An eye-derived growth factor regulates epithelial cell proliferation in the cultured lens

Differentiation

DifTercntiation (1985) 28:286290

fc, Springcr-Verlag1985

An eye-derived growth factor regulates epithelial cell proliferation in the cultured lens Cristina Arruti’ *, Angela Cirillo’ and Yves Courtois’ Laboratorio de Cultivo de Tejidos, Depto. de Histologia y Embriologia, Facultad de Medicina de Montevideo, Avda. Gral. Flores 2125, Montevideo, Uruguay Unite de Rcchcrches GCrontologiques, INSERM U. 118, CNRS ERA 842, Association Claude-Bernard, 29 rue Wilhem, Paris, France

Abstract. Lenses in organ culture permit an analysis of factors acting on epithelial cell growth, while keeping the normal steric constraints of the cell population. By employing this technique with radioautography of epithelial whole mounts, we showed that the DNA synthesis found in the epithelia of cultured bovine lenses follows an organized spatial and temporal pattern during culture. Within the first 48 h, active cells were located at the preequatorial region (“germinative zone”), a distribution consistent with the in vivo spatial organization of multiplying cells. Starting at about 48 h, cells from the central region of the epithelium - a nonproliferating population - were triggered to synthesize DNA in the presence of eye-derived growth factor (EDGF). When cultured in serum-free medium, only a small fraction of the cells was labeled, but when a low serum concentration was present, this fraction reached 50% of the cell population. The stimulatory effect of EDGF required a lag period, but its effect reached a maximum exceeding that found for serum. However, the cells from the germinative region, having a cell density three- to fourfold higher than the central region, were not stimulated to proliferate. This occurred irrespective of the presence of EDGF or serum. If this growth-stimulatory activity derived from the retina were the actual factor controlling cell proliferation in the lens in vivo, then the results presented here would point to the presence of a regulatory mechanism similar to that known for some other hormones.

Introduction The terminal differentiation of lens epithelial cells to fiber cells involves elongation and an increase in the volume of the cells. Ocular lens growth results from an increase in the number of these terminally differentiated cells (the lens fiber cells [7, 191). The epithelial sheet lying over the fiber mass possesses the renewing “stem-cell’’ population which will produce both epithelial cells and fibers [21]. In mature lens, these cycling cells reside just in front of the lens equator, in a broad band called the “germinative” or “proliferative” region [13], while the cells forming the central zone of the epithelium around the anterior pole are in a nonproliferative state [30]. When isolated and explanted in vitro, both the cycling and resting cells proliferate actively [8, 261. To whom reprint requests should be addressed

Some years ago, we employed bovine lens epithelial cells in monolayer cultures to investigate the existence and properties of a retinal growth factor [2]. This growth factor, a small protein with a molecular weight of about 17,000 daltons [5], is also a powerful mitogen and differentiation signal for other ocular cells and tissues, such as corneal endothelial cells [3], corneal epithelium [27], as well as an angiogenic factor [28]. As this growth-stimulatory activity is also found in ciliary bodies, vitreous body, iris, and choroid, and is usually prepared from neural retina [3, 5, 281, it has been named eye-derived growth factor (EDGF [4], or retina-derived growth factor (RDGF). The strong proliferative response induced in lens epithelial cell cultures by EDGF and the ubiquitous ocular localization of this factor suggest that it may be the signal regulating cell proliferation in the lens. Thus, as a first approach to the question of possible intraocular control of lens-epithelial-cell production, a study of its action on organ culture was undertaken. In this in vitro model, the cells retain their in vivo polarity with respect to the basement membrane and cellular neighborhood. Whole mounts of anterior epithelium permit observation of the total cell population, and the DNA-synthesizing cells can be carefully mapped by autoradiography [29]. If a possible physiological action of a growth factor on this organ were invoked, it would be expected that the proliferative response would follow a spatial pattern analogous to that shown by the lens epithelium in vivo [14]. The data presented in the present study indicate that EDGF sustains DNA synthesis in the cells residing in the germinative region, while it initiates DNA synthesis in the nonproliferating cells from the central zone some time later. A working hypothesis concerning a possible physiological mechanism for lens epithelium growth controlled by EDGF is constructed using analogies with the proliferative behavior of corneal endothelial cells in culture under the influence of EDGF.

Methods Lens incubution

Bovine eyes from Hereford cattle (ages ranging between 3 and 4 years) were processed at the laboratory within 3 h of enucleation. The lenses, carefully removed to avoid any damage to the capsule, were incubated in Eagle’s minimal

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essential medium (MEM) with gentamicin sulfate at 50 pg/ ml (Herix, Montevideo) at 35°C in a humidified atmosphere containing 5% CO, . In some experiments, the lenses were cultured in serum containing medium (foetal calf serum, Gibco, Grand Island, NY). EDGF was prepared as described elsewhere [2];the protein content was determined according to the method of Lowry et al. [16]. This preparation still contains several proteins but, when tested on bovine epithelial lens cells, it was shown to be optimally active at the concentration of 75 pg/ml in cultured medium [23. To exclude nonspecific effccts, some control experiments were performed using heat-inactivated factors. Tissue labeling and autoradiography

3H-Thymidine (0.76pCi/ml; 25 Ci/mM; C.E.A., France) was added to the culture medium during the intervals indicated in the experiments. This medium was discarded, and fresh medium containing nonradioactive thymidine was added. After 45 min of incubation at 35” C, the lenses were washed five times with culture medium and phosphate buffer (Dulbecco), and fixed in ethanol/acetic acid (3:l). Whole mounts of the lens epithelia were prepared and covered with Kodak stripping film (AR 10). After development of the radioautographs, the preparations were stained with hematoxylin. For morphological purposes, nonlabeled epithelia were stained with ferric hematoxylin.

Results -

Noncultured bovine lenses have a clear spatial distribution of cellular populations possessing different densities and nuclear morphologies. Figure 1 shows several different cell densities encountered along a line drawn diametrically across an epithelial mount. An ocular grid (130 x 130 pm) was placed sequentially along such lines from one end to the other. The cell number per field was obtained by averaging the cell numbers for four different lines. Only very slight variations in the general pattern of spatial distribution were found for the range of ages studied (3- to 4-yearold cattle). The two main regions, with the maximal and minimal cell density, were located at the preequatorial and central regions of the epithelium, respectively. These two

180

i

h Fig. 2A-C. Epithelial whole mount of a noncultured bovine lens. Photomicrographs from the germinative (A), precentral (B), and central (C) regions

2o

populations possessed very different nuclear morphologies (Fig. 2A, C). Mitotic figures were only found in the first region (“germinative region”). In the central part, the nuclei were much larger, and chromatin appeared to be more dispersed than in the germinative zone. The transition from large to dense nuclei occurred in an area between the central and the germinative zone. We refer to this region as the precentral zone (Fig. 2B). When the lenses were cultured in a chemically defined medium, the cells from the central region of the epithelium

t 25

50

75

100

125

150

Fields

Fig. 1. Cell distribution along a lens epithelium diameter. Each field is 130 x 130 pm

288 Table 1. Effect of EDGF on the 'H-thymidine incorporation in the central zone of cultured lens epithelium Medium composition

Percentage of labeled cells & SE

MEM MEM

0.00fO.00 (8) 0.77f0.10 (8)

+ EDGF

Table4. Comparison of the effects of EDGF and serum on 'Hthymidine incorporation in cells from the germinative plus prccentral regions

Interval of labeling (h)

Lenses were cultured in MEM alone or MEM + EDGF (75 pg/ml) as from explantation. Medium exchanges were made after 24 and 48 h of culture. In the last medium renewal, 'H-TdR was added at a concentration of 0.76 pCi/ml, and the lenses were fixed for autoradiography 24 h later. The number of experiments in each group is listed in parentheses

MEM

Table 2. Comparison of the effects of EDGF and serum on 'Hthymidinc incorporation in the central zone of cultured lens epithelium

0.5% serum

Interval of labeling (h)

MEM

+

0-24

2448

0.00 f0.00 (4)

0.00+_ 0.00(4)

0.07f0.00 (5)

0.00fO.00(4)

0.00+0.00(4)

0.61 k0.09 (5)

0.00+0.00 (4)

0.15f0.02 (4)

52.27f1.23 (5)

48-69

0.5% serum

+

MEM 10% serum MEM

+

0.5% serum

+

EDGF (75 pg/ml) Lenses were cultured and processed for autoradiography 24, 48, 69 h after explantation. Medium was renewed daily, and 3H-TdR

was added at a final concentration of 0.76pCi/ml. The number of experiments in each group is listed in parentheses. EDGF was added continuously Table 3. Time needed from initial exposure to EDGF to detect 'H-thymidine incorporation in the central epithelium of cultured lenses Treatment

Percentage of labeled cells

48 h MEM, 24 h EDGF 24 h MEM, 48 h EDGF 72 h EDGF

0.00f0.00 0.00f0 . 0

0-24

+ SE

0.72 kO.10

Lenses were cultured for 3 days and labeled during the last 24 h with 3H-TdR (0.76 pCi/ml). Percentages are thc mean of four experiments in each group

remained in an inactive state and did not incorporate the DNA precursor at any time during their culture which lasted for 72 h. I n the presence of EDGF, a small percentage of these cells were triggered to synthesize DNA (Table 1). Thus, in the absence of serum, a cell population - in a nonproliferative state from the embryonic stages - was induced by the retinal factor to enter the cell cycle 3 or 4 years later. When serum at a concentration of 0.5% was added to the culture medium in the presence of EDGF, about onehalf of the population incorporated thymidine (Table 2).

24-48

48-72

~

O.lOkO.04(6) 0.04fO.01(18) 0.01 kO.01 (2) 0.13f0.03 (6) 0.05&0.03 (14) 0.01fO.01 (4)

MEM + EDGF MEM + 0.5% serum MEM

+

0.10&0.06 (8) 0.04f0.02 (16)

+

0.02f0.01 (7)

0.05 & 0.02 (4) 0.03& 0.01 (1 6) NT

EDGF MEM + 5% serum MEM + 5% serum EDGF

Percentage of labeled cells & SE

Medium composition

Percentage of labeled cells & SE

Medium composition

0.10 f0.02(4) 0.04& 0.01 (4) 0.01 f0.00(2)

+

0.10f0.02 (4) 0.04+0.01 (4) 0.01 kO.00 (2)

Expcriments were performed as in Table 2. EDGF was added at a final concentration of 75 pg/ml. The mean values from each interval compared by Student's t-test give P>O.Ol. NT,not tested

When present at a high concentration (lo%), serum alone elicited a response comparable to EDGF (Table 2). There was a time lag after the lens had been placed in culture before the onset of DNA synthesis. As can be seen in Table 2, some incorporation was observed starting at the 24to 48-h interval, but the maximum value was only reached 24 h after this. To determine whether this time lag was a consequence of the culture conditions or whether it is inherent in the cell mechanisms for DNA synthesis, lenses were cultured for the first 48 or 24 h in medium without the factor. The factor was only added during the last 24 h. The data shown in Table 3 indicates that the presence of EDGF was necessary a long time before DNA synthesis could be detected, thus suggesting that this interval was the one actually needed to allow this activity. The germinative and precentral regions behaved differently from the central zone. The number of labeled cells found in these regions, reflecting the constitutive rate of division, corresponded to an extremely small fraction of the total cell population of this area (approximately l/l,OOO). Neither EDGF nor serum produced an increase in this fraction at any culture time (Table 4). Thus, in contrast to the central zone, the level of DNA synthesis found in the germinative region when the lenses were cultured in chemically defined medium was not modified by the same conditions which greatly stimulated cell proliferation in the central zone. Discussion

Different growth factors may trigger cell division in the epithelium of lenses cultured in vitro: serum [12], insulin [24], plasmoid aqueous humor [3t], insulin-like growth factors [23], and epidermal growth factor [22]. The existence of an ubiquitous growth factor in the eye (EDGF [4]) which has strong effects on the growth of tissues and cells derived

289

from the eye makes this factor a likely candidate for the control of cell division in the lens epithelium in vivo. Particularly relevant is the fact that epithelial lens cells are a very sensitive target for this factor, even in explants and when cultured as monolayers [9]. The results of the present study represent a discriminating analysis of the lens epithelium by regions, using morphological, morphometric, and kinetic differences. These results indicate that the cells from the central region are strongly stimulated to proliferate by EDGF, even in a serum-free medium. The presence of serum at low concentrations enhanced the intensity of the DNA-synthesis response in a quiescent population which remained in a nonproliferative (GO or G1 [ll]) state for 3 or 4 years. The results presented here do not make it possible to decide whether this amplification was due to a metabolic or a synergistic (cooperative) effect of the serum with the growth factor. After a time lag, this population was stimulated to proliferate by the growth factor or by serum. When bovine lenses were cultured in EDGF or serum-containing medium, the number of dividing cells throughout the central region increased, as is the case in other mammalian or amphibian lenses. On the other hand, the same stimuli (serum factors or EDGF) did not increase the proportion of dividing cells in the germinative zone, which is responsible in vivo for epithelial cell production. It is important to note that this region has a three- to fourfold-higher cell density than the central zone, representing the highest density possible within the confines of a monolayered structure. When higher cell densities are found, as in epithelial-lens-cell cultures, the cells are always multilayered [8]. Germinative cells appear to be at the limit of proliferation, considering the restriction imposed by cell-to-cell contact. We have previously reported a similar phenomenon in endothelial corneal cells in culture [l]. When these cells are cultured continuously in the presence of EDGF, they retain a very low proliferative rate at confluency; this is not modifiable by higher concentrations of EDGF or serum. However, should the factor be withdrawn, leading to a decrease in the already low proliferative rate, its readdition some days later triggers a wave of DNA synthesis. It is tempting to speculate on the analogy between the adult lens epithelium and the corneal endothelium in monolayer culture, as both cell populations are arranged in monolayers. If the germinative lens epithelium is controlled by EDGF in vivo, it seems likely that, once in organ culture in the presence of EDGF, it will maintain a low proliferation rate, since this is consistent with the low rate found in vivo [14]. The response of the central epithelium to the factor in organ culture was very different to that of the germinative region. In vivo, the spatial distribution of the factor may be such that it never reaches these central epithelial cells, but once the lens is explanted and cultured in the presence of the factor, this region of cells is greatly stimulated to proliferate. We propose that these cells, unlike the germinative cells, do not possess the internal mechanisms for regulating the proliferative response. The strong regulation existing at the germinative region could be also induced by the continuous presence of the factor. Some other cell systems are known to possess regulatory mechanisms to limit the response to a hormone or growth factor, e.g., in conditions involving hyperinsulinemia, tis-

sues have a markedly reduced number of receptors per cell (a “down regulation” caused by ligand binding [lo]), as is also the case in cells stimulated by epidermal growth factor (EGF) [15, 18, 321. The existence of receptors for EDGF [20] with some properties analogous to those of the EGF receptor increases the possibility of this kind of mechanism. It is difficult LO analyze the fate of cells in organ culture because, after 72 h, the cells start to migrate. It is possible that EDGF stimulation leads to an increase in differentiation in the adult bovine epithelium similar to the effects of lentropin on chick-embryo epithelium [6] or of retinal extracts on newborn-rat epithelium [17]. Our own analysis of the effect of EDGF on serially subcultured bovine lens cells did not show an increase in crystallin synthesis [25], but work on organ cultures may demonstrate such an effect. Acknowledgements. The authors would like to thank Frigorifico y Matadero Carrasco (Frimacar) Montevideo, for kindly providing the bovine eyes. This work has been supported in part by a cooperative research grant between INSERM and the Facultad de Medicina de Montevideo.

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25. Simonncau L, Herve B, Jacquemin E, Courtois Y (1983) State of differentiation of bovine epithelial lens cells in vitro. Relationship between the variation of the cell shape and the synthesis of crystallins. Cell Differ 13: 185190 26. Tassin J, Malaise E, Courtois Y (1979) Human lens cells have an in vitro proliferative capacity inversely proportional to the donor age. Exp Cell Res 123:388-392 27. Thompson P, Desbordes JM, Giraud J, Pouliquen Y, Barritault D, Courtois Y (1 982) The effect of an eyederived growth factor (EDGF) on corneal epithelium regeneration. Exp Eye Res 34:191-199 28. Thompson P, Arruti C, Maurice D, Plouet J, Barritault D, Courtois Y (1982) Angiogenic activity of a cell growth regulating factor derived from the retina. In: Clayton R, Haywood J, Reading HW, Wright A (eds) Problems of normal and genetically abnormal retinas. Academic Press, London 29. Treton J, Courtois Y (1981) Evolution of the distribution, proliferation and uv repair capacity of rat lens epithelium cells as a function of maturation and aging. Mech Ageing Dev 15:251-267 30. Von Sallman L (1952) Experimental studies on early lens changes after reentgen-irradiation. 111. Effect of X-irradiation on mitotic activity and nuclear fragmentation of lens epithelium in normal and cysteine-treated rabbit. Arch Ophthalmol 47: 305320 31. Weinsieder A, Reddan J, Wilson D (1976) Aqueous humor in lens repair and cell proliferation. Exp Eye Res 23 :355-363 32. Wolfe RA, Wu R, Sato GH (1980) Epidermal growth factor induced down regulation of the receptor does not occur in HeLa cells grown in defined medium. Proc Natl Acad Sci USA 77:2735-2739

Received July 1984 / Accepted in revised form November 1984