Genomic instability in the type II TGF-beta1 receptor gene in atherosclerotic and restenotic vascular cells.

Cells proliferating from human atherosclerotic lesions are resistant to the antiproliferative effect of TGF-beta1, a key factor in wound repair. DNA from human atherosclerotic and restenotic lesions was used to test the hypothesis that microsatellite instability leads to specific loss of the Type II receptor for TGF-beta1 (TbetaR-II), causing acquired resistance to TGF-beta1. High fidelity PCR and restriction analysis was adapted to analyze deletions in an A10 microsatellite within TbetaR-II. DNA from lesions, and cells grown from lesions, showed acquired 1 and 2 bp deletions in TbetaR-II, while microsatellites in the hMSH3 and hMSH6 genes, and hypermutable regions of p53 were unaffected. Sequencing confirmed that these deletions occurred principally in the replication error-prone A10 microsatellite region, though nonmicrosatellite mutations were observed. The mutations could be identified within specific patches of the lesion, while the surrounding tissue, or unaffected arteries, exhibited the wild-type genotype. This microsatellite deletion causes frameshift loss of receptor function, and thus, resistance to the antiproliferative and apoptotic effects of TGF-beta1. We propose that microsatellite instability in TbetaR-II disables growth inhibitory pathways, allowing monoclonal selection of a disease-prone cell type within some vascular lesions.

Translocation of protein kinase C-delta by PDGF in cultured vascular smooth muscle cells: inhibition by TGF-beta 1.

The migration and proliferation of vascular smooth muscle cells (SMC) into the neointima are important early events in the development of atherosclerosis and post-angioplasty restenosis. The stimulation of SMC migration by platelet derived growth factor (PDGF) involves the induction of protein kinase C activity. Using immunoblot techniques, we demonstrated that rat aortic SMC express a pattern of PKC isoforms which includes PKC-alpha, delta, epsilon, zeta and eta. Upon exposure to PDGF-BB, a fraction of PKC-delta was rapidly translocated from the cytosol to the post-nuclear particulate fraction at 15 seconds and reached an apparent maximum at 30 minutes. In contrast, PKC-alpha and zeta were not translocated by PDGF-BB, TGF-beta 1, which inhibits PDGF-induced DNA synthesis and chemotaxis, reduced the immunoreactive levels of PKC-delta and blocked the PDGF-induced translocation of PKC-delta to the particulate fraction. This suggests that the activation of PKC-delta by translocation to the particulate fraction may play an important role in the control of vascular smooth muscle cell migration by PDGF and TGF-beta 1.

Decreased type II/type I TGF-beta receptor ratio in cells derived from human atherosclerotic lesions. Conversion from an antiproliferative to profibrotic response to TGF-beta1.

Atherosclerosis and postangioplasty restenosis may result from abnormal wound healing. The present studies report that normal human smooth muscle cells are growth inhibited by TGF-beta1, a potent wound healing agent, and show little induction of collagen synthesis to TGF-beta1, yet cells grown from human vascular lesions are growth stimulated by TGF-beta1 and markedly increase collagen synthesis. Both cell types increase plasminogen activator inhibitor-1 production, switch actin phenotypes in response to TGF-beta1, and produce similar levels of TGF-beta activity. Membrane cross-linking of 125I-TGF-beta1 indicates that normal human smooth muscle cells express type I, II, and III receptors. The type II receptor is strikingly decreased in lesion cells, with little change in the type I or III receptors. RT-PCR confirmed that the type II TGF-beta1 receptor mRNA is reduced in lesion cells. Transfection of the type II receptor into lesion cells restores the growth inhibitory response to TGF-beta1, implying that signaling remains responsive. Because TGF-beta1 is overexpressed in fibroproliferative vascular lesions, receptor-variant cells would be allowed to grow in a slow, but uncontrolled fashion, while overproducing extracellular matrix components. This TGF-beta1 receptor dysfunction may be relevant for atherosclerosis, restenosis and related fibroproliferative diseases.

Protection of transforming growth factor-beta 1 activity by heparin and fucoidan.

The transforming growth factor-beta (TGF-beta) family of proteins exert diverse and potent effects on proliferation, differentiation, and extracellular matrix synthesis. However, relatively little is known about the stability or processing of endogenous TGF-beta activity in vitro or in vivo. Our previous work indicated that 1) TGF-beta 1 has strong heparin-binding properties that were not previously recognized because of neutralization by iodination, and 2) heparin, and certain other polyanions, could block the binding of TGF-beta 1 to alpha 2-macroglobulin (alpha 2-M). The present studies investigated the influence of heparin-like molecules on the stability of the TGF-beta 1 signal in the pericellular environment. The results indicate that heparin and fucoidan, a naturally occurring sulfated L-fucose polymer, suppress the formation of an initial non-covalent interaction between 125I-TGF-beta 1 and activated alpha 2-M. Electrophoresis of 125I-TGF-beta 1 showed that fucoidan protects TGF-beta 1 from proteolytic degradation by plasmin and trypsin. While plasmin caused little, if any, activation of latent TGF-beta derived from vascular smooth muscle cells (SMC), plasmin degraded acid-activated TGF-beta, and purified TGF-beta 1, and this degradation was inhibited by fucoidan. In vitro, heparin and fucoidan tripled the half-life of 125I-TGF-beta 1 and doubled the amount of cell-associated 125I-TGF-beta 1. Consistent with this protective effect, heparin- and fucoidan-treated SMC demonstrated elevated levels of active, but not latent, TGF-beta activity.

The temporal and spatial expression of basic fibroblast growth factor during ocular development in the chicken.

PURPOSE: The authors determined the temporal and spatial localization of basic fibroblast growth factor (bFGF) during ocular development in the chick embryo in an attempt to elucidate its role in this process. METHODS: These studies used monospecific polyclonal rabbit anti-human bFGF immunoglobulin G in conjunction with immunohistochemical techniques and western blot analysis. Embryonic eyes at 5-20 days of development were studied. RESULTS: The bFGF was not detected by western blot analysis until embryonic day 12. However, low levels of bFGF-immunoreactive material were present in pigmented and neural retina and the lenses of 5-day embryos. Lens epithelial and fiber cells remained weakly stained throughout development; neuroepithelial cells, ganglion cells, amacrine cells, and photoreceptors all contained significant levels of bFGF-immunoreactive material. Corneal epithelium consistently contained high levels of immunoreactive material. In the corneal endothelium, increasing levels of immunoreactive material were seen as development proceeded. CONCLUSION: It was hypothesized that bFGF may be involved in regulating the proliferation and differentiation of ocular cells in either an autocrine or paracrine fashion during development and that it may play a role in tissue maintenance in the adult eye. These conclusions are consistent with the temporal and spatial expression of bFGF described here.

Immunolocalization of basic fibroblast growth factor during chicken cardiac development.

Basic fibroblast growth factor (bFGF) has been identified in cultured cardiac myocytes as well as in myocardial tissue of both embryonic and adult organisms; bFGF has also been demonstrated to regulate proliferation and differentiation of these cells in culture. Such studies suggest a possible role for bFGF in cardiac myogenesis. In vitro studies using cultured endothelial and neuronal cells also suggest that myocyte-derived bFGF may be involved in the regulation of vascularization and/or innervation of the developing heart. We have generated a spatial and temporal map for bFGF in the developing chick heart using immunohistochemical techniques and our monospecific polyclonal rabbit antihuman bFGF IgG. A progressive decrease in bFGF expression was seen in the highly trabeculated region of the ventricular myocardium, relative to the myocardium directly underlying the epicardial tissue, with increasing developmental age. bFGF expression was limited to the cytoplasm of cardiac myocytes; neither vascular endothelium nor smooth muscle contained anti-bFGF immunoreactive material. A correlation between the temporal and spatial pattern of bFGF expression seen here, with the pattern of myocyte proliferation and differentiation reported by others, suggests a role for bFGF in the autocrine regulation of myocyte proliferation and differentiation.

Basic fibroblast growth factor in the chick embryo: immunolocalization to striated muscle cells and their precursors.

The identification of acidic and basic fibroblast growth factors (FGFs) in a number of embryonic tissue extracts has implicated these growth factors in the regulation of a variety of embryonic events including angiogenesis, eye development, and muscle differentiation. Lack of information concerning the cellular distribution of the growth factor within these tissues has made it extremely difficult to assign developmental roles to FGF. We have localized bFGF in the developing chick embryo using immunohistochemical techniques and our monospecific polyclonal rabbit anti-human bFGF IgG. The spatial pattern for bFGF localization was highly specific. The anti-human bFGF antibodies recognized striated muscle cells and their precursors in 2-6-d chick embryos. Myocardium, somite myotome, and limb bud muscle all stain positively for bFGF. In addition, the anti-human bFGF antibodies localized specifically to the cell, rather than to the extracellular matrix or nucleus of myotubes. The localization of bFGF demonstrated here provides further support for the hypothesis (Clegg et al., 1987; Seed et al., 1988) that this growth factor is involved in muscle development.