Guillermo Gimenez-Gallego

Fibroblast growth factors: broad spectrum mitogens with potent angiogenic activity

Abstract Fibroblast growth factors (FGFs) are protein mitogens, found in brain and pituitary, that induce division of a wide variety of cells in culture. Interest has focused on FGF, in part, because of their mitogenic activity for vascular endothelial cells in culture and their ability to induce blood vessel growth in vivo .

Human brain-derived acidic and basic fibroblast growth factors: Amino terminal sequences and specific mitogenic activities

Extended amino terminal sequence determinations, made on both acidic and basic fibroblast growth factors from human brain, showed extensive homology with each other and with their respective bovine counterparts. Both human growth factors in the presence of heparin have equivalent specific mitogenic activities on human umbilical vein endothelial cells in culture whereas in the absence of heparin, the acidic mitogen is less than 1% as active as the basic growth factor.

The complete amino acid sequence of human brain-derived acidic fibroblast growth factor

Acidic fibroblast growth factor is a potent mitogen for a variety of cells in culture, including vascular endothelial cells, and is angiogenic in vivo. The complete amino acid sequence of human brain-derived acidic fibroblast growth factor has been determined from amino terminal sequence analysis and carboxypeptidase A digestion of the whole protein and sequence analyses of peptides generated by tryptic, Staphylococcus aureus V8 protease and cyanogen bromide cleavages. A potential Asn-Gly-Ser glycosylation sequence is present in the human protein. The complete amino acid sequence is compared to that of the equivalent protein purified from bovine brain.

Disulfide bonds are neither required, present, nor compatible with full activity of human recombinant acidic fibroblast growth factor.

Human acidic fibroblast growth factor (aFGF) is a potent broad-spectrum mitogen that contains three Cys residues within its monomeric structure. We have found that site-directed mutants in which any one of these Cys residues is converted to serine remain highly active, although variably dependent on heparin, so none of the three possible intramolecular disulfide bonds that can be formed are required for mitogenic activity. Furthermore, a dispensable disulfide bond that might stabilize the active conformation is not present since all three Cys residues are accessible to chemical modification in recombinant as well as brain-derived aFGFs. Finally, formation of a disulfide bond between the two Cys residues conserved among all seven known members of the FGF family results in a virtually inactive product that can subsequently be reactivated by reduction. Thus, despite the extracellular function of aFGF, its Cys residues do not form intramolecular disulfide bonds in the active conformation.

Acidic Fibroblast Growth Factor Accelerates Dermal Wound Healing

Acidic fibroblast growth factor (aFGF) is a potent mitogen in vitro for many cells of ectodermal and mesodermal embryonic origin including skin-derived epidermal keratinocytes, dermal fibroblasts and vascular endothelial cells. Based on the mitogenic activity for these skin-derived cells, we tested the ability of topically applied aFGF to promote healing of full-thickness dermal wounds in healthy rodents. Low doses of aFGF can produce almost a two-fold maximum acceleration in the rate of closure of full-thickness dermal punch biopsy wounds in young healthy mice and rats. The mitogen also produces a 3 to 4 day acceleration in the time to complete closure in rats. Quantitative histomorphometric analysis of wound tissue shows that aFGF induces a marked stimulation of angiogenesis, granulation tissue formation and the growth of new epithelium, but does not promote dermal contraction. Application of aFGF to linear incisions in rat skin produces a transient increase in wound tensile strength accompanied by enhanced cellularity and deposition of collagen. Therefore, aFGF functions as a pharmacological agent that can accelerate dermal wound healing in rodents and could act therapeutically to promote dermal tissue repair in humans.

High-performance liquid chromatography of phenylthiocarbamyl-amino acids: application to carboxyl-terminal sequencing of proteins

A carboxyl-terminus microsequencing method has been developed on the basis of carboxypeptidase A digestion and derivatization of released amino acids with phenylisocyanate followed by subsequent high-performance liquid chromatographic analysis of the resulting phenylthiocarbamyl amino acids. This technique has been used to confirm the carboxyl-terminal sequence of bovine brain-derived acidic fibroblast growth factor. The method is of general application for the microsequencing of the carboxyl-termini of proteins. Since identification of derivatized amino acids was performed on the entire reaction mixture following digestion, a new chromatographic identification procedure for phenylthiocarbamyl amino acids was developed in order to resolve them from spurious peaks. This procedure can be applicable to amino acid analysis.

Occurrence of 3-Hydroxybutyrate Dehydrogenase Activity in the Rat Kidney Brush Border Membrane

3-Hydroxybutyrate dehydrogenase activity was found in both mitochondria and brush border membrane fractions of the rat kidney. Differences in properties provide evidence of a nonmitochondrial 3-hydroxybutyrate dehydrogenase in the brush border membranes.

Structure, Homologies and Activities of Acidic Fibroblast Growth Factor

Fibroblast growth factors (FGFS, reviewed by Thomas and Gimenez-Gallego, 1986) are mitogens for a wide variety of cells in culture including not only fibroblasts but also vascular endothelial cells, myoblasts, chondrocytes, osteoblasts and glial cells. In vivo, FGFS have been shown to promote blood vessel growth. Acidic and basic forms of FGFS have been identified, purified and characterized which, although structurally related to each other, are unique gene products. Alternative names for these molecules have been proposed based on their source (retina-derived growth factors, eye-derived growth factors, brain-derived growth factors), target cells (endothelial cell growth factor, astroglial growth factors, prostatropins) and ability to bind to heparin (heparin-binding growth factors). The amino acid sequence, homologies and activities of acidic FGF (aFGF) are reported.

Primary structure and mitogenic and angiogenic activities of brain-derived acidic fibroblast growth factor

Pure brain-derived acidic fibroblast growth factor (aFGF) is a protein that is mitogenic for a variety of types of cells in culture, including vascular endothelial cells, and is angiogenic in vivo. The complete amino acid sequence of the 140-residue bovine aFGF has been determined and recognized to be homologous to basic fibroblast growth factor and both interleukin-1α and-1β. These four growth factors define a new class of homologous proteins that presumably diverged from a common ancestor.