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(Investigative Ophthalmology and Visual Science. 2006;47:2390-2396.)
© 2006 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.05-1559
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Keratan Sulfate and Chondroitin/Dermatan Sulfate in Maximally Recovered Hypocellular Stromal Interface Scars of Postmortem Human LASIK Corneas

Yuntao Zhang,1,2 Ingo Schmack,2,3,4 Daniel G. Dawson,2,3 Hans E. Grossniklaus,3 Abigail H. Conrad,1 Yutaka Kariya,5 Kiyoshi Suzuki,5 Henry F. Edelhauser,3 and Gary W. Conrad1

1From the Division of Biology, Kansas State University, Manhattan, Kansas; the 3Department of Ophthalmology, Emory University, Atlanta, Georgia; the 4Department of Ophthalmology, Ruprecht-Karls-University, Heidelberg, Germany; and the 5Central Research Laboratories, Seikagaku Corp., Higashiyamato-shi, Japan.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. To analyze the amounts and distributions of nonsulfated and sulfated keratan sulfate (KS) and chondroitin/dermatan sulfate (CS/DS) disaccharides in the interface wound of human postmortem LASIK corneas in comparison with normal control corneas.

METHODS. Corneal stromal tissue samples from central and paracentral hypocellular primitive stromal interface scars of human LASIK corneas and from similar regions of normal control corneas were collected by laser capture microdissection (LCM) and subsequently were digested with specific glycosidase enzymes. Digests were directly analyzed by electrospray ionization tandem mass spectrometry (ESI-MS/MS).

RESULTS. Concentrations of both monosulfated GlcNAc(6S)-ß-1,3-Gal (MSD2) and disulfated Gal (6S)-ß-1,4-GlcNAc(6S) (DSD) KS disaccharides from the LASIK interface scars were significantly lower than in normal control corneal stromas. No significant difference was found for the concentration of nonsulfated (NSD) KS disaccharides in LASIK interface scars compared with normal controls. The concentration of {Delta}UA-ß-1,3-GalNAc(6S) ({Delta}di-6S) CS/DS disaccharides from the LASIK interface scar was significantly higher than normal corneal stroma, whereas concentrations of {Delta}UA-ß-1,3-GalNAc(4S) ({Delta}di-4S) and nonsulfated {Delta}di-0S CS/DS disaccharides demonstrated no significant differences from normal corneas.

CONCLUSIONS. The profiles of KS and CS/DS disaccharides in LASIK interface scars are significantly different from those in normal cornea stromal tissue, as revealed by LCM and ESI-MS/MS.

The human cornea is avascular. Therefore, wound healing in the human corneal stroma is much slower and less complete in repairing normal stromal tissue when compared with most other tissues in the body.1 In human corneas, wound healing after LASIK surgery does not stabilize until 3.5 years after surgery.2 Whereas animal corneas heal completely after microkeratome cuts and LASIK procedures,3 4 human corneas undergo only primitive and limited wound healing. Moreover, LASIK wound healing has been associated with numerous postoperative complications (e.g., trauma flap wound dehiscence, diffuse lamellar keratitis, interface fluid syndrome, and infections).5 Recent human LASIK studies have demonstrated that corneal stromal wounds heal in one of two reparative fashions: hypercellular fibrotic stromal scarring or hypocellular primitive stromal scarring.6 7 8 Corneal stromal wound healing at the LASIK flap margin is influenced by epithelial–stromal interactions and results in the formation of a hypercellular fibrotic stromal scar. The hypercellular fibrotic scar is functionally abnormal, in that it is more hazy than normal corneal stroma because of myofibroblast formation and, to a lesser extent, abnormal extracellular (ECM) matrix deposition. Yet, it is relatively strong, attaining 30% to 40% of normal stromal tensile strength.2 9 In contrast, the central and paracentral regions of the LASIK interface wound heal by producing a transparent hypocellular primitive stromal scar with a reduced tensile strength, attaining only 2% to 3% of normal stromal tensile strength.2 9 Ultrastructural and immunofluorescent analyses of the hypocellular primitive stromal scar have shown that it is mainly composed of electron-dense granular material that contains abnormally large (300 x 20 nm), noncollagen-fibril–bound proteoglycans (PGs), as well as keratocytes and very few interspersed collagen fibrils that are smaller than normal in diameter.6 7 8

The principal PGs found in the human corneal stroma are those of keratan sulfate (KSPG) and chondroitin/dermatan sulfate (CS/DSPG).10 In the cornea, these PGs consist of small leucine-rich core proteins (SLRPs) covalently bearing one or more long sugar side chains, glycosaminoglycans (GAGs), that project from the convex surfaces of the core proteins.11 12 KS GAG chains are composed of repeating disaccharides of residues of D-galactose (Gal) and N-acetyl-D-glucosamine (GlcNAc)–linked ß-1,4 and ß-1,3, respectively,13 14 15 16 whereas CS/DS GAG chains are composed of repeating disaccharides of N-acetyl-D-galactosamine (GalNAc) residues alternating in glycosidic linkages with glucuronic acid (in CS domains) or iduronic acid (in DS domains) residues.17 18 Specific sulfotransferases attach sulfate groups to some sugars as they are incorporated into GAG side chains during biosynthesis.19 Many factors influence sulfation, leading to the appearance of both nonsulfated and variously sulfated domains along individual chains. In KS, sulfation of the hydroxyl groups at the C-6 positions of either Gal or GlcNAc residues, or in some cases of both Gal and GlcNAc residues, can be demonstrated. In CS/DS, GalNAc residues are predominantly sulfated in the C-4- (chondroitin A) or C-6-hydroxyl (chondroitin C) positions, interspersed with a few nonsulfated residues.16 The SLRP core proteins are thought to wrap around collagen fibrils and constrain growth of their diameters, whereas the projecting heterogeneously sulfated GAG side chains repel one another, resulting in even spacing between collagen fibrils.11 It has been established that GAGs are responsible for hydration dynamics in the cornea.20 21

Because the hypocellular primitive LASIK scar has been found to display increased fluid retention and abnormally large PGs, it was determined that more detailed information about the proteoglycan composition of this scar was needed. Therefore, tissue samples from the central and paracentral hypocellular primitive stromal LASIK scar were precisely excised from human LASIK eye bank donors using laser capture microdissection (LCM), a recent technique developed to dissect homogenous tissue samples from heterogeneous tissue under microscopic observation. Subsequently, enzymatic digests of the extracted scar tissue were analyzed by electrospray ionization tandem mass spectrometry (ESI-MS/MS), to identify and quantify the amount and distribution of KS and CS/DS nonsulfated and sulfated disaccharides in the hypocellular primitive stromal LASIK scar compared with control corneas.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Tissue
All human cornea samples were collected in accordance with the guidelines in the Declaration of Helsinki for research involving human tissue. After approval by the Emory University Institutional Review Board (IRB), five corneoscleral specimens from four donors (mean age ± SD, 46.3 ± 2.9 years) with a prior history of LASIK (range, 4.0–6.5 years after surgery) and five control normal corneoscleral specimens from four donors (mean age ± SD, 53.0 ± 19.0 years) were obtained from various eye banks in North America. Thus, all LASIK corneas used in this study had undergone maximum wound healing at the time of their collection.2 All specimens were stored in preservative (Optisol-GS; Bausch & Lomb Surgical, Irvine, CA) at 4°C within 17 hours of death and were evaluated in our laboratory within 5 days of death. Review was performed of the donor’s ophthalmic history, when available. LASIK and control corneoscleral specimens were randomly paired to form five groups of sample pairs, with one LASIK and one normal control cornea specimen per pair. Detailed information about characteristics of the donors and corneal specimens in each group is provided in Table 1 .


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  		TABLE 1. Demographics of LASIK and Control Corneas

 
All 10 corneoscleral specimens (LASIK [n = 5]; controls [n = 5]) were evaluated for gross abnormalities with a dissecting microscope (SZ-40; Olympus Tokyo, Japan) using direct illumination and indirect retroillumination techniques, and subsequently were bisected (Fig. 1) . The bisected specimens were placed in 70% ethanol for 24-hour fixation, dehydrated through a graded series of alcohol solutions (80%, 95%, and 100%), cleared with xylene solution, infiltrated with paraffin, and embedded in paraffin blocks at 60°C for 2 hours. Six-micrometer-thick sections were cut from the blocks and placed on special PEN-membrane–coated histology glass slides (Arcturus Bioscience, Mountain View, CA). The unstained sections were dewaxed with a 120-second wash of xylene, air-dried in a laminar flow hood, and transferred for LCM.


Figure 1
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  		FIGURE 1. Diagram demonstrating how the LASIK (A) and normal control (B) corneas were bisected. Gray ring: sclera; white circle: cornea; blue-dashed semicircle: LASIK flap wound margin.

 
Laser Capture Microdissection Technique
The protocol for laser capture microdissection (LCM) has been well described previously.22 Our procedure, which was optimized for ESI-MS/MS analysis, was performed on a commercial LCM system (PixCell II; Arcturus Engineering). The protocol entailed microdissecting and capturing the hypocellular primitive stromal LASIK scar (central and paracentral stromal scars) from each LASIK corneal section on LCM caps (CapSure HS LCM caps; Arcturus) by using 200 contiguous laser spots (Figs. 2A 2B) . Because of the Health Insurance Portability and Accountability Act (HIPAA) guidelines, it was difficult to obtain refractive error and ablation depth from the donors’ ophthalmic records. However, the measured thickness of the LASIK flap in all the patients averaged 140 ± 12.6 µm (range, 120–160 µm). Corneal tissue was microdissected by LCM at the same depth in the control corneas (Fig. 2C) . The infrared laser settings were as follows: 55-mW power, 900-ms duration, and 7.5-µm diameter laser spot size. Replicate sets of each sample were taken, so that totals of 10 LASIK and 6 to 10 normal control sets were collected from sample pairs (five LASIK and three to five normal LCM caps per glycosaminoglycan analysis group).


Figure 2
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  		FIGURE 2. (A) The hypocellular primitive LASIK stromal interface scar (blue line) and the area of the interface wound, which was microdissected from the remaining LASIK corneal stroma (black line). P, paracentral scar; C, central scar. Two sections were used per specimen (LASIK and normal control cornea) to collect a sufficient amount of tissue. Overall, the final LCM cap contained tissue captured from 200 contiguous LCM laser spots. Photomicrographs show the areas of the microdissected LASIK scars (B) and normal stromal control tissue (C). (B) The thermal-responsive film is still in place in this LASIK corneal section, which highlights the 100 contiguous LCM laser spots and the underlying silhouette of the extracted tissue. (C) Section of normal control cornea that shows the silhouette of the microdissected tissue and the remaining corneal stromal tissue.

 
Reagents
KS disaccharides were obtained from Seikagaku Corp. (Tokyo, Japan) and V-LABS, Inc. (Covington, LA). CS/DS disaccharides were obtained from Sigma-Aldrich (St. Louis, MO), EMD Biosciences, Inc. (San Diego, CA), and V-LABS, Inc. {Delta}UA-ß-1,4-GlcNS, also called IV-S and used as the internal standard for quantification of CS/DS sulfated disaccharides, was purchased from EMD Biosciences, Inc. Gal-{alpha}-1,3-Gal, used as the internal standard for quantification of KS and CS/DS nonsulfated disaccharides, was purchased from V-LABS, Inc. Chondroitinase ABC (from Proteus vulgaris, protease-free, EC 4.2.2.4), chondroitinase AC (from Flavobacterium heparinum, protease-free, EC 4.2.2.5), ammonium acetate, and ammonium sulfate were obtained from Sigma-Aldrich. Keratanase II (from Bacillus sp.) and endo-ß-galactosidase (from Escherichia freundii) were purchased from Seikagaku America (East Falmouth, MA). High-pressure liquid chromatography (HPLC)–grade solvents and ultrafree-MC centrifugal filter units (5000 NMWL; Millipore, Bedford, MA) were purchased from Fisher (Santa Clara, CA).

Preparation of Internal Standard Solutions
All disaccharide standards were diluted in a series of 10 nmol/µL, 1 nmol/µL, 100 pmol/µL, and 10 pmol/µL of sterile water, and aliquots were stored at –20°C. For quantitative analysis, 10 µL of the 1-nmol/µL solution of each standard was diluted by adding 70 µL MeOH, 5 µL of 5 mM ammonium acetate buffer (pH 7.5), 5 µL of 2 mM ammonium sulfate, and 10 µL water, which made the solution 7:3 MeOH:H2O, containing 100 pmol/µL disaccharide.

Enzymatic Digestion of KS and CS/DS
The microdissected and captured tissue samples (five sets of LASIK and three to five sets of normal control human corneas) of each group were enzymatically digested and processed at the same time. Single LASIK or normal control samples of each pair were exposed to 10 µL digestion solution, wrapped with film, and incubated in a moist chamber at 37°C for 24 hours. For keratanase II digestion (releases Gal-ß-1,4-GlcNAc(6S) [MSD1] and Gal (6S)-ß-1,4-GlcNAc(6S) [DSD] KS disaccharides), the digest solution was composed of 0.1 M ammonium acetate buffer (pH 6.0), 0.1 mU/µL keratanase II, and 50 pmol/µL {Delta}UA-ß-1,3-GalNAc(2S) [{Delta}di-2S] (internal standard). For endo-ß-galactosidase digestion (releases GlcNAc(6S)-ß-1,3-Gal [MSD2] and GlcNAc-ß-1,3-Gal [NSD] KS disaccharides), the digest solution was composed of 0.05 M ammonium acetate buffer (pH 5.8), 0.1 mU/µL endo-ß-galactosidase, and 50 pmol/µL Gal-{alpha}-1,3-Gal (internal standard). For analysis of CS/DS disaccharides, the digest solution was composed of 50 mM ammonium acetate buffer (pH 7.5), 1 mU/µL chondroitinase ABC or 1 mU/µL chondroitinase ABC plus 1 mU/µL chondroitinase AC (releases {Delta}UA-ß-1,3-GalNAc [{Delta}di-0S], {Delta}di-4S, and {Delta}di-6S disaccharides), and 50 pmol/µL {Delta}UA-ß-1,4-GlcNS or Gal-{alpha}-1,3-Gal (internal standards). The digest solutions of each set were collected and diluted by adding 70 µL of MeOH, 5 µL of 5 mM ammonium acetate buffer (pH 7.5), 5 µL of 2 mM (NH4)2SO4, and 10 µL of water. The mixtures were centrifuged at 3800 rcf for 30 minutes at 4°C (Ultra free-MC Centrifugal Filter unit, 5000 NMWL; Millipore) with subsequent analysis of the filtrate by ESI-MS/MS.

ESI-MS/MS
Mass spectra were obtained with an electron spray ionization source on a quadrupole ion trap instrument (Esquire 3000; Bruker Daltonics, Billerica, MA). Parameters used for analysis of all internal standards, and corneal stromal tissue digests were as follows: spray voltage, 3.5 kV; dry gas, nitrogen, flow: 5.0 L/min; and drying temperature, 180°C. The mass range scanned was m/z 50 to 1000, and data were aquired (Data Analysis 3.0 acquisition software; Bruker Daltonics).

Quantitative Analysis of KS Disaccharides
Representative mass spectrometry traces of keratanase II digests of one control sample and one LASIK wound tissue sample, plus the internal standard, {Delta}di-2S, are shown in Figures 3A and 3B . The intensities of the MSD1 and DSD peaks were compared with the intensity of the internal standard peak, and a single-point normalization factor method was used to determine the amounts of MSD1 and DSD KS disaccharides as described previously.23 The same methods were used to determine the amounts of NSD and MSD2 from endo-ß-galactosidase digests of control and LASIK wound tissue.23


Figure 3
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  		FIGURE 3. Mass spectra of products released by keratanase II digestion of the normal and LASIK corneas by laser capture microdissection (LCM) in the negative ionization mode. (A) MS spectrum of the normal cornea sample. Molecular ions at m/z 462.0 and m/z 270.4 correspond to Gal-ß-1,4-GlcNAc(6S) (MSD1) and Gal(6S)-ß-1,4-GlcNAc(6S) (DSD), respectively. The ion at m/z 457.9 corresponds to the KS sulfated disaccharide internal standard {Delta}Di-2S. (B) MS spectrum of the LASIK cornea sample. Molecular ions at m/z 462.0 and m/z 270.4 correspond to Gal-ß-1,4-GlcNAc(6S) (MSD1) and Gal(6S)-ß-1,4-GlcNAc(6S) (DSD), respectively. The ion at m/z 457.9 corresponds to the KS sulfated disaccharide internal standard {Delta}Di-2S.

 
Quantitative Analysis of CS/DS Disaccharides
The molar concentrations of {Delta}di-0S, {Delta}di-4S, and {Delta}di-6S CS/DS disaccharides were determined using single-point normalization factors. A system of two equations that corrects for the presence of additional ions in the MS2 spectrum allows the relative molar percentages of {Delta}di-4S and {Delta}di-6S isomers in a mixture to be calculated from the observed relative intensities of the diagnostic ions for each isomer in the MS2 spectrum of that mixture.24


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Content of the KS Disaccharides
KS disaccharide sulfation profiles are summarized in Table 2 . Concentrations of the sulfated KS disaccharides MSD2 and DSD were significantly lower in LASIK interface scars than in normal human corneal stromal tissue (P < 0.01). Concentrations of MSD2 for LASIK interface scars averaged 2.25 ± 0.18 pmol/µm3x 10–3 (range, 2.09–2.35 pmol/µm3x 10–3) compared with normal control corneal samples, which averaged 2.96 ± 0.36 pmol/µm3x 10–3 (range, 2.7–3.21 pmol/µm3x 10–3). Concentrations of DSD for LASIK interface scars averaged 2.73 ± 0.69 pmol/µm3x 10–3 (range, 2.2–3.6 pmol/µm3x 10–3) compared with normal corneal stromal samples, which averaged 3.95 ± 0.79 pmol/µm3x 10–3 (range, 3.4–5.3 pmol/µm3x 10–3). In contrast, the concentrations of KS NSD and MSD1 disaccharides in the LASIK scar were not significantly different from those in normal stroma (P > 0.05).


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  		TABLE 2. Concentrations of KS Nonsulfated and Sulfated Disaccharides Released by Digestion with Endo-ß-Galactosidase and Keratanase-II

 
Content of CS/DS Disaccharides
CS/DS disaccharide sulfation profiles are summarized in Table 3 . The tissue concentration of the sulfated CS/DS disaccharide {Delta}di-6S was significantly increased in the LASIK stromal scar (mean, 0.23 ± 0.04 pmol/µm3x 10–3) compared with normal control corneal samples (mean, 0.07 ± 0.02 pmol/µm3x 10–3; P < 0.01). In contrast, the concentrations of {Delta}di-4S and of {Delta}di-0S in LASIK interface scars were not significantly different from corresponding areas of stromal tissue of the normal control corneas (P < 0.05). This resulted in an average 3.65-fold increase in the molar ratio of {Delta}di-6S to {Delta}di-4S CS/DS in LASIK scars compared with controls. Moreover, in the normal cornea, {Delta}di-6S represented 18.39% molar percentage of the total sulfated monosulfated CS/DS disaccharides, {Delta}di-6S + {Delta}di-4S, whereas in the LASIK stromal scars {Delta}di-6S represented 44.36%.


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  		TABLE 3. Concentrations of CS/DS Nonsulfated and Sulfated Disaccharides Released by Digestion with Chondroitinase ABC and Chondroitinase AC

 

   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
In this study, LCM and ESI-MS/MS techniques were used to quantify directly the KS and CS/DS disaccharides in human corneal tissue along the central and paracentral hypocellular primitive LASIK stromal interface scar and from similar areas of nonwounded normal control corneas. The recently developed LCM technique allows microdissection and capture of very small circumscribed regions of a tissue and thus permits previously unobtainable biochemical analyses of these very small regions. In this study, LCM coupled with ESI-MS/MS made it possible to determine unambiguously the KS and CS/DS disaccharide composition of the extremely thin LASIK interface scar. Without LCM, the KS and CS/DS disaccharide composition of the much larger stroma mass surrounding the scar would have overwhelmed resolution of the unique scar GAG composition.

Concentrations of KS MSD2 and DSD in LASIK interface scars were significantly lower than in normal corneal stroma. Although MSD1 concentration was not significantly lower in the LASIK scar compared with normal control stroma, it was the same as the concentration of MSD2. This finding supports the conclusion that the total concentration of mono- and disulfated KS disaccharides is slightly but significantly lower in the LASIK scar than in the normal corneal stroma. In contrast, the concentration of KS NSD in the LASIK scar was not significantly different from that in the normal corneal stroma. These LCM/ESI-MS/MS quantitative results are consistent with a previous human immunofluorescence study that used a primary monoclonal antibody directed against sulfated KS epitopes, which showed that the degree of KS fluorescence in the central and paracentral hypocellular primitive interface scar was slightly less than in normal corneal stroma.7 It is also consistent with the animal studies that show both a reduced amount of KS and transiently undersulfated KS in opaque corneal scars.25 26 27 These data suggest that sulfation of both Gal and GlcNAc in KS is reduced in the hypocellular primitive LASIK scar, presumably from reduced and altered KS biosynthesis. The data presented herein or in previous studies do not indicate, however, whether the LASIK corneal interface scar contains altered concentrations of any of the KS proteoglycan core proteins (e.g., keratocan, lumican, mimecan/osteoglycin).7 25 26 27 28 Their concentrations may be normal, despite the changes in the posttranslational modifications of attached KS side chains. Alternatively, the reduced concentration of sulfated KS in LASIK tissue may be caused by reduced synthesis of KS core proteins, thus providing fewer glycosylation sites on which KS chains could be assembled.

An increase in the CS/DS content was also found in the central and paracentral hypocellular primitive LASIK interface scar, predominantly due to an increase in the concentration of {Delta}di-6S, which was only minimally present in normal corneal stroma. In contrast, no significant changes in the LASIK scar concentrations of nonsulfated disaccharides and 4S-sulfated disaccharides were found compared with those in the normal corneal stroma. The disaccharide ratios found in the LASIK scar may reflect changes in specific activities of chondroitin sulfotransferases, specifically chondroitin 6-O-sulfotransferase.16 17 CS/DS that is enriched with {Delta}di-6S may be associated with the electron-dense granular material repeatedly detected in the hypocellular primitive LASIK stromal interface scar.6 7 8 Animal studies have shown that other types of corneal scars contain abnormally large chondroitin and dermatan sulfate PGs.25 26 27 28 29 30 31 Major alterations in the biochemical structure of PGs were seen in dermatan sulfate, which had a twofold increase in iduronic acid content and was in a more highly sulfated form than normal.27 A previous human immunofluorescence evaluation of the central and paracentral hypocellular primitive LASIK interface scar detected no DS proteoglycan, which is normally the major CS/DS proteoglycan of the corneal stoma.7 That study, however, used a primary monoclonal antibody directed against the CS/DS proteoglycan core protein, decorin, rather than specifically against the DS glycosaminoglycan side chains. Further study is needed to determine why decorin immunoreactivity could not be demonstrated in the hypocellular primitive LASIK scar, and whether expressions of any new CS/DS proteoglycan core proteins (e.g., serglycin, biglycan, and versican) are activated in resident keratocytes or invading inflammatory cells in LASIK scars. The current data highlight the striking increase in the concentration of {Delta}di-6S in the hypocellular primitive LASIK stromal interface scar, but do not resolve whether those specific disaccharides were originally derived from hybrid chains of chondroitin sulfate C or from dermatan sulfate. These data also do not identify to which core protein these CS/DS chains enriched in {Delta}di-6S are covalently bound. Enhanced presence of chondroitin sulfates, including {Delta}di-6S, has also been detected in glial scars and correlated with inhibition of neurite extension in these scars.32 Perhaps, a similar phenomenon occurs in LASIK corneas, where recovery of preoperative subbasal nerve density in the corneal LASIK flap is delayed by up to 5 years or more.33

The causes of the alterations in PG and GAG distribution observed in hypocellular central and paracentral LASIK primitive scars are likely to be complex. The LASIK procedure includes two main steps: creation of a thin LASIK flap composed of the overlying corneal epithelium and anterior stroma with mechanical or laser microkeratome and subsequent laser ablation of the exposed underlying corneal stromal tissue. Therefore, the measured changes in KS and CS/DS are most probably the result of both microkeratome cut and LASER ablation.

Decorin-, lumican-, and mimecan-null mice all show increased skin collagen fibril diameter heterogeneity and increased skin fragility due to reduced tensile strength, suggesting that loss of any of these SLRP proteoglycan core proteins in the LASIK scar could lead to reduced scar tensile strength.12 34 35 Thus, if the decrease in sulfated KS in the LASIK stromal interface scar tissue were caused by reduced synthesis of any of the core proteins to which KS chains are attached, it may also partially explain the decrease in tensile strength of the LASIK scar. More information about the concentration of PG core proteins in LASIK scars would be helpful, and newer mass spectrometry techniques are currently being developed to obtain these proteomic data.

Recent studies have shown that the proteoglycans in the interface of the LASIK wound, between the flap and bed, swell, with high IOP and corneal endothelial decompensation.36 Interface swelling also causes a high degree of reflectivity, as seen by confocal microscopy. Therefore, changes in KS and CS/DS may be responsible for this swelling. KS and CS/DS GAGs have different water-binding properties.21 Under normal hydration of the cornea, CS/DS is fully hydrated and holds its bound water very tightly, whereas KS is only partially hydrated and releases its bound water more readily.14 Greatly increased 6-sulfated CS/DS holding water very tightly in the hypocellular scar could explain the fluid accumulation often observed along the interface in human LASIK corneas.37 38 39 Previous animal cell culture work has shown that corneal wound healing also results in the production of hyaluronic acid.27 40 The hyaluronic acid content of LASIK wounds should be assessed in future studies, because hyaluronic acid is very hydrophilic and thus may contribute to the hydrophilic properties of the hypocellular primitive LASIK scar.

In summary, profiles of the concentrations of KS and CS/DS nonsulfated and sulfated disaccharides released from the hypocellular primitive LASIK scar and from normal control stroma were successfully determined with LCM and ESI-MS/MS. The concentration of sulfated KS disaccharides was reduced in the hypocellular primitive LASIK scar, whereas the concentration of {Delta}di-6S, a CS/DS-derived disaccharide, was enriched significantly along the hypocellular primitive LASIK scar, indicating that these ECM components may have an important role in corneal wound healing after refractive surgery.


   Footnotes
 
2 Contributed equally to the work and therefore should be considered equivalent authors. Back

Presented in part at the annual meeting of the of the American Academy of Ophthalmology, Chicago, Illinois, 2005.

Supported by National Eye Institute Grants EY 000933 (HFE), EY 000952 (GWC), P30-EY06360 (HFE), and T32-EY 07092 (HFE); an unrestricted departmental grant from Research to Prevent Blindness (HFE); and the Gertrud Kusen Foundation, Hamburg, Germany (IS).

Submitted for publication December 7, 2005; revised January 25, 2006; accepted March 20, 2006.

Disclosure: Y. Zhang, None; I. Schmack, None; D.G. Dawson, None; H.E. Grossniklaus, None; A.H. Conrad, None; Y. Kariya, Seikagaku Corporation (E); K. Suzuki, Seikagaku Corporation (E); H.F. Edelhauser, None; G.W. Conrad, None

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Yuntao Zhang, Division of Biology, Ackert Hall, Kansas State University, Manhattan, KS 66506-4901; ytz{at}ksu.edu.


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. Dawson DG, Watsky MA, Geroski DH, Edelhauser HF. Physiology of the eye and visual system: cornea and sclera. Tasman W Jaeger EA eds. Duane’s Foundation of Clinical Ophthalmology on CD-ROM. 2006;2c. 4:1–76. Lippincott Williams & Wilkins Philadelphia.
  2. Schmack I, Dawson DG, McCarey BE, Waring GO, III, Grossniklaus HE, Edelhauser HF. Cohesive tensile strength of human LASIK wounds with histologic, ultrastructural, and clinical correlations. J Refrac Surg. 2005;21:433–445.
  3. Goodman RL, Johnson DA, Dillon H, Edelhauser HF, Waller SG. Laser in situ keratomileusis flap stability during simulated aircraft ejection in a rabbit model. Cornea. 2003;22:142–145.[Medline][Order article via Infotrieve]
  4. Sundarraj N, Fite D, Belak R, et al. Proteoglycan distribution during healing of corneal stromal wounds in chick. Exp Eye Res. 1998;67:433–442.[CrossRef][ISI][Medline][Order article via Infotrieve]
  5. Melki SA, Azar DT. LASIK complications: etiology, management, and prevention. Surv Ophthalmol. 2001;46:95–116.[CrossRef][ISI][Medline][Order article via Infotrieve]
  6. Dawson DG, Edelhauser HF, Grossniklaus HE. Long-term histopathologic findings in human corneal wounds after refractive surgical procedures. Am J Ophthalmol. 2005;139:168–178.[CrossRef][ISI][Medline][Order article via Infotrieve]
  7. Dawson DG, Kramer TR, Grossniklaus HE, Waring GO, III, Edelhauser HF. Histologic, ultrastructural, and immunofluorescent evaluation of human laser-assisted in situ keratomileusis corneal wounds. Arch Ophthalmol. 2005;123:741–756.[Abstract/Free Full Text]
  8. Kramer TR, Chuckpaiwong V, Dawson DG, Hernault NL, Grossniklaus HE, Edelhauser HF. Pathologic findings in postmortem corneas after successful laser in situ keratomileusis. Cornea. 2005;24:92–102.[CrossRef][ISI][Medline][Order article via Infotrieve]
  9. Dawson DG, Holley GP, Geroski DH, Waring GO, III, Grossniklaus HE, Edelhauser HF. Ex Vivo confocal microscopy of human LASIK corneas with histologic and ultrastructural correlation. Ophthalmology. 2005;112:634–644.
  10. Friend J, Hassell JR. Biochemistry of the cornea. Smolin G Thoft RA eds. The Cornea. 1994;47–67. Little, Brown New York.
  11. Iozzo RV. The family of the small leucine-rich proteoglycans: key regulators of matrix assembly and cellular growth. Crit Rev Biochem Mol Biol. 1997;32:141–174.[ISI][Medline][Order article via Infotrieve]
  12. Iozzo RV. The biology of the small leucine-rich proteoglycans: functional network of interactive proteins. J Biol Chem. 1999;274:18843–18846.[Free Full Text]
  13. Wight TN, Heinegard DK, Hascall VC. Proteoglycans structure and function. Hay ED eds. Cell Biology of the Extracellular Matrix. 1991; 2nd ed. 45–78. Plenum Press New York.
  14. Funderburgh JL. Keratan sulfate: structure, biosynthesis, and function. Glycobiology. 2000;10:951–958.[Abstract/Free Full Text]
  15. Funderburgh JL. Keratan sulfate biosynthesis. IUBMB Life. 2002;54:187–194.[ISI][Medline][Order article via Infotrieve]
  16. Sugumaran G, Vertel BM. Biosynthesis of chondroitin sulfated and dermatan sulfated proteoglycans. Ernst B Hart GW Sina ÿ P eds. Carbohydrates in Chemistry and Biology. Part II. Biology of Saccharides. 2000;3:375–395. Wiley-VCH New York.
  17. Conrad GW. Biological role of keratan sulfate proteoglycan. Ernst B Hart GW Sinaÿ P eds. Carbohydrates in Chemistry and Biology. Part II. Biology of Saccharides. 2000;4:717–727. Wiley-VCH New York.
  18. Silbert JE, Sugumaran G. Biosynthesis of chondroitin/dermatan sulfate. IUBMB Life. 2002;54:177–186.[ISI][Medline][Order article via Infotrieve]
  19. Rodén L. Biosynthesis of acidic glycosaminoglycans. Fishman WH eds. Metabolic Conjugation and Metabolic Hydrolysis. 1970;2:345–442. Academic Press New York.
  20. Hedbys BO. The role of polysaccharides in corneal swelling. Exp Eye Res. 1961;1:81–91.[ISI][Medline][Order article via Infotrieve]
  21. Bettelheim FA, Plessy B. The hydration of proteoglycans of bovine cornea. Biochim Biophys Acta. 1975;381:203–214.[Medline][Order article via Infotrieve]
  22. Emmert-Buck MR, Bonner RF, Smith PD, et al. Laser capture microdissection. Science. 1996;274:998–1001.[Abstract/Free Full Text]
  23. Zhang Y, Kariya Y, Conrad AH, Conrad GW. Analysis of keratan sulfate oligosaccharides by electrospray ionization tandem mass spectrometry. Anal Chem. 2005;77:902–910.[Medline][Order article via Infotrieve]
  24. Zhang Y, Conrad AH, Tasheva ES, et al. Detection and quantification of sulfated disaccharides from keratan sulfate and chondroitin/dermatan sulfate during chick corneal development by ESI-MS/MS. Invest Ophthalmol Vis Sci. 2005;46:1604–1614.[Abstract/Free Full Text]
  25. Funderburg JL, Cintron C, Covington HI, Conrad GW. Immunoanalysis of keratan sulfate proteoglycan from corneal scars. Invest Ophthalmol Vis Sci. 1988;29:1116–1124.[Abstract/Free Full Text]
  26. Funderburg JL, Chandler JW. Proteoglycans of rabbit corneas with non-perforating wounds. Invest Ophthalmol Vis Sci. 1989;30:435–442.[Abstract/Free Full Text]
  27. Funderburg JL, Mann MM, Funderburgh ML. Keratocyte phenotype mediates proteoglycan structure: a role for fibroblasts in corneal fibrosis. J Biol Chem. 2003;278 45:629–637.
  28. Cintron C, Covington HI, Kublin CL. Morphologic analyses of proteoglycans in rabbit corneal scars. Invest Ophthalmol Vis Sci. 1990;31:1789–1798.[Abstract/Free Full Text]
  29. Kato T, Nakayasu K, Kanai A. Corneal wound healing. Immunohistological features of extracellular matrix following penetrating keratoplasty in rabbits. Jpn J Ophthalmol. 2000;44:334–341.[CrossRef][Medline][Order article via Infotrieve]
  30. Rawe IM, Tuft SJ, Meek KM. Proteoglycan and collagen morphology in superficially scarred rabbit cornea. Histochem J. 1992;24:311–318.[CrossRef][ISI][Medline][Order article via Infotrieve]
  31. Hassell JR, Cintron C, Kublin C, Newsome DA. Proteoglycan changes during restoration of transparency in corneal scars. Arch Biochem Biophys. 1983;222:362–369.[CrossRef][ISI][Medline][Order article via Infotrieve]
  32. Gilbert RJ, McKeon RJ, Darr A, Calabro A, Hascall VC, Bellamkonda RV. CS-4,6 is differentially upregulated in glial scar and is a potent inhibitor of neurite extension. Mol Cell Neurosci. 2005;29:545–558.[CrossRef][Medline][Order article via Infotrieve]
  33. Erie JC, McLaren JW, Hodge DO, Bourne WM. Recovery of corneal subbasal nerve density after PRK and LASIK. Am J Ophthalmol. 2005;140:1059–1064.[CrossRef][ISI][Medline][Order article via Infotrieve]
  34. Chakravarti S, Magnuson T, Lass JH, Jepsen KJ, LaMantia C, Carroll H. Lumican regulates collagen fibril assembly: skin fragility and corneal opacity in the absence of lumican. Arch Biochem Biophys. 1998;192:1277–1286.
  35. Tasheva ES, Koester A, Paulsen AP, et al. Mimecan/osteoglycin-deficient mice have collagen fibril abnormalities. Mol Vis. 2002;8:407–415.[ISI][Medline][Order article via Infotrieve]
  36. Wirbelauer C, Pham DT. Imaging interface fluid after laser in situ keratomileusis with corneal optical coherence tomography. J Cataract Refract Surg. 2005;31:853–856.[CrossRef][ISI][Medline][Order article via Infotrieve]
  37. Hamilton DR, Manche EE, Rich LF, et al. Steroid-induced glaucoma after laser in situ keratomileusis associated with interface fluid. Ophthalmology. 2002;109:659–665.
  38. Schmack I, Holley GP, Dawson DG, Grossniklaus HE, Edelhauser HF. Corneal endothelial decompensation in human LASIK corneas induces an interface fluid pocket. Poster presentation at the Annual Meeting of the American Academy of Ophthalmology, Chicago, IL. 2005; poster 045.
  39. Dawson DG, Hardten DR, Albert DM. Pocket of fluid in the lamellar interface after penetrating keratoplasty and laser in situ keratomileusis. Arch Ophthalmol. 2003;121:894–896.[Free Full Text]
  40. Conrad GW, Hamilton C, Haynes E. Differences in glycosaminoglycans synthesized by fibroblast-like cells from chick cornea, heart, and skin. J Biol Chem. 1977;252:6861–6870.[Free Full Text]




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