Altered Collagen Fibril Formation in the Sclera of Lumican-Deficient Mice (original) (raw)
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Ocular and Scleral Alterations in Gene-Targeted Lumican-Fibromodulin Double-Null Mice
2000
To elucidate the role of leucine-rich proteoglycans lumican and fibromodulin in the sclera. METHODS. Lumican-and fibromodulin-null heterozygous mice were intercrossed to obtain wild-type (Lum ϩ/ϩ Fmod ϩ/ϩ ), lumican-null (Lum Ϫ/Ϫ Fmod ϩ/ϩ ), fibromodulin-null (Lum ϩ/ϩ Fmod Ϫ/Ϫ ), and double-null (Lum Ϫ/Ϫ Fmod Ϫ/Ϫ ) littermates. Axial length was measured on enucleated whole eyes, and ocular structural changes were examined by histology. The morphology of collagen fibrils in the sclera was examined by transmission electron microscopy (TEM). RESULTS. Compared with the ocular axial length in wild type mice, the axial length was increased by 10% in Lum Ϫ/Ϫ Fmod Ϫ/Ϫ (P ϭ 0.02) mice. Retinal detachment was frequent in the double-null and rare in the lumican-null animals. Compared with the wild-type sclera, the sclera in all null mutants was significantly thinner with fewer lamellae (P Ͻ 0.05). The double-null sclera contained abnormally large-diameter (120 -160 nm) and small-diameter (30 -60 nm) collagen fibrils, whereas the fibromodulin-null sclera was enriched for the small-diameter fibrils. The collagen fibril diameter distribution in the lumican-null sclera was similar to that of the wild-type.
Developmental Dynamics, 2006
The transparent cornea is the outer barrier of the eye and is its major refractive surface. Development of a functional cornea requires a postnatal maturation phase involving development, growth and organization of the stromal extracellular matrix. Lumican, a leucine-rich proteoglycan, is implicated in regulating assembly of collagen fibrils and the highly organized extracellular matrix essential for corneal transparency. We investigated the regulatory role(s) of lumican in fibril assembly during postnatal corneal development using wild type (Lum ϩ/ϩ ) and lumican-null (Lum Ϫ/Ϫ ) mice. In Lum ϩ/ϩ mice, a regular architecture of small-diameter fibrils is achieved in the anterior stroma by postnatal day 10 (P10), while the posterior stroma takes longer to reach this developmental maturity. Thus, the anterior and the posterior stroma follow distinct developmental timelines and may be under different regulatory mechanisms. In Lum Ϫ/Ϫ mice, it is the posterior stroma where abnormal lateral associations of fibrils and thicker fibrils with irregular contours are evident as early as P10. In contrast, the anterior stroma is minimally perturbed by the absence of lumican. In Lum ϩ/ϩ mice, lumican is expressed throughout the developing stroma at P10, with strong expression limited to the posterior stroma in the adult. Therefore, the posterior stroma, which is most vulnerable to lumican-deficiency, demonstrates an early developmental defect in fibril structure and architecture in the Lum Ϫ/Ϫ mouse. These defects underlie the reported increased light scattering and opacity detectable in the adult. Our findings emphasize the early regulation of collagen structure by lumican during postnatal development of the cornea. Developmental Dynamics 235: 2493-2506, 2006.
Keratocan, a Cornea-specific Keratan Sulfate Proteoglycan, Is Regulated by Lumican
Journal of Biological Chemistry, 2005
Lumican is an extracellular matrix glycoprotein widely distributed in mammalian connective tissues. Corneal lumican modified with keratan sulfate constitutes one of the major proteoglycans of the stroma. Lumican-null mice exhibit altered collagen fibril organization and loss of corneal transparency. A closely related protein, keratocan, carries the remaining keratan sulfate of the cornea, but keratocan-null mice exhibit a less severe corneal phenotype. In the current study, we examined the effect of lumican overexpression in corneas of wild type mice. These mice showed no alteration in collagen organization or transparency but had increased keratocan expression at both protein and mRNA levels. Corneas of lumican-null mice showed decreased keratocan. This coupling of keratocan expression with lumican also was observed after intrastromal injection of a lumican expression minigene into the corneal stroma of Lum -/mice. Small interfering RNA knockdown of lumican in vitro reduced keratocan expression, whereas co-injection of a lumican-expressing minigene with a β-galactosidase reporter driven by the keratocan promoter demonstrated an increase of keratocan transcriptional activity in response to lumican expression in Lum -/corneas in vivo. These observations demonstrate that lumican has a novel regulatory role in keratocan expression at the transcriptional level. Such results help provide an explanation for the differences in severity of corneal manifestation found in Lum -/and Kera -/mice. The results also suggest a critical level of small proteoglycans to be essential for collagen organization but that overabundance is not detrimental to extracellular matrix morphogenesis.
Collagens and proteoglycans of the corneal extracellular matrix
Brazilian Journal of Medical and Biological Research, 2003
The cornea is a curved and transparent structure that provides the initial focusing of a light image into the eye. It consists of a central stroma that constitutes 90% of the corneal depth, covered anteriorly with epithelium and posteriorly with endothelium. Its transparency is the result of the regular spacing of collagen fibers with remarkably uniform diameter and interfibrillar space. Corneal collagen is composed of heterotypic fibrils consisting of type I and type V collagen molecules. The cornea also contains unusually high amounts of type VI collagen, which form microfibrillar structures, FACIT collagens (XII and XIV), and other nonfibrillar collagens (XIII and XVIII). FACIT collagens and other molecules, such as leucine-rich repeat proteoglycans, play important roles in modifying the structure and function of collagen fibrils. Proteoglycans are macromolecules composed of a protein core with covalently linked glycosaminoglycan side chains. Four leucine-rich repeat proteoglycans are present in the extracellular matrix of corneal stroma: decorin, lumican, mimecan and keratocan. The first is a dermatan sulfate proteoglycan, and the other three are keratan sulfate proteoglycans. Experimental evidence indicates that the keratan sulfate proteoglycans are involved in the regulation of collagen fibril diameter, and dermatan sulfate proteoglycan participates in the control of interfibrillar spacing and in the lamellar adhesion properties of corneal collagens. Heparan sulfate proteoglycans are minor components of the cornea, and are synthesized mainly by epithelial cells. The effect of injuries on proteoglycan synthesis is discussed.
Changes in scleral collagen organization in murine chronic experimental glaucoma
Investigative ophthalmology & visual science, 2014
The organization of scleral collagen helps to determine the eye's biomechanical response to intraocular pressure (IOP), and may therefore be important in glaucoma. This study provides a quantitative assessment of changes in scleral collagen fibril organization in bead-induced murine experimental glaucoma. Wide-angle X-ray scattering was used to study the effect of bead-induced glaucoma on posterior scleral collagen organization in one eye of 12 CD1 mice, with untreated fellow eyes serving as controls. Three collagen parameters were measured: the local preferred fibril directions, the degree of collagen anisotropy, and the total fibrillar collagen content. The mouse sclera featured a largely circumferential orientation of fibrillar collagen with respect to the optic nerve head canal. Localized alteration to fibril orientations was evident in the inferior peripapillary sclera of bead-treated eyes. Collagen anisotropy was significantly (P<0.05) reduced in bead-treated eyes in th...
Stability of collagen phenotype in morphologically modulated rabbit corneal endothelial cells
Investigative Ophthalmology & Visual Science
Primary monolayer cultures of rabbit corneal endothelial cells maintained characteristic polygonal morphology and synthesized type IV collagen as a major collagenous peptide. Upon serial passage, [ 3 H|-proline incorporation into proteins gradually decreased from the secondary subculture to a low of 71% of that in the primary culture in the fourth subculture, while type IV collagen synthesis significantly decreased from the tertiary subculture, and the fourth subculture retained only 22% of the primary culture value. When collagen molecules synthesized by the fourth subculture progeny were analyzed by SDS electrophoresis, no alteration was noted. The fourth subculture progeny continued to synthesize the characteristic type IV collagen, which migrates slightly faster than the "12 (I) band on SDS electrophoresis. However, the cellular morphology was changed dramatically from the characteristic polygonal shape to enlarged cells in the fourth subculture and mitotic activity was no longer apparent. Immunofluorescence studies showed that the enlarged fourth subculture progeny stained with anti-IV collagen antibodies, as did the primary polygonal endothelial cells. These findings confirmed type IV collagen synthesis in the enlarged endothelial cells. In addition, the morphologically altered cells continously deposited Descemet's membrane-like extracellular matrices between the basal cell layer and the plastic substratum. These results suggest that endothelial cells lose their proliferative capacity as they age, but compensate for the loss of cell numbers by enlargement while maintaining a normal collagen phenotype. Invest Ophthalmol Vis Sci 25:495-501, 1984 495 496
Structural Abnormalities of the Cornea and Lid Resulting from Collagen V Mutations
Investigative Ophthalmology & Visual Science, 2006
PURPOSE. Type V collagen forms heterotypic fibrils with type I collagen and accounts for 10% to 20% of corneal collagen. The purpose of this study was to define the ocular phenotype resulting from mutations in the type V collagen genes COL5A1 and COL5A2 and to study the pathogenesis of anomalies in a Col5a1-deficient mouse. METHODS. Seven patients with classic Ehlers-Danlos syndrome (EDS) due to COL5A1 haploinsufficiency and one with an exon-skipping mutation in COL5A2 underwent an ocular examination, corneal topography, pachymetry, and specular microscopy. A Col5a1-haploinsufficient mouse model of classic EDS was used for biochemical and immunochemical analyses of corneas. Light and electron microscopy were used to quantify stromal thickness, fibril density, fibril structure, and diameter. RESULTS. Five males and three females (mean age, 26 Ϯ 13.57 years; range, 11-52) were studied. All patients had "floppy eyelids." The corneas of all eyes were thinner (mean corneal thickness: 435.75 Ϯ 12.51 m) when compared with control corneas (568.89 Ϯ 28.46 m; P Ͻ 0.0001). In the Col5a1 ϩ/Ϫ mouse cornea, type V collagen content was reduced by ϳ49%, and stromal thickness was reduced by ϳ26%. Total collagen deposition in Col5a1 ϩ/Ϫ corneas also was reduced. Collagen fibril diameters were increased, but fibril density was decreased throughout the stroma at all developmental stages. CONCLUSIONS. In the eye, COL5A1 and COL5A2 mutations manifest as abnormally thin and steep corneas with floppy eyelids. Mechanisms involved in producing the latter anomalies probably involve altered regulation of collagen fibrillogenesis due to abnormalities in heterotypic type I/V collagen interactions sim-ilar to those observed in the Col5a1 ϩ/Ϫ mouse cornea. (Invest Ophthalmol Vis Sci. 2006;47:565-573) FIGURE 5. (A) Type V collagen was localized in wild-type (ϩ/ϩ) and haploinsufficient (ϩ/Ϫ) corneas using immunofluorescence microscopy. The haploinsufficient stromas have decreased reactivity for type V collagen relative to the wild-type control subject. Right: exposures of the same sections to DAPI, a nuclear counterstain. The controls without primary antibody were negative (not shown). Ep, epithelium; S, stroma. (B) Type V collagen expression in wild-type (ϩ/ϩ) and haploinsufficient (ϩ/Ϫ) corneas was analyzed using semiquantitative Western analyses. The haploinsufficient corneas had a 49% (34%-79%) reduction in type V collagen relative to the wild-type control subjects. Corneas from 5-month-old mice were extracted in equal volumes and serial dilutions, representing constant fractions of the corneas, were blotted with the anti-␣1(V) antibody. Numbers represent the corneal fraction loaded per well. 570 Segev et al.