Conditioned media (CMs) from many cell types contain a factor that can adsorb to a polycationic substratum and cause neurons plated on that substratum to extend neurites rapidly and profusely. The extracellular matrix glycoprotein laminin, when bound to a substratum, elicits a similar response by neurons. In this report, six CMs that contain a "neurite outgrowth-promoting factor" were studied. Immunoprecipitation with affinity-purified anti-laminin antibodies demonstrated that laminin is present in all of them, and when it was selectively removed, there was a corresponding loss of neurite outgrowth-promoting activity in each CM. Antibodies to purified laminin failed, however, to block the outgrowth-promoting activity of five of the CMs tested, even though these antibodies blocked the outgrowth-promoting activity of purified laminin in the same assay. This result could reflect differences in amino acid sequence or protein modification between CM-derived laminin and the purified laminin used in generating antibodies. Alternatively, it could reflect the fact that other molecules bind to laminin in CMs and could interfere with the binding of antibodies to sites on lanmini that are important for biological activity.gators have prepared antisera directed against laminin that completely block neuronal responses to laminin-treated substrata (6, 10, 11). These sera have failed to affect the activities of most CM-treated substrata, suggesting that the active material in most CMs is not laminin. Recently, however, we have completed the purification of the neurite outgrowthpromoting factor present in bovine corneal endothelial (BCE) CM. In agreement with earlier results (4, 10, 13), we found that the active factor is an aggregate involving a heparan sulfate proteoglycan and two large proteins. One of these proteins has been identified as laminin. The other components can be separated from laminin and do not promote neurite outgrowth on their own. Details of the purification will be presented elsewhere.In this report, we present evidence that laminin is a component not only of the active factor in BCE-CM but also of the active factors in CMs produced by several other cell types. We suggest that laminin is responsible for the neurite outgrowth-promoting activity of all CMs with this activity. Reasons for the failure of anti-laminin antibodies to block the activity of many CM-treated substrata are discussed.The role of the extracellular matrix in influencing axon outgrowth has been the subject of several recent in vitro studies (1-6). Neurons of various types, when plated on extracellular matrix deposited by cultured cells, respond dramatically with neurite growth that commences quickly and progresses rapidly and extensively (4). This response occurs even in the absence of nerve growth factor or other appropriate survival factors (4, 7). A similar response is seen when neurons are plated on polycationic substrata that have been exposed to media conditioned by almost any cell type (1-4). This observation suggests that t...

Rat sympathetic neurons, plated onto extracellular matrix produced by cultured bovine corneal endothelial cells, rapidly extended neurites in the absence of nerve growth factor (NGF). The response was unaffected by antiserum to NGF. Rapid outgrowth also occurred when sympathetic neurons were plated onto polylysine-coated surfaces that had been exposed to serum-free medium conditioned by corneal endothelial cells (CMsF). A response was seen even when the neurons were cultured without serum. When plated onto a polylysine-coated dish treated with CMsF over half its surface, only the neurons on the treated half extended neurites.The active factor in CMsF was destroyed by trypsin, acid (pH 1.6), base (pH 12.7), or heating to 80°C; it was stable to heating to 60°C, collagenase, deoxyribonuclease, and neuraminidase. The factor elutes just after the void volume of a Sepharose 6B column. In associative cesium chloride gradients, it sediments as a peak centered at a density of 1.36-1.37, corresponding to a peak of material that can be biosynthetically labeled with laSS]sulfate or [3H]leucine. Material from this fraction was inactivated by heparinase, but not chondroitinase ABC, implying that a heparan sulfate proteoglycan is essential for the factor's activity. Inactivation by contaminants in the heparinase preparation was ruled out. Further purification indicated that the active factor may exist as an aggregate containing a heparan sulfate proteoglycan and other molecules.CMsF also promoted neurite outgrowth by other types of neurons. Furthermore, a variety of cell types were shown to produce factors similar to that in CMsF.An important determinant in the development of multicellular organisms is the extracellular matrix (ECM) upon which cells attach, migrate, and differentiate. It seems likely that a class of substances that affect neuronal development will be found to be associated with this extracellular matrix. The importance of the interaction between neuron and substratum has already been demonstrated in vitro (1-3). In vivo, axons appear to follow routes determined by the substratum (4). In some cases, this reflects the association of axons with already oriented cells, e.g., radial glia in the cerebellum (5) and the pioneering optic nerve fibers of Daphnia (6).The existence of factors necessary for the normal developmerit of certain neurons is established. Sympathetic and sensory neurons require nerve growth factor (NGF) for survival and development in vivo and in vitro (7). Certain other factors appear to be necessary for the survival and development of 574 cultured parasympathetic neurons (8, 9). Other factors, few of which have been significantly purified, have recently been reported to promote the development of sympathetic, parasympathetic, sensory, and spinal cord neurons in culture (10-24). If such factors are supplied by the cells that innervate or are innervated by certain types of neurons, it may explain why appropriate intercellular contacts must be made in order for many types of neurons to su...

When culture medium, conditioned by any of several cell types, is applied to a polycationic substratum, a substance is adsorbed that causes neurons cultured on that substratum to extend processes (neurites) rapidly and profusely. We have purified the factor responsible for this effect from medium conditioned by bovine corneal endothelial cells, and have shown that it is composed of the glycoprotein laminin and two associated lamininbinding molecules: a sulfated protein known as entactin, and a large heparan sulfate proteoglycan. Of these molecules, only laminin was found to be present throughout the purification in all fractions possessing neurite outgrowth-promoting activity and absent from all fractions lacking activity. Laminin, purified from other sources, has been shown previously to promote extensive outgrowth by cultured neurons. These and other data presented here support the conclusion that laminin is responsible for the neurite outgrowth-promoting activity of the conditioned medium factor. Evidence is also presented that the association of a proteoglycan with laminin promotes efficient attachment of laminin to polycationic substrata, particularly in the presence of competing molecules.Little is known about the cellular mechanisms that generate the complex patterns of axon growth seen in nervous system development. The search for factors that may control axonal growth and guidance in vivo has relied heavily on the study of neurite growth in vitro (1). While it has long been appreciated that cell-substratum interactions influence the growth of neurites (2, 3), only recently have candidates emerged for molecules that might serve such a function in vivo. Helfand et al. (4) and Collins (5) were the first to report that cultured heart cells produce a substance that, when adsorbed to a substratum, dramatically enhances neurite outgrowth by neurons plated on that substratum. Since then, many other cell types have been shown to release into conditioned medium (CM) ~ a substance with a similar effect on neurons (6-9). It is characteristic of these "neurite outgrowth-promoting factors" found in CM to adsorb preferentially to polycationic substrata (i.e., substrata coated with polylysine or polyorni-J Abbreviations used in this paper: BCE, bovine corneal endothelial; CM, conditioned medium (media); DME-0.I, Dulbecco's modified Eagle's medium containing 0. 1% glucose; EIA, enzyme-linked immunoassay; HeS, heparan sulfate; TBS, Tris-buffered saline. thine) and cause many types of neurons to extend neurites both sooner and more rapidly than they would on untreated polycationic substrata or other conventional culture substrata (e.g., plastic, collagen). Interestingly, neurite outgrowth-promoting factors do not replace trophic factors in supporting the long-term survival of neurons, and are unrelated to nerve growth factor (7,8).Recently, considerable efforts have been made to characterize the neurite outgrowth-promoting factors found in CM (10). They are produced by a large number of cell types (6,8), are prot...

Bovine adrenal and brain cortex and corpus luteum-derived capillary endothelial cells have been established in culture, taking advantage of their ability to proliferate at clonal density when maintained on extracellular matrix (ECM) coated dishes in the presence of serum supplemented medium. All three cell types formed at confluency a monolayer of small, tightly packed, contact inhibited cells that express factor VIII related antigen. Their proliferative response to basic and acidic FGF when cells were maintained on plastic and exposed to serum supplemented medium was similar to that previously reported for endothelial cells derived from large vessels, with acidic FGF being 30-fold less potent than basic FGF. Their requirement for high density lipoproteins and transferrin in order to proliferate actively when maintained on ECM-coated dishes and exposed to serum-free conditions was also similar to that previously reported for endothelial cells derived from large vessels. Heparin strongly reduced the proliferative response of capillary endothelial cells to either basic or acidic FGF, as well as their response to serum alone, regardless of whether cells were maintained on plastic or on ECM-coated dishes. The present data indicate that bovine endothelial cells derived from large or small vessels are indistinguishable in so far as their response to growth factors, plasma factors, and substrata are concerned.

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.