Effect of substrata and fibroblast growth factor on the proliferation in vitro of bovine aortic endothelial cells

The hypothesis that, in the case of clonal or low-density cultures, cells which do not readily proliferate are those that do not produce an extracellular matrix (ECM), while those that proliferate actively are cells that have retained their ability to produce it, has been tested using low-density vascular endothelial cell cultures maintained on either plastic or ECM-coated dishes and exposed to various combinations of media and sera. Proliferation of low-density vascular endothelial cell cultures seeded on plastic and exposed to DMEM, RPMI-1640, or medium 199 plus thymidine is a function of the batch of calf serum used to supplement the various media. In all three cases, such cultures proliferated at a slow rate and fibroblast growth factor (FGF) greatly accelerated their proliferation. In contrast, when similar cultures were seeded on ECM-coated dishes, they actively proliferated regardless of the batch of calf serum to which they were exposed. FGF was no longer required in order for cultures to become confluent. In the case of cultures exposed to RPMI-1640 or medium 199 plus thymidine, it was even toxic. When cultures were exposed to either medium 199 or Waymouth medium, cells did not proliferate, regardless of the substrate (either plastic or ECM) upon which they were maintained and of the batch of serum to which they were exposed. Addition of FGF to such media had no effect. It is therefore likely that nutrient limitations in both of these media restrict the ability of low-density vascular endothelial cells to respond to the mitogenic stimuli provided by either serum or FGF. These restrictions cannot be relieved by maintaining cells on ECM-coated dishes, and modifications of the nutrient composition of both media is required in order to allow cells to respond to either FGF or serum when maintained on plastic or to serum alone when maintained on ECM. These results suggest that, when low-density cell cultures are maintained on plastic and exposed to an adequate medium, their proliferation will be a function of both serum and FGF. When maintained on ECM, their proliferation will depend only on serum. It is therefore possible that the inability of serum to stimulate optimal cell proliferation when cells are maintained on plastic results from an inability of the cells to produce an ECM, and that FGF could induce such production.     

Factors controlling the proliferative rate, final cell density, and life span of bovine vascular smooth muscle cells in culture

Low density vascular smooth muscle (VSM) cell cultures maintained on extracellular-matrix(ECM)-coated dishes and plated in the presence of either plasma or serum will proliferate actively when serum-containing medium is replaced by a synthetic medium supplemented with three factors: high density lipoprotein (HDL, 250 micrograms protein/ml); insulin (2.5 micrograms/ml) or somatomedin C (10 ng/ml); and fibroblast growth factor (FGF, 100 ng/ml) or epidermal growth factor (EGF, 50 ng/ml). The omission of any of these three factors from the synthetic medium results in a lower growth rate of the cultures, as well as in a lower final cell density once cultures reach confluence. When cells are plated in the total absence of serum, transferrin (10 micrograms/ml) is also required to induce optimal cell growth. The effects of the substrate and medium supplements on the life span of VSM cultures have also been analyzed. Cultures maintained on plastic and exposed to medium supplemented with 5% bovine serum underwent 15 generations. However, when maintained on ECM-coated dishes the serum-fed cultures had a life span of at least 88 generations. Likewise, when cultures were maintained in a synthetic medium supplemented with HDL and either FGF or EGF, an effect on the tissue culture life span by the substrate was observed. Cultures maintained on plastic underwent 24 generations, whereas those maintained on ECM-coated dishes could be passaged repeatedly for 58 generations. These experiments demonstrate the influence of the ECM-substrate only in promoting cell growth but also in increasing the longevity of the cultures.     

Factors involved in the control of proliferation of bovine corneal endothelial cells maintained in serum-free medium

Experimental conditions have been defined that allow bovine corneal endothelial (BCE) cells to grow in the complete absence of serum. Low density BCE cell cultures maintained on extracellular matrix (ECM)-coated dishes and plated in the total absence of serum proliferate actively when exposed to a synthetic medium supplemented with high density lipoprotein (HDL 500 μg protein/ml), transferrin (10 μg/ml), insulin (5 μg/ml), and fibroblast (FGP) or epidermal growth factor (EGF) added at concentrations of 100 or 50 ng/ml, respectively. Omission of any of these components results in a lower growth rate and/or final cell density of the cultures. BCE cell cultures plated on plastic dishes and exposed to the same synthetic medium grow very poorly. The longevity of BCE cultures maintained on plastic versus ECM and exposed to serum-free versus serum-containing medium has been studied. The use of ECM-coated dishes extended the life span of BCE cultures maintained in serum-supplemented medium to over 120 generations, as compared to less than 20 generations for cultures maintained on plastic. Likewise, BCE cells maintained on ECM and exposed to a synthetic medium supplemented with optimal concentrations of HDL, transferrin, insulin, and FGF underwent 85 generations, whereas control cultures maintained on plastic could not be passaged. The enhancing effect of ECM on BCE cell growth and culture longevity clearly illustrates the importance of the cell substrate in the control of proliferation of these cells.     

Hemopoiesis in spleen and bone marrow cultures

Four endothelial cell clones derived from adult bovine aorta were examined with respect to their proliferative characteristics in vitro. Three of these clones, derived in the absence of fibroblast growth factor (FGF), displayed variable basal proliferative rates. One of these non-FGF derived clones grew at a maximal rate which could not be further enhanced with FGF. The other two clones grew at a suboptimal rate which was stimulated by low doses of FGF (10-50 ng/ml) and inhibited by higher doses (100-250 ng/ml). The fourth clone, derived in the presence of FGF, was stimulated by FGF in a dose-dependent manner (10-250 ng/ml) and was not growth inhibited at high FGF concentrations (250-1,000 ng/ml). Growth of all four clones on extracellular matrix (ECM) derived from bovine aortic smooth muscle (BASM) cells was optimal in the absence of FGF. ECM-coated dishes also significantly increased the sensitivity of all clones by at least fivefold to mitogenic stimulation by serum. The proliferative lifespans of the clones ranged between 60 and 120 generations with the most actively proliferating clones attaining the greatest lifespan. Continuous subculture of two of the endothelial clones in the presence of FGF or on ECM-coated dishes did not induce a dependence of the cells on either factor for subsequent growth in its absence. The results indicate that aortic endothelial cells display considerable clonal variability in ther basal proliferative rate and in their response to FGF. This clonal variability is not observed when the cells are maintained on ECM-coated dishes derived from vascular smooth muscle cells.     

Effect of lipoproteins and growth factors on the proliferation of BHK-21 cells in serum free culture

Baby hamster kidney-derived cells (BHK-21 cell line), seeded at low density on gelatin coated dishes and exposed to a 1:1 (v/v) mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium, proliferate actively when exposed to high density lipoproteins (HDL), transferrin, and basic or acidic fibroblast growth factor (FGF). This serum free medium combination supported cell multiplication at a rate equal to that of serum supplemented medium, and at low cell input (10(3) cells/35-mm dish). Epidermal growth factor (EGF), although mitogenic for BHK-21 cells, was less efficient than either basic or acidic FGF in supporting cell growth. When the potency of basic and acidic FGF were compared, acidic FGF was 10-fold less potent than basic FGF. The requirement of BHK-21 cells for transferrin appears to be minimal since cells exposed to HDL and basic FGF could be serially transferred for at least 50 cumulative population doublings in the absence of transferrin.     

Factors involved in supporting the growth and steroidogenic functions of bovine adrenal cortical cells maintained on extracellular matrix and exposed to a serum-free medium

Bovine adrenal cortex cells maintained on extracellular matrix (ECM)-coated dishes will proliferate actively when serum is replaced by HDL (25 micrograms protein/ml), insulin (10 ng/ml), and FGF (100 ng/ml). The cells have an absolute requirement for HDL in order to survive and grow. The omission of insulin, FGF, or both results in a slower growth rate and lower final cell density of the cultures. A requirement for transferrin (1 microgram/ml) becomes apparent only when cells have been grown for at least four generations in the absence of serum. Early passage (P1-P3) bovine adrenal cortex cells cultured in serum-free medium responded to ACTH (10(-8)M) with increased 11-deoxycortisol production; this effect was not observed in later passage cells (P7-P15). The cells' ability to utilize LDL-derived cholesterol and to respond to db cAMP (1mM) by increased steroid release was preserved in cells cultured for over 60 generations in the serum-free medium. HDL, although also able to increase steroid production in early-passage cultures exposed to ACTH or to ACTH and dibutyryl cyclic AMP (db cAMP), was 10 fold less potent than LDL. It did not support steroidogenesis in cultures not exposed to these trophic agents. The life span of bovine adrenal cortex cells grown in the serum-free medium on fibronectin (FN)- versus ECM-coated dishes was compared. Cells seeded in serum-containing medium and grown in serum-free medium had a life span of 34 versus 60 generations when maintained on fibronectin- or ECM-coated dishes, respectively. Cells seeded in the complete absence of serum in the serum-free medium on ECM- or fibronectin-coated dishes could be passaged for 26 or 13 generations, respectively. While FGF was an absolute requirement for cells cultured on fibronectin-coated dishes, it was not required when cells were maintained on ECM. These observations demonstrate the influence of the ECM not only in promoting cell growth and differentiation but also on the life span of cultured cells.     

Growth requirements of low-density rabbit costal chondrocyte cultures maintained in serum-free medium

The factors required for the active proliferation of low-density rabbit costal chondrocytes exposed to 9:1 (v/v) mixture of Dulbecco's modified Eagle's medium and Ham's F12 medium have been defined. Low-density primary cultures of rabbit costal chondrocytes proliferated actively when the medium was supplemented with high-density lipoprotein (300 micrograms/ml), transferrin (60 micrograms/ml), fibroblast growth factor (FGF) (1 ng/ml), hydrocortisone (10(-6) M), and epidermal growth factor (EGF) (30 ng/ml). Insulin, although it slightly decreased the final cell density, was required for reexpression of the cartilage phenotype at confluence. Optimal proliferation of low-density chondrocyte cultures was only observed when dishes were coated with an extracellular matrix (ECM) produced by cultured corneal endothelial cells, but not on plastic. Furthermore, serum-free chondrocyte cultures seeded at low density and maintained on ECM-coated dishes gave rise to a homogeneous cartilage-like tissue composed of spherical cells. These chondrocytes therefore seem to provide a good experimental system for analyzing factors involved in supporting proliferation of chondrocytes and their phenotypic expression.     

Growth of myoblasts in lipoprotein-supplemented,serum-free medium: Regulation of proliferation by acidic and basic fibroblast growth factor

Summary BC₃H1 myoblast cells seeded at low density on gelatin-coated dishes and exposed to a 1∶1 (vol/vol) mixture of Dulbecco’s modified Eagle’s medium and Ham’s F12 medium, proliferate actively when exposed to high density lipoproteins (HDL), transferrin, insulin, and basic or acidic fibroblast growth factor (FGF). This serum-free medium combination supported cell multiplication at a rate equal to that of serum-supplemented medium, and at low cell input (10³ cells/35-mm dish). It also allowed serial transfer of the cultures under serum-free conditions. HDL seems to promote cell survival and to act as progression factor allowing cells to divide when exposed to either basic or acidic FGF. When the potency of basic and acidic FGF were compared, acidic FGF was 20-fold less potent than basic FGF.     

Effect of cell substratum on lateral mobility of lipids in the plasma membrane of vascular endothelial cells

Bovine vascular endothelial cells can be maintained in a highly differentiated state in vitro, either by the addition of fibroblast growth factor (FGF) to the culture medium or by plating the cells on extracellular matrix (ECM)-coated dishes. Under these conditions the cells proliferate actively and at confluence form a tightly packed monolayer composed of nonoverlapping polarized cells. A fluorescence recovery after photobleaching method was used to determine the lateral mobility coefficient D of the lipophilic fluorescent probe, 5N-(hexadecanoyl)-aminofluorescein (HEDAF), in the basal and apical plasma membranes of endothelial cells under various culture conditions (cells on glass coverslips in the presence or absence of FGF, or cells plated on ECM in the exponential growth phase or at confluence). A heterogeneous distribution of lateral diffusion coefficients D was found in a given cell population. Nevertheless, for the basal membrane, a "mean" D value close to 2.0 x 10(-9) cm2/s was found for all the culture conditions. The "mean" D value of HEDAF in the apical pole was slightly higher when sparse cells were exposed to FGF (D = 2.2 x 10(-9) cm2/s) and was further enhanced when cells were growing or confluent on ECM-coated coverslips (D = 2.7 x 10(-9) cm2/s). On the other hand, when the cells were maintained in the absence of FGF on glass coverslips, similar "mean" D values were found in both cell poles (D = 2.0 x 10(-9) cm2/s). These results show that lateral mobility of lipids in endothelial plasmalemma varies in response to external factors such as FGF and the ECM.     

Bovine brain and pituitary fibroblast growth factors: comparison of their abilities to support the proliferation of human and bovine vascular endothelial cells

The mitogenic effects of brain and pituitary fibroblast growth factors (FGF) on vascular endothelial cells derived from either human umbilical vein or bovine aortic arch have been compared. Both brain and pituitary FGF are mitogenic for low density human umbilical endothelial (HUE) cell cultures maintained on either fibronectin- or laminin-coated dishes or on biomatrices produced by cultured cells such as bovine corneal endothelial cells or the teratocarcinoma cell line PF-HR-9. Pituitary FGF triggered the proliferation of HUE cells at concentrations as low as 0.25 ng/ml, with a half-maximal response at 0.55 ng/ml and optimal effect at 2.5 to 5 ng/ml. It was 50,000-fold more potent than commercial preparations of endothelial cell growth factor and 40 times more potent than commercial preparations of pituitary FGF. Similar results were observed when the effect of pituitary FGF was tested on low density cultures of adult bovine aortic endothelial cells. When the activity of brain and pituitary FGF on low density HUE cell cultures was compared, both mitogens were active. To confirm the presence in brain extract of both acidic and neutral, as well as of basic mitogen, for HUE cells, brain tissues were extracted at acidic (4.5), neutral (7.2), and basic (8.5) pH. The three types of extracts were equally potent in supporting the proliferation of either HUE or adult bovine aortic endothelial cells. When the various extracts were absorbed at pH 6.0 on a carboxymethyl Sephadex C-50 column, the neutral and basic extracts had an activity after adsorption similar to that of unadsorbed extracts. In contrast, extracts prepared at pH 4.5 lost 90-95% of their activity which was recovered in the adsorbed fraction containing FGF.     

Are factors originating from serum,plasma, or cultured cells involved in the growth-promoting effect of the extracellular matrix produced by cultured bovine corneal endothelial cells?

The possibilities that the growth-promoting effect of the extracellular matrix (ECM) produced by cultured bovine corneal endothelial (BCE) cells could be due to: (1) adsorbed cellular factors released during the cell lysis process leading to the denudation of the ECM; (2) adsorbed serum or plasma factors: or (3) adsorbed exogenous growth factors have been examined. Exposure of confluent BCE cultures to 2 M urea in medium supplemented with 0.5% calf serum denudes the ECM without cell lysis. The ECM prepared by this procedure supports cell growth just as well as ECM prepared by denudation involving cell lysis. Thus, it is unlikely that the growth-promoting properties of ECM are due to adsorbed cellular factors. When the ECM produced by BCE cells grown in defined medium supplemented with high-density lipoprotein, transferrin, and insulin was compared to the ECMs produced by cells grown in the presence of serum- or plasma-supplemented medium, all were found to be equally potent in stimulating cell growth. It is therefore unlikely that the growth-promoting ability of the ECM is due to adsorbed plasma or serum components. When fibroblast growth factor (FGF)-coated and ECM-coated plastic dishes were submitted to a heat treatment (70 degrees C, 30 min) which results in the inactivation of FGF, the growth-supporting ability of FGF-coated dishes was lost, while the comparable ability of ECM-coated dishes was not affected significantly. This observation tends to demonstrate that the active factor present in the ECM is not FGF. Nor is it platelet-derived growth factor (PDGF), since treatment known to destroy the activity of PDGF, such as exposure to dithiothreitol (0.1 M, 30 min, 22 degrees C) or to beta-mercaptoethanol (10%) in the presence or absence of 6 M urea for 30 min at 22 degrees C, does not affect the growth-promoting activity of ECM. It is therefore unlikely that the growth-promoting effect of ECM is due to cellular growth-promoting agents or to plasma or serum factors adsorbed onto the ECM.     

Rapid purification and activity of apolipoprotein CI on the proliferation of bovine vascular endothelial cells in vitro

The growth-promoting activity of human high-density lipoproteins (HDL) and of their apolipoprotein components on bovine vascular endothelial cells in vitro has been compared. When maintained on plastic culture dishes and exposed to medium containing lipoprotein-deficient serum and fibroblast growth factor, these cells do not proliferate. Addition of either HDL or the total HDL apolipoproteins induces significant cell proliferation. Apolipoprotein C_I, purified by chromatography on the ion-exchanger resin Polybuffer exchanger 94, has an effect on the cell growth similar to that of the total apolipoproteins of HDL.     

Glycosaminoglycans synthesized by cultured bovine corneal endothelial cells

Bovine corneal endothelial (BCE) cells seeded and grown on plastic dishes were labeled with 35S-sulfate or 3H-glucosamine for 48 h at various phases of growth of the cultures. Newly synthesized proteoglycans were isolated from the culture medium and from the extracellular matrix (ECM) produced by the BCE cells, and the glycosaminoglycan (GAG) component of the proteoglycans was analyzed. Cells actively proliferating on plastic surfaces secreted an ECM that contained heparan sulfate as the major 35S-labeled GAG (86%) and dermatan sulfate as a minor component (13%). Upon reaching confluence, the BCE cells incorporated 35S-labeled chondroitin sulfate (20%), as well as heparan sulfate (66%) and dermatan sulfate (14%), into the EC. Seven-day postconfluent cells incorporated newly synthesized heparan sulfate and dermatan sulfate into the matrix in approximately equal proportions. Dermatan sulfate was the main 35S-labeled GAG (60-65%) in the medium of both confluent and postconfluent cultures. 35S-Labeled chondroitin sulfate (20-25%) and heparan sulfate (15%) were also secreted into the culture medium. The type of GAG incorporated into newly synthesized ECM was affected when BCE cells were seeded onto ECM-coated dishes instead of plastic. BCE cells actively proliferating on ECM-coated dishes incorporated newly synthesized heparan sulfate and dermatan sulfate into the ECM in a ratio that was very similar to the ratio of these GAGs in the underlying ECM. Addition of mitogens such as fibroblast growth factor (FGF) to the culture medium altered the type of GAG synthesized and incorporated into the ECM by BCE cells seeded onto ECM-coated dishes if the cells were actively growing, but had no effect on postconfluent cultures.     

Vasculogenesis and angiogenesis in embryonic-stem-cell-derived embryoid bodies

Embryonic stem cells (ESC) have been established previously from the inner cell mass cells of mouse blastocysts. In suspension culture, they spontaneously differentiate to blood-island-containing cystic embryoid bodies (CEB). The development of blood vessels from in situ differentiating endothelial cells of blood islands, a process which we call vasculogenesis, was induced by injecting ESC into the peritoneal cavity of syngeneic mice. In the peritoneum, fusion of blood islands and formation of an in vivo-like primary capillary plexus occurred. Transplantation of ESC and ESC-derived complex and cystic embryoid bodies (ESC-CEB) onto the quail chorioallantoic membrane (CAM) induced an angiogenic response, which was directed by nonyolk sac endoderm structures. Neither yolk sac endoderm from ESC-CEB nor normal mouse yolk sac tissue induced angiogenesis on the quail CAM. Extracts from ESC-CEB stimulated the proliferation of capillary endothelial cells in vitro. Mitogenic activity increase during in vitro culture and differentiation of ESC. Almost all growth factor activity was associated with the cells. The ESC-CEB derived endothelial cell growth factor bound to heparin-sepharose. The identification of acidic fibroblast growth factor (FGF)in heparin-sepharose-purified material was accomplished by immunoblot experiments involving antibodies against acidic and basic FGF. We conclude that vasculogenesis, the development of blood vessels from in situ differentiating endothelial cells, and angiogenesis, the sprouting of capillaries from preexisting vessels are very early events during embryogenesis which can be studied using ESC differentiating in vitro. Our results suggest that vasculogenesis and angiogenesis are differently regulated.     

Phosphatidyl choline and the growth in serum-free medium of vascular endothelial and smooth muscle cells,and corneal endothelial cells

Liposomes made by sonication of egg yolk phosphatidyl choline support the proliferation of low-density bovine vascular and corneal endothelial cells, and vascular smooth muscle cells maintained on basement laminacoated dishes and exposed to a defined medium supplemented with transferrin. The optimal growth-promoting effect of phosphatidyl choline was observed at concentrations of 25 μg/ml for low-density cultures of vascular smooth muscle cells, and 100 μg/ml for vascular and corneal endothelial cells. The growth rate and final cell density of vascular endothelial cells exposed to a synthetic medium supplemented with transferrin and either high-density lipoproteins or phosphatidyl choline has been compared. Although cultures exposed to phosphatidyl choline reached a final cell density similar to that of cultures exposed to high-density lipoproteins, they had a longer average doubling time (17 h vs. 12 h) during their logarithmic growth phase and a shorter lifespan (17 generations vs. 30 generations). Similar observations were made in the case of vascular smooth muscle cells or bovine corneal endothelial cells maintained in medium supplemented with transferrin, fibroblast growth factor (FGF) or epidermal growth factor (EGF), and insulin and exposed to either high-density lipoproteins or phosphatidyl choline. Since phosphatidyl choline can, for the most part, replace highdensity lipoproteins in supporting the proliferation of various cell types, it is likely that the growth stimulating signal conveyed by high-density lipoproteins is associated with its polar lipid fraction, which is composed mostly of phosphatidyl cholines.     

Control of proliferation of human vascular endothelial cells. Characterization of the response of human umbilical vein endothelial cells to fibroblast growth factor, epidermal growth factor, and thrombin

Because the response of human endothelial cells to growth factors and conditioning agents has broad implications for our understanding of wound healing angiogenesis, and human atherogenesis, we have investigated the responses of these cells to the fibroblast (FGF) and epidermal growth factors (EGF), as well as to the protease thrombin, which has been previously shown to potentiate the growth response of other cell types of FGF and EGF. Because the vascular endothelial cells that form the inner lining of blood vessels may be expected to be exposed to high thrombin concentrations after trauma or in pathological states associated with thrombosis, they are of particular interest with respect to the physiological role of this protease in potentiating cell proliferation. Our results indicate that human vascular endothelial cells respond poorly to either FGF or thrombin alone. In contrast, when cells are maintained in the presence of thrombin, their proliferative response to FGF is greatly increased even in cultures seeded at a density as low as 3 cells/mm2. Human vascular endothelial cells also respond to EGF and thrombin, although their rate of proliferation is much slower than when maintained with FGF and thrombin. In contrast, bovine vascular endothelial cells derived from vascular territories as diverse as the bovine heart, aortic arch, and umbilical vein respond maximally to FGF alone and neither respond to nor bind EGF. Furthermore, the response of bovine vascular endothelial cells to FGF was not potentiated by thrombin, indicating that the set of factors controlling the proliferation of vascular endothelial cells could be species-dependent. The requirement of cultured human vascular endothelial cells for thrombin could explain why the human cells, in contrast to bovine endothelial cells, are so difficult to maintain in tissue culture. Our results demonstrate that by using FGF and thrombin one can develop cultures of human vascular endothelial cells capable of being passage repeatedly while maintaining a high mitotic index. The stock cultures used for these studies have been passed weekly with a split ratio of 1 to 10 and are currently in their 30th passage. These cultures are indistinguishable from earlier passages when examined for the presence of Weibel-Palade bodies or Factor VIII antigen. We conclude that the use of FGF and thrombin can prevent the precocious senescence observed in most human endothelial cells cultures previously described.     

Heparin protects basic and acidic FGF from inactivation

The ability of heparin or that of hexuronyl hexosaminoglycan sulfate (HHS-4) to protect basic or acidic fibroblast growth factor (FGF) from acid or heat inactivation has been analyzed. Both freshly prepared basic and acidic FGF stimulate the growth of baby hamster kidney (BHK-21) cells exposed to medium supplemented with transferrin and insulin. Freshly prepared basic FGF was 10 fold more potent than acidic FGF. The addition of heparin to the medium decreased the potency of basic FGF while it potentiated that of acidic FGF. Upon storage of FGF at -80 degrees C, a decline in potency of both basic and acidic FGF was observed. Heparin, when added to the medium, potentiated their activities, which became similar to that of freshly prepared basic FGF. In order to test whether heparin could protect basic or acidic FGF from inactivation, both mitogens were exposed to acid conditions (1% trifluoroacetic acid, pH 1.08, 2 h) or heat (65 degrees C, 5 min) which inactivate basic or acidic FGF. When exposed to such treatment in the presence of heparin or HHS-4, basic and acidic FGF retained their potency. The effect of heparin and HHS-4 on the bioactivity of basic and acidic FGF is truly of a protective nature, since they had no effect when added after inactivation of the mitogens. Potentiation of the bioactivity of the protected mitogens or of the inactivated one could only be observed when cells were exposed to high heparin or HHS-4 concentrations. This indicates that heparin and HHS-4, in addition to protecting FGF from inactivation, also acts at another locus, as yet unidentified.     

Retina- and eye-derived endothelial cell growth factors: partial molecular characterization and identity with acidic and basic fibroblast growth factors

Two retina-derived growth factors have been isolated on the basis of their ability to stimulate the proliferation of capillary endothelial cells in vitro. Gas-phase sequence analysis identified the amino-terminal sequence of the major form of the mitogen as being identical with residues 1-35 of bovine basic fibroblast growth factor (FGF). Amino-terminal sequence analysis of the second form identified 28 residues that are indistinguishable from those of brain acidic FGF (residues 1-28). The possibility that these retina-derived endothelial cell growth factors are related to, if not identical with, basic and acidic FGF is supported by observations that they have similar molecular weights (15000-16000), similar retention behavior on all steps of chromatography (ion-exchange, heparin-Sepharose), and similar amino acid compositions and that they cross-react with antibodies to basic and acidic FGF. The eye-derived growth factors, like FGF, are potent stimulators of capillary endothelial cell growth in vitro. The results identify the major retina-derived endothelial cell growth factor as indistinguishable from basic FGF and demonstrate the presence of an acidic FGF in the eye. They suggest that at least some of the mitogenic, angiogenic, and neovascularizing activities described as being present in the retina are due to the existence of FGF in this tissue. The implications of this finding on the etiology and pathophysiology of vasoproliferative diseases of the eye are discussed.     

Sulfated proteoglycan synthesis by confluent cultures of rabbit costal chondrocytes grown in the presence of fibroblast growth factor

We examined the effect of fibroblast growth factor (FGF) on proteoglycan synthesis by rabbit costal chondrocyte cultures maintained on plastic tissue culture dishes. Low density rabbit costal chondrocyte cultures grown in the absence of FGF gave rise at confluency to a heterogeneous cell population composed of fibroblastic cells and poorly differentiated chondrocytes. When similar cultures were grown in the presence of FGF, the confluent cultures organized into a homogenous cartilage-like tissue composed of rounded cells surrounded by a refractile matrix. The cell ultrastructure and that of the pericellular matrix were similar to those seen in vivo. The expression of the cartilage phenotype in confluent chondrocyte cultures grown from the sparse stage in the presence vs. absence of FGF was reflected by a fivefold increase in the rate of incorporation of [35S]sulfate into proteoglycans. These FGF effects were only observed when FGF was present during the cell logarithmic growth phase, but not when it was added after chondrocyte cultures became confluent. High molecular weight, chondroitin sulfate proteoglycans synthesized by confluent chondrocyte cultures grown in the presence of FGF were slightly larger in size than that produced by confluent cultures grown in the absence of FGF. The major sulfated glycosaminoglycans associated with low molecular weight proteoglycan in FGF-exposed cultures were chondroitin sulfate, while in cultures not exposed to FGF they were chondroitin sulfate and dermatan sulfate. Regardless of whether or not cells were grown in the presence or absence of FGF, the 6S/4S disaccharide ratio of chondroitin sulfate chains associated with high and low molecular weight proteoglycans synthesized by confluent cultures was the same. These results provide evidence that when low density chondrocyte cultures maintained on plastic tissue culture dishes are grown in the presence of FGF, it results in a stimulation of the expression and stabilization of the chondrocyte phenotype once cultures become confluent.     

Fibroblast growth factors are present in the extracellular matrix produced by endothelial cells in vitro: implications for a role of heparinase-like enzymes in the neovascular response

Salt extracts of the extracellular matrix (ECM) that is produced by vascular and capillary endothelial cells contain mitogens that are indistinguishable from basic and acidic fibroblast growth factors (FGFs). The biological activity found in these extracts is retained by heparin-Sepharose affinity columns and elutes with salt concentrations similar to those required to elute FGFs (i.e. 1.1 - 2M NaCl). Antisera raised against synthetic fragments of basic and acidic FGF crossreact with the ECM-derived mitogens. Radioiodinated basic FGF binds to the ECM formed by both vascular and capillary endothelial cells, a result that is consistent with the observation that FGF-like mitogens are found on the ECM. The binding of FGF to the ECM is negligible when the ECM has been pretreated with heparinase or heparitinase suggesting that the mitogen is interacting with a heparin-like glycosaminoglycan in the ECM. The digestion of the ECM with several grades of hyaluronidase, chondroitinase or chondro-4-sulfatase or chondro-6-sulfatase has little or no effect on 125I-FGF binding to the ECM. In view of the fact that many, if not all cells, produce heparan sulfates and that these glycosaminoglycans are associated with the external surface of the cell and the ECM, a model is proposed suggesting that the neovascular response induced by tumours and some normal tissues may be mediated at least in part, by the initial release of heparinase-like enzymes rather than angiogenic factors (FGFs) per se. The release of these enzymes would effectively mobilize a secondary local release of FGF from the ECM which then induces a proliferative response.