertrophy and proliferation occurs during the first few weeks of postnatal life.35 38 Medial SMCs in the newborn aorta have a high replication index that declines gradually as postnatal development proceeds,39-41 reaching a minimal value of
