Effects of ultraviolet-A and riboflavin on the interaction of collagen and proteoglycans during corneal cross-linking.

Corneal cross-linking using riboflavin and ultraviolet-A (RFUVA) is a clinical treatment targeting the stroma in progressive keratoconus. The stroma contains keratocan, lumican, mimecan, and decorin, core proteins of major proteoglycans (PGs) that bind collagen fibrils, playing important roles in stromal transparency. Here, a model reaction system using purified, non-glycosylated PG core proteins in solution in vitro has been compared with reactions inside an intact cornea, ex vivo, revealing effects of RFUVA on interactions between PGs and collagen cross-linking. Irradiation with UVA and riboflavin cross-links collagen α and ß chains into larger polymers. In addition, RFUVA cross-links PG core proteins, forming higher molecular weight polymers. When collagen type I is mixed with individual purified, non-glycosylated PG core proteins in solution in vitro and subjected to RFUVA, both keratocan and lumican strongly inhibit collagen cross-linking. However, mimecan and decorin do not inhibit but instead form cross-links with collagen, forming new high molecular weight polymers. In contrast, corneal glycosaminoglycans, keratan sulfate and chondroitin sulfate, in isolation from their core proteins, are not cross-linked by RFUVA and do not form cross-links with collagen. Significantly, when RFUVA is conducted on intact corneas ex vivo, both keratocan and lumican, in their natively glycosylated form, do form cross-links with collagen. Thus, RFUVA causes cross-linking of collagen molecules among themselves and PG core proteins among themselves, together with limited linkages between collagen and keratocan, lumican, mimecan, and decorin. RFUVA as a diagnostic tool reveals that keratocan and lumican core proteins interact with collagen very differently than do mimecan and decorin.

Thyroxine increases the rate but does not alter the pattern of innervation during embryonic chick corneal development.

PURPOSE: Embryonic chick corneal nerves reach limbal mesenchyme by embryonic day (E)5, encircle the cornea in several days, then defasciculate into the stroma simultaneously from all sides, while extracellular keratan sulfate proteoglycan (KSPG) accumulates from posterior to anterior stroma. Precocious thyroxine (T4)-induced increases in corneal thinning/transparency are blocked by 2-thiouracil (2-TU) inhibition of T3 synthesis. The hypothesis for this study was that precocious T4 exposure increases corneal innervation similarly. METHODS: E8 embryos received T4, 2-TU, T4+2-TU, or buffer; corneas were harvested on E12. Corneal nerves were stained with neuronal beta-tubulin-specific TuJ1 antibody or chick nerve-specific CN antibody. Corneal thickness was determined from cryostat sections, and mRNA expression was measured by real-time PCR. RESULTS: Nerves avoided the cornea until E9, then entered the anterior stroma, extended toward and reached the cornea center by E14, and never invaded posterior stroma. E7 to E18 corneal expressions of nerve growth factor and neurotrophin-3 genes were unchanged; receptor gene expressions rose. E7 to E12 semaphorin 3A and 3F and ephrin A2 and A5 expressions did not change significantly; semaphorin and ephrin/eph expressions increased from E9 to E18. E8 T4 administration increased nerve extension by E11, but did not alter circumferential penetration, anterior-only penetration, or neurotrophin expressions. 2-TU prevented T4-induced precocious corneal thinning, but augmented T4 nerve stimulation. CONCLUSIONS: No changes in corneal neurotrophin or nerve pathfinding gene expressions accompany corneal transition to nerve growth cone permissiveness. T4 increases corneal nerve penetration rates by a non-T3-dependent mechanism. Results are consistent with possible roles for corneal KSPGs in regulating corneal nerve growth.

On-target derivatization of keratan sulfate oligosaccharides with pyrenebutyric acid hydrazide for MALDI-TOF/TOF-MS.

In the present work, a rapid and novel method of on-target plate derivatization of keratan sulfate (KS) oligosaccharides for subsequent analysis by matrix-assisted laser desorption and ionization (MALDI) mass spectrometry is described. MALDI-(time-of-flight)-TOF spectra of labeled KS oligosaccharides revealed that significantly improved ionization can be accomplished through derivatization with pyrenebutyric acid hydrazide (PBH), and the most abundant peak in each spectrum corresponds to the singly charged molecular ion [M - H]- or [M + (n - 1)Na - nH]-, where n = the number of sulfates (n = 1, 2, 3...). The high-energy collision-induced dissociation (heCID) spectra of labeled KS oligosaccharides displayed fragments of compounds similar to those observed with laser-induced dissociation (LID) analysis, suggesting that both heCID and LID fragmentations can be used to analyze KS oligosaccharides. Moreover, fragmentation analysis of all labeled KS oligosaccharides was performed by MALDI-TOF/TOF-MS. With LID mode, sodium adducts showed fragmentation of glycosidic linkages with mainly Y/B/C ions, as well as various cross-ring cleavages providing exact information for the positions of sulfate groups along the KS oligosaccharide chains. This one-step on-target derivatization method makes MALDI-TOF/TOF-MS identification of KS fast, simple and highly throughput for trace amounts of biological samples.

Keratan sulfate and chondroitin/dermatan sulfate in maximally recovered hypocellular stromal interface scars of postmortem human LASIK corneas.

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)-beta-1,3-Gal (MSD2) and disulfated Gal (6S)-beta-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 DeltaUA-beta-1,3-GalNAc(6S) (Deltadi-6S) CS/DS disaccharides from the LASIK interface scar was significantly higher than normal corneal stroma, whereas concentrations of DeltaUA-beta-1,3-GalNAc(4S) (Deltadi-4S) and nonsulfated Deltadi-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.

Thyroxine affects expression of KSPG-related genes, the carbonic anhydrase II gene, and KS sulfation in the embryonic chicken cornea.

PURPOSE: Opaque chick corneas become thin and transparent from embryonic day (E)9 to E20 of incubation. Thyroxine (T4) injected in ovo on E9 induces precocious transparency by E12. The present study was conducted to determine whether corneal cells differentially express genes for T4 regulation, keratan sulfate proteoglycan (KSPG) synthesis, crystallins, and endothelial cell ion transporters during transparency development and whether these expressions are altered when E9 embryos are treated with T4. METHODS: E9 eggs received T4 or buffer; corneas were dissected on E12. Corneal transparency was measured digitally and thickness was determined from cryostat cross sections. mRNA expressions were determined by real-time PCR using cDNA synthesized from whole-cell RNA, cells expressing T4 receptor mRNAs assessed by in situ hybridization, and KS disaccharide sulfation measured by electrospray ionization tandem mass spectrometry (ESI-MS/MS). RESULTS: All corneal layers expressed T4 receptor alpha (THRA) mRNA; keratocytes and endothelial cells expressed T4 receptor beta (THRB) mRNA. During normal development, THRB expression increased 20-fold from E12 to E20; THRA expression remained constant. Expressions of most genes involved in KS synthesis increased from E9 to E16, and then decreased from E16 to E20. From E9 to E20, expressions of crystallin genes increased; T4/3-deiodinase DIII (DIO3) increased 10-fold; and sodium-potassium ATPase transporter (ATP1A1), sodium-bicarbonate transporter (NBC), and carbonic anhydrase II (CA2) increased 5- to 10-fold. E9 T4 administration decreased corneal thickness by E12; increased DIO3, THRB, and CA2 expressions 5- to 20-fold; decreased KSPG core protein genes and galactose sulfotransferase CHST1 expressions 2-fold; and reduced KS disulfated/monosulfated disaccharide (DSD/MSD) ratios. CONCLUSIONS: Thyroxine modifies expressions of KSPG synthesis and carbonic anhydrase genes.

Differentiation of diiodothyronines using electrospray ionization tandem mass spectrometry.

Diiodothyronines 3,5-diiodothyronine (3,5-T2), 3',5'-diiodothyronine (3',5'-T2), and 3,3'-diiodothyronine (3,3'-T2) are important metabolites of 3,5,3'-triiodothyronine (T3) and 3,3',5'-triiodothyronine (rT3; reverse T3). In this paper, a novel and rapid method for identifying and quantifying 3,5-T2, 3',5'-T2 and 3,3'-T2 has been introduced using electrospray ionization tandem mass spectrometry (ESI-MS/MS). Fragmentation patterns were proposed on the basis of our data obtained by ESI-MS/MS. MS2 spectra in either negative ionization mode or positive ionization mode can be used to differentiate 3,5-T2, 3',5'-T2 and 3,3'-T2. On the basis of the relative abundance of fragment ions in MS2 spectra under the positive ionization mode, quantification of the 3,5-T2, 3',5'-T2 and 3,3'-T2 isomers in mixtures is also achieved without prior separation.

Detection and quantification of sulfated disaccharides from keratan sulfate and chondroitin/dermatan sulfate during chick corneal development by ESI-MS/MS.

PURPOSE: To identify and quantify changes in keratan sulfate (KS) and chondroitin/dermatan sulfate (CS/DS) sulfated disaccharides in the developing chick cornea using electrospray ionization tandem mass spectrometry (ESI-MS/MS). METHODS: Cryostat sections of fresh nonfixed corneas were obtained from White Leghorn embryonic day (E)8 to E20 chicks, and from 4- and 70-week-old chickens. Tissue sections on glass slides were incubated with selected glycosidase enzymes. Digest solutions were analyzed directly by ESI-MS/MS. RESULTS: The concentration of KS monosulfated disaccharide (MSD) Gal-beta-1,4-GlcNAc(6S) in E8 cornea equaled that at E20, declined to its lowest level by E10, increased to a second peak by E14, decreased to a second low by E18, peaked again by E20, and remained high in adult corneas. A similar concentration profile was observed for KS disulfated disaccharide (DSD) Gal(6S)-beta-1,4-GlcNAc(6S), and thus also for total sulfated KS disaccharides. The molar percent of DSD was higher than that of MSD from E8 to E18, equivalent at E20, and less than that of MSD in adult corneas. In contrast, total concentration of CS/DS Deltadi-4S plus Deltadi-6S decreases as development progresses and is lowest in adult corneas. Concentration and molar percent of Deltadi-6S is highest at E8, then decreases through development as the concentration and molar percent of Deltadi-4S increases from E8 and exceeds that of Deltadi-6S after E14. CONCLUSIONS: New, rapid, direct chemical analysis of extracellular matrix components obtained from sections from embryonic and adult chick corneas reveals heretofore undetected changes in sulfation characteristics of KS and CS/DS disaccharides during corneal development.

Detection and quantification of 3,5,3'-triiodothyronine and 3,3',5'-triiodothyronine by electrospray ionization tandem mass spectrometry.

A novel and rapid method for identifying and quantifying 3,5,3'-triiodothyronine (T3) and 3,3',5'-triiodothyronine (rT3; reverse T3) has been introduced using electrospray ionization tandem mass spectrometry (ESI-MS/MS). MS(2) spectra in either negative ionization mode or positive ionization mode can be used to differentiate T3 and rT3. Quantification of the T3 and rT3 isomers under the negative ionization mode is also achieved without prior separation by HPLC.

The keratocan gene is expressed in both ocular and non-ocular tissues during early chick development.

Extracellular matrix (ECM) keratan sulfate proteoglycans (KSPGs) are core proteins with sulfated polylactosamine side chains (KS). The KSPG core protein keratocan gene (Kera) is expressed almost exclusively in adult vertebrate cornea, but its embryonic expression is little known. Embryonic chick in situ hybridization reveals Kera mRNA expression in corneal endothelium from embryonic day (E) 4.5, Hamburger-Hamilton (HH) 25, in stromal keratocytes from E6.5, HH30, and in iris distal surface cells from E8, HH34. As highly sulfated, antibody I22-positive KS increases extracellularly from posterior to anterior across the stroma, nerves enter and populate only anterior stroma and epithelium. RT-PCR and in situ hybridization demonstrate that developmentally regulated Kera mRNA expression initiates in midbrain and dorsolateral mesenchyme at E1, HH7, then spreads caudally in hindbrain and cranial and trunk mesenchyme flanking the neural tube through E2, HH20. Cranial expression extends ventrally through the developing head, and concentrates in mesenchyme surrounding eye anterior regions and cranial ganglia, and in subepidermal pharyngeal arch mesenchyme by E3.5, HH22. Kera expression in the trunk at E3.5, HH22 and E4.5, HH25, is strong in dorsolateral subepidermal, sclerotomal and nephrogenic mesenchymes, but absent in neural tube, dorsal root ganglia, nerve outgrowths, notochord, heart and gut. Early limb buds express Kera mRNA throughout their mesenchyme, then in restricted proximal and distal mesenchymes. I22-positive KS appears only in notochord in E3.5, HH22 and E4.5, HH25, embryos. Results suggest the hypothesis that keratocan, or keratocan with minimally sulfated KS chains, may play a role in structuring ECM for early embryonic cell and neuronal migrations.

Proteomic analysis of potential keratan sulfate, chondroitin sulfate A, and hyaluronic acid molecular interactions.

PURPOSE: Corneal stroma extracellular matrix (ECM) glycosaminoglycans (GAGs) include keratan sulfate (KS), chondroitin sulfate A (CSA), and hyaluronic acid (HA). Embryonic corneal keratocytes and sensory nerve fibers grow and differentiate according to chemical cues they receive from the ECM. This study asked which of the proteins that may regulate keratocytes or corneal nerve growth cone immigration interact with corneal GAGs. METHODS: Biotinylated KS (bKS), CSA (bCSA), and HA (bHA) were prepared and used in microarray protocols to assess their interactions with 8268 proteins and a custom microarray of 85 extracellular epitopes of nerve growth-related proteins. Surface plasmon resonance (SPR) was performed with bKS and SLIT2, and their ka, kd, and KD were determined. RESULTS: Highly sulfated KS interacted with 217 microarray proteins, including 75 kinases, several membrane or secreted proteins, many cytoskeletal proteins, and many nerve function proteins. CSA interacted with 24 proteins, including 10 kinases and 2 cell surface proteins. HA interacted with 6 proteins, including several ECM-related structural proteins. Of 85 ECM nerve-related epitopes, KS bound 40 proteins, including SLIT, 2 ROBOs, 9 EPHs, 8 Ephrins (EFNs), 8 semaphorins (SEMAs), and 2 nerve growth factor receptors. CSA bound nine proteins, including ROBO2, 2 EPHs, 1 EFN, two SEMAs, and netrin 4. HA bound no ECM nerve-related epitopes. SPR confirmed that KS binds SLIT2 strongly. The KS core protein mimecan/osteoglycin bound 15 proteins. CONCLUSIONS: Corneal stromal GAGs bind, and thus could alter the availability or conformation of, many proteins that may influence keratocyte and nerve growth cone behavior in the cornea.

Embryonic corneal Schwann cells express some Schwann cell marker mRNAs, but no mature Schwann cell marker proteins.

PURPOSE: Embryonic chick nerves encircle the cornea in pericorneal tissue until embryonic day (E)9, then penetrate the anterior corneal stroma, invade the epithelium, and branch over the corneal surface through E20. Adult corneal nerves, cut during transplantation or LASIK, never fully regenerate. Schwann cells (SCs) protect nerve fibers and augment nerve repair. This study evaluates SC differentiation in embryonic chick corneas. METHODS: Fertile chicken eggs were incubated from E0 at 38 degrees C, 45% humidity. Dissected permeabilized corneas plus pericorneal tissue were immunostained for SC marker proteins. Other corneas were paraffin embedded, sectioned, and processed by in situ hybridization for corneal-, nerve-related, and SC marker gene expression. E9 to E20 corneas, dissected from pericorneal tissue, were assessed by real-time PCR (QPCR) for mRNA expression. RESULTS: QPCR revealed unchanging low to moderate SLIT2/ROBO and NTN/UNC5 family, BACE1, and CADM3/CADM4 expressions, but high NEO1 expression. EGR2 and POU3F1 expressions never surpassed PAX3 expression. ITGNA6/ITGNB4 expressions increased 20-fold; ITGNB1 expression was high. SC marker S100 and MBP expressions increased; MAG, GFAP, and SCMP expressions were very low. Antibodies against the MPZ, MAG, S100, and SCMP proteins immunostained along pericorneal nerves, but not along corneal nerves. In the cornea, SLIT2 and SOX10 mRNAs were expressed in anterior stroma and epithelium, whereas PAX3, S100, MBP, and MPZL1 mRNAs were expressed only in corneal epithelium. CONCLUSIONS: Embryonic chick corneas contain SCs, as defined by SOX10 and PAX3 transcription, which remain immature, at least in part because of stromal transcriptional and epithelial translational regulation of some SC marker gene expression.

Differentiation of monoiodothyronines using electrospray ionization tandem mass spectrometry.

In this work two monoiodothyronines, 3-T1 and 3'-T1, have been analyzed using electrospray ionization tandem mass spectrometry (ESI-MS/MS). Fragmentation patterns were proposed based on our data obtained by ESI-MS/MS. MS2 spectra in either negative or positive ion mode can be used to differentiate 3-T1 and 3'-T1. Based on the relative abundance of fragment ions in MS2 spectra in the negative ion mode, quantification of the 3-T1 and 3'-T1 isomers in mixtures is achieved without prior separation. Solid-phase extraction in combination with ESI-MS/MS provides a practicable procedure that can be used to determine the molar ratio of 3-T1 and 3'-T1 in human serum with an error less than 3%. The detection limits for 3-T1 and 3'-T1 were 0.5 and 0.7 pg/microL, respectively.

Analysis of keratan sulfate oligosaccharides by electrospray ionization tandem mass spectrometry.

Keratan sulfate (KS) is a glycosaminoglycan consisting of repeating disaccharide units composed of alternating residues of d-galactose and N-acetyl-d-glucosamine linked beta-(1-4) and beta-(1-3), respectively. In this study, electrospray ionization tandem mass spectrometry (ESI-MS/MS) was employed to identify keratan sulfate oligosaccharides. Two nonsulfated disaccharide isomers and two monosulfated disaccharide isomers were distinguished through MS/MS. In MS(1) spectra of multiply sulfated KS oligosaccharides, the charge state of the most abundant molecular ion equals the number of sulfates. Subsequent MS(2) and MS(3) spectra of mono-, di-, tri-, and tetrasulfated KS oligosaccharides and sialylated tetrasaccharides reveal diagnostic ions that can be used as fingerprint maps to identify unknown KS oligosaccharides. Based on the pattern of fragment ions, the compositions of an oligosaccharide mixture from shark cartilage KS and of two enzyme digests of bovine corneal KS were determined directly, without prior isolation of individual oligosaccharides by HPLC or other methods.