Background-The present article is a report of our animal experiments and also of the first clinical results of a new treatment for coronary heart disease using the human growth factor FGF-I (basic fibroblast growth factor) to induce neoangiogenesis in the ischemic myocardium. Methods and Results-FGF-I was obtained from strains of Escherichia coli by genetic engineering, then isolated and highly purified.Several series of animal experiments demonstrated the apathogenic action and neoangiogenic potency of this factor. After successful conclusion of the animal experiments, it was used clinically for the first time. FGF-I (0.01 mg/kg body weight) was injected close to the vessels after the completion of internal mammary artery (IMA)/left anterior descending coronary artery (LAD) anastomosis in 20 patients with three-vessel coronary disease. All the patients had additional peripheral stenoses of the LAD or one of its diagonal branches. Twelve weeks later, the IMA bypasses were selectively imaged by intra-arterial digital subtraction angiography and quantitatively evaluated. In all the animal experiments, the development of new vessels in the ischemic myocardium could be demonstrated angiographically. The formation of capillaries could also be demonstrated in humans and was found in all cases around the site of injection. A capillary network sprouting from the proximal part of the coronary artery could be shown to have bypassed the stenoses and rejoined the distal parts of the vessel. Conclusions-We believe that the use of FGF-I for myocardial revascularization is in principle a new concept and that it may be particularly suitable for patients with additional peripheral stenoses that cannot be revascularized surgically. (Circulation. 1998;97:645-650.)

The three‐dimensional structure of the holo form of recombinant cellular bovine heart fatty‐acid‐binding protein (H‐FABPc), a polypeptide of 133 amino acid residues with a molecular mass of 15 kDa, has been determined by multidimensional homonuclear and heteronuclear NMR spectroscopy applied to uniformly 15N‐labeled and unlabeled protein. A nearly complete set of 1H and 15N chemical shift assignments was obtained. A total of 2329 intramolecular distance constraints and 42 side‐chain χi dihedral‐angle constraints were derived from cross‐relaxation and J coupling information. 3D nuclear Overhauser enhancement and exchange spectroscopy combined with heteronuclear multiple‐quantum coherence (NOESY‐HMQC) experiments, performed on a sample of uniformly 13C‐labeled palmitic acid bound to unlabeled cellular heart fatty‐acid‐binding protein revealed 10 intermolecular contacts that determine the orientation of the bound fatty acid. An ensemble of protein conformations was calculated with the distance‐geometry algorithm for NMR applications (DIANA) using the redundant dihedral‐angle constraint (REDAC) strategy. After docking the fatty acid into the protein, the protein‐ligand arrangement was subject to distance‐restrained energy minimization. The overall conformation of the protein is a β‐barrel consisting of 10 antiparallel β‐strands which form two nearly orthogonal β‐sheets of five strands each. Two short helices form a helix‐turn‐helix motif in the N‐terminal region of the polypeptide chain. The palmitic acid is bound within the protein in a U‐shaped conformation close to the two helices. The obtained solution structure of the protein is consistent with a number of fatty‐acid‐binding‐protein crystal structures.

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Cytoplasmic heart-type fatty acid-binding protein has recently gained much attention in clinical diagnosis as a very early marker of acute myocardial infarction. Immunoassays have been developed for determination of this protein in plasma and urine samples. In the present study it is shown that those types of fatty acid-binding proteins which are abundant in tissues other than heart and muscle do not interfere with immunochemical determination of heart-type fatty acid-binding protein. To provide sufficient protein of consistent quality as standard in these immunoassays, human heart-type fatty acid-binding protein was cloned, expressed in Escherichia coli and purified to homogeneity. For quantitation of the recombinant protein its extinction coefficient was determined. Comparison of the recombinant and tissue-derived proteins by a variety of methods revealed both proteins to show similar kinetic as well as equilibrium constants with respect to two monoclonal antibodies currently applied in immunochemical detection of heart-type fatty acid-binding protein. Both preparations were indistiguishable in sandwich-ELISA and immunosensor measurements. A high stability of the recombinant protein was proven by ELISA measurements during storage and several freeze and thaw cycles. Thus, recombinant and tissue-derived heart-type fatty acid-binding proteins are immunochemically equivalent. The recombinant human heart-type fatty acid-binding protein is now available as standard for immunoassays.

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.