In Situ Inner Lumen Attachment of Heparin to Poly(ether sulfone) Hollow Fiber Membranes Used for Microdialysis Sampling.

An in situ chemical surface modification method to attach heparin to the inner lumen of a single hollow fiber poly(ether sulfone) (PES) membrane incorporated into a commercial microdialysis sampling device is described. The immobilization process uses gentle, room-temperature conditions with the enzyme laccase (E.C. 1.10.3.2) and 4-hydroxybenzoic acid (4HBA). The resulting functionalized inner membrane surface with a carboxylic acid functional groups allowed for (1-ethyl-3-(3-(dimethylamino)propyl) carbodimide)/ N-hydroxysuccinimide) EDC/NHS chemistry to attach heparin to the membrane surface. X-ray photoelectron spectroscopy measurements suggested successful attachment of 4HBA polymers and heparin onto the PES membrane. The microdialysis extraction efficiency after membrane surface modification was measured with model compounds fluorescein isothiocyanate (FITC)-labeled dextrans and lysozyme and the cytokines acidic fibroblast growth factor (aFGF) and CXCL1 (KC/GRO). This work demonstrates an in situ method to modify commercially available PES hollow fiber microdialysis membranes with amine or carboxylic acid functional groups.

Heparin-immobilized microspheres for the capture of cytokines.

The preparation and characterization of heparin-immobilized microspheres which were used to bind acidic fibroblast growth factor (aFGF), vascular endothelial growth factor (VEGF), monocyte chemoattractant protein 1 (MCP-1/CCL2), and regulation upon activation normal T cell express sequence (RANTES/CCL5) is described. These beads were used as trapping agents in microdialysis sampling experiments in a separate study. Both free heparin and a synthesized heparin-albumin conjugate were immobilized onto microspheres and compared for their effectiveness. The heparin-albumin conjugate microspheres exhibited significant nonspecific adsorption which appeared to be due to the albumin content. The prepared heparin-immobilized microspheres were stable for 3 months at 4 °C. A bead-based flow cytometric assay was developed to study the binding capacity and specificity of the heparin-immobilized microspheres to cytokines. These heparin-immobilized microspheres exhibited broad dynamic ranges for binding to the four cytokines (aFGF, 1.0-1,000 ng/mL; VEGF, 0.5-1,000 ng/mL; CCL2, 1.95-1,000 ng/mL; CCL5, 1.95-500 ng/mL). Fast binding kinetics of the cytokines to the heparin-immobilized beads suggests that these beads may be useful as affinity agents in microfluidic flow systems.

Strategy for fluorescent labeling of human acidic fibroblast growth factor without impairment of mitogenic activity: a bona fide tracer.

Here we describe, for the first time, the design and characterization of a bona fide fluorescently labeled mutant of the human acidic fibroblast growth factor (aFGF). The aFGF-Cys2 mutant was recombinantly synthesized by substituting the second amino acid with a reactive cysteine whose sulfhydryl group's side chain reactivity facilitated the covalent binding of a fluorescent probe as a thiolyte monobromobimane. Using a combination of biophysical and functional assays, we found that the fluorescently labeled mutant aFGF is characterized by essentially the same global folding, mitogenic activity, and association behavior with heparin, its physiological activator, as the unlabeled wild-type protein. We used this new tracer protein mutant to determine the association behavior of aFGF with heparin in the presence of high concentrations of albumin that mimicked more closely the plasma medium in which aFGF is naturally located and in which it has evolved to function. By exposing the aFGF-Cys2-heparin complex to increasing concentrations of albumin up to physiological plasma levels, we were able to demonstrate that macromolecular crowding does not affect the stoichiometry of the interaction. In summary, the dimeric aFGF-Cys2-heparin complex might represent a biologically relevant complex in physiological media.

In vitro and in vivo affinity microdialysis sampling of cytokines using heparin-immobilized microspheres.

Heparin-immobilized microspheres were included in microdialysis sampling perfusion fluids under both in vitro and in vivo conditions to improve the recovery of different cytokines, acidic fibroblast growth factor, vascular endothelial growth factor, monocyte chemoattractant protein-1 (or CCL2), and regulation upon activation normal T cell express sequence (or CCL5). Different strategies to dissociate captured CCL2 and CCL5 from the immobilized heparin were attempted, and both cytokines could be quantitatively eluted from the beads using a phosphate buffer (pH 7.4) containing 25% (v/v) acetonitrile which did not interfere with the subsequent detection of cytokine using an ELISA assay. Using these heparin-immobilized microspheres, a two to fivefold increase of microdialysis relative recovery (RR) was achieved for the four cytokines from a quiescent solution. Enhanced microdialysis RR of CCL2 using the heparin-immobilized microspheres from microdialysis probes implanted into the peritoneal cavity of a rat was performed to test the in vivo application. This work suggests that the heparin-immobilized microspheres provide an alternative affinity agent to the previously used antibody-immobilized microspheres for enhanced microdialysis sampling of cytokines.

Affinity capillary electrophoresis employing immobilized glycosaminoglycan to resolve heparin-binding peptides.

A new capillary electrophoresis technique has been developed for the affinity resolution of synthetic heparin-binding peptides using an immobilized glycosaminoglycan. Heparin and heparan sulfate were immobilized onto fused silica capillaries using biotin-neutravidin conjugation. These capillaries exhibited markedly reduced electroosmotic flow and were able to distinguish peptides based on the heparin binding domain of acidic fibroblast growth factor (residues 125-144, GLKKNGSCKRGPRTHYGQKA) that differed only in the stereochemistry of the proline amino acid residue. The peptide based on the native sequence was retarded compared to the peptide having unnatural stereochemistry, consistent with its stronger interaction for immobilized glycosaminoglycan. Improved resolution is also obtained for additional arginine and lysine containing heparin-binding peptides.

Production of acidic and basic fibroblast growth factor by the hormone-independent breast cancer cell line MDA-MB-231.

Normal, as well as the majority of malignant, mammary epithelial cells will proliferate upon stimulation by FGFs. However estrogen-independent MDA-MB-231 cells (which are able to grow in vitro in serum-free medium) are not significantly stimulated by exogenous FGFs even though they possess high and low affinity receptors for these growth factors. Biological assays, measuring CCL39 fibroblast proliferation or PC12 pheochromocytoma cell neurite outgrowth, demonstrated the presence of FGF activity in MDA-MB-231 cell extracts and also in the culture medium conditioned by these cells. This biological activity decreased in the presence of neutralizing anti-FGF antibodies. Using immunohistochemical methods FGF1 and FGF2 immunoreactivity was detected in MDA-MB-231 cells. After purification by heparin-Sepharose chromatography and Western blot analysis, M(r) 18000 molecules showed the same physicochemical characteristics as FGFI and FGF2. These results demonstrate the production and release of FGF related molecules by MDA-MB-231 cells and suggest an autocrine stimulation of these cells.

Growth of "new" coronary vascular structures by angiogenetic growth factors.

The efficacy of the human angiogenetic heparin-binding growth factor I (HBGF-I) to initiate site-directed growth of new blood vessels from the aorta into the myocardium was studied. First, manipulated Escherichia coli bacteria, which had received the human mRNA-transcript for HBGF I into their genetic material, were cultivated. The growth factor derived was purified using heparin-Sepharose affinity chromatography. The separation and characterization of biologically active alpha- and beta-chains was carried out using high pressure liquid chromatography (HPLC) of dialyzed and lyophilized samples from the heparin-Sepharose column. One microgram HBGF I (alpha-ECGF) was bound to polytetrafluoroethylene (PTFE) sponges, precoated with collagen type I, and implanted between the aorta and the myocardium of the left ventricle in experimental rats. Twelve growth factor implants in the experimental group were compared to six controls receiving uncoated PTFE sponges for 9 weeks. Digitized computed angiography showed new blood vessels between the aorta and the myocardium in 11 of the 12 experimental animals, and retrograde coronary perfusion by these "new" vascular structures could be seen. Histology showed no specific structures in the control group (without HBGF I). In the experimental group (with HBGF I) individual vessels with highly differentiated endothelial and smooth muscle cell layers were evident. Our experiments proved the feasibility of induced, site-directed angiogenesis. It is possible to initiate in vivo growth of new "coronary" vascular structures between the aorta and the myocardium.

Single-step purification on DEAE-sephacel of recombinant polypeptides produced in Escherichia coli.

We describe a method for the purification of recombinant proteins based upon the selective interaction of the choline-binding domain of the pneumococcal murein hydrolase and tertiary amines. Proteins of interest, fused to the binding domain by a peptide linker, containing the cleaving sequence recognized by blood coagulation factor Xa, can either be assayed for biological activities in vitro and in vivo or have the binding moiety removed to yield a totally unmodified form, suitable for clinical and functional studies. The method can also be applied to the production of low molecular mass peptides. The principle of the technique is illustrated with acidic fibroblast growth factor and with a neuropeptide-like fragment of ten amino acids contained within its sequence.

Purification and characterization of fibroblast growth factors from the opossum, Monodelphis domestica, brain.

An extract from the brain of the opossum Monodelphis domestica was fractionated by heparin affinity chromatography. A major peak of mitogenic activity (heparin binding growth factor 2, HBGF-2) eluted from heparin-Sepharose between 1.7 and 2.0 M NaCl. Antisera specific for bovine bFGF detected four polypeptides of 17.5-23 kDa in opossum brain HBGF-2 preparations. Opossum brain heparin binding growth factor 1 (HBGF-1), a minor peak of activity, eluted from heparin-Sepharose at 1.1 NaCl and contained a 16.2 kDa protein that cross-reacted with antiserum against bovine aFGF.

Cytotoxic activity of chimeric proteins composed of acidic fibroblast growth factor and Pseudomonas exotoxin on a variety of cell types.

Chimeric proteins composed of acidic fibroblast growth factor (acidic FGF) and several forms of Pseudomonas exotoxin (PE) that cannot bind to the PE receptor have been produced in Escherichia coli by expressing chimeric genes in which DNA encoding acidic FGF is fused to various mutant forms of PE. These acidic FGF-PE fusion proteins were found to be cytotoxic to a variety of tumor cell lines including hepatocellular (PLC/PRF/5 and HEPG2), prostatic (LNCaP), colon (HT29), and breast (MCF-7) carcinomas at concentrations of 1-70 ng/ml. The cytotoxic effects of acidic FGF-PE were FGF-receptor specific as demonstrated by competition with excess acidic FGF and by showing that acidic FGF-PE bound to the FGF receptor with the same affinity as acidic FGF. Furthermore, the cell-killing activity of acidic FGF-PE was toxin-mediated, as an acidic FGF-PE mutant, which does not possess ADP-ribosylation activity, failed to kill cells. These findings demonstrate that acidic FGF-PE is a potent cytotoxic molecule that can be targeted to FGF receptor-bearing cells. Because acidic FGF is a potent angiogenic molecule, cytotoxic acidic FGF-PE chimeras may have utility as anti-angiogenic agents. These molecules could be helpful in determining the functional role of FGF receptors in cellular processes.

High-level production of human acidic fibroblast growth factor in E. coli cells: inhibition of DNA synthesis in rat mammary fibroblasts at high concentrations of growth factor.

Recombinant human acidic fibroblast growth factor has been produced in E. coli cells at a level of at least 50 mg/l culture. The recombinant and natural acidic fibroblast growth factors are almost identical to one another when tested on rat mammary fibroblasts for their ability to stimulate DNA synthesis, to bind to the high-affinity surface receptors of the cells and to inhibit DNA synthesis when present in the culture medium at high concentrations. The recombinant acidic fibroblast growth factor binds to two cell-surface polypeptides of molecular masses 160 kDa and 140 kDa, which are the same size as the receptors for basic fibroblast growth factor, and it binds preferentially to the smaller polypeptide.

Soluble heparin-binding lectins from human brain: purification, specificity and relationship to an heparin-binding growth factor.

1. An heparin-binding lectin activity was detected in soluble extracts of human brain. Three polypeptide chains were purified by affinity chromatography on heparin-Sepharose. Their Mrs estimated by polyacrylamide gel electrophoresis were 13,000, 14,500 and 16,000. 2. Several glycosaminoglycans were potent inhibitors of their hemagglutination activity. 3. From the pool of purified lectins three peaks were separated by reversed-phase high-performance liquid chromatography. They were indistinguishable by activity criteria (hemagglutination, stimulation of endothelial cell growth), and immunological relationship was found between one of them and acidic fibroblast growth factor (aFGF).

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.