Extracellular matrix production by cat retinal pigment epithelium in vitro: Characterization of type IV collagen synthesis

Feline retinal pigment epithelial cells (RPE) produced an extracellular matrix (ECM) in vitro which was located between the basal surface of the RPE and the culture plate. This ECM had three morphological components: bundle, granular and fibrillar. After 14 days in culture the basal extracellular space contained small amounts of bundle material; granular and fibrillar material were infrequently observed at this time. The amount of ECM material increased with increasing time in culture. The accumulation of the granular component extracellularly was greatest between 60 and 108 days. Fibrillar material, although occasionally observed in the ECM, appeared to be an infrequent component. By 145 days, the ECM filled the extracellular space between the RPE and the culture plate. The time-dependent increase of the ECM indicated continued synthesis and secretion of ECM into the basal extracellular space by the RPE.Confluent RPE cultures, or choroidal/scleral fibroblasts, were incubated for 24 hr with [¹⁴C]-proline. Newly synthesized collagen, either in the culture medium or the cell layer, was co-precipitated with added carrier collagen by (NH₄)₂SO₄. The samples, with or without reduction and alkylation, were digested with pepsin and fractioned by selective salt precipitation and carboxymethyl(CM)-cellulose chromatography. The resulting fractions were further analyzed, or purified for thin layer chromatography (TLC) amino acid analysis, by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Cultured RPE cells, but not choroidal/scleral fibroblasts, produced labelled peptides which were characterized as α₁ (IV), and α₂ (IV) collagen chains by CM-cellulose chromatography, SDS-PAGE, proline:hydroxyproline ratios and sensitivity to bacterial collagenase. In contrast, choroidal/scleral fibroblasts produced labelled α₁ (I), β₁₂ (I) and α₂ (I) collagen chains. The synthesis of type IV collagen by RPE cells may reflect the production of ECM observed by electron microscopy in cultured feline RPE cells.     

Collagen heterogeneity in the avian eye: lens, vitreous body, cornea and sclera

In order to further investigate the molecular heterogeneity of the primary structure of collagen, we have extracted, purified and characterized collagens from the various component tissues in the eyes of lathyritic and normal chickens. The eye tissues were dissected arbitrarily into four portions; the cornea, the lens, the vitreous body and the sclera.The collagen isolated from the cornea as well as the fibrous portion of the sclera was shown to consist of [α1(I)]₂α2 by the criteria of CM-cellulose chromatography, and amino acid analysis. The collagens isolated from the lens and the vitreous body contained predominantly α1 chains whose amino acid composition differs significantly from that previously obtained for chick α1(I), α1(II) or α2. Pepsin-solubilization of the cartilaginous portion of the sclera and subsequent CM-cellulose chromatography revealed, in addition to the anticipated α1(II), another fraction eluting after α2.Thus, we conclude that the avian eye contains several distinct species of collagens. In addition to the [α1(I)]₂α2 and [α1(II)]₃ in the cornea, fibrous sclera and cartilaginous sclera respectively, our data indicate the presence in the lens and the vitreous body of a species of collagen composed of α1-chains previously not described and in the scleral cartilage an α2-like molecule with an unusual amino acid composition.     

Biochemical investigation of cells from keratoconus and normal cornea

Collagen is the major structural protein in the cornea. In keratoconus the central cornea is thin, opaque and weak. Collagen synthesis was investigated in cells derived from the stroma of cornea with keratoconus and from controls. No difference was found in the ratio of collagens type I and type III synthesized, which were investigated as procollagens and after conversion to collagen as well. The $\text{α1}\text{α2}$ ratio in type I collagen was similar in keratoconus cells and controls.     

Corneal ulceration and the serum antiproteases. II. Complexes of corneal collagenases and alpha-macroglobulins.

Previous work has demonstrated that whole rabbit or human serum and the human α₂-macroglobulin (Hα₂-m) inhibit corneal collagenases. In the present study, the ability of rabbit and human serum to cause rabbit corneal collagenase, mol. wt 45 000, to elute in high molecular weight fractions from sieving columns is taken as presumptive evidence for the formation of collagenase-serum protein complexes. The recovery of increased collagenase activity by thiocyanate treatment of serum effluent fractions containing the human α₂-macroglobulin indicates that it is the α₂-m in human serum that complexes rabbit corneal collagenase. This conclusion is supported by the demonstration that purified Hα₂-m transports the rabbit corneal collagenase through a molecular sieve. Moreover, the chromatography of day one culture media from ulcerating rabbit corneas has demonstrated the presence of a significant amount of the total collagenase activity in the high molecular weight (850 000 – 1 000 000) fractions in which rabbit α-macroglobulin (Rα₁-m) was also demonstrated. These observations support the hypothesis that the α-macroglobulins play an important role in the regulation of corneal collagenase activity.Crossed-gel immunoelectrophoretic methods have shown that rabbit and human corneal collagenase preparations perturb the patterns of their respective α-macroglobulins. The perturbed patterns are taken as evidence for the formation of collagenase-α-macroglobulin complexes. The application of crossed-gel methods to the tears of human ulcer patients shows the utility of such methods for examining the status of α₂-m in tears.     

Promotion of minTBP-1-PRGDN on the attachment, proliferation and collagen I synthesis of human keratocyte on titanium

AIM:To investigate the influence of minTBP-1-PRGDN on the attachment, proliferation and collagen I synthesis of human keratocyte on titanium (Ti) surface.METHODS:The chimeric peptide RKLPDAPRGDN (minTBP-1-PRGDN) was synthesized by connecting RKLPDA (minTBP-1) to the N-terminal of PRGDN , the influence of minTBP-1-PRGDN on the attachment, proliferation and collagen I synthesis of human keratocyte on Ti surface were tested using PRGDN and minTBP-1as controls. The keratocytes attached to the surface of Ti were either stained with FITC-labeled phalloidin and viewed with fluorescence microscope or quantified with alamar Blue method. The proliferation of keratocytes on Ti were quantified with 3-(4,5-dim- ethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide up-taking methods. The secretion of type I collagen were determined using an ELISA kit.RESULTS:The results showed that minTBP-1-PRGDN at a concentration of 100ng/mL was the most potent peptide to enhance the attachment of human keratocytes to the surface of Ti (1.40±0.03 folds, P=0.003), to promote the proliferation (1.26±0.05 folds, P=0.014) and the synthesis of type I collagen (1.530±0.128, P=0.008). MinTBP-1 at the same concentration could only promote the attachment (1.13±0.04 folds, P=0.020) and proliferation(1.15±0.06 folds, P=0.021), while PRGDN had no significant influence (P＞0.05).CONCLUSION:Our data shows that the novel chimeric peptide minTBP-1-PRGDN could promote the attachment, proliferation and type I collagen synthesis of human keratocytes on the surface of Ti.     

IGF-II and collagen expression by keratocytes during postnatal development

Keratocytes produce the extensive stromal matrix of the cornea during the late embryonic and neonatal time periods. We propose to test the hypothesis that their biosynthetic activity declines during this process. Keratocytes were isolated from corneas of 6-8-week-old rabbits and corneas of 1-2-year-old cows and their ability to proliferate and synthesize collagen in serum-free media was determined. Rabbit keratocyte cultures increased 38% in DNA content after one week and deposited collagen type I and IGF-II in the media. Bovine keratocyte cultures, in contrast, did not increase in DNA or produce detectable collagen and IGF-II. Bovine keratocytes cultured in media previously conditioned by rabbit keratocytes, however, increased 56% in DNA content, and deposited collagen type I into the media. Microarray analysis of mRNA from neonatal and adult mouse keratocytes was used to confirm these differences. Compared to adult mouse keratocytes, neonatal keratocytes showed high expression levels of IGF-I, IGF-II and collagen types III and V. Since previous studies showed that IGFs stimulate bovine keratocytes to proliferate and to synthesize procollagen type I, we therefore propose that the results of this study suggests that the IGFs may play an important role in regulating early corneal growth in vivo.     

Type II collagen in the early embryonic chick cornea and vitreous: Immunoradiochemical evidence

The relative proportion of collagen types I and II was examined in early chick corneas and vitrous bodies by radioimmunoprecipitation. Embryos (4–5 days of incubation) were labelled in ovo with ³H-proline. After an additional 1–2 days, corneas and vitreous bodies were removed and the collagen was extracted and treated by limited pepsinization. Then, the labelled collagens were reacted with affinity purified antibodies against collagen types I and II, and the immune complexes precipitated with protein A-containing Staphylococcus aureus. In some cases, the precipitated collagen was further analysed by SDS-polyacrylamide gel electrophoresis. The results for the vitreous showed that greater than 90% of the labelled material precipitated by the antibodies was in type II. In the corneal samples, on the other hand, both types of antibodies precipitated appreciable labelled collagen with somewhat less than half being type II (30–46%). These results are in agreement with our earlier biochemical observations on the relative content of type II collagen in these extracellular matrix-rich structures from embryonic avian eyes.     

A comparative study of elasmobranch corneal and scleral collagens

Corneas, dissected from young and adult spiny dogfish sharks (Squalus acanthias), were prepared for transmission electron microscopy and immunofluorescence. In the latter case, tissues were fixed in formaldehyde solutions, sectioned with a cryostat, incubated with antibodies specific for collagen types I and II, and examined by indirect immunofluorescence. Collagen α- and β- chains were separated by sodium dodecylsulfate-slab gel electrophoresis and characterized by two-dimensional mapping of ¹²⁵I-labeled peptides generated by tryptic and chymotryptic digestion.The corneal stroma, the sutural fibers which span the stroma, and the surrounding limbus were positive for type I collagen, as judged by immunofluorescence. The corneal stroma was negative for type II collagen. Scleral cartilage matrix was intensely positive for type II collagen, but was negative for type I. In addition, the perichondrium of the scleral cartilage was positive for type I collagen. In confirmation of these results, slab gel electrophoresis revealed α1, and α2-like bands from shark corneal stroma, but only an α1-like band from shark cartilage collagen. Two-dimensional peptide mapping revealed some degree of resemblance between the α1 band of shark corneal stroma and the α1 band of chick type I collagen. Likewise, the α1 band of shark cartilage collagen somewhat resembled the α1 band of chick type II collagen. The α2-like band of shark corneal stroma did not closely resemble the α2 band of chick type I collagen. The most prominent β band of shark corneal stroma appeared to be a dimer composed of one α1 chain and one α2 chain. The collagen of shark corneal stroma was very susceptible to degradation by pepsin, whereas that from shark cartilage was much less susceptible.     

Isoelectric focusing of embryonic cow lens crystallins

Soluble lens crystallins from several embryonic stages and also from adult cow lens cortex are compared by thin layer isoelectric focusing, immunoelectrophoresis, immuno-osmophoresis and antigen/antibody crossed electrophoresis. A gradual change in crystallin composition is found between the lens proteins of 1·5–7 month embryos.Thin layer isoelectric focusing studies of various embryonic stages from 1·5 to 7 months show a decrease of γ-crystallins along with an increase of β-crystallins and a slight increase of α-crystallins. When compared with the adult lens nucleus, the cortex shows a sharp decrease in concentration of γ-crystallin components. In addition, the composition of γ-crystallin of embryonic lens is similar to that of the adult lens nucleus, but different from that of the adult lens cortex. This difference appears due to certain components of γ-crystallin that are absent or present in lower amounts in the adult lens cortex compared with the embryonic lens. The same number of α-, β- and γ-crystallin antigens are present, they differ only in the relative concentration of the protein components.In all embryonic stages two pre-α-crystallins could be detected. These pre-α-crystallins have a high anodic mobility and appear to have a molecular weight lower than α-crystallin itself; the pre-α-crystallin is found to be eluted with the low molecular weight β-crystallins in Sepharose gel chromatography.By gel chromatography procedure, crystallins of cow lens cortex could be separated into five fractions; α₁-crystallins of a molecular weight > 2,000,000, α₂-crystallins, two β-crystallin fractions and γ-crystallin. Immuno-osmophoresis analysis shows that α₁-crystallin is present in higher amounts in embryonic lens, and in adult lens nucleus it is higher than in adult lens cortex, while the α₂-crystallin is present both in cortex and nucleus.     

Effects of the Matrix Metalloproteinase Inhibitor GM6001 on the Destruction and Alteration of Epithelial Basement Membrane During the Healing of Post-alkali Burn in Rabbit Cornea

Purpose

To examine the alteration in structure and matrix composition of epithelial basement membrane (BM) during the healing of alkali-burned rabbit cornea, and the roles of matrix metalloproteinases (MMPs) in these alterations.

Methods

The central cornea of one eye of 78 albino rabbits was exposed to 1?N NaOH for 180?s under general and topical anesthesia and allowed to heal with or without subconjunctival injection of GM6001 (an MMP inhibitor). Cryosections of affected corneas were observed by H&;E staining, immunohistochemistry for type IV collagen subtypes, or in situ zymography for detection of localization of MMP activity.

Results

Uninjured corneal epithelial BM exhibited α5 (IV)-immunoreactivity, but lacked the α1/α2-immunoreactivity of collagen IV. Epithelial BM in healing burned cornea transiently exhibited α1/α2-immunoreactivity. Examination by in situ zymography showed an upregulation of MMP activity in the regenerated central epithelium and anterior stroma of the burned corneas at days 7 and 14. GM6001 suppressed degradation of α5-containing epithelial BM in vivo and also in organ culture.

Conclusions

Epithelial BM was degraded by endogenous MMPs during healing following an alkali burn in rabbit cornea. GM6001 had an inhibitory effect on the degradation of the epithelial basement membrane in burned cornea in vivo.?Jpn J Ophthalmol 2006;50:90–95 © Japanese Ophthalmological Society 2006     

Transforming growth factor-β2 induces morphological alteration of human corneal endothelial cells in vitro

AIM:To investigate the morphological altering effect of transforming growth factor-β2 (TGF-β2) on untransfected human corneal endothelial cells (HCECs) in vitro.METHODS: After untransfected HCECs were treated with TGF-β2 at different concentrations, the morphology, cytoskeleton distribution, and type IV collagen expression of the cells were examined with inverted contrast light microscopy, fluorescence microscopy, immunofluorescence or Western Blot.RESULTS:TGF-β2 at the concentration of 3-15 μg/L had obviously alterative effects on HCECs morphology in dose and time-dependent manner, and 9 μg/L was the peak concentration. TGF-β2 (9 μg/L) altered HCE cell morphology after treatment for 36h, increased the mean optical density (P<0.01) and the length of F-actin, reduced the mean optical density (P<0.01) of the collagen type IV in extracellular matrix (ECM) and induced the rearrangement of F-actin, microtubule in cytoplasm and collagen type IV in ECM after treatment for 72h. CONCLUTION:TGF-β2 has obviously alterative effect on the morphology of HCECs from polygonal phenotype to enlarged spindle-shaped phenotype, in dose and time-dependence manner by inducing more, elongation and alignment of F-actin, rearrangement of microtubule and larger spread area of collagen type IV.     

The collagens of the developing bovine cornea

The morphology of the developing bovine eye has been examined and the collagens in fetal bovine eyes from three months' gestation to maturity have been solubilized by pepsin treatment and analyzed to determine the ratios of the predominant types of collagen. The type I collagen decreased, while the type V collagen increased with age. Type III collagen comprised less than 1% of all the corneas, except for the three-month fetal calf. The anterior to posterior thickness of the paraffin-embedded fetal calf cornea increased from the third to the seventh month, decreased from the seventh month to birth, and then increased after birth. Descemet's membrane increased in thickness with age. Analysis of dissected regions of the calf cornea showed a uniform distribution of the collagen populations from the center to the limbus (89% type I, 10% type V and less than 1% type III collagen) and uniformity through the depth of the stroma, except that type III was concentrated around Bowman's layer, and type IV in Descement's membrane. The localization of the different collagens was consistent with the immunofluorescent staining studies with anticollagen antibodies, but the ratios of the intensities of the fluorescence did not correspond to the quantitative analyses. These results are concordant with other studies that have shown that antibody binding may be masked or diminished in certain tissues and therefore immunofluorescence cannot be used reliably for quantitative measurements.     

Fractionation and characterization of the polypeptide chains of low molecular weight calf lens alpha-crystallin

Purified polypeptides of low molecular weight calf alpha-crystallin have been obtained by a combination of affinity chromatography upon a p-aminophenylmercuric acetate-Bio-Gel A-5m column followed by DEAE cellulose chromatography. The affinity chromatography provides a very rapid procedure for obtaining A chains completely free of B polypeptides. Subsequent fractionation upon DEAE cellulose yields pure B₂, B₁, A₂ and A₁. Atypical A chains have also been observed and fractionated. Characterization of the isolated polypeptides indicates that on the basis of size, amino acid composition and fingerprinting of tryptic and chymotryptic peptides, B₂ and B₁ are very closely related as are A₂ and A₁. The data suggest a ratio of approximately 2 : 1 for A to B chains.     

Quantitative analysis of the collagens in the bovine cornea

Calf corneas were treated with pepsin at 4°C in acetic acid and 95% of the tissue was solubilized. The resulting preparation was separated into major collagen fractions by salting out at neutral pH and these fractions were sub-fractionated as necessary to provide a quantitative estimate of the different types of collagen in the tissue. The collagen types were identified by electophoretic analysis of the α-chains and cyanogen bromide peptides. The major component was type I collagen which comprised up to 94% of the solubilized preparation; type II and III collagen were not detected; a fraction which yielded type VI SLS aggregated comprised at least 6%. Gel electrophoretic and solubility studies on the latter fraction showed it is similar to the collagen fraction found by Burgeson et al. (1976) in placental membranes. Examination of the small, pepsin-resistant residue revealed two additional components which may indicate collagen α-chains different from any of the types identified in this study.     

Angiotensin II-induced hypertension regulates AT1 receptor subtypes and extracellular matrix turnover in mouse retinal pigment epithelium

Accumulation of specific deposits and extracellular molecules under the retinal pigment epithelium (RPE) has been previously observed in eyes with age-related macular degeneration (AMD) and may play a role in the pathogenesis of AMD. Even though age is the major determinant for developing AMD, clinical studies have revealed hypertension (HTN) as another systemic risk factor. Angiotensin II (Ang II) is considered the most important hormone associated with HTN. To evaluate the relationship of Ang II to AMD, we studied whether mouse RPE expresses functional Ang II receptor subtypes and whether HTN-induced Ang II regulates expression of these receptors as well as critical ECM molecules (MMP-2 and type IV collagen) involved in ECM turnover in RPE. We used 9-month-old C57BL/6 male mice infused with Ang II alone or Ang II in combination with the AT1 receptor antagonist candesartan or the AT2 receptor antagonist PD123319 for 4 weeks to determine whether HTN-associated Ang II was important for ECM regulation in RPE. We found that mouse RPE expressed both Ang II receptor subtypes at the mRNA and protein levels. Infusion with Ang II induced HTN and elevated plasma and ocular Ang II levels. Ang II also regulated AT1a and AT1b receptor mRNA expression, the intracellular concentration of calcium [Ca²⁺]_i, MMP-2 activity, and type IV collagen accumulation. Concurrent administration of Ang II with the AT1 receptor blocker prevented the increase in blood pressure and rise in ocular Ang II levels, as well as the calcium and MMP-2 responses. In contrast, the type IV collagen response to Ang II was prevented by blockade of AT2 receptors, but not AT1 receptors. Plasma Ang II levels were not modified by the AT1 or AT2 receptor blockade. Since the effects of Ang II on MMP-2 and type IV collagen require inhibition of both Ang II receptor subtypes, these receptors may play a role as a potential therapeutic targets to prevent ECM turnover dysregulation in the RPE basement membrane, suggesting a pathogenic mechanism to explain the link between HTN and AMD.     

Interference of Y-27632 on the signal transduction of transforming growth factor beta type 1 in ocular Tenon capsule fibroblasts

AIM: To investigate the interfering effect of Y-27632, a ROCK-I selective inhibitor, on the signal transduction pathway of transforming growth factor-β1 (TGF-β1) in ocular Tenon capsule fibroblasts (OTFS) in vitro. METHODS: After OTFS from passages 4 to 6 in vitro were induced by TGF-β1 and then treated by Y-27632, the changes of the OTFS cell cycles were analyzed via flow cytometry, and the proteins expression of the α-smooth muscular actin (α-SMA), connective tissue growth factor (CTGF), collagen I were calculated by Western blot. After OTFS treated by the different concentrations of Y-27632, the expression levels of the α-SMA, CTGF and collagen I mRNA were assayed by RT-PCR. RESULTS: Y-27632 had no markedly effect on the OTFS cell cycles. After treated by TGF-β1, OTFS in G1 period significantly increased. The cell cycles distribution by both TGF-β1 and Y-27632 had no remarkable difference from that in control group. Y-27632 significantly inhibited the proteins expressions of both α-SMA and CTGF, while to some extent inhibited that of collagen I. TGF-β1 significantly promoted the proteins expressions of α-SMA, CTGF and collagen I. After OTFS treated by both TGF-β1 and Y-27632, of α-SMA, the protein expression was similar with that in control group (P=0.066>0.05), but the protein expression of CTGF or collagen I, respectively, was significantly different from that in control group (P=0.000<0.01). The differences of expressions of the α-SMA, CTGF and collagen I mRNA in 30, 150, 750μmol/L Y-27632 group were statistically significant, compared with those in control group, respectively (α-SMA, P=0.002, 0.000, 0.000; CTGF, P=0.014, 0.002, 0.001; collagen I, P=0.003, 0.002, 0.000). CONCLUSION: Blocking the Rho/ROCK signaling pathway by using of Y-27632 could inhibit the cellular proliferation and the expression of both CTGF and α-SMA whatever OTFS induced by TGF-β1 or not. Y-27632 suppressed the expression of collagen I mRNA without induction.     

Identification of collagens isolated from bovine Descemet's membrane

In this study collagens were isolated and identified from a morphologically pure preparation of bovine Descemet's membrane (DM) which was obtained by gentle scraping of the cornea, sieving and subsequent treatment with detergents. An alternative procedure of DM isolation with forceps resulted in stromal contamination of the preparation as verified by light and transmission electron microscopy, and gel electrophoresis. The amino acid profile of collagens isolated by pepsinization and salt precipitation from the pure sample was similar to the analysis of the intact bovine DM. Polyacrylamide gel electrophoresis of this collagen under non-reducing conditions resulted in five major bands: 300 000 daltons (300 K), 200 000 daltons (200 K), 100 000 daltons (100 K) and lower molecular weight fractions (50 K1 and 50 K2). Individual collagen chains were isolated from preparative polyacrylamide gels and characterized by 125I two dimensional peptide mapping patterns. This data suggests that (1) the majority of collagen fragments seen in bovine DM pepsin supernatant are derived from a single genetically distinct collagen molecule, and (2) that type I and V collagens are not major components of bovine DM.     

Collagen composition and turnover in ocular tissues of the rabbit

The changes in the collagen composition of the rabbit sclera, cornea and eyelid with maturation were determined. Type I collagen was the major constituent. The cornea, sclera and eyelid contained about 2%, 7% and 25% type III respectively after birth, dropping to 0, 0 and 5% in the mature rabbit. The AB collagen in the sclera and eyelid decreased from about 2% and 3% to near 0% during the same time period. The AB collagen in the cornea, however, increased from about 5% at birth to about 9% at 4 years of age. A pulse of [³H]proline into the collagen of the cornea of newborn rabbits was used to demonstrate that the collagen on average has a half-life of 50 hr or less.     

Expressions of type I collagen, α2 integrin and β1 integrin in sclera of guinea pig with defocus myopia and inhibitory effects of bFGF on the formation of myopia

AIM: Toinvestigate the expressions of type I collagen, α2 integrin and β1 integrin in the posterior sclera of guinea pigs with defocus myopia and whether basic fibroblast growth factor (bFGF) injection inhibits the formation and development of myopia by upregulating the expression of type I collagen, α2 integrin and β1 integrin.METHODS: After 14 days of treatment, the refractive state and axial length were measured and the levels of type I collagen, α2 integrin and β1 integrin were assayed in the posterior sclerae of groups of guinea pigs that wore a monocular -7D polymethylmethacrylate (PMMA) lens or had -7D lens wear followed by the peribulbar injection of Phosphate Buffer Solution (PBS) or bFGF. The untreated fellow eye served as a control. Guinea pigs with no treatment served as normal group.RESULTS: The results showed that 14 days of monocular defocus increased axial eye length and refraction, while bFGF delivery inhibited them markedly. Further, it was also found that the monocular -7D lens could decrease the levels of type I collagen, α2 integrin and β1 integrin expressions, while, unlike PBS, bFGF increased them significantly in comparison to contralateral control eyes and normal eyes.CONCLUSION: bFGF can prevent the formation and development of defocus myopia by upregulating the expressions of type I collagen, α2 integrin and β1 integrin. Taken together, our results demonstrate that bFGF promotes sclera remodeling to prevent myopia in guinea pigs.     

Characterization of the Descemet's membrane/posterior collagenous layer isolated from Fuchs' endothelial dystrophy corneas

The combined Descemet's membrane (DM) and posterior collagenous layer (PCL) of Fuchs' endothelial dystrophy corneas were isolated and characterized by biochemical and immunofluorescence methods. The amino acid composition of the Fuchs' DM-PCL was similar to age-matched normal Descemet's membranes (DM). As determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and ¹²⁵I two-dimensional peptide mapping, normal DM and Fuchs' DM-PCL contained the same collagen types [type IV and endothelial cell (EC) collagen], but a slight discrepancy was seen in the electrophoretic mobility of some collagen chains. Immunofluorescence staining localized fibrinogen/fibrin to Fuchs' DM-PCL but not to normal DM. These data suggest (1) that the appearance of 110 nm banded material in sheets and fusiform bundles characteristic of Fuchs' PCL is not due to the presence of a new (abnormal) collagen type but may represent altered assembly of collagen molecules, and (2) that the fibrinolytic system may play a role in the degenerative process of Fuchs' endothelial dystrophy.