Angiogenic bFGF expression from gas-plasma treated implants (original) (raw)
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In Vitro Cellular & Developmental Biology - Animal, 1994
During studies comparing 12Sl-bFGF internalization between endothelial cells and other cell types, we found, unexpectedly, internalization and nuclear translocation of exogenously added 12Sl-bFGF in two cell lines: Chinese hamster ovary cells (CHO) and rat L6 myoblasts. These cell lines were previously reported to be devoid of FGF receptors. Furthermore, CHO cells showed a weak mitogenic response to added bFGF, while L6 cells were mitogenically unresponsive. By comparison, coronary venular endothelial ceils (CVEC), BALB/c 3T3 fibroblasts, and BHK-21 cells, demonstrated internalization and nuclear translocation of added t2Sl-bFGF, and mitogenic responsiveness to the growth factor. Insulin alone stimulated DNA synthesis in all cell types, yet augmented bFGF-dependent DNA synthesis only in CVEC, 3T3, and BHK. All five cell types expressed FGF receptors as assessed by covalent crosslinking with 12SI-bFGF and immunoblotting with anti-FGF receptor antibodies. Differing rates of cytoplasmic and nuclear accumulation of lzSI-bFGF and partial inhibition of internalization by pretreatment of CVEC with chlorate support a recent model that bFGF can internalize by two mechanisms. Insulin did not significantly affect 12SI-bFGF internalization or metabolism in any cell type. bFGF treatment resulted in weak inhibition of RNA synthesis in L6 cells, bFGF appears firmly bound to the nuclear matrix as little nuclear-bound 12SI-bFGF in CVEC is released by DNAse I or RNAse A digestion, while washes with 0.5 M NaC1 result in partial release. Nuclear bFGF may thus be involved in regulation of nuclear events (e.g., gene transcription and/or DNA replication).
Journal of Immunological Methods, 1988
Polyclonal antibodies which have the capacity to neutralize the biological activity of basic fibriblast growth factor (bFGF) in vitro, have been raised in rabbits against the 157 amino acid form of bFGF purified from human placenta. In a dot blot assay the anti-bFGF antibodies do not recognize the acidic form of FGF (aFGF) with which the basic form shares significant amino acid sequence homology. As determined by immunoblotting, bFGF antibodies recognized only bFGF in a mixture of placentally derived heparin-binding proteins, demonstrating the specificity of these antibodies. Using the anti-human bFGF antibodies, we have developed a solid-phase competitive radioimmunoassay sensitive to 7.8 ng/ml (0.4 pmol/ml) for bFGF. aFGF does not compete with bFGF for binding to the antibodies in the radioimmunoassay even at 2.04/~g/ml. The specificity of the assay was further demonstrated by a lack of competition of cytochrome C, myoglobin, epidermal growth factor or bovine serum albumin with bFGF for binding to the antibodies. We have identified bFGF in extracts of adherent thioglycoUate-stimulated mouse peritoneal macrophages by immunological criteria including the ability of the extract to compete with a25I-bFGF for binding to affinity-purified anti-human bFGF antibodies in the RIA and the ability of these antibodies in inhibit the bFGF-like biological activity of the macrophage extract.
Journal of Cellular Physiology, 1994
The single-copy gene of human basic fibroblast growth factor (bFGF) encodes four co-expressed isoforms, with an apparent molecular weight (M,) of 24 kD, 22.5 kD, 22 kD, and 18 kD, co-translated from a single mRNA. As a tool for the study of the role exerted by the different bFGF isoforms in the biology of endothelial cells, human recombinant 24-kD bFGF was produced and purified from transformed Escherichia coli cells. To this purpose, the novel CUG start codon present in human bFGF cDNA and responsible for the synthesis of 24-kD bFGF was mutagenized to the classic AUG start codon. Transient expression of the mutagenized cDNA in simian COS-1 cells, followed by immunolocalization and subcellular fractionation, resulted in the synthesis of high levels of 24-kD bFGF, which localizes in the cell nucleus as an intact protein. When the same 24-kD bFGF cDNA was expressed in E. coli, the recombinant protein was purified to homogeneity by heparin-Sepharose and ion-exchange chromatography. Recombinant 24-kD bFGF was similar to recombinant 18-kD bFGF in receptor-binding activity and in inducing cell proliferation, plasminogen activator production, and chemotactic movement in cultured endothelial cells. In agreement with the in vitro observations, 24-kD bFGF and 18-kD bFGF exerted a similar angiogenic response when assayed in vivo in the rabbit cornea. Experiments performed with the radiolabeled molecule demonstrated that 24-kD bFGF has an intrinsic ability to bind to high-affinity receptors when added to endothelial GM 7373 cell cultures. Receptor-bound 24-kD bFGF is internalized within the cell and associates with the nucleus with kinetics similar to 18-kD bFGF. Internalized 24-kD bFGF is first processed to the 18-kD form via a chloroquine-insensitive pathway and then to smaller fragments into the lysosomal compartment. At variance with the data obtained in transfected COS-1 cells, only limited amounts of exogenous internalized 24-kD bFGF associates with the nucleus in the intact form, mostly of the
Basic Fibroblast Growth Factor
Encyclopedia of Cancer, 2011
Angiogenesis is a complex process that involves the activation of quiescent endothelial cells (ECs) to a proliferative and migratory phenotype and, subsequently, their redifferentiation to form vascular tubes. We hypothesized that NO contributes to angiogenesis by terminating the proliferative action of angiogenic growth factors and initiating a genetic program of EC differentiation. Human umbilical vein ECs (HUVECs) and calf pulmonary artery ECs (CPAECs) were grown directly on plastic dishes or on three-dimensional fibrin matrices. In the absence of fibrin, treatment with NO-donor compounds, such as S-nitroso-N-acetylpenicillamine (SNAP, 0.1 and 0.4 mmol/L), produced a dose-dependent inhibition of proliferation in both cell lines, whereas the inhibition of endogenous NO production using N G-nitro-L-arginine methyl ester (L-NAME, 1 mmol/L) or N G-monomethyl-L-arginine (L-NMMA, 1 mmol/L) significantly increased proliferation of the CPAECs. The addition of basic fibroblast growth factor (bFGF, 30 ng/mL) increased the expression of endothelial NO synthase mRNA and the production of NO in both cell types when cultured on three-dimensional fibrin gels and produced profound morphological changes characterized by the appearance of extensive capillary-like vascular structures and the loss of EC monolayers. These changes were quantified by measuring total tube length per low-power field (ϫ100), and a differentiation index was derived using the ratio of tube length over area covered by residual EC monolayer. In the absence of additional angiogenic factors, the differentiation index was low for both HUVECs and CPAECs (control, 1.16Ϯ0.19 and 2.07Ϯ0.87, respectively). Treatment with bFGF increased the differentiation index significantly in both cell types (10.59Ϯ2.03 and 20.02Ϯ5.01 for HUVECs and CPAECs, respectively; PϽ.05 versus control), and the addition of SNAP (0.4 mmol/L) mimicked the angiogenic response to bFGF (8.57Ϯ1.34 and 12.20Ϯ3.49 for HUVECs and CPAECs, respectively; PϽ.05 versus control). Moreover, L-NAME inhibited EC tube formation in response to bFGF in a dose-response manner, consistent with a role of endogenous NO production in EC differentiation in this angiogenic model. These findings suggest that NO may act as a crucial signal in the angiogenic response to bFGF, terminating the proliferative actions of angiogenic growth factors and promoting EC differentiation into vascular tubes.