Proc. Natl. Acad. Sd. USA Vol. 77, No. 7, pp. 4094-4098, July 1980 Permissive effect of the extracellular on in vitro ( /granulosa cells/adrenal cortex cells/vascular endothelial cells/) D. GOSPODAROWICZ, D. DELGADO, AND I. VLODAVSKY Research Institute and the Departments of Medicine and Ophthalmology, University of California Medical Center, San Francisco, California 94143 Communicated by Clifford Grobstein, March 28,1980

ABSTRACT Corneal endothelial cells maintained in exposed to FGF. In contrast, when maintained on a corneal culture retain the ability to synthesize and secrete an extracel- ECM, these same cell types proliferated actively and no longer lular matrix (ECM) along their basal cell surface. Treatment of required FGF to reach confluence. These results demonstrated confluent cultures with 0.5% Triton X-100 results in the removal of the cell monolayer, thereby exposing the ECM, which adheres that the ECM, which is the natural substrate upon which cells strongly to the tissue culture dish. Dishes coated with ECM were rest in vivo, can greatly affect their proliferation in vitro. used to study the permissive effect of such a substrate on cell proliferation. The proliferation of bovine granulosa and adrenal MATERIALS AND METHODS cortex cells maintained on plastic tissue culture dishes was Materials. FGF was purified as described from bovine brains compared to that on dishes coated with ECM. Neither cell type, even when exposed to optimal serum concentration, replicated (8), and was purified as described from bovine when seeded at low cell density on plastic. In contrast, when plasma (9). Purified (types I, II, III, and IV) was do- seeded on ECM they proliferated actively. None of the cultures nated by S. Tseng (University of California, San Francisco). maintained on ECM required in order Dulbecco's modified Eagle's medium H-16 and F-12 medium to reach confluence, although when maintained on plastic they were obtained from GIBCO. Calf serum and fetal calf serum were totally dependent on fibroblast growth factor for prolif- were obtained from Irvine Serum (Irvine, CA). Tissue culture eration. Because cells maintained on plastic do not respond to dishes were from Falcon Plastics. Dextran (Mr 40,000) was from factors present in serum or plasma, although they do so respond when maintained on ECM, it is likely that the close contact of Sigma, gentamycin was from Schering, and Fungizone was the cells with the ECM restores their sensitivity to agents present from Squibb. in serum and plasma. Conditions. Cultures of bovine corneal endo- thelial cells were established from steer eyes as described (10, The extracellular matrix (ECM) or produced by 11). Stock cultures were maintained on tissue culture dishes in cells is the natural substrate upon which cells migrate, prolif- Dulbecco's modified Eagle's medium supplemented with 10% erate, and differentiate in vivo. Although the exact nature and (vol/vol) fetal calf serum, 5% (vol/vol) calf serum, gentamycin composition of ECMs are still to be elucidated, they are com- (50 ,tg/ml), and Fungizone (2.5 jig/ml). FGF (100 ng/ml) was posed in large part of different types of collagen (1), glycosa- added every other day until the cells were nearly confluent. minoglycans, (2), and , among which Cultures of bovine granulosa cells were established from ovarian is fibronectin-a ubiquitous component of various types of follicles as described (12). Stock cultures were maintained in ECMs (3). F-12 medium supplemented with 10% (vol/vol) calf serum, The notion, first proposed by Grobstein in 1953 (4), that gentamycin (50,gg/ml), Fungizone (2.5 ,ug/ml), and FGF (100 ECMs promote and stabilize the of the epithelia ng/ml added every other day). Cultures of bovine adrenal associated with them has since been confirmed (5, 6). However, cortex cells were established from adrenal cortex as described although numerous studies have dealt with the differentiation (13). Stock cultures were maintained in F-12 medium supple- of tissue induced by the ECMs and basal lamina, few studies mented with 10% (vol/vol) horse serum, gentamycin (50 have dealt with their effect on cell proliferation. Because cell ,gg/ml), Fungizone (2.5 ,gg/ml), and FGF (100 ng/ml added proliferation precedes cell differentiation in most organs, it is every other day). likely that both proliferation and differentiation could be Preparation of Plates Coated with an ECM, Different controlled directly or indirectly by the substrate upon which Types of Collagen, or Fibronectin. Corneal endothelial cells the cells rest. This is particularly true of tissues that have only were plated at an initial density of 104 cells per 35-mm dish and one developmental option. were maintained in Dulbecco's modified Eagle's medium Earlier studies in our laboratory on the control of cellular supplemented with 10% (vol/vol) fetal calf serum, 5% (vol/vol) proliferation by fibroblast growth factor (FGF) have shown that calf serum, 5% (wt/vol) dextran T-40, gentamycin (50 ,g/ml), cells that proliferate best in tissue culture retain their ability to and Fungizone (2.5 Atg/ml). FGF (100 ng/ml) was added every produce an ECM, whereas cells that proliferate poorly, or not other day. Once the cultures became confluent (ordinarily at all, have lost that ability (7). In this study, we examined the within 6 days), the media were renewed and the cultures were proliferative behavior of two different cell types of bovine or- further incubated for 6 days. The cultures then were washed igin when maintained on plastic or on an ECM produced by with phosphate-buffered saline and exposed for 30 min to 0.5% corneal endothelial cells. When maintained on plastic and ex- Triton X-100 in the same buffer. Once the nuclei and the ECM posed to optimal serum concentration, bovine adrenal cortex became visible, the cultures were washed three times with cells and granulosa cells did not proliferate unless they were phosphate-buffered saline. After these washings, only a few and nuclei were associated with the intact ECM The publication costs of this article were defrayed in part by page that coated the entire dish (see Figs. 1 and 2). To coat dishes charge payment. This article must therefore be hereby marked "ad- vertisement" in accordance with 18 U. S. C. §1734 solely to indicate Abbreviations: ECM, extracellular matrix; FGF, fibroblast growth this fact. factor. 4094 Downloaded by guest on September 27, 2021 : Gospodarowicz et al. Proc. Nati. Acad. Sci. USA 77 (1980) 4095 with different types of collagen, 50,ul of a solution of collagen in 0.5 M acetic acid (5 mg/ml) was applied to a 35-mm dish and spread uniformly with a rubber policeman. The plates were exposed to ammonia vapor for 30 min and then washed ex- tensively with phosphate-buffered saline. To coat the dish with fibronectin, 2 ml of a solution of fibronectin in 0.05 M urea (30 ug/ml) was added to the dishes. After an incubation period of 60 min at 220C, the dishes were washed five times with phos- phate-buffered saline (14). Cell Growth Measurement. Cells were plated at an initial density of 1 or 2 X 104 cells per 35-mm dish. FGF (100 ng/ml) was added every other day. Triplicate cultures were trypsinized, and cells were counted with a Coulter Counter every other day. The morphological appearance of the cultures was analyzed by phase-contrast , and pictures were taken when they became confluent. RESULTS Morphological Appearance of the ECM Produced by Corneal Endothelial Cells When confluent cultures of corneal endothelial cells were examined by scanning electron micros- copy, the cells resembled those in vlvo-closely apposed, highly flattened, and hexagonal in pattern (Fig. 1A). Exposure of the cell monolayer to phosphate-buffered saline containing 0.5% Triton X-100 resulted in the permeabilization of the cells, and the ECM underneath the cells became partially visible (Fig. 1 B and D). In some areas cytoskeletons and associated nuclei spontaneously detached from it and thereby exposed it (Fig. 1C). When the ECM was examined, after removal of the cy- FIG. 2. Scanning electron micrographs of the ECM produced by corneal endothelial cell cultures. (A) Area of light deposit ofthe ECM; toskeletons and associated nuclei, by washing with phos- the matrix has been scratched with a needle to release it from the phate-buffered saline, two areas were distinguished. One tissue culture dish, which it coats uniformly. (X500.) (B) Same as A, consisted of a uniform layer of amorphous material that was but the area has not been scratched. (X1000.) (C) Area of heavy de- closely associated with the plastic (Fig. 2 A and B). Scratching posit of the ECM, which uniformly coats the tissue culture dish; the plate with a needle allowed partial detachment of that polygonal ridges of amorphous material can be seen superimposed amorphous material and enabled one to appreciate its thickness on it. (X150.) (D) Same as C, but at a higher magnification. and homogeneity (Fig. 2A). In other areas, ridges forming a (X750.) hexagonal pattern were seen (Fig. 2 C and D). The composition of the ECM coating the dish has been analyzed by column chromatography (16) and by immunofluorescence (17). The collagen distribution in the was mainly collagen types III, IV, and V. Fibronectin was also present in large quantities (18), as well as (19, unpublished data). Growth and Appearance of Cultured Cells Maintained on Plastic or ECM; Effect of Absence or Addition of FGF. Granulosa cells. Low-density granulosa cell cultures main- tained on plastic and exposed to either high (10%) or low (1%) serum concentrations hardly proliferated (Fig. 3 A and B). After a few days, the cells had enlarged considerably (Fig. 4A), and a high percentage (20%) of binucleated cells was observed, which suggested that the cells had gone once through the pro- cess of endomitosis (unpublished results). Following the addition of FGF to the cultures, cells proliferated actively (Fig. 3 A and B). Within a week, this resulted in the formation of a monolayer ~ ~ ~ ~ ~ ~ j of closely apposed mononucleated cells (Fig. 4B). In contrast to cells maintained on plastic, cells maintained on the ECM no longer required the presence of FGF to proliferate actively. Within 5 days the cultures became confluent (Fig. 3 A and B). FIG. 1. Scanning electron micrographs of a monolayer of bovine corneal endothelial cells before and after exposure to Triton X-100. Addition of FGF to such cultures did not decrease the mean (A) A monolayer composed ofpolygonal, highly flattened, and closely doubling time of the cultures but resulted in a final cell density apposed cells before Triton treatment. (X250.) (B) Posttreatment that was twice as high as that of cultures maintained in the monolayer composed ofnuclei and cytoskeletons that no longer attach absence of FGF. The differences in the growth rate of granulosa firmly to the ECM. (X90.) (C) ECM exposure in some areas. (X250.) cells maintained with or without FGF on plastic or ECM were (D) Nuclei and their associated cytoskeletons with the ECM under- neath. Processing of the samples for scanning electron microscopy seen best when the cultures were maintained in the medium was as described (15). Scanning electron micrographs were obtained supplemented with a low (1%) serum concentration. Under with a JOEL scanning electron microscope (model 35). these conditions, cells maintained on plastic alone did not Downloaded by guest on September 27, 2021 4096 Cell Biology: Gospodarowicz et al. Proc. Natl. Acad. Sci. USA 77 (1980)

A.

FIG. 4. Granulosa cells maintained on plastic substrate (A and B) or on an ECM (C and D). Cultures were plated and maintained as described in Fig. 3A with (B and D) or without (A and C) FGF. Pic- tures were taken on day 8. (Phase-contrast; X45.) was added to the cultures (Fig. 3 C and D). As described for granulosa cells, within 2-3 days these cells enlarged consider- ably (Fig. 5A), and by 7 days a large proportion of binucleated cells could be observed in the cultures. When FGF was added to the media, active proliferation was observed, but the mean doubling time of the cultures was lower than that of cultures maintained on the ECM and not exposed to FGF. Addition of FGF to cultures maintained on the ECM and exposed to high serum concentrations did not result in a faster growth rate but did result in a higher (2-fold) final cell density. As with gran- ulosa cells, the differences in growth rate between cultures maintained with or without FGF on plastic versus ECM were best seen when the cultures were maintained in low (1%) serum Days concentration (Fig. 3D). Cultures maintained on plastic plus FIG. 3. Proliferation of granulosa cells (A and B) and adrenal FGF, on the ECM alone, and on the ECM plus FGF showed no cortex cells (C and D) when maintained with (0, A) or without (0, obvious changes in cell morphology except that cells maintained *) FGF on plastic or ECM. Granulosa cells were plated at 1 X 104 cells on the ECM (Fig. 5C) or the ECM plus FGF (Fig. 5D) had a (A) or 2 X 104 cells (B) per 35-mm dish and maintained in Dulbecco's smaller average size and a higher density of lipid inclusions than modified Eagle's medium supplemented with either 10% (A) or 1% did cells maintained on plastic plus FGF (Fig. 5B). (B) calf serum. The cells were maintained on plastic (0,0) or ECM (A, A). FGF (100 ng/ml) was added every other day to half of the Proliferation of Granulosa Cells and Adrenal Cortex Cells dishes (0, A). Adrenal cortex cells were plated at 1 X 104 cells per on Dishes Coated with Different Types of Collagen or with 35-mm dish and maintained in Dulbecco's modified Eagle's medium Fibronectin. To test the possibility that collagen or fibronectin supplemented with either 10% (C) or 1% calfserum (D). The cells were alone could be the component of the ECM responsible for the maintained on plastic (0, 0) or ECM (-, A). FGF (100 ng/ml) was increased rate of proliferation of cells plated on it, the growths added every other day to half of the dishes (0, A). of granulosa and adrenal cortex cells plated on dishes coated with purified collagen type I, II, III, or IV or with fibronectin proliferate, whereas addition of FGF caused a 7-fold increase were compared. In no case did the cultures significantly in- in cell number over an 8-day period. Cells maintained on the ECM reached a cell density 22-fold higher than those main- tained on plastic, and addition of FGF caused a 44-fold increase A. t I., in cell number over that in the control (Fig. 3 A and B). i i not ex- k Granulosa cells maintained on the ECM alone and I posed to FGF had the same appearance as that of cells main- I tained on plastic and exposed to FGF; the cultures were com- posed of a monolayer of cells that were more tightly packed when maintained on the ECM than on plastic (Fig. 4C). Cul- I 'I tures maintained on ECM and grown in the presence of FGF ISB i I p formed a monolayer composed of extremely small and tightly packed cells whose contained numerous lipid in- clusions (Fig. 4D). Adrenal cortex cells. The growth characteristics and ap- pearance of adrenal cortex cells maintained on plastic or ECM and exposed to high (10%) or low (1%) serum concentrations with or without FGF added to the medium were essentially FIG. 5. Appearance of adrenal cortex cells maintained on plastic similar to those already reported for granulosa cells. Adrenal substrate (A and B) or ECM (C and D). Cultures were plated and cortex cells maintained on plastic hardly proliferated, even maintained as described in Fig. 3C with (B and D) or without (A and when exposed to a high (10%) serum concentration, unless FGF C) FGF. Pictures were taken on day 7. (Phase-contrast; X45.) Downloaded by guest on September 27, 2021 Cell Biology: Gospodarowicz et al. Proc. Natl. Acad. Sci. USA 77(1980) 4097

A flattened and lose their columnar appearance. If, on the other hand, the cells are maintained on the proper substrate (in this 6rrB case refrozen thawed , tropocollagen, or Millipore filters) EM and exposed to chicken embryo extract, the cells remain co- lumnar and mitotically active. This indicates that the orienta- 5 tion of the cells, their shape, and their proliferative activity are Ca closely linked properties of the basal germinal population and 4 will depend on the inert substrate upon which the cells are r- maintained. x Evidence that the substrate upon which the cells are main- Gog 3[ tained is important for their proliferation is now plentiful (for

Uc) a review, see ref. 28). The earlier work by Ehrman and Gey 2 showed that various tissues demonstrate enhanced growth and differentiation when cultured on collagen gels (29). In the case of primary cultures of mouse mammary epithelial cells, the cells 1 I have a limited growth capacity; although they undergo a few rounds of division, proliferation cannot be sustained nor can these cells be passaged (30). When the same cells are embedded within collagen gel, sustained growth of primary cells can be FIG. 6. Proliferation of adrenal cortex (A) and granulosa cells avo (B) maintained on different substrates. The cells (2 X 104) were seeded observed (30). In implantation of a collagenous matrix on dishes coated with collagen type 1,11, m, or IV, with collagen types results in the proliferation of adjacent mesenchymal cells which I and III, with fibronectin (Fib), or with an ECM (EM). After 5 days, differentiate into , thereby implying a role for this the cultures were trypsinized and the cells were counted. The final ECM in anchorage-dependent events in cell growth and dif- cell density was compared to that of cultures maintained for the same ferentiation (31). period of time on plastic without FGF (P) or with FGF (P + FGF). However, even the use of collagen-coated dishes or collagen gel may not replace the need for a normally produced ECM. crease their rate of growth when maintained on these different In addition to the difficulties involved in getting proper poly- substrates (Fig. 6), and an aberrant appearance was observed merization of purified collagen, one has to keep in mind that in all cases-the cultures were composed of large cells. Only the ECM is composed not only of collagens but also of proteo- cells maintained on the ECM proliferated actively (Fig. 6), glycans, , and glycoproteins, all of which reaching confluence within 5 days. This observation therefore could affect the proliferation and subsequent morphogenesis excludes the possibility that the component in the ECM pro- of cells and tissue placed in contact with it (5, 23). Therefore, duced by corneal endothelial cells that could have a permissive it may well be that the proper steric recombination in vitro of effect on the proliferation of other cell types maintained on it all these components will be a difficult, if not impossible, is either collagen or fibronectin alone. task. Corneal endothelial cells in tissue culture produce an ex- DISCUSSION tensive ECM whose appearance has been shown to correlate Early studies on the control of proliferation and differentiation with the acquisition by the cells of their normal "in vdvo" shape, of embryonic epithelia demonstrated a requirement for em- cell surface polarity, and function (32). This ECM could sub- bryonic if epithelia are to proliferate and dif- stitute for the ECM produced by other cell types and could help ferentiate (5), although the mechanism through which the us to understand its role in the modulation of the mitogenic mesenchyme exerts its permissive effect is still largely unknown. response of other cell types when maintained on it and exposed It is likely, however, that one of the mechanisms involved is to to the plethora of growth factors that have been discovered provide a proper substratum for epithelial proliferation. The during the past 10 years and are now routinely tested on cells possibility has also been raised that direct contact between maintained on plastic. mesenchymal and epithelial cells could be involved (20-22). When the proliferation of two different cell types such as Investigation of the role of extracellular materials at the ep- bovine granulosa and adrenal cortex cells on plastic is compared ithelial-mesodermal interface has shown that collagens and to that on ECM, none of the cultures maintained on plastic and mucopolysaccharides-major molecular species at the junction exposed to serum proliferated but when maintained on an ECM of interacting tissues-could be implicated in epithelial mor- they proliferated quite actively and no longer required FGF phogenesis (5,23). Because the mesoderm provides the bulk of in order to become confluent. In contrast, when maintained on the collagens to the epitheliomesoderm surface, it could indi- plastic, both cell types exhibited an absolute requirement for rectly modulate proliferation and morphogenesis of the epi- FGF in order to reach confluence. Therefore, one may conclude thelium (5). Equally possible is that epithelia interacting with that the close contact of the cells with the ECM can make them the ECM produced by another tissue could produce their own responsive to factors present in serum and that adherence to collagens and mucopolysaccharides (24). This newly produced plastic prevents such a response. That this is a likely explanation ECM could in turn be responsible for the control of proliferation can be inferred from the observation that cells maintained on of the basal epithelial cell layer, possibly by affecting the cell an ECM had a faster growth rate when maintained in high than shape (25). in low serum concentrations, thereby exhibiting a dependence The need for a proper substratum in order for to on serum in order to proliferate. The simple change of substrate proliferate was first demonstrated by Dodson (26) and Wessels from plastic to ECM restores the normal sensitivity of these cells (27), who used as a model the basal layer of chicken embryonic to agents present in serum as well as in plasma (33). epidermis, which is normally columnar and mitotically active. Although FGF is clearly mitogenic for a number of meso- A loss of mitotic activity results when the dermis and epidermis derm-derived cells, its action on some of the cell types could be are separated in such a way that their previous orientation to indirect. It could either replace the cellular requirement for a each other is destroyed. In such a case the cells also become substrate such as the ECM and thereby make the cells fully Downloaded by guest on September 27, 2021 4098 Cell Biology: Gospodarowicz et al. Proc. Natl. Acad. Sci. USA 77 (1980)

responsive to growth factors present in serum or plasma, even 9. Engvall, E. & Ruoslahti, E. (1973) Int. J. Cancer 20, 1-5. when the cells are maintained on plastic, or it could control the 10. Gospodarowicz, D., Mescher, A. L. & Birdwell, C. R. (1977) Exp. synthesis and secretion of the ECM produced by the cells. Such Eye Res. 25, 75-87. control could in turn make the cells sensitive to factors present 11. Gospodarowicz, D. & Greenburg, G. (1979) Exp. Eye Res. 28, in serum and plasma. That the latter alternative could occur 249-266. is supported by earlier observations which demonstrate that 12. Gospodarowicz, D., Ill, C. R. & Birdwell, C. R. (1977) Endocri- cells, nology 100, 1108-1122. FGF can control the production, by vascular endothelial 13. Gospodarowicz, D., Ill, C. R., Hornsby, P. J. & Gill, G. N. (1977) of their extracellular and cell surface components such as fi- Endocrinology 100, 1080-1089. bronectin and various types of collagen (15, 34). Evidence that 14. Orly, J. & Sato, G. (1979) Cell 17,295-305. FGF can also directly affect cell proliferation is provided by 15. Greenburg, G., Vlodavsky, I., Foidart, J. M. & Gospodarowicz, human umbilical endothelial cells which, even when main- D. (1980) J. Cell Physiol. 103, 333-347. tained on the ECM, still required FGF in order to become 16. Tseng, S., Savion, N., Stern, R. & Gospodarowicz, D. (1980) Exp. confluent (35). Eye Res., in press. The ways in which the ECM exerts its permissive effect on 17. Vlodavsky, I., Lui, G. M. & Gospodarowicz, D. (1980) Cell 19, cell proliferation can only be the object of speculation. One 607-616. possible effect could be to modify the cell shape in order to 18. Gospodarowicz, D., Greenburg, G., Vlodavsky, I., Alvarado, J. make the cells responsive to factor(s) to which they cannot re- & Johnson, L. K. (1979) Exp. Eye Res. 29,485-509. spond unless they adopt an appropriate shape. Recently, 19. Gospodarowicz, D. & Tauber, J. P. (1980) Endocrine Rev., in Folkman and Moscona (36), using vascular endothelial cells press. maintained on tissue culture dishes coated with an agent that 20. Saxen, L., Lehtonen, E., Karkinen-Jaaskelainen, M., Nordling, S. & Wartiovaara, J. (1976) Nature (London) 259,662-663. modifies the adhesiveness of the cells to the dish, were able to 21. Saxen, L. (1977) in Celland Tissue Interactions, eds. Lash, J. W. control precisely the cellular shape, ranging from highly flat- & Burger, M. M. (Raven, New York), pp. 1-9. tened to almost spheroidal. The extent of cell spreading was 22. Slavkin, H. C., Trump, G. N., Brownell, A. & Sorgente, N. (1977) found to be correlated with DNA synthesis or cell growth. in Cell and Tissue Interaction, eds. Lash, J. W. & Burger, M. M. Whereas highly flattened cells responded to serum factors, (Raven, New York), pp. 29-46. spheroidal cells no longer responded, and intermediate degrees 23. Bernfield, M. R. (1978) in Birth Defects, eds. Littlefield, J. E. & of response could be observed depending upon how flattened de Grouchy, J. (Excerpta Medica, Amsterdam), Int. Cong. Series the cells were. Likewise, with corneal epithelial cells, changes 432, pp. 111-125. in cell shape that depend on the substrate upon which the cells 24. Hay, E. D. (1977) in International Cell Biology, eds. Brinkley, are maintained correspond to drastically altered sensitivities B. R. & Porter, K. R. (Rockefeller Univ. Press, New York), pp. of the cells to growth factors (7, 37, 38). 50-57. The results presented above emphasize how drastically one 25. Hay, E. D. (1978) in Birth Defects, eds. Littlefield, J. W. & de can modify the proliferative response of a given cell type to Grouchy, J. (Excerpta Medica, Amsterdam), Int. Cong. Series 432, serum factors, depending upon the substrate upon which the pp. 126-140. 26. Dodson, J. W. (1963) Exp. Cell Res. 31, 233-235. cells are maintained. it is possible that the lack of response of 27. Wessels, N. K. (1964) Proc. Natl. Acad. Sci. USA 52,252-259. many cell types maintained under tissue culture conditions to 28. Reid, L. N. & Rojkind, H. (1979) Methods Enzymol. 58,263- naturally occurring physiological agents responsible in vivo for 278. their proliferation and differentiation may be directly attrib- 29. Ehrmann, R. L. & Gey, G. 0. (1956) J. Natl. Cancer Inst. 16, uted to the artificial substrate, whether plastic or glass, upon 1375-1402. which the cells rest. It is likely that the study of cell cultures, 30. Yang, J., Richards, J., Bowman, P., Guzman, R., Ehami, J., which for the past 50 years, in Wilt's words, is "almost a history McCormick, K., Hamamoto, S., Pitelka, D. & Nandi, S. (1979) of the adequacy of media," may become a history of the in- Proc. Natl. Acad. Sci. USA 76,3401-3405. adequacy of substrates. 31. Rath, N. C. & Reddi, A. H. (1979) Nature (London) 278,855- 857. We thank Mr. Harvey Scodel for his invaluable assistance in the 32. Gospodarowicz, D., Vlodavsky, I., Greenburg, G., Alvarado, J., preparation of this manuscript. This work was supported by grants Johnson, L. K. & Moran, J. (1979) Recent Prog. Horm. Res. 35, from the National Institutes of Health (EY 02196 and HL 20197). 315-377. 1. Miller, E. J. (1977) in Cell and Tissue Interactions, eds. Lash, 33. Gospodarowicz, D. & Ill, C. R. (1980) Proc. Natl. Acad. Sct. USA J. W. & Burger, M. M. (Raven, New York), Vol. 32, pp. 71-86. 77,2726-2730. 2. Muir, H. (1977) in Cell and Tissue Interactions, eds. Lash, J. W. 34. Vlodavsky, I., Johnson, L. K., Greenburg, G. & Gospodarowicz, & Burger, M. M. (Raven, New York), Vol. 32, pp. 32-42. D. (1979) J. Cell Biol. 83,468-486. 3. Stenman, S. & Vaheri, A. (1978) J. Exp. Med. 147, 1054-1063. 35. Gospodarowicz, D. & Ill, C. R. (1980) J. Clin. Invest. 65, 4. Grobstein, C. (1953) Nature (London) 172,869-871. 1357-1364. 5. Grobstein, C. (1967) Natl. Cancer Inst. Monogr. 26,279-299. 36. Folkman, J. & Moscona, A. (1978) Nature (London) 273,345- 6. Grobstein, C. (1975) in Extracellular Matrix Influences on 349. Expression, eds. Slavkin, H. C. & Grenlich, R. C. (Academic, New 37. Gospodarowicz, D., Greenburg, G. & Birdwell, C. R. (1978) York), pp. 9-16; 804-814. Cancer Res. 38, 4155-4171. 7. Gospodarowicz, D., Greenburg, G. & Birdwell, C. R. (1978) 38. Gospodarowicz, D., Vlodavsky, I., Greenburg, G. & Johnson, L. Cancer Res. 38, 4155-4171. K. (1979) in Hormones and Cell Culture, Cold Spring Harbor 8. Gospodarowicz, D., Bialecki, H. & Greenburg, G. (1978) J. Biol. Conferences on Cell Proliferation (Cold Spring Harbor Labo- Chem. 253, 3737. ratory, Cold Spring Harbor, NY), Vol. 6, pp. 561-592. Downloaded by guest on September 27, 2021