Biomedical subjects
Y Shing
Publications and source records attributed to Y Shing.
Control of angiogenesis by heparin and other sulfated polysaccharides.
Heparin and its related polysaccharides are revealed to have important new functions as regulators of blood vessel growth and regression. This regulatory activity may be explained in part by at least five mechanisms in which heparin and heparan sulfate interact with peptide growth factors: (1) Heparin and heparan sulfate have a high affinity for angiogenic growth factors such as the fibroblast growth factors and VEGF, as well as for angiogenic inhibitors such as thrombospondin and platelet factor 4. (2) Heparin and its related polysaccharides stabilize bFGF and other growth factors. (3) FGFs and thrombospondin are stored in the extracellular matrix bound to heparan sulfate; fragments of heparin or heparan sulfate may act as natural chaperones to shuttle bFGF or other growth factors to different cellular compartments. (5) Heparin-like low-affinity receptors on the surface of endothelial cells (and other cells), prepare FGFs for binding to their specific high affinity receptors; and (6) Heparin and its related polysaccharides potentiate angiostatic steroids. It is likely that future investigations will uncover even more fundamental regulatory roles for heparin as well as for other polysaccharides in the normal function of growth factors, especially in the complex process of angiogenesis.
Biaffinity chromatography of fibroblast growth factors.
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Duodenal ulcer. Discovery of a new mechanism and development of angiogenic therapy that accelerates healing.
The complete purification of the first angiogenic molecule, basic fibroblast growth factor (bFGF), was carried out in the authors' laboratory in 1983. Application of this peptide to chronic wounds enhances angiogenesis and accelerates wound healing. The authors showed that an acid-stable form of bFGF (i.e., bFGF-CS23) could be administered orally to rats with duodenal ulcers. The peptide promoted a ninefold increase of angiogenesis in the ulcer bed and accelerated ulcer healing more potently than cimetidine. Basic fibroblast growth factor did not reduce gastric acid. The authors now show that bFGF exists as a naturally occurring peptide in rat and human gastric and duodenal mucosa. This endogenous bFGF is present also in the bed of chronic ulcers in rats. Sucralfate binds bFGF and protects it from acid degradation. The sucralfate is angiogenic, based on its affinity for bFGF. When sucralfate is administered orally to rats, it significantly elevates the level of bFGF in the ulcer bed. Cimetidine, by its capacity to reduce gastric acid, also elevates bFGF in the ulcer bed. A hypothetical model is proposed in which prevention of ulcer formation or accelerated healing of ulcers by conventional therapies may be FGF dependent. Acid-stable bFGF-CS23 may be considered as a form of replacement therapy in the treatment of duodenal ulcers.
Induction of tumor necrosis factor by macrophage colony-stimulating factor in vivo.
The effect of human urinary colony-stimulating factor (CSF-1) on the production of tumor necrosis factor (TNF) in vivo was assessed. Purified CSF-1 was administered i.v. to rabbits 4 days prior to injection with lipopolysaccharide (LPS). TNF in the serum prepared from rabbits bled 90 min after LPS injection was measured using cytotoxicity assays employing mouse L929 cells and antirabbit TNF monoclonal antibody. The results indicated that CSF-1 was able to induce the production of TNF in vivo and had a synergistic effect with Propionibacterium acnes.
A new method for purifying heparin-binding growth factors.
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The fate of intravenously administered bFGF and the effect of heparin.
The fate and effects of intravascular bFGF are unknown. We have investigated the fate of bFGF administered intravenously to rats in the presence and absence of heparin, and evaluated the effect of a 3-day IV infusion of bFGF on proliferation of endothelial and vascular smooth muscle cells in situ. [125I]bFGF, administered as an IV bolus, was rapidly cleared from the circulation (t1/2 = 1.5 min) by the liver. Nevertheless, it was maintained at a constant, predictable concentration in the blood (9.7 +/- 4% of the amount infused) by continuous IV infusion. Heparin consistently altered the pattern: slowing the rate of clearance (t1/2 = 4.5 min), increasing the plateau concentration in the blood during continuous infusion (32.5 +/- 14.3% of the amount infused), and allowing intact (as determined by gel analysis) bFGF to cross from the circulation into the urine. A 3-day infusion of bFGF alone (2.5 ng/kg/min) and with adenosine (11.6 microM/kg/hr) did not increase [3H]thymidine incorporation in either endothelial cells or vascular smooth muscle cells, suggesting that they are refractory to this factor when it is administered intravascularly.
Multiple forms of basic fibroblast growth factor: amino-terminal cleavages by tumor cell- and brain cell-derived acid proteinases.
Basic fibroblast growth factor (FGF) was purified by heparin-Sepharose chromatography from two sources, brain and hepatoma cells. Brain cell-derived basic FGF (brFGF) and hepatoma cell-derived basic FGF (heFGF) were found to exist in multiple forms whose molecular weights depended on whether they were extracted from their respective tissue or cells at neutral or acid pH. When extracted at pH 7.0 brFGF and heFGF comigrated on NaDodSO4/PAGE with a Mr of approximately 18,400. When extracted at pHs 3.5-4.5, acid proteinases cleaved brFGF and heFGF to lower molecular weight forms but to different extents. brFGF was cleaved to a Mr 18,000 form at acid pH by a brain-derived acid proteinase that could be inhibited by pepstatin. heFGF was cleaved mostly to a Mr 16,500 form at acid pH by a hepatoma cell-derived acid proteinase that was inhibited by leupeptin. Electrophoretic transfer blot analysis using site-specific anti-FGF antibodies suggested that the cleavages occurred at the amino-terminal ends of brFGF and heFGF. Cleavage to lower molecular weight forms of brFGF and heFGF did not affect growth factor activity or chromatographic behavior on heparin-Sepharose columns.
Purification and characterization of a bovine colostrum-derived growth factor.
A growth factor in bovine colostrum was purified to homogeneity by a combination of acid extraction, boiling, cation exchange chromatography, isoelectric focusing, and reverse phase HPLC. The bovine colostrum growth factor (BCGF) had an isoelectric point of about 10, a native mol wt of about 30,000, was resistant to inactivation by boiling and exposure to pH 1, but was inactivated by dithiothreitol. BCGF appeared to be structurally related to human platelet-derived growth factor (PDGF) and competed with human PDGF in a radioreceptor assay. However, while human PDGF appeared to be a heterodimer of 17,000 and 14,000 mol wt subunits, BCGF appeared to be a homodimer of 20,000 mol wt subunits. Purified BCGF had a specific activity in stimulating 3T3 cell proliferation of about 3-6 U/ng and was active at about 1-2 ng/ml.
Purification and characterization of heparin-binding endothelial cell growth factors.
Thirteen endothelial cell growth factors have been purified to homogeneity by heparin affinity and reversed-phase high performance liquid chromatography, and their chromatographic and electrophoretic properties were compared. The amino acid compositions of 10 of these mitogens have also been determined. The results indicate that these heparin-binding growth factors (HBGFs) can be subdivided into two classes. Class 1 HBGFs are anionic mitogens of molecular weight 15,000-17,000 found in high levels in neural tissue and include acidic brain fibroblast growth factor and retina-derived growth factor. Class 2 HBGFs are cationic mitogens of molecular weight 18,000-20,000 found in a variety of normal tissues and are typified by pituitary fibroblast growth factor and cartilage-derived growth factor. Typical class 2 HBGFs have also been isolated from a rat chondrosarcoma, a human melanoma, and a human hepatoma, suggesting that tumors do not make a structurally distinct HBGF class. These results provide a sound basis for the evaluation of the HBGFs purified from a variety of tissues and species and for the delineation of their normal and pathological functions in vivo.
Angiogenesis is stimulated by a tumor-derived endothelial cell growth factor.
A growth factor mitogenic for BALB/C 3T3 cells and capillary endothelial cells was isolated from a rat chondrosarcoma and purified to homogeneity. Purification was accomplished by a combination of BioRex 70 cation exchange chromatography and heparin affinity chromatography. The pure chondrosarcoma-derived growth factor (ChDGF) had a molecular weight of about 18,000. The angiogenesis activity of pure ChDGF was tested by measuring its ability to vascularize the chorioallantoic membrane (CAM) and yolk sac membrane of the developing chick. The ability of ChDGF to induce the growth of limbal vessels in the rat cornea was also measured. To quantitate the angiogenesis response, a unit system based on the growth factor activity of ChDGF for 3T3 cells was adopted. ChDGF was found to have a specific activity of about 5 units/ng when applied to 3T3 cells. About 300-600 units of ChDGF in the two types of developing chick membrane and 30-5 units of ChDGF in the rat cornea were found to stimulate noninflammatory angiogenesis.