Judith A. Abraham

Modulation of Wound Repair by Members of the Fibroblast Growth Factor Family

The fibroblast growth factors (FGFs) comprise a family of at least nine structurally homologous polypeptides that are found in a variety of cells and tissues (Baird and Bohlen, 1990; Brem and Klagsbrun, 1993; Burgess and Maciag, 1989; Folkman and Klagsbrun, 1987; Klagsbrun, 1989; Klagsbrun and D’Amore, 1991; Klagsbrun and Folkman, 1990; Rifkin and Moscatelli, 1989; Tanaka et al., 1992; Miyamoto et al., 1993). This family includes acidic FGF (aFGF), basic FGF (bFGF), int-2 protein, HST/K-FGF, FGF-5, FGF-6, keratinocyte growth factor (KGF), androgen-induced growth factor (AIGF), and glia-activating factor (GAF) (see Table I). These growth factors have been enumerated as FGF-1 through FGF-9, respectively, in order to simplify the nomenclature (Baird and Klagsbrun, 1991). However, since the name KGF is still widely used, the designation “KGF/FGF-7” will be used here in referring to this factor. The aFGF (FGF-1) and bFGF (FGF-2) proteins are the most extensively characterized FGF family members in terms of detailed knowledge concerning structure and biological activity.

Peptides derived from the complementarity-determining regions of anti-Mac-1 antibodies block intercellular adhesion molecule-1 interaction with Mac-1.

Peptides or small molecules that can block the interaction of the integrin Mac-1 with its receptor, intercellular adhesion molecule-1 (ICAM-1), have not previously been developed. We studied this interaction by measuring the adherence of ICAM-1-expressing Chinese hamster ovary (CHO) cells to immobilized, purified Mac-1. Nucleotide sequence information was obtained for the complementarity determining regions (CDRs) of three antibodies (44aacb, MY904, and 118.1) shown to block Mac-1-mediated cell adherence. Peptides were synthesized based on the predicted amino acid sequences of the CDRs and tested for the ability to block cell adhesion to Mac-1. Peptides derived from CDR1 of 44aacb, CDR2 of 118.1, and CDRs 1 and 3 of MY904 heavy chains were found to possess blocking activity at 10–100 μm. This may indicate that one or two CDRs contribute disproportionately to the antibody binding affinity. The binding of ligands to Mac-1 has been shown to require a region of the α-chain known as the I- or A-domain. We have recombinantly produced Mac-1 I-domain, and show that it is also capable of supporting the adherence of ICAM-1-expressing CHO cells. The adherence of ICAM-1-CHO cells to the I-domain is inhibited by 44aacb and 118.1 and by the CDR peptides from 44aacb and 118.1. By using phage display of peptide libraries based on the 118.1 CDR peptide with five residues randomized, we were able to identify a novel peptide inhibitor of Mac-1 with substitutions at all five positions. These peptides provide lead structures for development of Mac-1 antagonists.

Heparin-Binding EGF-Like Growth Factor Synthesis by Smooth Muscle Cells

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) has been previously demonstrated to be a potent mitogen for smooth muscle cells. Evidence is now presented that these cells synthesize HB-EGF as well. Cultured fetal human vascular smooth muscle cells express 2.5-kb HB-EGF mRNA. These cells also release an HB-EGF-like activity that (i) stimulates smooth muscle cell and BALB/c 3T3 cell but not endothelial cell proliferation; (ii) binds to TSK heparin affinity columns and is eluted with 0.9-1.2 M NaCl, and (iii) triggers phosphorylation of a protein with the same molecular weight as the 170-kD EGF receptor. In addition, 125I-HB-EGF can be cross-linked to the EGF receptor on fetal human vascular smooth muscle cells. These results suggest that smooth muscle cells can both synthesize and respond to HB-EGF, and that HB-EGF may therefore be involved in autocrine regulation of these cells.

Insulin-Like Growth Factor-Binding Protein-3 Induces Fetalization in Neonatal Rat Cardiomyocytes

We employed cDNA microarrays representing 4000 distinct sequences to profile changes in gene expression in a rodent model of heart disease, namely, progression to heart failure after myocardial infarction. Differential gene expression in the left ventricle was examined at 4-week intervals over a 12-week period after coronary artery ligation in rats. Over this time course, insulin-like growth factor-binding protein-3 (IGFBP-3) was found to have a greater expression than in nondiseased tissues. We then employed quantitative real-time PCR to analyze gene expression in neonatal rat cardiac myocytes that had been treated with recombinantly expressed IGFBP-3 to examine a number of transcriptional responses designed to reflect the heart failure phenotype. The IGFBP-3 protein was shown to induce transcription of atrial natriuretic factor (ANF) and beta-myosin heavy chain (B-MHC). Analysis of conditioned media taken from IGFBP-3-treated cardiac myocyte cultures demonstrated an increase in ANF protein as well as in protein synthesis, as determined by metabolic incorporation of a radiolabeled amino acid. However, transcriptional changes of troponin-1, endothelin-1, or angiotensin-II by IGFBP-3 were not observed.