|Molecular Vision 2004;
Received 27 January 2004 | Accepted 9 July 2004 | Published 15 July 2004
Fibroblast growth factor 2: Roles of regulation of lens cell proliferation and epithelial-mesenchymal transition in response to injury
Shizuya Saika,1 Yoshitaka
Ohnishi,1 Akira Ooshima,2 John W. McAvoy,3,4 Chia-Yang
Liu,5 Muhamad Azhar,6 Thomas Doetschman,6 Winston
Departments of 1Ophthalmology and 2Pathology, Wakayama Medical University, 811-1 Kimiidera, Wakayama, 641-0012, Japan; 3Save Sight Institute and 4Department of Anatomy and Histology, University of Sydney, NSW, Australia; 5Bascom Palmer Eye Institute, University of Miami School of Medicine, Miami, FL; Departments of 6Molecular Genetics and 7Ophthalmology, University of Cincinnati Medical Center, Cincinnati, OH
Correspondence to: Takeshi Tanaka, M.D., Ph.D., Department of Ophthalmology, Wakayama Medical University, 811-1 Kimiidera, Wakayama, 641-0012, Japan; Phone: 81-73-447-2300; FAX: 81-73-448-1991; email: firstname.lastname@example.org
Purpose: To examine the role of fibroblast growth factor 2 (FGF2) in regulating lens cell proliferation and epithelial-mesenchymal transition (EMT) in response to injury.
Methods: The amount of FGF2 protein was determined in healing, injured rat lenses by enzyme immunoassay. The effects of FGF2 and transforming growth factor β2 (TGFβ2) on cell proliferation of αTN4 cells (a mouse lens epithelial cell line) were determined. FGF2-knockout mice were used to further examine the role of endogenous FGF2 on injury-induced epithelial cell proliferation and EMT. The anterior lens capsule was injured by a hypodermic needle under both general and topical anesthesia in one eye of 34 fgf2+/+ mice and 42 fgf2-/- mice. At days 2, 5, and 10 post-injury the mice were sacrificed following a 2 h labeling period with bromo-deoxyuridine (BrdU). The number of BrdU-positive cells in each specimen was determined.
Results: A capsular break caused a 10 fold increase of FGF2 protein accumulated in rat lens 14 days after injury. Addition of 3.43 ng/ml FGF2 enhanced proliferation of αTN4 cells. This occurred in the presence or absence of exogenous TGFβ2, that has an inhibitory effect on αTN4 cell proliferation. Significantly fewer BrdU-labeled cells were found in fgf2-/- mice than in fgf2+/+ mice during healing post-injury. However, lacking FGF2 did not alter the expression patterns of α-smooth muscle actin and collagen type I, markers of EMT in lens cells.
Conclusions: Endogenous FGF2 is required for increased cell proliferation but not essential for EMT during the lens response to injury.
Hyper-proliferation of lens epithelial cells (LECs) and accumulation of extracellular matrix in the residual lens capsule after cataract extraction and implantation of an intraocular lens can lead to capsular opacification (post-operative capsular opacification, PCO) [1-6].
Various growth factors are believed to orchestrate the responses of LECs after injury. Among them, members of the transforming growth factor β (TGFβ) family are abundant in aqueous humor and appear to have leading roles in regulating the behavior of LECs [7-11]. For example, studies have shown that TGFβ induces explanted rat lens epithelial cells to undergo an epithelial-mesenchymal transition (EMT) characteristic of PCO [12-16]. We also previously reported that human post-operative LECs were positive for nuclear Smad3/Smad4, the key molecules conveying signals after cell surface receptor binding to transforming growth factor β (TGFβ). In experimental animal models, an anterior capsular injury induces transient nuclear translocation of Smad3/Smad4, which is effectively blocked by the administration of TGFβ2 neutralizing antibody . This finding suggested that endogenous TGFβ2 may modulate LEC behavior. Our previous study demonstrated that the administration of neutralizing antibodies against TGFβ2 induces 5-bromo-2'-deoxyuridine (BrdU)-labeled LECs at 24 h post-injury . At this timepoint no significant increment of cell proliferation was seen in the injured, compared with the uninjured, lens in the absence of the antibody. This suggested that endogenous TGFβ2 might actually suppress LEC proliferation in the relatively early phase of healing. However, LECs do eventually proliferate and undergo changes that result in the formation of PCO in the later healing phase. Therefore, other factor(s) present in the injured lens may eventually counteract the suppression by endogenous TGFβ2. FGFs are possible candidates for such mitogenic factors. The FGF growth factor family consists of 23 members, and the classical FGFs, namely acidic FGF and basic FGF, are now known as FGF1 and FGF2, respectively . It is known that FGF2 can promote growth of LECs in vitro . However, the role of this factor in modulating in vivo proliferation of LECs in response to injury has not yet been explored.
The present study was undertaken to examine the role of endogenous FGF2 in injury-induced proliferation of LECs. We first determined the total amount of FGF2 protein in uninjured and healing, injured, rat lenses and showed that the level of FGF2 in an injured lens at day 14 is 10 fold greater than that of an uninjured lens. This marked accumulation of FGF2 in an injured rat lens prompted us to hypothesize that FGF2 may have a major role in promoting LEC proliferation in response to injury. We then showed that exogenous FGF2 enhances proliferation of αTN4 cells, an SV40-transformed mouse lens epithelial cell line, in the presence and absence of TGFβ2 which has an inhibitory effect on cell proliferation. To further elucidate the role of FGF2 in vivo, we examined injury-induced proliferation and EMT  of LECs in FGF2-knockout mice. The results indicate that loss of FGF2 reduces injury-induced proliferation of LECs in vivo, but does not show any effect on EMT of LECs.
Animal experiments were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and also with approvals of Institutional Animal Care and Use Committees of Wakayama Medical University and the University of Cincinnati.
Protein levels of FGF2 in healing and injured rat lens in vivo
Aqueous humor and uninjured lenses were obtained from four month old adult Wistar rats (n=3, body weight 400 g) sacrificed by an overdose of sodium pentobarbital administered intraperitoneally. Each lens (39.8±3.4 mg) was sonicated with an ultrasound tissue homogenizer in phosphate-buffered saline (PBS, 1 ml/g of tissue). Anterior capsular injury was produced with a hypodermic needle in one eye of adult Wistar rats (6 rats per healing interval) under both general and topical anesthesia as previously reported . The injured animals were allowed to heal for 2 h, and 1, 3, 5, 7, and 14 days. The healing lens and aqueous humor (at day 14 only) were obtained and the lens was treated similarly. All the specimens were then stored at -80 °C until use for FGF2 protein content. The amount of FGF2 protein in each specimen was measured by using an enzyme immunoassay kit (R & D system, Minneapolis, MN) according to the manufacturer's protocol.
Proliferation of αTN4 cells in the presence of FGF2 and TGFβ2
Cell growth was evaluated by using a BrdU incorporation assay kit (Cell Proliferation kit, Amersham, Buckinghamshire, UK) according the protocol recommended by the manufacturer. An SV40-transformed mouse lens epithelial cell line, αTN4 cells (1.0x104 cells/well), were cultured in 96-well culture plates (Corning-Iwaki Glass, Corning, NY) in serum-free Eagle's minimum essential medium for 24 h. Six wells were prepared for each of the individual culture conditions. Cultures were then treated with human recombinant FGF2 (R & D systems, 0, 0.26, or 3.43 ng/ml) or with a combination of human recombinant TGFβ2 (R & D systems, 0.43 ng/ml) and FGF2 (3.43 ng/ml) for 24 h in 100 μl serum-free medium. The cells were labeled with BrdU for 2 h, washed, and reacted with anti-BrdU antibody adequately diluted by the manufacturer's suggestion. After a wash, secondary antibody reaction, the antibody complex was visualized with the substrate from the kit. Absorbance at 450 nm was measured by BioRad plate reader model 450.
Cell proliferation and EMT in injured lens epithelia of FGF2-knockout mice
The experimental fgf2+/+ (n=34) and fgf2-/- (n=42) mice  were anesthetized by an intraperitoneal injection of sodium pentobarbital (70 mg/kg body weight) and topical application of oxybuprocaine. A small incision in the central anterior capsule of the lens was made with a 26 G hypodermic needle in one eye (right or left) after topical application of mydriatics as previously reported (following a method approved by NCI/NIH, Bethesda, MD) [5,6,15]. After instillation of ofloxacin ointment, the fgf2-/- mice were allowed to heal for 2 day (12 mice), 5 day (10 mice), and 10 day (12 mice), and fgf2+/+ mice for 2 day (8 mice), 5 day (12 mice), and 10 day (8 mice). Uninjured eyes served as control. The animals were labeled with BrdU 2 h prior to sacrifice . Mice were killed by CO2 asphyxia and cervical dislocation. Enucleated globes were fixed and embedded in paraffin . Three sagittal sections, 25 μm apart from each other, were prepared by cutting each paraffin embedded eye through the optic nerve. Deparaffinized sections were treated with 2 N HCl for 60 min and then processed for immunohistochemistry for BrdU (1:10 dilution in PBS, Boehringer Mannheim, Mannheim, Germany), αSMA, and collagen I as previously reported . The sections were counter stained with methyl green and mounted with balsam. Non-immune mouse IgG (10 μg/ml) was used as negative control. The mean number of BrdU-positive cells from these three sections of one eye represented the value of the eye, and data from all eyes in fgf2+/+ and fgf2-/- groups were statistically analyzed at each timepoint by using an unpaired t test.
FGF2 and TGFβ2 protein in uninjured and injured rat lens
The amount of FGF2 increased in an injured lens time-dependently during the healing interval. Figure 1 shows that the amount of FGF2 protein in an uninjured adult rat lens was 13.0±3.6 pg. Following anterior capsular injury, the level of FGF2 protein began to increase at day 3 post-injury and then reached a level of over 10 fold (171.5±1.5 pg/lens) above that of the normal lens at day 14 of injury. We also measured FGF2 protein concentration in aqueous humor and showed that it was 70±10 pg/ml in an uninjured eye and 30±10 pg/ml or 40±10 pg/ml in an eye with an injured lens at day 1 or 14, respectively.
Proliferation of αTN4 cells in the presence of FGF2 and/or TGFβ2
Addition of FGF2 at a concentration of 0.26 ng/ml did not affect the proliferation of αTN4 cells (data not shown), whereas at 3.43 ng/ml cell proliferation was promoted (Figure 2). The addition of TGFβ2 (0.43 ng/ml) to culture medium did not stimulate but rather had a significant (p<0.01) inhibitory effect on proliferation of αTN4 cells. Combining TGFβ2 (0.43 ng/ml) with FGF2 (3.43 ng/ml) did not abolish the stimulatory effect of FGF2 on proliferation of αTN4 cells.
Loss of FGF2 reduced cell proliferation, but did not affect EMT with in vivo lens epithelium in response to injury
Histological analysis of injured lenses showed that at day 2, the epithelial cells remained in a monolayer around the break in the central anterior capsule, whereas at 5 and 10 days of injury multilayers of elongated, fibroblast-like cells were present at the capsular break (not illustrated). The number of BrdU-labeled LECs increased post-injury with a peak at day 5 in both fgf2+/+ and fgf2-/- mice. However, fewer BrdU-labeled cells were detected in fgf2-/- mice compared with fgf2+/+ mice. This labeling difference was statistically significant at the timepoints of day 2, 5, and 10 following injury (Figure 3 and Table 1).
To assess phenotypic changes in the lens epithelium post-injury, immunohistochemistry was carried out to look for markers of epithelial-mesenchymal transition in the lens. Using antibodies for αSMA, immunoreactivity was not detected in cells around the capsular injury at day 2 (not illustrated) but at 5 (Figure 4) and 10 days (data not shown) when the cells had multilayered, αSMA immunoreactivity was detected in both fgf2+/+ and fgf2-/- mice. Type I collagen immunoreactivity was not detected in the cells around the capsular break at either day 2 or 5 but was similarly observed at day 10 in both fgf2+/+ and fgf2-/- mice (not illustrated). No immunoreactivity was detected when non-immune IgG was substituted for the primary antibody (not illustrated).
In the present study, we showed that FGF2 accumulated in the healing, injured rat lens and that addition of exogenous FGF2 enhances the proliferation of αTN4 cells. Interestingly, we observed fewer BrdU-labeled cells in healing lenses of fgf2-/- mice than in fgf2+/+ mice. A significant difference appears only in response to wounding and is accompanied by an increase in endogenous FGF2. These results indicate that endogenous FGF2 contributes to the injury-induced proliferation of LECs. The increase in proliferation was observed both in the vicinity of injury and in the germinative zone.
We have previously shown that an injury in the anterior capsule induces TGFβ-Smad3/TGFβ-Smad4 signaling as early as 12 h post-injury . Although previous studies have indicated that endogenous TGFβ2 suppresses LEC proliferation , the present data from our cell culture experiment suggest that up-regulation of endogenous FGF2 upon capsular injury may overcome the growth inhibition by TGFβ2 and then lead to acceleration of cell proliferation. In a cultured mouse lens epithelial cell line, αTN4, FGF2 at a lower concentration (0.26 ng/ml) did not show a significant effect on cell proliferation, but a higher concentration (3.43 ng/ml) could overcome the growth-inhibitory effect of TGFβ2 and stimulat cell proliferation. Our immunohistochemistry showed that immunoreactivity for both FGF2 and TGFβ2 is more prominent in epithelial cells as compared with lens fibers in rat (data not shown), indicating that the concentration of growth factors inside the lens in vivo may not be uniform. Nevertheless, the results obtained from cell culture experiments suggest that the balance of TGFβ2 and FGF2 may also be one of the components regulating post-injury lens epithelial cell proliferation.
It has been reported that TGFβ1 up-regulates TGFβ1 and FGF2 in corneal fibroblasts . However, the exact mechanism of up-regulation of FGF2 in healing, injured rat lens remains unknown. We have also previously reported that administration of TGFβ2 neutralizing antibody induces cell proliferation in an injured mouse lens at 24 h post-injury, although no significant increment of cell proliferation was observed without antibody administration as compared with uninjured lens . The present study of enzyme immunoassay of in vivo specimens shows that FGF2 protein levels in an injured rat lens began to increase at day 3, suggesting that the interval of 1 healing day might not be enough for endogenous TGFβ2 to increase LEC's FGF2 production enough to accelerate cell proliferation. This notion might be confirmed that our previous study showed no increment of cell proliferation at day 1 post-injury without administration of the antibody and that lens epithelial cell proliferation in fgf2-/- mice was not significantly less than that in fgf2+/+ mice at day 2 post-injury. Therefore, it may be hypothesized that cell proliferation induced by TGFβ2 neutralizing antibody in relatively early stage post-injury and late stage may be regulated by different mechanisms in terms of FGF2-dependency. The result from administration of TGFβ2 neutralizing antibody in that study, therefore, may not exclude the possibility that increment of FGF2 in lens tissue may be regulated by endogenous TGFβs.
Although ligand binding to TGFβ-receptor activates various signaling cascades (i.e., mitogen-activated kinase [MAPK], p38 MAPK, or c-Jun-N-terminal kinase cascade) signaling through Smad prototypes TGFβ-signaling [23-25]. On the other hand, in vivo LECs express FGF-receptor 1 (FGFR1), FGFR2, FGFR3, and FGFR4. Although FGFR1 is known to be required for lens fiber differentiation, the exact receptor(s) essential for promotion of cell proliferation is (are) not fully investigated [26,27]. However, FGF2 reportedly enhances cell proliferation by activating Ras/MAPK cascade, resulting in nuclear translocation of Erk-1/2 . It is hypothesized that some cross-talk(s) between TGFβ-Smad signal and FGF2-MAPK may locate above the level before affecting gene promoters during modulation of LEC's proliferation .
Mice lacking FGF2 grow normally, but their blood pressure is lower than wild type mice suggesting that the tonus of the blood vessel is attenuated with some abnormalities in vessel wall structure . Wound healing in the blood vessel is reportedly normal . In the present study injury induced proliferation of LECs in fgf2-/- mice is suppressed compared with wild type mice although the lens of the adult fgf2-/- mouse looks morphologically normal. The discrepancy between blood vessel and lens in terms of cell proliferation regulation is not known, it may be attributed to the differences of intrinsic cellular contents between endothelia and epithelial cell lineages. However, the exact factor(s) that account for this phenomenon is not known.
We have shown that loss of Smad3, a key signaling molecule upon TGFβ stimulation, blocks EMT in in vivo LECs upon injury . On the other hand, the present study revealed that loss of FGF2, a growth factor capable of activating Ras/MAPK, does not affect the process of such EMT, although both Ras/MAPK and Smad signal are required for EMT in some neoplastic cell types . LECs in injured lenses of both fgf2+/+ and fgf2-/- mice showed a fibroblastic appearance at day 5 with expression of αSMA.
Further detailed study is needed to clarify the mechanism of regulation of proliferation of LECs by growth factor networks in response to injury. Such studies may help find a new strategy to prevent or treat PCO.
This study was supported by a Grant from the Ministry of Education, Science, Sports and Culture of Japan and Uehara Memorial Foundation (SS), and a Research Grant on Priority Areas from Wakayama Medical University (SS and AO), and NIH grant EY13755, Research to Prevent Blindness, Ohio Lions Eye Research Foundation (WWYK). WWYK is the recipient of an RPB Senior Investigator Award.
1. Saika S, Ohmi S, Kanagawa R, Tanaka S, Ohnishi Y, Ooshima A, Yamanaka A. Lens epithelial cell outgrowth and matrix formation on intraocular lenses in rabbit eyes. J Cataract Refract Surg 1996; 22 Suppl 1:835-40.
2. Saika S, Yamanaka A, Tanaka S, Ohmi S, Ohnishi Y, Ooshima A. Extracellular matrix on intraocular lenses. Exp Eye Res 1995; 61:713-21.
3. Saika S, Kawashima Y, Miyamoto T, Okada Y, Tanaka SI, Ohmi S, Minamide A, Yamanaka O, Ohnishi Y, Ooshima A, Yamanaka A. Immunolocalization of prolyl 4-hydroxylase subunits, alpha-smooth muscle actin, and extracellular matrix components in human lens capsules with lens implants. Exp Eye Res 1998; 66:283-94.
4. Wormstone IM. Posterior capsule opacification: a cell biological perspective. Exp Eye Res 2002; 74:337-47.
5. Saika S, Miyamoto T, Ishida I, Ohnishi Y, Ooshima A. Osteopontin: a component of matrix in capsular opacification and subcapsular cataract. Invest Ophthalmol Vis Sci 2003; 44:1622-8.
6. Saika S, Miyamoto T, Tanaka S, Tanaka T, Ishida I, Ohnishi Y, Ooshima A, Ishiwata T, Asano G, Chikama T, Shiraishi A, Liu CY, Kao CW, Kao WW. Response of lens epithelial cells to injury: role of lumican in epithelial-mesenchymal transition. Invest Ophthalmol Vis Sci 2003; 44:2094-102.
7. Massague J. The transforming growth factor-beta family. Annu Rev Cell Biol 1990; 6:597-641.
8. Grainger DJ, Kemp PR, Metcalfe JC, Liu AC, Lawn RM, Williams NR, Grace AA, Schofield PM, Chauhan A. The serum concentration of active transforming growth factor-beta is severely depressed in advanced atherosclerosis. Nat Med 1995; 1:74-9.
9. Streilein JW, Cousins SW. Aqueous humor factors and their effect on the immune response in the anterior chamber. Curr Eye Res 1990; 9 Suppl:175-82.
10. Jampel HD, Roche N, Stark WJ, Roberts AB. Transforming growth factor-beta in human aqueous humor. Curr Eye Res 1990; 9:963-9.
11. de Boer JH, Limpens J, Orengo-Nania S, de Jong PT, La Heij E, Kijlstra A. Low mature TGF-beta 2 levels in aqueous humor during uveitis. Invest Ophthalmol Vis Sci 1994; 35:3702-10.
12. Liu J, Hales AM, Chamberlain CG, McAvoy JW. Induction of cataract-like changes in rat lens epithelial explants by transforming growth factor beta. Invest Ophthalmol Vis Sci 1994; 35:388-401.
13. Hales AM, Schulz MW, Chamberlain CG, McAvoy JW. TGF-beta 1 induces lens cells to accumulate alpha-smooth muscle actin, a marker for subcapsular cataracts. Curr Eye Res 1994; 13:885-90.
14. Saika S, Saika S, Liu CY, Azhar M, Sanford LP, Doetschman T, Gendron RL, Kao CW, Kao WW. TGFbeta2 in corneal morphogenesis during mouse embryonic development. Dev Biol 2001; 240:419-32.
15. Saika S, Okada Y, Miyamoto T, Ohnishi Y, Ooshima A, McAvoy JW. Smad translocation and growth suppression in lens epithelial cells by endogenous TGFbeta2 during wound repair. Exp Eye Res 2001; 72:679-86.
16. Saika S, Miyamoto T, Ishida I, Shirai K, Ohnishi Y, Ooshima A, McAvoy JW. TGFbeta-Smad signalling in postoperative human lens epithelial cells. Br J Ophthalmol 2002; 86:1428-33.
17. Yamashita T, Yoshioka M, Itoh N. Identification of a novel fibroblast growth factor, FGF-23, preferentially expressed in the ventrolateral thalamic nucleus of the brain. Biochem Biophys Res Commun 2000; 277:494-8.
18. McAvoy JW, Chamberlain CG. Fibroblast growth factor (FGF) induces different responses in lens epithelial cells depending on its concentration. Development 1989; 107:221-8.
19. Zuk A, Hay ED. Expression of beta 1 integrins changes during transformation of avian lens epithelium to mesenchyme in collagen gels. Dev Dyn 1994; 201:378-93.
20. Shirai K, Okada Y, Saika S, Senba E, Ohnishi Y. Expression of transcription factor AP-1 in rat lens epithelial cells during wound repair. Exp Eye Res 2001; 73:461-8.
21. Zhou M, Sutliff RL, Paul RJ, Lorenz JN, Hoying JB, Haudenschild CC, Yin M, Coffin JD, Kong L, Kranias EG, Luo W, Boivin GP, Duffy JJ, Pawlowski SA, Doetschman T. Fibroblast growth factor 2 control of vascular tone. Nat Med 1998; 4:201-7.
22. Song QH, Klepeis VE, Nugent MA, Trinkaus-Randall V. TGF-beta1 regulates TGF-beta1 and FGF-2 mRNA expression during fibroblast wound healing. Mol Pathol 2002; 55:164-76.
23. Wrana JL, Attisano L, Wieser R, Ventura F, Massague J. Mechanism of activation of the TGF-beta receptor. Nature 1994; 370:341-7.
24. Massague J, Hata A, Liu F. TGF-β signalling through the Smad pathway. Trends Cell Biol 1997; 7:187-192.
25. Itoh S, Itoh F, Goumans MJ, Ten Dijke P. Signaling of transforming growth factor-beta family members through Smad proteins. Eur J Biochem 2000; 267:6954-67.
26. de Iongh RU, Lovicu FJ, Chamberlain CG, McAvoy JW. Differential expression of fibroblast growth factor receptors during rat lens morphogenesis and growth. Invest Ophthalmol Vis Sci 1997; 38:1688-99.
27. de Iongh RU, Lovicu FJ, Hanneken A, Baird A, McAvoy JW. FGF receptor-1 (flg) expression is correlated with fibre differentiation during rat lens morphogenesis and growth. Dev Dyn 1996; 206:412-26.
28. Lovicu FJ, McAvoy JW. FGF-induced lens cell proliferation and differentiation is dependent on MAPK (ERK1/2) signalling. Development 2001; 128:5075-84.
29. Kretzschmar M, Doody J, Massague J. Opposing BMP and EGF signalling pathways converge on the TGF-beta family mediator Smad1. Nature 1997; 389:618-22.
30. Saika S, Kono-Saika S, Ohnishi Y, Sato M, Muragaki Y, Ooshima A, Flanders KC, Yoo J, Anzano M, Liu CY, Kao WW, Roberts AB. Smad3 signaling is required for epithelial-mesenchymal transition of lens epithelium after injury. Am J Pathol 2004; 164:651-63.
31. Oft M, Akhurst RJ, Balmain A. Metastasis is driven by sequential elevation of H-ras and Smad2 levels. Nat Cell Biol 2002; 4:487-94.