Kenneth A. Thomas

Vascular Endothelial Growth Factor, a Potent and Selective Angiogenic Agent

The vascular system is essential for providing oxygen and nutrients, removing metabolic waste products, and furnishing efficient access of leukocytes to tissues throughout larger animals. Angiogenesis, the sprouting of new capillaries, is required for the development of the vascular system and, consequently, the growth of vertebrates. Angiogenic proteins, including several from the fibroblast growth factor family, were identified and purified in the 1980s. They were, however, found to be mitogenic not only for vascular endothelial cells but also for a wide variety of other types of cells and appeared to promote angiogenesis as part of coordinated tissue growth and repair. In the late 1980s the first selective angiogenic growth factor was purified on the basis of its ability to induce transient vascular leakage (vascular permeability factor) and endothelial cell mitogenesis (vascular endothelial growth factor (VEGF) or vasculotropin). By amino acid and cDNA sequencing, these proteins were subsequently demonstrated to be identical. The identification of VEGF set the stage for a rapid expansion in the understanding of what now appears to be one of the most important mediators of physiologic and pathologic angiogenesis yet discovered. Previous reviews have documented some of the initial characterization of VEGF structure and activities (1–3).

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 .

The structure of human acidic fibroblast growth factor and its interaction with heparin

The secondary and tertiary structure of recombinant human acidic fibroblast growth factor (aFGF) has been characterized by a variety of spectroscopic methods. Native aFGF consists of ca. 55% beta-sheet, 20% turn, 10% alpha-helix, and 15% disordered polypeptide as determined by laser Raman, circular dichroism, and Fourier transform infrared spectroscopy; the experimentally determined secondary structure content is in agreement with that calculated by the semi-empirical methods of Chou and Fasman (Chou, P. Y., and Fasman, G. C., 1974, Biochemistry 13, 222-244) and Garnier et al. (Garnier, J. O., et al., 1978, J. Mol. Biol. 120, 97-120). Using the Garnier et al. algorithm, the major secondary structure components of aFGF have been assigned to specific regions of the polypeptide chain. The fluorescence spectrum of native aFGF is unusual in that it is dominated by tyrosine fluorescence despite the presence of a tryptophan residue in the protein. However, tryptophan fluorescence is resolved upon excitation above 295 nm. The degree of tyrosine and tryptophan solvent exposure has been assessed by a combination of ultraviolet absorption, laser Raman, and fluorescence spectroscopy; the results suggest that seven of the eight tyrosine residues are solvent exposed while the single tryptophan is partially inaccessible to solvent in native aFGF, consistent with recent crystallographic data. Denaturation of aFGF by extremes of temperature or pH leads to spectroscopically distinct conformational states in which contributions of tyrosine and tryptophan to the fluorescence spectrum of the protein vary. The protein is unstable at physiological temperatures. Addition of heparin or other sulfated polysaccharides does not affect the spectroscopic characteristics of native aFGF. These polymers do, however, dramatically stabilize the native protein against thermal and acid denaturation as determined by differential scanning calorimetry, circular dichroism, and fluorescence spectroscopy. The interaction of aFGF with such polyanions may play a role in controlling the activity of this growth factor 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.

Angiogenic potential of perivascularly delivered aFGF in a porcine model of chronic myocardial ischemia

A number of heparin-binding growth factors, including basic (bFGF) and acidic (aFGF) fibroblast growth factors have been shown to promote angiogenesis in vivo. In this study, we employed a sustained-release polymer extravascular delivery system to evaluate the angiogenic efficacy of a novel form of genetically modified aFGF in the setting of chronic myocardial ischemia. Fifteen Yorkshire pigs subjected to Ameroid occluder placement on the left circumflex (LCX) artery were treated with perivascularly administered aFGF in ethylene vinyl acetate (EVAc) polymer (10 μg, n = 7) or EVAc alone (controls, n = 8). Seven to nine weeks later, after coronary angiography to document Ameroid-induced coronary occlusion, all animals underwent studies of coronary flow and global and regional left ventricular function. Microsphere-determined coronary flow in the Ameroid-compromised territory was significantly increased in aFGF-treated compared with control animals, and this improvement in perfusion was maintained during ventricular pacing. Left ventricular function studies demonstrated improved global and regional function in aFGF-treated animals. We conclude that local perivascular delivery of genetically modified aFGF results in significant improvement in myocardial flow and regional and global left ventricular function.A number of heparin-binding growth factors, including basic (bFGF) and acidic (aFGF) fibroblast growth factors have been shown to promote angiogenesis in vivo. In this study, we employed a sustained-release polymer extravascular delivery system to evaluate the angiogenic efficacy of a novel form of genetically modified aFGF in the setting of chronic myocardial ischemia. Fifteen Yorkshire pigs subjected to Ameroid occluder placement on the left circumflex (LCX) artery were treated with perivascularly administered aFGF in ethylene vinyl acetate (EVAc) polymer (10 micrograms, n = 7) or EVAc alone (controls, n = 8). Seven to nine weeks later, after coronary angiography to document Ameroid-induced coronary occlusion, all animals underwent studies of coronary flow and global and regional left ventricular function. Microsphere-determined coronary flow in the Ameroid-compromised territory was significantly increased in aFGF-treated compared with control animals, and this improvement in perfusion was maintained during ventricular pacing. Left ventricular function studies demonstrated improved global and regional function in aFGF-treated animals. We conclude that local perivascular delivery of genetically modified aFGF results in significant improvement in myocardial flow and regional and global left ventricular function.

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.

Localization of Acidic Fibroblast Growth Factor within the Mouse Brain Using Biochemical and Immunocytochemical Techniques

The localization of acidic fibroblast growth factor (aFGF) in the male mouse brain was studied with biochemical and immunocytochemical techniques. Using two peptide-based aFGF antisera directed against independent epitopes, Western gel analysis of dissected brain demonstrated significant levels of aFGF immunoreactivity in the pons-medulla, hypothalamus and cerebellum. The cortex contained much less immunoreactivity. Consistent with the biochemical data, immunocytochemical analysis with the same two antisera demonstrated that aFGF immunoreactivity is localized in neuronal cell bodies in these regions. Numerous immunoreactive neurons were observed in the reticular formation of the pons and medulla, as well as in several other brainstem nuclei and areas. Immunoreactive neurons were also present in the lateral and medial hypothalamus, and some thalamic, subthalamic and epithalamic nuclei. In the basal ganglia, immunoreactive neurons were present in the amygdala and septum. Few intensely stained immunoreactive neurons were observed in the striatum, pallidum and neocortex. Limbic cortices contained more numerous immunoreactive neurons than neocortex. These results support the concept that aFGF is present in the brain, where it is heterogeneously distributed in neuronal cell bodies in regions involved in sensory, extrapyramidal motor, limbic and autonomic functions. The results are consistent with various neurotrophic, mitogenic, and neuromodulatory functions associated with aFGF in the mammalian central nervous system.

A novel orally bioavailable inhibitor of kinase insert domain-containing receptor induces antiangiogenic effects and prevents tumor growth in vivo.

A strategy for antagonizing vascular endothelial growth factor (VEGF) -induced angiogenesis is to inhibit the kinase activity of its receptor, kinase insert domain-containing receptor (KDR), the first committed and perhaps the last unique step in the VEGF signaling cascade. We synthesized a novel ATP-competitive KDR tyrosine kinase inhibitor that potently suppresses human and mouse KDR activity in enzyme (IC50 = 7.8–19.5 nm) and cell-based assays (IC50 = 8 nm). The compound was bioavailable in vivo, leading to a dose-dependent decrease in basal- and VEGF-stimulated KDR tyrosine phosphorylation in lungs from naïve and tumor-bearing mice (IC50 = 23 nm). Pharmacokinetics and pharmacodynamics guided drug dose selection for antitumor efficacy studies. HT1080 nude mice xenografts were treated orally twice daily with vehicle, or 33 or 133 mg/kg of compound. These doses afforded trough plasma concentrations approximately equal to the IC50 for inhibition of KDR autophosphorylation in vivo for the 33 mg/kg group, and higher than the IC99 for the 133 mg/kg group. Chronic treatment at these doses was well-tolerated and resulted in dose-dependent inhibition of tumor growth, decreased tumor vascularization, decreased proliferation, and enhanced cell death. Antitumor efficacy correlated with inhibition of KDR tyrosine phosphorylation in the tumor, as well as in a surrogate tissue (lung). Pharmacokinetics and pharmacodynamics assessment indicated that the degree of tumor growth inhibition correlated directly with the extent of inhibition of KDR tyrosine phosphorylation in tumor or lung at trough. These observations highlight the need to design antiangiogenic drug regimens to ensure constant target suppression and to take advantage of PD end points to guide dose selection.

Property-based design of KDR kinase inhibitors.

Small molecule inhibitors of KDR kinase activity have typically possessed poor intrinsic physical properties including low aqueous solubility and high lipophilicity. These features have often conferred limited cell permeability manifested in low levels of cell-based KDR inhibitory activity and oral bioavailability. Thus, the design of inhibitors with appropriate physical properties has played a critical role in the development of clinical candidates. We present a variety of structural modifications that have afforded improvements in physical properties and thereby have addressed suboptimal cellular potency and pharmacokinetics for three unique classes of KDR kinase inhibitors.