Differential expression and processing of transforming growth factor beta induced protein (TGFBIp) in the normal human cornea during postnatal development and aging.

Transforming growth factor beta induced protein (TGFBIp, also named keratoepithelin) is an extracellular matrix protein abundant in the cornea. The purpose of this study was to determine the expression and processing of TGFBIp in the normal human cornea during postnatal development and aging. TGFBIp in corneas from individuals ranging from six months to 86 years of age was detected and quantified by immunoblotting. The level of TGFBIp in the cornea increases about 30% between 6 and 14 years of age, and adult corneas contain 0.7-0.8 microg TGFBIp per mg wet tissue. Two-dimensional (2-D) immunoblots of the corneal extracts showed a characteristic "zig-zag" pattern formed by different lower-molecular mass TGFBIp isoforms (30-60 kDa). However, the relative abundance of the different isoforms was different between infant corneas (<1 year) and the child/adult corneas (>6 years). Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) data of TGFBIp isoforms separated on large 2-D gels show that TGFBIp is proteolytically processed from the N-terminus. This observation was supported by in silico 2-D gel electrophoresis showing that sequential proteolytical trimming events from the N-terminus of mature TGFBIp generate TGFBIp isoforms which form a similar "zig-zag" pattern when separated by 2-D polyacrylamide gel electrophoresis (PAGE). This study shows that in humans TGFBIp is more abundant in mature corneas than in the developing cornea and that the processing of TGFBIp changes during postnatal development of the cornea. In addition, TGFBIp appears to be degraded in a highly orchestrated manner in the normal human cornea with the resulting C-terminal fragments being retained in the cornea. The age-related changes in the expression and processing of corneal TGFBIp suggests that TGFBIp may play a role in the postnatal development and maturation of the cornea. Furthermore, these observations may be relevant to the age at which mutant TGFBIp deposits in the cornea in those dystrophies caused by mutations in the transforming growth factor beta induced gene (TGFBI) as well as the mechanisms of corneal protein deposition.

Evidence against a blood derived origin for transforming growth factor beta induced protein in corneal disorders caused by mutations in the TGFBI gene.

PURPOSE: Several inherited corneal disorders in humans result from mutations in the transforming growth factor beta induced gene (TGFBI), which encodes for the extracellular transforming growth factor beta induced protein (TGFBIp) that is one of the most abundant proteins in the cornea. We previously reported a significant amount of TGFBIp in plasma by immunoblotting using the only TGFBIp antiserum (anti-p68(beta ig-h3)) available at that time (anti-p68(beta ig-h3) was generated against residues Val210-His683 of TGFBIp). This observation raised the possibility that a fraction of corneal TGFBIp may originate from the plasma. However, recent experiments in our laboratory indicated that the anti-p68(beta ig-h3) antiserum cross-reacts with an environmental protein contaminant. Therefore, we investigated the specificity of the originally utilized anti-p68(beta ig-h3) antiserum and re-evaluated the amount of TGFBIp in human plasma by immunoblotting using a new specific antiserum. METHODS: The observed cross-reactivity of the previously utilized anti-p68(beta ig-h3) antiserum was tested by immunoblotting and the antigen identity was determined by mass spectrometry. A part of human TGFBI encoding an NH2-terminal 11.4 kDa fragment of TGFBIp (residues Gly134-Ile236) was amplified by polymerase chain reaction (PCR) and cloned in E. coli. The TGFBIp fragment was expressed in E. coli, purified by Ni2+-affinity chromatography, and used to immunize rabbits to produce a specific antiserum (anti-TGFBIp(134-236)). To enhance the detection of possible TGFBIp in plasma by allowing a higher sample load, albumin and immunoglobulin G (IgG) were specifically depleted from normal human plasma by affinity chromatography. The presence of TGFBIp in plasma was investigated by immunoblotting using the anti-TGFBIp(134-236) antiserum. Purified TGFBIp from porcine corneas was used for estimation of the TGFBIp detection limit. RESULTS: The previously utilized TGFBIp antiserum, anti-p68(beta ig-h3), cross-reacted with human keratin-1, a common environmental protein contaminant. Thus, the anti-p68(beta ig-h3) antiserum recognizes both TGFBIp and keratin-1. In contrast, the anti-TGFBIp(134-236) antiserum reacted with TGFBIp but showed no indication of reactivity with other proteins in plasma. Using this antiserum, TGFBIp was not detected in crude or albumin/IgG-depleted human plasma and the detection limit of TGFBIp using immunoblotting was estimated to be 10 ng. CONCLUSIONS: Our failure to detect TGFBIp in human plasma using a highly specific antiserum suggests that TGFBIp is not present in a physiologically relevant concentration in human plasma. The previous impression that normal human plasma contains a significant amount of TGFBIp by immunoblotting was due to the utilization of a less specific antiserum that recognizes both TGFBIp and human keratin-1. Together with other results, our observation makes it unlikely that TGFBIp is imported into the cornea from the circulation as reported for other abundant extracellular corneal proteins and suggests corneal origin of TGFBIp deposits in individuals with inherited corneal diseases caused by mutations in the TGFBI gene.

The human cornea proteome: bioinformatic analyses indicate import of plasma proteins into the cornea.

Increased biochemical knowledge of normal and diseased corneas is essential for the understanding of corneal homeostasis and pathophysiology. In a recent study, we characterized the proteome of the normal human cornea and identified 141 distinct proteins. This dataset represents the most comprehensive protein study of the cornea to date and provides a useful reference for further studies of normal and diseased human corneas. The list of identified proteins is available at the Cornea Protein Database. In the present paper, we review the utilized procedures for extraction and fractionation of corneal proteins and discuss the potential roles of the identified proteins in relation to homeostasis, diseases, and wound-healing of the cornea. In addition, we compare the list of identified proteins with high quality gene expression libraries (cDNA libraries) and Serial Analysis of Gene Expression (SAGE) data. Of the 141 proteins, 86 (61%) were recognized in cDNA libraries from the corneas of dogs and rabbits, or humans with keratoconus, and 98 (69.5%) were recognized in SAGE data of mouse and human corneas. However, the percentages of identified genes in each of the protein functional groups differed markedly. Thus, exceptionally few of the traditional blood/plasma proteins and immune defense proteins that were identified in the human cornea were recognized in the gene expression libraries of the cornea. This observation strongly indicates that these abundant corneal proteins are not expressed in the cornea but originate from the surrounding pericorneal tissue.

LASIK induces minimal regrowth and no haze development in rabbit corneas.

PURPOSE: To quantify central corneal regrowth and haze development after LASIK in rabbits. METHODS: New Zealand White rabbits received an 89 microm (-8 diopters) myopic LASIK and were evaluated during 4 months using slit-lamp and in vivo confocal microscopy to monitor changes in central corneal morphology, epithelial and stromal thickness, flap and bed thickness, and corneal light backscattering (haze). At various time-points, corneas were processed for histology. RESULTS: Using in vivo confocal microscopy, LASIK induced no detectable morphological changes besides a slightly elevated light backscattering at the interface. Correspondingly, all corneas remained clear with no haze development by slit-lamp biomicroscopy. Corneal thickness was stable by 8 weeks after an increase of 17 +/- 4 microm that consisted of a 13 +/- 3 microm stromal regrowth and a 4 +/- 2 microm epithelial hyperplasia. At the LASIK interface, less than 4 microm new extracellular matrix was deposited. Accordingly, all LASIK flaps were easily pulled off by 6 months. CONCLUSIONS: LASIK induces a minimal wound healing response in rabbit corneas with no haze development and a regrowth (regression) of only 17 microm of an 89-microm photoablation. Three main factors contributed to the observed regrowth: epithelial hyperplasia (approximately 4 microm), matrix deposition at the LASIK interface (approximately 4 microm), and stromal growth outside the interface within the flap and wound bed (approximately 9 microm).

Nordic research in ophthalmology.

Nordic ophthalmologists and vision scientists are active in many fields of eye research. This is most evident at the biannual Nordic Congress of Ophthalmology, most recently held in Malmö in June 2004. The authors here review some of the research in vision and ophthalmology presented at this meeting or published recently by Nordic scientists. This paper does not represent a comprehensive review of all Nordic research in the field, but attempts to give an overview of some of the activities underway in eye research in this part of the world.

A dataset of human cornea proteins identified by Peptide mass fingerprinting and tandem mass spectrometry.

Diseases of the cornea are extremely common and cause severe visual impairment worldwide. To explore the basic molecular mechanisms involved in corneal health and disease, the present study characterizes the proteome of the normal human cornea. All proteins were extracted from the central 7-mm region of 12 normal human donor corneas containing all layers: epithelium, Bowman's layer, stroma, Descemet's membrane, and endothelium. Proteins were fractionated and identified using two different procedures: (i) two-dimensional gel electrophoresis and protein identification by MALDI-MS and (ii) strong cation exchange or one-dimensional SDS gel electrophoresis followed by LC-MS/MS. All together, 141 distinct proteins were identified of which 99 had not previously been identified in any mammalian corneas by direct protein identification methods. The characterized proteins are involved in many processes including antiangiogenesis, antimicrobial defense, protection from and transport of heme and iron, tissue protection against UV radiation and oxidative stress, cell metabolism, and maintenance of intracellular and extracellular structures and stability. This proteome study of the healthy human cornea provides a basis for further analysis of corneal diseases and the design of bioengineered corneas.

Corneal power, thickness, and stiffness: results of a prospective randomized controlled trial of PRK and LASIK for myopia.

PURPOSE: To compare the short-, medium-, and long-term changes in corneal optical power and corneal aberrations, central corneal thickness, and corneal "stiffness" assessed by pneumotonometry readings in patients having laser in situ keratomileusis (LASIK) or photorefractive keratectomy (PRK) for myopia. SETTING: Department of Ophthalmology, Arhus University Hospital, Arhus, Denmark. METHODS: One eye of each of 45 patients with myopia ranging from -6.00 to -8.00 diopters (D) (spherical equivalent spectacle refraction [SER]) was randomized to LASIK (n=25; mean SER -7.12 D +/- 0.57 [SD]) or PRK (n=20; mean SER -6.91 +/- 0.57 D). Data were collected prospectively before and 1, 3, 6, 12, and 36 months after surgery. Measurements included corneal topography (TMS-1, Tomey), corneal thickness (ultrasound pachymetry), and apparent intraocular pressure (IOP) (pneumotonometry). Retreatments were not performed during the first year, and retreated eyes were excluded from the 3-year follow-up. Changes in corneal power and aberrations, thickness, and apparent IOP were calculated in a pair-wise manner for 3 time periods: short term (preoperative to 1 month after surgery), medium term (1 to 12 months after surgery), and long term (1 to 3 years after surgery). RESULTS: In the short term, corneal power decreased equally in LASIK and PRK eyes. Spherical aberrations and coma-like aberrations increased equally, while corneal thickness decreased significantly less in LASIK eyes than in PRK eyes. The apparent IOP decreased more in LASIK eyes than in PRK eyes. In the medium term, corneal power increased significantly in both groups. Spherical aberrations decreased significantly in PRK eyes but not in LASIK eyes. From 1 to 12 months, corneal thickness increased more in PRK eyes than in LASIK eyes. During this period, the apparent IOP increased significantly in LASIK eyes. In the long term, corneal power and corneal aberrations did not change significantly in either group. Corneal thickness increased slightly but significantly in both groups. The apparent IOP increased significantly more in PRK eyes. CONCLUSIONS: Differences between LASIK and PRK related to time-dependent events affecting corneal shape and structural integrity were present. Peripheral changes in flap hydration in LASIK eyes and epithelial and/or stromal thickening in PRK eyes appeared to be the most important factors in optical power changes in the first year after treatment. The changes in apparent IOP suggest that some interlamellar healing occurred during the first year after LASIK. After LASIK and PRK, corneal bending stiffness seemed permanently decreased, although some restiffening may occur in PRK eyes in the long term.

Purification and structural characterization of transforming growth factor beta induced protein (TGFBIp) from porcine and human corneas.

Mutations in the TGFBI (BIGH3) gene that encodes for transforming growth factor beta induced protein (TGFBIp) are the cause of several phenotypically different corneal dystrophies. While the genetics of these protein misfolding diseases are well documented, relatively little is known about this extracellular matrix protein itself. In this study, we have purified TGFBIp from normal human and porcine corneas using nondenaturing conditions and standard chromatography techniques. The two homologues were shown to be monomers, and we did not find evidence for posttranslational additions. The C-terminal of both human and porcine TGFBIp is truncated predominantly after the integrin binding sequence Arg(642)-Gly(643)-Asp(644) (RGD). However, using an antibody against the C-terminal fragment (residues 648-683), we also detected a small amount of full-length TGFBIp in corneal extracts. Approximately 60% of TGFBIp was covalently associated with insoluble components of the extracellular matrix in both human and porcine corneas through a disulfide bridge.

Role of keratocyte loss on corneal wound repair after LASIK.

PURPOSE: To investigate whether an initial keratocyte loss intensifies central corneal wound repair after LASIK in rabbits. METHODS: New Zealand White rabbits received either conventional LASIK (-8 D, 6-mm diameter) or LASIK combined with a 7-mm diameter, epithelial denudation (LASIK-scrape). Animals were examined during 4 months by slit lamp and in vivo confocal microscopy to monitor changes in central corneal morphology, light backscattering (haze), and sublayer thickness. At various time points, corneas were processed for histology and stained for nuclei; F-actin; ED-A fibronectin; alpha-smooth muscle actin; TGF-beta1, -beta2, and -beta receptor II; and connective tissue growth factor (CTGF). RESULTS: In vivo confocal microscopy identified no major acellular zones or changes in cell morphology or reflectivity after conventional LASIK. By contrast, a complete loss of keratocytes was observed in the anterior 77 +/- 25 microm stroma 1 week after LASIK-scrape. Highly reflective, migratory fibroblasts gradually repopulated the acellular zone, and by week 8, quiescent-appearing keratocytes were observed throughout the stroma. Correspondingly, stromal light backscattering peaked at 2 weeks after LASIK-scrape (2200 +/- 620 U) followed by a decline to approximately 60 U from week 8; comparable to the slightly increased reflectivity (approximately 50 U) observed after conventional LASIK (ns). Stromal thickness appeared stable 8 weeks after both LASIK and LASIK-scrape, after a regrowth of 13 +/- 3 and 20 +/- 11 microm, respectively (ns). In addition, both procedures induced a minor and comparable epithelial hyperplasia of 4 +/- 2 and 7 +/- 5 microm, respectively (ns). No myofibroblast transformation or TGF-beta growth factor expression was observed below the flap after either treatment. CONCLUSIONS: LASIK-scrape induces an anterior keratocyte loss, leading to development of temporary haze during cell repopulation. However, 8 weeks after both LASIK and LASIK-scrape, only a slightly increased reflectivity is noted at the interface. Corneal thickness is stable by week 8, and stromal regrowth and epithelial hyperplasia are comparable after both treatments. Thus, an initial loss of stromal keratocytes does not appear to intensify corneal wound repair after LASIK.

Keratocyte reflectivity and corneal haze.

Corneal transparency is a remarkable characteristic that is essential for vision. Biophysical models of corneal transparency have entirely focused on the stromal extracellular matrix and disruption of the regular array of collagen fibres as the main reason for corneal haziness. Therefore, disorder of corneal transparency has traditionally been explained by a combination of three main factors: (1) abnormal water content (i.e. swelling or edema); (2) abnormal collagen fibre diameter, spacing, and orientation (i.e. scar tissue or fibrosis); and (3) abnormal accumulation of macromolecules (proteins, glycosaminoglycans, lipids, etc.) as in many corneal dystrophies. Here, clinical and experimental data are provided to support the concept that corneal keratocytes, which are normally invisible and transmit light, may show intense light scattering in injured corneas. Thus, the existence of a fourth group of corneal transparency disorders is proposed that predominantly are associated with abnormal cellular-based reflections from multiple layers of stromal keratocytes. In this group of patients, the light scattering structures (keratocyte nuclei, cell-body, and cell-processes) cannot be discriminated using standard slit-lamp biomicroscopy but requires a confocal microscopic examination. Despite their importance, almost nothing is known about the physical basis for the invisibility and haziness of the keratocytes. A more comprehensive model to understand corneal transparency is needed and should include the interaction of visible light with the physical structure of the keratocyte and its subcellular constituents.

Proteomic analysis of the soluble fraction from human corneal fibroblasts with reference to ocular transparency.

The transparent corneal stroma contains a population of corneal fibroblasts termed keratocytes, which are interspersed between the collagen lamellae. Under normal conditions, the keratocytes are quiescent and transparent. However, after corneal injury the keratocytes become activated and transform into backscattering wound-healing fibroblasts resulting in corneal opacification. At present, the most popular hypothesis suggests that particular abundant water-soluble proteins called enzyme-crystallins are involved in maintaining corneal cellular transparency. Specifically, corneal haze development is thought to be related to low levels of cytoplasmic enzyme-crystallins in reflective corneal fibroblasts. To further investigate this hypothesis, we have used a proteomic approach to identify the most abundant water-soluble proteins in serum-cultured human corneal fibroblasts that represent an in vitro model of the reflective wound-healing keratocyte phenotype. Densitometry of one-dimensional gels revealed that no single protein isoform exceeded 5% of the total water-soluble protein fraction, which is the qualifying property of a corneal enzyme-crystallin according to the current definition. This result indicates that wound-healing corneal fibroblasts do not contain enzyme-crystallins. A total of 254 protein identifications from two-dimensional gels were performed representing 118 distinct proteins. Proteins protecting against oxidative stress and protein misfolding were prominent, suggesting that these processes may participate in the generation of cytoplasmic light-scattering from corneal fibroblasts.

Plastic particles at the LASIK interface.

PURPOSE: To investigate the origin, composition, and persistence of the interface particles that frequently are observed after LASIK. DESIGN: Small case series and experimental animal study. METHODS: Four patients received LASIK using a Schwind Supratome (Schwind, Kleinostheim, Germany) and a MEL 70 G-Scan excimer laser (Asclepion, Jena, Germany) and were examined over the course of 1 year using slit-lamp and in vivo confocal microscopy. Four rabbits received a monocular microkeratome incision and were examined immediately after surgery without lifting the flap. After monthly evaluation for 4 months using in vivo confocal microscopy, 2 corneas were processed for histologic analysis and were sectioned serially. To measure the iron content, atomic absorption spectrometry was performed on 2 operated and 2 unoperated rabbit corneas. The chemical composition of the metal and plastic parts of the microkeratome blade was identified using energy dispersive x-ray fluorescence (metal part), and Raman and infrared spectroscopy (plastic part). Before and after oscillation in air, the microkeratome blade and motor-head were examined using light and fluorescence microscopy. In serial sections, interface particles were identified by fluorescence microscopy and their chemical composition was determined using Coherent Antistokes Raman Scattering microscopy. RESULTS: In LASIK patients, thousands of brightly reflecting particles (up to 30 micro m) were observed throughout the interface. The highest particle density was detected where the microkeratome blade had first entered the cornea. Both in the center and at the flap edge, the morphologic features, distribution, and density of these particles remained unaltered throughout the 1-year observation period. In rabbit corneas, interface particles were observed immediately after the microkeratome incision, even though the flap had not been lifted. These particles were similar to those observed in humans and persisted unaltered throughout the study. The operated and unoperated rabbit corneas had comparable iron content, demonstrating that the particles were not fragments of the uncoated steel blade. Only a few particles were observed on the unused microkeratome motor head and blade, whereas numerous fluorescent particles were detected after oscillation in air, the amount of particles increasing with oscillation time. Interestingly, the only fluorescent part of the microkeratome was the plastic segment of the blade. This plastic (polyetherimide) emitted fluorescence identical to that of the observed particles, whereas all metal parts of the microkeratome blade and motor head were nonfluorescent. In serial sections, interface particles showed fluorescent properties equivalent to polyetherimide and exhibited molecular resonance at 1780 and 3100 cm(-1), in accordance with the Raman spectrum of polyetherimide. CONCLUSIONS: Numerous plastic particles are generated during microkeratome oscillation and are deposited at the interface during LASIK. The particles persist unaltered for at least 1 year.

Transforming growth factor beta induced protein accumulation in granular corneal dystrophy type III (Reis-Bücklers dystrophy). Identification by mass spectrometry in 15 year old two-dimensional protein gels.

PURPOSE: To investigate the accumulation of TGFBIp in GCDIII and to demonstrate the ability to perform mass spectrometry on old two-dimensional protein gels. METHODS: Proteins were extracted from one cornea with GCDIII from a person with an Arg124Leu mutation in the TGFBI (BIGH3) gene and from one normal human cornea in 1987 and subjected to two-dimensional (2D) gel electrophoresis. After keeping the gels at room temperature for 15 years, protein spots of interest were excised and digested with trypsin. The tryptic-derived peptides were analyzed using mass spectrometry. RESULTS: Four areas of interest were examined and four different proteins (TGFBIp, aldehyde dehydrogenase class 3, actin, and albumin) were identified in the 15-year-old gels. Using image analysis, the amount of TGFBIp was found to be 77 fold higher in the GCDIII affected cornea relative to the normal tissue. In both situations, TGFBIp migrated on the 2D gels as a 63 kDa protein. Mass spectrometry revealed the same nine peptides in TGFBIp from both the normal and the GCDIII affected corneas, including one peptide situated at the amino terminus. Moreover, the cornea with GCDIII contained abundant 40 kDa TGFBIp fragments that were lacking sequences in both the amino and carboxy termini. CONCLUSIONS: Mass spectrometry can be performed on old 2D polyacrylamide gels. In both normal and GCDIII affected corneas, the majority of TGFBIp migrated on 2D gels as a 63 kDa protein with an intact amino terminus. However, the amount of the 63 kDa TGFBIp was 77 fold higher in the GCDIII affected cornea. Furthermore, the GCDIII affected cornea contained abundant 40 kDa fragments that were trunctated in both the amino and carboxy termini.

Validation of confocal microscopy through focusing for corneal sublayer pachymetry.

PURPOSE: To evaluate the accuracy and precision of confocal microscopy through focusing (CMTF) for corneal sublayer pachymetry. METHODS: A tandem scanning confocal microscope equipped with a nonapplanating contact objective was used to perform CMTF. The accuracy of CMTF measurements was evaluated using nine custom-made calibration contact lenses (PMMA) with varying thickness (300-600 microm) and radius of curvature (7.0-9.0 mm). The influence of immersion fluid stabilization and the consequence of prolonged corneal examination were assessed by performing CMTF in rabbits. Additionally, factors related to the instrumental setup and to sedation of experimental animals were examined. RESULTS: For all calibration contact lenses, the thickness measured by CMTF was within +/-1.0 microm of the certified value. Varying the target thickness or radius of curvature had no consistent impact on the high accuracy of CMTF. When performing CMTF in vivo, z-axis motion was readily identified by sampling and comparing both in- and out-scans. Apart from involuntary eye movements, z-axis motion was due to initial thinning of the immersion fluid with stabilization obtained after approximately 1.5 minutes. Continued confocal examination led to slight but significant swelling of both the stroma (0.5 microm/min) and epithelium (0.1 microm/min). CONCLUSIONS: CMTF is an accurate and precise technique for corneal sublayer pachymetry with concurrent display of cellular morphology. The precision of CMTF can be improved by allowing time for methylcellulose stabilization and by performing repeated two-way (in and out) scans to account for z-axis motion.

[Supervision]. / Supersyn.

Within the past year, the idea of supervision has been introduced and promoted in the media by the manufacturers of wavefront-guided excimer lasers for refractive surgery. The concept is related to the ability of obtaining supernormal visual acuity and contrast sensitivity by correction of higher order aberrations in the biological optics of the human eye. However, the ocular aberrations fluctuate continuously and dynamically over time with the functional state of several of the individual optical components, including: thickness of the tear film, size of the pupil, and the degree of lens accommodation. These temporal variations represent major obstacles for achieving aberration-free images. In addition, the central perception of ideal aberration-free images may be hindered by neuronal limitations in the retina and brain. So far, only a few patients have experienced an increase in best corrected visual acuity following wavefront-guided refractive surgery. Also in the first prospective clinical trials with myopic individuals, only modest differences in visual performance have been detected between wavefront-guided and conventional treatment. Thus, immediate expectations from the new technology should be tuned down; supervision is not waiting around the corner.