Endothelial cells and angiogenic growth factors in cancer growth and metastasis. Introduction.
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Animals↗
Biomedical subjects
J Folkman
Publications and source records attributed to J Folkman.
A proposed cause for the hepatic dysfunction associated with parenteral nutrition.
Total parenteral nutrition (TPN) is associated with cholestasis and hepatic steatosis, which can be lethal in infants who cannot be fed orally. It was determined that route of administration was not the critical variable in the development of hepatic steatosis. Two groups of young rats received equivalent amounts of a standard TPN solution either orally or intravenously for an 8- to 10-day period during which they received no other nutrition. Both groups gained equivalent weight and developed marked hepatic steatosis. To test whether the solution was toxic or deficient, three groups of rats were given TPN solution orally and a fraction of their usual daily intake of rat chow. Rats receiving less than 10% of their usual chow intake developed steatosis; rats receiving more than that did not. To determine the solubility of the protective material in chow, two groups of rats were given TPN solution orally and chow that been extracted with either water or the organic solvent chloroform. Rats eating the water-extracted chow developed steatosis, rats eating chloroform-extracted chow did not. Although the protective component in chow was apparently water soluble, addition of a water soluble extract of chow to the TPN solution fed another two groups of rats did not prevent steatosis at 0.1 mg/mL and only partially, if at all, at 10 mg/mL. TPN-related hepatic dysfunction, as measured by the development of hepatic steatosis in this model, may be due to a deficiency in the TPN solution. The missing constituent(s) appears to be present in rat chow and can be extracted with water, but not with an organic solvent.
Animals↗
How the field of controlled-release technology began, and its central role in the development of angiogenesis research.
The first controlled-release methodology was developed in 1962 and was based upon diffusion of small molecules (molecular weight less than 500) through the wall of silicone rubber tubing. The problem of controlled-release of large molecules such as proteins was not solved until more than a decade later, when it was discovered that microchannels formed around lyophilized proteins embedded in polymers such as ethylene vinyl acetate copolymer (EVA) and poly(hydroxyethylmethacrylate) (pHEMA), as water permeated through the polymer. These studies led to the development of a 5-year contraceptive implant, e.g., Norplant; rapid progress in the field of angiogenesis research; and a method for regulating cell shape and growth control in vitro.
Animals↗
Isolation, characterization, and localization of heparin-binding growth factors in the heart.
Acidic and basic fibroblast growth factors (aFGF and bFGF) are angiogenic polypeptide mitogens for cells of mesodermal and neuroectodermal origin. In this report we describe the purification from several normal human hearts (including a very fresh, nonischemic sample) of heparin-binding, acid-, heat- and trypsin-sensitive 14-18-kD peptides that crossreact with antisera against aFGF and bFGF. Further evidence includes (a) prevention of mitogenicity by protamine and by anti-bFGF, (b) displacement of 125I-bFGF from cell membranes, and (c) stimulation of capillary endothelial cell migration. Specific immunohistochemistry localized bFGF to endothelial cells and, surprisingly, to cardiac myocytes, with almost no immunoreactivity in smooth muscle cells. These peptides may function in cardiac embryogenesis, hypertrophy, atherogenesis, angiogenesis, and wound healing, and may also have endocrine, neurotropic, or vasomotor functions.
Aged↗
Conditioned medium from mouse sarcoma 180 cells contains vascular endothelial growth factor.
Medium conditioned by mouse sarcoma 180 cells stimulates the growth of capillary endothelial cells. The growth factor produced by mouse sarcoma 180 cells is heparin-binding, dithiothreitol-sensitive, endothelial cell specific, and secreted into the medium. The characteristics of this mouse sarcoma-derived growth factor are very similar to those of vascular endothelial growth factor (VEGF) first described by Ferrara and Henzel (1989). The N-terminal amino acid sequences of the two growth factors are similar. Since the amino acid sequence of vascular permeability factor (VPF) is essentially identical to that of VEGF, a Western blot of mouse sarcoma 180-derived endothelial growth factor was probed with a polyclonal antibody raised against human VPF. This antibody reacted with several proteins of approximately 23 kDa, suggesting the presence of multiple forms of a VEGF-like protein. A full length cDNA probe for bovine VEGF reacted strongly with RNA isolated from mouse sarcoma 180 cells. We conclude that an endothelial growth factor found in conditioned medium from mouse sarcoma 180 cells is VEGF.
Amino Acid Sequence↗
Expression of acidic and basic fibroblast growth factors in human and bovine vascular smooth muscle cells.
The expression and synthesis of acidic and basic fibroblast growth factors (aFGF and bFGF) in cultures of bovine and human vascular smooth muscle cells (BSMC and HSMC) was studied. BSMC express and synthesize only bFGF, whereas HSMC express and synthesize both bFGF and aFGF. The presence of bFGF in BSMC is shown by the following criteria: (1) the growth factor activity in BSMC lysates binds to a heparin-affinity column and elutes as a single peak at 1.5-1.7 M NaCl, characteristic for bFGF; (2) this extract is mitogenic for smooth muscle cells; (3) Northern blot analysis demonstrates three distinct bFGF mRNAs of 7.0, 4.0, and 1.9 kb; no aFGF mRNA species were detected. Analysis of human umbilical vein endothelial cells yielded similar results: Heparin-affinity chromatography and Northern blot analysis failed to demonstrate the presence of aFGF despite the detection of bFGF by these techniques. In contrast, HSMC synthesize two growth factor activities: First, they bind to an immobilized heparin column and elute as two separate peaks at 1.2 and 1.5-1.7 M NaCl, characteristic for aFGF and bFGF; and second. Northern blot analysis demonstrates the expression of aFGF mRNA of 4.6 kb and bFGF mRNAs of 7.0, 4.0 and 1.9 kb. Furthermore, it is shown that aFGF and bFGF are potent mitogens for smooth muscle cells in vitro.
Animals↗
How does extracellular matrix control capillary morphogenesis?
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Animals↗
Induction of angiogenesis during the transition from hyperplasia to neoplasia.
It is now well established that unrestricted growth of tumours is dependent upon angiogenesis. Previous studies on tumour growth, however, have not revealed when or how the transition to an angiogenic state occurs during early tumour development. The advent of transgenic mice carrying oncogenes that reproducibly elicit tumours of specific cell types is providing a new format for studying multi-step tumorigenesis. In one of these models, transgenic mice expressing an oncogene in the beta-cells of the pancreatic islets heritably recapitulate a progression from normality to hyperplasia to neoplasia. We report here that angiogenic activity first appears in a subset of hyperplastic islets before the onset of tumour formation. A novel in vitro assay confirms that hyperplasia per se does not obligate angiogenesis. Rather, a few hyperplastic islets become angiogenic in vitro at a time when such islets are neovascularized in vivo and at a frequency that correlates closely with subsequent tumour incidence. These findings suggest that induction of angiogenesis is an important step in carcinogenesis.
Adenoma, Islet Cell↗
Successful treatment of an angiogenic disease.
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Capillaries↗
Importance of size, sulfation, and anticoagulant activity in the potentiation of acidic fibroblast growth factor by heparin.
Heparin was previously reported to potentiate the mitogenic activity of endothelial cell mitogens in a crude extract of bovine hypothalami (Thornton, S. C., Mueller, S. N., and Levine, E. M. (1983) Science 222, 623-625). We and others (Gospodarowicz, D., and Cheng, J. (1986) J. Cell. Physiol. 128, 475-484) have reported that the growth stimulatory effects of acidic fibroblast growth factor (aFGF) are potentiated in a similar manner. We have used these observations as the basis of an assay to characterize the importance of size, sulfation, and anticoagulant activity of heparin in mediating this effect. Partial nitrous acid depolymerization of heparin from porcine intestinal mucosa resulted in a mixture of heparin fragments, containing oligosaccharides ranging from disaccharides to polysaccharides of about 40 monosaccharides in length. This mixture was fractionated by ion exchange chromatography and gel permeation chromatography to obtain size-homogeneous oligosaccharides with different degrees of sulfation. Assay of these heparin-derived saccharides in the presence of a suboptimal concentration of aFGF revealed that a minimum chain length and a certain degree of sulfation is required in order to potentiate the action of aFGF. Low sulfate oligosaccharides (4-16 units) were unable to potentiate aFGF, whereas medium sulfate fractions of octadecasaccharides and larger were able to moderately potentiate aFGF. The potentiation of aFGF by the high sulfate fraction correlated with the saccharide size: 12 or more monosaccharide units were necessary to achieve potentiation equivalent to whole heparin, octa- and decasaccharides were mildly stimulatory, and hexasaccharides were without effect. In the absence of aFGF, intact heparin as well as all the oligosaccharides examined, inhibited the proliferation of capillary endothelial cells to approximately the same degree, between 20 and 50% inhibition. When a tetradecasaccharide was separated into a binding and a nonbinding fraction on matrix-bound antithrombin III, no difference was seen for these fractions in the endothelial cell proliferation assay. These results indicate that both size and sulfation of a heparin-derived oligosaccharide contribute to its ability to interact with aFGF and/or endothelial cells and that this interaction is independent of anticoagulant activity. In addition, our findings suggest that the inhibitory and potentiating effects of heparin on capillary endothelial cells have different structural requirements.
Animals↗
Control of angiogenesis with synthetic heparin substitutes.
Many diseases are dominated by persistent growth of capillary blood vessels. Tumor growth is also angiogenesis-dependent. Safe and effective angiogenesis inhibitors are needed to determine whether control of angiogenesis would be therapeutic. Heparin and certain steroids, administered together, can inhibit angiogenesis in a synergistic manner. This "pair" effect suggested that specific hydrophilic cycloamyloses may be suitable heparin substitutes. beta-Cyclodextrin tetradecasulfate administered with a steroid inhibits angiogenesis at 100 to 1000 times the effectiveness of heparin in the chick embryo bioassay. This cyclic oligosaccharide also augments the anti-angiogenic effect of angiostatic steroids against corneal neovascularization in rabbits when beta-cyclodextrin tetradecasulfate and a steroid are inserted into the cornea or applied topically as eyedrops.
Animals↗
Basic fibroblast growth factor binds to subendothelial extracellular matrix and is released by heparitinase and heparin-like molecules.
Basic fibroblast growth factor (bFGF) exhibits specific binding to the extracellular matrix (ECM) produced by cultured endothelial cells. Binding was saturable as a function both of time and of concentration of 125I-bFGF. Scatchard analysis of FGF binding revealed the presence of about 1.5 X 10(12) binding sites/mm2 ECM with an apparent kD of 610nM. FGF binds to heparan sulfate (HS) in ECM as evidenced by (i) inhibition of binding in the presence of heparin or HS at 0.1-1 micrograms/mL, but not by chondroitin sulfate, keratan sulfate, or hyaluronic acid at 10 micrograms/mL, (ii) lack of binding to ECM pretreated with heparitinase, but not with chondroitinase ABC, and (iii) rapid release of up to 90% of ECM-bound FGF by exposure to heparin, HS, or heparitinase, but not to chondroitin sulfate, keratan sulfate, hyaluronic acid, or chondroitinase ABC. Oligosaccharides derived from depolymerized heparin, and as small as the tetrasaccharide, released the ECM-bound FGF, but there was little or no release of FGF by modified nonanticoagulant heparins such as totally desulfated heparin, N-desulfated heparin, and N-acetylated heparin. FGF released from ECM was biologically active, as indicated by its stimulation of cell proliferation and DNA synthesis in vascular endothelial cells and 3T3 fibroblasts. Similar results were obtained in studies on release of endogenous FGF-like mitogenic activity from Descemet's membranes of bovine corneas. It is suggested that ECM storage and release of bFGF provide a novel mechanism for regulation of capillary blood vessel growth. Whereas ECM-bound FGF may be prevented from acting on endothelial cells, its displacement by heparin-like molecules and/or HS-degrading enzymes may elicit a neovascular response.
Animals↗
Mechanochemical switching between growth and differentiation during fibroblast growth factor-stimulated angiogenesis in vitro: role of extracellular matrix.
The angiogenic factor, basic fibroblast growth factor (FGF), either stimulates endothelial cell growth or promotes capillary differentiation depending upon the microenvironment in which it acts. Analysis of various in vitro models of spontaneous angiogenesis, in combination with time-lapse cinematography, demonstrated that capillary tube formation was greatly facilitated by promoting multicellular retraction and cell elevation above the surface of the rigid culture dish or by culturing endothelial cells on malleable extracellular matrix (ECM) substrata. These observations suggested to us that mechanical (i.e., tension-dependent) interactions between endothelial cells and ECM may serve to regulate capillary development. To test this hypothesis, FGF-stimulated endothelial cells were grown in chemically defined medium on bacteriological (nonadhesive) dishes that were precoated with different densities of fibronectin. Extensive cell spreading and growth were promoted by fibronectin coating densities that were highly adhesive (greater than 500 ng/cm2), whereas cell rounding, detachment, and loss of viability were observed on dishes coated with low fibronectin concentrations (less than 100 ng/cm2). Intermediate fibronectin coating densities (100-500 ng/cm2) promoted cell extension, but they could not completely resist cell tractional forces. Partial retraction of multicellular aggregates resulted in cell shortening, cessation of growth, and formation of branching tubular networks within 24-48 h. Multicellular retraction and subsequent tube formation also could be elicited on highly adhesive dishes by overcoming the mechanical resistance of the substratum using higher cell plating numbers. Dishes coated with varying concentrations of type IV collagen or gelatin produced similar results. These results suggest that ECM components may act locally to regulate the growth and pattern-regulating actions of soluble FGF based upon their ability to resist cell-generated mechanical loads. Thus, we propose that FGF-stimulated endothelial cells may be "switched" between growth, differentiation, and involution modes during angiogenesis by altering the adhesivity or mechanical integrity of their ECM.
Animals↗
A new method for purifying heparin-binding growth factors.
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Animals↗
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.
Animals↗
Angiogenic activity of adipose tissue.
Adipose tissue has been used to promote wound healing and to revascularize ischemic myocardium. We explored whether fat from various sources was angiogenic in the cornea. Rabbit subcutaneous and omental fat induced grossly visible neovascularization of all rabbit corneas studied, and at a similar rate and intensity. Neovascularization was not observed in any cornea following control implantation of liver or muscle. Neovascularization was blocked in all rabbits in which indomethacin was administered orally 3 days before implantation of fat and continued following implantation, suggesting that prostaglandins are associated with fat induced angiogenesis.
Adipose Tissue↗
Inhibition of angiogenesis through modulation of collagen metabolism.
Combinations of angiostatic steroids and heparin induce basement membrane breakdown, capillary retraction, and endothelial cell rounding as part of their antiangiogenic action (Ingber DE, Madri JA, Folkman J: Endocrinology 119:1768, 1986). Specific modulators of matrix metabolism were examined for their effects and on capillary development in the growing chick chorioallantoic membrane to explore the possibility that structural alterations of extracellular matrix (ECM) could be causally involved in the antiangiogenic mechanism. Regression of growing capillaries was induced by proline analogs (1-azetidine-2-carboxylic acid, cis-hydroxyproline,d,L-3,4-dehydroproline, thioproline) and an inhibitor of prolyl hydroxylase (alpha,alpha-dipyridyl) which interfere with triple helix formation and prevent collagen deposition. Beta-aminopropionitrile, an inhibitor of collagen cross-linking, was also antiangiogenic although an inhibitor of glycosaminoglycan deposition (beta-methyl d-xyloside) was not. Combination of suboptimal doses of active collagen modulators with either angiostatic steroids or heparin resulted in great potentiation of their antiangiogenic effects. Coadministration of proline analogs, angiostatic steroids, and heparin resulted in complete inhibition of angiogenesis (avascular zones in 100% of chorioallantoic membranes) and larger and more extensive avascular zones than previously observed with steroid-heparin combinations. The antiangiogenic effects of these compounds (alone and in combination) were found to be directly related to their ability to inhibit collagen accumulation. Screening of additional compounds that are known to alter ECM turnover resulted in identification of a new angiogenesis inhibitor, all trans-retinoic acid. These findings suggest that growing capillaries may be "switched" into a regressive mode through alterations of collagen metabolism that result in loss of ECM structural integrity. Identification of ECM turnover as a major control point in angiogenesis may also provide a more rational approach for development of new antiangiogenic regimens.
Allantois↗
Potentiation of angiostatic steroids by a synthetic inhibitor of arylsulfatase.
Sodium 2-hydroxy-5-nitro-alpha-toluenesulfonate (HNT, compound II, Fig. 7) is a synthetic inhibitor of arylsulfatase (E.C. 3.1.6.1.). At 1 to 10 mM, HNT increased the clotting time of heparinized rabbit blood by 7-fold. In the 6-day chick embryo, mixtures of heparin and hydrocortisone applied to the chorioallantoic membrane, are known to inhibit specifically the growth of capillary blood vessels. HNT potentiated this antiangiogenic activity in a dose-dependent manner when the concentration of heparin was suboptimal. Potentiation of the antiangiogenic activity of steroids by HNT correlated inversely with the concentration of exogenous heparin. In the absence of exogenous heparin, hydrocortisone did not inhibit angiogenesis. Hydrocortisone and HNT, however, inhibited angiogenesis to the same extent as hydrocortisone and heparin. An arylsulfatase with a Km of 1.5 mM for nitrocatechol sulfate as substrate and a Ki of 10.0 microM for the inhibition of HNT, was identified in the chick embryo chorioallantoic membrane. Preincubation of heparin with a commercially available arylsulfatase caused a 50% reduction in antiangiogenic activity of heparin-steroid mixtures applied to the chorioallantoic membrane. This loss of activity was prevented completely by addition of HNT to the arylsulfatase-heparin incubation mixture. These results suggest that HNT (a) potentiates the anticoagulant function of heparin, (b) prevents the inactivation of antiangiogenic activity of heparin by an endogenous arylsulfatase in the chorioallantoic membrane and by a commercial arylsulfatase, and (c) in the presence of angiostatic steroids can inhibit angiogenesis in the chick embryo without the addition of exogenous heparin. On the basis of these data, we propose that this inhibitor of arylsulfatase acts to potentiate angiostatic steroids by suppressing the desulfation of tissue heparin.
Animals↗