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From the Departments of Ophthalmology*
and
Pathology
and the Graduate Institute
of Clinical Medicine,
National Taiwan
University Hospital, Taipei, Taiwan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Two recent studies showed that transformed limbal epithelium tissue expressing vimentin and fibroblasts with transformed cell characteristics are responsible for the formation of the corneal part in a pterygium.9,10 But these two studies emphasized the importance of the corneal part in the formation of a pterygium and provided little information on the pinguecular part. Accumulation of elastin in solar elastosis in photodamaged skin has been demonstrated in transgenic mice, by both immunohistochemistry and molecular biology techniques.11-13 It is likely that the pathological changes in conjunctiva in response to chronic UV irradiation are similar to those in chronically sun-damaged skin. For example, elastodysplasia and elastodystrophy are known to be present in the subepithelial connective tissue of the pinguecular part in a pterygium, and a similar pathological elastin accumulation is found in chronically sun-damaged skin.14 In the present study, we compared the expression pattern of elastin in the pinguecular part of a pterygium and in UV-damaged conjunctival tissue to determine whether ultraviolet B (UVB) irradiation is associated with the accumulation of elastin in the pinguecular part. Our results indicated that a UV-induced pterygium is characterized by modification of elastin gene expression at the posttranscriptional level.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Surgical specimens of pterygia and normal conjunctival specimens from age-matched cataract patients were obtained from the Department of Ophthalmology, National Taiwan University Hospital, Taipei, Taiwan, according to the tenets of the Declaration of Helsinki. Specimens from the patients, which included seven women and 10 men treated for pterygia and two women and three men treated for cataracts, were used for immunohistochemical stain. Written informed consent was obtained from all patients.
The pinguecular parts of pterygial specimens were aligned parallel to the axis of pterygial movement and the sections were taken perpendicular to this axis, fixed in 3.7% formaldehyde in 0.1 mol/L of phosphate-buffered saline (pH 7.4), and embedded in paraffin after graded dehydration. After deparaffinization and rehydration, serial tissue sections (4-µm thick) were used for immunohistochemical stains and incubated overnight with anti-elastin antibody (1:4,000 dilution; Elastin Products, Ovensville, MO) at 4°C as previously described.15 The immunoreaction was detected with avidin-biotin-horseradish peroxidase complex (Vector Laboratories, Burlingame, CA), using 4-diamethylaminoazobenzene as a chromogen. Nonimmune mouse IgG was used as the negative control. Routine hematoxylin and eosin (H&E), Masson’s trichrome, and Verhoeff’s elastic stains were also performed for studies of subepithelial connective tissue.
Cell Cultures
Because of the limited amount of tissue available for further studies, we used explant cultures, established from normal conjunctival and pterygial tissue samples obtained from surgical specimens from age-matched patients for the following experiments. The subepithelial connective tissue in the pinguecular part of the pterygium was dissected out and used for the following studies. Twenty specimens used for explant culture were different from those used for immunohistochemistry. Five strains of conjunctival fibroblasts, five strains of normal conjunctival fibroblasts with 3-mJ/cm2 UV irradiation, five strains with 6-mJ/cm2 UV irradiation, and five strains of pterygial fibroblasts were used for protein analysis. Three strains from each group were also used for in vitro translation. From these strains, we chose randomly three of five normal conjunctival fibroblasts, two of five pterygial fibroblasts, and another two strains of normal conjunctival fibroblasts with UV irradiation for the Northern hybridization. Furthermore, four strains of control fibroblasts, four strains with 3 mJ/cm2, four strains with 6 mJ/cm2, and five strains of pterygial fibroblasts were used for reverse transcriptase-polymerase chain reaction (RT-PCR) and DNA sequencing. The epithelial layer and subepithelial connective tissue layer of pinguecular tissue from pterygia and normal conjunctiva were dissected under a surgical microscope. Subepithelial connective tissues of the pinguecular part of pterygia and subconjunctival connective tissue of normal conjunctiva, were cut into 0.5 x 0.5-mm fragments, and then cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum and 2 mmol/L L-glutamine (all from Life Technologies, Inc., Grand Island, NY) at 37°C for 1 week. Fibroblasts that migrated outside the tissue fragments were subcultured in the same Dulbecco’s modified Eagle’s medium culture medium at 37°C. The early passages (passages 1 to 4) of pinguecular and conjunctival fibroblasts were used for the subsequent experiments. Contamination of epithelial cells was prevented by meticulous surgical excision and almost disappeared after the first passage because of their limited replicative capacity and culture medium used.
UV Source
UVB (312 nm) light was supplied by a Westinghouse Fs-40 sunlamp (Westinghouse Electric Corp., Pittsburgh, PA), which delivers uniform irradiation at a distance of 38 cm. The output of the Fs-40 sunlamp is 23.4 units UVB light/hour UVB at a distance of 38 cm, where each unit is equivalent to 21 mJ/cm2 of erythema-effective energy. For the following experiments, the total energy used ranged from 0 mJ/cm2 to 6 mJ/cm2. Cells were irradiated after immediately reaching confluence and the culture medium was replaced with phosphate-buffered saline (PBS). After irradiation, phosphate buffered saline was substituted with fresh culture medium.
RNA Extraction, Preparation of Elastin cDNA, and Northern Hybridization
Total RNA was isolated from cell cultures with an Ultraspec RNA isolation kit according to the manufacturer’s instructions (Biotecx Laboratories, Houston, TX).
The RT-PCR was performed according to a published method.16 A pair of 23-nucleotide residue primers (upstream primer, 5'-TTTCTCCCCGAGATGGCGGGTCT-3'; downstream primer, 5'-TCTCTTCCGGCCACAAGCTTTCC-3') were designed for RT-PCR amplification of the full-length coding sequence of elastin mRNA.13 After initial denaturation for 6 minutes at 94°C, 34 amplification cycles were performed as follows: first cycle at 92°C for 2 minutes, annealing at 60°C for 2 minutes, and extension at 72°C for 2 minutes. The PCR products were cloned into a TA-cloning vector (pGEM4Z-T, Promega, Madison, MI). The sequence of the cDNA insert was confirmed with an ABI PRISMTM dRhodamine Terminator Cycle Sequencing Ready Reaction kit and an ABI Prism 310 Genetic Analyzer (Perkin-Elmer-Cetus, Emeryville, CA) after purification of the insert through a spin column (Qiagen Inc., Valencia, CA).
32P-labeled probes were made by means of a random-priming labeling method (Boehringer Mannheim, Indianapolis, IN). Twenty micrograms of RNA sample was mixed with loading buffer and heated at 65°C for 10 minutes. The mixture was then chilled in an ice bath and electrophoresed though 1% agarose/formaldehyde (Amresco, Solon, OH) at 100 V. After electrophoresis, the gel was equilibrated twice in 20x standard saline citrate for 15 minutes. Capillary transfer of the RNA onto a nylon membrane (Amersham, Arlington Heights, IL) that had been prepared by washing in methanol and distilled water was performed at 4°C overnight or 4 hours at room temperature. The transferred RNA was cross-linked to the membrane in a UV cross-linking box. Glyceraldehyde-3-phosphate dehydrogenase cDNA contained in a plasmid (pHcGAP; American Type Culture Collection, Rockville, MD) served as an internal control.
Prehybridization was performed in prehybridization buffer (5x Denhardt’s solution, 5x sodium chloride/sodium phosphate/ethylenediaminetetraacetic acid, 50% formamide, 1% sodium dodecyl sulfate, 100 µg/ml tRNA) at 42°C for 90 minutes. Hybridization was performed at 42°C overnight. The membrane was washed twice with washing buffer 1 (2x standard saline citrate and 0.2% sodium dodecyl sulfate) at room temperature for 15 minutes and twice in washing buffer 2 (0.2x standard saline citrate and 0.5% sodium dodecyl sulfate) at 75°C for 30 minutes, and then blotted onto 3 mol/L paper. X-ray film (Amersham) was exposed to the membrane in a dark cassette at -70°C for 2.5 days. The quantities of RNA were determined with a FUJIX PhosphoImager scanner (FUJI Inc., Tokyo, Japan).
Quantification of Radiolabeled Tropoelastin
Radiolabeled tropoelastin was quantified according to the methods
described by Schwartz et al.17
Four days after
treatment with UV irradiation, the cell cultures were incubated with
fresh medium containing 5 µCi [14C]-proline
(Amersham) per 75-cm2
dish. After 24 hours,
protease inhibitors (phenylmethylsulfonyl fluoride, 2 mmol/L;
N-ethyl maleimide, 1 mmol/l; aprotinin, 1 µg/ml;
pepstatin, 1 µg/ml; and leupeptin, 1 µg/ml, final
concentration; all from Sigma Chemical Co., St. Louis, MO) were added
to the medium. The medium was decanted and stored at -20°C until
use. The cell layers were then rinsed with PBS containing proteolytic
inhibitors and extracted at 4°C overnight with PBS containing 0.05%
Tween-20 and protease inhibitors. The cell lysate was centrifuged at
12,000 x g for 5 minutes and the residue was extracted
overnight with 1% sodium dodecyl sulfate, 0.05 mol/l Tris-HCl (pH7.4),
0.33 mol/L mercaptoethanol, and protease inhibitors. An aliquot was
taken for the determination of proteins by the method of
Bradford.18
The accumulation of radiolabeled tropoelastin
was determined by the amount bound to an affinity column (Aminolink;
Pierce Chemical Co., Rockford, IL) prepared with IgG directed against
human 
elastin (Elastin Products).
Translation in Vitro
In vitro translation was performed by using a rabbit reticulocyte lysate (FlexiLysate, Promega) for 60 minutes at 30°C. The translation reaction (50 µl) contained 35 µl of reticulocyte lysate, 1 µl of 0.5 mmol/L amino acids (lacking methionine), 1 µl of 16.5 mmol/L of MgCl2, 2 µl of 1 mol/L KCl, 0.25 µl of ascorbic acid (5 mg/ml), 5 µCi of [14C]-proline (Amersham), 2 µl of translation cocktails, and 2 µg of transcribed RNA. After 60 minutes of translation, cycloheximide was added to 5 mmol/L to stop the reaction. The translated products were measure as above.
Detection of Mutations in the 3'-Untranslated Region (UTR) of Tropoelastin mRNA
Total RNA was extracted as described above from normal conjunctival fibroblasts, UV-irradiated conjunctival fibroblasts, and pinguecular fibroblasts from pterygial tissue. Twenty microliters of RT mixture containing 400 ng of total RNA was used for RT-PCR. The PCR reaction was performed with SuperTaq DNA polymerase as described above. Amplification of a 660-bp portion of the 3'-UTR of human tropoelastin mRNA was done using a pair of primers: upstream primer, 5'-CCCTGACTCACGACTCATCAACG-3'; downstream primer, 5'-AACTGGGACATGGATGGACGGACC-3'. PCR was performed in a Perkin-Elmer PCR thermocycler for 40 cycles at the following conditions: denaturation for 1 minute at 94°C, annealing at 63°C for 1 minute, and primer extension at 72°C for 2 minutes. The products were separated on 2% agarose gels and visualized by ethidium bromide staining. The PCR products were purified with a PCR purification kit (Qiagen), and the sequence of the 660-bp 3'-UTR was determined with an ABI Prism dRhodamine Terminator Cycle Sequencing Ready Reaction kit and ABI Prism 310 Genetic Analyzer (Perkin-Elmer-Cetus).
Statistical Analysis
Data from quantification of radiolabeled tropoelastin and Northern hybridization were analyzed by using analysis of variance and Student’s t-test. Means were considered to be significantly different from controls when P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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On H&E staining, the subepithelial connective tissues and vessels
of the pinguecular part in pterygia were more prominent than those of
normal conjunctiva tissue (Figure 1, A and B)
. An amorphous subepithelial superficial hyalinized zone and
coarse eosinophilic granular materials were observed in the pinguecular
parts of pterygia, but not in normal conjunctiva specimens (Figure 1, A and B)
. Coarse fibers were visible only in the deeper subepithelial
connective tissues of pterygial samples (Figure 1, A and B)
. On
Masson’s trichrome staining, the dense staining of collagen fibers was
also more prominent in the pinguecular part of pterygia than in the
subepithelial connective tissues of conjunctiva (Figure 1, C and D)
. On
Verhoeff’s elastic staining, the dark coarse elastic fibers were found
only in the subepithelial deep connective tissues of pinguecula in the
pterygia but not in the conjunctiva (Figure 1, E and F)
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, whereas the subepithelial connective tissues of
the normal conjunctival specimens from the five cataract patients did
not stain positively for elastin (Figure 1H)
. The staining of
elastin by anti-elastin monoclonal antibodies was localized in
massive quantities of thickened, tangled, coarse and tortuous fibers,
which is characteristic of elastic fibers. Northern Hybridization
The quantities of tropoelastin mRNA in cultured cells were
determined by Northern hybridization. Tropoelastin mRNA levels did not
increase in pinguecular or UV-treated conjunctival fibroblasts, as
compared with glyceraldehyde-3-phosphate dehydrogenase mRNA levels
(one-way analysis of variance, P > 0.05) (Figure 2, A and B)
. The amounts of mRNA in
steady-state and UV-treated conjunctival fibroblasts were also similar.
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When the UV irradiation dose increased to 6
mJ/cm2, a large quantity of radiolabeled
tropoelastin in the culture media of UV-treated conjunctival
fibroblasts was found to be gradually increased, as determined by the
use of affinity chromatography (n = 5;
Student’s t-test; P < 0.05) (Figure 3)
. The quantity did not increase at 3
mJ/cm2
of UVB irradiation
(n = 5; Student’s t-test;
P > 0.05). The quantity of tropoelastin in culture
medium of pinguecular fibroblasts was increased and had a quantity
similar to that of conjunctival fibroblasts treated with 6.0
mJ/cm2
UV irradiation (n =
5; Student’s t-test; P > 0.05). The levels
of tropoelastin in cellular extracts did not increase in any group
(n = 5; one-way analysis of variance;
P > 0.05).
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The amount of radiolabeled tropoelastin after in vitro
translation was consistent with the results of cellular cultures
(Figure 4)
. Levels of radiolabeled
tropoelastin in reticulolysates from cytoplasmic mRNA from conjunctival
fibroblasts were not different from fibroblasts irradiated with UVB at
3 mJ/cm2
(n = 3;
Student’s t-test; P > 0.05), but increased
after irradiation with UVB at 6 mJ/cm2
(n = 3; Student’s t-test;
P < 0.05 compared with normal conjunctival
fibroblasts). The levels of radiolabeled tropoelastin in
reticulolysates from cytoplasmic mRNA irradiated with UVB at 6
mJ/cm2
were less than those in pinguecular
fibroblasts (n = 3; Student’s
t-test; P < 0.05).
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We checked the mutations of 3'-UTR and coding sequences in five
strains of pinguecular fibroblasts, four strains of
3-mJ/cm2
UV-irradiated conjunctival fibroblasts,
four strains 6-mJ/cm2
UV-irradiated conjunctival
fibroblasts, and four stains of normal conjunctival fibroblasts (Table 1)
. There was no mutation in the 3'-UTR
of normal conjunctival fibroblasts. We found that there were 10
mutations in the 3-mJ/cm2
group, 20 mutations in
the 6-mJ/cm2
group, and 29 mutations in five
strains of pinguecular fibroblasts. There were on average 2.5, 5, and
5.8 mutations in the 3-mJ/cm2,
6-mJ/cm2, and pinguecular groups, respectively.
We also found that insertion occurred more frequently in pinguecular
fibroblasts. For example, in one strain of pinguecular fibroblasts,
there were two nucleotide insertions in the 3'- UTR of tropoelastin
mRNA: a C insertion after nucleotide 2409 and an A insertion after
nucleotide 2832. In another strain, we found the same C insertion after
nucleotide 2409 (Figure 5)
.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The mechanisms of the development of conjunctival lesions and their growth onto the cornea are unknown, but the relationship between chronic sun exposure and the formation of pterygia is well established.8,26,27 Chronic exposure to sunlight, especially UV irradiation, is considered to be the main cause of pterygia as well as solar elastosis. In the present study we used a model of solar elastosis to investigate the mechanism of abnormal elastin expression or elastoid degeneration in pterygia.
Electron microscopy studies have revealed elastogenesis in the substantia propria as well as in deeper episcleral and Tenon’s-level connective tissues in normal conjunctiva.14 Ultrastructural examination also disclosed evidence of elastogenesis in the pinguecular part of pterygia, but the morphogenetic sequence of fiber formation was distorted.14 Therefore, elastodysplasia (immature formation of elastic fibers) and elastodystrophy (degenerative changes of elastic fibers and formation of electron-dense inclusions) were found in the substantia propria.14 The amorphous materials in the pinguecular parts of pterygia are composed of excessive numbers of hollow-centered microfibrils, precursors of elastic fibers that tend to clump centrally in large aggregated sheets and to acquire electron-dense inclusions.14 In previous studies, these changes were considered to reflect an increased accumulation of elastic fibers in normal bulbar conjunctiva and were thought to result from degeneration of collagen fibers in response to sun exposure and subsequent insensitivity to elastase digestion.28-30 However, Austin et al14 reported that these abnormal elastic fibers exist in the substantia propria of the pinguecular parts in pterygia, and that pinguecular fibroblasts in pterygia might play a role in their formation. In the present study, we used immunohistochemical staining to confirm that these abnormal tortuous fibers are composed of elastin; these fibers are elastic and do not result from elastotic degeneration of collagen fibers. In contrast, there was no expression of elastin in the substantia propria of normal conjunctiva. The accumulation of abnormal elastic fibers was the same as that reported in solar elastosis of photodamaged skin.12,13 These findings confirm the appropriateness of our model of photodamaged or photoaged skin for studies of the pathological changes in the pinguecular part of a pterygium. An increase in elastin mRNA levels has been demonstrated in fibroblasts of photoaged skin, compared with those from healthy skin.31,32 This increased level of elastin mRNA in sun-damaged skin results from enhanced elastin promoter activity, as shown by transient transfections of fibroblasts with a DNA construct composed of the human elastin promoter linked to the chloramphenicol acetyl transferase reporter gene.31 This finding was further confirmed in transgenic mice carrying a human elastin promoter exposed to UV radiation in vivo and in vitro.11 However, the study of Schwartz et al17 indicated that the increased tropoelastin accumulation that results from UV radiation is because of a posttranscriptional mechanism rather than increased synthesis of mRNA. There are multiple base substitutions in the noncoding domain of tropoelastin mRNA that may be responsible for the posttranscriptional increase in tropoelastin accumulation after UV irradiation. In addition, other reports describing the posttranscriptional regulation of tropoelastin mRNA, as in cultured bovine chondrocytes treated with phorbol ester or vitamin D3, provide strong evidence to support posttranscriptional regulation of tropoelastin mRNA under specific conditions.33,34 In the present study the levels of elastin mRNA were similar in normal conjunctival fibroblasts, UV-irradiated conjunctival fibroblasts, and pinguecular fibroblasts of pterygia, whereas tropoelastin gene expression was enhanced in pinguecular and UV-irradiated conjunctival fibroblasts. These observations suggest that the increased elastin accumulation in pinguecular or UV-damaged conjunctival fibroblasts is not because of elevated steady-state levels of mRNA.
In the present study, UV-irradiation of conjunctival fibroblasts at 3 and 6 mJ/cm2 resulted in, on average, 2.5 and five mutations, respectively. There were on average 5.8 mutations in pinguecular fibroblasts of pterygia. The mutations increased with UV dosage and in pinguecular fibroblasts. Furthermore, we found that the insertion occurred more frequently either in pinguecular fibroblasts or after UV-irradiation than in control fibroblasts. For example, we found that there was a constant C insertion after nucleotide 2409 in two strains of pinguecular fibroblasts in pterygia and there was another A insertion after nucleotide 2832 in one strain of pinguecular fibroblasts in pterygia. The total base substitution rates in our study were <5%. The regulation of gene expression at the level of translation is important for the control of protein synthesis, and specific sequences in the 5'- and 3'-UTRs are known to control translation.35-38 Stable secondary structures (stem loops) in the 5'-UTR can interfere with the initiation of translation.39 The regulation of cytoplasmic polyadenylation in the 3'-UTR is controlled by two unique sequences, AAUAAA and the poly (U) tract.40 Mutations in these sequences can cause deadenylation and decay of mRNA. In addition, RNA-binding proteins can interact with specific sequences in the 3'-UTR and therefore regulate mRNA stability and translational efficiency.41 Our findings suggest that mutations in the 3'-UTR can alter the translational efficiency of tropoelastin mRNA in pterygial and UV-treated conjunctival fibroblasts. We also speculate that the number of mutations will affect the translational efficiency of tropoelastin mRNA. When the number of mutations increase, the secondary structure of 3'-UTR will be destabilized more heavily. Therefore, we found that the quantity of tropoelastin did not increase in the 3-mJ/cm2 group, but did increase in the 6-mJ/cm2 group and in pinguecular fibroblasts of pterygia.
Although UVB irradiation has been reported to specifically target G-C bp, resulting in G-C to A-T transversion mutations, we found that UVB irradiation resulted in both transition and transversion mutations.42 Furthermore, insertion of nucleotides was found only in pinguecular fibroblasts of pterygia, although there were no base substitutions in pinguecular fibroblasts of pterygia. Although the patterns of mutations in the 3'-UTR in our study were not completely consistent with those of previous reports of UVB-induced mutations, we could not find any mutation in the conjunctival fibroblasts and these mutations are still significant in the control of tropoelastin mRNA expression. These multiple mutations result from gap filling by an error-prone DNA polymerase activity during repair.43 One possible explanation for the lack of mutations in the coding region is that the structure of mRNA in the coding region is protected from UV radiation. Alternatively, another repair mechanism responsible for the coding region, such as the transcription-coupled DNA repair system, may be involved.43,44
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Footnotes |
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Supported in part by Grants NSC-86-2314-B-002-076, NSC-87-2314-B-002-071 and NSC-88-2314-B-002-380 from the National Science Council, Taiwan.
Accepted for publication June 30, 2000.
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