- Email: [email protected]
Overaccumulation of transforming growth factor-β1 and basic fibroblast growth factor in lens epithelial cells of congenital cataract
Overaccumulation of transforming growth factor-b1 and basic fibroblast growth factor in lens epithelial cells of congenital cataract Ying Xiao,* MD, PhD; Bojun Zhao,† MD, PhD; Zhijuan Gao,* MD; Qingmin Pan,* MD TGF- b1 and bFGF in congenital cataract—Xiao et al. !"342!#4 s 2»35-» Objective: The aim of this study was to evaluate the accumulation of transforming growth factor (TGF)-β1 and basic fibroblast growth factor (bFGF) at gene and protein levels in lens epithelial cells of anterior subcapsular congenital cataracts (ASCC). Design: Case–control study and analysis. Participants: Twenty-six eyes with ASCC were studied, and 14 eyes with transparent lenses were used as controls. -ETHODS Anterior epithelium capsules with ASCC were obtained from patients who had undergone extracapsular cataract extraction.The accumulation of TGF-β1 and bFGF gene and protein was determined by reverse transcription-real time polymerase chain reaction (RT-PCR) and Western blotting. Results: The results from RT-PCR showed that the accumulation of TGF-β1 and bFGF mRNA in lens epithelial cells of ASCC was significantly higher compared with controls in both male and female samples. Furthermore, there was a positive correlation between the accumulation of bFGF mRNA and TGF-β1 mRNA.Western blotting results demonstrated that the accumulation of TGF-β1 and bFGF proteins in lens epithelial cells with ASCC was also highly elevated. #ONCLUSIONS The overaccumulation of TGF-β1 and bFGF in lens epithelial cells may play an important role in the formation of subcapsular congenital cataract. Objet : Cette étude a pour objet d’évaluer l’accumulation du facteur de transformation de la croissance (FTC)-β1 et du facteur de base de la croissance du fibroblast (FbCF) aux niveaux des gènes et des protéines des cellules épithéliales du cristallin dans les cataractes sous-capsulaires antérieures congénitales (CSAC). Nature : Étude contrôlé et analyse. Participants : Vingt-six yeux avec CSAC ont été étudiés et 14 yeux avec cristallin transparent ont servi de témoins. -£THODES : Les capsules de l’épithélium antérieur avec CSAC ont été obtenues de patients qui avaient subi l’extraction d’une cataracte extracapsulaire. L’accumulation des gènes et des protéines FTC-β1 et FbCF a été établie par transcription inverse – réaction en chaîne de la polymérase (TI-RCP) en temps réel et buvardage de western. 2£SULTATS : Les résultats de la TI-RCP montrent que les accumulations d’ARN messager (ARNm) du FTC-β1 et FbCF dans les cellules épithéliales étaient significativement plus élevées comparativement aux échantillons témoins, masculins et féminins. En outre, il y avait une corrélation positive entre l’accumulation d’ARNm du FbCF et d’ARNm du FTC-β1. Les résultats du buvardage de western ont démontré que l’accumulation de protéines FTC-β1 et FbCF dans les cellules épithéliales du cristallin avec CSAC était aussi très élevée. #ONCLUSIONS : La suraccumulation de FTC-β1 et FbCF dans les cellules épithéliales du cristallin peut jouer un rôle important dans la formation de la cataracte sous-capsulaire congénitale.
N
ormal lens development and maintenance is dependent on the tight spatial and temporal regulation of lens cell proliferation and fiber cell differentiation, in which growth factors play a pivotal role. Imbalance in this process may result in the formation of cataract, and studies using rats or rat lens explants show that ectopic addition of transforming growth factor (TGF)-β and basic fibroblast growth factor (bFGF) induces cataractous change.1–4 Whole rat lenses cultured with TGF-β develop anterior opacities corresponding to fibrotic plaques that are analogous to human anterior subcapsular cataract, and these abnormal changes can be blocked by a pan-specific antibody against TGF-β.2,5
In vitro, bFGF affects lens epithelial cell survival, migration, and differentiation in a dose-dependent manner in humans and mammals, and involves the formation of posterior capsule opacification.1,6 Congenital cataract is an opacity in the lens of the eye that presents at, or develops shortly after, birth. Approximately 0.03% of newborns have some form of congenital cataract. Congenital cataract may be associated with hereditary disorders, metabolic diseases, and also infections that the mother had during pregnancy. However, the exact mechanism of the development of this disease has not been fully elucidated. There are different types of congenital cataract,
From *the Department of Ophthalmology, Second Hospital of Shandong University, and †the Department of Ophthalmology, Shandong Provincial Hospital, Jinan, China
Correspondence to Bojun Zhao, MD, Department of Ophthalmology, Shandong Provincial Hospital, Jinan, China; [email protected]
Originally received Apr. 5, 2008. Revised July 28, 2008 Accepted for publication Aug. 8, 2008 Published online Feb. 27, 2009
This article has been peer-reviewed. Cet article a été évalué par les pairs. Can J Ophthalmol 2009;44:189–92 doi:10.3129/i09-006 CAN J OPHTHALMOL—VOL. 44, NO. 2, 2009
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TGF- b1 and bFGF in congenital cataract—Xiao et al. such as nuclear congenital cataract, cortical congenital cataract, and subcapsular congenital cataract. Significant vision impairment owing to congenital cataract can lead to permanent amblyopia. Treatment of congenital cataract remains a challenge. Therefore, the further elucidation of the mechanism of this disease is a prerequisite for its prevention. Overaccumulation of TGF-β mRNA was reported in lens epithelial cells of adult anterior polar cataract.7 In this study, we investigated whether there is an elevation in the accumulation of TGF-β1 and bFGF in lens epithelial cells of anterior subcapsular congenital cataracts (ASCC). The results demonstrate that the accumulation of TGF-β1 and bFGF was increased at both gene and protein levels and that the accumulation of TGF-β1 mRNA and bFGF mRNA were positively correlated. METHODS Sample collection
The study was approved by the Ethical Review Board of Shandong University. Capsulotomy specimens with ASCC were obtained from patients who had undergone extracapsular cataract extraction. Anterior epithelium capsules from transparent lenses obtained from eyeballs enucleated because of trauma or retinoblastoma were used as controls. Informed consent was obtained in accordance with the Declaration of Helsinki before samples were taken for analysis. For reverse transcription-real time polymerase chain reaction (RT-PCR), 12 samples from males and 8 samples from females with ASCC were used with 6 male and 3 female samples as controls. Western blotting was performed using 6 samples with ASCC and 5 samples as control. Patients with other eye diseases were excluded from the study. The ages of patients with ASCC ranged from 3 months to 9 years. The ages of controls were from 1 to 10 years. Circular pieces of the anterior capsules with lens epithelial cells attached were put into RNase-free EP tubes after capsulotomy and stored at –80 °C immediately. RNA extraction, reverse transcription, and real-time quantitative PCR
Total cellular RNA was isolated from lens epithelial cells attached to the anterior capsules using TRIzol (Invitrogen, Shanghai, China). RNA concentrations were determined spectrophotometrically, and 1 μg RNA from each sample was reverse transcribed using the First Strand Synthesis Kit (Abgene, Shanghai, China) according to the manufacturer’s instructions. Quantitative-PCR (q-PCR) primer pairs were designed using the software of Primer Premier 5.0 (PREMIER Biosoft International, Palo Alto, Calif.). The specificity of the primers was confirmed by a Basic Local Alignment Search Tool search. The oligonucleotide primers used for the amplification of human bFGF cDNA were 5′-ACAGCAGCAGCCTAGCAACTC-3′ (sense) and 5′-CGGTTCGAGAAGTTTTTGAAGAG-3′ (antisense). The resultant
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PCR product was 123 bp. The sequences for TGF-β1 were 5′-CAACACATCAGAGCTCCGAGAA-3′ (sense) and 5′AAGGCGAAAGCCCTCAATTT-3′ (antisense), and the target fragment was 112 bp. The sequences of β-actin were 5′-ACGTCTGCTGGAAGGTGGAC-3′ (sense) and 5′GGTACCACCATGTACCCAGG-3′ (antisense) and gave an 89 bp amplimer. Quantitative PCR was carried out on an ABI Prism 7900 HT device (Applied Biosystems, Foster City, Calif.) using the SYBR Green PCR Master Mix (Applied Biosystems) and specific oligonucleotide primers at a concentration of 300 nmol/L. It was performed at 95 °C for 10 minutes, followed by 40 cycles of 95 °C for 15 seconds, and 60 °C for 1 minute. The relative quantification was calculated using a β-actin housekeeping gene as a normalizer. The reactions were conducted in triplicate to minimize the error. The difference between the cycle time (Ct) of the gene of interest and that of the normalizer gene was then calculated: Ct = Ct value of gene of interest – Ct value of normalizer. Relative mRNA expression = 2 Ct.8 The specificity of PCR amplifications from the different sets of oligonucleotide primers was examined routinely by agarose gel electrophoresis. Western blotting
Total cell lysates were isolated from lens epithelial cells attached to the anterior capsules by using RIPA buffer (50 mmol/L Tris–HCl, 1% NP-40, 0.25% Na-deoxycholate, 150 mmol/L NaCl, 1 mmol/L Na3VO4, and NaF) containing protease inhibitors (1 μg/mL each of aprotinin, leupeptin, pepstatin, EDTA, phenyl methyl sulfonyl fluoride). Lysates were centrifuged at 12 000g for 15 minutes at 4 °C. Total protein was determined by the bicinchoninic acid protein assay (Pierce, Rockford, Ill.), and proteins with equal concentration were subjected to SDS-PAGE. Resolved proteins were transferred to a nitrocellulose membrane and were probed with mouse anti-human TGF-β1, bFGF, and β-actin antibodies (all from Santa Cruz Biotechnology Inc, Santa Cruz, Calif.) followed by incubation with secondary antibody conjugated with horseradish peroxide. The enhanced chemiluminescence reaction system was used to visualize the bands. Statistical analysis
Statistical analysis was carried out using an unpaired Student’s t test. Values were expressed as means ± SEs. Statistical significance was defined as p < 0.05. A Pearson correlation was analyzed between the accumulation of TGF-β1 mRNA and bFGF mRNA. RESULTS The accumulation of TGF-b1 and bFGF mRNA increases in lens epithelial cells with ASCC
The results demonstrated that the accumulation of TGF-β1 mRNA was 32.89 ± 4.86 and 32.25 ± 4.51 in male and female lens epithelial cells of ASCC, which was
TGF- b1 and bFGF in congenital cataract—Xiao et al. significantly greater than in controls (10.3 ± 3.62 and 8.00 ± 3.31; Figs. 1A and 1B). A similar profile was obtained from the accumulation of bFGF mRNA, which was 7.10 ± 1.13 and 7.68 ± 1.04 in male and female lens epithelial cells of ASCC, respectively, significantly higher than the 3.55 ± 0.60 and 3.50 ± 0.96 in controls (Figs. 1C and 1D). A housekeeping gene of β-actin was employed as a normalizer in the study. Furthermore, the overall accumulation of bFGF mRNA and TGF-β1 mRNA in the 2 groups combined was positively correlated (r = 0.7985, p < 0.01). The accumulation of TGF-b1 and bFGF proteins increases in lens epithelial cells with ASCC
To confirm the accumulation of corresponding proteins, cell lysates were extracted from the lens epithelial cells attached to the anterior capsules, and Western blot analysis was performed. A complete epithelial specimen was used for each protein preparation. As shown in Fig. 2, the result was consistent with the RT-PCR data and demonstrated an increased accumulation of TGF-β1 protein in lens epithelial cells with ASCC as compared with non-cataractous controls. Also in correspondence with gene accumulation, bFGF protein level was increased in lens epithelial cells with ASCC as compared with controls.
phenotypic markers, including E-cadherin and connexin 43; they multilayer, and subsequently differentiate into myofibroblastic and (or) fiber-like cells, accompanied by accumulation of extracellular matrix.11 The myofibroblastic cells express α-smooth muscle actin, which is normally absent in the lens. Basic fibroblast growth factor has a function in promoting the fibrogenesis of lens epithelial cells. Our study demonstrates that the accumulation of TGF-β1 mRNA and bFGF mRNA was significantly higher than in controls in both male and female subjects, and the accumulation had a positive correlation. In correspondence with the RT-PCR data, our Western blotting data show that there was an increased accumulation of TGF-β1 and bFGF protein level. As the accumulation patterns of TGF-β1 and bFGF mRNA were similar, we presented our Western blotting data for males and females together. Several studies show that TGF-β and bFGF have either an additive or a synergistic effect on the formation of cataract. Cerra et al.1
CONCLUSIONS
TGF-β plays an important role in tissue repair and fibrogenesis. TGF-β induces the aberrant changes in lens cells that mimic events in the development of human subcapsular cataract and posterior capsule opacification.2,3,5,9,10 In TGFβ-induced subcapsular plaques, lens epithelial cells lose key
Fig. 1—The accumulation of TGF-β1 and bFGF mRNA is significantly elevated in lens epithelial cells of anterior subcapsular congenital cataracts. (A) The accumulation of TGF-β1 mRNA in male subjects. (B) The accumulation of TGF-β1 mRNA in female subjects. (C) The accumulation of bFGF mRNA in male subjects. (D) The accumulation of bFGF mRNA in female subjects. (TGF-β1, transforming growth factor β1; bFGF, basic fibroblast growth factor.)
Fig. 2—The accumulation of TGF-β1 and bFGF proteins is greatly increased in lens epithelial cells of anterior subcapsular congenital cataracts. (A) The accumulation of TGF-β1 and bFGF was assessed by Western blotting.The lens epithelial cell lysates were subjected to SDS-PAGE followed by blotting on a nitrocellulose membrane. Positive bands were visualised by an ECL detection system. (B) The band intensity was quantified by laser densitometry, and the amount of TGF-β1/bFGF was normalized against the amount of β-actin present. (TGF-β1, transforming growth factor β1; bFGF, basic fibroblast growth factor.) CAN J OPHTHALMOL—VOL. 44, NO. 2, 2009
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TGF- b1 and bFGF in congenital cataract—Xiao et al. demonstrated that supplementing TGF-β at a barely cataractogenic dose with bFGF in cultured rat lenses resulted in a very strong opacification response. In another study they showed that the survival of TGF-β-treated rat lens epithelial cells was promoted by bFGF but not by epidermal growth factor, platelet-derived growth factor, insulin-like growth factor, or hepatocyte growth factor, and in the absence of bFGF all cells were lost from cultured explants within 5 days.12 Moreover, standard explants from 10-day-old rats responded to TGF-β only in the presence of FGF,9 indicating that FGF may be more important in the development of subcapsular opacity in the prenatal or postnatal lens than the adult lens. We propose that in congenital cataract, TGF-β may initially induce transdifferentiation of lens epithelial cells, and those TGF-β-affected cells are further sustained by bFGF. In accordance with other studies,1,9,12 our results implicate TGF-β and bFGF in the formation of ASCC and suggest that TGF-β and bFGF may have either an additive or a synergistic effect on this formation. Our results might also indicate that high levels of TGF-β and bFGF are a consequence of cataract. The aetiology of congenital cataract has not been fully elucidated, and the different types of congenital cataract may have different causes. Many other factors may also contribute to the development of this disease. Obtaining a better understanding of the molecular aspects of the formation of congenital cataract will aid the development of strategies for combating this disease. This study was supported by the Foundation of Shandong Sciences and Technology, China. The authors thank Dr. James Heath for his critical reading. The authors have no proprietary or commercial interest in any materials discussed in this article.
REFERENCES 1. Cerra A, Mansfield KJ, Chamberlain CG. Exacerbation of TGFbeta-induced cataract by FGF-2 in cultured rat lenses. Mol Vis 2003;9:689–700.
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2. Gordon-Thomson C, de Iongh RU, Hales AM, Chamberlain CG, McAvoy JW. Differential cataractogenic potency of TGF-beta1, -beta2, and -beta3 and their expression in the postnatal rat eye. Invest Ophthalmol Vis Sci 1998;39:1399–409. 3. Hales AM, Chamberlain CG, Dreher B, McAvoy JW. Intravitreal injection of TGF beta induces cataract in rats. Invest Ophthalmol Vis Sci 1999;40:3231–6. 4. Symonds JG, Lovicu FJ, Chamberlain CG. Posterior capsule opacification-like changes in rat lens explants cultured with TGF beta and FGF: effects of cell coverage and regional differences. Exp Eye Res 2006;82:693–9. 5. Hales AM, Chamberlain CG, McAvoy JW. Cataract induction in lenses cultured with transforming growth factor-beta. Invest Ophthalmol Vis Sci 1995;36:1709–13. 6. McAvoy JW, Chamberlain CG. Fibroblast growth factor (FGF) induces different responses in lens epithelial cells depending on its concentration. Development 1989;107:221–8. 7. Lee EH, Joo CK. Role of transforming growth factor-beta in transdifferentiation and fibrosis of lens epithelial cells. Invest Ophthalmol Vis Sci 1999;40:2025–32. 8. Rasmussen R. Quantification on the lightcycler real-time PCR. In Meuer S, Witter C, Nakagawara K, eds. Rapid Cycle RealTime PCR, Methods and Applications. Springer Press, Heidelberg; 2001:21–30. 9. 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. 10. Srinivasan Y, Lovicu FJ, Overbeek PA. Lens-specific expression of transforming growth factor beta1 in transgenic mice causes anterior subcapsular cataracts. J Clin Invest 1998;101:625–34. 11. Lovicu FJ, Ang S, Chorazyczewska M, McAvoy JW. Deregulation of lens epithelial cell proliferation and differentiation during the development of TGF beta-induced anterior subcapsular cataract. Dev Neurosci 2004;26:446–55. 12. Mansfield KJ, Cerra A, Chamberlain CG. FGF-2 counteracts loss of TGF beta affected cells from rat lens explants: implications for PCO (after cataract). Mol Vis 2004;10:521–32. Keywords: congenital cataract, lens epithelial cells, transforming growth factor-β1, basic fibroblast growth factor
N
ormal lens development and maintenance is dependent on the tight spatial and temporal regulation of lens cell proliferation and fiber cell differentiation, in which growth factors play a pivotal role. Imbalance in this process may result in the formation of cataract, and studies using rats or rat lens explants show that ectopic addition of transforming growth factor (TGF)-β and basic fibroblast growth factor (bFGF) induces cataractous change.1–4 Whole rat lenses cultured with TGF-β develop anterior opacities corresponding to fibrotic plaques that are analogous to human anterior subcapsular cataract, and these abnormal changes can be blocked by a pan-specific antibody against TGF-β.2,5
In vitro, bFGF affects lens epithelial cell survival, migration, and differentiation in a dose-dependent manner in humans and mammals, and involves the formation of posterior capsule opacification.1,6 Congenital cataract is an opacity in the lens of the eye that presents at, or develops shortly after, birth. Approximately 0.03% of newborns have some form of congenital cataract. Congenital cataract may be associated with hereditary disorders, metabolic diseases, and also infections that the mother had during pregnancy. However, the exact mechanism of the development of this disease has not been fully elucidated. There are different types of congenital cataract,
From *the Department of Ophthalmology, Second Hospital of Shandong University, and †the Department of Ophthalmology, Shandong Provincial Hospital, Jinan, China
Correspondence to Bojun Zhao, MD, Department of Ophthalmology, Shandong Provincial Hospital, Jinan, China; [email protected]
Originally received Apr. 5, 2008. Revised July 28, 2008 Accepted for publication Aug. 8, 2008 Published online Feb. 27, 2009
This article has been peer-reviewed. Cet article a été évalué par les pairs. Can J Ophthalmol 2009;44:189–92 doi:10.3129/i09-006 CAN J OPHTHALMOL—VOL. 44, NO. 2, 2009
189
TGF- b1 and bFGF in congenital cataract—Xiao et al. such as nuclear congenital cataract, cortical congenital cataract, and subcapsular congenital cataract. Significant vision impairment owing to congenital cataract can lead to permanent amblyopia. Treatment of congenital cataract remains a challenge. Therefore, the further elucidation of the mechanism of this disease is a prerequisite for its prevention. Overaccumulation of TGF-β mRNA was reported in lens epithelial cells of adult anterior polar cataract.7 In this study, we investigated whether there is an elevation in the accumulation of TGF-β1 and bFGF in lens epithelial cells of anterior subcapsular congenital cataracts (ASCC). The results demonstrate that the accumulation of TGF-β1 and bFGF was increased at both gene and protein levels and that the accumulation of TGF-β1 mRNA and bFGF mRNA were positively correlated. METHODS Sample collection
The study was approved by the Ethical Review Board of Shandong University. Capsulotomy specimens with ASCC were obtained from patients who had undergone extracapsular cataract extraction. Anterior epithelium capsules from transparent lenses obtained from eyeballs enucleated because of trauma or retinoblastoma were used as controls. Informed consent was obtained in accordance with the Declaration of Helsinki before samples were taken for analysis. For reverse transcription-real time polymerase chain reaction (RT-PCR), 12 samples from males and 8 samples from females with ASCC were used with 6 male and 3 female samples as controls. Western blotting was performed using 6 samples with ASCC and 5 samples as control. Patients with other eye diseases were excluded from the study. The ages of patients with ASCC ranged from 3 months to 9 years. The ages of controls were from 1 to 10 years. Circular pieces of the anterior capsules with lens epithelial cells attached were put into RNase-free EP tubes after capsulotomy and stored at –80 °C immediately. RNA extraction, reverse transcription, and real-time quantitative PCR
Total cellular RNA was isolated from lens epithelial cells attached to the anterior capsules using TRIzol (Invitrogen, Shanghai, China). RNA concentrations were determined spectrophotometrically, and 1 μg RNA from each sample was reverse transcribed using the First Strand Synthesis Kit (Abgene, Shanghai, China) according to the manufacturer’s instructions. Quantitative-PCR (q-PCR) primer pairs were designed using the software of Primer Premier 5.0 (PREMIER Biosoft International, Palo Alto, Calif.). The specificity of the primers was confirmed by a Basic Local Alignment Search Tool search. The oligonucleotide primers used for the amplification of human bFGF cDNA were 5′-ACAGCAGCAGCCTAGCAACTC-3′ (sense) and 5′-CGGTTCGAGAAGTTTTTGAAGAG-3′ (antisense). The resultant
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PCR product was 123 bp. The sequences for TGF-β1 were 5′-CAACACATCAGAGCTCCGAGAA-3′ (sense) and 5′AAGGCGAAAGCCCTCAATTT-3′ (antisense), and the target fragment was 112 bp. The sequences of β-actin were 5′-ACGTCTGCTGGAAGGTGGAC-3′ (sense) and 5′GGTACCACCATGTACCCAGG-3′ (antisense) and gave an 89 bp amplimer. Quantitative PCR was carried out on an ABI Prism 7900 HT device (Applied Biosystems, Foster City, Calif.) using the SYBR Green PCR Master Mix (Applied Biosystems) and specific oligonucleotide primers at a concentration of 300 nmol/L. It was performed at 95 °C for 10 minutes, followed by 40 cycles of 95 °C for 15 seconds, and 60 °C for 1 minute. The relative quantification was calculated using a β-actin housekeeping gene as a normalizer. The reactions were conducted in triplicate to minimize the error. The difference between the cycle time (Ct) of the gene of interest and that of the normalizer gene was then calculated: Ct = Ct value of gene of interest – Ct value of normalizer. Relative mRNA expression = 2 Ct.8 The specificity of PCR amplifications from the different sets of oligonucleotide primers was examined routinely by agarose gel electrophoresis. Western blotting
Total cell lysates were isolated from lens epithelial cells attached to the anterior capsules by using RIPA buffer (50 mmol/L Tris–HCl, 1% NP-40, 0.25% Na-deoxycholate, 150 mmol/L NaCl, 1 mmol/L Na3VO4, and NaF) containing protease inhibitors (1 μg/mL each of aprotinin, leupeptin, pepstatin, EDTA, phenyl methyl sulfonyl fluoride). Lysates were centrifuged at 12 000g for 15 minutes at 4 °C. Total protein was determined by the bicinchoninic acid protein assay (Pierce, Rockford, Ill.), and proteins with equal concentration were subjected to SDS-PAGE. Resolved proteins were transferred to a nitrocellulose membrane and were probed with mouse anti-human TGF-β1, bFGF, and β-actin antibodies (all from Santa Cruz Biotechnology Inc, Santa Cruz, Calif.) followed by incubation with secondary antibody conjugated with horseradish peroxide. The enhanced chemiluminescence reaction system was used to visualize the bands. Statistical analysis
Statistical analysis was carried out using an unpaired Student’s t test. Values were expressed as means ± SEs. Statistical significance was defined as p < 0.05. A Pearson correlation was analyzed between the accumulation of TGF-β1 mRNA and bFGF mRNA. RESULTS The accumulation of TGF-b1 and bFGF mRNA increases in lens epithelial cells with ASCC
The results demonstrated that the accumulation of TGF-β1 mRNA was 32.89 ± 4.86 and 32.25 ± 4.51 in male and female lens epithelial cells of ASCC, which was
TGF- b1 and bFGF in congenital cataract—Xiao et al. significantly greater than in controls (10.3 ± 3.62 and 8.00 ± 3.31; Figs. 1A and 1B). A similar profile was obtained from the accumulation of bFGF mRNA, which was 7.10 ± 1.13 and 7.68 ± 1.04 in male and female lens epithelial cells of ASCC, respectively, significantly higher than the 3.55 ± 0.60 and 3.50 ± 0.96 in controls (Figs. 1C and 1D). A housekeeping gene of β-actin was employed as a normalizer in the study. Furthermore, the overall accumulation of bFGF mRNA and TGF-β1 mRNA in the 2 groups combined was positively correlated (r = 0.7985, p < 0.01). The accumulation of TGF-b1 and bFGF proteins increases in lens epithelial cells with ASCC
To confirm the accumulation of corresponding proteins, cell lysates were extracted from the lens epithelial cells attached to the anterior capsules, and Western blot analysis was performed. A complete epithelial specimen was used for each protein preparation. As shown in Fig. 2, the result was consistent with the RT-PCR data and demonstrated an increased accumulation of TGF-β1 protein in lens epithelial cells with ASCC as compared with non-cataractous controls. Also in correspondence with gene accumulation, bFGF protein level was increased in lens epithelial cells with ASCC as compared with controls.
phenotypic markers, including E-cadherin and connexin 43; they multilayer, and subsequently differentiate into myofibroblastic and (or) fiber-like cells, accompanied by accumulation of extracellular matrix.11 The myofibroblastic cells express α-smooth muscle actin, which is normally absent in the lens. Basic fibroblast growth factor has a function in promoting the fibrogenesis of lens epithelial cells. Our study demonstrates that the accumulation of TGF-β1 mRNA and bFGF mRNA was significantly higher than in controls in both male and female subjects, and the accumulation had a positive correlation. In correspondence with the RT-PCR data, our Western blotting data show that there was an increased accumulation of TGF-β1 and bFGF protein level. As the accumulation patterns of TGF-β1 and bFGF mRNA were similar, we presented our Western blotting data for males and females together. Several studies show that TGF-β and bFGF have either an additive or a synergistic effect on the formation of cataract. Cerra et al.1
CONCLUSIONS
TGF-β plays an important role in tissue repair and fibrogenesis. TGF-β induces the aberrant changes in lens cells that mimic events in the development of human subcapsular cataract and posterior capsule opacification.2,3,5,9,10 In TGFβ-induced subcapsular plaques, lens epithelial cells lose key
Fig. 1—The accumulation of TGF-β1 and bFGF mRNA is significantly elevated in lens epithelial cells of anterior subcapsular congenital cataracts. (A) The accumulation of TGF-β1 mRNA in male subjects. (B) The accumulation of TGF-β1 mRNA in female subjects. (C) The accumulation of bFGF mRNA in male subjects. (D) The accumulation of bFGF mRNA in female subjects. (TGF-β1, transforming growth factor β1; bFGF, basic fibroblast growth factor.)
Fig. 2—The accumulation of TGF-β1 and bFGF proteins is greatly increased in lens epithelial cells of anterior subcapsular congenital cataracts. (A) The accumulation of TGF-β1 and bFGF was assessed by Western blotting.The lens epithelial cell lysates were subjected to SDS-PAGE followed by blotting on a nitrocellulose membrane. Positive bands were visualised by an ECL detection system. (B) The band intensity was quantified by laser densitometry, and the amount of TGF-β1/bFGF was normalized against the amount of β-actin present. (TGF-β1, transforming growth factor β1; bFGF, basic fibroblast growth factor.) CAN J OPHTHALMOL—VOL. 44, NO. 2, 2009
191
TGF- b1 and bFGF in congenital cataract—Xiao et al. demonstrated that supplementing TGF-β at a barely cataractogenic dose with bFGF in cultured rat lenses resulted in a very strong opacification response. In another study they showed that the survival of TGF-β-treated rat lens epithelial cells was promoted by bFGF but not by epidermal growth factor, platelet-derived growth factor, insulin-like growth factor, or hepatocyte growth factor, and in the absence of bFGF all cells were lost from cultured explants within 5 days.12 Moreover, standard explants from 10-day-old rats responded to TGF-β only in the presence of FGF,9 indicating that FGF may be more important in the development of subcapsular opacity in the prenatal or postnatal lens than the adult lens. We propose that in congenital cataract, TGF-β may initially induce transdifferentiation of lens epithelial cells, and those TGF-β-affected cells are further sustained by bFGF. In accordance with other studies,1,9,12 our results implicate TGF-β and bFGF in the formation of ASCC and suggest that TGF-β and bFGF may have either an additive or a synergistic effect on this formation. Our results might also indicate that high levels of TGF-β and bFGF are a consequence of cataract. The aetiology of congenital cataract has not been fully elucidated, and the different types of congenital cataract may have different causes. Many other factors may also contribute to the development of this disease. Obtaining a better understanding of the molecular aspects of the formation of congenital cataract will aid the development of strategies for combating this disease. This study was supported by the Foundation of Shandong Sciences and Technology, China. The authors thank Dr. James Heath for his critical reading. The authors have no proprietary or commercial interest in any materials discussed in this article.
REFERENCES 1. Cerra A, Mansfield KJ, Chamberlain CG. Exacerbation of TGFbeta-induced cataract by FGF-2 in cultured rat lenses. Mol Vis 2003;9:689–700.
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2. Gordon-Thomson C, de Iongh RU, Hales AM, Chamberlain CG, McAvoy JW. Differential cataractogenic potency of TGF-beta1, -beta2, and -beta3 and their expression in the postnatal rat eye. Invest Ophthalmol Vis Sci 1998;39:1399–409. 3. Hales AM, Chamberlain CG, Dreher B, McAvoy JW. Intravitreal injection of TGF beta induces cataract in rats. Invest Ophthalmol Vis Sci 1999;40:3231–6. 4. Symonds JG, Lovicu FJ, Chamberlain CG. Posterior capsule opacification-like changes in rat lens explants cultured with TGF beta and FGF: effects of cell coverage and regional differences. Exp Eye Res 2006;82:693–9. 5. Hales AM, Chamberlain CG, McAvoy JW. Cataract induction in lenses cultured with transforming growth factor-beta. Invest Ophthalmol Vis Sci 1995;36:1709–13. 6. McAvoy JW, Chamberlain CG. Fibroblast growth factor (FGF) induces different responses in lens epithelial cells depending on its concentration. Development 1989;107:221–8. 7. Lee EH, Joo CK. Role of transforming growth factor-beta in transdifferentiation and fibrosis of lens epithelial cells. Invest Ophthalmol Vis Sci 1999;40:2025–32. 8. Rasmussen R. Quantification on the lightcycler real-time PCR. In Meuer S, Witter C, Nakagawara K, eds. Rapid Cycle RealTime PCR, Methods and Applications. Springer Press, Heidelberg; 2001:21–30. 9. 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. 10. Srinivasan Y, Lovicu FJ, Overbeek PA. Lens-specific expression of transforming growth factor beta1 in transgenic mice causes anterior subcapsular cataracts. J Clin Invest 1998;101:625–34. 11. Lovicu FJ, Ang S, Chorazyczewska M, McAvoy JW. Deregulation of lens epithelial cell proliferation and differentiation during the development of TGF beta-induced anterior subcapsular cataract. Dev Neurosci 2004;26:446–55. 12. Mansfield KJ, Cerra A, Chamberlain CG. FGF-2 counteracts loss of TGF beta affected cells from rat lens explants: implications for PCO (after cataract). Mol Vis 2004;10:521–32. Keywords: congenital cataract, lens epithelial cells, transforming growth factor-β1, basic fibroblast growth factor